Section (i): Brief account of...

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

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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.

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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.

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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.

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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.

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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


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

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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.

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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-



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-


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-


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-


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.


1.8.1. 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


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.


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.


2.1. 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.


2.3.1. 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


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


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


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


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


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


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


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


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


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


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


Linearity graph for repaglinide

y=72149.03 x- 125.923, r=0.999

161

2.3.5. Robustness


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.


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.


2.1 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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2. European pharmacopeia, 5th edn., Strasbourg council of Europe, 2007.

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11. C.B.Sekaran., R.A.Prameela.,A. Archana., T.P.Siva., J. Pharm. Res., 2010; 3(2):201-

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12. R.A. Prameela., C.B.Sekaran., Ch.Srilakshmi., Biomed.Pharmacol. J., 2009; 2(1): 91-

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13. V.P. Rane., D.B. Shinde., Chromatographia., 2007; 66:583-587.

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