INTRODUCTION:

Pregabalin (PGB), (S) - 3 - amino methyl hexanoic acid, is an antiepileptic drug and a structural analogues of α- amino butyric acid (GABA). It is a white to off- white, non- hygroscopic, crystalline and water soluble powder. PGB is a new anticonvulsant and analgesic medication that was recently approved for adjunctive treatment of partial seizures in adults in both the United States and Europe and for the treatment of neuropathic pain from postherpetic neuralgia and diabetic neuropathy ^[1-4]. It is both structurally and pharmacologically related to the anticonvulsant and analgesic medication gabapentin (Figure 1). Like gabapentin, pregabalin binds to the alpha2-delta site (an auxiliary subunit of voltage-gated calcium channels) in central nervous system tissues, reducing depolarization-induced calcium influx and, thereby reducing release of excitatory neurotransmitters such as glutamate, noradrenaline, and substance P. These actions are thought to be responsible for their antiseizure effects. Gabapentin shows saturable absorption kinetics thus limiting its systemic availability. Hence, pregabalin was developed for its better pharmacokinetic properties over those of gabapentin.

Pregabalin’s oral bioavailability is approximately 90% and it is independent of dose and frequency of administration. Pregabalin undergoes minimal metabolism in human with unchanged parent representing the majority (≥90 %) of drug - derived material ^[5]. This contrasts with gabapentin, which is absorbed via a capacity limited L - amino acid transport system from the proximal small bowel into the blood stream ^{[6, 7]}.

Fig. 1. Structures of gabapentin, pregabalin and gama aminobutyric acid.

Currently, there is no official analytical procedure for pregabalin in any pharmacopoeia. Few reports are there in literature for PGB determination based on chromatographic methods, i.e., gas chromatography-mass spectrophotometry (GC-MS), LC-MS-MS^[8,9], HPLC^[10-12] coupled with varying detection techniques like tandem mass spectrometry^[13], fluorometry^[14] and enantiospecific analysis^[15]. These methods may include variations in procedures including pre- and post- column derivatization^[15]. Recently, capillary electrophoresis and nuclear magnetic resonance technique was reported for PGB involving complexation with cyclodextrins^[16]. All these methods require long and tedious pretreatment of the samples and laborious cleanup procedures prior to analysis, which eventually leads to cost constraints. So, there is a need for the development of a simple HPLC method for the analysis of PGB in bulk and pharmaceutical formulations^[17]. The proposed method requires no derivatization steps. HPLC instrument with UV detector that is commonly available in most analytical and pharmaceutical laboratories was used. A total run time of less than 5 minutes was achieved.

The method was successfully used to evaluate 2 marketed PGB drug products.

MATERIALS AND METHODS:

Materials

i) Pregabalin (Rubicon Research Pvt Ltd.) used as a standard. ii) Pharmaceutical formulations of pregabalin such as Pevesca 75 (USV Ltd., Mumbai, India) and Pregalin 75 (Torrent Pharmaceutical Ltd., Baddi, India) were purchased from local markets. iii) Di- potassium hydrogen orthophosphate was purchased from Merck Ltd., Mumbai, India. iv) Acetonitrile was HPLC grade purchased from Merck Ltd., Mumbai, India. v.) Orthophosphoric acid was LR grade purchased from Merck Ltd., Mumbai, India. vi.) All other chemicals were of analytical grade and used without any further purification.

Instrumentation

i) Waters 2690 series LC system with UV detector with inbuilt auto injector (Waters, Alliance) was used for method development and validation. ii) Data acquisition and system suitability calculations were carried out using Waters Empower software. iii) The HPLC column used was Kromasil C18, (100 × 4.6) mm, 5 μ, (Thermo Fisher Scientific, Waltham MA, USA). iv) UV/VIS spectrophotometer, model no. UV 3000+ (LABINDIA®, India).

Chromatographic systems and conditions

The proposed method was performed using a Waters 2690 series LC system. Separation was operated on C18 5 μm kromasil column (100 mm × 4.6 mm) using a mixture of di – potassium hydrogen orthophosphate (K₂HPO₄) (pH – 6.9) and acetonitrile in a ratio of 90: 10 v/v mobile phase at a flow rate of 1.0 ml/min. Di- potassium hydrogen orthophosphate solution was prepared by dissolving 680 mg K₂HPO₄ in 500 ml double distilled water. Final pH of the mobile phase was adjusted to 6.9 with 0.01 M orthophosphoric acid, prepared daily and degassed by passing through a 0.45 μm Ultipor filter and ultrasonication for 10 min. All separations were performed at room temperature with detection at 274 nm. The column oven was maintained at 30° and injection volume was 20 μl.

Standard solutions

Stock standard solution of pregabalin was prepared by dissolving an appropriate amount of the compound in buffer to give a final concentration of 1000 µg/ml. Standard solutions of pregabalin (10, 20, 30, 40, 50, 100 μg/ml) were prepared by subsequent dilution.

Determination of appropriate UV wavelength

A suitable wavelength was required for the determination of pregabalin. The appropriate wavelength for the detection of drug was determined by wavelength scanning over the range of 200 – 400 nm with a UV 3000+ (LABINDIA®, India) UV/VIS spectrophotometer.

Procedure for determination of pregabalin

Accurately weighed sample of pregabalin (50 mg) working standard was transferred to a 50 ml volumetric flask and 30 ml of buffer was added and sonicated to dissolve the sample completely. The solution was made up to the mark and filtered through 0.45 μ nylon membrane filter.

Procedure for determination of pregabalin in pharmaceutical formulations

Capsule powder equivalent to 10 mg of pregabalin (about 51.67 mg of pregabalin capsule powder of 75 mg strength) was accurately weighed and transferred in to a 10 ml volumetric flask; 6 ml of buffer was added and sonicated with occasional shaking for 30 min. The solution was cooled to room temperature and diluted to volume with the diluent. The final solution was filtered through a 0.45 μ nylon membrane filter.

In order to develop an accurate and suitable chromatographic method for the determination of PGB various chromatographic conditions were employed using columns of different dimensions like C18 5 μm kromasil column (100 mm × 4.6 mm) and C18 5 μm kromasil column (250 mm × 4.6 mm) and different mobile phase containing buffers like acetate and phosphate with different pH ranging from 4.5 to 6.9 and using organic modifiers like methanol and acetonitrile in the mobile phase. Also, the flow rate ranging from 0.8ml/min to 1.2ml/min was checked. Finally, the mobile phase consisting of a mixture of di – potassium hydrogen orthophosphate (K₂HPO₄) (pH – 6.9) and acetonitrile in a ratio of 90: 10 v/v at a flow rate of 1ml/min using C18 5 μm kromasil column (100 mm × 4.6 mm) was found to be appropriate. The column oven was maintained at 30°. The compound would give no characteristic spectra as it lacks a chromophore (Figure 1). Hence, 274 nm wavelength has been chosen for the detection in the analysis for better sensitivity. The analyte peak was found to be symmetrical under these optimized conditions. The typical HPLC chromatogram was scanned at 274 nm (Figure 2). The developed method was validated according to the ICH guidelines.

Method validation

Method validation is the process used to confirm that the analytical procedure developed for a specific test is suitable for its intended use. Results from method validation can be used to judge the quality, accuracy, reliability and consistency of analytical results. It is an integral part of any good analytical practice. USP defines eight steps for validation such as accuracy, precision, specificity, limit of detection, limit of quantitation, linearity and range, ruggedness, robustness ^[18].

Solution stability

The stability of the reference pregabalin sample solutions at room temperature was evaluated with the help of HPLC systems.

Specificity and selectivity

The specificity and selectivity of the developed method was evaluated by estimating the amount of pregabalin in presence of common excipients such as glucose, fructose, starch, lactose. By assessing the resolution between the peaks corresponding to various substances, the ability of the developed method to separate all the compounds from PGB in the sample was demonstrated.

Linearity

Six serial dilutions were prepared in concentration range from 10 µg/ml to 100 µg/ml. A volume of 20 µl from each concentration of the solution was injected and chromatograms were recorded; three independent determinations were performed at each concentration.

Accuracy

To ensure the accuracy of the analytical method, the recovery studies were carried out. Known amount of pregabalin was added to a pre quantified sample solution and the amounts of pregabalin was estimated by measuring the peak area ratios and by fitting these values to the straight line equation of straight line equation of calibration curve. The recovery studies were carried out three times over the specified concentration range and amount of pregabalin was estimated by measuring the peak area ratios by fitting these values to the straight line equation of calibration curve. Accuracy was evaluated at three different concentrations equivalent to 80, 100 and 120% of the active ingredient by calculating the recovery of pregabalin with RSD (%) and %.

Precision

The within-day precision of the method was determined for both peak area and retention time by repeat analysis (three identical injections) at three concentration levels. The between day precision was established by performing the analysis over a 5-day period on solution prepared freshly on each day.

Repeatability

Robustness

The robustness was assessed by altering the following experimental conditions such as, by changing the flow rate from 0.8 to 1.2 ml/min.

RESULTS AND DISCUSSION:

Method development

By monitoring varying columns and mobile systems, optimization of chromatographic condition was achieved. Silica column such as C18 5 μm kromasil column (250 mm × 4.6 mm) did not gave a proper peak shape for analysis. Whereas, C18 5 μm kromasil column (100 mm × 4.6 mm) gave better results. Different mobile phase combinations in different ratios were also evaluated such as acetonitrile : water at 80 : 20 v/v, acetonitrile : di – potassium hydrogen orthophosphate (K₂HPO₄) (pH – 4.5) at 50 : 50, 90 : 10, 80 : 20, 5 : 95, 10 : 90 v/v, methanol : di – potassium hydrogen orthophosphate (K₂HPO₄) (pH – 4.5) at 5 : 95 v/v, but the best results were achieved by using a mixture of di – potassium hydrogen orthophosphate (K₂HPO₄) (pH – 6.9) and acetonitrile in a ratio of 90: 10 v/v. Also, a flow rate of 1ml/min was found to be suitable over a flow rate of 0.8ml/min and 1.2ml/min for the developed method. Excellent chromatogramphic specificity with no interference from dosage form excipients was observed. Moreover, a suitable retention time for PGB was achieved. Under the chromatographic conditions described, PGB was well resolved and eluted at about 2.32 min (Figure 2), the total run time was within 5 min. Good baseline resolution and peak shape can be observed.

Fig. 2. HPLC chromatogram of pregabalin

Determination of suitable UV wavelength

For determining the appropriate wavelength for determination of pregabalin, solution of pregabalin in mobile phase was scanned by UV spectroscopy in the range of 200 – 400 nm. The maximum absorbance was observed at 200 nm. Also, solution of pregabalin in the same mobile phase was also injected to HPLC directly at different wavelengths. But, the maximum peak area was observed at 274 nm. Therefore, it was concluded that 274 nm is the most appropriate wavelength for the analysis of PGB with suitable sensitivity.

Specificity

The specificity and selectivity of the proposed method was evaluated by estimating the amount of pregabalin in the presence of common excipients such as glucose, fructose, starch, lactose. The HPLC chromatograms recorded for the mixture of the drug excipients revealed almost no peaks within a retention time range of 5 min. The study of the absence of excipients showed that none of the peaks appears at the retention time of PGB and it was concluded that the developed method is selective in relation to the excipients of the final preparation.

Solution stability

The solution stability was ascertained from HPLC peak area of reference standard samples. The peak area was obtained at 2.32 min retention time with a UV detector of wavelength of 274 nm. The standard sample solutions were kept at room temperature for 15 days, it was observed that there was no change in peak area of these solutions.

Linearity

A linear calibration graph (figure 3) (y = 1730x + 2792.5; where y and x are peak area and concentration, respectively) was obtained over six concentrations 10, 20, 30, 40, 50 and 100 µg/ml. Correlation coefficient was found to be 0.9963.

Fig. 3. Linearity curve

Accuracy

The accuracy of method was calculated with 2 marketed formulations of pregabalin, Pevesca 75 and Pregalin 75 at three concentrations such as 80, 100 and 120 µg/mlin triplicate. Table 1 and 2 indicates that the recoveries at three different concentrations were found to be within the range of 98 to 102% as per ICH guidelines. Mean % recovery (mean ± SD) was found to be 99.46 ± 0.31.

Table 1. Recovery study, Pevesca 75 (n=3)

Amount added (µg/ml)	Amount recovered (µg/ml)	% Recovered
80	77.96	98.87
100	99.97	99.98
120	122.32	101.05

Table 2. Recovery study, Pregalin 75 (n=3)

Amount added (µg/ml)	Amount recovered (µg/ml)	% Recovered
80	79.91	99.95
100	99.30	99.65
120	119.56	99.80

Precision

The low RSD values as shown in table 3 indicate the ruggedness of the method.

Table 3. Precision study (n=3)

Concentration	Mean Peak area	SD	%RSD
Interday
10 µg/ml	18626.51	21.89	0.118
20 µg/ml	30239.07	67.95	0.225
40 µg/ml	63996.10	605.73	0.947
Intraday
10 µg/ml	20194.77	183.51	0.909
20 µg/ml	29788.39	177.62	0.596
40 µg/ml	63690.63	227.79	0.357

Repeatability

Table 4 indicates the repeatability data of the study.

Table 4. Repeatability study (n=6)

Concentration	% RSD^a	%RSD^b
20 µg/ml	0.31	0.22

RSD^a- Based on peak area, RSD^b- Based on retention time

Robustness

As seen in table 5 it can be said that in all varied chromatographic conditions, there was no significant change in chromatographic parameters.

Table 5. Robustness study (n=3)

Concentration	Conditions changed	% RSD	Mean RT
100 µg/ml	Flow rate
	0.8 ml/min	0.69	2.84
	1.2 ml/min	0.40	1.89

CONCLUSION:

The proposed method does not require any laborious clean up procedure before measurement. In addition, the method has wider linear dynamic range with good accuracy and precision. The method shows no interference from the common excipients and additives. Thus, the described HPLC method is suitable for routine analysis and quality control of pharmaceutical preparation containing pregabaline active pharmaceutical ingredient.

ACKNOWLEDGEMENTS:

The authors wish to thank Dr. Shirish Deshpande, Associate Dean, SPTM NMIMS for providing excellent research facilities.

REFERENCES:

1. Tassone DM, Boyce E, Guyer J, Pregabalin: a novel gammaaminobutyric acid analogue in the treatment of neuropathic pain, partial-onset seizures, and anxiety disorders, Clin Ther 2007, 29(1):26-48.

2. Hamandi K, Sander JW, Pregabalin: a new antiepileptic drug for refractory epilepsy, Seizure 2006, 15(2):73-78.

3. Freynhagen R, Strojek K, Griesing T, Efficacy of pregabalin in neuropathic pain evaluated in a 12- week, randomized, double blind, multicentre, placebo controlled trial of flexible and fixed dose regimens, Pain 2005; 115: 254- 263.

4. Ralph W, Uma S, Linda L, Relief of painful diabetic peripheral neuropathy with pregabalin: A randomized, placebo controlled trial, Pain 2005: 253- 260.

5. Bockbrader HN, Hunt T, Pregabalin pharmacokinetics and safety in healthy volunteers: Results from two phase one studies, Neurology 2000;54:421.

6. Stewart BH, Kugler AR, Thompson PR, Pharm. Res. 1993; 10: 226-228.

7. Turck D, Vollmer KO, Bockbrader H, Eur. J. Clin. Pharmacol. 1989; 36: 310.

8. Vaidya VV, Yetal SM, LC-MS-MS Determination of Pregabalin in Human Plasma, Chromatographia 2007, 66(11-12):925-928.

9. Mandal U, Agarwal S, Bose A, Determination of Pregabalin in Human Plasma Using LC-MS-MS, Chromatographia 2008, 67(3-4):237-243.

10. David B, Millington C. Analysis of Pregabalin at Therapeutic Concentrations in Human Plasma/Serum by Reversed- Phase HPLC, Ther Drug Monit 2005, 27(4):451-456.

11. Gujral RS. A novel method for the determination of pregabalin in bulk pharmaceutical formulations and human urine samples, Afr J Pharm Pharmacol 2009, 3(6):327-334.

12. Kasawar GB, Farooqui MN. Development and validation of HPLC method for the determination of pregabalin in capsules, Indian J Pharm Sci 2010, 72(4):517-519.

13. Shah GR, Ghosh C, Thaker BT. Determination of pregabalin in human plasma by electrospray ionisation tandem mass spectroscopy, J Adv Pharm Tech Res 2010, 1:354-357.

14. Vermeij TAC, Edelbroek PM. Simultaneous high-performance liquid chromatographic analysis of pregabalin, gabapentin and vigabatrin in human serum by precolumn derivatization with o-phtaldialdehyde and fluorescence detection, J Chromatogr B Analyt Technol Biomed Life Sci 2004, 810(2):297-303.

15. Jadhav AS, Pathare DB, Shingare MS. Validated enantioselective LC method, with precolumn derivatization with Marfey’s reagent, for analysis of the antiepileptic drug pregabalin in bulk drug samples, Chromatographia 2007, 65(3-4):253-256.

16. Béni S, Sohajda T, Separation and characterization of modified pregabalins in terms of cyclodextrin complexation, using capillary electrophoresis and nuclear magnetic resonance, J Pharm Biomed Anal 2010, 51(4):842-852.

17. Rasha Abdel-Aziz Shaalan. Spectrofluorimetric and Spectrophotometric Determination of Pregabalin in Capsules and Urine Samples, Int J Biomed Sci 2010, 6(3):260-267.

18. Food and Drug Administration of the United States. Guidance for industry - Bioanalytical Method Validation, U.S. Department of Health and Human Services, Centre for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM). 2001.

19. http://www.fda.gov/cder/guidance/index.htm.

Received on 08.05.2012 Modified on 28.05.2012

Research J. Pharm. and Tech. 5(6): June 2012; Page 829-833