127
Section (i): Brief account of Repaglinide
Repaglinide (RPL) single enantiomer is chemically (S)-(+)2-ethoxy-4-[N [1-(2-
piperidinophenyl)-3-methyl-1-butyl] aminocarbonylmethyl] benzoic acid. The empirical
formula and molecular weight of RPL are C27H36N2O4 and 452.59 respectively. It is a white
to off-white powder. Its structure is:
USFDA approved RPL (PRANDIN®,NOVO NORDISK INC) for the treatment of
benign prostatic hyperplasia in December 1997. RPL tablets are supplied as 0.5 mg, 1 mg, or 2
mg of RPL.
It is a fast-acting prandial glucose regulator used in the treatment of type II diabetes.
RPL and sulphonyl ureas such as tolbutamide and glibenclamide, share the common property
that they are capable of closing ATP (Adenosine-5'-triphosphate) sensitive potassium
channels. The ATP channel plays a key role in glucose-dependent insulin secretion from
pancreatic beta cells. RPL, a new carbamoylmethyl benzoic acid derivative, is the first of a
new class of oral antidiabetic agents designed to normalize postprandial glucose excursions in
patients with type II diabetes mellitus. RPL exerts its effects by binding to a site on plasma
membrane of beta cells, thereby closing ATP-sensitive potassium channels. RPL, a novel
compound with a nonsulphonylurea structure, is clinically tested as a therapeutic agent. The
hypoglycemic effects of RPL are investigated. Whereas the (R)-enantiomer of RPL shows
only weak hypoglycemic activity, the (S)-enantiomer has turned out to be a potent
hypoglycemic compound.
RPL drug substance and drug product is official in United States pharmacopeia
(USP)[1] and drug substance is official in European pharmacopeia [EP][2]. USP described
128
only three impurities namely Imp-B, Imp-D and other process related impurity in the drug
substance monograph and only imp-D in the drug product monograph. EP captured four
impurities Imp-A, Imp-B, Imp-D, Imp-E and enantiomer. All the process related impurities
and degradation products are captured in neither USP nor EP.
Literature survey revealed few methods have been reported for the quantitative
determination of RPL in pharmaceutical preparations such as an HPLC method [3] for
impurity profile study of RPL, but force degradation study, method development and method
validation is not reported. Assay methods are reported by HPLC [4-8] , HPTLC[9], RP-TLC
with UV detection[10], UV spectrophotometric[11], enzymatic method[12] for the
determination of RPL in pharmaceutical formulations and in combination with other actives.
A chiral LC method [13] reported for enantiomeric separation of RPL. Two more methods are
reported for the quantification of RPL in biological samples by HPLC with coulometric
detection [14] and UV detection [15]. So far no method is reported for forced degradation
study and no method capturing all process related impurities and degradation products of
RPL. Existing pharmacopoeial or literature methods are not able to separate all known
impurities and unknown degradants.
Two unknown impurities (degradation products) present at a level below 0.1% in the
initial samples of RPL tablets increased to a level of 0.5% in 3 M/40 ◦C/75%RH stability
studies. The assay methods reported in the literature is not able to separate all the known
impurities and degradation products from the analyte peak.
This prompted the author to develop stability indicating HPLC methods for the assay
and impurities. The two degradation impurities were enriched and isolated by using reverse-
phase preparative liquid chromatography and characterized.
This chapter describes development and validation of a stability indicating methods
for assay and impurities along with the identification, isolation and characterization of two
unknown degradation products formed in RPL tablets during the stability studies. Though
some of the impurities and degradation products were reported in the literature, identification,
isolation and characterization of these degradation products is not reported to the best of our
knowledge.
129
Based on the spectral data (LS-MS, 1H NMR, 13C NMR and DEPT) the structures
of these impurities are characterized as 4-(2-((1-(2-(4- carboxybutanamido) phenyl)-3-
methylbutyl) amino)-2-oxoethyl)-2-ethoxybenzoic acid(degradation product-I) and 2-ethoxy-
4-(2-((3-methyl-1-(2-(5-oxopentanamido) phenyl) butyl)amino)-2-oxoethyl)benzoic acid.
130
Section (ii): Stability Indicating HPLC Assay method for Repaglinide tablets.
This section reports the various aspects relating to the development and validation of
stability indicating HPLC method for assay of repaglinide (RPL) tablets.
1. Experimental
1.1. Chemicals
Samples of RPL API are received from Process R&D, Dr Reddy’s Laboratories,
Hyderabad, India. RPL tablets of 0.5, 1 and 2 mg & all excipients are received from
formulation R&D, Dr Reddy’s Laboratories, Hyderabad, India. HPLC grade acetonitrile, and
potassium dihydrogen phosphate, NaOH, HCl, H2O2 are supplied by Merck, Darmstadt,
Germany. High purity water is prepared by using millipore milli Q plus purification system.
1.2. Determination of appropriate UV wavelength
The suitable wavelength for the determination of RPL in diluent is identified by scanning
over the range 200–400 nm with a Shimadzu UV-160 (Shimadzu, Japan) double beam
spectrophotometer.
1.3. Instrumentation and chromatographic conditions
The Waters HPLC System with a photo diode array detector is used for the method
development and force degradation studies .The out put signal is monitored and processed
using Waters Empower Networking software. The HPLC system used for method validation
is Agilent 1100 series LC system with variable wavelength detector (VWD).The
chromatographic column used is an ACE,C-8, 250mm x 4.6 mm column, with 5µ particle
size. The mobile phase is a mixture of 30mM pH 3.2 phosphate buffer and acetonitrile in the
ratio of 30:70 (v/v) .The flow rate of the mobile phase is 1.0 ml min-1
. The column
temperature is maintained at 30ºC and the detection wavelength is 240 nm. The injection
volume is 20µl.
1.4. Diluent:
Mobile phase is used as diluent.
131
1.5. Preparation of standard drug solution:
The stock solution of RPL standard equivalent to 0.8 mg ml-1
of RPL is prepared
in diluent. The working solution of standard having 80µg ml-1
of RPL for 1 and 2mg & 40µg
ml-1
of RPL for 0.5mg is obtained by dilution of the stock solution in diluent. The Specimen
overlay chromatogram of diluent and standard is shown in fig.3.2.1.
Fig 3.2.1: Overlay chromatogram of diluent and RPL standard 80µg ml-1
.
1.6. Test Preparation for RPL pharmaceutical formulations:
Twenty tablets of RPL are weighed and transferred into a clean dry 500ml volumetric
flask for 2mg and 250 ml volumetric flask for 1mg and 0.5mg. Diluent is added up 80% of the
volumetric flask , sonicated for 20 min with frequent intermediate shaking, made up to
volume with diluent and mixed .The resulting solution is centrifuged at 4000 rpm for 5 min.
About 2 ml of the solution is filtered by using 0.45µm nylon 66 membrane filters. Placebo
sample is prepared in the same way by taking the placebo equivalent its weight present in a
test preparation .The Specimen overlay chromatogram of placebo and test samples is shown
in fig.3.2.2.
132
Fig 3.3.2: Overlay chromatogram of placebo and RPL tablets 2mg.
1.7. Specificity:
Regulatory guidances in ICH Q2A, Q2B, Q3B and FDA 21 CFR section 211, require the
development and validation of stability-indicating potency of assays. However, the current
guidance documents do not indicate detailed degradation conditions in stress testing. The
forced degradation conditions, stress agent concentration and time of stress, are found to
effect the % degradation. Preferably not more than 20% is recommended for active materials
to make the right assessment of stability indicating nature of the chromatographic methods.
The optimisation of such stress conditions which can yield not more than 20% degradation is
based on experimental studies. Chromatographic runs of placebo solution and samples
subjected to force degradation are performed in order to provide an indication of the stability
indicating properties and specificity of the method. The stress conditions employed are acid,
base, neutral and oxidant media, moisture, heat and light. After the degradation is completed,
the samples are allowed to equilibrate to room temperature, neutralized with acid or base (as
necessary), and diluted with diluent to get solutions having RPL at 80 µg ml-1
concentration.
The samples are analyzed against a freshly prepared control sample (with no degradation
treatment) and evaluated for peak purity by using photo diode array detector. Specific
conditions are described below.
1.7.1. Placebo (excipients) interference:
Samples are prepared in triplicate by taking the weight of placebo approximately
equivalent to its weight in the test sample preparation portion as described in the Test
preparation.
133
1.7.2. Effect of acid, base and neutral hydrolysis
10 ml of the 0.5 mg ml-1
of
RPL test solution is transferred to three different round
bottomed flasks and 5 ml of 5N HCl , 1N NaOH and water, respectively are added. The acidic
solution, alkaline solution and neutral solutions are stressed for 3 hours at 60 ºC. Then all
samples are allowed to equilibrate to room temperature, neutralized with base or acid as
appropriate and then diluted with diluent to 80µg ml-1
and filtered by using 0.45 µm nylon 66
membrane filters.
1.7.3. Effect of oxidation
A 0.5 mg ml-1
RPL test solution is prepared from the tablets. 5 ml of this solution are
added to 5 ml of 3%H2O2 and heated for 2 hours at 60ºC. The solution is, then diluted with
diluent to 80 µg ml-1
and filtered by using 0.45 µm nylon 66 membrane filter.
1.7.4. Effect of moisture and heat
To evaluate the effect of moisture and heat, thin layers of tablets powder are distributed
over a glass plate. The plate is stored at 25ºC/90% relative humidity for 7 days. A similar
sample is kept in a oven at 105ºC for 48 hours. Then, the samples are prepared in diluent as
described in the test preparation.
1.7.5. Effect of UV and visible light
To study the photochemical stability of the drug product the tablet’s powder is exposed
to 1200 K Lux hours of visible light and 200 Watt hours/ m2 of UV light by using photo
stability chamber. After exposure the samples are prepared in diluent as described in test
preparation.
1.8. Method validation
1.8.1. Precision
Precision (intra-day precision) of the assay method is evaluated by carrying out six
independent assays of test sample of RPL tablets against qualified reference standard. The %
RSD of six assays obtained is calculated. The intermediate precision (inter day precision) of
the method is also evaluated using two different HPLC systems and different HPLC columns
in different days in the same laboratory.
134
1.8.2. Linearity
Linearity test for assay is made for different concentration levels in the range of about
10-120 µg ml-1
of RPL (corresponding to LOQ to 150% of assay of highest sample
concentration).
The
peak area versus concentration data is performed by least-square
regression analysis.
1.8.3. Accuracy
A study of recovery of RPL from spiked placebo is conducted. Samples are prepared by
mixing placebo with RPL API equivalent to about 25%, 50%, 75%, 100%, 125%, and 150%
of the assay of highest sample concentration. Sample solutions are prepared in triplicate for
each spike level as described in the sample preparation. The % recovery is calculated.
1.8.4. Robustness
To determine the robustness of the developed method, experimental conditions are
purposely altered one after the other to estimate their effect. Five replicate injections of
standard solution are injected under each parameter change. The effect of flow rate, column
temperature, organic phase composition (acetonitrile) in mobile phase and pH of the buffer in
mobile phase, on the tailing factor of RPL peak and %RSD for peak areas of replicate
injections of standard is studied at flow rates of 0.8 ml min-1
and 1.2 ml min-1
, column
temperatures of 25ºC and 35ºC, organic phase compositions (acetonitrile) in mobile phase at
+ 10% and pH 3.0 and 3.4 respectively.
1.8.5. Solution stability and mobile phase stability
The solution stability of RPL in the assay method is carried out by leaving solutions of
both the test sample and reference standard in tightly capped volumetric flasks at room
temperature for 48 hours.The same sample solutions is assayed at 24 hours interval up to the
study period.
The mobile phase stability is also carried out by injecting freshly prepared standard
solutions at 24hours interval for 48 hours. Mobile phase prepared is kept constant during the
study period. The tailing factor and % RSD of RPL standard area is calculated.
135
2. Results and discussion
2.1. Determination of suitable wavelength
The UV spectrum of RPL recorded in the range 200-400 nm is illustrated in fig.3.2.3.
The spectrum indicates that 245 nm gives a good sensitivity for the assay.
Fig 3.2.3: UV Spectra of RPL.
2.2. Optimization of chromatographic conditions
The HPLC procedure is optimized with a view to develop a stability indicating assay
method. Pure drug and stressed samples are injected and run in different solvent systems.
preliminary experiments using different compositions of water, acetonitrile and methanol on
different reversed phase stationary phases did not give good peak shape and ‘k’ value is found
to be more than 20 in C-18 column. When methanol is replaced by acetonitrile into the mobile
phase, ‘k’ value decreased to less than 10 in C18 column, but still the peak shape is not
optimal and the separation between RPL and degradants is not satisfactory. Eventually, in a
mobile phase consisting of mixture of buffer and acetonitrile in the ratio of 30: 70(v/v) on
ACE C-8, 250mm x 4.6 mm, with 5µ particle size column with a flow rate of 1.0 ml min-1
, a
retention time of about 8 min is achieved for RPL with good peak symmetry and also the
separation between RPL and degradants is found to be satisfactory.
136
2.3. Method validation
2.3.1. Precision
Method repeatability (intra-day precision) is evaluated by assaying six samples,
prepared as described in the sample preparation. The mean % assay and %RSD for assay
values are found to be well within the acceptance criteria i.e. mean % assay between 97.0 -
103.0 and RSD not more than 2.0 %. The intermediate precision (inter day precision) is
performed by assaying six samples on different HPLC systems and different HPLC columns
in different days as described in the test preparation. The result shows good precision of the
method (table 3.2.1).
Table 3.2.1: Results of precision of test method
Sample No. Assay of RPL as % of labeled amount
Intra-day precision Inter-day precision
1 99.2 97.6
2 99.1 98.5
3 98.8 98.0
4 98.8 98.3
5 99.3 98.1
6 99.4 98.3
Mean 99.1 98.1
%RSD 0.3 0.3
2.3.2. LOQ and LOD
The LOQ and LOD are determined for RPL based on signal-to-noise ratio at analytical
responses of 10 and 3 times the background noise, respectively. The LOQ is found to be
0.1 μg ml -1
with a resultant %RSD of 0.4 (n = 5). The LOD is found to be 0.03 μg ml.-1
2.3.3. Linearity
A linear calibration plot for assay of RPL is obtained over the calibration range of
about 10-120 µg ml-1
and the correlation co-efficient is found to be greater than 0.999. The
result shown in fig.3.2.4 indicates that an excellent correlation exists between the peak area
and concentration of the analyte.
137
Fig.3.2.4: Linearity of detector response graph for RPL.
2.3.4. Accuracy
The percentage recovery of RPL in pharmaceutical dosage forms shown in table 3.2.2
ranged from 99.1 to 100.7% indicating high accuracy of the method.
Table 3.2.2: Recovery results of RPL in pharmaceutical dosage form.
Spike
level (%)
Average
‘mg’ added
Average
‘mg’ found
Mean %
recovery
%
RSD
25 2.520 2.498 99.1 0.3
50 5.012 5.045 100.7 0.3
75 7.497 7.485 99.8 0.1
100 10.012 10.056 100.4 0.2
125 12.548 12.497 99.6 0.2
150 14.950 14.938 99.9 0.1
2.3.5. Robustness
In all the deliberately varied chromatographic conditions studied (flow rate, column
temperature, ratio of acetonitrile and pH of the buffer in mobile phase), the tailing factor and
the %RSD for the RPL peak area for five replicate injections of standard is found to be within
the acceptable limit of not more than 2.0%, illustrating the robustness of the method (table
3.2.3).
y = 32303x + 8966. R = 0.999
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
0 20 40 60 80 100 120 140
Pe
ak A
rea
Concentration µg/mL
Linearity graph for RPL
138
Table3.2.3: Results of Robustness study
Parameter
Observed value
Variation Tailing factor of
RPL
%RSD for five
injections of standard
1.Flow rate 0.8 ml min-1
1.0 0.35
1.0 ml min-1
1.0 0.42
1.2 ml min-1
1.0 0.44
2.Column temperature 25ºC 1.0 0.53
30ºC 1.0 0.42
35ºC 1.0 0.57
3.Mobile phase
composition(acetonitrile)
90% 1.1 0.45
100% 1.0 0.42
110% 1.0 0.61
4. pH of the buffer pH 3.0 1.0 0.51
pH 3.2 1.0 0.42
pH 3.4 1.0 0.49
2.3.6. Solution stability and mobile phase stability
The difference in % assay of test and standard preparations upon storage on bench top
is found to be less than 1.0% up to 48 hours. Mobile phase stability experiments showed that
tailing factor and % RSD are less than 1.1 and 0.7% respectively up to 48 hours. The solution
stability and mobile phase stability experimental data confirmed that sample solutions and
mobile phase used during assay determination are stable up to 48 hours.
2.3.7. Results of specificity studies
All the placebo and stressed samples prepared are injected into the HPLC system
with photodiode array detector as per the described chromatographic conditions.
Chromatograms of placebo solutions have shown no peaks at the retention time of RPL. This
indicates that the excipients used in the formulation do not interfere in estimation of RPL in
tablets.
139
Degradation is not observed in light exposure or heating or moisture studies. In acid
hydrolysis, base hydrolysis, water hydrolysis and oxidative studies significant degradation is
observed. All degradant peaks are well resolved from RPL peak in the chromatograms of all
stressed samples. The chromatograms of the stressed samples are evaluated for peak purity of
RPL using Waters Empower Networking software. For all forced degradation samples, the
purity angle (the weighted average of all spectral contrast angles calculated by comparing all
spectra in the integrated peak against the peak apex spectrum) is found to be less than
threshold angle (the sum of the purity noise angle and solvent angle, the purity noise angles
across the integrated peak) for RPL peak (table 3.2.4). This indicates that there is no
interference from degradants in quantitation the RPL in tablets. Thus, this method is
considered "Stability indicating”. The typical chromatogram and purity plots of all stressed
samples are shown in figs 3.2.5 to 3.2.12.
Table 3.2.4: Table of results of specificity
Stress Condition
Details of Stressed Drug Product
% Assay Purity
angle
Purity
threshold
Stressed with 5 N HCl solution for about 3 hours at 60ºC. 97.9 0.074 0.236
Stressed with 1 N NaOH solution for about 3 hours at 60ºC. 96.7 0.037 0.225
Stressed with 3% H2O2 for about 2 hours at 60ºC. 97.2 0.168 0.234
Stressed with purified water for about 3 hours at 60ºC. 98.4 0.053 0.247
Exposed to Sunlight for 1.2 Million Lux hours for 7 days. 99.5 0.052 0.261
Exposed to UV light for 200 watt Hours/m2 for 7 days. 99.0 0.049 0.260
Stressed to dry heat at 105°C for about 48 hours. 99.4 0.050 0.259
Exposed to humidity at 25°C, 90% RH for about 7 days. 99.3 0.051 0.258
140
Fig 3.2.5: Chromatogram and purity plot of acid stressed RPL tablets.
Fig 3.2.6: Chromatogram and purity plot of base stressed RPL tablets.
141
Fig 3.2.7: Chromatogram and purity plot of H2O2 stressed RPL tablets.
Fig 3.2.8: Chromatogram and purity plot of water stressed RPL tablets.
142
Fig 3.2.9: Chromatogram and purity plot of visible light stressed RPL tablets.
Fig 3.2.10: Chromatogram and purity plot of UV light stressed RPL tablets.
143
Fig 3.2.11: Chromatogram and purity plot of heat stressed RPL tablets.
Fig 3.2.12: Chromatogram and purity plot of humidity stressed RPL tablets.
144
3. Conclusion:
A validated stability-indicating HPLC analytical method has been developed for the
determination of RPL in formulation dosage form. The results of stress testing reveal that the
method is selective and stability-indicating. The proposed method is simple, accurate, precise,
specific and has the ability to separate the drug from degradation products and excipients
found in the RPL tablet dosage form. The method is suitable for the routine analysis of RPL
in either bulk powder or in pharmaceutical dosage forms. The HPLC procedure can be applied
to the analysis of samples obtained during accelerated stability experiments to predict the
expiry dates of pharmaceuticals in bulk and in formulations.
145
Section (iii): Stability Indicating HPLC method for impurities in Repaglinide tablets.
This section reports the various aspects relating to the development and validation of
stability indicating HPLC method for impurities in repaglinide (RPL)tablet dosage form.
1. Experimental
1.1. Chemicals
RPL tablets are formulated in Dr Reddy’s laboratories Ltd, Hyderabad, India. The
standards of RPL and its eight impurities namely -A, imp-B, imp-C, imp-D, imp-E, imp-F,
imp-G and imp-H are supplied by Dr. Reddy’s laboratories limited, Hyderabad, India. The
HPLC grade acetonitrile, methanol and analytical grade KH2PO4, sodium perchlorate,
trifluoroacetic acid and ortho phosphoric acid are purchased from Merck, Darmstadt,
Germany. High purity water was prepared by using Milli Q Plus water purification system
(Millipore, Milford, MA, USA). The chemical names and structures of RPL and its impurities
are shown below.
S.No Name of the
impurity
IUPAC name Structure
1 Repaglinide (S)-2-ethoxy-4-(2-((3-
methyl-1-(2-(piperidin-1-
yl)phenyl)butyl)amino)-2-
oxoethyl)benzoic acid
2 Imp-A 4-(carboxymethyl)-2-
ethoxybenzoic acid
3 Imp-B 2-(3-ethoxy-4-
(ethoxycarbonyl)phenyl)a
cetic acid
4 Imp-C 2-hydroxy-4-(2-
(methyl(1-(2-(piperidin-1-
yl)phenyl)butyl)amino)-2-
oxoethyl)benzoic acid
146
5 Imp-D (S)-3-methyl-1-(2-
(piperidin-1-
yl)phenyl)butan-1-amine
6 Imp-E (S)-ethyl 2-ethoxy-4-(2-
((3-methyl-1-(2-
(piperidin-1-
yl)phenyl)butyl)amino)-2-
oxoethyl)benzoate
7 Imp-F
2-ethoxy-4-(2-((4aS,6S)-
6-isobutyl-2,3,4,4a-
tetrahydro-1H-pyrido[1,2-
a]quinazolin-5(6H)-yl)-2-
oxoethyl)benzoic acid
8 Imp-G 2-ethoxy-4-(2-oxo-2-
((4aR,6S)-6-propyl-
2,3,4,4a-tetrahydro-1H-
pyrido[1,2-a]quinazolin-
5(6H)-yl)ethyl)benzoic
acid
9 Imp-H (S)-4-(2-((1-(2-
aminophenyl)-3-
methylbutyl)amino)-2-
oxoethyl)-2-
ethoxybenzoic acid
10 Degradant-1 (S)-4-(2-((1-(2-(4-
carboxybutanamido)pheny
l)-3-methylbutyl)amino)-
2-oxoethyl)-2-
ethoxybenzoic acid
147
11 Degradant-2 (S)-2-ethoxy-4-(2-((3-
methyl-1-(2-(5-
oxopentanamido)phenyl)b
utyl)amino)-2-
oxoethyl)benzoic acid
1.2. Determination of appropriate UV wavelength
The suitable wavelength for the determination of RPL and its impurities is identified by
taking the overlay spectra from 200–400 nm of all known impurities and RPL from PDA
detector.
1.3. Instrumentation and chromatographic conditions
The Waters HPLC System with a photo diode array detector is used for the
method development and force degradation studies .The output signal is monitored and
processed using Waters Empower Networking software. The HPLC system used for method
validation is Agilent 1100 series LC system with variable wavelength detector (VWD). The
chromatographic column used is an Zodiac AQ C18, 250 x 4.6 mm, with 5µ particle size. The
mobile phase consists of a gradient mixture of solvent A and B. 0.015 M sodium per chlorate
buffer, pH adjusted to 3.1 with trifluoro acetic acid is used as solvent A. pH 3.1perchlorate
buffer and acetonitrile in the ratio 32:68, v/v is s used as solvent B. The gradient program
(T/%B) is 0/35, 5/35, 30/70, 31/65, 40/65, 45/80, 55/85, 75/80, 76/35 and 82/35. The flow
rate of the mobile phase is T/Flow : 0/1.0, 5/1.0, 30/1.0, 31/0.8, 40/1.0 45/1.0, 55/1.0, 75/1.0,
76/1.0 and 82/1.0. The column temperature is maintained at 55 °C and the wavelength is
monitored at 240 nm. The injection volume is 50µl.
1.4. Diluent:
0.05 M potassium dihydrogen phosphate buffer (pH adjusted to 1.8 with phosphoric
acid) and acetonitrile in the ratio 50:50 v/v/ is used as a diluent.
1.5. Preparation of Repaglinide standard drug solution:
The stock solution of RPL standard equivalent to 0.5 mg ml-1
of RPL is prepared
in diluent. The working diluted standard solution of 100 µg ml-1
and 1.5 µg ml-1
of RPL is
148
obtained by dilution of the stock solution in diluent. The overlay chromatogram of diluent and
standard is shown in fig.3.3.1.
Fig 3.3.1: Overlay chromatogram of diluent as blank and RPL standard.
1.6. Test Preparation for RPL pharmaceutical formulations:
Twenty tablets of RPL are weighed and transferred into a clean dry mortar and made it
into a fine powder. Tablet powder equivalent to about 100 mg of RPL is transferred into a 200
ml volumetric flask. 40 ml of acetonitrile is added to the flask, kept on rotary shaker for 20
min and then 50 ml of water is added and sonicated for 30 min with frequent intermediate
shaking. The resultant solution is made up to volume with diluent and mixed. The solution is
then centrifuged at 3000 rpm for 5 min. This gives a test preparation of 0.5 mg ml-1
of RPL.
About 2 ml of the solution is filtered by using 0.45µm nylon 66 membrane filter. Placebo
sample is prepared in the same way by taking the placebo equivalent its weight present in a
test preparation .The overlay chromatogram of placebo and test samples is shown in fig.3.3.2.
149
Fig 3.3.2: Overlay chromatogram of placebo and RPL tablets test spiked with impurities.
1.7. Specificity:
Regulatory guidances ICH Q2A, Q2B, Q3B and FDA 21 CFR section 211, require the
development and validation of stability-indicating impurities method for all pharmaceutical
dosage forms. However, the current guidance documents do not indicate detailed degradation
conditions in stress testing. The forced degradation conditions, stress agent concentration and
time of stress, are found to effect the % degradation. Not more than 20% degradation is
recommended for active materials to make the right assessment of stability indicating nature
of the chromatographic methods. The optmisation of such stress conditions which can yield
not more than 20% degradation is based on experimental study. Chromatographic runs of
placebo and samples subjected to force degradation are performed in order to provide an
indication of the stability indicating properties and to establish the specificity of the method.
The stress conditions employed are acid, base, neutral and oxidant media, moisture, heat and
light. After the degradation treatments are completed, the samples are allowed to equilibrate
to room temperature, neutralized with acid or base (as necessary), and diluted with diluent to
get the working concentrations equivalent to test preparation. The stressed samples are
subjected to assay analysis to assess the mass balance. The samples are analyzed against a
freshly prepared control sample (with no degradation treatment) and evaluated for peak purity
by using photo diode array detector. Specific conditions are described below.
150
1.7.1. Placebo (excipients) interference:
Samples are prepared in triplicate by taking the weight of placebo approximately
equivalent to its weight in the test as described in the test preparation.
1.7.2. Effect of acid, base and neutral hydrolysis
20 ml of the 1.0 mg ml-1
of
RPL test solution is transferred to three different round
bottomed flasks and 5 ml of 5 N HCl , 5 N NaOH and water, respectively are added. The
acidic solution, alkaline solution and neutral solutions are refluxed for 16 hours at 80 ºC in a
heating mantle. All samples are allowed to equilibrate to room temperature, neutralized with
base or acid as appropriate and diluted with diluents to obtain a test concentration of about 0.5
mg ml-1
. The resultant solutions are filtered by using 0.45 µm nylon 66 membrane filters. The
test solutions are further diluted to obtain Spectral absorbance less than 1000 mAU to
facilitate peak purity assessment and assay analysis to calculate mass balance. Peak purity test
is carried out for the RPL peak by using PDA detector in stress samples. Assay studies are
carried out on all stressed samples at 100 µg ml-1
to get the assay of RPL for mass balance
study.
1.7.3. Effect of oxidation
A 1.0 mg ml-1
RPL test solution is prepared from the tablets. 20 ml of this
solution is taken and 5 ml of 1%H2O2 is added and heated for 30 min at 60ºC. The solution is
equilibrated at room temperature and appropriately diluted to obtain a test solution having
about 0.5 mg ml-1
of RPL. The resultant solution is filtered by using 0.45 µm nylon 66
membrane filter. Peak purity test is carried out for the RPL peak by using PDA detector in
stress samples. Assay studies are carried out at 100 µg ml-1
to get the assay of RPL for mass
balance study.
1.7.4. Effect of moisture and heat
To evaluate the effect of moisture and heat, thin layers of tablets powder are
distributed over a glass plate. The plate is stored at 25ºC/90% relative humidity for 7days. A
similar sample is kept in a oven at 105ºC for 12 hours. Then test solutions are prepared from
the samples in diluent as described in the test preparation and filtered by using 0.45 µm nylon
66 membrane filters. Peak purity test is carried out for the RPL peak by using PDA detector in
151
stress samples. Assay studies are carried out on all stressed samples at 100 µg ml-1
to get the
assay of RPL for mass balance study.
1.7.5. Effect of UV and visible light
To study the photochemical stability of the drug product, the tablets powder is exposed
to 1200 K Lux hours of visible light and 200 Watt hours/ m2 of UV light by using photo
stability chamber. After exposure, the test solutions are prepared in diluent as per the
procedure and filtered by using 0.45 µm nylon 66 membrane filters. Peak purity test was
carried out for the RPL peak by using PDA detector in stress samples. Assay studies are
carried out on stressed samples at 100 µg ml-1
to get the assay of RPL for mass balance study.
1.8. Method validation
1.8.1. Relative retention times and relative response factors:
The relative retention times (RRTs) and relative response factors (RRFs) of all known
impurities are established against RPL. Different concentrations of RPL and its known
impurities are injected into the chromatographic conditions developed. The linearity graphs
are drawn for RPL and all its known impurities individually. The relative response factors are
then calculated by dividing the slope of impurity by slope of RPL. The Relative retention
times (RRT’s) and Relative response factors (RRF’s) of known impurities are summarized in
table 3.3.1.
Table 3.3.1: RRT and RRF values of impurities of RPL
Name Compound RRT RRF
Impurity -A 0.18 1.79
Impurity -B 0.53 1.58
Degradant-1 0.58 0.99
Degradant-2 0.66 0.88
Impurity -H 0.72 1.06
Impurity -C 0.78 1.14
Impurity -D 0.81 0.41
Repaglinide 1.00 1.00
Impurity -E 1.41 0.82
Impurity -F 1.61 1.62
Impurity -G 1.69 1.70
152
1.8.2. Precision
Precision (intra-day precision) of the impurities method is evaluated by preparing six
different solutions of test sample of RPL tablets spiked with known impurities at about
0.3%to 0.5% level (with respect to 0.5 mg ml-1
RPL). Solutions of placebo spiked with RPL
at 0.3% level are prepared. The solutions are then injected into the developed
chromatographic conditions described above. % of impurities are calculated against a
qualified RPL standard. RSD is then calculated for % of impurities individually obtained for
six different preparations. The intermediate precision (inter day precision) of the method is
also evaluated using different HPLC systems and different HPLC columns on different day in
the same laboratory.
1.8.3 Limits of Detection (LOD) and Quantification (LOQ)
The LOD and LOQ for RPL and its ten impurities are determined at a signal-to-noise
ratio of 3:1 and 10:1 respectively, by injecting a series of dilute solutions with known
concentrations. A Precision study is also carried out at the LOQ level by injecting six
individual preparations. % RSD is calculated.
1.8.4. Linearity
Linearity test solutions for RPL and 10 known impurities are prepared by diluting
stock solutions to the required concentrations. The solutions are prepared at different
concentration levels from LOQ to 300% of the specification level.
1.8.5. Accuracy
A study of recovery of RPL and its 10 known impurities from placebo is conducted.
Samples are prepared by mixing placebo with RPL as per the formulation composition and
then spiking all the known impurities at different spike levels starting from LOQ to 150% of
the specification level. Sample solutions are prepared in triplicate for each spike level as
described in the test preparation and injected into the chromatographic conditions developed.
The % recovery is then calculated against RPL diluted standard and by using relative response
factor and compared against the known amounts of impurities spiked.
153
1.8.6. Robustness
To determine the robustness of the developed method, experimental conditions are
deliberately altered and the elution patterns, tailing factor and resolution between its
impurities are evaluated. The effect of flow rate is studied by changing it by 0.2 units from 0.8
to 1.2 ml min-1
. The effect of the column temperature is studied at 50ºC and 60ºC instead of
55ºC. The effect of pH is studied using mobile phase containing buffer with pH 3.1 ± 0.1.The
effect of the percent organic strength is studied by varying acetonitrile by −10 to +10% while
other mobile phase components are held constant.
1.8.7. Solution stability and mobile phase stability
The stability of RPL and its impurities in solution for the impurities method is
determined by leaving spiked sample solution in a tightly capped volumetric flask at room
temperature on bench top for 6 hours and measuring the amounts of the impurities at every 3
hours. The stability of mobile phase is also determined by analysing freshly prepared solution
of RPL and its impurities at 24 hour interval for 48 hours. The mobile phase is not changed
during the study period.
2. Results and discussion
2.1. Determination of suitable wavelength
The UV spectrum of RPL and its impurities are extracted in PDA detector from 200-
400 nm is illustrated in fig.3.3.3. The spectrum indicates that 240 nm gives a good sensitivity
for the impurities an RPL.
Fig 3.3.3: UV Spectra of RPL and its impurities
154
2.2. Optimization of chromatographic conditions
When the Pharmacopoeal methods are used the separations are not found to be
satisfactory. Known and unknown degradants are not getting separated satisfactorily. Hence,
the main objective of the chromatographic method is to separate critical closely eluting pair of
compounds Imp-B and Degradant-1, Degradant-1 and Imp-H, Deg-2 and Imp-H, Imp-C and
Imp-D, Imp-F and Imp-G and to elute RPL as a symmetrical peak. The blend solution
containing 500 µg/ml of RPL and 1.5µg/ml (equivalent to 0.3%) of each impurity is prepared
in diluent.
Attempts are made with gradient elution with solvent A and B using different C18
columns (Inertsil ODS-3, 250 mm × 4.6 mm, 5μm particles and Zorbax XDB C-18,
250 mm × 4.6 mm, containing 5μm particles,) and using different buffer pH (2.5 and 7.0)
conditions. But at all above conditions, separation of impurities is not satisfactory.
All the impurities of RPL are subjected to separation by reversed-phase LC on a
Zodiac C18 AQ, 250 x 4.6 mm, 5μm column with 0.015 M sodium per chlorate buffer pH
adjusted to 3.5 with trifluoroacetic acid is used as solvent A and pH 3.5 per chlorate buffer
and acetonitrile in the ratio 30:70, v/v is used as solvent B. The gradient program (T/%B) is
set as 0/35, 5/35, 30/70, 31/65, 40/65, 45/80, 55/85, 75/80, 76/35 and 82/35. The flow rate of
the mobile phase is set at 1.0 ml min-1
. The compounds viz., Imp-B and Deg-1, Deg-2 and
Imp-H are co-eluted and Imp-C and Imp-D are found to be merging together and also Imp-F
is co-eluted with Imp-G. For above chromatographic conditions, solvent B ratio is modified
as 32:68 v/v instead of ratio 30:70v/v. Two compounds viz., Imp-C and Imp-D are well
separated but still Imp-B and Deg-1, Deg-2 and Imp-H are merging together and also Imp-F
and Imp-G are co-eluting. The pH of the buffer is then changed to pH 3.1 from 3.5 and flow
rate is modified to flow gradient keeping in view the key separations needed. The flow rate of
the mobile phase is set as T/Flow: 0/1.0, 5/1.0, 30/1.0, 31/0.8, 40/1.0 45/1.0, 55/1.0, 75/1.0,
76/1.0 and 82/1.0. With modification of buffer pH from 3.5 to 3.1 and flow rate as flow
gradient, the compounds Imp-B and Deg-1, Deg-2 and Imp-H are found to be well separated
with resolution more than 5 and also Imp-F and Imp-G are well separated with resolution
more than 4. Different column oven temperatures are employed, but at 55 ºC column oven
temperature, all impurities and RPL peaks are separating satisfactorily as symmetrical peaks.
155
After several sets of experiments as described above, it is observed that using Zodiac
C18 AQ, 250 x 4.6 mm, 5μm column with solvent A (0.015 M sodium per chlorate buffer,
pH adjusted to 3.1 with trifluoro acetic acid) and solvent B (pH 3.1 perchlorate buffer and
acetonitrile in the ratio 32:68, v/v) using a mobile phase gradient program of T/%B : 0/35,
5/35, 30/70, 31/65, 40/65, 45/80, 55/85, 75/80, 76/35 and 82/35 and flow rate gradient of the
mobile phase T/Flow : 0/1.0, 5/1.0, 30/1.0, 31/0.8, 40/1.0 45/1.0, 55/1.0, 75/1.0, 76/1.0 and
82/1.0, gives best separation for all pair compounds and eluted RPL as a symmetrical peak .
Interference from the excipients is verified with the above final conditions and found to be
satisfactory.
2.3. Method validation
2.3.1. Precision
The Precision of impurities method (Intra-day precision) is evaluated for RPL and its
10 known impurities by injecting the solutions prepared as described under section 1.8.2.
%RSD for RPL and its 10 known impurities is found to be less than 6.3. For Intermediate
precision (Inter-day precision) for RPL and its 10 known impurities is found to be less than
2.0. The data is summarized in table 3.3.2 and 3.3.3. %RSD values of less than 15% confirms
good precision of the method.
156
Table 3.3.2: Results of precision of test method for RPL and its impurities.
Table 3.3.3: Results of intermediate precision for RPL and its impurities.
Sample
No.
RPL Imp-A Imp-B Degradant-
1
Degrdant-
2 Imp-H Imp-C Imp-D Imp-E Imp-F Imp-G
1 0.310 0.329 0.307 0.532 0.509 0.510 0.312 0.352 0.327 0.447 0.458
2 0.308 0.319 0.317 0.529 0.502 0.501 0.310 0.356 0.338 0.437 0.459
3 0.314 0.325 0.313 0.531 0.499 0.485 0.310 0.374 0.334 0.413 0.459
4 0.319 0.329 0.306 0.532 0.496 0.470 0.313 0.387 0.330 0.409 0.477
5 0.311 0.324 0.313 0.533 0.489 0.459 0.313 0.399 0.341 0.397 0.482
6 0.309 0.328 0.309 0.532 0.485 0.444 0.312 0.412 0.335 0.384 0.488
Avg 0.312 0.326 0.311 0.532 0.497 0.478 0.312 0.380 0.334 0.415 0.471
%RSD 1.3 1.2 1.4 0.3 1.8 5.3 0.4 6.3 1.5 5.7 2.9
Sample
No.
RPL Imp-A Imp-B Degradant-
1
Degrdant-
2 Imp-H Imp-C Imp-D Imp-E Imp-F Imp-G
1 0.321 0.334 0.310 0.520 0.495 0.543 0.351 0.346 0.331 0.469 0.526
2 0.325 0.329 0.308 0.510 0.493 0.546 0.344 0.339 0.319 0.460 0.524
3 0.338 0.333 0.306 0.514 0.494 0.542 0.354 0.346 0.331 0.469 0.532
4 0.326 0.333 0.308 0.527 0.490 0.540 0.344 0.329 0.323 0.462 0.524
5 0.327 0.333 0.312 0.525 0.487 0.538 0.345 0.338 0.330 0.463 0.530
6 0.322 0.332 0.307 0.520 0.490 0.536 0.348 0.335 0.324 0.460 0.527
Avg 0.327 0.332 0.309 0.519 0.492 0.541 0.348 0.339 0.326 0.464 0.527
%RSD 1.9 0.5 0.7 1.2 0.6 0.7 1.2 1.9 1.5 0.9 0.6
157
2.3.2. LOD and LOQ
The limit of detection (LOD), limit of quantification (LOQ) and precision at LOQ
values for RPL and its ten impurities are summarized in Table 3.3.4.
Table 3.3.4: LOD, LOQ data for RPL and its impurities.
Name of the
Impurity
LOD LOQ
Concentration in
‘%’
S/N
ratio
Concentration
in ‘%’
S/N
ratio % RSD LOQ*
Imp-A 0.0022 3.10 0.005 10.24 1.2
Imp-B 0.0021 2.96 0.005 10.28 0.5
Imp-C 0.0081 3.04 0.026 10.43 0.6
Imp-D 0.0063 3.20 0.018 9.96 0.4
Imp-E 0.0031 2.98 0.010 9.98 0.3
Degradant-1 0.0054 3.04 0.015 10.04 1.1
Degradant-2 0.0062 3.13 0.021 10.12 0.9
Imp-H 0.0044 3.11 0.008 10.18 1.7
Imp-F 0.0032 2.99 0.014 10.22 0.7
Imp-G 0.0040 2.97 0.015 10.16 2.1
Repaglinide 0.0052 3.01 0.010 9.95 1.8
* RSD for 6 deteterminations.
2.3.3. Linearity
A linear calibration plot for RPL is obtained over the calibration range LOQ to 4.5 µg
ml-1
and the correlation co-efficient is found to be greater than 0.997. The result shown in
fig.3.3.4 to 3.3.6 indicate an excellent correlation between the peak area and concentration of
the analyte for RPL and all the impurities.
2.3.4. Accuracy
The percentage recovery of RPL and its 10 impurities from placebo matrix is found to
be in the range of 91.2 to 110.8%. The LC chromatogram of spiked sample at 0.30% level of
all ten impurities in RPL tablets sample is shown in Fig. 3.3.2. The % recovery values for
RPL and its 10 impurities are presented in Table 3.3.5. The data shows that the method is
having capability to estimate impurities of RPL accurately in RPL tablets.
158
Figure 3.3.4. : linearity graphs of RPL impurities
0
50000
100000
150000
200000
250000
300000
0 1 2 3 4 5 6
Pe
ak A
rea
Concentration µg/mL
Linearity graph for Imp-A
y=113035 x +216.542, r=0.999
0
50000
100000
150000
200000
250000
300000
0 1 2 3 4 5 6
Pe
ak A
rea
Concentration µg/mL
Linearity graph for Imp-B
y=99386.44 x -488.983, r=0.999
0
50000
100000
150000
200000
250000
300000
0 1 2 3 4 5 6
Pe
ak A
rea
Concentration µg/mL
Linearity graph for Imp-C
y=30661.71 x -596.024, r=0.999
0
50000
100000
150000
200000
250000
300000
0 1 2 3 4 5 6
Pe
ak A
rea
Concentration µg/mL
Linearity graph for Imp-D
y=58169.34 x -1089.572, r=0.999
159
Figure 3.3.5. : linearity graphs of RPL impurities
0
50000
100000
150000
200000
250000
300000
0 1 2 3 4 5 6
Pe
ak A
rea
Concentration µg/mL
Linearity graph for Imp-E
y=82286.86 x- 2478.32, r=0.999
0
50000
100000
150000
200000
250000
300000
0 1 2 3 4 5 6
Pe
ak A
rea
Concentration µg/mL
Linearity graph for Imp-F
y=72155.61 x- 2477.42, r=0.999
0
50000
100000
150000
200000
250000
300000
0 1 2 3 4 5 6
Pe
ak A
rea
Concentration µg/mL
Linearity graph for Imp-G
y=99532.11 x+ 87.393, r=0.999
0
50000
100000
150000
200000
250000
300000
0 1 2 3 4 5 6
Pe
ak A
rea
Concentration µg/mL
Linearity graph for Imp-H
y=62024.13 x- 419.789, r=0.999
160
Figure 3.3.6. : linearity graphs of RPL and its impurities
0
50000
100000
150000
200000
250000
300000
0 1 2 3 4 5 6
Pe
ak A
rea
Concentration µg/mL
Linearity graph for Degradant-1
y=59710.29 x- 3103.398, r=0.999
0
50000
100000
150000
200000
250000
300000
0 1 2 3 4 5 6
Pe
ak A
rea
Concentration µg/mL
Linearity graph for Degradant-2
y=47490.38 x- 2454.942, r=0.999
0
50000
100000
150000
200000
250000
300000
0 1 2 3 4 5 6
Pe
ak A
rea
Concentration µg/mL
Linearity graph for repaglinide
y=72149.03 x- 125.923, r=0.999
161
2.3.5. Robustness
To determine the robustness of the developed method, experimental conditions are
deliberately altered and the elution pattern, separation between RPL and its impurities and
tailing factor for RPL and its impurities are recorded. The effect of flow rate is studied at 1.0
± 0.2 ml min-1
. The effect of the column temperature is studied at of 55⁰C±5⁰C. The effect of
pH is studied at 3.1± 0.1. The effect of the percent organic strength is studied by varying
acetonitrile by −10 to +10% while other mobile phase components are held constant.
In all the deliberately varied chromatographic conditions (flow rate, column
temperature, pH and composition of organic solvent), all analytes are adequately resolved and
elution orders remained unchanged. RRT of all the known impurities for all deliberately
varied conditions along with original conditions are summarized in table 3.3.6. The resolution
between all critical pair components is found to be greater than 1.5 and tailing factor for RPL
and its impurities is found to be less than 1.5.
2.3.6. Solution stability and mobile phase stability
The stability of RPL in diluted standard solution is estimated against freshly prepared
standard each time. The standard solution is found to be stable up to 2 days as the difference
in assay from initial is found to be less than 1.0%. RPL test preparation prepared as per test
method by spiking with 10 known impurities is found to be stable up to 3 hours. The results
are summarized in table 3.3.7.The stability of mobile phase-A & B on bench top is conducted
for a period of about 2 days. RPL test solution prepared as per test method by spiking with
known Impurities is injected into the chromatographic conditions developed by preparing the
solution freshly each time and by using the stored mobile phase up to 2 days. The results are
summarized in table 3.3.8. The elution pattern, RRT’s of all 10 known impurities are found to
be comparable between zero day and other days during the study. From the above study it was
established that the mobile phase is stable for a period of 2 days on bench top.
162
Table 3.3.5: Recovery results of RPL impurities in pharmaceutical dosage forms
‡ 0.3% of the 0.5 mg ml-1 is 100% spike level for impurities, † 0.3% Spiked on placebo preparation is 100% for RPL,
b Mean ± % RSD for three determinations
Spike
Level ‡
%Recoveryb
RPL † Imp-A Imp-B Degradant-1 Degradant-2 Imp-H Imp-C Imp-D Imp-E Imp-F Imp-G
LOQ 98.6± 2.82 96.9±1.89 95.0±1.22 96.4±1.15 92.0±0.25 97.0±1.31 100.4±0.33 96.0±2.64 92.0±0.25 100.3±0.35
99.3±0.58
50% 100.4± 3.21
107.2±1.37
101.2±1.72 96.9±1.29 95.0±2.20 94.9±0.43 110.8±4.26 95.5±1.87 94.7±2.33 92.2±0.71 92.0±0.75
75% 101.7± 2.55
107.4±1.46
91.7±1.63 101.4±0.40 93.0±0.65 94.1±0.32 102.9±1.15 97.9±1.18 101.3±3.40 94.8±2.43 95.0±1.44
100% 100.9± 1.16 105.5±1.28 92.4±4.33 99.3±0.25 95.0±1.10 91.2±0.39 92.6±2.74 104.0±0.97 97.3±0.88 93.0±1.25
95.9±0.93
125% 102.5± 1.77 100.3±0.98 110.8±0.26 98.0± 0.75 94.0± 0.72
99.3±0.18
95.0±1.28
106.2±1.37
102.2±1.11 98.1±1.63
99.8±0.76
150% 103.3± 3.91 102.7±0.70 102.9±1.15 95.6±1.17 101.2±0.66 95.0±0.53 101.2±1.72
103.8±1.44
92.6±3.72 100.7±0.91 101.5±1.04
163
Table 3.3.6: Results of Robustness study.
Impurity
Name
RRT’s of the impurities
As per the
method
conditions
1.2ml/min 0.8 ml/min 50°C 60°C pH 3.0 pH 3.2 90% 110%
IMP-A 0.19 0.18 0.20 0.20 0.18 0.21 0.18 0.20 0.18
IMP-B 0.54 0.54 0.53 0.55 0.53 0.59 0.51 0.55 0.53
IMP-D 0.83 0.85 0.80 0.86 0.80 0.90 0.79 0.83 0.80
IMP-D 1.40 1.33 1.47 1.41 1.31 1.47 1.37 1.44 1.38
Imp-C 0.79 0.77 0.81 0.80 0.78 0.84 0.76 0.82 0.77
Degrdant-1 0.58 0.60 0.56 0.59 0.57 0.63 0.55 0.59 0.57
Degradant-2 0.66 0.63 0.67 0.66 0.65 0.71 0.62 0.68 0.64
Imp-H 0.72 0.74 0.70 0.72 0.71 0.76 0.69 0.70 0.73
Imp-F 1.60 1.54 1.66 1.63 1.58 1.75 1.68 1.61 1.62
Imp-G 1.68 1.63 1.73 1.72 1.65 1.84 1.61 1.71 1.70
164
Table 3.3.7: Results of solution stability on bench top.
Table 3.3.8: Results of mobile phase stability
Acceptance limit for individual impurities is ±0.04% and for total impurities is ±0.2%.
Duration % impurities
Imp-A Imp-B Degradant-1 Degradant-2 Imp-H Imp-C Imp-D Imp-E Imp-F Imp-G Total
Initial 0.3283 0.3022 0.4970 0.4949 0.5662 0.3404 0.3616 0.2943 0.4485 0.5776 4.34
After 3 h 0.3289 0.3032 0.4976 0.4836 0.5323 0.3398 0.3865 0.2892 0.4193 0.5955 4.36
After 6 h 0.3333 0.3028 0.4968 0.4731 0.5061 0.3404 0.4090 0.2892 0.4011 0.6135 4.45
Max. Diff.
from Initial 0.01 0.00 0.00 0.02 0.06 0.00 0.05 0.01 0.05 0.04 0.11
Duration % impurities
Imp-A Imp-B Degradant-1 Degradant-2 Imp-H Imp-C Imp-D Imp-E Imp-F Imp-G Total
Initial 0.3062 0.3008 0.4995 0.4680 0.5191 0.3108 0.3073 0.3004 0.4478 0.5184 4.17
After 24 h 0.3172 0.3052 0.5213 0.5035 0.5282 0.3159 0.3015 0.3075 0.4468 0.5448 4.24
After 48 h 0.3083 0.2952 0.4909 0.4519 0.5107 0.3027 0.2878 0.2805 0.4411 0.5305 4.12
Max .Diff.
from Initial 0.00 0.01 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.02 0.07
165
2.3.7. Results of specificity studies
All the placebo and stressed samples prepared are injected into the HPLC system
with photodiode array detector as per the described chromatographic conditions.
Chromatograms of placebo solutions have shown no peaks at the retention time of RPL and
its impurities. This indicates that the excipients used in the formulation do not interfere in
estimation of impurities in RPL tablets
Degradation results and mass balance data is tabulated in table 3.3.9. All degradant
peaks are well resolved from RPL peak and from each other in the chromatograms of all
stressed samples. The chromatograms of the stressed samples are evaluated for peak purity of
RPL. For all forced degradation samples, the purity angle is found to be less than Purity
threshold for RPL peak. The sum of total % of impurities and assay is presented as Mass
balance, which is found to be in the range of 97.2 to 99.4% for the stressed samples. The data
indicates that there is no co elution of any degradants in the RPL peak and no impurity which
is missing, as the mass balance is close to 100%. Thus, this method is considered as specific
and "Stability indicating”. The chromatogram and purity plots of all stressed samples are
shown in figs 3.3.7 to 3.3.14.
166
Table 3.3.9: Summary of forced degradation studies
Stress
condition
% of impurities formed %
Assay
Mass
Balance Imp-A Imp-B Imp-C Imp-D Imp-E Imp-F Imp-G Imp-H Deg-1 Deg-2 SIM TI
unstressed 0.18 ND ND 0.05 ND ND ND 0.01 0.03 0.02 0.02 0.47 98.6 99.1
Acid 0.30 ND ND ND 0.09 ND ND 0.02 0.10 0.11 1.78 2.36 94.8 97.2
Base 0.08 ND 0.02 ND ND 0.11 0.30 0.13 0.07 0.03 0.03 0.95 97.6 98.6
Peroxide 0.18 ND 0.06 ND 0.05 0.68 2.68 0.10 0.18 0.12 0.35 5.29 93.1 98.4
Thermal 0.07 ND 0.01 ND 0.21 0.83 0.98 0.44 0.17 0.15 0.17 3.19 94.5 97.7
Water 0.02 ND 0.03 ND ND 0.07 0.07 0.22 0.09 0.07 0.03 0.68 98.4 99.1
UV 0.02 ND 0.03 ND ND 0.09 0.09 0.21 0.08 0.07 0.03 0.72 99.2 99.9
Sunlight 0.03 ND 0.01 ND 0.02 0.04 0.33 0.04 0.10 0.06 0.05 0.74 97.8 98.5
Humidity 0.03 ND 0.02 ND ND 0.08 0.07 0.18 0.08 0.06 0.02 0.54 98.9 99.4
ND: Not Detected; Deg-1: Degrdant-1; Deg-2: Degrdant-2; TI: Total impurities
167
Fig 3.3.7: Chromatogram and purity plot of acid stressed RPL tablets.
168
Fig 3.3.8: Chromatogram and purity plot of base stressed RPL tablets.
169
Fig 3.3.9: Chromatogram and purity plot of H2O2 stressed RPL tablets..
170
Fig 3.3.10: Chromatogram and purity plot of water stressed RPL tablets.
171
Fig 3.3.11: Chromatogram and purity plot of visible light stressed RPL tablets.
172
Fig 3.3.12: Chromatogram and purity plot of UV light stressed RPL tablets.
173
Fig 3.3.13: Chromatogram and purity plot of heat stressed RPL tablets.
174
Fig 3.3.14: Chromatogram and purity plot of humidity stressed RPL tablets.
175
3. Conclusion:
A sensitive, specific, accurate, validated and well-defined stability indicating HPLC
method for the determination of degradation products and its process- related impurities of
RPL is described. The behavior of RPL under various stress conditions is studied. All of the
degradation products and process impurities are well separated from the RPL and from each
other. This demonstrates the stability indicating power of the method. The information
presented in this study could be very useful for quality monitoring of RPL pharmaceutical
dosage forms and can be used to check drug product quality during stability studies.
176
Section (iv): Isolation and Characterization of Repaglinide degradation impurities
This section reports the various aspects relating to the isolation and characterization of
two degradation impurities found in Repaglinide (RPL) tablets.
1. Experimental
1.1. Samples and chemicals
Samples of RPL API are received from Process R&D, Dr Reddy’s Laboratories,
Hyderabad, India. RPL tablets of 2 mg are received from formulation R&D, Dr Reddy’s
Laboratories, Hyderabad, India. HPLC grade acetonitrile, KH2PO4 are supplied by Merck,
Darmstadt, Germany. High purity water is prepared by using millipore milli Q plus
purification system.
1.2 Analytical liquid chromatography conditions
The Waters HPLC system used consists of a quaternary solvent pumping system, a
sample manager and a PDA detector using empower software. The pH of the solutions is
measured by a pH meter (Thermo Orion Model 420 A, USA). All solutions are degassed by
ultra sonication (Power sonic 420, Labtech, Korea) and filtered through a 0.45μm Nylon 66
filter (PALL life sciences, USA). The method employs ZodiacAQ C18, 250 x 4.6 mm, 5μm
column with mobile phase containing a gradient mixture of solvent A and B. 0.015 M sodium
per chlorate buffer, pH adjusted to 3.1 with trifluoro acetic acid is used as solvent A. pH
3.1perchlorate buffer and acetonitrile in the ratio 32:68 v/v is used as solvent B. The gradient
program is (T/%B) : 0/35, 5/35, 30/70, 31/65, 40/65, 45/80, 55/85, 75/80, 76/35 and 82/35.
The mobile phase is filtered through a nylon 0.45 µm membrane filter. The flow rate of the
mobile phase is (T/Flow) : 0/1.0, 5/1.0, 30/1.0, 31/0.8, 40/1.0 45/1.0, 55/1.0, 75/1.0, 76/1.0
and 82/1.0. The column temperature is maintained at 55 °C and the wavelength is monitored
at 240 nm. The injection volume is 50µl.
1.3. Preparative HPLC instrumentation and chromatographic conditions
A Shimadzu LC-8A (from Shimadzu Corporation, Kyoto, Japan) preparative HPLC
equipped with PDA detector is used for the isolation unknown impurities. ZodiacSIL AQ-C18
250mm ×20 mm preparative column packed with 5µ particle size is employed for isolation of
impurities. The mobile phase consisted of (A) 0.05M potassium dihydrogen phosphate buffer
pH adjusted to 3.2 with ortho phosphoric acid (B) mobile phase A and acetonitrile in the ratio
177
of 30:70(v/v). Flow rate is kept at 10 ml/min and UV detection is carried out at 240 nm. The
gradient program for degradation product I is: time (min)/A (v/v): B (v/v); 0.01/65:35,
25.0/30:70, 30.0/30:70, 32.0/0:100, 45.0/0:100, 46.0/65:35, 55.0/65:35 and for degradation
product II is: time (min)/A (v/v):B (v/v); 0.01/65:35, 20.0/35:65, 25.0/30:70,30.0/30:70,
32.0/0:100, 48.0/0:100, 49.0/65:35, 55.0/65:35.
1.4. Mass spectrometry
An LC-MS analysis is performed on AB-4000 Q-trap LC-MS/MS mass spectrometer.
The analysis is performed in positive ionization mode with turbo ion spray interface with the
following conditions. Ion source voltage 5500V, declustering potential 80V, entrance
potential 10V, with the nebulizer gas as nitrogen at 30 psi.
1.5. NMR spectroscopy
The 1H NMR,
13C NMR (proton decoupled) and DEPT spectra are recorded on
400MHz and 100MHz on Varian Mercury plus 400MHz FT NMR spectrometer using
DMSO-d6 as solvent for degradation impurity-I and II and CDCl3 for RPL. The 1H chemical
shift values are reported on the δ scale in ppm, relative to TMS (δ = 0.00 ppm) and in the 13
C
NMR the chemical shift values are reported relative to DMSO-d6 (δ = 39.50 ppm) as internal
standards.
1.6 Enrichment of degradation impurity- I
1g of RPL drug substance is dissolved in a mixture of 400ml of acetonitrile and water
in the ratio 90:10(v/v), 50mg of methylene blue is added and stirred with the help of magnetic
bead under 250w tungsten bulb at room temperature for about 24 hrs. After 24 hrs a pinch of
acidic carbon black is added to the solution and filtered through hip flow bed to eliminate
methylene blue. The sample is analyzed by the HPLC method mentioned in section 1.2.The
degradation product is enriched to about 40% by area normalization. The resulting clear
solution is subjected for rota-evoporation to knock out the acetonitrile.
1.7 Enrichment of degradation impurity- II
1g of RPL drug substance is dissolved in a mixture of 400mL of acetonitrile and water
in the ratio 50:50(v/v), 50mg of potassium permanganate is added and stirred with the help of
magnetic bead at room temperature for about 2hrs. The sample is analyzed by the HPLC
178
method mentioned in section 1.2.The degradation product is enriched to about 5% by area
normalization. The resulting clear solution is subjected for rotavoporation to knock out the
acetonitrile.
1.8 Isolation of degradation impurity by preparative HPLC
The concentrated solution of degradation impurity I and II are loaded into the
preparative column using the conditions mentioned in section 1.3. Fractions collected are
analyzed by analytical HPLC as per the conditions mentioned in section 1.2. Fractions of
>95% are pooled together, concentrated on rotavapour to remove solvent mixture.
Concentrated fractions are passed through the preparative column by using water: acetonitrile
in the ratio 50:50 (v/v) as mobile phase to remove the buffer used for isolation. Again the
eluate is concentrated using rotavapour to remove acetonitrile. The aqueous solution is then
lyophilized using freeze dryer (Virtis advantage 2XL). The impurity is obtained as pale
yellow powder found to have chromatographic purity of about 96.0%, determined by the
HPLC method mentioned in section 1.2.
2. Results and discussion
2.1 Structural elucidation of degradation impurity-I :
The electro spray ionization mass spectrum of degradation impurity I exhibited a
molecular ion peak at m/z 499.5 [(MH)+] in positive ion mode(Figure3.4.1), indicating the
molecular weight of the compound as 498.5, which is 46 amu more than that of RPL. In 1H
NMR spectrum of RPL the signal assigned to CH2 at 25&29 positions has disappeared and
the CH2 signals at 26&28 signals are shifted to deshilded region. The broad 1H NMR signal at
12 ppm indicates the –OH of carboxylic acid. In 13
C NMR spectrum signal at 53.69 ppm
assigned to 25&29 CH2 has disappeared and new signals appeared at 163.2 and 169.1 ppm in
13C NMR. This observation indicates that the CH2 group at 25 and 29 positions is converted
to carbonyl group. Based on LC-MS, 1H NMR,
13C NMR, DEPT, DQCOSY, HSQC and
HMBC data ( fig 3.4.4 to 3.4.12), the degradation impurity I is characterized as 4-(2-((1-(2-(4-
carboxybutanamido) phenyl)-3-methylbutyl) amino)-2-oxoethyl)-2-ethoxybenzoic acid, with
molecular formula C27H34N2O7 and molecular weight 498.5. The 1H and
13C NMR chemical
shift values of RPL and its degradation impurity-I are given in table 3.4.1 and 3.4.2.
179
Fig 3.4.1: ESI Mass spectrum of RPL degradation impurity-I.
180
Fig 3.4.2: Structure of Repaglinide.
Fig 3.4.3: Structure of RPL degradation impurity-I
181
Table3.4.1: Chemical shift values of RPL.
Position1
1H J(Hz)
2 13C DEPT gHSQC
1 - - - 157.04 - -
2
- - - 120.49 - -
3
1H 7.56 d(7.5) 130.36 CH (3H,7.56)
4 1H 6.85 d(7.5) 121.53 CH (4H,6.85)
5 - 142.12 -
6 1H 6.99 s 113.81 CH (6H,6.99)
7 2H 4.02 63.64 CH2 (7H,4.02)
8 3H 1.33 m 13.97 CH3 (8H,1.33)
9
- 167.03 -
10 OH 12.38 s - -
11 2H 3.55 42.45 CH2 (11H,3.55)
12 - - - 168.64 - -
13 NH 8.45 - - - -
14 1H 5.41 m 45.54 CH (14H,5.41)
15 Ha 1.50 m
46.29 CH2 ??
Hb 1.34
16 1H 1.58 m 24.83 CH ??
17# 3H 0.90 d(4.5) 21.92 CH3 (17H,0.91)
18# 3H 0.91 d(4.5) 21.73 CH3 (18H,0.91)
19 - - - 140.38 - -
20 - - - 151.43 - -
21 1H 7.10 d(7.5) 120.15 CH (21H,7.08)
22 1H 7.16 t(7.5) 127.16 CH (22H,7.16)
23 1H 7.05 t(7.5) 123.63 CH (23H,7.05)
24 1H 7.30 d(7.5) 125.73 CH (24H,7.30)
25,29 2Ha 3.07 br s
53.69 CH2 (25&29H,3.07)
2Hb 2.52 br s (25&29H,2.52)
26 & 28 Ha 1.53 m 26.29 CH2 (26H,1.53)
Hb 1.70 m
27 Ha 1.67 m
23.80 CH2 Hb 1.56 m
182
Table3.4.2: Chemical shift values of RPL degradation impurity-I
Position1
1H δ(ppm) J(Hz)
2 13C DEPT gHSQC
1 - 157.86
2
- 119.61
3
1H 7.52 m 130.68 CH (3H,7.52)
4 1H 6.82 d(8.0) 120.69 CH (4H,6.82)
5 - - - 142.26 -
6 1H 6.99 d 113.90 CH (6H,6.99)
7 2H 4.02 dd(8.0,16.5) 63.94 CH2 (7H,4.0)
8 3H 1.31 T(8.5) 14.55
9
- 173.8
10 OH 12.0
11 2H 3.45 42.34 CH2
12 - 167.1
13 NH 8.05
14 1H 4.85 45.94 CH
15 2H 2.21 46.0 CH2
16 1H 1.66 24.7 CH
17 3H 0.82 20.93 CH3
18 3H 0.86 23.01 CH3
19 - 130.6
20 - 141.7
21 1H 7.47 127.0 CH
22 1H 7.40 129.0 CH
23 1H 7.34 127.71 CH
24 1H 7.18 129.50 CH
25 NH 8.05
26 - 163.2
27 2H 2.21 CH2
28 2H 1.66 22.65 CH2
29 2H 2.22 30.89 CH2
30 - 169.1
31 OH 12.0
183
Fig 3.4.4: 1H NMR spectra of RPL in CDCl3 .
184
Fig 3.4.5: 13
C NMR spectra of RPL in CDCl3 .
185
Fig 3.4.6: DEPT spectra of RPL in CDCl3
186
Fig 3.4.7: 1H NMR spectra of RPL degradation impurity-I in DMSO
187
Fig 3.4.8: 13
C NMR spectra of RPL degradation impurity-I in DMSO
188
Fig 3.4.9: DEPT spectra of RPL degradation impurity-I in DMSO
189
Fig 3.4.10: gDQCOSY spectra of RPL degradation impurity-I in DMSO
190
Fig 3.4.11: gHSQC spectra of RPL degradation impurity-I in DMSO
191
Fig 3.4.12: gHMBC spectra of RPL degradation impurity-I in DMSO
192
2.2 Structural elucidation of degradation impurity-II
The degradation impurity II exhibited a molecular ion peak at m/z 483.5 [(MH)+] in
LC-MS analysis, indicating the molecular weight of the impurity as 482.5, which is 30 amu
more than that of RPL. In 1H NMR spectrum of RPL the signal assigned to CH2 at 25&29
positions has disappeared and the CH2 signals at 26&28 signals are shifted to deshilded
region. The 1H NMR signal appeared at 9.6 ppm indicates the aldehyde proton. In
13C NMR
spectrum signal at 53.69 ppm assigned to 25&29 CH2 has disappeared and new signals
appeared at 163.2 and 202.5 ppm. This observation indicates that the CH2 group at 25 and 29
positions is converted to carbonyl group. Based on the LC-MS, 1H NMR, 13C NMR, DEPT,
DQCOSY, HSQC and HMBC data, the degradation product II is characterized as 2-ethoxy-4-
(2-((3-methyl-1-(2-(5-oxopentanamido) phenyl) butyl)amino)-2-oxoethyl)benzoic acid, with
molecular formula C27H34N2O7 and molecular weight 482.5. The 1H and
13C NMR chemical
shift values of RPL and its degradation impurity-II are given in table 3.4.3.
Fig 3.4.13: Chemical structure of RPL degradation impurity-II
193
Table3.4.3: Chemical shift values of RPL degradation impurity-II
Position1 1
H δ(ppm) J(Hz)2 13
C DEPT
1 - 158.25
2
- 119.6
3
1H 7.54 130.24 CH
4 4H 6.82 120.70 CH
5 - 142.85
6 1H 6.94 113.91 CH
7 2H 4.00 63.95 CH2
8 3H 1.31 14.55 CH3
9
- 169.1
10 OH -
11 2H 3.46 42.36 CH2
12 - 167.2
13 NH 8.69
14 1H 4.85 46.05 CH
15 Ha 1.54 44.98 CH2
Hb 1.31
16 1H 1.65 24.70
17 3H 0.79 21.20 CH3
18 3H 0.85 22.80
19 - 137.80
20 - 143.62
21 1H 7.50 126.83 CH
22 1H 7.30 127.72 CH
23 1H 7.46 128.38 CH
24 1H 7.19 129.24 CH
25 NH 8.05
26 - 162.7
27 2H 42.3
28 2H 1.68 20.9
29 2H 2.48 40.02
30 9.62 S 202.54 CH
194
Fig 3.4.14: ESI Mass spectrum of RPL degradation impurity-II.
195
Fig 3.4.15: 1H NMR spectra of RPL degradation impurity-II in DMSO
196
Fig 3.4.16: 13C NMR spectra of RPL degradation impurity-II in DMSO
197
Fig 3.4.17: DEPT spectra of RPL degradation impurity-II in DMSO
198
Fig 3.4.18: gDQCOSY spectra of RPL degradation impurity-II in DMSO
199
Fig 3.4.19: gHSQC spectra of RPL degradation impurity-II in DMSO
200
Fig 3.4.20: gHMBC spectra of RPL degradation impurity-II in DMSO
201
3.0. Conclusion:
Two new degradation products, which are increasing in stability, are isolated using
preparative HPLC. Based on the spectral data (LC-MS, 1H NMR, 13
C NMR, DEPT,
DQCOSY, HSQC and HMBC) the structures of these impurities are characterized as 4-(2-
((1-(2-(4- carboxybutanamido) phenyl)-3-methylbutyl) amino)-2-oxoethyl)-2-ethoxybenzoic
acid (degradation impurity-I) and 2-ethoxy-4-(2-((3-methyl-1-(2-(5-oxopentanamido)
phenyl) butyl)amino)-2-oxoethyl)benzoic acid (degradation impurity-II).
202
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