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CHAPTER- 5

2.5 DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-HPLCMETHOD FOR THE DETERMINATON OF PREGABALIN IN ITS CAPSULES DOSAGE FORMS

CONTENTS

1. Drug profile

2. Review of the past work on the analytical methods for Pregabalin.

3. Experimental and results

a. Material and methods

b. Optimization of chromatographic conditions and method development

c. Validation of the proposed method

4. Summary of the results and Conclusion

5. References

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1. DRUG PROFILE- PREGABALIN

Pregabalin1-6 is an anticonvulsant drug for neuropathic pain. It is also an adjunct therapy

for partial seizures, and it is reported as being used for general anxiety disorder and for

treatment of epilepsy. neuropathic pain. It was designed as a more potent successor to a

related drug, gabapentin. Pregabalin binds to the alpha2-delta subunit of the voltage-

gated calcium channel in the central nervous system. While pregabalin is a structural

derivative of the inhibitory neurotransmitter gamma- aminobutyric acid (GABA), it

does not bind directly to GABAA, GABAB, or benzodiazepine receptors, does not

augment GABAA responses in cultured neurons, does not alter rat brain GABA

concentration or have acute effects on GABA uptake or degradation. However, in

cultured neurons prolonged application of pregabalin increases the density of GABA

transporter protein and increases the rate of functional GABA transport. Pregabalin does

not block sodium channels, is not active at opiate receptors, and does not alter

cyclooxygenase enzyme activity. It is inactive at serotonin and dopamine receptors and

does not inhibit dopamine, serotonin, or noradrenaline reuptake

Mechanism of Action

Pregabalin binds with high affinity to the alpha2-delta site (an auxiliary subunit of

voltage-gated calcium channels) in central nervous system tissues. Although the

mechanism of action of pregabalin is unknown, results with genetically modified mice

and with compounds structurally related to pregabalin (such as gabapentin) suggest that

binding to the alpha2-delta subunit may be involved in pregabalinís antinociceptive and

antiseizure effects in animal models. In vitro, pregabalin reduces the calcium-dependent

release of several neurotransmitters, possibly by modulation of calcium channel

function.

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Chemical Structure:

Chemical name (IUPAC ) : (S)-3-(aminomethyl)-5-methylhexanoic

Chemical formula : C8H17 NO2

Molecular weight : 159.23

Physical state : A white crystalline powder

Melting point : 186-1880 C

Solubility : Slightly solublw in Ethanol, DMSO and Soluble

in Phosphate buffer

Official Status of the drug :The drug is official in Merck Index

Table 2.5.1 Important brand names of Pregabalin formulations

S.No

Brand

Name Company Composition Packing

1 Gabanext ABBOTT HC Pregabalin 75mg 10 SG-acp

Pregabalin 150mg 10 SG-acp

2 Galinerve SUN (ARIAN) Pregabalin 75mg 10 cap

Pregabalin 150mg 10 cap

3

Nervup-

PG

ABBOTT HC

Each hard gelatin cap

cont:Pregabalin 75mg,

Methylcobalamin 750 mcg,

Alpha lipoic acid100mg 10 cap

(as 2 film coated tablets each

contain 50 mg of Alpha lipoic

acid. Colour:Quinoline yellow

4 Nuramed ZYDUS (CND) Pregabalin 75mg 10 cap


5 Pregamet ABBOTT HC

Pregabalin 75mg,

Methylcobalamin 750 mcg 10 cap

6 Pregatar LUPIN(PINNACLE)



220

2. Review of the past work on the analytical methods for Pregabalin

T.A.C. Vermeij et al 7 , proposed a HPLC method for simultaneous determination of

the γ-amino-n-butyric acid (GABA) derivatives pregabalin (PGB), gabapentin (GBP)

and vigabatrin (VGB) in human serum. Separation is achieved on a Alltima 3C18

column using isocratic elution; the drugs are monitored using fluorescence detection.

Norvaline is used as an internal standard. Within-day precision (COV; n = 10) is 1.2%

for PGB (serum concentration 10.0 mg/l), 1.1% for GBP (serum concentration 15.8

mg/l) and 0.3% for VGB (serum concentration 15.5 mg/l). The method is linear up to at

least 63 mg/l for PGB, 40 mg/l for GBP and 62 mg/l for VGB. Lower limits of

quantitation (LOQ) are 0.13 mg/l for PGB, 0.53 mg/l for GBP and 0.06 mg/l for VGB.

Berry et al 8, proposed a HPLC method for determination of pregabalin in

serum/plasma. Using C8 column The assay is calibrated over the range 0.5 mg/L to 8

mg/L. concentration measurements in predose samples from a group of patients with

dose escalated to 600 mg/d pregabalin are presented. The drug concentrations measured

were in the range 2.8-8.2 mg/L at steady state.

A. S. Jadhav et al 9, proposed a HPLC method for the determination of pregabalin in

bulk drugs using reversed-phase ODS column with a 60:40 (v/v) mixture of aqueous

0.2% triethylamine (pH adjusted to 3.5 with dilute orthophosphoric acid) and

acetonitrile as mobile phase. Concentration over the range 750 (LOQ) to

7,500 ng L−1 for the Renantiomer. The limits of detection and quantification of

the R enantiomer were 250 and 750 ng L−1, respectively, for an injection volume of

10 µL. Recovery of the R enantiomer from bulk drug samples of pregabalin ranged from

97.5 to 101.76%. Solutions of pregabalin in water and in the mobile phase were found

to be stable for at least 48 h.

Yizhong Zhang et al 10, proposed a method for direct chiral separation of pregabalin

from its R-enantiomer and HPLC/MS/MS assays have been validated to support isolated

perfused rat kidney studies. The separation was developed through serial coupling of

various macrocyclic glycopeptide stationary phases until partial separation of the

enantiomers was achieved. Identification of the resolving stationary phase followed by

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optimization of the mobile phase enabled the baseline resolution of the enantiomers

using mass spectrometry compatible solvents and modifiers.

Önal Armağan et al 11, proposed a spectrophotometric method for determination of

pregabalin (Pgb) in pharmaceutical preparations. The method is based on the reaction of

Pgb as n-electron donors with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and

7,7,8,8-tetracyanoquinodimethane (TCNQ) as π-acceptors to give highly colored

complex species. The colored products were quantitated spectrophotometrically at 494

and 841 nm for DDQ and TCNQ, respectively. Optimization of the different

experimental conditions was conducted. Beer's law was obeyed in the concentration

ranges 2.0–30.0 and 1.5–10 g·mL−1 for DDQ and TCNQ methods, respectively. The

third method is based on the interaction of ninhydrin (NN) with primary amine present

in the pregabaline. This reaction produces a blue coloured product in N,N-

dimethylformamide (DMF) medium, which absorbs maximally at 573 nm. Beer's law

was found in the concentration range 40.0–180.0 µg·mL−1.

Ramakrishna Nirogi et al 12, proposed a HPLC positive ion atmospheric pressure

chemical ionization tandem mass spectrometry method for the quantification of

pregabalin in human plasma. Following liquid–liquid extraction, the analyte was

separated using an isocratic mobile phase on a reverse-phase column and analyzed by

MS/MS in the multiple reaction monitoring mode using the respective

[M+H] + ions, m/z 160–142 for pregabalin and m/z 482–258 for the internal standard.

The assay exhibited a linear dynamic range of 1–10,000 ng/mL for pregabalin in human

plasma. The lower limit of quantification was 1 ng/mL with a relative standard

deviation of less than 11.4%.

M. N. Farooqui et al 13, proposed a HPLC for the determination of pregabalin in

capsule dosage form. Using Hypersil BDS, C8, 150×4.6 mm, 5 µm column,

photodiode array detector. The mobile phase consisting of phosphate buffer pH 6.9 and

acetonitrile in the ratio of 95:05 with flow rate of 1 ml/min. Lower limit of

quantification is 0.6 mg/l. The sample solution was stable at room temperature for about

26 h.

M. I. Walash et al 14 , proposed a spectrofluorimetric method for the determination of

pregabalin (PG) in capsules. The method is based on the reaction between pregabalin

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and fluorescamine in borate buffer solution of pH 10 to give a highly fluorescent

derivative that is measured at 487 nm after excitation at 390 nm. The fluorescence

intensity concentration plot was rectilinear over the range of 0.01–0.3µg/mL−1 with a

lower detection limit of 0.0017µg/mL−1 and limit of quantitation of 0.005µg/mL−1. The

developed method was successfully applied to the analysis of the drug in its commercial

capsules. The mean percentage recovery of PG in its capsule was 99.93±1.24 (n = 3).

Ashu M et al 15, proposed a RP-HPLC method for the determination of pregabalin in

the capsule dosage form. Stationary phase as waters spherisorb 5µ ODS 24.6mm x

250mm column using a mobile phase of acetonitrile:buffer (30:70 V/V) at a flow rate of

1ml/min with detection of analyte at 210 nm. The retention time for pregabalini is

3.1±0.3 min. Peak width 5.26s and SD 1.3152 for the sample peak. Linearity in the

range of 200-800 µg/ml. The intra and inter day R.S.D ranged from 0.79-1.85%. The

recovery (mean ±S.D.) of low, middle and high concentrations were 100.02± 0.80,

100.05 ± 0.42, 100.03 ± 0.35 respectively.

The present investigation by the author describes the development of a rapid, accurate

and precise RP-HPLC method for the determination of Pregabalin in capsule dosage

forms

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3) EXPERIMENTAL AND RESULTS

a) MATERIALS AND METHODS Instrumentation

The author had attempted to develop a liquid chromatographic method for

simultaneous estimation of Pregabalin. The separation of the analyte was done by using

an isocratic Agilent HPLC instrument, on a Grace Kromasil C8 column (150 x 4.6mm;

5µ).The instrument was equipped with a pump (G1311A), injector, DAD (G13158)

Detector and column oven. Data acquisition was done by using Agilent software.

Degassing of the mobile phase was done by using a Spectra lab model DGA

20A3 ultrasonic bath sonicator. A Sartorious electronic balance was used for weighing

the materials. Class ‘A’ Borosil glassware was employed for volumetric and general

purpose in the study.

Drugs

The reference sample of Pregabalin was gifted by M/s LUPIN Ltd. The samples of

branded formulations of pregabalin (Pregastar capsules of Lupin) were procured from

the local market.

Reagents

Potassium dihydrogen phosphate : GR grade

Potassium hydroxide pellets : GR grade

Acetonitrile : HPLC grade

Water : Milli-Q / HPLC grade

Preparation of 5N Potassium hydroxide solution

28g of Potassium hydroxide pellets was dissolved in 100 mL of water.

Preparation of Buffer

1.2g of Potassium dihydrogen phosphate was dissolved in 1000 mL of water. The pH

was adjusted to 6.7 ± 0.05 with 5N Potassium hydroxide solution. This solution was

filtered through a 0.45µm membrane filter.

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Preparation of Mobile phase:

The above buffer solution and acetonitrile were mixed in the ratio of 970: 30 (v/v),

filtered and degassed.

Diluent:

The above buffer used as a diluent also in preparing drug solutions.

Preparation of working standard solution:

About 50mg of Pregabalin standard was accurately weighed and transferred into a 50

mL volumetric flask, about 30 mL of the diluent was added and sonicated to dissolve,

dilute to volume with the diluent. The working standard solution containing 1000

mcg/mL. The solution was filtered through a 0.45µm Nylon membrane filter.

Preparation of formulation sample solution

Determined 20 capsules average fill weight of the Pregabalin Capsules (Pregastar of

Lupin Pharmaceutical Ltd.).A quantity of equivalent to 100 mg of Pregabalin was

transferred into a 100 mL volumetric flask, about 60 mL of the diluent was added and

the contents were sonicated for about 30 min with intermittent shaking. The flask was

cooled to room temperature and the solution was made up to the volume with the

diluent and mixed well. The solution was filtered through a 0.45µ Millipore nylon

membrane filter.

b) OPTIMIZATION OF THE CHROMATOGRAPHIC

CONDITIONS AND METHOD DEVELOPMENT

For developing the HPLC method, a systematic study of the effect of various

factors for ideal separation of the drugs was undertaken. This was done by varying one

parameter at a time and keeping all other conditions constant. The following studies

were conducted for this purpose. A non-polar C8 column was chosen as the stationary

phase for this study.

The mobile phase and the flow rate

In order to get sharp peak and good base line separation of the components, the

author carried out a number of experiments by varying the commonly used solvents,

their compositions and flow rate.

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To find out the most suitable mobile phase to effect ideal separation of the drug

under isocratic conditions, mixtures of commonly used solvents like water, methanol

and acetonitrile with or without different buffers in different combinations were tested

as mobile phases on a C8 stationary phase. 1.2 g of potassium dihydrogen phospahate

was dissolved into 1000 mL of water and the pH was adjusted to 6.7 (±0.05) with 5N

potassium hydroxide. Buffer and acetonitrile in a ratio of 97:3 v/v was proved to be the

most suitable of all the combinations since the chromatographic peaks obtained were

better defined and resolved and almost free from tailing.

A mobile phase flow rate of 1.0 mL/min was found to be suitable in the study

range of 0.5 -2.0 mL/min.

Detection wave length

The UV absorption spectrum of the drug was taken in methanol and the λ max

found to be at 200 nm. Hence detection of the drug was made at 200 nm.

Retention time of Pregabalin

A model chromatogram showing the separation of Pregabalin is presented in Fig

2.5.1. Under the above optimized conditions retention time of Pregabalin was obtained

at about 4.75 min.

After a thorough study of the various parameters the following optimized

conditions mentioned in Table 2.5.2 were followed for the determination of Pregabalin

bulk samples and pharmaceutical formulations.

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Fig 2.5.1 A Model Chromatogram showing the separation of Pregabalin peak

Table 2.5.2 Optimized Chromatographic Conditions

Parameter Value

Column Grace Kromasil C8 (150 x 4.6mm;

5µ)

Mobile Phase Buffer(pH 6.7):acetonitrile

(97:03)

Flow Rate 1.0 mL/min

Run Time 8 min

Column Temperature 30±1 ˚C

Volume Of Injection 20 µL

Detection Wave Length 200 nm

Retention Time 4.75 min

227

c) VALIDATION OF THE PROPOSED METHOD

The method was validated in compliance with ICH guidelines16-19. The

parameters determined for validation were specificity, precision, accuracy, robustness,

Linearity, Forced Degradation, Limit of quantification and Limit of detection, system

suitability and stability of analytical solution.

1. Specificity

The method specificity was assessed by comparing the chromatograms obtained from a

placebo solution containing a mixture of most commonly used excipients without the

drug and another solution containing the excpeints with the drug. These solutions were

prepared in the diluent. The drug to excipient ratio used was similar to that in the

commercial formulation. The commonly used excipients in formulations like lactose,

starch, microcrystalline cellulose, ethyl cellulose, hydroxyl propyl methylcellulose,

magnesium stearate and colloidal silicon dioxide were taken up for the study. The

mixtures were filtered through 0.45µ membrane filter before injection. The placebo

solution and the sample solution (placebo and the drug) were injected into HPLC

system separately in triplicate and the relevant chromatograms observed. There was no

interference from blank and placebo at the retention time of analyte peak. The absence

of additional peaks in the chromatogram indicates non interference of the commonly

used excipients in the tablets and hence the method is specific. The relevant

chromatograms are given in Fig 2.5.2, 2.5.3 and 2.5.4 for chromatograms of blank,

placebo and placebo with drug sample solutions respectively.

Acceptance criterion

No interfering peak should appear retention time of Pregabalin peak from blank and

placebo. Peak purity of Pregabalin peak should pass.

Conclusion

The proposed method is specific for estimation of Pregabalin in its Capsules

formulations, as the method meets acceptance criterion.

228

2. Forced degradation study

Forced degradation study was carried out by treating placebo and sample to the

following conditions.

1. Treatment with hydrochloric acid.

2. Treatment with sodium hydroxide.

3. Treatment with hydrogen peroxide.

4. Thermal exposure.

5. Photolytic exposure.

6. Exposure to humidity

Acid degradation

The placebo and sample powders equivalent to 100 mg of Pregabalin were accurately

weighed and transferred into two separate 100 mL volumetric flasks. 60 mL of the

diluent was added to each flask and sonicated for 30 min with intermittent shaking. To

each flask 10.0 mL of 5N hydrochloric acid was added and the solutions were kept in

water bath at 80°C. After 30 min flasks were removed from water bath and cooled to

room temperature. The resulting solutions were neutralized with 10.0 mL of 5N sodium

hydroxide. The solutions were diluted up to the mark with the diluent and mixed well.

The solutions were filtered through a 0.45µ membrane filter.

Alkali degradation




each flask 10.0 mL of 5N sodium hydroxide was added and the solutions were kept in

water bath at 80°C. After 30 min flasks were removed from water bath and cooled to

room temperature. The resulting solutions were neutralized with 10.0 mL of 5N

hydrochloric acid was added to each flask. The solutions were diluted up to the mark

with the diluent and mixed well. The solutions were filtered through 0.45µ membrane

filter.

Peroxide degradation



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each flask 10.0 mL of 30% solution of hydrogen peroxide was added and the solutions

were kept in water bath at 80°C. After 1hr flasks were removed from water bath and

cooled to room temperature. The solutions were diluted up to the mark with the diluent

and mixed well. The solutions were filtered through 0.45µ membrane filter.

Thermal degradation


weighed and transferred into two separate 100 mL volumetric flasks and exposed to

heat at 80°C for about 24 hrs. The exposed samples were diluted with 60 mL of the

diluent and sonicated for 30 min with intermittent shaking. The solutions were diluted

up to the mark with the diluent and mixed well. The solutions were filtered through

0.45µ membrane filter.

Photolytic degradation


weighed and transferred into two separate 100 mL volumetric flasks and exposed

photolytic treatment for about 22 hr. (1.2 million lux hr). 60 mL of the diluent was

added to each flask and sonicated for 30 min with intermittent shaking. The solutions

were diluted up to the mark with the diluent and mixed well. The solutions were

filtered through 0.45µ membrane filter.

Humidity degradation


weighed and transferred into two separate 100 mL volumetric flasks and exposed to

humidity at 40°C/75%RH about 73 hrs. 60 mL of the diluent was added to each flask

and sonicated for 30 min with intermittent shaking. The solutions were diluted up to the

mark with the diluent and mixed well. The solutions were filtered through 0.45µ

membrane filter.

% Assay values with respect to untreated sample and Peak Purity data of Pregabalin at

each condition were tabulated in Table 2.5.3. Refer in Fig 2.5.5, 2.5.6, 2.5.7, 2.5.8,

2.5.9, 2.5.10 and 2.5.11 for chromatograms and purity plots of untreated and treated

sample solutions respectively.

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Peak purity for Pregabalin peak should pass.

Table 2.5.3 Forced degradation study data

Sr.No.

Condition % Assay % Degradation w.r.t. Untreated sample

Peak Purity

Purity Angle

Purity Threshold

Purity Flag

1 Untreated Sample 101.10* - 0.258 0.490 No

2 Acid Degradation 94.94 6.09 0.261 1.124 No

3 Alkali Degradation 100.31 0.78 0.239 1.106 No

4 Peroxide Degradation 86.96 13.99 0.817 1.130 No

5 Thermal Degradation 99.37 1.71 0.468 1.438 No

6 Photolytic Degradation 97.96 3.11 0.135 1.116 No

7 Humidity Degradation 100.61 0.48 0.188 1.088 No

*Data taken from method precision

Conclusion

As the method meets acceptance criterion, the method can be considered stability

indicating for determination of Pregabalin in its capsule formulation.

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3. Precision 3.1 System precision

Six replicate injections of standard solution were injected into HPLC system. Mean, SD

and % RSD were calculated. Results are tabulated in Table 2.5.4.


% Relative standard deviation for Pregabalin peak area counts should not be more than

2.0

Table 2.5.4 System precision data

.

Sr. No. Pregabalin peak area

counts

1. 1374829

2. 1374945

3. 1372518

4. 1368497

5. 1371838

6. 1370900

Mean 1372255

SD 2452.1

% RSD 0.18

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3.2 Method precision

Six Sample preparations were made from a single batch of Pregabalin capsules and

analyzed as per the proposed method. % Assay of Pregabalin for six samples was

calculated. Results are tabulated in Table 2.5.5.


% Relative Standard Deviation (RSD) for % Assay of Pregabalin should not be more

than 2.0.

Table 2.5.5 Method precision data

Sr. No. Assay of Pregabalin

1. 101.89

2. 101.03

3. 104.04

4. 100.31

5. 99.29

6. 100.03

Mean 101.10

SD 1.692

% RSD 1.67

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3.3 Intermediate precision (Ruggedness)

Ruggedness of method was verified by analyzing six sample preparations of same batch

used under method precision as per proposed method by different analysts using

different instrument and different column on different day. The amount of Pregabalin in

Pregabalin Capsules was determined. %RSD for %Assay of Pregabalin and overall

%RSD for above results of the method precision was calculated. The results are

tabulated in Table 2.5.6.

Acceptance criteria

% Relative Standard Deviation (RSD) for % Assay of Pregabalin should not be more

than 2.0 and overall %RSD should be not more than 2.0

Table 2.5.6 Ruggedness data

Sr. No. Assay of Pregabalin

Method precision Ruggedness

1. 101.89 98.40 2. 101.03 98.68 3. 104.04 99.52 4. 100.31 100.14 5. 99.29 99.73 6. 100.03 99.09

Mean 101.10 99.26 SD 1.69 0.66 % RSD 1.67 0.66 Overall Mean 100.18 Overall SD 1.556 Overall %RSD 1.55

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4. Accuracy

The placebo was spiked with known amounts of Pregabalin API at 50%, 100% and

150% of test concentration of 50 mg strength capsules were prepared in triplicate at

each level. Amount of Pregabalin was quantified and % recovery was calculated from

amount found and actual amount added. The results are tabulated in Table 2.5.7.


% Recovery of Pregabalin at each spiked level should be in between 98.0 and 102.0

Table 2.5.7 Accuracy data

Conclusion

Analytical method meets pre-established acceptance criterion. Hence, method is

accurate and precise.

Spike level (%)

Actual Amount of Pregabalin added in mg

Amount of Pregabalin found in mg

%Recovery Mean SD %

RSD

50

49.88 50.26 100.76

100.72 0.032 0.03 50.12 50.47 100.70

50.39 50.75 100.71

100

100.20 100.59 100.39

100.95 0.605 0.60 100.00 100.86 100.86

100.20 101.79 101.59

150

149.70 151.09 100.93

100.70 0.202 0.20 149.90 150.79 100.59

150.09 150.95 100.57

Overall mean 100.79

Overall SD 0.341

Overall % RSD

0.34

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5. Linearity

Linearity range of response was performed using the standard solution in a range of

500.28 to 1500.84 mcg/mL [about 50% - 150% of the test concentration].The results are

tabulated in Table 2.5.8 and represented graphically in Fig 2.5 12.


Correlation coefficient (r) value should not be less than 0.99

Table 2.5.8 Linearity data

Conclusion

Response of Pregabalin was found to be linear in the range of 500.28 mcg/mL

to1500.84 mcg/mL.

Spike level (%)

Concentration in mcg/mL

Pregabalin average peak area

counts 50 500.28 676017 60 600.34 806589 80 800.45 1070235 90 900.50 1215657 100 1000.56 1358510 110 1100.62 1505016 120 1200.67 1623262 140 1400.78 1893146 150 1500.84 2018357

Slope 1353 Intercept -1763

Correlation coefficient (r)

0.99982

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6. Stability in analytical solution

Stability of Pregabalin in analytical solution was verified by analyzing sample solution

initial and also at different time intervals up to 26 hrs and 09 min by storing sample

solution at room temperature. Cumulative % RSD for peak area counts of Pregabalin

was calculated. The results are tabulated in Table 2.5.9.


Cumulative %RSD should not be more than 2.0 at each time interval.

Table 2.5.9 Stability in analytical solution data

Time Pregabalin peak

area counts Cumulative % RSD

Initial 1415203 - 0 hr 09 min 1416518 0.07 0 hr 18 min 1413914 0.09 0 hr 27 min 1413200 0.10 0 hr 36 min 1412677 0.11 0 hr 45 min 1411237 0.13 2 hr 39 min 1421012 0.23 2 hr 48 min 1420696 0.26 3 hr 49 min 1427926 0.38 3 hr 58 min 1425113 0.40 4 hr 57 min 1430342 0.46 5 hr 06 min 1429502 0.49 08 h. 34 min 1431582 0.52 08 hr 43 min 1432480 0.55 13 hr 10 min 1428315 0.54 13 hr 19 min 1427439 0.53 17 hr 47 min 1415123 0.53 17 hr 56 min 1416089 0.52 22 hr 23 min 1419814 0.51 22 hr 32 min 1421889 0.50 26 hr 00 min 1427096 0.49 26 hr 09 min 1426766 0.49

Conclusion

The solution was found to be stable up to 26 hrs at room temperature and hence, it is

concluded that the proposed analytical method meets the pre-established acceptance

criterion.

237

7. Robustness

To evaluate its robustness, following small deliberate variations were made in the

method and the samples were analyzed in triplicate.

1. Changing flow rate by (±10%)

2. Changing organic content in mobile phase by (±10% relative)

3. Changing column oven temperature by (± 5°C)

4. Changing wavelength by (± 5 nm)

5. Changing pH of buffer of mobile phase by (± 0.1 units)

6.

System suitability was evaluated in each condition and results were compared with

method precision results. The results are tabulated in Table 2.5.10.


Overall %RSD should not be more than 2.0 for individual experiment.

Table 2.5.10 Robustness data

Sr.No. M.P. -Flow +Flow -Temp +Temp - nm +nm -Org +Org -pH +pH

1 101.89 99.33 98.30 99.12 100.84 101.39 102.19 100.52 100.87 99.59 100.67

2 101.03 100.30 98.32 99.46 101.20 100.79 101.37 99.39 99.14 98.53 98.26

3 104.04 100.77 99.33 100.66 102.65 103.95 104.31 100.21 97.75 98.26 99.87

4 100.31 - - - - - - - - - -

5 99.29 - - - - - - - - - -

6 100.03 - - - - - - - - - -

Overall mean 100.78 100.28 100.65 101.25 101.41 101.61 100.75 100.48 100.33 100.60

Overall SD 1.469 1.837 1.552 1.440 1.648 1.717 1.468 1.803 1.800 1.652

Overall % RSD 1.46 1.83 1.54 1.42 1.63 1.69 1.46 1.79 1.79 1.64

238

M.P. Method precision data

-Flow Flow rate (0.9 mL/min)

+Flow Flow rate (1.1 mL/min)

-Temp Column oven temperature (25°C)

+Temp Column oven temperature (35°C)

-nm Wavelength (195 nm)

+nm Wavelength (205 nm)

-Org Organic content variation - 10 % relative

+Org Organic content variation + 10 % relative

- pH PH of buffer in mobile phase - 6.60

+ pH PH of buffer in mobile phase – 6.80

Conclusion

Method meets pre-established acceptance criteria for small changes in flow rate,

wavelength, column oven temperature, pH of Buffer organic content in mobile phase.

Hence, it is concluded that method is robust.

8. Limit of Detection and Limit of Quantification

Limit of detection (LOD) is defined as the lowest concentration of analyte that gives a

measurable response. LOD is determined based on signal to noise ratio (S/N) of three

times typically for HPLC methods. The limit of quantification (LOQ) is defined as the

lowest concentration that can be quantified reliably with a specified level of accuracy

and precision. It is the lowest concentration at which the precision expressed by an RSD

of less than 2%. In this study the analyte response is 10 times greater than the noise

response. For this study six replicates of the analyte at lowest concentration in the

calibration range were measured and quantified. The LOD and LOQ of Pregabalin

obtained by the proposed method were 0.122 and 0.432 µg/mL respectively.

239

9. Summary of system suitability

System suitability was evaluated by injecting Standard solution during different days of

validation. Tailing factor and theoretical plates for Pregabalin peak from standard

solution and % relative standard deviation for the peak area counts of Pregabalin from

five replicate injections of standard solution was verified at every stage. The results are

tabulated in Table 2.5.11.

Acceptance criteria

1. Column efficiency determined for the Pregabalin peak should not be less than 3500

theoretical plates and tailing factor for the same peak should not be more than 2.0 from

standard solution.

2. Percentage relative standard deviation for Pregabalin peak area counts from five

replicate injections of standard solution should not be more than 2.0.

240

Table 2.5.11 Summary of system suitability data

S.No. Name of Experiment Theoretical plates

Tailing factor

%RSD

1 System precision, Method precision and Solution stability

6449 1.1 0.19

2 Robustness (- Wavelength) 6304 1.1 0.21

3 Robustness (+ Wavelength) 6482 1.1 0.21

4 Linearity 7335 1.0 0.30

5 Ruggedness 7405 1.1 0.36

6 Robustness (- Flow) 6995 1.1 0.46

7 Robustness (+ Flow) 4440 1.1 0.30

8 Robustness (- Temperature) 4583 1.1 0.13

9 Robustness (+ Temperature) 5918 1.0 0.45

10 Recovery and Specificity 6160 1.1 0.08

11 Robustness (-pH) 5548 1.1 0.44

12 Robustness (+pH) 4246 1.2 0.71

13 Forced Degradation-1 5903 1.1 0.17

14 Forced Degradation-2 6557 1.2 0.16

15 Robustness (- Organic) 6241 1.2 1.10

16 Robustness (+ Organic) 6459 1.1 1.64

241

4) SUMMARY OF THE RESULTS AND CONCLUSION

The present study was aimed at developing a simple, precise and accurate

HPLC method for the analysis of Pregabalin from its capsule dosage forms. A non-polar

C8 analytical chromatographic column was chosen as the stationary phase for the

separation and determination of Pregabalin. For the selection of the mobile phase a

number of eluting systems were examined. Mixtures of commonly used solvents like

water, and acetonitrile with or without different buffers in different combinations were

tested as mobile phases on a C8 stationary phase. The choice of the optimum

composition is based on the chromatographic response factor, a good peak shape with

minimum tailing. A buffer containing potassium dihydrogen phosphate Monohydrate

(pH 6.7± 0.05 ) and acetonitrile in a ratio of 970:30 v/v was proved to be the most

suitable of all the combinations since the chromatographic peak obtained was better

defined and resolved and almost free from tailing. The retention time of the drug was

found at 4.75 min.

Summary of validation:

Specificity

No interfering peak was observed at the retention time of Pregabalin from blank and

placebo samples. Thus, the Peak purity for the analyte passed.

Hence, it is concluded that method is specific for determination of Pregabalin in

Pregabalin capsules.

Forced Degradation

Forced degradation study was carried out by subjecting the placebo and sample to the

following conditions.

1. Treatment with hydrochloric acid.

2. Treatment with sodium hydroxide.

3. Treatment with hydrogen peroxide.

4. Thermal exposure.

5. Photolytic exposure.

6. Exposure to humidity

242

In all the above conditions, the method met the acceptance criteria for peak purity.

Hence, the method can be considered as stability indicating for determination of

Pregabalin in its capsule formulation.

System Precision

% RSD of Pregabalin peak area counts from six replicate injections of standard solution

was less than 2.0 and it meets the acceptance criterion.

Method precision

Six samples from a single batch were prepared and analyzed as per test method and

their % RSD for % assay were calculated. The % RSD for % assay of Pregabalin was

less than 2.0 and meets the acceptance criterion.

Ruggedness

Six samples from a single batch (same batch used under Method precision) were

prepared and analysed as per test method by different analyst by using different column,

different HPLC system and on different day.

% RSD for % assay of Pregabalin was calculated, for six preparations.

% RSD for % assay of Pregabalin was less than 2.0 and meets the acceptance criteria.

Overall % RSD for % assay of Pregabalin obtained from ruggedness and method

precision was less than 2.0 and meets the acceptance criteria.

The proposed analytical method meets acceptance criteria for precision. Hence, the

method is precise.

Accuracy (Recovery)

The sample solutions were prepared in triplicate at each level by spiking the placebo

with Pregabalin API at about 50%, 100 % and 150 % of test concentration. % Recovery

at each level was calculated.

Analytical method meets acceptance criterion for recovery study. Hence, the method is

accurate and precise.

243

Linearity

Linearity range for Pregabalin was determined using solutions containing about 50% to

150% of test concentration. It was found that response for Pregabalin was linear in the

range of 500.28 mcg/mL to1500.84 mcg/mL and the relevant correlation coefficient

value is more than 0.99

Stability in analytical solution

By analysing sample solution, at different time intervals, stability in analytical solution

was carried out. It was found that the sample solution is stable up to 26 hrs at room

temperature.

Robustness

Robustness of analytical method was carried out by deliberately varying optimized

chromatographic conditions of flow rate (±10%), column oven temperature (±5°C),

organic content of mobile phase (±2 % absolute), pH of the buffer in mobile phase

(±0.1unit) and wavelength (±5nm). The method was found to be robust for change in

flow rate, change in column oven temperature, change in organic content in mobile

phase, change in pH of buffer in mobile phase and change in wavelength, as method

meets acceptance criteria.

Limit of detection and Limit of Quantification

The lowest values of LOD and LOQ as obtained by the proposed method indicate the

method is sensitive

Conclusion

The validation data proves that the proposed method for determination of pregabalin in

pregabalin capsules is specific, precise, accurate, linear and robust under the given

conditions of methodology and is suitable for use.

244

Fig 2.5.2: HPLC Chromatogram of Blank

245

Fig 2.5.3: HPLC Chromatogram of Placebo

246

Fig 2.5.4: HPLC Chromatogram and purity plot of placebo with drug sample solution

247

Fig 2.5.5: HPLC Chromatogram and purity plot of untreated sample

248

Fig 2.5.6: HPLC Chromatogram and purity plot of Acid treated sample

249

Fig 2.5.7: HPLC Chromatogram and purity plot of Alkali treated sample

250

Fig 2.5.8: HPLC Chromatogram and purity plot of Peroxide treated sample

251

Fig 2.5.9: HPLC Chromatogram and purity plot of Thermal treated sample

252

Fig 2.5.10: HPLC Chromatogram and purity plot of Photolytic treated sample

253

Fig 2.5.11: HPLC Chromatogram and purity plot of Humidity treated sample

254

Fig 2.5.12: Linearity plot for Pregabalin

Spike level (%)

Concentration in mcg/mL

Pregabalin average peak area counts

50 500.28 676017 60 600.34 806589 80 800.45 1070235 90 900.50 1215657 100 1000.56 1358510 110 1100.62 1505016 120 1200.67 1623262 140 1400.78 1893146 150 1500.84 2018357

Slope 1353 Intercept -1763 Correlation coefficient (r) 0.99982

255

5. REFERENCES

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256

10. Yihong Zhang, Christopher Holliman, Daniel Tang, Douglas Fast, and

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257

16. ICH Guidelines Q2B, Validation of Analytical Procedure: Definitions,

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Tripartite Guideline Q2 (R1), Commission of the European Communities 2005.

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