LC-MSMS Determination of Pregabalin in Human Plasma

2007, 66, 925928

Vikas V. Vaidya&, Santosh M. Yetal, Shikha M. N. Roy, Noel A. Gomes, Santosh S. Joshi
Department of Chemistry, Ramnarain Ruia College, Matunga, Mumbai 400 019, India; E-Mail: vaidya_vikas@yahoo.com

Received: 25 April 2007 / Revised: 1 September 2007 / Accepted: 10 September 2007 Online publication: 26 October 2007

Abstract
A rapid, sensitive and specic method to quantify pregabalin in human plasma using metaxalone as the internal standard is described. Sample preparation involved simple protein precipitation by using acetronitrile as solvent. The extract was analyzed by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry (LC-MSMS). Chromatography was performed isocratically on Thermo Hypurity C18 5 lm analytical column, (50 mm 4.6 mm i.d.). The assay of pegabalin was linear calibration curve over the range 10.00010000.000 ng mL1. The lower limit of quantication was 10.000 ng mL1 in plasma. The method was successfully applied to the bioequivalence study of pregabalin capsules (150.0 mg) administered as a single oral dose.

present paper describes the validated method for the quantication of pregabalin in human plasma with high-pressure liquid chromatography coupled to tandem mass spectrometry. The method was based on a simple sample preparation, rapid LC separation and selective MS MS detection. The applicability of the proposed method was demonstrated for routine therapeutic drug monitoring of pregabalin used in clinical practice.

Experimental

Keywords
Column liquid chromatographymass spectrometry Pregabalin in human plasma

Chemicals and Reagents

Working standard of pregabalin was obtained from Sai Life Sciences, Hyderabad, India. Working standard of metaxalone was obtained from Lannett Company, Philadelphia, USA. Methanol and acetonitrile of HPLC grade J.T. Baker were used. Ammonium acetate and formic acid of HPLC grade were obtained from BDH (BDH Laboratory Reagents, UK), HPLC grade water was used.

Introduction
Pregabalin, chemically (S)-3-(amino methyl)-5-methylhexanoic acid has the molecular formula C8H17NO2 and molecular weight 159.23 g mol1 [1]. Pregabalin is an alpha-2-delta (a2d) ligand that has analgesic, anxiolytic and anticonvulsant activity. Pregabalin is an anticonvulsant drug used for neuropathic pain, as an adjunct therapy for partial seizures, and in generalized anxiety disorder. It was designed as a more potent successor to gabapentin. Pregabalin binds potentently to the a2d subunit of the voltage-dependent calcium channel in the central nervous system. However, the Full Short Communication DOI: 10.1365/s10337-007-0430-4 0009-5893/07/12

exact mechanism of action is unknown. Potent binding at this site reduces calcium inux at nerve terminals and, therefore, reduces the release of several neurotransmitters, including glutamate, noradrenalin and the substance pregabalin. These activities and eects result in the analgesic, anxiolytic and anticonvulsant activity exhibited by pregabalin [28]. Pregabalin has been determined in biological uids such as human plasma with high-pressure liquid chromatography coupled to uorescence detection [912]. Similarly, biological uids such as urine and rat plasma with high-pressure liquid chromatography coupled to tandem mass spectrometry [13, 14]. The

Instrumentation and Chromatographic Conditions

A Shimadzu LC-20AD system (Kyoto, Japan), equipped with two pumps, a vacuum degasser and an auto-sampler (SIL-HTc) were used in the study. Chromatographic separation was performed on Thermo Hypurity C18, 5 lm analytical column (50 mm 4.6 mm i.d.) at ambi-

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Table 1. Calculated concentration of pregabalin in calibration standard in human plasma

Theoretical concentration (ng mL1) (n = 4) Nominal conc. Mean CV (%) Accuracy (%) 9.920 11.16 6.72 112.50 99.200 93.18 4.06 93.93 992.000 1001.63 1.35 100.97 1984.000 1878.92 2.62 94.70 3968.000 3746.43 1.29 94.42 5952.000 6026.11 2.01 101.25 7936.000 7824.33 2.21 98.59 9920.000 10279.37 1.39 103.62 Slope Intercept r

0.000184

0.000131

0.993

ent temperature. The mobile phase was consisted of acetronitrile and 2 mM ammonium acetate in a ratio of 80:20 (v/v). A triple quadrapole tandem mass spectrometer API 2000 instrument (APISCIEX, Toronta, Canada) was equipped with turbo ionspray source and operated in positive ionization mode. Analyst 1.4 software package was used for instrument control and data acquisition. The ion spray voltage was set at 5.5 kv and source temperature at 400 C. The collision activation dissociation (CAD) was set at 4.0, using nitrogen as the collision gas. Instrument was set up in multiple reactions monitoring (MRM), monitoring the transitions for pregabalin 160.2 > 142.2 and metaxalone 222.2 > 161.1.

Sample Preparation
To 1.5 mL polypropylene centrifuge tube, 200 lL of plasma sample was spiked with 50 lL of internal standard (10 lg mL1) solution, 20 lL diluent (or pregabalin working calibration standard solution or quality control solution) and 25 lL 10% orthophosphoric acid. Deproteinized the acidied plasma with 1.0 mL of acetonitrile and the contents of the tube were vortexed for 10 min followed by centrifugation at 15,000g for 10 min. The supernatant layer was separated and evaporated to dryness under a stream of nitrogen at 50 C. The dry residue was dissolved with 250 lL of mobile phase, vortexed for 1 min and 10 lL of the sample was injected.

solution at 48 C) for a month, freezethaw stability at three cycle, short-term stability, an autosampler and long-term stability. For freeze-thaw stability, QC plasma samples were subjected to three cycles from 20 C to room temperature. Short-term bench top stability was performed by placing samples on the bench top at ambient temperature for 24 h. An autosampler stability was assessed by placing processed QC samples in an autosampler at 10 C for 24 h, and longterm stability was evaluated by freezing QC samples at 20 C for a month, then comparing the concentration with those of QC before the storage period. Recovery was assessed by comparing the peak areas of the neat analyte standards, standard spiked before and after extraction at three concentrations.

Preparation of the Stock Solution and Validation Calibration and Quality Control Samples
Standard stock solutions of pregabalin (1000.00 lg mL1) and metaxalone (1000.00 lg mL1) were prepared separately in methanol. For calibration curve and quality control sample, series of working calibration standard solutions at concentrations of 0.10, 1.00, 10.00, 20.00, 40.00, 60.00, 80.00 and 100.00 lg mL1 were prepared by appropriate dilution of its stock with diluent. Similarly, low, medium and high quality control working standard solutions of 3.00, 15.00 and 90.00 lg mL1, respectively were prepared in diluent. Working internal standard solution (10 lg mL1) was also prepared in diluent. Diluent used for preparation of working solution is the mixture of methanol and water in the ratio 60:40 (v/v). Stock solution was stored at 48 C and used within 30 days. Calibration curves were prepared by spiking blank plasma at concentration of 10, 100, 1,000, 2,000, 4,000, 6,000, 8,000 and 10,000 ng mL1 and quality control 30, 1,500 and 90,000 ng mL1 of pregabalin.

Method Validation
All assay validation steps were carried out according to the 2001 version of the FDA guidance for bioanalytical method validation [15]. Linearity was evaluated using a 1/x2 weighed linear regression method between wide range from subjectto over-therapeutic concentration in plasma at clinical practice. The sensitivity of the analytical procedure was expressed as the lower limit of quantication (LLOQ) or the lowest concentration on the calibration curve that can be quantitatively determined with acceptable accuracy and precision, and should be at least ve times the response compared to blank response. The specicity of assay was determined by analysis of six blank plasmas from dierent subjects. There should not be any interference from endogenous or exogenous materials observed at the retention time. The precision and accuracy of the method was determined at three QC samples levels for six duplicates together with calibration samples of dierent validation batches. The basic fundamental parameter of the method validation were determined by stock solution stability (kept the stock

Results
Linearity
Linearity was determined to assess the performance of the method. A linear least-squares regression with a weighing index of 1/x2 was performed on the peak area ratios of pregabalin drug to internal standard vs concentrations of 10, 100, 1,000, 2,000, 4,000, 6,000, 8,000 and 10,000 ng mL1 to generate a calibration curve. Regression coecient (r) greater than 0.99 and low CV (%) indicated the repeatability of the method (Table 1).

Recovery
Extraction recoveries of pregabalin in human plasma were determined by comparison of response (area) from extracted QC level samples containing a known amount of drug and internal standard to those from unextracted samples directly spiked with the same amount of the QC standard solutions of the respective concentration. The extraction recovery of the developed method determined at low, Full Short Communication

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medium and high QCs levels was 67.38, 74.01 and 68.59%. Absolute mean % recovery of the pregabalin and internal standard metaxalone was found to be 69.99 and 79.73%, respectively.

Table 2. Precision and accuracy of pregabalin in quality control samples (four dierent batches)
Intra-day (n = 6) QC (ng mL 1) Mean CV (%) Accuracy (%) 29.760 30.603 13.88 102.83 1488.000 1492.96 8.45 100.33 8928.000 9620.71 5.18 107.76 Inter-day (n = 6) 29.760 31.247 9.99 105.00 1488.000 1487.79 8.30 99.99 8928.000 9443.66 6.47 105.78

Precision and Accuracy

The intra- and inter-day precision and accuracy were evaluated by assaying the QC samples. In this assay, the inter-day precision was 9.99, 8.30, 6.47% and intraday precision was 3.88, 8.45, 5.18% at low, medium and high QCs levels for pregabalin. The inter-day accuracy was 105.00, 99.99, 105.78% and intra-day accuracy was 102.83, 100.33, 107.76% at low, medium and high QCs levels for pregabalin. Table 2 results demonstrate that the values were within the acceptable range and the method was reproducible accurate and precise.
Fig. 1. Representative chromatograms of plasma blank for pregabalin drug

Specicity and Sensitivity

Six dierent sources of blank plasma samples were extracted and analyzed for the assessment of potential interference with endogenous substances. The apparent response at the retention time of pregabalin was compared to the lower limit of quantication (10 ng mL1), Figs. 1 and 2 show representatitive chromatograms of specicity. The average signal-to-noise ratio obtained is greater than 5.0.

Stabilities
To determine the inuence of temperature on the stability of drugs in plasma matrix, the plasma quality control samples spiked with drug and analyzed under dierent condition: Drug stability after three freeze and thaw cycles was performed at LQC and HQC concentrations and the percent bias was evaluated to be as low as 1.18 and 1.51%, respectively, in comparison with freshly prepared samples. Bench top stability was done for 24 h at LQC and HQC concentrations and the % bias calculated by comparing with freshly prepared sample was found to be 0.01 and 1.14%, respectively. Auto sampler stability was also done for 24 h at the two QC concentrations LQC and HQC, respectively. The percent bias was found to be about 1.08 and 0.60%, Full Short Communication
Fig. 2. Representative chromatograms of plasma spiked with pregabalin drug at the lower limit of quantication

respectively. Long-term stability was evaluated at a percent bias of about 4.09 and 9.63% by freezing two QC concentration samples, LQC and HQC, at 20 C for a month, then comparing the concentration with those of QC before the storage period.

Discussion
The electrospray ionization of pregabalin and metaxalone produced the abundant protonated molecules ([MH+]) at m/z 160.2 and 222.2, respectively under positive ionization conditions, with evidence

of adduct formation. The quantication of the analysis was performed using the MRM mode due to high selectivity and sensitivity of MRM data acquisitions: Pregabalin 160.2 > 142.2 and metaxalone 222.2 > 161.1. LC-MSMS methods operated with the revere phase C18 column and low aqueous-high organic mobile phase have been proven to be ideal for the analysis of polar compounds in biological uids [1620]. The higher organic content in the mobile phase resulted in the sensitivity improvement via enhancement of ionization yield. It is desirable to use isotope-labled or a structure similar internal standard in an

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LC-MSMS procedure. However such compounds are not commercially available. In this study, a simple deproteinized procedure was used to treat the sample, therefore, metaxalone with similar chromatographic retention to its analyte was used as internal standard. Protein precipitation with acetronitrile was used with 500 lL plasma. The proposed method, proved to be accurate and selective, meeting the standards of bioanalysis method validation accepted by FDA.

Conclusion
A rapid, sensitive and reliable LC-MS MS method for the determination of pregabalin has been successfully developed and validated using one-step deprotenization. This assay method demonstrated acceptable sensitivity, recovery, precision, accuracy and stability. The validated method was successfully applied to assay human plasma samples from the bioequivalence study of pregabalin.

Application of Method
This method has been successfully applied to the bioequivalence study of pregabalin (150 mg capsule) in twelve healthy volunteers. After the oral administration of the pregabalin capsule to volunteers, the observed peak plasma concentration (Cmax) were 4109.06 for test and 4040.85 for reference. The time values taken to achieved (Tmax) were 1.25 for test and 1.17 for reference. In addition, the calculated 90% CI for mean Cmax, AUClast and AUC0inf individual ratio within the 80125% interval dened by the US Food and Drug Administration.

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