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141 Chapter 3
[1 2-a] pyridin-3-yl] methanol
Imp-5
Imp-7
32 Literature Search
A thorough literature search revealed that only a few HPLC methods had
been discussed for the determination of ZT in pharmaceutical preparations [7ndash
10] Quantifications of ZT by liquid chromatographic methods have been
officially published in USP and EP [11-12] A potentiometric method was
reported for the estimation of ZT in human serum and tablet formulation [13]
determination of ZT tablets by means of spectrophotometric methods had been
published [14-15] Two methods describing the testing of zolpidem in oral fluid
using Liquid chromatographyndashtandem mass spectrometric methods have been
reported [16-17] HPLC method pertaining to chromatographic behavior of ZT
and its degradation products has been published [18]
Only a few stability-indicating HPLC methods are reported in the literature
The official monograph methods are not capable of separating the potential
impurities and degradation products The existing isocratic HPLC methods
cannot give adequate resolution between Imp-5 amp Imp-6 Furthermore the
approximate run time is 35 min According to our extensive survey there is no
single analytical method reported in the literature that is capable of separating
all the potential impurities and degradation products of ZT in drug substance
and drug product at shorter run times
142 Chapter 3
33 Ultra Performance Liquid Chromatography (UPLC)
Ultra-performance liquid chromatography (UPLC) is a new class of
separation technique based upon deep-rooted principles of liquid
chromatography which utilizes sub-2microm particles for column stationary phase
(CSP) These particles function at elevated mobile phase linear velocities
resulting in an amazing increase in resolution sensitivity and speed of
analysis
The increase in the number of impurities in the drug poses challenges in
regular chromatographic method development while achieving adequate
resolutions at shorter run times However recent advances in the use of LC
technology have significantly facilitated progress to prevail over these
challenges UPLC is such an advancement that accentuates chromatographic
high throughput in terms of speed sensitivity and resolution Because of these
attributes UPLC has gained immense importance in pharmaceutical analysis
Hence the present work is aimed at developing a selective fast cost-effective
and stability indicating method using the contemporary UPLC technique and
subsequently the method is validated as per ICH guidelines [19]
34 pH Gradient
A theory of pH gradient has been applied in the current work A
programmed increase of eluent strength during chromatographic procedure by
altering mobile phase pH is known in the prior art [20-21] A narrow range
143 Chapter 3
pH gradient together with organic modifier has been used in the present UPLC
method to achieve separation between the critical pairs
35 Experimental
351 Chemicals and Reagents
Zolpidem tartrate and its related compounds were obtained from Dr
Reddyrsquos Laboratories Limited IPDO Hyderabad India Commercially available
brand Ambienreg tablets were used for dosage form analysis Analytical grade
sodium dihydrogen ortho phosphate monohydrate (NaH2PO4H2O) ortho-
phosphoric acid (H3PO4) HPLC-grade solvents ie methanol (MeOH) and
acetonitrile (ACN) were procured from Merck Darmstadt Germany
Triethylamine (TEA) was bought from JT Baker Mallinckrodt Inc Phillipsburg
NJ USA High purity water has been prepared by using Millipore Milli Q plus
water purification equipment (Millipore Corporate Headquarters Billerica MA)
352 Chromatographic Conditions and Equipment
Liquid chromatographic analysis was performed on Waters Acquity UPLC
equipped with photodiode array detector (PDA) at a wavelength of 254 nm using
Empower-2 software Separation was achieved on C18 column ie Acquity
UPLC HSS T3-C18 of dimensions 100 x 21mm 18microm at a flow rate of 03
mLmin using a gradient program (TB) set as 0015 255 3560 5060
8270 855 and 1005 Buffer solution was prepared by adding 1mL of
Triethyl amine (TEA) to 1000mL of 10 mM NaH2PO4 H2O Mobile phase A was a
mixture of buffer (pH adjusted to 55 with H3PO4) ACN and MeOH in the ratio
of 602020 (vvv) Mobile phase B was a mixture of buffer (pH adjusted to 73
144 Chapter 3
with H3PO4) and ACN in the ratio of 4555 (vv) The column oven temperature
was maintained at 25ordmC The injection volume was 10 microL
353 LC-MSMS conditions
An LCndashMSMS system (Agilent 1100 series liquid chromatograph coupled
with Applied Biosystems 4000 Q Trap triple quadrupole mass spectrometer with
Analyst 14 software MDS SCIEX USA) was utilized for mass identification of
degradation products A Novapak C18 column with dimensions 150 x 39 mm
4μm was employed as a stationary phase An ammonium acetate (Merck
Darmstadt Germany) solution of concentration 002 mM was used as buffer A
mixture of Ammonium acetate buffer MeOH and ACN (590230180 vvv) was
used as mobile phase The column oven temperature was maintained at 25degC
ACN was used as diluent The flow rate was set as 15 mLmin The analysis
was carried out in positive electro-spraypositive ionization mode the ion
source voltage was 5000 V and the source temperature was 450degC GS1 and
GS2 were set to 30 and 35 psi respectively The curtain gas flow was set at 20
psi
36 Preparation of solutions
361 Preparation of Stock solution
A stock solution of ZT of concentration 500 microgmL was prepared by
dissolving an appropriate quantity of the drug in a diluent containing a mixture
of acetonitrile and water in the ratio of 8020 (vv) Working solutions of
concentrations 50microgmL and 5microgmL were prepared from this stock solution
for the determination of related compounds and assay respectively Composite
145 Chapter 3
and individual stock solutions of impurities having concentration 05microgmL
each were prepared in the diluent
362 Preparation of sample solution
Brand Ambienreg 10 mg of quantity 20 tablets was weighed and the average
tablet weight was determined These tablets were transferred into a clean dry
mortar and grounded into fine powder The fine powdered sample equivalent to
50 mg of the drug was weighed and dissolved in 100 mL of diluent (MeOH
Buffer 7030 vv) to make a solution of concentration 500microgmL The solution
was subjected to sonication for 30min and mechanical shaking for each 30
minutes The solution was filtered from which a 10mL of filtered solution was
diluted to 10 mL with the aid of mobile phase A The resulting solution of
concentration 50microgmL was filtered through 022μm nylon membrane filter
The solution was then analyzed in UPLC for related substances analysis The
solution was further diluted to obtain the concentration of 5microgmL for assay
determination Similar concentration solutions were prepared for API analysis
363 Generation of Stress sample solutions
A qualified sample of ZT has been chosen to conduct the stress study
According to ICH stability guidelines (Q1AR2) stress studies are likely to be
performed to study the intrinsic stability of the molecule ZT has been exposed
to various stress conditions such as heat light acid (HCl) base (NaOH)
oxidative (H2O2) and water hydrolysis The final stress conditions were tabulated
below
Table 31 Stress study conditions for ZT
146 Chapter 3
37 Method development
371 Objectives of method development
1 Rapid separation of ZT and its eight potential impurities
2 Identification of possible degradation products by means of LCMS analysis
and evaluation of peak purity
SNo Stressed Agent Stressing Condition
1 Heat
API and tablets were exposed to dry heat
at 105degC for about 7 days
2 Light API and tablets were exposed to UV light
at 254nm for 7 days
3 Acid (5N HCl) API and tablets extracted solutions were
treated with 5N HCl 60degC for 24 hrs
4 Base (5N NaOH)
API and tablets solutions were treated
with 5N NaOH at 60degC for 90mins
5 Oxidation (50 H2O2) API and tablets solutions were treated
with 5 H2O2 at 60degC for 2 hrs
6 Water hydrolysis API and tablets solutions were treated
with water at 60degC for 24h
147 Chapter 3
3 Single analytical method for the determination of assay and related
substances in bulk actives and dosage forms
4 Targeted for a minimum resolution of 15 between impurities and tailing
factor lt 20 for ZT peak at assay method
5 Aimed at LOQ values ie 0375microgmL (50 of the specification level which
is equivalent to 0375microgmL) for all the impurities of ZT
372 Method development strategy
A systematic method development approach had been followed to achieve
successful separation The key steps are mentioned below
Fig 33 Flow diagram of method development strategy
148 Chapter 3
373 Classification of the sample
Zolpidem tartrate and its properties
1 Chemical name N N 6-trimethyl-2-p-tolylimidazo [1 2-a] pyridine-3-
acetamide L-(+)-tartrate (21)
2 Chemical structure The polar groups in the structure are highlighted in
circles
3 Molecular weight 76488 as tartrate salt
30739 as base
4 Solubility Slightly soluble in water and sparingly soluble in
alcohol
5 pKa 62
6 Log P 12
7 Nature of the
molecule
Basic in nature and ionic compound
8 UV sensitivity
UV active since ZT has phenyl ring systems with
extended conjugated double bonds in its chemical
structure
149 Chapter 3
374 Impurity details
Zolpidem tartrate has eight potential impurities as mentioned in the section
31 The source and classifications of the impurities are tabulated below Table
32
Table 32 Origin of impurities for ZT
SNo Name of the impurity Source of impurity
Process related Degradation related
1 Imp-1 No Yes
2 Imp-2 Yes Yes
3 Imp-3 Yes No
4 Imp-4 Yes Yes
5 Imp-5 Yes No
6 Imp-6 Yes No
7 Imp-7 Yes No
8 Imp-8 Yes No
375 Preliminary chromatographic conditions
3751 Selection of detection wavelength
A composite solution containing 50 μgmL of drug and 1 μgmL of each of
the eight impurities was prepared in the diluent All the samples were analyzed
in HPLC- PDA system and the UV spectrums for all the components were
extracted
150 Chapter 3
Fig 34 Individual and overlaid UV spectra of ZT and its impurities
151 Chapter 3
152 Chapter 3
153 Chapter 3
Selection of wavelength to monitor zolpidem tartrate and its impurities
An optimal wavelength of detection for ZT and its impurities was selected as
254 nm based on UV spectra using PDA detector for LC analysis Fig 34
3752 Selection of UPLC column
Initial experiments were carried out on different commercially available RP-
UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH
shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to
determine the selectivity and symmetry of the individual components In all the
columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline
separation and imp-5 amp imp-6 peaks were merged The remaining peaks were
separated with resolution (RS)gt15 and the peak tailing was greater than 15 for
ZT Due to the structural similarities co-elution of above mentioned impurity
0970 Imp-1
1134 Imp-2
1978 Imp-3
2486 Imp-4
3443 Imp-5
3619 Imp-6
3897 ZT
4796 Imp-7
5671 Imp-8
2631
3438
20672114
3200
3578
3057
3710
2114
2443
3854
2443
3105
2114
2443
3057
nm
22000 24000 26000 28000 30000 32000 34000 36000 38000
mA
u
154 Chapter 3
pairs was observed Additionally peak tailing was also observed due to the
secondary interactions with residual silanol groups Although separation was
similar in all the three columns HSS T3 column had shown relatively good
peak symmetry and efficiency so it was thus selected for further optimization
Addition of triethylamine in the buffer improved the peak shape of ZT but the
resolution between imp-5 amp imp-6 remained critical
3753 Effect of buffer pH
The important parameters buffer pH and gradient mode of elution that
would likely to have significant effect on critical pair resolution were
investigated There was no substantial change in the resolution between Imps-1
amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly
decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp
6 (RS2) and Imp-6 amp ZT (RS
3) were improved when the pH altered from 60 to 70
The increase in Rs1 in acidic mobile phase and RS
2 RS3 in basic mobile phase
can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of
adequately low pH will remain unionized and get increased retentions On the
other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be
more retained From table 33 acceptable resolutions were obtained at pH 70
with isocratic elution and pH 75 with gradient elution but in both the cases
the run time was longer
155 Chapter 3
Table 33 Effect of Buffer pH on resolution of critical pairs
Mode of
elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time
Isocratic 40 21 lt10 17 15min
Isocratic 50 22 lt10 17 14min
Isocratic 60 21 11 18 15min
Isocratic 70 20 18 22 gt15min
Gradient 55 21 lt10 17 8min
Gradient 75 18 22 23 gt15min
Gradient A 55 B
73 25 21 22 8 min
a Rs1 Resolution between Imp-1 and Imp-2
b Rs2 Resolution between Imp-5 and Imp-6
c Rs3Resolution between Imp-6 and ZT
3754 Optimization of gradient program
Gradient mode of elution containing mobile phase-A (1mL of TEA1L
phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile
phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555
vv) were chosen to separate both the critical pairs in a single run and this
resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)
156 Chapter 3
Fig 35 Co elution of imp-5 and imp-6 at pH 55
Based on the experimental data it was predicted that increase in mobile
phase pH during the gradient elution could resolve the Imp-5 and Imp-6
without compromising the total run time Hence two more experiments were
designed with a mobile phase-B as combination of water ACN (4555 vv) for
experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-
2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase
the solvent strength thereby to elute strongly retained compounds (Imp-8)
faster The RS2 was less than 10 for experiment-1 and RS
2 was 15 for
experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value
60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from
60 to 70) between 50min and 82min was added to the program to reduce the
run time So Imp-8 was eluted at 8min To further improve the RS2 gradient
program was optimized with a linear increase of mobile phase-B with varying
157 Chapter 3
time points An optimum resolution was obtained (Fig 36) when the gradient
program of section 22 was used In addition to this the elution time of imp-8
was reduced to 6 min in the modified gradient method
Fig 36 Baseline separations of zolpidem and its impurities
Placebo interference study was verified by injecting sample solutions of
placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-
8 and ZT analyte peak was found
Fig 37 Placebo chromatogram for zolpidem tablets
Imp-
1 - 1
051
Imp-
2 - 1
231
Imp-
3 - 2
139
Imp-
4 - 2
692
Imp-
5 - 3
720
Imp-
6 - 3
892
ZT -
414
7
Imp-
7 - 5
096
Imp-
8 - 5
981
AU
-0020
-0015
-0010
-0005
0000
0005
0010
0015
0020
0025
0030
0035
0040
0045
0050
0055
0060
Minutes
050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850
158 Chapter 3
Three different lots of mobile phases were prepared and the consistency in
the resolution was verified (Table 34) A system suitability check (RS1 RS
2 amp
RS3) was incorporated to ensure the adequate system performance during the
regular analysis and method validation
Table 34 Reproducibility in separation of ZT and its impurities
Compound Average
RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing
factor
(n=6)c (n=6)c
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT) of
Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6)
159 Chapter 3
Fig 38 Peak purity plot for ZT in optimized conditions
Where PA = Purity angle and TH = Purity threshold
3755 The finalized RP-UPLC conditions for the estimation of assay and
related substances of ZT
Apparatus An UPLC with VWD and integrator
Column Acquity UPLC HSS T3 C-18 100 mm length
21 mm id 18 micron particle size
Flow Rate 03 ml min
Wave length 254 nm
Load 10microL
Column temperature 250C
Diluent Acetonitrile (ACN) Water (82)
Buffer Buffer solution was prepared by adding 1mL
of Triethyl amine (TEA) to 1000mL of 10 mM
NaH2PO4 H2O
Mobile phase-A Mixture of buffer (pH adjusted to 55 with
H3PO4) ACN and MeOH in the ratio of
602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with
H3PO4) and ACN in the ratio of 4555 (vv)
160 Chapter 3
Table 35 Gradient program
Time Mobile phase-A Mobile phase -B
001 95 5
25 95 5
35 40 60
50 40 60
82 30 70
85 95 5
100 95 5
Table 36 Retention times and relative retention times wrto ZT
SNo ImpurityAnalyte
name
Retention time ~Relative
retention time
1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100
8 Imp-7 5118 121 9 Imp-8 6037 142
3756 Degradation behavior
The purpose of this study is to establish the fact that the inherent chemical
stability of the molecule remains intact during its existence in the solid dosage
form along with other excipients So forced degradation studies should be
conducted on the drug to generate product related variants which in turn can
help to establish degradation pathways and thereby to develop and validate
suitable analytical procedure [22]
In this perspective a series of induced degradation experiments were
conducted at a concentration of 50 μgmL of ZT in active pharmaceutical
161 Chapter 3
ingredients (API) and tablets Degradation was induced by external physico-
chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)
oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal
treatment (105oC for 7 days)
The purity of the peaks obtained from the stressed samples was verified by
using the PDA detector The purity angle and purity threshold parameters were
evaluated for all the stressed samples to demonstrate the homogeneity of the
analyte peaks Assay of stressed samples was performed by comparison with
reference standards and the mass balance was calculated
37561 Degradation in Acidic solution
A single major degradation product Imp-2 (Zolpidem Acid) was formed when
ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The
degradation pathway is based on the premise that acid or base catalyzed
reaction between water and tertiary amide gives its parent carboxylic acid and
an amine Here the other hydrolytic product was dimethylamine Using LC-MS
in positive ion ESI the degradation product was found to have an mz value of
281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2
and UV spectral match
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
142 Chapter 3
33 Ultra Performance Liquid Chromatography (UPLC)
Ultra-performance liquid chromatography (UPLC) is a new class of
separation technique based upon deep-rooted principles of liquid
chromatography which utilizes sub-2microm particles for column stationary phase
(CSP) These particles function at elevated mobile phase linear velocities
resulting in an amazing increase in resolution sensitivity and speed of
analysis
The increase in the number of impurities in the drug poses challenges in
regular chromatographic method development while achieving adequate
resolutions at shorter run times However recent advances in the use of LC
technology have significantly facilitated progress to prevail over these
challenges UPLC is such an advancement that accentuates chromatographic
high throughput in terms of speed sensitivity and resolution Because of these
attributes UPLC has gained immense importance in pharmaceutical analysis
Hence the present work is aimed at developing a selective fast cost-effective
and stability indicating method using the contemporary UPLC technique and
subsequently the method is validated as per ICH guidelines [19]
34 pH Gradient
A theory of pH gradient has been applied in the current work A
programmed increase of eluent strength during chromatographic procedure by
altering mobile phase pH is known in the prior art [20-21] A narrow range
143 Chapter 3
pH gradient together with organic modifier has been used in the present UPLC
method to achieve separation between the critical pairs
35 Experimental
351 Chemicals and Reagents
Zolpidem tartrate and its related compounds were obtained from Dr
Reddyrsquos Laboratories Limited IPDO Hyderabad India Commercially available
brand Ambienreg tablets were used for dosage form analysis Analytical grade
sodium dihydrogen ortho phosphate monohydrate (NaH2PO4H2O) ortho-
phosphoric acid (H3PO4) HPLC-grade solvents ie methanol (MeOH) and
acetonitrile (ACN) were procured from Merck Darmstadt Germany
Triethylamine (TEA) was bought from JT Baker Mallinckrodt Inc Phillipsburg
NJ USA High purity water has been prepared by using Millipore Milli Q plus
water purification equipment (Millipore Corporate Headquarters Billerica MA)
352 Chromatographic Conditions and Equipment
Liquid chromatographic analysis was performed on Waters Acquity UPLC
equipped with photodiode array detector (PDA) at a wavelength of 254 nm using
Empower-2 software Separation was achieved on C18 column ie Acquity
UPLC HSS T3-C18 of dimensions 100 x 21mm 18microm at a flow rate of 03
mLmin using a gradient program (TB) set as 0015 255 3560 5060
8270 855 and 1005 Buffer solution was prepared by adding 1mL of
Triethyl amine (TEA) to 1000mL of 10 mM NaH2PO4 H2O Mobile phase A was a
mixture of buffer (pH adjusted to 55 with H3PO4) ACN and MeOH in the ratio
of 602020 (vvv) Mobile phase B was a mixture of buffer (pH adjusted to 73
144 Chapter 3
with H3PO4) and ACN in the ratio of 4555 (vv) The column oven temperature
was maintained at 25ordmC The injection volume was 10 microL
353 LC-MSMS conditions
An LCndashMSMS system (Agilent 1100 series liquid chromatograph coupled
with Applied Biosystems 4000 Q Trap triple quadrupole mass spectrometer with
Analyst 14 software MDS SCIEX USA) was utilized for mass identification of
degradation products A Novapak C18 column with dimensions 150 x 39 mm
4μm was employed as a stationary phase An ammonium acetate (Merck
Darmstadt Germany) solution of concentration 002 mM was used as buffer A
mixture of Ammonium acetate buffer MeOH and ACN (590230180 vvv) was
used as mobile phase The column oven temperature was maintained at 25degC
ACN was used as diluent The flow rate was set as 15 mLmin The analysis
was carried out in positive electro-spraypositive ionization mode the ion
source voltage was 5000 V and the source temperature was 450degC GS1 and
GS2 were set to 30 and 35 psi respectively The curtain gas flow was set at 20
psi
36 Preparation of solutions
361 Preparation of Stock solution
A stock solution of ZT of concentration 500 microgmL was prepared by
dissolving an appropriate quantity of the drug in a diluent containing a mixture
of acetonitrile and water in the ratio of 8020 (vv) Working solutions of
concentrations 50microgmL and 5microgmL were prepared from this stock solution
for the determination of related compounds and assay respectively Composite
145 Chapter 3
and individual stock solutions of impurities having concentration 05microgmL
each were prepared in the diluent
362 Preparation of sample solution
Brand Ambienreg 10 mg of quantity 20 tablets was weighed and the average
tablet weight was determined These tablets were transferred into a clean dry
mortar and grounded into fine powder The fine powdered sample equivalent to
50 mg of the drug was weighed and dissolved in 100 mL of diluent (MeOH
Buffer 7030 vv) to make a solution of concentration 500microgmL The solution
was subjected to sonication for 30min and mechanical shaking for each 30
minutes The solution was filtered from which a 10mL of filtered solution was
diluted to 10 mL with the aid of mobile phase A The resulting solution of
concentration 50microgmL was filtered through 022μm nylon membrane filter
The solution was then analyzed in UPLC for related substances analysis The
solution was further diluted to obtain the concentration of 5microgmL for assay
determination Similar concentration solutions were prepared for API analysis
363 Generation of Stress sample solutions
A qualified sample of ZT has been chosen to conduct the stress study
According to ICH stability guidelines (Q1AR2) stress studies are likely to be
performed to study the intrinsic stability of the molecule ZT has been exposed
to various stress conditions such as heat light acid (HCl) base (NaOH)
oxidative (H2O2) and water hydrolysis The final stress conditions were tabulated
below
Table 31 Stress study conditions for ZT
146 Chapter 3
37 Method development
371 Objectives of method development
1 Rapid separation of ZT and its eight potential impurities
2 Identification of possible degradation products by means of LCMS analysis
and evaluation of peak purity
SNo Stressed Agent Stressing Condition
1 Heat
API and tablets were exposed to dry heat
at 105degC for about 7 days
2 Light API and tablets were exposed to UV light
at 254nm for 7 days
3 Acid (5N HCl) API and tablets extracted solutions were
treated with 5N HCl 60degC for 24 hrs
4 Base (5N NaOH)
API and tablets solutions were treated
with 5N NaOH at 60degC for 90mins
5 Oxidation (50 H2O2) API and tablets solutions were treated
with 5 H2O2 at 60degC for 2 hrs
6 Water hydrolysis API and tablets solutions were treated
with water at 60degC for 24h
147 Chapter 3
3 Single analytical method for the determination of assay and related
substances in bulk actives and dosage forms
4 Targeted for a minimum resolution of 15 between impurities and tailing
factor lt 20 for ZT peak at assay method
5 Aimed at LOQ values ie 0375microgmL (50 of the specification level which
is equivalent to 0375microgmL) for all the impurities of ZT
372 Method development strategy
A systematic method development approach had been followed to achieve
successful separation The key steps are mentioned below
Fig 33 Flow diagram of method development strategy
148 Chapter 3
373 Classification of the sample
Zolpidem tartrate and its properties
1 Chemical name N N 6-trimethyl-2-p-tolylimidazo [1 2-a] pyridine-3-
acetamide L-(+)-tartrate (21)
2 Chemical structure The polar groups in the structure are highlighted in
circles
3 Molecular weight 76488 as tartrate salt
30739 as base
4 Solubility Slightly soluble in water and sparingly soluble in
alcohol
5 pKa 62
6 Log P 12
7 Nature of the
molecule
Basic in nature and ionic compound
8 UV sensitivity
UV active since ZT has phenyl ring systems with
extended conjugated double bonds in its chemical
structure
149 Chapter 3
374 Impurity details
Zolpidem tartrate has eight potential impurities as mentioned in the section
31 The source and classifications of the impurities are tabulated below Table
32
Table 32 Origin of impurities for ZT
SNo Name of the impurity Source of impurity
Process related Degradation related
1 Imp-1 No Yes
2 Imp-2 Yes Yes
3 Imp-3 Yes No
4 Imp-4 Yes Yes
5 Imp-5 Yes No
6 Imp-6 Yes No
7 Imp-7 Yes No
8 Imp-8 Yes No
375 Preliminary chromatographic conditions
3751 Selection of detection wavelength
A composite solution containing 50 μgmL of drug and 1 μgmL of each of
the eight impurities was prepared in the diluent All the samples were analyzed
in HPLC- PDA system and the UV spectrums for all the components were
extracted
150 Chapter 3
Fig 34 Individual and overlaid UV spectra of ZT and its impurities
151 Chapter 3
152 Chapter 3
153 Chapter 3
Selection of wavelength to monitor zolpidem tartrate and its impurities
An optimal wavelength of detection for ZT and its impurities was selected as
254 nm based on UV spectra using PDA detector for LC analysis Fig 34
3752 Selection of UPLC column
Initial experiments were carried out on different commercially available RP-
UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH
shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to
determine the selectivity and symmetry of the individual components In all the
columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline
separation and imp-5 amp imp-6 peaks were merged The remaining peaks were
separated with resolution (RS)gt15 and the peak tailing was greater than 15 for
ZT Due to the structural similarities co-elution of above mentioned impurity
0970 Imp-1
1134 Imp-2
1978 Imp-3
2486 Imp-4
3443 Imp-5
3619 Imp-6
3897 ZT
4796 Imp-7
5671 Imp-8
2631
3438
20672114
3200
3578
3057
3710
2114
2443
3854
2443
3105
2114
2443
3057
nm
22000 24000 26000 28000 30000 32000 34000 36000 38000
mA
u
154 Chapter 3
pairs was observed Additionally peak tailing was also observed due to the
secondary interactions with residual silanol groups Although separation was
similar in all the three columns HSS T3 column had shown relatively good
peak symmetry and efficiency so it was thus selected for further optimization
Addition of triethylamine in the buffer improved the peak shape of ZT but the
resolution between imp-5 amp imp-6 remained critical
3753 Effect of buffer pH
The important parameters buffer pH and gradient mode of elution that
would likely to have significant effect on critical pair resolution were
investigated There was no substantial change in the resolution between Imps-1
amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly
decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp
6 (RS2) and Imp-6 amp ZT (RS
3) were improved when the pH altered from 60 to 70
The increase in Rs1 in acidic mobile phase and RS
2 RS3 in basic mobile phase
can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of
adequately low pH will remain unionized and get increased retentions On the
other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be
more retained From table 33 acceptable resolutions were obtained at pH 70
with isocratic elution and pH 75 with gradient elution but in both the cases
the run time was longer
155 Chapter 3
Table 33 Effect of Buffer pH on resolution of critical pairs
Mode of
elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time
Isocratic 40 21 lt10 17 15min
Isocratic 50 22 lt10 17 14min
Isocratic 60 21 11 18 15min
Isocratic 70 20 18 22 gt15min
Gradient 55 21 lt10 17 8min
Gradient 75 18 22 23 gt15min
Gradient A 55 B
73 25 21 22 8 min
a Rs1 Resolution between Imp-1 and Imp-2
b Rs2 Resolution between Imp-5 and Imp-6
c Rs3Resolution between Imp-6 and ZT
3754 Optimization of gradient program
Gradient mode of elution containing mobile phase-A (1mL of TEA1L
phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile
phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555
vv) were chosen to separate both the critical pairs in a single run and this
resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)
156 Chapter 3
Fig 35 Co elution of imp-5 and imp-6 at pH 55
Based on the experimental data it was predicted that increase in mobile
phase pH during the gradient elution could resolve the Imp-5 and Imp-6
without compromising the total run time Hence two more experiments were
designed with a mobile phase-B as combination of water ACN (4555 vv) for
experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-
2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase
the solvent strength thereby to elute strongly retained compounds (Imp-8)
faster The RS2 was less than 10 for experiment-1 and RS
2 was 15 for
experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value
60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from
60 to 70) between 50min and 82min was added to the program to reduce the
run time So Imp-8 was eluted at 8min To further improve the RS2 gradient
program was optimized with a linear increase of mobile phase-B with varying
157 Chapter 3
time points An optimum resolution was obtained (Fig 36) when the gradient
program of section 22 was used In addition to this the elution time of imp-8
was reduced to 6 min in the modified gradient method
Fig 36 Baseline separations of zolpidem and its impurities
Placebo interference study was verified by injecting sample solutions of
placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-
8 and ZT analyte peak was found
Fig 37 Placebo chromatogram for zolpidem tablets
Imp-
1 - 1
051
Imp-
2 - 1
231
Imp-
3 - 2
139
Imp-
4 - 2
692
Imp-
5 - 3
720
Imp-
6 - 3
892
ZT -
414
7
Imp-
7 - 5
096
Imp-
8 - 5
981
AU
-0020
-0015
-0010
-0005
0000
0005
0010
0015
0020
0025
0030
0035
0040
0045
0050
0055
0060
Minutes
050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850
158 Chapter 3
Three different lots of mobile phases were prepared and the consistency in
the resolution was verified (Table 34) A system suitability check (RS1 RS
2 amp
RS3) was incorporated to ensure the adequate system performance during the
regular analysis and method validation
Table 34 Reproducibility in separation of ZT and its impurities
Compound Average
RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing
factor
(n=6)c (n=6)c
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT) of
Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6)
159 Chapter 3
Fig 38 Peak purity plot for ZT in optimized conditions
Where PA = Purity angle and TH = Purity threshold
3755 The finalized RP-UPLC conditions for the estimation of assay and
related substances of ZT
Apparatus An UPLC with VWD and integrator
Column Acquity UPLC HSS T3 C-18 100 mm length
21 mm id 18 micron particle size
Flow Rate 03 ml min
Wave length 254 nm
Load 10microL
Column temperature 250C
Diluent Acetonitrile (ACN) Water (82)
Buffer Buffer solution was prepared by adding 1mL
of Triethyl amine (TEA) to 1000mL of 10 mM
NaH2PO4 H2O
Mobile phase-A Mixture of buffer (pH adjusted to 55 with
H3PO4) ACN and MeOH in the ratio of
602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with
H3PO4) and ACN in the ratio of 4555 (vv)
160 Chapter 3
Table 35 Gradient program
Time Mobile phase-A Mobile phase -B
001 95 5
25 95 5
35 40 60
50 40 60
82 30 70
85 95 5
100 95 5
Table 36 Retention times and relative retention times wrto ZT
SNo ImpurityAnalyte
name
Retention time ~Relative
retention time
1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100
8 Imp-7 5118 121 9 Imp-8 6037 142
3756 Degradation behavior
The purpose of this study is to establish the fact that the inherent chemical
stability of the molecule remains intact during its existence in the solid dosage
form along with other excipients So forced degradation studies should be
conducted on the drug to generate product related variants which in turn can
help to establish degradation pathways and thereby to develop and validate
suitable analytical procedure [22]
In this perspective a series of induced degradation experiments were
conducted at a concentration of 50 μgmL of ZT in active pharmaceutical
161 Chapter 3
ingredients (API) and tablets Degradation was induced by external physico-
chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)
oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal
treatment (105oC for 7 days)
The purity of the peaks obtained from the stressed samples was verified by
using the PDA detector The purity angle and purity threshold parameters were
evaluated for all the stressed samples to demonstrate the homogeneity of the
analyte peaks Assay of stressed samples was performed by comparison with
reference standards and the mass balance was calculated
37561 Degradation in Acidic solution
A single major degradation product Imp-2 (Zolpidem Acid) was formed when
ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The
degradation pathway is based on the premise that acid or base catalyzed
reaction between water and tertiary amide gives its parent carboxylic acid and
an amine Here the other hydrolytic product was dimethylamine Using LC-MS
in positive ion ESI the degradation product was found to have an mz value of
281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2
and UV spectral match
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
143 Chapter 3
pH gradient together with organic modifier has been used in the present UPLC
method to achieve separation between the critical pairs
35 Experimental
351 Chemicals and Reagents
Zolpidem tartrate and its related compounds were obtained from Dr
Reddyrsquos Laboratories Limited IPDO Hyderabad India Commercially available
brand Ambienreg tablets were used for dosage form analysis Analytical grade
sodium dihydrogen ortho phosphate monohydrate (NaH2PO4H2O) ortho-
phosphoric acid (H3PO4) HPLC-grade solvents ie methanol (MeOH) and
acetonitrile (ACN) were procured from Merck Darmstadt Germany
Triethylamine (TEA) was bought from JT Baker Mallinckrodt Inc Phillipsburg
NJ USA High purity water has been prepared by using Millipore Milli Q plus
water purification equipment (Millipore Corporate Headquarters Billerica MA)
352 Chromatographic Conditions and Equipment
Liquid chromatographic analysis was performed on Waters Acquity UPLC
equipped with photodiode array detector (PDA) at a wavelength of 254 nm using
Empower-2 software Separation was achieved on C18 column ie Acquity
UPLC HSS T3-C18 of dimensions 100 x 21mm 18microm at a flow rate of 03
mLmin using a gradient program (TB) set as 0015 255 3560 5060
8270 855 and 1005 Buffer solution was prepared by adding 1mL of
Triethyl amine (TEA) to 1000mL of 10 mM NaH2PO4 H2O Mobile phase A was a
mixture of buffer (pH adjusted to 55 with H3PO4) ACN and MeOH in the ratio
of 602020 (vvv) Mobile phase B was a mixture of buffer (pH adjusted to 73
144 Chapter 3
with H3PO4) and ACN in the ratio of 4555 (vv) The column oven temperature
was maintained at 25ordmC The injection volume was 10 microL
353 LC-MSMS conditions
An LCndashMSMS system (Agilent 1100 series liquid chromatograph coupled
with Applied Biosystems 4000 Q Trap triple quadrupole mass spectrometer with
Analyst 14 software MDS SCIEX USA) was utilized for mass identification of
degradation products A Novapak C18 column with dimensions 150 x 39 mm
4μm was employed as a stationary phase An ammonium acetate (Merck
Darmstadt Germany) solution of concentration 002 mM was used as buffer A
mixture of Ammonium acetate buffer MeOH and ACN (590230180 vvv) was
used as mobile phase The column oven temperature was maintained at 25degC
ACN was used as diluent The flow rate was set as 15 mLmin The analysis
was carried out in positive electro-spraypositive ionization mode the ion
source voltage was 5000 V and the source temperature was 450degC GS1 and
GS2 were set to 30 and 35 psi respectively The curtain gas flow was set at 20
psi
36 Preparation of solutions
361 Preparation of Stock solution
A stock solution of ZT of concentration 500 microgmL was prepared by
dissolving an appropriate quantity of the drug in a diluent containing a mixture
of acetonitrile and water in the ratio of 8020 (vv) Working solutions of
concentrations 50microgmL and 5microgmL were prepared from this stock solution
for the determination of related compounds and assay respectively Composite
145 Chapter 3
and individual stock solutions of impurities having concentration 05microgmL
each were prepared in the diluent
362 Preparation of sample solution
Brand Ambienreg 10 mg of quantity 20 tablets was weighed and the average
tablet weight was determined These tablets were transferred into a clean dry
mortar and grounded into fine powder The fine powdered sample equivalent to
50 mg of the drug was weighed and dissolved in 100 mL of diluent (MeOH
Buffer 7030 vv) to make a solution of concentration 500microgmL The solution
was subjected to sonication for 30min and mechanical shaking for each 30
minutes The solution was filtered from which a 10mL of filtered solution was
diluted to 10 mL with the aid of mobile phase A The resulting solution of
concentration 50microgmL was filtered through 022μm nylon membrane filter
The solution was then analyzed in UPLC for related substances analysis The
solution was further diluted to obtain the concentration of 5microgmL for assay
determination Similar concentration solutions were prepared for API analysis
363 Generation of Stress sample solutions
A qualified sample of ZT has been chosen to conduct the stress study
According to ICH stability guidelines (Q1AR2) stress studies are likely to be
performed to study the intrinsic stability of the molecule ZT has been exposed
to various stress conditions such as heat light acid (HCl) base (NaOH)
oxidative (H2O2) and water hydrolysis The final stress conditions were tabulated
below
Table 31 Stress study conditions for ZT
146 Chapter 3
37 Method development
371 Objectives of method development
1 Rapid separation of ZT and its eight potential impurities
2 Identification of possible degradation products by means of LCMS analysis
and evaluation of peak purity
SNo Stressed Agent Stressing Condition
1 Heat
API and tablets were exposed to dry heat
at 105degC for about 7 days
2 Light API and tablets were exposed to UV light
at 254nm for 7 days
3 Acid (5N HCl) API and tablets extracted solutions were
treated with 5N HCl 60degC for 24 hrs
4 Base (5N NaOH)
API and tablets solutions were treated
with 5N NaOH at 60degC for 90mins
5 Oxidation (50 H2O2) API and tablets solutions were treated
with 5 H2O2 at 60degC for 2 hrs
6 Water hydrolysis API and tablets solutions were treated
with water at 60degC for 24h
147 Chapter 3
3 Single analytical method for the determination of assay and related
substances in bulk actives and dosage forms
4 Targeted for a minimum resolution of 15 between impurities and tailing
factor lt 20 for ZT peak at assay method
5 Aimed at LOQ values ie 0375microgmL (50 of the specification level which
is equivalent to 0375microgmL) for all the impurities of ZT
372 Method development strategy
A systematic method development approach had been followed to achieve
successful separation The key steps are mentioned below
Fig 33 Flow diagram of method development strategy
148 Chapter 3
373 Classification of the sample
Zolpidem tartrate and its properties
1 Chemical name N N 6-trimethyl-2-p-tolylimidazo [1 2-a] pyridine-3-
acetamide L-(+)-tartrate (21)
2 Chemical structure The polar groups in the structure are highlighted in
circles
3 Molecular weight 76488 as tartrate salt
30739 as base
4 Solubility Slightly soluble in water and sparingly soluble in
alcohol
5 pKa 62
6 Log P 12
7 Nature of the
molecule
Basic in nature and ionic compound
8 UV sensitivity
UV active since ZT has phenyl ring systems with
extended conjugated double bonds in its chemical
structure
149 Chapter 3
374 Impurity details
Zolpidem tartrate has eight potential impurities as mentioned in the section
31 The source and classifications of the impurities are tabulated below Table
32
Table 32 Origin of impurities for ZT
SNo Name of the impurity Source of impurity
Process related Degradation related
1 Imp-1 No Yes
2 Imp-2 Yes Yes
3 Imp-3 Yes No
4 Imp-4 Yes Yes
5 Imp-5 Yes No
6 Imp-6 Yes No
7 Imp-7 Yes No
8 Imp-8 Yes No
375 Preliminary chromatographic conditions
3751 Selection of detection wavelength
A composite solution containing 50 μgmL of drug and 1 μgmL of each of
the eight impurities was prepared in the diluent All the samples were analyzed
in HPLC- PDA system and the UV spectrums for all the components were
extracted
150 Chapter 3
Fig 34 Individual and overlaid UV spectra of ZT and its impurities
151 Chapter 3
152 Chapter 3
153 Chapter 3
Selection of wavelength to monitor zolpidem tartrate and its impurities
An optimal wavelength of detection for ZT and its impurities was selected as
254 nm based on UV spectra using PDA detector for LC analysis Fig 34
3752 Selection of UPLC column
Initial experiments were carried out on different commercially available RP-
UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH
shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to
determine the selectivity and symmetry of the individual components In all the
columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline
separation and imp-5 amp imp-6 peaks were merged The remaining peaks were
separated with resolution (RS)gt15 and the peak tailing was greater than 15 for
ZT Due to the structural similarities co-elution of above mentioned impurity
0970 Imp-1
1134 Imp-2
1978 Imp-3
2486 Imp-4
3443 Imp-5
3619 Imp-6
3897 ZT
4796 Imp-7
5671 Imp-8
2631
3438
20672114
3200
3578
3057
3710
2114
2443
3854
2443
3105
2114
2443
3057
nm
22000 24000 26000 28000 30000 32000 34000 36000 38000
mA
u
154 Chapter 3
pairs was observed Additionally peak tailing was also observed due to the
secondary interactions with residual silanol groups Although separation was
similar in all the three columns HSS T3 column had shown relatively good
peak symmetry and efficiency so it was thus selected for further optimization
Addition of triethylamine in the buffer improved the peak shape of ZT but the
resolution between imp-5 amp imp-6 remained critical
3753 Effect of buffer pH
The important parameters buffer pH and gradient mode of elution that
would likely to have significant effect on critical pair resolution were
investigated There was no substantial change in the resolution between Imps-1
amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly
decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp
6 (RS2) and Imp-6 amp ZT (RS
3) were improved when the pH altered from 60 to 70
The increase in Rs1 in acidic mobile phase and RS
2 RS3 in basic mobile phase
can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of
adequately low pH will remain unionized and get increased retentions On the
other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be
more retained From table 33 acceptable resolutions were obtained at pH 70
with isocratic elution and pH 75 with gradient elution but in both the cases
the run time was longer
155 Chapter 3
Table 33 Effect of Buffer pH on resolution of critical pairs
Mode of
elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time
Isocratic 40 21 lt10 17 15min
Isocratic 50 22 lt10 17 14min
Isocratic 60 21 11 18 15min
Isocratic 70 20 18 22 gt15min
Gradient 55 21 lt10 17 8min
Gradient 75 18 22 23 gt15min
Gradient A 55 B
73 25 21 22 8 min
a Rs1 Resolution between Imp-1 and Imp-2
b Rs2 Resolution between Imp-5 and Imp-6
c Rs3Resolution between Imp-6 and ZT
3754 Optimization of gradient program
Gradient mode of elution containing mobile phase-A (1mL of TEA1L
phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile
phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555
vv) were chosen to separate both the critical pairs in a single run and this
resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)
156 Chapter 3
Fig 35 Co elution of imp-5 and imp-6 at pH 55
Based on the experimental data it was predicted that increase in mobile
phase pH during the gradient elution could resolve the Imp-5 and Imp-6
without compromising the total run time Hence two more experiments were
designed with a mobile phase-B as combination of water ACN (4555 vv) for
experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-
2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase
the solvent strength thereby to elute strongly retained compounds (Imp-8)
faster The RS2 was less than 10 for experiment-1 and RS
2 was 15 for
experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value
60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from
60 to 70) between 50min and 82min was added to the program to reduce the
run time So Imp-8 was eluted at 8min To further improve the RS2 gradient
program was optimized with a linear increase of mobile phase-B with varying
157 Chapter 3
time points An optimum resolution was obtained (Fig 36) when the gradient
program of section 22 was used In addition to this the elution time of imp-8
was reduced to 6 min in the modified gradient method
Fig 36 Baseline separations of zolpidem and its impurities
Placebo interference study was verified by injecting sample solutions of
placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-
8 and ZT analyte peak was found
Fig 37 Placebo chromatogram for zolpidem tablets
Imp-
1 - 1
051
Imp-
2 - 1
231
Imp-
3 - 2
139
Imp-
4 - 2
692
Imp-
5 - 3
720
Imp-
6 - 3
892
ZT -
414
7
Imp-
7 - 5
096
Imp-
8 - 5
981
AU
-0020
-0015
-0010
-0005
0000
0005
0010
0015
0020
0025
0030
0035
0040
0045
0050
0055
0060
Minutes
050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850
158 Chapter 3
Three different lots of mobile phases were prepared and the consistency in
the resolution was verified (Table 34) A system suitability check (RS1 RS
2 amp
RS3) was incorporated to ensure the adequate system performance during the
regular analysis and method validation
Table 34 Reproducibility in separation of ZT and its impurities
Compound Average
RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing
factor
(n=6)c (n=6)c
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT) of
Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6)
159 Chapter 3
Fig 38 Peak purity plot for ZT in optimized conditions
Where PA = Purity angle and TH = Purity threshold
3755 The finalized RP-UPLC conditions for the estimation of assay and
related substances of ZT
Apparatus An UPLC with VWD and integrator
Column Acquity UPLC HSS T3 C-18 100 mm length
21 mm id 18 micron particle size
Flow Rate 03 ml min
Wave length 254 nm
Load 10microL
Column temperature 250C
Diluent Acetonitrile (ACN) Water (82)
Buffer Buffer solution was prepared by adding 1mL
of Triethyl amine (TEA) to 1000mL of 10 mM
NaH2PO4 H2O
Mobile phase-A Mixture of buffer (pH adjusted to 55 with
H3PO4) ACN and MeOH in the ratio of
602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with
H3PO4) and ACN in the ratio of 4555 (vv)
160 Chapter 3
Table 35 Gradient program
Time Mobile phase-A Mobile phase -B
001 95 5
25 95 5
35 40 60
50 40 60
82 30 70
85 95 5
100 95 5
Table 36 Retention times and relative retention times wrto ZT
SNo ImpurityAnalyte
name
Retention time ~Relative
retention time
1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100
8 Imp-7 5118 121 9 Imp-8 6037 142
3756 Degradation behavior
The purpose of this study is to establish the fact that the inherent chemical
stability of the molecule remains intact during its existence in the solid dosage
form along with other excipients So forced degradation studies should be
conducted on the drug to generate product related variants which in turn can
help to establish degradation pathways and thereby to develop and validate
suitable analytical procedure [22]
In this perspective a series of induced degradation experiments were
conducted at a concentration of 50 μgmL of ZT in active pharmaceutical
161 Chapter 3
ingredients (API) and tablets Degradation was induced by external physico-
chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)
oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal
treatment (105oC for 7 days)
The purity of the peaks obtained from the stressed samples was verified by
using the PDA detector The purity angle and purity threshold parameters were
evaluated for all the stressed samples to demonstrate the homogeneity of the
analyte peaks Assay of stressed samples was performed by comparison with
reference standards and the mass balance was calculated
37561 Degradation in Acidic solution
A single major degradation product Imp-2 (Zolpidem Acid) was formed when
ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The
degradation pathway is based on the premise that acid or base catalyzed
reaction between water and tertiary amide gives its parent carboxylic acid and
an amine Here the other hydrolytic product was dimethylamine Using LC-MS
in positive ion ESI the degradation product was found to have an mz value of
281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2
and UV spectral match
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
144 Chapter 3
with H3PO4) and ACN in the ratio of 4555 (vv) The column oven temperature
was maintained at 25ordmC The injection volume was 10 microL
353 LC-MSMS conditions
An LCndashMSMS system (Agilent 1100 series liquid chromatograph coupled
with Applied Biosystems 4000 Q Trap triple quadrupole mass spectrometer with
Analyst 14 software MDS SCIEX USA) was utilized for mass identification of
degradation products A Novapak C18 column with dimensions 150 x 39 mm
4μm was employed as a stationary phase An ammonium acetate (Merck
Darmstadt Germany) solution of concentration 002 mM was used as buffer A
mixture of Ammonium acetate buffer MeOH and ACN (590230180 vvv) was
used as mobile phase The column oven temperature was maintained at 25degC
ACN was used as diluent The flow rate was set as 15 mLmin The analysis
was carried out in positive electro-spraypositive ionization mode the ion
source voltage was 5000 V and the source temperature was 450degC GS1 and
GS2 were set to 30 and 35 psi respectively The curtain gas flow was set at 20
psi
36 Preparation of solutions
361 Preparation of Stock solution
A stock solution of ZT of concentration 500 microgmL was prepared by
dissolving an appropriate quantity of the drug in a diluent containing a mixture
of acetonitrile and water in the ratio of 8020 (vv) Working solutions of
concentrations 50microgmL and 5microgmL were prepared from this stock solution
for the determination of related compounds and assay respectively Composite
145 Chapter 3
and individual stock solutions of impurities having concentration 05microgmL
each were prepared in the diluent
362 Preparation of sample solution
Brand Ambienreg 10 mg of quantity 20 tablets was weighed and the average
tablet weight was determined These tablets were transferred into a clean dry
mortar and grounded into fine powder The fine powdered sample equivalent to
50 mg of the drug was weighed and dissolved in 100 mL of diluent (MeOH
Buffer 7030 vv) to make a solution of concentration 500microgmL The solution
was subjected to sonication for 30min and mechanical shaking for each 30
minutes The solution was filtered from which a 10mL of filtered solution was
diluted to 10 mL with the aid of mobile phase A The resulting solution of
concentration 50microgmL was filtered through 022μm nylon membrane filter
The solution was then analyzed in UPLC for related substances analysis The
solution was further diluted to obtain the concentration of 5microgmL for assay
determination Similar concentration solutions were prepared for API analysis
363 Generation of Stress sample solutions
A qualified sample of ZT has been chosen to conduct the stress study
According to ICH stability guidelines (Q1AR2) stress studies are likely to be
performed to study the intrinsic stability of the molecule ZT has been exposed
to various stress conditions such as heat light acid (HCl) base (NaOH)
oxidative (H2O2) and water hydrolysis The final stress conditions were tabulated
below
Table 31 Stress study conditions for ZT
146 Chapter 3
37 Method development
371 Objectives of method development
1 Rapid separation of ZT and its eight potential impurities
2 Identification of possible degradation products by means of LCMS analysis
and evaluation of peak purity
SNo Stressed Agent Stressing Condition
1 Heat
API and tablets were exposed to dry heat
at 105degC for about 7 days
2 Light API and tablets were exposed to UV light
at 254nm for 7 days
3 Acid (5N HCl) API and tablets extracted solutions were
treated with 5N HCl 60degC for 24 hrs
4 Base (5N NaOH)
API and tablets solutions were treated
with 5N NaOH at 60degC for 90mins
5 Oxidation (50 H2O2) API and tablets solutions were treated
with 5 H2O2 at 60degC for 2 hrs
6 Water hydrolysis API and tablets solutions were treated
with water at 60degC for 24h
147 Chapter 3
3 Single analytical method for the determination of assay and related
substances in bulk actives and dosage forms
4 Targeted for a minimum resolution of 15 between impurities and tailing
factor lt 20 for ZT peak at assay method
5 Aimed at LOQ values ie 0375microgmL (50 of the specification level which
is equivalent to 0375microgmL) for all the impurities of ZT
372 Method development strategy
A systematic method development approach had been followed to achieve
successful separation The key steps are mentioned below
Fig 33 Flow diagram of method development strategy
148 Chapter 3
373 Classification of the sample
Zolpidem tartrate and its properties
1 Chemical name N N 6-trimethyl-2-p-tolylimidazo [1 2-a] pyridine-3-
acetamide L-(+)-tartrate (21)
2 Chemical structure The polar groups in the structure are highlighted in
circles
3 Molecular weight 76488 as tartrate salt
30739 as base
4 Solubility Slightly soluble in water and sparingly soluble in
alcohol
5 pKa 62
6 Log P 12
7 Nature of the
molecule
Basic in nature and ionic compound
8 UV sensitivity
UV active since ZT has phenyl ring systems with
extended conjugated double bonds in its chemical
structure
149 Chapter 3
374 Impurity details
Zolpidem tartrate has eight potential impurities as mentioned in the section
31 The source and classifications of the impurities are tabulated below Table
32
Table 32 Origin of impurities for ZT
SNo Name of the impurity Source of impurity
Process related Degradation related
1 Imp-1 No Yes
2 Imp-2 Yes Yes
3 Imp-3 Yes No
4 Imp-4 Yes Yes
5 Imp-5 Yes No
6 Imp-6 Yes No
7 Imp-7 Yes No
8 Imp-8 Yes No
375 Preliminary chromatographic conditions
3751 Selection of detection wavelength
A composite solution containing 50 μgmL of drug and 1 μgmL of each of
the eight impurities was prepared in the diluent All the samples were analyzed
in HPLC- PDA system and the UV spectrums for all the components were
extracted
150 Chapter 3
Fig 34 Individual and overlaid UV spectra of ZT and its impurities
151 Chapter 3
152 Chapter 3
153 Chapter 3
Selection of wavelength to monitor zolpidem tartrate and its impurities
An optimal wavelength of detection for ZT and its impurities was selected as
254 nm based on UV spectra using PDA detector for LC analysis Fig 34
3752 Selection of UPLC column
Initial experiments were carried out on different commercially available RP-
UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH
shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to
determine the selectivity and symmetry of the individual components In all the
columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline
separation and imp-5 amp imp-6 peaks were merged The remaining peaks were
separated with resolution (RS)gt15 and the peak tailing was greater than 15 for
ZT Due to the structural similarities co-elution of above mentioned impurity
0970 Imp-1
1134 Imp-2
1978 Imp-3
2486 Imp-4
3443 Imp-5
3619 Imp-6
3897 ZT
4796 Imp-7
5671 Imp-8
2631
3438
20672114
3200
3578
3057
3710
2114
2443
3854
2443
3105
2114
2443
3057
nm
22000 24000 26000 28000 30000 32000 34000 36000 38000
mA
u
154 Chapter 3
pairs was observed Additionally peak tailing was also observed due to the
secondary interactions with residual silanol groups Although separation was
similar in all the three columns HSS T3 column had shown relatively good
peak symmetry and efficiency so it was thus selected for further optimization
Addition of triethylamine in the buffer improved the peak shape of ZT but the
resolution between imp-5 amp imp-6 remained critical
3753 Effect of buffer pH
The important parameters buffer pH and gradient mode of elution that
would likely to have significant effect on critical pair resolution were
investigated There was no substantial change in the resolution between Imps-1
amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly
decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp
6 (RS2) and Imp-6 amp ZT (RS
3) were improved when the pH altered from 60 to 70
The increase in Rs1 in acidic mobile phase and RS
2 RS3 in basic mobile phase
can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of
adequately low pH will remain unionized and get increased retentions On the
other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be
more retained From table 33 acceptable resolutions were obtained at pH 70
with isocratic elution and pH 75 with gradient elution but in both the cases
the run time was longer
155 Chapter 3
Table 33 Effect of Buffer pH on resolution of critical pairs
Mode of
elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time
Isocratic 40 21 lt10 17 15min
Isocratic 50 22 lt10 17 14min
Isocratic 60 21 11 18 15min
Isocratic 70 20 18 22 gt15min
Gradient 55 21 lt10 17 8min
Gradient 75 18 22 23 gt15min
Gradient A 55 B
73 25 21 22 8 min
a Rs1 Resolution between Imp-1 and Imp-2
b Rs2 Resolution between Imp-5 and Imp-6
c Rs3Resolution between Imp-6 and ZT
3754 Optimization of gradient program
Gradient mode of elution containing mobile phase-A (1mL of TEA1L
phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile
phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555
vv) were chosen to separate both the critical pairs in a single run and this
resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)
156 Chapter 3
Fig 35 Co elution of imp-5 and imp-6 at pH 55
Based on the experimental data it was predicted that increase in mobile
phase pH during the gradient elution could resolve the Imp-5 and Imp-6
without compromising the total run time Hence two more experiments were
designed with a mobile phase-B as combination of water ACN (4555 vv) for
experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-
2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase
the solvent strength thereby to elute strongly retained compounds (Imp-8)
faster The RS2 was less than 10 for experiment-1 and RS
2 was 15 for
experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value
60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from
60 to 70) between 50min and 82min was added to the program to reduce the
run time So Imp-8 was eluted at 8min To further improve the RS2 gradient
program was optimized with a linear increase of mobile phase-B with varying
157 Chapter 3
time points An optimum resolution was obtained (Fig 36) when the gradient
program of section 22 was used In addition to this the elution time of imp-8
was reduced to 6 min in the modified gradient method
Fig 36 Baseline separations of zolpidem and its impurities
Placebo interference study was verified by injecting sample solutions of
placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-
8 and ZT analyte peak was found
Fig 37 Placebo chromatogram for zolpidem tablets
Imp-
1 - 1
051
Imp-
2 - 1
231
Imp-
3 - 2
139
Imp-
4 - 2
692
Imp-
5 - 3
720
Imp-
6 - 3
892
ZT -
414
7
Imp-
7 - 5
096
Imp-
8 - 5
981
AU
-0020
-0015
-0010
-0005
0000
0005
0010
0015
0020
0025
0030
0035
0040
0045
0050
0055
0060
Minutes
050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850
158 Chapter 3
Three different lots of mobile phases were prepared and the consistency in
the resolution was verified (Table 34) A system suitability check (RS1 RS
2 amp
RS3) was incorporated to ensure the adequate system performance during the
regular analysis and method validation
Table 34 Reproducibility in separation of ZT and its impurities
Compound Average
RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing
factor
(n=6)c (n=6)c
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT) of
Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6)
159 Chapter 3
Fig 38 Peak purity plot for ZT in optimized conditions
Where PA = Purity angle and TH = Purity threshold
3755 The finalized RP-UPLC conditions for the estimation of assay and
related substances of ZT
Apparatus An UPLC with VWD and integrator
Column Acquity UPLC HSS T3 C-18 100 mm length
21 mm id 18 micron particle size
Flow Rate 03 ml min
Wave length 254 nm
Load 10microL
Column temperature 250C
Diluent Acetonitrile (ACN) Water (82)
Buffer Buffer solution was prepared by adding 1mL
of Triethyl amine (TEA) to 1000mL of 10 mM
NaH2PO4 H2O
Mobile phase-A Mixture of buffer (pH adjusted to 55 with
H3PO4) ACN and MeOH in the ratio of
602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with
H3PO4) and ACN in the ratio of 4555 (vv)
160 Chapter 3
Table 35 Gradient program
Time Mobile phase-A Mobile phase -B
001 95 5
25 95 5
35 40 60
50 40 60
82 30 70
85 95 5
100 95 5
Table 36 Retention times and relative retention times wrto ZT
SNo ImpurityAnalyte
name
Retention time ~Relative
retention time
1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100
8 Imp-7 5118 121 9 Imp-8 6037 142
3756 Degradation behavior
The purpose of this study is to establish the fact that the inherent chemical
stability of the molecule remains intact during its existence in the solid dosage
form along with other excipients So forced degradation studies should be
conducted on the drug to generate product related variants which in turn can
help to establish degradation pathways and thereby to develop and validate
suitable analytical procedure [22]
In this perspective a series of induced degradation experiments were
conducted at a concentration of 50 μgmL of ZT in active pharmaceutical
161 Chapter 3
ingredients (API) and tablets Degradation was induced by external physico-
chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)
oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal
treatment (105oC for 7 days)
The purity of the peaks obtained from the stressed samples was verified by
using the PDA detector The purity angle and purity threshold parameters were
evaluated for all the stressed samples to demonstrate the homogeneity of the
analyte peaks Assay of stressed samples was performed by comparison with
reference standards and the mass balance was calculated
37561 Degradation in Acidic solution
A single major degradation product Imp-2 (Zolpidem Acid) was formed when
ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The
degradation pathway is based on the premise that acid or base catalyzed
reaction between water and tertiary amide gives its parent carboxylic acid and
an amine Here the other hydrolytic product was dimethylamine Using LC-MS
in positive ion ESI the degradation product was found to have an mz value of
281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2
and UV spectral match
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
145 Chapter 3
and individual stock solutions of impurities having concentration 05microgmL
each were prepared in the diluent
362 Preparation of sample solution
Brand Ambienreg 10 mg of quantity 20 tablets was weighed and the average
tablet weight was determined These tablets were transferred into a clean dry
mortar and grounded into fine powder The fine powdered sample equivalent to
50 mg of the drug was weighed and dissolved in 100 mL of diluent (MeOH
Buffer 7030 vv) to make a solution of concentration 500microgmL The solution
was subjected to sonication for 30min and mechanical shaking for each 30
minutes The solution was filtered from which a 10mL of filtered solution was
diluted to 10 mL with the aid of mobile phase A The resulting solution of
concentration 50microgmL was filtered through 022μm nylon membrane filter
The solution was then analyzed in UPLC for related substances analysis The
solution was further diluted to obtain the concentration of 5microgmL for assay
determination Similar concentration solutions were prepared for API analysis
363 Generation of Stress sample solutions
A qualified sample of ZT has been chosen to conduct the stress study
According to ICH stability guidelines (Q1AR2) stress studies are likely to be
performed to study the intrinsic stability of the molecule ZT has been exposed
to various stress conditions such as heat light acid (HCl) base (NaOH)
oxidative (H2O2) and water hydrolysis The final stress conditions were tabulated
below
Table 31 Stress study conditions for ZT
146 Chapter 3
37 Method development
371 Objectives of method development
1 Rapid separation of ZT and its eight potential impurities
2 Identification of possible degradation products by means of LCMS analysis
and evaluation of peak purity
SNo Stressed Agent Stressing Condition
1 Heat
API and tablets were exposed to dry heat
at 105degC for about 7 days
2 Light API and tablets were exposed to UV light
at 254nm for 7 days
3 Acid (5N HCl) API and tablets extracted solutions were
treated with 5N HCl 60degC for 24 hrs
4 Base (5N NaOH)
API and tablets solutions were treated
with 5N NaOH at 60degC for 90mins
5 Oxidation (50 H2O2) API and tablets solutions were treated
with 5 H2O2 at 60degC for 2 hrs
6 Water hydrolysis API and tablets solutions were treated
with water at 60degC for 24h
147 Chapter 3
3 Single analytical method for the determination of assay and related
substances in bulk actives and dosage forms
4 Targeted for a minimum resolution of 15 between impurities and tailing
factor lt 20 for ZT peak at assay method
5 Aimed at LOQ values ie 0375microgmL (50 of the specification level which
is equivalent to 0375microgmL) for all the impurities of ZT
372 Method development strategy
A systematic method development approach had been followed to achieve
successful separation The key steps are mentioned below
Fig 33 Flow diagram of method development strategy
148 Chapter 3
373 Classification of the sample
Zolpidem tartrate and its properties
1 Chemical name N N 6-trimethyl-2-p-tolylimidazo [1 2-a] pyridine-3-
acetamide L-(+)-tartrate (21)
2 Chemical structure The polar groups in the structure are highlighted in
circles
3 Molecular weight 76488 as tartrate salt
30739 as base
4 Solubility Slightly soluble in water and sparingly soluble in
alcohol
5 pKa 62
6 Log P 12
7 Nature of the
molecule
Basic in nature and ionic compound
8 UV sensitivity
UV active since ZT has phenyl ring systems with
extended conjugated double bonds in its chemical
structure
149 Chapter 3
374 Impurity details
Zolpidem tartrate has eight potential impurities as mentioned in the section
31 The source and classifications of the impurities are tabulated below Table
32
Table 32 Origin of impurities for ZT
SNo Name of the impurity Source of impurity
Process related Degradation related
1 Imp-1 No Yes
2 Imp-2 Yes Yes
3 Imp-3 Yes No
4 Imp-4 Yes Yes
5 Imp-5 Yes No
6 Imp-6 Yes No
7 Imp-7 Yes No
8 Imp-8 Yes No
375 Preliminary chromatographic conditions
3751 Selection of detection wavelength
A composite solution containing 50 μgmL of drug and 1 μgmL of each of
the eight impurities was prepared in the diluent All the samples were analyzed
in HPLC- PDA system and the UV spectrums for all the components were
extracted
150 Chapter 3
Fig 34 Individual and overlaid UV spectra of ZT and its impurities
151 Chapter 3
152 Chapter 3
153 Chapter 3
Selection of wavelength to monitor zolpidem tartrate and its impurities
An optimal wavelength of detection for ZT and its impurities was selected as
254 nm based on UV spectra using PDA detector for LC analysis Fig 34
3752 Selection of UPLC column
Initial experiments were carried out on different commercially available RP-
UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH
shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to
determine the selectivity and symmetry of the individual components In all the
columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline
separation and imp-5 amp imp-6 peaks were merged The remaining peaks were
separated with resolution (RS)gt15 and the peak tailing was greater than 15 for
ZT Due to the structural similarities co-elution of above mentioned impurity
0970 Imp-1
1134 Imp-2
1978 Imp-3
2486 Imp-4
3443 Imp-5
3619 Imp-6
3897 ZT
4796 Imp-7
5671 Imp-8
2631
3438
20672114
3200
3578
3057
3710
2114
2443
3854
2443
3105
2114
2443
3057
nm
22000 24000 26000 28000 30000 32000 34000 36000 38000
mA
u
154 Chapter 3
pairs was observed Additionally peak tailing was also observed due to the
secondary interactions with residual silanol groups Although separation was
similar in all the three columns HSS T3 column had shown relatively good
peak symmetry and efficiency so it was thus selected for further optimization
Addition of triethylamine in the buffer improved the peak shape of ZT but the
resolution between imp-5 amp imp-6 remained critical
3753 Effect of buffer pH
The important parameters buffer pH and gradient mode of elution that
would likely to have significant effect on critical pair resolution were
investigated There was no substantial change in the resolution between Imps-1
amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly
decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp
6 (RS2) and Imp-6 amp ZT (RS
3) were improved when the pH altered from 60 to 70
The increase in Rs1 in acidic mobile phase and RS
2 RS3 in basic mobile phase
can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of
adequately low pH will remain unionized and get increased retentions On the
other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be
more retained From table 33 acceptable resolutions were obtained at pH 70
with isocratic elution and pH 75 with gradient elution but in both the cases
the run time was longer
155 Chapter 3
Table 33 Effect of Buffer pH on resolution of critical pairs
Mode of
elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time
Isocratic 40 21 lt10 17 15min
Isocratic 50 22 lt10 17 14min
Isocratic 60 21 11 18 15min
Isocratic 70 20 18 22 gt15min
Gradient 55 21 lt10 17 8min
Gradient 75 18 22 23 gt15min
Gradient A 55 B
73 25 21 22 8 min
a Rs1 Resolution between Imp-1 and Imp-2
b Rs2 Resolution between Imp-5 and Imp-6
c Rs3Resolution between Imp-6 and ZT
3754 Optimization of gradient program
Gradient mode of elution containing mobile phase-A (1mL of TEA1L
phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile
phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555
vv) were chosen to separate both the critical pairs in a single run and this
resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)
156 Chapter 3
Fig 35 Co elution of imp-5 and imp-6 at pH 55
Based on the experimental data it was predicted that increase in mobile
phase pH during the gradient elution could resolve the Imp-5 and Imp-6
without compromising the total run time Hence two more experiments were
designed with a mobile phase-B as combination of water ACN (4555 vv) for
experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-
2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase
the solvent strength thereby to elute strongly retained compounds (Imp-8)
faster The RS2 was less than 10 for experiment-1 and RS
2 was 15 for
experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value
60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from
60 to 70) between 50min and 82min was added to the program to reduce the
run time So Imp-8 was eluted at 8min To further improve the RS2 gradient
program was optimized with a linear increase of mobile phase-B with varying
157 Chapter 3
time points An optimum resolution was obtained (Fig 36) when the gradient
program of section 22 was used In addition to this the elution time of imp-8
was reduced to 6 min in the modified gradient method
Fig 36 Baseline separations of zolpidem and its impurities
Placebo interference study was verified by injecting sample solutions of
placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-
8 and ZT analyte peak was found
Fig 37 Placebo chromatogram for zolpidem tablets
Imp-
1 - 1
051
Imp-
2 - 1
231
Imp-
3 - 2
139
Imp-
4 - 2
692
Imp-
5 - 3
720
Imp-
6 - 3
892
ZT -
414
7
Imp-
7 - 5
096
Imp-
8 - 5
981
AU
-0020
-0015
-0010
-0005
0000
0005
0010
0015
0020
0025
0030
0035
0040
0045
0050
0055
0060
Minutes
050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850
158 Chapter 3
Three different lots of mobile phases were prepared and the consistency in
the resolution was verified (Table 34) A system suitability check (RS1 RS
2 amp
RS3) was incorporated to ensure the adequate system performance during the
regular analysis and method validation
Table 34 Reproducibility in separation of ZT and its impurities
Compound Average
RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing
factor
(n=6)c (n=6)c
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT) of
Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6)
159 Chapter 3
Fig 38 Peak purity plot for ZT in optimized conditions
Where PA = Purity angle and TH = Purity threshold
3755 The finalized RP-UPLC conditions for the estimation of assay and
related substances of ZT
Apparatus An UPLC with VWD and integrator
Column Acquity UPLC HSS T3 C-18 100 mm length
21 mm id 18 micron particle size
Flow Rate 03 ml min
Wave length 254 nm
Load 10microL
Column temperature 250C
Diluent Acetonitrile (ACN) Water (82)
Buffer Buffer solution was prepared by adding 1mL
of Triethyl amine (TEA) to 1000mL of 10 mM
NaH2PO4 H2O
Mobile phase-A Mixture of buffer (pH adjusted to 55 with
H3PO4) ACN and MeOH in the ratio of
602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with
H3PO4) and ACN in the ratio of 4555 (vv)
160 Chapter 3
Table 35 Gradient program
Time Mobile phase-A Mobile phase -B
001 95 5
25 95 5
35 40 60
50 40 60
82 30 70
85 95 5
100 95 5
Table 36 Retention times and relative retention times wrto ZT
SNo ImpurityAnalyte
name
Retention time ~Relative
retention time
1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100
8 Imp-7 5118 121 9 Imp-8 6037 142
3756 Degradation behavior
The purpose of this study is to establish the fact that the inherent chemical
stability of the molecule remains intact during its existence in the solid dosage
form along with other excipients So forced degradation studies should be
conducted on the drug to generate product related variants which in turn can
help to establish degradation pathways and thereby to develop and validate
suitable analytical procedure [22]
In this perspective a series of induced degradation experiments were
conducted at a concentration of 50 μgmL of ZT in active pharmaceutical
161 Chapter 3
ingredients (API) and tablets Degradation was induced by external physico-
chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)
oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal
treatment (105oC for 7 days)
The purity of the peaks obtained from the stressed samples was verified by
using the PDA detector The purity angle and purity threshold parameters were
evaluated for all the stressed samples to demonstrate the homogeneity of the
analyte peaks Assay of stressed samples was performed by comparison with
reference standards and the mass balance was calculated
37561 Degradation in Acidic solution
A single major degradation product Imp-2 (Zolpidem Acid) was formed when
ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The
degradation pathway is based on the premise that acid or base catalyzed
reaction between water and tertiary amide gives its parent carboxylic acid and
an amine Here the other hydrolytic product was dimethylamine Using LC-MS
in positive ion ESI the degradation product was found to have an mz value of
281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2
and UV spectral match
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
146 Chapter 3
37 Method development
371 Objectives of method development
1 Rapid separation of ZT and its eight potential impurities
2 Identification of possible degradation products by means of LCMS analysis
and evaluation of peak purity
SNo Stressed Agent Stressing Condition
1 Heat
API and tablets were exposed to dry heat
at 105degC for about 7 days
2 Light API and tablets were exposed to UV light
at 254nm for 7 days
3 Acid (5N HCl) API and tablets extracted solutions were
treated with 5N HCl 60degC for 24 hrs
4 Base (5N NaOH)
API and tablets solutions were treated
with 5N NaOH at 60degC for 90mins
5 Oxidation (50 H2O2) API and tablets solutions were treated
with 5 H2O2 at 60degC for 2 hrs
6 Water hydrolysis API and tablets solutions were treated
with water at 60degC for 24h
147 Chapter 3
3 Single analytical method for the determination of assay and related
substances in bulk actives and dosage forms
4 Targeted for a minimum resolution of 15 between impurities and tailing
factor lt 20 for ZT peak at assay method
5 Aimed at LOQ values ie 0375microgmL (50 of the specification level which
is equivalent to 0375microgmL) for all the impurities of ZT
372 Method development strategy
A systematic method development approach had been followed to achieve
successful separation The key steps are mentioned below
Fig 33 Flow diagram of method development strategy
148 Chapter 3
373 Classification of the sample
Zolpidem tartrate and its properties
1 Chemical name N N 6-trimethyl-2-p-tolylimidazo [1 2-a] pyridine-3-
acetamide L-(+)-tartrate (21)
2 Chemical structure The polar groups in the structure are highlighted in
circles
3 Molecular weight 76488 as tartrate salt
30739 as base
4 Solubility Slightly soluble in water and sparingly soluble in
alcohol
5 pKa 62
6 Log P 12
7 Nature of the
molecule
Basic in nature and ionic compound
8 UV sensitivity
UV active since ZT has phenyl ring systems with
extended conjugated double bonds in its chemical
structure
149 Chapter 3
374 Impurity details
Zolpidem tartrate has eight potential impurities as mentioned in the section
31 The source and classifications of the impurities are tabulated below Table
32
Table 32 Origin of impurities for ZT
SNo Name of the impurity Source of impurity
Process related Degradation related
1 Imp-1 No Yes
2 Imp-2 Yes Yes
3 Imp-3 Yes No
4 Imp-4 Yes Yes
5 Imp-5 Yes No
6 Imp-6 Yes No
7 Imp-7 Yes No
8 Imp-8 Yes No
375 Preliminary chromatographic conditions
3751 Selection of detection wavelength
A composite solution containing 50 μgmL of drug and 1 μgmL of each of
the eight impurities was prepared in the diluent All the samples were analyzed
in HPLC- PDA system and the UV spectrums for all the components were
extracted
150 Chapter 3
Fig 34 Individual and overlaid UV spectra of ZT and its impurities
151 Chapter 3
152 Chapter 3
153 Chapter 3
Selection of wavelength to monitor zolpidem tartrate and its impurities
An optimal wavelength of detection for ZT and its impurities was selected as
254 nm based on UV spectra using PDA detector for LC analysis Fig 34
3752 Selection of UPLC column
Initial experiments were carried out on different commercially available RP-
UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH
shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to
determine the selectivity and symmetry of the individual components In all the
columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline
separation and imp-5 amp imp-6 peaks were merged The remaining peaks were
separated with resolution (RS)gt15 and the peak tailing was greater than 15 for
ZT Due to the structural similarities co-elution of above mentioned impurity
0970 Imp-1
1134 Imp-2
1978 Imp-3
2486 Imp-4
3443 Imp-5
3619 Imp-6
3897 ZT
4796 Imp-7
5671 Imp-8
2631
3438
20672114
3200
3578
3057
3710
2114
2443
3854
2443
3105
2114
2443
3057
nm
22000 24000 26000 28000 30000 32000 34000 36000 38000
mA
u
154 Chapter 3
pairs was observed Additionally peak tailing was also observed due to the
secondary interactions with residual silanol groups Although separation was
similar in all the three columns HSS T3 column had shown relatively good
peak symmetry and efficiency so it was thus selected for further optimization
Addition of triethylamine in the buffer improved the peak shape of ZT but the
resolution between imp-5 amp imp-6 remained critical
3753 Effect of buffer pH
The important parameters buffer pH and gradient mode of elution that
would likely to have significant effect on critical pair resolution were
investigated There was no substantial change in the resolution between Imps-1
amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly
decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp
6 (RS2) and Imp-6 amp ZT (RS
3) were improved when the pH altered from 60 to 70
The increase in Rs1 in acidic mobile phase and RS
2 RS3 in basic mobile phase
can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of
adequately low pH will remain unionized and get increased retentions On the
other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be
more retained From table 33 acceptable resolutions were obtained at pH 70
with isocratic elution and pH 75 with gradient elution but in both the cases
the run time was longer
155 Chapter 3
Table 33 Effect of Buffer pH on resolution of critical pairs
Mode of
elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time
Isocratic 40 21 lt10 17 15min
Isocratic 50 22 lt10 17 14min
Isocratic 60 21 11 18 15min
Isocratic 70 20 18 22 gt15min
Gradient 55 21 lt10 17 8min
Gradient 75 18 22 23 gt15min
Gradient A 55 B
73 25 21 22 8 min
a Rs1 Resolution between Imp-1 and Imp-2
b Rs2 Resolution between Imp-5 and Imp-6
c Rs3Resolution between Imp-6 and ZT
3754 Optimization of gradient program
Gradient mode of elution containing mobile phase-A (1mL of TEA1L
phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile
phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555
vv) were chosen to separate both the critical pairs in a single run and this
resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)
156 Chapter 3
Fig 35 Co elution of imp-5 and imp-6 at pH 55
Based on the experimental data it was predicted that increase in mobile
phase pH during the gradient elution could resolve the Imp-5 and Imp-6
without compromising the total run time Hence two more experiments were
designed with a mobile phase-B as combination of water ACN (4555 vv) for
experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-
2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase
the solvent strength thereby to elute strongly retained compounds (Imp-8)
faster The RS2 was less than 10 for experiment-1 and RS
2 was 15 for
experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value
60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from
60 to 70) between 50min and 82min was added to the program to reduce the
run time So Imp-8 was eluted at 8min To further improve the RS2 gradient
program was optimized with a linear increase of mobile phase-B with varying
157 Chapter 3
time points An optimum resolution was obtained (Fig 36) when the gradient
program of section 22 was used In addition to this the elution time of imp-8
was reduced to 6 min in the modified gradient method
Fig 36 Baseline separations of zolpidem and its impurities
Placebo interference study was verified by injecting sample solutions of
placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-
8 and ZT analyte peak was found
Fig 37 Placebo chromatogram for zolpidem tablets
Imp-
1 - 1
051
Imp-
2 - 1
231
Imp-
3 - 2
139
Imp-
4 - 2
692
Imp-
5 - 3
720
Imp-
6 - 3
892
ZT -
414
7
Imp-
7 - 5
096
Imp-
8 - 5
981
AU
-0020
-0015
-0010
-0005
0000
0005
0010
0015
0020
0025
0030
0035
0040
0045
0050
0055
0060
Minutes
050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850
158 Chapter 3
Three different lots of mobile phases were prepared and the consistency in
the resolution was verified (Table 34) A system suitability check (RS1 RS
2 amp
RS3) was incorporated to ensure the adequate system performance during the
regular analysis and method validation
Table 34 Reproducibility in separation of ZT and its impurities
Compound Average
RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing
factor
(n=6)c (n=6)c
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT) of
Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6)
159 Chapter 3
Fig 38 Peak purity plot for ZT in optimized conditions
Where PA = Purity angle and TH = Purity threshold
3755 The finalized RP-UPLC conditions for the estimation of assay and
related substances of ZT
Apparatus An UPLC with VWD and integrator
Column Acquity UPLC HSS T3 C-18 100 mm length
21 mm id 18 micron particle size
Flow Rate 03 ml min
Wave length 254 nm
Load 10microL
Column temperature 250C
Diluent Acetonitrile (ACN) Water (82)
Buffer Buffer solution was prepared by adding 1mL
of Triethyl amine (TEA) to 1000mL of 10 mM
NaH2PO4 H2O
Mobile phase-A Mixture of buffer (pH adjusted to 55 with
H3PO4) ACN and MeOH in the ratio of
602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with
H3PO4) and ACN in the ratio of 4555 (vv)
160 Chapter 3
Table 35 Gradient program
Time Mobile phase-A Mobile phase -B
001 95 5
25 95 5
35 40 60
50 40 60
82 30 70
85 95 5
100 95 5
Table 36 Retention times and relative retention times wrto ZT
SNo ImpurityAnalyte
name
Retention time ~Relative
retention time
1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100
8 Imp-7 5118 121 9 Imp-8 6037 142
3756 Degradation behavior
The purpose of this study is to establish the fact that the inherent chemical
stability of the molecule remains intact during its existence in the solid dosage
form along with other excipients So forced degradation studies should be
conducted on the drug to generate product related variants which in turn can
help to establish degradation pathways and thereby to develop and validate
suitable analytical procedure [22]
In this perspective a series of induced degradation experiments were
conducted at a concentration of 50 μgmL of ZT in active pharmaceutical
161 Chapter 3
ingredients (API) and tablets Degradation was induced by external physico-
chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)
oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal
treatment (105oC for 7 days)
The purity of the peaks obtained from the stressed samples was verified by
using the PDA detector The purity angle and purity threshold parameters were
evaluated for all the stressed samples to demonstrate the homogeneity of the
analyte peaks Assay of stressed samples was performed by comparison with
reference standards and the mass balance was calculated
37561 Degradation in Acidic solution
A single major degradation product Imp-2 (Zolpidem Acid) was formed when
ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The
degradation pathway is based on the premise that acid or base catalyzed
reaction between water and tertiary amide gives its parent carboxylic acid and
an amine Here the other hydrolytic product was dimethylamine Using LC-MS
in positive ion ESI the degradation product was found to have an mz value of
281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2
and UV spectral match
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
147 Chapter 3
3 Single analytical method for the determination of assay and related
substances in bulk actives and dosage forms
4 Targeted for a minimum resolution of 15 between impurities and tailing
factor lt 20 for ZT peak at assay method
5 Aimed at LOQ values ie 0375microgmL (50 of the specification level which
is equivalent to 0375microgmL) for all the impurities of ZT
372 Method development strategy
A systematic method development approach had been followed to achieve
successful separation The key steps are mentioned below
Fig 33 Flow diagram of method development strategy
148 Chapter 3
373 Classification of the sample
Zolpidem tartrate and its properties
1 Chemical name N N 6-trimethyl-2-p-tolylimidazo [1 2-a] pyridine-3-
acetamide L-(+)-tartrate (21)
2 Chemical structure The polar groups in the structure are highlighted in
circles
3 Molecular weight 76488 as tartrate salt
30739 as base
4 Solubility Slightly soluble in water and sparingly soluble in
alcohol
5 pKa 62
6 Log P 12
7 Nature of the
molecule
Basic in nature and ionic compound
8 UV sensitivity
UV active since ZT has phenyl ring systems with
extended conjugated double bonds in its chemical
structure
149 Chapter 3
374 Impurity details
Zolpidem tartrate has eight potential impurities as mentioned in the section
31 The source and classifications of the impurities are tabulated below Table
32
Table 32 Origin of impurities for ZT
SNo Name of the impurity Source of impurity
Process related Degradation related
1 Imp-1 No Yes
2 Imp-2 Yes Yes
3 Imp-3 Yes No
4 Imp-4 Yes Yes
5 Imp-5 Yes No
6 Imp-6 Yes No
7 Imp-7 Yes No
8 Imp-8 Yes No
375 Preliminary chromatographic conditions
3751 Selection of detection wavelength
A composite solution containing 50 μgmL of drug and 1 μgmL of each of
the eight impurities was prepared in the diluent All the samples were analyzed
in HPLC- PDA system and the UV spectrums for all the components were
extracted
150 Chapter 3
Fig 34 Individual and overlaid UV spectra of ZT and its impurities
151 Chapter 3
152 Chapter 3
153 Chapter 3
Selection of wavelength to monitor zolpidem tartrate and its impurities
An optimal wavelength of detection for ZT and its impurities was selected as
254 nm based on UV spectra using PDA detector for LC analysis Fig 34
3752 Selection of UPLC column
Initial experiments were carried out on different commercially available RP-
UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH
shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to
determine the selectivity and symmetry of the individual components In all the
columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline
separation and imp-5 amp imp-6 peaks were merged The remaining peaks were
separated with resolution (RS)gt15 and the peak tailing was greater than 15 for
ZT Due to the structural similarities co-elution of above mentioned impurity
0970 Imp-1
1134 Imp-2
1978 Imp-3
2486 Imp-4
3443 Imp-5
3619 Imp-6
3897 ZT
4796 Imp-7
5671 Imp-8
2631
3438
20672114
3200
3578
3057
3710
2114
2443
3854
2443
3105
2114
2443
3057
nm
22000 24000 26000 28000 30000 32000 34000 36000 38000
mA
u
154 Chapter 3
pairs was observed Additionally peak tailing was also observed due to the
secondary interactions with residual silanol groups Although separation was
similar in all the three columns HSS T3 column had shown relatively good
peak symmetry and efficiency so it was thus selected for further optimization
Addition of triethylamine in the buffer improved the peak shape of ZT but the
resolution between imp-5 amp imp-6 remained critical
3753 Effect of buffer pH
The important parameters buffer pH and gradient mode of elution that
would likely to have significant effect on critical pair resolution were
investigated There was no substantial change in the resolution between Imps-1
amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly
decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp
6 (RS2) and Imp-6 amp ZT (RS
3) were improved when the pH altered from 60 to 70
The increase in Rs1 in acidic mobile phase and RS
2 RS3 in basic mobile phase
can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of
adequately low pH will remain unionized and get increased retentions On the
other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be
more retained From table 33 acceptable resolutions were obtained at pH 70
with isocratic elution and pH 75 with gradient elution but in both the cases
the run time was longer
155 Chapter 3
Table 33 Effect of Buffer pH on resolution of critical pairs
Mode of
elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time
Isocratic 40 21 lt10 17 15min
Isocratic 50 22 lt10 17 14min
Isocratic 60 21 11 18 15min
Isocratic 70 20 18 22 gt15min
Gradient 55 21 lt10 17 8min
Gradient 75 18 22 23 gt15min
Gradient A 55 B
73 25 21 22 8 min
a Rs1 Resolution between Imp-1 and Imp-2
b Rs2 Resolution between Imp-5 and Imp-6
c Rs3Resolution between Imp-6 and ZT
3754 Optimization of gradient program
Gradient mode of elution containing mobile phase-A (1mL of TEA1L
phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile
phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555
vv) were chosen to separate both the critical pairs in a single run and this
resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)
156 Chapter 3
Fig 35 Co elution of imp-5 and imp-6 at pH 55
Based on the experimental data it was predicted that increase in mobile
phase pH during the gradient elution could resolve the Imp-5 and Imp-6
without compromising the total run time Hence two more experiments were
designed with a mobile phase-B as combination of water ACN (4555 vv) for
experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-
2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase
the solvent strength thereby to elute strongly retained compounds (Imp-8)
faster The RS2 was less than 10 for experiment-1 and RS
2 was 15 for
experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value
60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from
60 to 70) between 50min and 82min was added to the program to reduce the
run time So Imp-8 was eluted at 8min To further improve the RS2 gradient
program was optimized with a linear increase of mobile phase-B with varying
157 Chapter 3
time points An optimum resolution was obtained (Fig 36) when the gradient
program of section 22 was used In addition to this the elution time of imp-8
was reduced to 6 min in the modified gradient method
Fig 36 Baseline separations of zolpidem and its impurities
Placebo interference study was verified by injecting sample solutions of
placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-
8 and ZT analyte peak was found
Fig 37 Placebo chromatogram for zolpidem tablets
Imp-
1 - 1
051
Imp-
2 - 1
231
Imp-
3 - 2
139
Imp-
4 - 2
692
Imp-
5 - 3
720
Imp-
6 - 3
892
ZT -
414
7
Imp-
7 - 5
096
Imp-
8 - 5
981
AU
-0020
-0015
-0010
-0005
0000
0005
0010
0015
0020
0025
0030
0035
0040
0045
0050
0055
0060
Minutes
050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850
158 Chapter 3
Three different lots of mobile phases were prepared and the consistency in
the resolution was verified (Table 34) A system suitability check (RS1 RS
2 amp
RS3) was incorporated to ensure the adequate system performance during the
regular analysis and method validation
Table 34 Reproducibility in separation of ZT and its impurities
Compound Average
RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing
factor
(n=6)c (n=6)c
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT) of
Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6)
159 Chapter 3
Fig 38 Peak purity plot for ZT in optimized conditions
Where PA = Purity angle and TH = Purity threshold
3755 The finalized RP-UPLC conditions for the estimation of assay and
related substances of ZT
Apparatus An UPLC with VWD and integrator
Column Acquity UPLC HSS T3 C-18 100 mm length
21 mm id 18 micron particle size
Flow Rate 03 ml min
Wave length 254 nm
Load 10microL
Column temperature 250C
Diluent Acetonitrile (ACN) Water (82)
Buffer Buffer solution was prepared by adding 1mL
of Triethyl amine (TEA) to 1000mL of 10 mM
NaH2PO4 H2O
Mobile phase-A Mixture of buffer (pH adjusted to 55 with
H3PO4) ACN and MeOH in the ratio of
602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with
H3PO4) and ACN in the ratio of 4555 (vv)
160 Chapter 3
Table 35 Gradient program
Time Mobile phase-A Mobile phase -B
001 95 5
25 95 5
35 40 60
50 40 60
82 30 70
85 95 5
100 95 5
Table 36 Retention times and relative retention times wrto ZT
SNo ImpurityAnalyte
name
Retention time ~Relative
retention time
1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100
8 Imp-7 5118 121 9 Imp-8 6037 142
3756 Degradation behavior
The purpose of this study is to establish the fact that the inherent chemical
stability of the molecule remains intact during its existence in the solid dosage
form along with other excipients So forced degradation studies should be
conducted on the drug to generate product related variants which in turn can
help to establish degradation pathways and thereby to develop and validate
suitable analytical procedure [22]
In this perspective a series of induced degradation experiments were
conducted at a concentration of 50 μgmL of ZT in active pharmaceutical
161 Chapter 3
ingredients (API) and tablets Degradation was induced by external physico-
chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)
oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal
treatment (105oC for 7 days)
The purity of the peaks obtained from the stressed samples was verified by
using the PDA detector The purity angle and purity threshold parameters were
evaluated for all the stressed samples to demonstrate the homogeneity of the
analyte peaks Assay of stressed samples was performed by comparison with
reference standards and the mass balance was calculated
37561 Degradation in Acidic solution
A single major degradation product Imp-2 (Zolpidem Acid) was formed when
ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The
degradation pathway is based on the premise that acid or base catalyzed
reaction between water and tertiary amide gives its parent carboxylic acid and
an amine Here the other hydrolytic product was dimethylamine Using LC-MS
in positive ion ESI the degradation product was found to have an mz value of
281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2
and UV spectral match
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
148 Chapter 3
373 Classification of the sample
Zolpidem tartrate and its properties
1 Chemical name N N 6-trimethyl-2-p-tolylimidazo [1 2-a] pyridine-3-
acetamide L-(+)-tartrate (21)
2 Chemical structure The polar groups in the structure are highlighted in
circles
3 Molecular weight 76488 as tartrate salt
30739 as base
4 Solubility Slightly soluble in water and sparingly soluble in
alcohol
5 pKa 62
6 Log P 12
7 Nature of the
molecule
Basic in nature and ionic compound
8 UV sensitivity
UV active since ZT has phenyl ring systems with
extended conjugated double bonds in its chemical
structure
149 Chapter 3
374 Impurity details
Zolpidem tartrate has eight potential impurities as mentioned in the section
31 The source and classifications of the impurities are tabulated below Table
32
Table 32 Origin of impurities for ZT
SNo Name of the impurity Source of impurity
Process related Degradation related
1 Imp-1 No Yes
2 Imp-2 Yes Yes
3 Imp-3 Yes No
4 Imp-4 Yes Yes
5 Imp-5 Yes No
6 Imp-6 Yes No
7 Imp-7 Yes No
8 Imp-8 Yes No
375 Preliminary chromatographic conditions
3751 Selection of detection wavelength
A composite solution containing 50 μgmL of drug and 1 μgmL of each of
the eight impurities was prepared in the diluent All the samples were analyzed
in HPLC- PDA system and the UV spectrums for all the components were
extracted
150 Chapter 3
Fig 34 Individual and overlaid UV spectra of ZT and its impurities
151 Chapter 3
152 Chapter 3
153 Chapter 3
Selection of wavelength to monitor zolpidem tartrate and its impurities
An optimal wavelength of detection for ZT and its impurities was selected as
254 nm based on UV spectra using PDA detector for LC analysis Fig 34
3752 Selection of UPLC column
Initial experiments were carried out on different commercially available RP-
UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH
shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to
determine the selectivity and symmetry of the individual components In all the
columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline
separation and imp-5 amp imp-6 peaks were merged The remaining peaks were
separated with resolution (RS)gt15 and the peak tailing was greater than 15 for
ZT Due to the structural similarities co-elution of above mentioned impurity
0970 Imp-1
1134 Imp-2
1978 Imp-3
2486 Imp-4
3443 Imp-5
3619 Imp-6
3897 ZT
4796 Imp-7
5671 Imp-8
2631
3438
20672114
3200
3578
3057
3710
2114
2443
3854
2443
3105
2114
2443
3057
nm
22000 24000 26000 28000 30000 32000 34000 36000 38000
mA
u
154 Chapter 3
pairs was observed Additionally peak tailing was also observed due to the
secondary interactions with residual silanol groups Although separation was
similar in all the three columns HSS T3 column had shown relatively good
peak symmetry and efficiency so it was thus selected for further optimization
Addition of triethylamine in the buffer improved the peak shape of ZT but the
resolution between imp-5 amp imp-6 remained critical
3753 Effect of buffer pH
The important parameters buffer pH and gradient mode of elution that
would likely to have significant effect on critical pair resolution were
investigated There was no substantial change in the resolution between Imps-1
amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly
decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp
6 (RS2) and Imp-6 amp ZT (RS
3) were improved when the pH altered from 60 to 70
The increase in Rs1 in acidic mobile phase and RS
2 RS3 in basic mobile phase
can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of
adequately low pH will remain unionized and get increased retentions On the
other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be
more retained From table 33 acceptable resolutions were obtained at pH 70
with isocratic elution and pH 75 with gradient elution but in both the cases
the run time was longer
155 Chapter 3
Table 33 Effect of Buffer pH on resolution of critical pairs
Mode of
elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time
Isocratic 40 21 lt10 17 15min
Isocratic 50 22 lt10 17 14min
Isocratic 60 21 11 18 15min
Isocratic 70 20 18 22 gt15min
Gradient 55 21 lt10 17 8min
Gradient 75 18 22 23 gt15min
Gradient A 55 B
73 25 21 22 8 min
a Rs1 Resolution between Imp-1 and Imp-2
b Rs2 Resolution between Imp-5 and Imp-6
c Rs3Resolution between Imp-6 and ZT
3754 Optimization of gradient program
Gradient mode of elution containing mobile phase-A (1mL of TEA1L
phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile
phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555
vv) were chosen to separate both the critical pairs in a single run and this
resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)
156 Chapter 3
Fig 35 Co elution of imp-5 and imp-6 at pH 55
Based on the experimental data it was predicted that increase in mobile
phase pH during the gradient elution could resolve the Imp-5 and Imp-6
without compromising the total run time Hence two more experiments were
designed with a mobile phase-B as combination of water ACN (4555 vv) for
experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-
2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase
the solvent strength thereby to elute strongly retained compounds (Imp-8)
faster The RS2 was less than 10 for experiment-1 and RS
2 was 15 for
experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value
60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from
60 to 70) between 50min and 82min was added to the program to reduce the
run time So Imp-8 was eluted at 8min To further improve the RS2 gradient
program was optimized with a linear increase of mobile phase-B with varying
157 Chapter 3
time points An optimum resolution was obtained (Fig 36) when the gradient
program of section 22 was used In addition to this the elution time of imp-8
was reduced to 6 min in the modified gradient method
Fig 36 Baseline separations of zolpidem and its impurities
Placebo interference study was verified by injecting sample solutions of
placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-
8 and ZT analyte peak was found
Fig 37 Placebo chromatogram for zolpidem tablets
Imp-
1 - 1
051
Imp-
2 - 1
231
Imp-
3 - 2
139
Imp-
4 - 2
692
Imp-
5 - 3
720
Imp-
6 - 3
892
ZT -
414
7
Imp-
7 - 5
096
Imp-
8 - 5
981
AU
-0020
-0015
-0010
-0005
0000
0005
0010
0015
0020
0025
0030
0035
0040
0045
0050
0055
0060
Minutes
050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850
158 Chapter 3
Three different lots of mobile phases were prepared and the consistency in
the resolution was verified (Table 34) A system suitability check (RS1 RS
2 amp
RS3) was incorporated to ensure the adequate system performance during the
regular analysis and method validation
Table 34 Reproducibility in separation of ZT and its impurities
Compound Average
RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing
factor
(n=6)c (n=6)c
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT) of
Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6)
159 Chapter 3
Fig 38 Peak purity plot for ZT in optimized conditions
Where PA = Purity angle and TH = Purity threshold
3755 The finalized RP-UPLC conditions for the estimation of assay and
related substances of ZT
Apparatus An UPLC with VWD and integrator
Column Acquity UPLC HSS T3 C-18 100 mm length
21 mm id 18 micron particle size
Flow Rate 03 ml min
Wave length 254 nm
Load 10microL
Column temperature 250C
Diluent Acetonitrile (ACN) Water (82)
Buffer Buffer solution was prepared by adding 1mL
of Triethyl amine (TEA) to 1000mL of 10 mM
NaH2PO4 H2O
Mobile phase-A Mixture of buffer (pH adjusted to 55 with
H3PO4) ACN and MeOH in the ratio of
602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with
H3PO4) and ACN in the ratio of 4555 (vv)
160 Chapter 3
Table 35 Gradient program
Time Mobile phase-A Mobile phase -B
001 95 5
25 95 5
35 40 60
50 40 60
82 30 70
85 95 5
100 95 5
Table 36 Retention times and relative retention times wrto ZT
SNo ImpurityAnalyte
name
Retention time ~Relative
retention time
1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100
8 Imp-7 5118 121 9 Imp-8 6037 142
3756 Degradation behavior
The purpose of this study is to establish the fact that the inherent chemical
stability of the molecule remains intact during its existence in the solid dosage
form along with other excipients So forced degradation studies should be
conducted on the drug to generate product related variants which in turn can
help to establish degradation pathways and thereby to develop and validate
suitable analytical procedure [22]
In this perspective a series of induced degradation experiments were
conducted at a concentration of 50 μgmL of ZT in active pharmaceutical
161 Chapter 3
ingredients (API) and tablets Degradation was induced by external physico-
chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)
oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal
treatment (105oC for 7 days)
The purity of the peaks obtained from the stressed samples was verified by
using the PDA detector The purity angle and purity threshold parameters were
evaluated for all the stressed samples to demonstrate the homogeneity of the
analyte peaks Assay of stressed samples was performed by comparison with
reference standards and the mass balance was calculated
37561 Degradation in Acidic solution
A single major degradation product Imp-2 (Zolpidem Acid) was formed when
ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The
degradation pathway is based on the premise that acid or base catalyzed
reaction between water and tertiary amide gives its parent carboxylic acid and
an amine Here the other hydrolytic product was dimethylamine Using LC-MS
in positive ion ESI the degradation product was found to have an mz value of
281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2
and UV spectral match
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
149 Chapter 3
374 Impurity details
Zolpidem tartrate has eight potential impurities as mentioned in the section
31 The source and classifications of the impurities are tabulated below Table
32
Table 32 Origin of impurities for ZT
SNo Name of the impurity Source of impurity
Process related Degradation related
1 Imp-1 No Yes
2 Imp-2 Yes Yes
3 Imp-3 Yes No
4 Imp-4 Yes Yes
5 Imp-5 Yes No
6 Imp-6 Yes No
7 Imp-7 Yes No
8 Imp-8 Yes No
375 Preliminary chromatographic conditions
3751 Selection of detection wavelength
A composite solution containing 50 μgmL of drug and 1 μgmL of each of
the eight impurities was prepared in the diluent All the samples were analyzed
in HPLC- PDA system and the UV spectrums for all the components were
extracted
150 Chapter 3
Fig 34 Individual and overlaid UV spectra of ZT and its impurities
151 Chapter 3
152 Chapter 3
153 Chapter 3
Selection of wavelength to monitor zolpidem tartrate and its impurities
An optimal wavelength of detection for ZT and its impurities was selected as
254 nm based on UV spectra using PDA detector for LC analysis Fig 34
3752 Selection of UPLC column
Initial experiments were carried out on different commercially available RP-
UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH
shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to
determine the selectivity and symmetry of the individual components In all the
columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline
separation and imp-5 amp imp-6 peaks were merged The remaining peaks were
separated with resolution (RS)gt15 and the peak tailing was greater than 15 for
ZT Due to the structural similarities co-elution of above mentioned impurity
0970 Imp-1
1134 Imp-2
1978 Imp-3
2486 Imp-4
3443 Imp-5
3619 Imp-6
3897 ZT
4796 Imp-7
5671 Imp-8
2631
3438
20672114
3200
3578
3057
3710
2114
2443
3854
2443
3105
2114
2443
3057
nm
22000 24000 26000 28000 30000 32000 34000 36000 38000
mA
u
154 Chapter 3
pairs was observed Additionally peak tailing was also observed due to the
secondary interactions with residual silanol groups Although separation was
similar in all the three columns HSS T3 column had shown relatively good
peak symmetry and efficiency so it was thus selected for further optimization
Addition of triethylamine in the buffer improved the peak shape of ZT but the
resolution between imp-5 amp imp-6 remained critical
3753 Effect of buffer pH
The important parameters buffer pH and gradient mode of elution that
would likely to have significant effect on critical pair resolution were
investigated There was no substantial change in the resolution between Imps-1
amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly
decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp
6 (RS2) and Imp-6 amp ZT (RS
3) were improved when the pH altered from 60 to 70
The increase in Rs1 in acidic mobile phase and RS
2 RS3 in basic mobile phase
can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of
adequately low pH will remain unionized and get increased retentions On the
other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be
more retained From table 33 acceptable resolutions were obtained at pH 70
with isocratic elution and pH 75 with gradient elution but in both the cases
the run time was longer
155 Chapter 3
Table 33 Effect of Buffer pH on resolution of critical pairs
Mode of
elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time
Isocratic 40 21 lt10 17 15min
Isocratic 50 22 lt10 17 14min
Isocratic 60 21 11 18 15min
Isocratic 70 20 18 22 gt15min
Gradient 55 21 lt10 17 8min
Gradient 75 18 22 23 gt15min
Gradient A 55 B
73 25 21 22 8 min
a Rs1 Resolution between Imp-1 and Imp-2
b Rs2 Resolution between Imp-5 and Imp-6
c Rs3Resolution between Imp-6 and ZT
3754 Optimization of gradient program
Gradient mode of elution containing mobile phase-A (1mL of TEA1L
phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile
phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555
vv) were chosen to separate both the critical pairs in a single run and this
resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)
156 Chapter 3
Fig 35 Co elution of imp-5 and imp-6 at pH 55
Based on the experimental data it was predicted that increase in mobile
phase pH during the gradient elution could resolve the Imp-5 and Imp-6
without compromising the total run time Hence two more experiments were
designed with a mobile phase-B as combination of water ACN (4555 vv) for
experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-
2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase
the solvent strength thereby to elute strongly retained compounds (Imp-8)
faster The RS2 was less than 10 for experiment-1 and RS
2 was 15 for
experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value
60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from
60 to 70) between 50min and 82min was added to the program to reduce the
run time So Imp-8 was eluted at 8min To further improve the RS2 gradient
program was optimized with a linear increase of mobile phase-B with varying
157 Chapter 3
time points An optimum resolution was obtained (Fig 36) when the gradient
program of section 22 was used In addition to this the elution time of imp-8
was reduced to 6 min in the modified gradient method
Fig 36 Baseline separations of zolpidem and its impurities
Placebo interference study was verified by injecting sample solutions of
placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-
8 and ZT analyte peak was found
Fig 37 Placebo chromatogram for zolpidem tablets
Imp-
1 - 1
051
Imp-
2 - 1
231
Imp-
3 - 2
139
Imp-
4 - 2
692
Imp-
5 - 3
720
Imp-
6 - 3
892
ZT -
414
7
Imp-
7 - 5
096
Imp-
8 - 5
981
AU
-0020
-0015
-0010
-0005
0000
0005
0010
0015
0020
0025
0030
0035
0040
0045
0050
0055
0060
Minutes
050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850
158 Chapter 3
Three different lots of mobile phases were prepared and the consistency in
the resolution was verified (Table 34) A system suitability check (RS1 RS
2 amp
RS3) was incorporated to ensure the adequate system performance during the
regular analysis and method validation
Table 34 Reproducibility in separation of ZT and its impurities
Compound Average
RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing
factor
(n=6)c (n=6)c
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT) of
Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6)
159 Chapter 3
Fig 38 Peak purity plot for ZT in optimized conditions
Where PA = Purity angle and TH = Purity threshold
3755 The finalized RP-UPLC conditions for the estimation of assay and
related substances of ZT
Apparatus An UPLC with VWD and integrator
Column Acquity UPLC HSS T3 C-18 100 mm length
21 mm id 18 micron particle size
Flow Rate 03 ml min
Wave length 254 nm
Load 10microL
Column temperature 250C
Diluent Acetonitrile (ACN) Water (82)
Buffer Buffer solution was prepared by adding 1mL
of Triethyl amine (TEA) to 1000mL of 10 mM
NaH2PO4 H2O
Mobile phase-A Mixture of buffer (pH adjusted to 55 with
H3PO4) ACN and MeOH in the ratio of
602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with
H3PO4) and ACN in the ratio of 4555 (vv)
160 Chapter 3
Table 35 Gradient program
Time Mobile phase-A Mobile phase -B
001 95 5
25 95 5
35 40 60
50 40 60
82 30 70
85 95 5
100 95 5
Table 36 Retention times and relative retention times wrto ZT
SNo ImpurityAnalyte
name
Retention time ~Relative
retention time
1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100
8 Imp-7 5118 121 9 Imp-8 6037 142
3756 Degradation behavior
The purpose of this study is to establish the fact that the inherent chemical
stability of the molecule remains intact during its existence in the solid dosage
form along with other excipients So forced degradation studies should be
conducted on the drug to generate product related variants which in turn can
help to establish degradation pathways and thereby to develop and validate
suitable analytical procedure [22]
In this perspective a series of induced degradation experiments were
conducted at a concentration of 50 μgmL of ZT in active pharmaceutical
161 Chapter 3
ingredients (API) and tablets Degradation was induced by external physico-
chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)
oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal
treatment (105oC for 7 days)
The purity of the peaks obtained from the stressed samples was verified by
using the PDA detector The purity angle and purity threshold parameters were
evaluated for all the stressed samples to demonstrate the homogeneity of the
analyte peaks Assay of stressed samples was performed by comparison with
reference standards and the mass balance was calculated
37561 Degradation in Acidic solution
A single major degradation product Imp-2 (Zolpidem Acid) was formed when
ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The
degradation pathway is based on the premise that acid or base catalyzed
reaction between water and tertiary amide gives its parent carboxylic acid and
an amine Here the other hydrolytic product was dimethylamine Using LC-MS
in positive ion ESI the degradation product was found to have an mz value of
281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2
and UV spectral match
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
150 Chapter 3
Fig 34 Individual and overlaid UV spectra of ZT and its impurities
151 Chapter 3
152 Chapter 3
153 Chapter 3
Selection of wavelength to monitor zolpidem tartrate and its impurities
An optimal wavelength of detection for ZT and its impurities was selected as
254 nm based on UV spectra using PDA detector for LC analysis Fig 34
3752 Selection of UPLC column
Initial experiments were carried out on different commercially available RP-
UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH
shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to
determine the selectivity and symmetry of the individual components In all the
columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline
separation and imp-5 amp imp-6 peaks were merged The remaining peaks were
separated with resolution (RS)gt15 and the peak tailing was greater than 15 for
ZT Due to the structural similarities co-elution of above mentioned impurity
0970 Imp-1
1134 Imp-2
1978 Imp-3
2486 Imp-4
3443 Imp-5
3619 Imp-6
3897 ZT
4796 Imp-7
5671 Imp-8
2631
3438
20672114
3200
3578
3057
3710
2114
2443
3854
2443
3105
2114
2443
3057
nm
22000 24000 26000 28000 30000 32000 34000 36000 38000
mA
u
154 Chapter 3
pairs was observed Additionally peak tailing was also observed due to the
secondary interactions with residual silanol groups Although separation was
similar in all the three columns HSS T3 column had shown relatively good
peak symmetry and efficiency so it was thus selected for further optimization
Addition of triethylamine in the buffer improved the peak shape of ZT but the
resolution between imp-5 amp imp-6 remained critical
3753 Effect of buffer pH
The important parameters buffer pH and gradient mode of elution that
would likely to have significant effect on critical pair resolution were
investigated There was no substantial change in the resolution between Imps-1
amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly
decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp
6 (RS2) and Imp-6 amp ZT (RS
3) were improved when the pH altered from 60 to 70
The increase in Rs1 in acidic mobile phase and RS
2 RS3 in basic mobile phase
can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of
adequately low pH will remain unionized and get increased retentions On the
other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be
more retained From table 33 acceptable resolutions were obtained at pH 70
with isocratic elution and pH 75 with gradient elution but in both the cases
the run time was longer
155 Chapter 3
Table 33 Effect of Buffer pH on resolution of critical pairs
Mode of
elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time
Isocratic 40 21 lt10 17 15min
Isocratic 50 22 lt10 17 14min
Isocratic 60 21 11 18 15min
Isocratic 70 20 18 22 gt15min
Gradient 55 21 lt10 17 8min
Gradient 75 18 22 23 gt15min
Gradient A 55 B
73 25 21 22 8 min
a Rs1 Resolution between Imp-1 and Imp-2
b Rs2 Resolution between Imp-5 and Imp-6
c Rs3Resolution between Imp-6 and ZT
3754 Optimization of gradient program
Gradient mode of elution containing mobile phase-A (1mL of TEA1L
phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile
phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555
vv) were chosen to separate both the critical pairs in a single run and this
resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)
156 Chapter 3
Fig 35 Co elution of imp-5 and imp-6 at pH 55
Based on the experimental data it was predicted that increase in mobile
phase pH during the gradient elution could resolve the Imp-5 and Imp-6
without compromising the total run time Hence two more experiments were
designed with a mobile phase-B as combination of water ACN (4555 vv) for
experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-
2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase
the solvent strength thereby to elute strongly retained compounds (Imp-8)
faster The RS2 was less than 10 for experiment-1 and RS
2 was 15 for
experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value
60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from
60 to 70) between 50min and 82min was added to the program to reduce the
run time So Imp-8 was eluted at 8min To further improve the RS2 gradient
program was optimized with a linear increase of mobile phase-B with varying
157 Chapter 3
time points An optimum resolution was obtained (Fig 36) when the gradient
program of section 22 was used In addition to this the elution time of imp-8
was reduced to 6 min in the modified gradient method
Fig 36 Baseline separations of zolpidem and its impurities
Placebo interference study was verified by injecting sample solutions of
placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-
8 and ZT analyte peak was found
Fig 37 Placebo chromatogram for zolpidem tablets
Imp-
1 - 1
051
Imp-
2 - 1
231
Imp-
3 - 2
139
Imp-
4 - 2
692
Imp-
5 - 3
720
Imp-
6 - 3
892
ZT -
414
7
Imp-
7 - 5
096
Imp-
8 - 5
981
AU
-0020
-0015
-0010
-0005
0000
0005
0010
0015
0020
0025
0030
0035
0040
0045
0050
0055
0060
Minutes
050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850
158 Chapter 3
Three different lots of mobile phases were prepared and the consistency in
the resolution was verified (Table 34) A system suitability check (RS1 RS
2 amp
RS3) was incorporated to ensure the adequate system performance during the
regular analysis and method validation
Table 34 Reproducibility in separation of ZT and its impurities
Compound Average
RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing
factor
(n=6)c (n=6)c
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT) of
Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6)
159 Chapter 3
Fig 38 Peak purity plot for ZT in optimized conditions
Where PA = Purity angle and TH = Purity threshold
3755 The finalized RP-UPLC conditions for the estimation of assay and
related substances of ZT
Apparatus An UPLC with VWD and integrator
Column Acquity UPLC HSS T3 C-18 100 mm length
21 mm id 18 micron particle size
Flow Rate 03 ml min
Wave length 254 nm
Load 10microL
Column temperature 250C
Diluent Acetonitrile (ACN) Water (82)
Buffer Buffer solution was prepared by adding 1mL
of Triethyl amine (TEA) to 1000mL of 10 mM
NaH2PO4 H2O
Mobile phase-A Mixture of buffer (pH adjusted to 55 with
H3PO4) ACN and MeOH in the ratio of
602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with
H3PO4) and ACN in the ratio of 4555 (vv)
160 Chapter 3
Table 35 Gradient program
Time Mobile phase-A Mobile phase -B
001 95 5
25 95 5
35 40 60
50 40 60
82 30 70
85 95 5
100 95 5
Table 36 Retention times and relative retention times wrto ZT
SNo ImpurityAnalyte
name
Retention time ~Relative
retention time
1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100
8 Imp-7 5118 121 9 Imp-8 6037 142
3756 Degradation behavior
The purpose of this study is to establish the fact that the inherent chemical
stability of the molecule remains intact during its existence in the solid dosage
form along with other excipients So forced degradation studies should be
conducted on the drug to generate product related variants which in turn can
help to establish degradation pathways and thereby to develop and validate
suitable analytical procedure [22]
In this perspective a series of induced degradation experiments were
conducted at a concentration of 50 μgmL of ZT in active pharmaceutical
161 Chapter 3
ingredients (API) and tablets Degradation was induced by external physico-
chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)
oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal
treatment (105oC for 7 days)
The purity of the peaks obtained from the stressed samples was verified by
using the PDA detector The purity angle and purity threshold parameters were
evaluated for all the stressed samples to demonstrate the homogeneity of the
analyte peaks Assay of stressed samples was performed by comparison with
reference standards and the mass balance was calculated
37561 Degradation in Acidic solution
A single major degradation product Imp-2 (Zolpidem Acid) was formed when
ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The
degradation pathway is based on the premise that acid or base catalyzed
reaction between water and tertiary amide gives its parent carboxylic acid and
an amine Here the other hydrolytic product was dimethylamine Using LC-MS
in positive ion ESI the degradation product was found to have an mz value of
281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2
and UV spectral match
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
151 Chapter 3
152 Chapter 3
153 Chapter 3
Selection of wavelength to monitor zolpidem tartrate and its impurities
An optimal wavelength of detection for ZT and its impurities was selected as
254 nm based on UV spectra using PDA detector for LC analysis Fig 34
3752 Selection of UPLC column
Initial experiments were carried out on different commercially available RP-
UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH
shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to
determine the selectivity and symmetry of the individual components In all the
columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline
separation and imp-5 amp imp-6 peaks were merged The remaining peaks were
separated with resolution (RS)gt15 and the peak tailing was greater than 15 for
ZT Due to the structural similarities co-elution of above mentioned impurity
0970 Imp-1
1134 Imp-2
1978 Imp-3
2486 Imp-4
3443 Imp-5
3619 Imp-6
3897 ZT
4796 Imp-7
5671 Imp-8
2631
3438
20672114
3200
3578
3057
3710
2114
2443
3854
2443
3105
2114
2443
3057
nm
22000 24000 26000 28000 30000 32000 34000 36000 38000
mA
u
154 Chapter 3
pairs was observed Additionally peak tailing was also observed due to the
secondary interactions with residual silanol groups Although separation was
similar in all the three columns HSS T3 column had shown relatively good
peak symmetry and efficiency so it was thus selected for further optimization
Addition of triethylamine in the buffer improved the peak shape of ZT but the
resolution between imp-5 amp imp-6 remained critical
3753 Effect of buffer pH
The important parameters buffer pH and gradient mode of elution that
would likely to have significant effect on critical pair resolution were
investigated There was no substantial change in the resolution between Imps-1
amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly
decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp
6 (RS2) and Imp-6 amp ZT (RS
3) were improved when the pH altered from 60 to 70
The increase in Rs1 in acidic mobile phase and RS
2 RS3 in basic mobile phase
can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of
adequately low pH will remain unionized and get increased retentions On the
other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be
more retained From table 33 acceptable resolutions were obtained at pH 70
with isocratic elution and pH 75 with gradient elution but in both the cases
the run time was longer
155 Chapter 3
Table 33 Effect of Buffer pH on resolution of critical pairs
Mode of
elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time
Isocratic 40 21 lt10 17 15min
Isocratic 50 22 lt10 17 14min
Isocratic 60 21 11 18 15min
Isocratic 70 20 18 22 gt15min
Gradient 55 21 lt10 17 8min
Gradient 75 18 22 23 gt15min
Gradient A 55 B
73 25 21 22 8 min
a Rs1 Resolution between Imp-1 and Imp-2
b Rs2 Resolution between Imp-5 and Imp-6
c Rs3Resolution between Imp-6 and ZT
3754 Optimization of gradient program
Gradient mode of elution containing mobile phase-A (1mL of TEA1L
phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile
phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555
vv) were chosen to separate both the critical pairs in a single run and this
resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)
156 Chapter 3
Fig 35 Co elution of imp-5 and imp-6 at pH 55
Based on the experimental data it was predicted that increase in mobile
phase pH during the gradient elution could resolve the Imp-5 and Imp-6
without compromising the total run time Hence two more experiments were
designed with a mobile phase-B as combination of water ACN (4555 vv) for
experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-
2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase
the solvent strength thereby to elute strongly retained compounds (Imp-8)
faster The RS2 was less than 10 for experiment-1 and RS
2 was 15 for
experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value
60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from
60 to 70) between 50min and 82min was added to the program to reduce the
run time So Imp-8 was eluted at 8min To further improve the RS2 gradient
program was optimized with a linear increase of mobile phase-B with varying
157 Chapter 3
time points An optimum resolution was obtained (Fig 36) when the gradient
program of section 22 was used In addition to this the elution time of imp-8
was reduced to 6 min in the modified gradient method
Fig 36 Baseline separations of zolpidem and its impurities
Placebo interference study was verified by injecting sample solutions of
placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-
8 and ZT analyte peak was found
Fig 37 Placebo chromatogram for zolpidem tablets
Imp-
1 - 1
051
Imp-
2 - 1
231
Imp-
3 - 2
139
Imp-
4 - 2
692
Imp-
5 - 3
720
Imp-
6 - 3
892
ZT -
414
7
Imp-
7 - 5
096
Imp-
8 - 5
981
AU
-0020
-0015
-0010
-0005
0000
0005
0010
0015
0020
0025
0030
0035
0040
0045
0050
0055
0060
Minutes
050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850
158 Chapter 3
Three different lots of mobile phases were prepared and the consistency in
the resolution was verified (Table 34) A system suitability check (RS1 RS
2 amp
RS3) was incorporated to ensure the adequate system performance during the
regular analysis and method validation
Table 34 Reproducibility in separation of ZT and its impurities
Compound Average
RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing
factor
(n=6)c (n=6)c
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT) of
Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6)
159 Chapter 3
Fig 38 Peak purity plot for ZT in optimized conditions
Where PA = Purity angle and TH = Purity threshold
3755 The finalized RP-UPLC conditions for the estimation of assay and
related substances of ZT
Apparatus An UPLC with VWD and integrator
Column Acquity UPLC HSS T3 C-18 100 mm length
21 mm id 18 micron particle size
Flow Rate 03 ml min
Wave length 254 nm
Load 10microL
Column temperature 250C
Diluent Acetonitrile (ACN) Water (82)
Buffer Buffer solution was prepared by adding 1mL
of Triethyl amine (TEA) to 1000mL of 10 mM
NaH2PO4 H2O
Mobile phase-A Mixture of buffer (pH adjusted to 55 with
H3PO4) ACN and MeOH in the ratio of
602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with
H3PO4) and ACN in the ratio of 4555 (vv)
160 Chapter 3
Table 35 Gradient program
Time Mobile phase-A Mobile phase -B
001 95 5
25 95 5
35 40 60
50 40 60
82 30 70
85 95 5
100 95 5
Table 36 Retention times and relative retention times wrto ZT
SNo ImpurityAnalyte
name
Retention time ~Relative
retention time
1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100
8 Imp-7 5118 121 9 Imp-8 6037 142
3756 Degradation behavior
The purpose of this study is to establish the fact that the inherent chemical
stability of the molecule remains intact during its existence in the solid dosage
form along with other excipients So forced degradation studies should be
conducted on the drug to generate product related variants which in turn can
help to establish degradation pathways and thereby to develop and validate
suitable analytical procedure [22]
In this perspective a series of induced degradation experiments were
conducted at a concentration of 50 μgmL of ZT in active pharmaceutical
161 Chapter 3
ingredients (API) and tablets Degradation was induced by external physico-
chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)
oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal
treatment (105oC for 7 days)
The purity of the peaks obtained from the stressed samples was verified by
using the PDA detector The purity angle and purity threshold parameters were
evaluated for all the stressed samples to demonstrate the homogeneity of the
analyte peaks Assay of stressed samples was performed by comparison with
reference standards and the mass balance was calculated
37561 Degradation in Acidic solution
A single major degradation product Imp-2 (Zolpidem Acid) was formed when
ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The
degradation pathway is based on the premise that acid or base catalyzed
reaction between water and tertiary amide gives its parent carboxylic acid and
an amine Here the other hydrolytic product was dimethylamine Using LC-MS
in positive ion ESI the degradation product was found to have an mz value of
281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2
and UV spectral match
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
152 Chapter 3
153 Chapter 3
Selection of wavelength to monitor zolpidem tartrate and its impurities
An optimal wavelength of detection for ZT and its impurities was selected as
254 nm based on UV spectra using PDA detector for LC analysis Fig 34
3752 Selection of UPLC column
Initial experiments were carried out on different commercially available RP-
UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH
shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to
determine the selectivity and symmetry of the individual components In all the
columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline
separation and imp-5 amp imp-6 peaks were merged The remaining peaks were
separated with resolution (RS)gt15 and the peak tailing was greater than 15 for
ZT Due to the structural similarities co-elution of above mentioned impurity
0970 Imp-1
1134 Imp-2
1978 Imp-3
2486 Imp-4
3443 Imp-5
3619 Imp-6
3897 ZT
4796 Imp-7
5671 Imp-8
2631
3438
20672114
3200
3578
3057
3710
2114
2443
3854
2443
3105
2114
2443
3057
nm
22000 24000 26000 28000 30000 32000 34000 36000 38000
mA
u
154 Chapter 3
pairs was observed Additionally peak tailing was also observed due to the
secondary interactions with residual silanol groups Although separation was
similar in all the three columns HSS T3 column had shown relatively good
peak symmetry and efficiency so it was thus selected for further optimization
Addition of triethylamine in the buffer improved the peak shape of ZT but the
resolution between imp-5 amp imp-6 remained critical
3753 Effect of buffer pH
The important parameters buffer pH and gradient mode of elution that
would likely to have significant effect on critical pair resolution were
investigated There was no substantial change in the resolution between Imps-1
amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly
decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp
6 (RS2) and Imp-6 amp ZT (RS
3) were improved when the pH altered from 60 to 70
The increase in Rs1 in acidic mobile phase and RS
2 RS3 in basic mobile phase
can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of
adequately low pH will remain unionized and get increased retentions On the
other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be
more retained From table 33 acceptable resolutions were obtained at pH 70
with isocratic elution and pH 75 with gradient elution but in both the cases
the run time was longer
155 Chapter 3
Table 33 Effect of Buffer pH on resolution of critical pairs
Mode of
elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time
Isocratic 40 21 lt10 17 15min
Isocratic 50 22 lt10 17 14min
Isocratic 60 21 11 18 15min
Isocratic 70 20 18 22 gt15min
Gradient 55 21 lt10 17 8min
Gradient 75 18 22 23 gt15min
Gradient A 55 B
73 25 21 22 8 min
a Rs1 Resolution between Imp-1 and Imp-2
b Rs2 Resolution between Imp-5 and Imp-6
c Rs3Resolution between Imp-6 and ZT
3754 Optimization of gradient program
Gradient mode of elution containing mobile phase-A (1mL of TEA1L
phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile
phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555
vv) were chosen to separate both the critical pairs in a single run and this
resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)
156 Chapter 3
Fig 35 Co elution of imp-5 and imp-6 at pH 55
Based on the experimental data it was predicted that increase in mobile
phase pH during the gradient elution could resolve the Imp-5 and Imp-6
without compromising the total run time Hence two more experiments were
designed with a mobile phase-B as combination of water ACN (4555 vv) for
experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-
2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase
the solvent strength thereby to elute strongly retained compounds (Imp-8)
faster The RS2 was less than 10 for experiment-1 and RS
2 was 15 for
experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value
60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from
60 to 70) between 50min and 82min was added to the program to reduce the
run time So Imp-8 was eluted at 8min To further improve the RS2 gradient
program was optimized with a linear increase of mobile phase-B with varying
157 Chapter 3
time points An optimum resolution was obtained (Fig 36) when the gradient
program of section 22 was used In addition to this the elution time of imp-8
was reduced to 6 min in the modified gradient method
Fig 36 Baseline separations of zolpidem and its impurities
Placebo interference study was verified by injecting sample solutions of
placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-
8 and ZT analyte peak was found
Fig 37 Placebo chromatogram for zolpidem tablets
Imp-
1 - 1
051
Imp-
2 - 1
231
Imp-
3 - 2
139
Imp-
4 - 2
692
Imp-
5 - 3
720
Imp-
6 - 3
892
ZT -
414
7
Imp-
7 - 5
096
Imp-
8 - 5
981
AU
-0020
-0015
-0010
-0005
0000
0005
0010
0015
0020
0025
0030
0035
0040
0045
0050
0055
0060
Minutes
050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850
158 Chapter 3
Three different lots of mobile phases were prepared and the consistency in
the resolution was verified (Table 34) A system suitability check (RS1 RS
2 amp
RS3) was incorporated to ensure the adequate system performance during the
regular analysis and method validation
Table 34 Reproducibility in separation of ZT and its impurities
Compound Average
RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing
factor
(n=6)c (n=6)c
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT) of
Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6)
159 Chapter 3
Fig 38 Peak purity plot for ZT in optimized conditions
Where PA = Purity angle and TH = Purity threshold
3755 The finalized RP-UPLC conditions for the estimation of assay and
related substances of ZT
Apparatus An UPLC with VWD and integrator
Column Acquity UPLC HSS T3 C-18 100 mm length
21 mm id 18 micron particle size
Flow Rate 03 ml min
Wave length 254 nm
Load 10microL
Column temperature 250C
Diluent Acetonitrile (ACN) Water (82)
Buffer Buffer solution was prepared by adding 1mL
of Triethyl amine (TEA) to 1000mL of 10 mM
NaH2PO4 H2O
Mobile phase-A Mixture of buffer (pH adjusted to 55 with
H3PO4) ACN and MeOH in the ratio of
602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with
H3PO4) and ACN in the ratio of 4555 (vv)
160 Chapter 3
Table 35 Gradient program
Time Mobile phase-A Mobile phase -B
001 95 5
25 95 5
35 40 60
50 40 60
82 30 70
85 95 5
100 95 5
Table 36 Retention times and relative retention times wrto ZT
SNo ImpurityAnalyte
name
Retention time ~Relative
retention time
1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100
8 Imp-7 5118 121 9 Imp-8 6037 142
3756 Degradation behavior
The purpose of this study is to establish the fact that the inherent chemical
stability of the molecule remains intact during its existence in the solid dosage
form along with other excipients So forced degradation studies should be
conducted on the drug to generate product related variants which in turn can
help to establish degradation pathways and thereby to develop and validate
suitable analytical procedure [22]
In this perspective a series of induced degradation experiments were
conducted at a concentration of 50 μgmL of ZT in active pharmaceutical
161 Chapter 3
ingredients (API) and tablets Degradation was induced by external physico-
chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)
oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal
treatment (105oC for 7 days)
The purity of the peaks obtained from the stressed samples was verified by
using the PDA detector The purity angle and purity threshold parameters were
evaluated for all the stressed samples to demonstrate the homogeneity of the
analyte peaks Assay of stressed samples was performed by comparison with
reference standards and the mass balance was calculated
37561 Degradation in Acidic solution
A single major degradation product Imp-2 (Zolpidem Acid) was formed when
ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The
degradation pathway is based on the premise that acid or base catalyzed
reaction between water and tertiary amide gives its parent carboxylic acid and
an amine Here the other hydrolytic product was dimethylamine Using LC-MS
in positive ion ESI the degradation product was found to have an mz value of
281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2
and UV spectral match
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
153 Chapter 3
Selection of wavelength to monitor zolpidem tartrate and its impurities
An optimal wavelength of detection for ZT and its impurities was selected as
254 nm based on UV spectra using PDA detector for LC analysis Fig 34
3752 Selection of UPLC column
Initial experiments were carried out on different commercially available RP-
UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH
shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to
determine the selectivity and symmetry of the individual components In all the
columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline
separation and imp-5 amp imp-6 peaks were merged The remaining peaks were
separated with resolution (RS)gt15 and the peak tailing was greater than 15 for
ZT Due to the structural similarities co-elution of above mentioned impurity
0970 Imp-1
1134 Imp-2
1978 Imp-3
2486 Imp-4
3443 Imp-5
3619 Imp-6
3897 ZT
4796 Imp-7
5671 Imp-8
2631
3438
20672114
3200
3578
3057
3710
2114
2443
3854
2443
3105
2114
2443
3057
nm
22000 24000 26000 28000 30000 32000 34000 36000 38000
mA
u
154 Chapter 3
pairs was observed Additionally peak tailing was also observed due to the
secondary interactions with residual silanol groups Although separation was
similar in all the three columns HSS T3 column had shown relatively good
peak symmetry and efficiency so it was thus selected for further optimization
Addition of triethylamine in the buffer improved the peak shape of ZT but the
resolution between imp-5 amp imp-6 remained critical
3753 Effect of buffer pH
The important parameters buffer pH and gradient mode of elution that
would likely to have significant effect on critical pair resolution were
investigated There was no substantial change in the resolution between Imps-1
amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly
decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp
6 (RS2) and Imp-6 amp ZT (RS
3) were improved when the pH altered from 60 to 70
The increase in Rs1 in acidic mobile phase and RS
2 RS3 in basic mobile phase
can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of
adequately low pH will remain unionized and get increased retentions On the
other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be
more retained From table 33 acceptable resolutions were obtained at pH 70
with isocratic elution and pH 75 with gradient elution but in both the cases
the run time was longer
155 Chapter 3
Table 33 Effect of Buffer pH on resolution of critical pairs
Mode of
elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time
Isocratic 40 21 lt10 17 15min
Isocratic 50 22 lt10 17 14min
Isocratic 60 21 11 18 15min
Isocratic 70 20 18 22 gt15min
Gradient 55 21 lt10 17 8min
Gradient 75 18 22 23 gt15min
Gradient A 55 B
73 25 21 22 8 min
a Rs1 Resolution between Imp-1 and Imp-2
b Rs2 Resolution between Imp-5 and Imp-6
c Rs3Resolution between Imp-6 and ZT
3754 Optimization of gradient program
Gradient mode of elution containing mobile phase-A (1mL of TEA1L
phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile
phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555
vv) were chosen to separate both the critical pairs in a single run and this
resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)
156 Chapter 3
Fig 35 Co elution of imp-5 and imp-6 at pH 55
Based on the experimental data it was predicted that increase in mobile
phase pH during the gradient elution could resolve the Imp-5 and Imp-6
without compromising the total run time Hence two more experiments were
designed with a mobile phase-B as combination of water ACN (4555 vv) for
experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-
2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase
the solvent strength thereby to elute strongly retained compounds (Imp-8)
faster The RS2 was less than 10 for experiment-1 and RS
2 was 15 for
experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value
60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from
60 to 70) between 50min and 82min was added to the program to reduce the
run time So Imp-8 was eluted at 8min To further improve the RS2 gradient
program was optimized with a linear increase of mobile phase-B with varying
157 Chapter 3
time points An optimum resolution was obtained (Fig 36) when the gradient
program of section 22 was used In addition to this the elution time of imp-8
was reduced to 6 min in the modified gradient method
Fig 36 Baseline separations of zolpidem and its impurities
Placebo interference study was verified by injecting sample solutions of
placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-
8 and ZT analyte peak was found
Fig 37 Placebo chromatogram for zolpidem tablets
Imp-
1 - 1
051
Imp-
2 - 1
231
Imp-
3 - 2
139
Imp-
4 - 2
692
Imp-
5 - 3
720
Imp-
6 - 3
892
ZT -
414
7
Imp-
7 - 5
096
Imp-
8 - 5
981
AU
-0020
-0015
-0010
-0005
0000
0005
0010
0015
0020
0025
0030
0035
0040
0045
0050
0055
0060
Minutes
050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850
158 Chapter 3
Three different lots of mobile phases were prepared and the consistency in
the resolution was verified (Table 34) A system suitability check (RS1 RS
2 amp
RS3) was incorporated to ensure the adequate system performance during the
regular analysis and method validation
Table 34 Reproducibility in separation of ZT and its impurities
Compound Average
RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing
factor
(n=6)c (n=6)c
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT) of
Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6)
159 Chapter 3
Fig 38 Peak purity plot for ZT in optimized conditions
Where PA = Purity angle and TH = Purity threshold
3755 The finalized RP-UPLC conditions for the estimation of assay and
related substances of ZT
Apparatus An UPLC with VWD and integrator
Column Acquity UPLC HSS T3 C-18 100 mm length
21 mm id 18 micron particle size
Flow Rate 03 ml min
Wave length 254 nm
Load 10microL
Column temperature 250C
Diluent Acetonitrile (ACN) Water (82)
Buffer Buffer solution was prepared by adding 1mL
of Triethyl amine (TEA) to 1000mL of 10 mM
NaH2PO4 H2O
Mobile phase-A Mixture of buffer (pH adjusted to 55 with
H3PO4) ACN and MeOH in the ratio of
602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with
H3PO4) and ACN in the ratio of 4555 (vv)
160 Chapter 3
Table 35 Gradient program
Time Mobile phase-A Mobile phase -B
001 95 5
25 95 5
35 40 60
50 40 60
82 30 70
85 95 5
100 95 5
Table 36 Retention times and relative retention times wrto ZT
SNo ImpurityAnalyte
name
Retention time ~Relative
retention time
1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100
8 Imp-7 5118 121 9 Imp-8 6037 142
3756 Degradation behavior
The purpose of this study is to establish the fact that the inherent chemical
stability of the molecule remains intact during its existence in the solid dosage
form along with other excipients So forced degradation studies should be
conducted on the drug to generate product related variants which in turn can
help to establish degradation pathways and thereby to develop and validate
suitable analytical procedure [22]
In this perspective a series of induced degradation experiments were
conducted at a concentration of 50 μgmL of ZT in active pharmaceutical
161 Chapter 3
ingredients (API) and tablets Degradation was induced by external physico-
chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)
oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal
treatment (105oC for 7 days)
The purity of the peaks obtained from the stressed samples was verified by
using the PDA detector The purity angle and purity threshold parameters were
evaluated for all the stressed samples to demonstrate the homogeneity of the
analyte peaks Assay of stressed samples was performed by comparison with
reference standards and the mass balance was calculated
37561 Degradation in Acidic solution
A single major degradation product Imp-2 (Zolpidem Acid) was formed when
ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The
degradation pathway is based on the premise that acid or base catalyzed
reaction between water and tertiary amide gives its parent carboxylic acid and
an amine Here the other hydrolytic product was dimethylamine Using LC-MS
in positive ion ESI the degradation product was found to have an mz value of
281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2
and UV spectral match
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
154 Chapter 3
pairs was observed Additionally peak tailing was also observed due to the
secondary interactions with residual silanol groups Although separation was
similar in all the three columns HSS T3 column had shown relatively good
peak symmetry and efficiency so it was thus selected for further optimization
Addition of triethylamine in the buffer improved the peak shape of ZT but the
resolution between imp-5 amp imp-6 remained critical
3753 Effect of buffer pH
The important parameters buffer pH and gradient mode of elution that
would likely to have significant effect on critical pair resolution were
investigated There was no substantial change in the resolution between Imps-1
amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly
decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp
6 (RS2) and Imp-6 amp ZT (RS
3) were improved when the pH altered from 60 to 70
The increase in Rs1 in acidic mobile phase and RS
2 RS3 in basic mobile phase
can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of
adequately low pH will remain unionized and get increased retentions On the
other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be
more retained From table 33 acceptable resolutions were obtained at pH 70
with isocratic elution and pH 75 with gradient elution but in both the cases
the run time was longer
155 Chapter 3
Table 33 Effect of Buffer pH on resolution of critical pairs
Mode of
elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time
Isocratic 40 21 lt10 17 15min
Isocratic 50 22 lt10 17 14min
Isocratic 60 21 11 18 15min
Isocratic 70 20 18 22 gt15min
Gradient 55 21 lt10 17 8min
Gradient 75 18 22 23 gt15min
Gradient A 55 B
73 25 21 22 8 min
a Rs1 Resolution between Imp-1 and Imp-2
b Rs2 Resolution between Imp-5 and Imp-6
c Rs3Resolution between Imp-6 and ZT
3754 Optimization of gradient program
Gradient mode of elution containing mobile phase-A (1mL of TEA1L
phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile
phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555
vv) were chosen to separate both the critical pairs in a single run and this
resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)
156 Chapter 3
Fig 35 Co elution of imp-5 and imp-6 at pH 55
Based on the experimental data it was predicted that increase in mobile
phase pH during the gradient elution could resolve the Imp-5 and Imp-6
without compromising the total run time Hence two more experiments were
designed with a mobile phase-B as combination of water ACN (4555 vv) for
experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-
2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase
the solvent strength thereby to elute strongly retained compounds (Imp-8)
faster The RS2 was less than 10 for experiment-1 and RS
2 was 15 for
experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value
60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from
60 to 70) between 50min and 82min was added to the program to reduce the
run time So Imp-8 was eluted at 8min To further improve the RS2 gradient
program was optimized with a linear increase of mobile phase-B with varying
157 Chapter 3
time points An optimum resolution was obtained (Fig 36) when the gradient
program of section 22 was used In addition to this the elution time of imp-8
was reduced to 6 min in the modified gradient method
Fig 36 Baseline separations of zolpidem and its impurities
Placebo interference study was verified by injecting sample solutions of
placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-
8 and ZT analyte peak was found
Fig 37 Placebo chromatogram for zolpidem tablets
Imp-
1 - 1
051
Imp-
2 - 1
231
Imp-
3 - 2
139
Imp-
4 - 2
692
Imp-
5 - 3
720
Imp-
6 - 3
892
ZT -
414
7
Imp-
7 - 5
096
Imp-
8 - 5
981
AU
-0020
-0015
-0010
-0005
0000
0005
0010
0015
0020
0025
0030
0035
0040
0045
0050
0055
0060
Minutes
050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850
158 Chapter 3
Three different lots of mobile phases were prepared and the consistency in
the resolution was verified (Table 34) A system suitability check (RS1 RS
2 amp
RS3) was incorporated to ensure the adequate system performance during the
regular analysis and method validation
Table 34 Reproducibility in separation of ZT and its impurities
Compound Average
RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing
factor
(n=6)c (n=6)c
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT) of
Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6)
159 Chapter 3
Fig 38 Peak purity plot for ZT in optimized conditions
Where PA = Purity angle and TH = Purity threshold
3755 The finalized RP-UPLC conditions for the estimation of assay and
related substances of ZT
Apparatus An UPLC with VWD and integrator
Column Acquity UPLC HSS T3 C-18 100 mm length
21 mm id 18 micron particle size
Flow Rate 03 ml min
Wave length 254 nm
Load 10microL
Column temperature 250C
Diluent Acetonitrile (ACN) Water (82)
Buffer Buffer solution was prepared by adding 1mL
of Triethyl amine (TEA) to 1000mL of 10 mM
NaH2PO4 H2O
Mobile phase-A Mixture of buffer (pH adjusted to 55 with
H3PO4) ACN and MeOH in the ratio of
602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with
H3PO4) and ACN in the ratio of 4555 (vv)
160 Chapter 3
Table 35 Gradient program
Time Mobile phase-A Mobile phase -B
001 95 5
25 95 5
35 40 60
50 40 60
82 30 70
85 95 5
100 95 5
Table 36 Retention times and relative retention times wrto ZT
SNo ImpurityAnalyte
name
Retention time ~Relative
retention time
1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100
8 Imp-7 5118 121 9 Imp-8 6037 142
3756 Degradation behavior
The purpose of this study is to establish the fact that the inherent chemical
stability of the molecule remains intact during its existence in the solid dosage
form along with other excipients So forced degradation studies should be
conducted on the drug to generate product related variants which in turn can
help to establish degradation pathways and thereby to develop and validate
suitable analytical procedure [22]
In this perspective a series of induced degradation experiments were
conducted at a concentration of 50 μgmL of ZT in active pharmaceutical
161 Chapter 3
ingredients (API) and tablets Degradation was induced by external physico-
chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)
oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal
treatment (105oC for 7 days)
The purity of the peaks obtained from the stressed samples was verified by
using the PDA detector The purity angle and purity threshold parameters were
evaluated for all the stressed samples to demonstrate the homogeneity of the
analyte peaks Assay of stressed samples was performed by comparison with
reference standards and the mass balance was calculated
37561 Degradation in Acidic solution
A single major degradation product Imp-2 (Zolpidem Acid) was formed when
ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The
degradation pathway is based on the premise that acid or base catalyzed
reaction between water and tertiary amide gives its parent carboxylic acid and
an amine Here the other hydrolytic product was dimethylamine Using LC-MS
in positive ion ESI the degradation product was found to have an mz value of
281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2
and UV spectral match
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
155 Chapter 3
Table 33 Effect of Buffer pH on resolution of critical pairs
Mode of
elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time
Isocratic 40 21 lt10 17 15min
Isocratic 50 22 lt10 17 14min
Isocratic 60 21 11 18 15min
Isocratic 70 20 18 22 gt15min
Gradient 55 21 lt10 17 8min
Gradient 75 18 22 23 gt15min
Gradient A 55 B
73 25 21 22 8 min
a Rs1 Resolution between Imp-1 and Imp-2
b Rs2 Resolution between Imp-5 and Imp-6
c Rs3Resolution between Imp-6 and ZT
3754 Optimization of gradient program
Gradient mode of elution containing mobile phase-A (1mL of TEA1L
phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile
phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555
vv) were chosen to separate both the critical pairs in a single run and this
resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)
156 Chapter 3
Fig 35 Co elution of imp-5 and imp-6 at pH 55
Based on the experimental data it was predicted that increase in mobile
phase pH during the gradient elution could resolve the Imp-5 and Imp-6
without compromising the total run time Hence two more experiments were
designed with a mobile phase-B as combination of water ACN (4555 vv) for
experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-
2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase
the solvent strength thereby to elute strongly retained compounds (Imp-8)
faster The RS2 was less than 10 for experiment-1 and RS
2 was 15 for
experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value
60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from
60 to 70) between 50min and 82min was added to the program to reduce the
run time So Imp-8 was eluted at 8min To further improve the RS2 gradient
program was optimized with a linear increase of mobile phase-B with varying
157 Chapter 3
time points An optimum resolution was obtained (Fig 36) when the gradient
program of section 22 was used In addition to this the elution time of imp-8
was reduced to 6 min in the modified gradient method
Fig 36 Baseline separations of zolpidem and its impurities
Placebo interference study was verified by injecting sample solutions of
placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-
8 and ZT analyte peak was found
Fig 37 Placebo chromatogram for zolpidem tablets
Imp-
1 - 1
051
Imp-
2 - 1
231
Imp-
3 - 2
139
Imp-
4 - 2
692
Imp-
5 - 3
720
Imp-
6 - 3
892
ZT -
414
7
Imp-
7 - 5
096
Imp-
8 - 5
981
AU
-0020
-0015
-0010
-0005
0000
0005
0010
0015
0020
0025
0030
0035
0040
0045
0050
0055
0060
Minutes
050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850
158 Chapter 3
Three different lots of mobile phases were prepared and the consistency in
the resolution was verified (Table 34) A system suitability check (RS1 RS
2 amp
RS3) was incorporated to ensure the adequate system performance during the
regular analysis and method validation
Table 34 Reproducibility in separation of ZT and its impurities
Compound Average
RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing
factor
(n=6)c (n=6)c
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT) of
Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6)
159 Chapter 3
Fig 38 Peak purity plot for ZT in optimized conditions
Where PA = Purity angle and TH = Purity threshold
3755 The finalized RP-UPLC conditions for the estimation of assay and
related substances of ZT
Apparatus An UPLC with VWD and integrator
Column Acquity UPLC HSS T3 C-18 100 mm length
21 mm id 18 micron particle size
Flow Rate 03 ml min
Wave length 254 nm
Load 10microL
Column temperature 250C
Diluent Acetonitrile (ACN) Water (82)
Buffer Buffer solution was prepared by adding 1mL
of Triethyl amine (TEA) to 1000mL of 10 mM
NaH2PO4 H2O
Mobile phase-A Mixture of buffer (pH adjusted to 55 with
H3PO4) ACN and MeOH in the ratio of
602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with
H3PO4) and ACN in the ratio of 4555 (vv)
160 Chapter 3
Table 35 Gradient program
Time Mobile phase-A Mobile phase -B
001 95 5
25 95 5
35 40 60
50 40 60
82 30 70
85 95 5
100 95 5
Table 36 Retention times and relative retention times wrto ZT
SNo ImpurityAnalyte
name
Retention time ~Relative
retention time
1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100
8 Imp-7 5118 121 9 Imp-8 6037 142
3756 Degradation behavior
The purpose of this study is to establish the fact that the inherent chemical
stability of the molecule remains intact during its existence in the solid dosage
form along with other excipients So forced degradation studies should be
conducted on the drug to generate product related variants which in turn can
help to establish degradation pathways and thereby to develop and validate
suitable analytical procedure [22]
In this perspective a series of induced degradation experiments were
conducted at a concentration of 50 μgmL of ZT in active pharmaceutical
161 Chapter 3
ingredients (API) and tablets Degradation was induced by external physico-
chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)
oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal
treatment (105oC for 7 days)
The purity of the peaks obtained from the stressed samples was verified by
using the PDA detector The purity angle and purity threshold parameters were
evaluated for all the stressed samples to demonstrate the homogeneity of the
analyte peaks Assay of stressed samples was performed by comparison with
reference standards and the mass balance was calculated
37561 Degradation in Acidic solution
A single major degradation product Imp-2 (Zolpidem Acid) was formed when
ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The
degradation pathway is based on the premise that acid or base catalyzed
reaction between water and tertiary amide gives its parent carboxylic acid and
an amine Here the other hydrolytic product was dimethylamine Using LC-MS
in positive ion ESI the degradation product was found to have an mz value of
281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2
and UV spectral match
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
156 Chapter 3
Fig 35 Co elution of imp-5 and imp-6 at pH 55
Based on the experimental data it was predicted that increase in mobile
phase pH during the gradient elution could resolve the Imp-5 and Imp-6
without compromising the total run time Hence two more experiments were
designed with a mobile phase-B as combination of water ACN (4555 vv) for
experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-
2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase
the solvent strength thereby to elute strongly retained compounds (Imp-8)
faster The RS2 was less than 10 for experiment-1 and RS
2 was 15 for
experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value
60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from
60 to 70) between 50min and 82min was added to the program to reduce the
run time So Imp-8 was eluted at 8min To further improve the RS2 gradient
program was optimized with a linear increase of mobile phase-B with varying
157 Chapter 3
time points An optimum resolution was obtained (Fig 36) when the gradient
program of section 22 was used In addition to this the elution time of imp-8
was reduced to 6 min in the modified gradient method
Fig 36 Baseline separations of zolpidem and its impurities
Placebo interference study was verified by injecting sample solutions of
placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-
8 and ZT analyte peak was found
Fig 37 Placebo chromatogram for zolpidem tablets
Imp-
1 - 1
051
Imp-
2 - 1
231
Imp-
3 - 2
139
Imp-
4 - 2
692
Imp-
5 - 3
720
Imp-
6 - 3
892
ZT -
414
7
Imp-
7 - 5
096
Imp-
8 - 5
981
AU
-0020
-0015
-0010
-0005
0000
0005
0010
0015
0020
0025
0030
0035
0040
0045
0050
0055
0060
Minutes
050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850
158 Chapter 3
Three different lots of mobile phases were prepared and the consistency in
the resolution was verified (Table 34) A system suitability check (RS1 RS
2 amp
RS3) was incorporated to ensure the adequate system performance during the
regular analysis and method validation
Table 34 Reproducibility in separation of ZT and its impurities
Compound Average
RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing
factor
(n=6)c (n=6)c
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT) of
Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6)
159 Chapter 3
Fig 38 Peak purity plot for ZT in optimized conditions
Where PA = Purity angle and TH = Purity threshold
3755 The finalized RP-UPLC conditions for the estimation of assay and
related substances of ZT
Apparatus An UPLC with VWD and integrator
Column Acquity UPLC HSS T3 C-18 100 mm length
21 mm id 18 micron particle size
Flow Rate 03 ml min
Wave length 254 nm
Load 10microL
Column temperature 250C
Diluent Acetonitrile (ACN) Water (82)
Buffer Buffer solution was prepared by adding 1mL
of Triethyl amine (TEA) to 1000mL of 10 mM
NaH2PO4 H2O
Mobile phase-A Mixture of buffer (pH adjusted to 55 with
H3PO4) ACN and MeOH in the ratio of
602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with
H3PO4) and ACN in the ratio of 4555 (vv)
160 Chapter 3
Table 35 Gradient program
Time Mobile phase-A Mobile phase -B
001 95 5
25 95 5
35 40 60
50 40 60
82 30 70
85 95 5
100 95 5
Table 36 Retention times and relative retention times wrto ZT
SNo ImpurityAnalyte
name
Retention time ~Relative
retention time
1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100
8 Imp-7 5118 121 9 Imp-8 6037 142
3756 Degradation behavior
The purpose of this study is to establish the fact that the inherent chemical
stability of the molecule remains intact during its existence in the solid dosage
form along with other excipients So forced degradation studies should be
conducted on the drug to generate product related variants which in turn can
help to establish degradation pathways and thereby to develop and validate
suitable analytical procedure [22]
In this perspective a series of induced degradation experiments were
conducted at a concentration of 50 μgmL of ZT in active pharmaceutical
161 Chapter 3
ingredients (API) and tablets Degradation was induced by external physico-
chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)
oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal
treatment (105oC for 7 days)
The purity of the peaks obtained from the stressed samples was verified by
using the PDA detector The purity angle and purity threshold parameters were
evaluated for all the stressed samples to demonstrate the homogeneity of the
analyte peaks Assay of stressed samples was performed by comparison with
reference standards and the mass balance was calculated
37561 Degradation in Acidic solution
A single major degradation product Imp-2 (Zolpidem Acid) was formed when
ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The
degradation pathway is based on the premise that acid or base catalyzed
reaction between water and tertiary amide gives its parent carboxylic acid and
an amine Here the other hydrolytic product was dimethylamine Using LC-MS
in positive ion ESI the degradation product was found to have an mz value of
281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2
and UV spectral match
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
157 Chapter 3
time points An optimum resolution was obtained (Fig 36) when the gradient
program of section 22 was used In addition to this the elution time of imp-8
was reduced to 6 min in the modified gradient method
Fig 36 Baseline separations of zolpidem and its impurities
Placebo interference study was verified by injecting sample solutions of
placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-
8 and ZT analyte peak was found
Fig 37 Placebo chromatogram for zolpidem tablets
Imp-
1 - 1
051
Imp-
2 - 1
231
Imp-
3 - 2
139
Imp-
4 - 2
692
Imp-
5 - 3
720
Imp-
6 - 3
892
ZT -
414
7
Imp-
7 - 5
096
Imp-
8 - 5
981
AU
-0020
-0015
-0010
-0005
0000
0005
0010
0015
0020
0025
0030
0035
0040
0045
0050
0055
0060
Minutes
050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850
158 Chapter 3
Three different lots of mobile phases were prepared and the consistency in
the resolution was verified (Table 34) A system suitability check (RS1 RS
2 amp
RS3) was incorporated to ensure the adequate system performance during the
regular analysis and method validation
Table 34 Reproducibility in separation of ZT and its impurities
Compound Average
RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing
factor
(n=6)c (n=6)c
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT) of
Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6)
159 Chapter 3
Fig 38 Peak purity plot for ZT in optimized conditions
Where PA = Purity angle and TH = Purity threshold
3755 The finalized RP-UPLC conditions for the estimation of assay and
related substances of ZT
Apparatus An UPLC with VWD and integrator
Column Acquity UPLC HSS T3 C-18 100 mm length
21 mm id 18 micron particle size
Flow Rate 03 ml min
Wave length 254 nm
Load 10microL
Column temperature 250C
Diluent Acetonitrile (ACN) Water (82)
Buffer Buffer solution was prepared by adding 1mL
of Triethyl amine (TEA) to 1000mL of 10 mM
NaH2PO4 H2O
Mobile phase-A Mixture of buffer (pH adjusted to 55 with
H3PO4) ACN and MeOH in the ratio of
602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with
H3PO4) and ACN in the ratio of 4555 (vv)
160 Chapter 3
Table 35 Gradient program
Time Mobile phase-A Mobile phase -B
001 95 5
25 95 5
35 40 60
50 40 60
82 30 70
85 95 5
100 95 5
Table 36 Retention times and relative retention times wrto ZT
SNo ImpurityAnalyte
name
Retention time ~Relative
retention time
1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100
8 Imp-7 5118 121 9 Imp-8 6037 142
3756 Degradation behavior
The purpose of this study is to establish the fact that the inherent chemical
stability of the molecule remains intact during its existence in the solid dosage
form along with other excipients So forced degradation studies should be
conducted on the drug to generate product related variants which in turn can
help to establish degradation pathways and thereby to develop and validate
suitable analytical procedure [22]
In this perspective a series of induced degradation experiments were
conducted at a concentration of 50 μgmL of ZT in active pharmaceutical
161 Chapter 3
ingredients (API) and tablets Degradation was induced by external physico-
chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)
oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal
treatment (105oC for 7 days)
The purity of the peaks obtained from the stressed samples was verified by
using the PDA detector The purity angle and purity threshold parameters were
evaluated for all the stressed samples to demonstrate the homogeneity of the
analyte peaks Assay of stressed samples was performed by comparison with
reference standards and the mass balance was calculated
37561 Degradation in Acidic solution
A single major degradation product Imp-2 (Zolpidem Acid) was formed when
ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The
degradation pathway is based on the premise that acid or base catalyzed
reaction between water and tertiary amide gives its parent carboxylic acid and
an amine Here the other hydrolytic product was dimethylamine Using LC-MS
in positive ion ESI the degradation product was found to have an mz value of
281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2
and UV spectral match
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
158 Chapter 3
Three different lots of mobile phases were prepared and the consistency in
the resolution was verified (Table 34) A system suitability check (RS1 RS
2 amp
RS3) was incorporated to ensure the adequate system performance during the
regular analysis and method validation
Table 34 Reproducibility in separation of ZT and its impurities
Compound Average
RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing
factor
(n=6)c (n=6)c
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT) of
Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6)
159 Chapter 3
Fig 38 Peak purity plot for ZT in optimized conditions
Where PA = Purity angle and TH = Purity threshold
3755 The finalized RP-UPLC conditions for the estimation of assay and
related substances of ZT
Apparatus An UPLC with VWD and integrator
Column Acquity UPLC HSS T3 C-18 100 mm length
21 mm id 18 micron particle size
Flow Rate 03 ml min
Wave length 254 nm
Load 10microL
Column temperature 250C
Diluent Acetonitrile (ACN) Water (82)
Buffer Buffer solution was prepared by adding 1mL
of Triethyl amine (TEA) to 1000mL of 10 mM
NaH2PO4 H2O
Mobile phase-A Mixture of buffer (pH adjusted to 55 with
H3PO4) ACN and MeOH in the ratio of
602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with
H3PO4) and ACN in the ratio of 4555 (vv)
160 Chapter 3
Table 35 Gradient program
Time Mobile phase-A Mobile phase -B
001 95 5
25 95 5
35 40 60
50 40 60
82 30 70
85 95 5
100 95 5
Table 36 Retention times and relative retention times wrto ZT
SNo ImpurityAnalyte
name
Retention time ~Relative
retention time
1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100
8 Imp-7 5118 121 9 Imp-8 6037 142
3756 Degradation behavior
The purpose of this study is to establish the fact that the inherent chemical
stability of the molecule remains intact during its existence in the solid dosage
form along with other excipients So forced degradation studies should be
conducted on the drug to generate product related variants which in turn can
help to establish degradation pathways and thereby to develop and validate
suitable analytical procedure [22]
In this perspective a series of induced degradation experiments were
conducted at a concentration of 50 μgmL of ZT in active pharmaceutical
161 Chapter 3
ingredients (API) and tablets Degradation was induced by external physico-
chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)
oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal
treatment (105oC for 7 days)
The purity of the peaks obtained from the stressed samples was verified by
using the PDA detector The purity angle and purity threshold parameters were
evaluated for all the stressed samples to demonstrate the homogeneity of the
analyte peaks Assay of stressed samples was performed by comparison with
reference standards and the mass balance was calculated
37561 Degradation in Acidic solution
A single major degradation product Imp-2 (Zolpidem Acid) was formed when
ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The
degradation pathway is based on the premise that acid or base catalyzed
reaction between water and tertiary amide gives its parent carboxylic acid and
an amine Here the other hydrolytic product was dimethylamine Using LC-MS
in positive ion ESI the degradation product was found to have an mz value of
281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2
and UV spectral match
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
159 Chapter 3
Fig 38 Peak purity plot for ZT in optimized conditions
Where PA = Purity angle and TH = Purity threshold
3755 The finalized RP-UPLC conditions for the estimation of assay and
related substances of ZT
Apparatus An UPLC with VWD and integrator
Column Acquity UPLC HSS T3 C-18 100 mm length
21 mm id 18 micron particle size
Flow Rate 03 ml min
Wave length 254 nm
Load 10microL
Column temperature 250C
Diluent Acetonitrile (ACN) Water (82)
Buffer Buffer solution was prepared by adding 1mL
of Triethyl amine (TEA) to 1000mL of 10 mM
NaH2PO4 H2O
Mobile phase-A Mixture of buffer (pH adjusted to 55 with
H3PO4) ACN and MeOH in the ratio of
602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with
H3PO4) and ACN in the ratio of 4555 (vv)
160 Chapter 3
Table 35 Gradient program
Time Mobile phase-A Mobile phase -B
001 95 5
25 95 5
35 40 60
50 40 60
82 30 70
85 95 5
100 95 5
Table 36 Retention times and relative retention times wrto ZT
SNo ImpurityAnalyte
name
Retention time ~Relative
retention time
1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100
8 Imp-7 5118 121 9 Imp-8 6037 142
3756 Degradation behavior
The purpose of this study is to establish the fact that the inherent chemical
stability of the molecule remains intact during its existence in the solid dosage
form along with other excipients So forced degradation studies should be
conducted on the drug to generate product related variants which in turn can
help to establish degradation pathways and thereby to develop and validate
suitable analytical procedure [22]
In this perspective a series of induced degradation experiments were
conducted at a concentration of 50 μgmL of ZT in active pharmaceutical
161 Chapter 3
ingredients (API) and tablets Degradation was induced by external physico-
chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)
oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal
treatment (105oC for 7 days)
The purity of the peaks obtained from the stressed samples was verified by
using the PDA detector The purity angle and purity threshold parameters were
evaluated for all the stressed samples to demonstrate the homogeneity of the
analyte peaks Assay of stressed samples was performed by comparison with
reference standards and the mass balance was calculated
37561 Degradation in Acidic solution
A single major degradation product Imp-2 (Zolpidem Acid) was formed when
ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The
degradation pathway is based on the premise that acid or base catalyzed
reaction between water and tertiary amide gives its parent carboxylic acid and
an amine Here the other hydrolytic product was dimethylamine Using LC-MS
in positive ion ESI the degradation product was found to have an mz value of
281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2
and UV spectral match
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
160 Chapter 3
Table 35 Gradient program
Time Mobile phase-A Mobile phase -B
001 95 5
25 95 5
35 40 60
50 40 60
82 30 70
85 95 5
100 95 5
Table 36 Retention times and relative retention times wrto ZT
SNo ImpurityAnalyte
name
Retention time ~Relative
retention time
1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100
8 Imp-7 5118 121 9 Imp-8 6037 142
3756 Degradation behavior
The purpose of this study is to establish the fact that the inherent chemical
stability of the molecule remains intact during its existence in the solid dosage
form along with other excipients So forced degradation studies should be
conducted on the drug to generate product related variants which in turn can
help to establish degradation pathways and thereby to develop and validate
suitable analytical procedure [22]
In this perspective a series of induced degradation experiments were
conducted at a concentration of 50 μgmL of ZT in active pharmaceutical
161 Chapter 3
ingredients (API) and tablets Degradation was induced by external physico-
chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)
oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal
treatment (105oC for 7 days)
The purity of the peaks obtained from the stressed samples was verified by
using the PDA detector The purity angle and purity threshold parameters were
evaluated for all the stressed samples to demonstrate the homogeneity of the
analyte peaks Assay of stressed samples was performed by comparison with
reference standards and the mass balance was calculated
37561 Degradation in Acidic solution
A single major degradation product Imp-2 (Zolpidem Acid) was formed when
ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The
degradation pathway is based on the premise that acid or base catalyzed
reaction between water and tertiary amide gives its parent carboxylic acid and
an amine Here the other hydrolytic product was dimethylamine Using LC-MS
in positive ion ESI the degradation product was found to have an mz value of
281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2
and UV spectral match
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
161 Chapter 3
ingredients (API) and tablets Degradation was induced by external physico-
chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)
oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal
treatment (105oC for 7 days)
The purity of the peaks obtained from the stressed samples was verified by
using the PDA detector The purity angle and purity threshold parameters were
evaluated for all the stressed samples to demonstrate the homogeneity of the
analyte peaks Assay of stressed samples was performed by comparison with
reference standards and the mass balance was calculated
37561 Degradation in Acidic solution
A single major degradation product Imp-2 (Zolpidem Acid) was formed when
ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The
degradation pathway is based on the premise that acid or base catalyzed
reaction between water and tertiary amide gives its parent carboxylic acid and
an amine Here the other hydrolytic product was dimethylamine Using LC-MS
in positive ion ESI the degradation product was found to have an mz value of
281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2
and UV spectral match
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
162 Chapter 3
Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis
Peak purity was evaluated for ZT peak and the resulting plot had shown that
the peak is pure as shown in Fig 310
Fig 310 Peak purity plot for ZT acid hydrolysis
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
163 Chapter 3
37562 Oxidative conditions
Zolpidem experienced a total degradation of 18 when the drug treated with
peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation
product was at RRT~127 Fig 311
Fig 311 Typical UPLC chromatogram of ZT oxidative degradation
Mass spectrum had shown the mz value 227 in positive ESI mode This
predominant degradation product with a molecular weight of 226 could be
zolpyridine as depicted in Fig 312
Fig 312 LC-MS data of ZT oxidative degradation
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
164 Chapter 3
The formation of zolpyridine could be due to the simultaneous cleavage of
tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks
were also formed during the peroxide degradation which was confirmed by
spiking analysis followed by spectral match with the known standards Imp-1
was the secondary hydroxylated degradation product originated from Imp-2 In
addition a low level degradation product at RRT~11 with mz 324 indicating
the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt
323) (Fig 313)
Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity
Peak purity was evaluated for the final stressed solution in peroxide and the
plot had been shown in Fig 314 The data had revealed that peak was
homogeneous and no other peaks were masked under ZT
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
165 Chapter 3
Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample
37563 Base hydrolysis
Similar to acid hydrolysis single major degradation product Imp-2
(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N
NaOH at 60oC for 90 min) conditions (Fig 315)
Fig 315 ZT Base degradation chromatogram
But there was a difference in the rate at which hydrolysis occurred
Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
166 Chapter 3
LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)
Fig 316 LCMS data for base hydrolysis
Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)
Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
167 Chapter 3
37564 Water hydrolysis
The drug was not much susceptible to water hydrolysis however Imp-2 was
formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig
318)
Fig 318 Typical UPLC chromatogram of ZT water hydrolysis
Peak purity data had shown that the peak was pure in water hydrolysis
Fig 319 Peak purity plot for ZT Water hydrolysis
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
168 Chapter 3
37565 Heat and light conditions
Degradation was not observed when ZT was subjected to light and heat
conditions Purity angle was less than purity threshold in all the stressed
samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal
peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the
stressed conditions
The peak homogeneity test was performed for the ZT drug substance and drug
product by using DAD The data was shown below (Table 37)
Table 37 Purity Angle Purity Threshold and Purity Flag values
Stressed Condition Purity
Angle
Purity
Threshold
Purity
Flag
Normal 0278 0485 No
Acid degradation 0482 0669 No
Base degradation 1732 2687 No
Water hydrolysis 0301 1485 No
Oxidative degradation 1117 4584 No
Thermal degradation 0589 0678 No
Photolytic degradation 0405 0721 No
Relative rates of degradation were plotted in all the stress conditions as shown
in Fig 320
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
169 Chapter 3
Fig 320 Rate of degradation in various stress conditions
376 Mass balance study
The mass balance ( assay + sum of all compounds (impurities +
degradation products)) results were calculated for all of the stressed samples
and were found to be more than 988 (Table 38) The purity and assay of ZT
were unaffected by the presence of its impurities and degradation products
demonstrating the stability-indicating nature of the developed UPLC method
80
85
90
95
100
105
0 3 6 9 12 15 18 21 24
A
ssa
y
Time (in hours)
Rate of degradation in various stress conditions
Acid hydrolysis
Base hydrolysis
Peroxide degradation
Water hydrolysis
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
170 Chapter 3
Table 38 Summary of mass balance data in various stress conditions
Degradation
condition
Time RS by UPLC
degradation
Assay
(ww on
anhydrous
basis)
Mass balance
(assay+deg
Products)
Acid degradation 24 hrs 57 934 991
Base degradation 90 mins 83 909 992
Water hydrolysis 24 hrs 02 996 998
Oxidative
degradation
2 hrs 180 808 988
Thermal
degradation
7 days 01 996 997
Photolytic
degradation
7 days 01 996 998
Mass balance data close to 100 indicates good correlation between assay and
degradation products formed
The data shown above indicating the developed RP-UPLC method that was
found to be specific and selective for ZT analyte peak in the presence of its
impurities and degradation products
38 Analytical method validation
The developed method was completely validated as per ICH and USP [11-12]
381 System suitability test
A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution
was injected into the chromatographic system and good resolutions (Rs)
between all impurities and ZT peak was observed as shown in (Fig 321 to Fig
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
171 Chapter 3
323) The system suitability RS1 RS
2 and RS3 were found to be greater than 15
(Table 39)
Fig 321 Typical chromatogram of Blank
Fig 322 Typical chromatogram of test sample
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
172 Chapter 3
Fig 323 Chromatogram of test sample spiked with impurities
Assay standard solution (50microgmL) was also injected n=6 times and the RSD
for ZT peak area and tailing factor was recorded
Table 39 System suitability data
Compound Retention time (min)
RRT a Resolution b
(n=6) Tailing factor c
(n=6)
Imp-1 1019 024plusmn002 - 12plusmn010
Imp-2 1217 029plusmn002 22plusmn05 12plusmn010
Imp-3 2203 052plusmn002 95plusmn080 12plusmn010
Imp-4 2815 066plusmn002 50plusmn050 13plusmn009
Imp-5 3879 091plusmn002 81plusmn080 11plusmn010
Imp-6 4035 095plusmn002 18plusmn02 11plusmn005
ZT 4244 095plusmn002 22plusmn030 13plusmn005
Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009
Imp-8 6037 142plusmn005 52plusmn10 11plusmn010
a Relative retention times (RRT) were calculated against the retention time (RT)
of Zolpidem tartrate
b Resolutions were calculated between two adjacent peaks
c Mean plusmn RSD (n=6) for ZT peak at assay concentration level
RSD for ZT peak area at assay concentration level was less than 08
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
173 Chapter 3
382 Precision
The precision study was carried out for assay using UPLC method by
analyzing six individual solutions of ZT test sample and standard solution The
RSD of six assay determinations was less than 04 (Table 310) The above
UPLC precision data had shown good precision and therefore the method was
reproducible
Table 310 Assay by UPLC precision results
Preparation Assay
1 999
2 991
3 991
4 993
5 999
6 990
Mean 993
Stdev 040
RSD 04
Precision was checked for related substances by injecting six different
preparations (n=6) of ZT (50microgmL) drug substance which was spiked with
impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test
concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)
preparations was calculated (Table 311)
These results had confirmed that the developed UPLC method for related
substances was precise
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
174 Chapter 3
Table 311 Related substances by UPLC precision results
PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
1 17968 17995 12867 25650 16722 12189 10181 10713
2 17361 17514 11614 24793 16979 11797 9627 10428
3 17549 17559 12123 25358 16230 11379 10532 11164
4 17943 18096 12675 24985 16050 12183 9680 10199
5 17271 16847 12939 24626 16553 12212 9965 10379
6 17530 16754 12267 24573 16865 12760 9452 10553
Mean 17604 17461 12414 24998 16567 12087 9906 10573
Stdev 292 562 509 428 364 463 402 337
RSD 17 32 41 17 22 38 41 32
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
175 Chapter 3
Impurity precision was determined on 3 days and ANOVA was calculated for all
the impurities RSD was also calculated for inter-day precision and results
were tabulated below (Table 312)
Table 312 ANOVA results for precision
SNo Name F critical F calculated RSD
1 Imp-1 36823 14302 18
2 Imp-2 36823 21217 34
3 Imp-3 36823 10732 29
4 Imp-4 36823 23109 19
5 Imp-5 36823 27039 21
6 Imp-6 36823 24048 18
7 Imp-7 36823 03180 34
8 Imp-8 36823 09564 31
Inter day precision (RSD) for assay method was found to be 03
Intermediate precision study was carried out for ZT impurities by a different
analyst using a different instrument and a different column The RSD values
were calculated and tabulated as shown below Table 313
Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
RSD 36 28 27 09 16 39 62 34
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
176 Chapter 3
The result had shown the excellent precision of the method and was thus
proved to be rugged
Intermediate precision for ZT assay was performed on a different column
with a different instrument by a different analyst The results had shown
excellent precision as shown in Table 314
Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)
Preparation Assay
1 992
2 993
3 990
4 994
5 991
6 993
Mean 992
Stdev 015
RSD 015
383 Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were determined for impurities and ZT based on signal to
noise ratio A series of diluted solutions were injected to achieve SN ratio
about 100 for LOQ and 30 for LOD The final concentrations and SN ratios
were reported below (Table 315)
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
177 Chapter 3
Table 315 LOD and LOQ values of ZT and its impurities
SNo
Name of the
impurity LOQ in microgmL LOD in microgmL
10 Imp-1 000053 000016
20 Imp-2 000056 000017
30 Imp-3 000122 000037
40 Imp-4 000070 000021
50 Imp-5 000080 000024
60 Imp-6 000097 000029
70 Imp-7 000162 000049
80 Imp-8 000165 000050
90 ZT 000058 000017
3831 Precision at Limit of quantification
The precision at quantification level for related substances method was also
checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-
3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results
had shown consistent peak areas The method was found to be repeatable and
reproducible at limit of quantification level with RSD lower than 44 (Table
316)
Table 316 LOQ level precision results
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT
RSD 44 28 17 36 28 29 20 41 13
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
178 Chapter 3
3832 Accuracy at LOQ
LOQ recovery experiments were conducted to determine the accuracy of
developed method at limit of quantification LOQ blend solution LOQ recovery
solution and test sample solutions were prepared separately Each solution was
injected in triplicate (n=3) and the recovery was determined Excellent
recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries
were shown in Table 317
Fig 324 Accuracy at LOQ chromatogram for ZT impurities
Table 317 Mean recovery data for ZT impurities
SNo Name of the impurity Mean Recovery
1 Imp-1 955
2 Imp-2 1012
3 Imp-3 1044
4 Imp-4 993
5 Imp-5 1028
6 Imp-6 1044
7 Imp-7 987
8 Imp-8 1030
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
179 Chapter 3
384 Linearity
3841 Linearity of assay method
The linearity test solutions of assay method were prepared from stock
standard solution Six solutions of concentrations ranging from 25 to 150
(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each
solution was injected in triplicate The peak areas were recorded and the mean
peak area was calculated (Table 318) The calibration curve was plotted
between concentration and mean peak area
Table 318 Linearity data for ZT assay method
SNo Level of
concentration Area mean area
Inj-1 50 387491
Inj -2 387640
Inj -3 386495 387209
Inj -1 75 555238
Pre-2 558542
Inj -3 559897 557892
Inj -1 100 761747
Inj -2 757099
Inj -3 760110 759652
Inj -1 125 935749
Inj -2 933310
Inj -3 932900 933986
Inj -1 150 1121624
Inj -2 1112798
Inj -3 1103885 1112769
Correlation coefficient 09998
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
180 Chapter 3
Fig 325 Linearity plot for ZT assay method
3842 Linearity of related compounds method
Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by
analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ
00075 001875 00375 005625 0075 009375 01125 013375 and 015
microgmL) of the specification Each solution was injected (n=6) times and the
mean peak area was considered for linearity assessment Regression analysis
was performed for concentration versus peak area to determine R2 value and
correlation coefficient (r) The data were also treated with ANOVA to calculate
the F-significance value The results were tabulated in Table 319
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
181 Chapter 3
Table 319 Linearity results for ZT and its impurities
SNo Name Corrleation(r) Co-efficient of
determination (R2) F-Significance
1 ZT 09996 09991 221x10-10
2 Imp-1 09998 09995 376x10-11
3 Imp-2 09999 09998 453 x10-12
4 Imp-3 09996 09992 179 x10-10
5 Imp-4 09996 09993 129 x10-10
6 Imp-5 09993 09985 953 x10-10
7 Imp-6 09990 09980 263 x10-09
8 Imp-7 09991 09982 193 x10-09
9 Imp-8 09996 09991 221 x10-10
The results shown above had shown excellent linearity of the analyte peaks
The much lower values of p lt 005 from ANOVA test indicated the significant
relationship between the concentration and the peak response
385 AccuracyRecovery
3851 Accuracy of the assay method
The recovery of the ZT assay method was determined by injecting five
individual sample preparations (n=5) at levels 50 100 and 150 of analyte
with concentration 50microgmL for drug substance and drug product Each
solution was injected in triplicate (n=3) and the mean peak area was considered
for the calculation of amount recovered The amount recovered for all the test
solutions was determined against ZT reference standard Percent recovery was
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
182 Chapter 3
calculated in terms of amount recovered versus amount added The mean
recoveries were shown below (Table 320)
Table 320 Accuracy results of ZT assay method
SNo levels () mean recovery (n=3)
(For drug substance)
() mean recovery (n=3)
(For drug product)
1 50 997 988
2 100 992 989
3 150 989 985
RSD at 100level for drug substance and drug product were 058 and 055
The data had shown very good recoveries at all three point concentration
(50 100 and 150) levels wherein the mean recovery lay between 989
and 997 for drug substance and 985 to 989 for drug product Thus the
developed UPLC method had shown good accuracy
3852 The accuracy of the related substance method
The recovery of impurities in related compounds method was studied at
50 100 and 150 of the specification level (015)
Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the
level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and
each prepared solution was injected three times into the liquid chromatographic
system The mean recoveries of each impurity were calculated (Table 321)
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
183 Chapter 3
Table 321 Accuracy study data for ZT impurities
Name
of the impurity
(n=3) Mean recovery
50 level 100 level 150 level
Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD
Imp-1 9697plusmn12 9797plusmn40 9683plusmn13
Imp-2 9423plusmn07 9879plusmn01 9567plusmn08
Imp-3 9760plusmn060 10015plusmn83 9780plusmn07
Imp-4 9527plusmn05 9866plusmn16 9777plusmn07
Imp-5 10083plusmn03 10250plusmn35 10133plusmn09
Imp-6 10157plusmn08 10346plusmn17 10040plusmn09
Imp-7 9777plusmn06 9785plusmn43 9837plusmn09
Imp-8 10213plusmn08 10165plusmn49 10120plusmn10
The related substances by the UPLC method had shown consistent recoveries
at all three level concentrations
386 Solution stability
The solution stability for ZT in diluent medium for the assay method and
related substances by UPLC method was carried out by leaving the test
solutions of samples in tightly closed flasks (VF) on bench top at room
temperature for five days The sample solutions shown above were determined
for assay content and impurity content with an interval of 1 day The
percentage RSD for assay content of ZT during solution stability experiments
was not more than 05 (Table 322) Thus the sample solutions were found to
be stable up to 5 days
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
184 Chapter 3
Table 322 Solution stability study of ZT assay
SNo Time interval ZT Assay
1 Initial 998
2 Day1 997
3 Day2 998
4 Day3 999
5 Day4 992
6 Day5 993
RSD 029
No significant change was observed in the impurity content (Imp-1 to Imp-8)
of spiked solutions during the study period Hence the sample solutions were
considered to be stable up to 5 days
Table 323 Solution stability results for ZT impurities
SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD
1 Imp-1 019 019 020 019 019 018 33
2 Imp-2 019 021 021 020 021 020 40
3 Imp-3 018 019 018 017 018 018 35
4 Imp-4 016 015 015 016 015 016 35
5 Imp-5 018 018 018 019 018 017 35
6 Imp-6 018 019 018 020 020 018 52
7 Imp-7 016 015 015 014 015 015 42
8 Imp-8 020 021 022 020 021 020 40
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
185 Chapter 3
387 Robustness
Experiments were carried out with deliberately varied method parameters
and the system suitability was evaluated The results were tabulated below
(Table 324)
Table 324 System suitability-Robustness study
Parameters Conditions Resolution
RS1 a RS
2 b
Plate counts
Temperature
(plusmn 5ordmC)
32ordmC 18 23 12500
27ordmC 16 23 12932
22ordmC 18 22 13895
Different flow
(plusmn 10 )
033 ml 18 21 10298
030 ml 18 23 12932
027ml 19 24 13580
Different organic ratio
Acetonitrile
(plusmn 10)
110 17 23 9685
100 18 23 12932
90 17 23 14254
Buffer pH
(plusmn 01) in
mobile phase-A
54 17 23 9356
55 18 23 12932
56 18 24 14268
Buffer pH
(plusmn 01) in mobile phase-B
72 19 23 11874
73 18 23 12932
74 18 23 12864
aRS1 Resolution between imp-5 and imp-6
bRS2 Resolution between imp-6 and ZT
Tailing factor Tf was less than 12 in all the varied experimental conditions
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
186 Chapter 3
Table 325 Summary of ZT analytical method validation
Related substances by UPLC
Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8
F critical 36823 36823 36823 36823 36823 36823 36823 36823
F calculated 14302 21217 10732 23109 27039 24048 003180 09564
p-value 02700 01543 03667 01333 00993 012419 07323 04064
Precision (RSD)
17 32 41 17 22 38 41 38
LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050
LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165
Inter day
precision 18 34 29 19 21 18 34 31
Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996
ANOVA
p-value 376
x10-11 453
x10-12 179
x10-10 129
x10-10
953 x10-10
263 x10-09
193 x10-09
221 x10-10
R2 value 09995 09998 09992 09993 09985 09980 09982 09991
Mean
recovery 980 988 1002 987 1025 1035 979 1017
Solution stability
5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
187 Chapter 3
39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process
samples using the finalized method conditions The results were
tabulated below (Table 326)
Table 326 Impurity levels in different samples of ZT
Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI
1 002 005 002 004 006 001 004 003
2 001 006 002 002 004 001 003 002
3 002 005 002 004 006 001 003 003
4 002 007 004 004 004 002 004 003
5 002 006 002 006 005 001 004 005
6 002 004 004 004 005 ND 003 004
7 001 005 003 002 006 ND 004 002
8 001 005 005 003 007 ND 003 002
9 002 004 003 002 006 002 002 003
10 002 006 004 002 006 001 003 004
Mean 0017 0053 0031 0033 0055 00129 0033 0031
SD 00048 00095 0011 00134 00097 00049 00067 00099
Imp-7 was not detected in all the samples shown above
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
188 Chapter 3
Fig 326 Graphical representation of impurity profiles of ZT samples
310 Conclusion
A reversed phase-UPLC gradient method was developed for the determination
of ZT its related process impurities and degradation products This method can
be applicable for both ZT drug substance and drug product The method was
found to be selective precise accurate linear rugged and robust The total run
time was 100 minutes which enabled fast estimation of the drug substance
The developed method was proved to be stability-indicating This method can be
used for regular analysis of bulk and formulation samples from production area
and for the analysis of ZT stability samples
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
189 Chapter 3
References
[1]
Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29
[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ
Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I
Neuropharmacological and behavioral effects J Pharmacol Exp Ther
237(2) 1986 649-58
[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02
2lblpdf Prescribing Information from official Gazette of Food and Drug
Administration
[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel
nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels
and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65
[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that
affects electronic transfer alters synaptic activity through potential GABA
receptors in the nervous system without significant free radical
generation Oxid Med Cell Longev 2009 2(1) 52ndash57
[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda
Satyanarayana Synthesis and spectral characterization of process-
related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009
143-149
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
190 Chapter 3
[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification
of zolpidem tartrate in human plasma by high performance liquid
chromatography with fluorescence detection J Chromat B 2004 811 59ndash
63
[8] Laviana L Mangas C Mari FF Bayod M Determination and in process
control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36
925ndash928
[9] Ring PR Bostick JM Validation of a method for the determination of
zolpidem tartrate in human plasma using LC with fluorescence detection
J Pharm Biomed Anal 2000 22 495-504
[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem
hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal
2003 33 393-401
[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487
[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257
[13] Kelani KM Selective potentiometric determination of zolpidem
hemitartrate in tablets and biological fluids by using polymeric membrane
electrode J AOAC Int 2004 87 1309-1318
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
191 Chapter 3
[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of
Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4
[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods
for determination of zolpidem tartrate in tablet formulation J Pharm
Bioallied Sci 2010 2(4) 365ndash368
[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral
fluid by liquid chromatographyndashtandem mass spectrometry Journal of
Chromatography B 2004 811 59ndash63
[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of
zolpidem in urine and hair application to two drug facilitated sexual
assaults Forensic Science International 2004 143 157-161
[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic
Multiobjective Optimization Approach in Evaluation of Chromatographic
Behaviour of Zolpidem Tartrate and Its Degradation Products
Chromatographia 2011 74 197ndash208
[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology
2005
[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient
high-performance liquid chromatography theory and applications
Journal of Chromatography A 2004 1060 165-175
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87
192 Chapter 3
[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change
of mobile phase pH during gradient reversed-phase chromatography with
222-trifluoroethanol-water as mobile phase and its effect on the
chromatographic hydrophobicity index determination Journal of
Chromatography A 2002 954 77ndash87