WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Vaishali et al. World Journal of Pharmacy and Pharmaceutical Sciences

Volume 3, Issue 1, 703-711. Research Article ISSN 2278 – 4357

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR

THE ASSAY OF PREGABALIN CAPSULE

Vaishali1*,Vikas Singh2, Rajnish Kumar Singh1, Ramesh Kumar Gupta1, Sudhansu

Ranjan Swain3, Jagannath Sahoo4

1
Moradabad Educational Trust, Group of Institution Faculty of Pharmacy, Moradabad -
244001, Uttar Pradesh, India.
2
Teva Pharma India, Plot no.189, Verna Industrial estate, Verna-403602, Goa, India
3
Sherwood College of Pharmacy, Barabanki-225001, Uttar Pradesh, India.
4
Shri Ram Murti Smarak College of Engineering & Technology, Bareilly 243202, Uttar
Pradesh, India.

Article Received on ABSTRACT

23 October 2013,
Revised on 20 November
Background: Pregabalin is S-3-(amino methyl)-5-methylhexanoic
2013, acid. It is an anticonvulsant drug for neuropathic pain and adjunct for
Accepted on 28 December
2013 partial seizures. It can be used in generalised anxiety disorders.1,2
Methylcobalamin (MC;carbanide; cobalt; [5-5, 6-dimethyl
*Correspondence for benzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl) oxolan-3-yl] 1-[3-
Author: [2,13,18-tris (2-amino-2- oxoethyl)- 7,12,17- tris(3-amino- 3-
Vaishali oxopropyl)-3,5,8,8,13,15,18,19-octamethy-2,7,12,17 tetrahydrocorrin-
Moradabad Educational Trust,
3-yl]-propanoylamino]propan-2-yl hydrogen phosphate. It is a form of
Group of Institution Faculty of
Pharmacy, Moradabad-244001,
Vit-B. Information collected from previous research has played an
Uttar Pradesh, India. important role to develop for quantitative estimation of pregabalin
vaishalihi009@gmail.com from capsule dosage form.
Aim: The present study focused to developand validate the RP-HPLC
method for the assay of pregabalin capsule.
Methods: The method was validated according to ICH guidelines with respect to specificity,
linearity, accuracy, precision and robustness. The chromatography was set on Hypersil
BDS,C18 column (250mm×4.6mm, 5µ) column using fluorescence detector set at Ex 220nm
and Em 280nm. The mobile phase consisting of Water and Methanol in the ratio of 90:10 was
used.

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Result: The mobile phase consisting of Water and Methanol in the ratio of 90:10 which gave
better resolution and sensitivity with flow rate of 1 ml/min at temperature 40°C.The retention
time of Pregabalin was 6.4min .The method was found to be linear in the range of 14000-
26000 mcg/ml for pregabalin. Lower limit of quantification is 0.5 mg/l.
Conclusion:A simple, precise, specific, accurate highly selective reverse phase high
performance liquid chromatographic (RP-HPLC) method has been developed for the
determination of pregabalin in capsule dosage form.There is no generally accepted method
for the determination of pregabalin.This method was used successfully for the quality
assessment of pregabalin capsules with good precision and low cost. The pregabalin sample
solution was found to be stable at room temperature for about 24 h.
Key Words: Pregabalin capsule, RP-HPLC, Method development and validation.

INTRODUCTION
Pregabalin (Lyrica TM) is an antiepileptic drug approved for a number of indications in the
US and Europe that include adjunctive therapy of partial seizures in adults, pain from diabetic
neuropathy or post-herpetic neuralgia in adults, and the treatment of anxiety
disorders.([1])Pregabalin chemically known as (3S)-3-(aminomethyl)-5-methylhexanoic acid is
an antiepileptic, anticonvulsant and neurotransmitter. This drug produces its actions by
binding to the alpha2-delta (α2δ) subunit of the voltage-gated calcium channels. It is freely
soluble in water both in acid and basic aqueous solution. It is well absorbed after oral
administration and largely excreted by renal excretion.([2])Pregabalin is the 3-isobutyl
substituted analogue of _-amino butyric acid (GABA) but is inactive at GABA receptors.([3])
The pharmacological activities of pregabalin result from its binding to the alpha-2-delta (_2_)
protein, an auxiliary protein associated with voltage-gated calcium channels in the central
nervous system.([4]) More recently pregabalin has been approved by the FDA for the treatment
of spinal cord injury and as the first drug indicated for the treatment of fibromyalgia and
pregabalin received U.S.FDA approval for use in treating epilepsy, diabetic neuropathy pain
and post herpetic neuralgia.It is considered to have a low potential for abuse, and a limited
([5])
dependence liability if misused, and is thus classified as a Schedule V drug in the U.S.
Although various bio-analytical methods for estimation of pregabalin in human serum and
spectrophotometric methods for estimation of pregabalin in dosge form([6,7])and HPLC
methods for development & validation of pregabalin in capsuleshave been reported in the
([8, 9])
literature. The results of analysis were validated using International Conference on
Harmonization (ICH) guidelines.The present work describes a simple, precise and accurate

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Vaishali et al. World Journal of Pharmacy and Pharmaceutical Sciences

RP HPLC method for estimation of pregabalin in commercial dosage form. The main
objective of this study was to develop a fast, sensitive, robust and cheap method to validate
pregabalin using isotopically labeled internal standard (ISTD), in order to extend its
application to assess the bioequivalence of two lamotrigine 50 mg tablet formulations in
healthy volunteers.

MATERIAL AND METHODS

1. Chemical and Reagents
Ultra gradient grade Glacial acetic acid, Methanol and Acetonitrile was purchased from
Merck Ltd. Methanol of HPLC grade was obtained from Sigma-Aldrich Pvt. Ltd.
Orthophosphoric acid (AR grade) and Triethyl amine was supplied from Fisher Scientific Ltd
(Mumbai, India) and Merck specialties Pvt.Ltd respectively. All aqueous solutions and
buffers were prepared using water that was purified using Millipore-Qs Gradient A10s
(Millipore). Pregabalin Working standard and test sample ware procured from Central Drug
Laboratories and Akums Drugs & Pharmaceuticals Ltd. respectively.

2. HPLC Instrumentation and analytical conditions

The liquid chromatography separation was performed using a Shimadzu scientific
instruments (Shimadzu Corporation; Kyoto, Japan) UFLC-Auto sampler and HPLC-1500
series (Water 1515) Isocratic Pump and (Water 2487) dual Lambda absorbance detector
(WATERS) with Model no. Alliance e 2695 and FTIR (8400S). Liquid chromatographic
separations were achieved using Lichro CART C-18, 250 mm × 4.6 mm, 5µm (Merck
Scientific, USA) and Inertsil ODS Column (150 x4.6mm), 5µm OHS10077, consisting of
C18 which is manufactured by G.L.Sciences,USA. Magnetic stirrer (Remi equipments Pvt.
ltd.) , Electronic Balance (Mettler), KBr Press (Technosearch ltd.), Ultra Sonicator (Spectral
Lab), PH Meter (Eutech instruments Ltd), Digital Weighing Balance(Sartorius) were used
during the process of Validation. An injection volume of 20 µL was used for each analysis.
Mobile phase consisted of water and methanol (90:10) V/V. The flow rate of the mobile
phase was set at 1.0mL/min.

3. Preparation of Mobile phase

A mixture of water and methanol prepared in the ratio of 90:10. The solution was filtered
through 0.45µ nylon membrane filter and degas.

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4. Preparation of Standard solution and Test solution

Standard solution- 200mg of Pregabalin working standard was weighed accurately and
transferred into a 10ml. volumetric flask. Volumetric flask was sonicated to dissolve the
contents and make up the volume up to mark with mobile phase. (Concentration 20,000ppm).
Test solution- Equivalent of 200mg of pregabalin was weighed and transferred into 10ml
volumetric flask. And 5ml. of water was added and sonicated to dissolve, cool and make up
the volume 10ml. with mobile phase & was mixed. The solution was filtered through 0.45µ
nylon membrane filter. (Concentration 20,000ppm).

5. Method validation
A full method validation was performed according to guide- lines set by the USFDA&ICH
([10,11])
Guidelines. The validation of this procedure was performed in order to evaluate the
method in terms of selectivity, sensitivity, range, linearity of response, accuracy, precision
and intermediate precision.

Specificity-Equal volume (about 20µl.) of standard preparation and test preparation were
separately injected into the chromatograph. Chromatograms were recorded and measured the
responses for major peaks.

Linearity-Equal volume (about 20µl.) of standard preparation and test preparation were
separately injected at different conenteration14000 mcg to 26000 mcg into the
chromatograph. Chromatograms were recorded and measured the responses for major peaks.

Accuracy-Itwas obtained by Recovery studying using standard addition method, Equal

volume (about 20µl.) of standard preparation and test preparation were separately injected
into the chromatograph. Chromatograms were recorded and measured the responses for major
peaks.

Precision-Equal volume (about 20µl.) of standard preparation and test preparation were
separately injected into the chromatograph. Chromatograms were recorded and measured the
responses for major peaks.

Intermediate Precision-Equal volume (about 20µl) of standard preparation and test

preparation were separately injected into the chromatograph. Chromatograms were recorded
and measured the responses for major peaks.

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RESULTS
1. Optimization of the chromatographic condition
To optimize the chromatographic conditions, the effect of chromatographic variables such as
mobile phase, pH, flow rate and solvent ratio were studied. Various solvent systems were
tried for the development of a suitable HPLC method for determination of pregabalin in
pharmaceutical formulations. Mobile phase tried for this purpose were acetonitrile: Water
(30:70 V/V), acetonitrile: Water (50:50 V/V), methanol: water (70:30 V/V), methanol: water
(50:50 V/V), water: methanol (90:10V/V). The condition that gave the best resolution and
symmetry was selected. Same solvent system was used for the extraction of the drug from the
formulation containing excipients which was used for quantification. (Table1)

2. Method optimization
Selection of detection wavelength: Pregabalin showed absorbance at 220nm. So the
wavelength selected for the determination of Pregabalin was 220nm.
Selection of proper column: Lichro CART C-18, 250 mm × 4.6 mm, 5µm
Selection of chromatographic conditions: Optimized chromatographic conditions for
estimation of Pregabalin are finalized as shown in Table 2.

3. Method validation
Selectivity and specificity
Selectivity is the ability of an analytical method to differentiate and quantify the analyte in
the presence of other components in the sample. Selectivity was ascertained in different
samples of pregabalin by comparing the chromatograms of pregabalin standard. All the peaks
were properly resolved from each other and peak purity of all the peaks in the spiked sample
was passed. Besides, no peak of placebo was found at the RT of the compound which showed
the specificity of the method. The specificity was found to be under limit. Specificity for
Pregabalin was 0.031.

Linearity and sensitivity

The method was validated and calibration standard curve containing pregabalin was linear
over the concentration range of 14000 mcg -26000 mcg with a correlation coefficient (r) of
0.999904 The intercept with the y-axis was not significantly different from zero. (Table 3)

Accuracy
Accuracy of the method was determined by analyzing quality control samples at three

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concentrations within the calibration curve range to validate reproducibility. The accuracy
limit is the percentage recovery should be the range of 98.0%-102%. The validation of the
development method shows that the accuracy is well within the limit. The accuracy of the
method was determined by performing recovery studies by standard addition method in
whichpre-analyzed samples were taken and standard drug was added at 3 different levels. The
% recovery lies in the range of 99.76% - 100.36%. Table summarizes accuracy values for
quality control samples.(Table 4).

4. Intermediate Precision
Intermediate Precision of the method was determined by analyzing quality control samples at
four concentrations within the calibration curve range to validate reproducibility.
Intermediate precision was done at inter day analysis by two analysts by HPLC. The
maximum variation against 1 is 1.731 (limit is less than 2%). These results above indicate
that the present method has good accuracy, precision and reproducibility. (Table 5)
Table (1): Trial Taken For Optimization Condition
Sr. No. Trails Taken Observation Remarks
1 Mobile Phase: Tailing and Not Satisfactory
Buffer : Solvent Mixture: (40:60) v/v Retention
Flow rate 1.0 ml/min time High
Detector wavelength :230 nm
Injection volume :10µL
Column:- :- Inertsil ODS-2, (150 x
4.6mm),5µm
2 Mobile phase: Retention time Not Satisfactory
Buffer : Solvent Mixture: (60:40) v/v satisfactory but
Flow rate 1.0 ml/min low
Column:- :- Inertsil ODS-2, (150 x theoretical plate
4.6mm),5µm
3 Mobile phase : Retention time
Buffer : Solvent Mixture: (40:60) v/v satisfactory but
Flow rate :1.3 ml/min low
Column :- Inertsil ODS-2, (150 x theoretical plate
4.6mm),5µm
4 Column: Inertsil ODS-2, (150 x Tailing low, Not Satisfactory
4.6mm),5µm Retention
Flow rate: 1.0 mL/minute time good, USP
Detector : UV Detector Plate
wavelength: 230 nm High, Area
Injection volume: 10µL High
Run time : 8 minute
Diluent : Buffer: Methanol (50:50) v/v
Mobile phase:
Buffer : Solvent Mixture: (50:50) v/v

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5. Robustness
Robustness of the method was investigated by varying the instrumental conditions such
aswavelength of detection (± 5 nm), column oven temperature (+ 5 °C), pH of buffer (± 0.2
Ph unit), % organic (±2mL absolute). System suitability of the standard solution was checked
ateach variable condition and data was found to be within the acceptable range.(% RSD
NMT2)

Table (2): Optimized chromatographic conditions

Column LichroCART C-18, (250 x 4.6mm),5µm

Flow rate 1.0 ml/minute
Detector Water 2487 dual Lamda absorbance detector.
Detector wavelength 220 nm
Injection volume 20µl
Run time 15 minutes
Mobile phase water and methanol (90:10) V/V

Table (3): Observation for Linearity and Range

Concentration
S.No. Area
( In mcg )
14000 1951221
16000 2194673
18000 2436173
20000 2664755
22000 2887756
24000 3117242
26000 3354157
Correlation coefficient 0.999904

Table (4): Observation for Accuracy

S.No. Known amount Recovery in mg. % of

added in the placebo Individual Average Recovery
(in mg.) value value
1 1 80% 61.36 61.41
2 80% 61.36 61.63
61.58 100.36
3 80% 61.36 61.70
2 1 100% 74.32 74.12
2 100% 74.32 74.47
74.42 100.13
3 100% 74.32 74.67
3 1 120% 89.16 89.47

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2 120% 89.16 88.86

120% 89.16 88.52 88.95 99.76
3

Table (5): Observation for Intermediate Precision

Parameter Maximum
Analyst Acceptance
which Analyst II variation against
S.No. I Criteria
are validated Analyst 1 in %
1 Assay 100.54 100.45 -0.090
2 Assay 99.95 99.82 -0.130
3 Assay 100.1 99.94 -0.160
NMT 2%
4 Assay 99.72 101.17 1.433
5 Assay 98.80 100.54 1.731
6 Assay 99.07 99.67 0.602

DISCUSSION
A simple, rapid, highly sensitive, selective and reproducible RP-HPLC method for the
quantitation of pregabalin capsules was developed and validated. The results indicate the
method to be sensitive, selective, accurate and reproducible. Sample preparation is very
simple and it involves mixing of Water and Methanol. From the results of all the validation
parameters and applicability of the assay, we can conclude that the present method can be
useful for Assay of Pregabalin Capsule with desired precision and accuracy along with high
throughput. The assay was linear from 14000mcg- 26000mcg.The developed method it will
be validate as per ICH guidelines. The developed method can be used for the analysis of
routine quality control sample. The proposed method shows good agreement with all
validation parameters. In the Specificity There should not be any interference from diluents
and blank with main peak.

CONCLUSION
In the accuracy % recovery is 98.70 and % RSD is 0.655 it meets criteria according to ICH
Guideline. In the present study we observed the linear relation between the concentration and
the result. Result of this study showed the stability of pregabalinduring storage, processing
and throughout the validation.

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pregabalin: the calcium channel alpha2-delta (alpha2-delta) subunit as a target for
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5. Anonymous, Drug Enforcement Administration, Department of Justice. Schedules of
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