Gabapentin Bioequivalence Study: Quantification by Liquid Chromatography Coupled to Mass Spectrometry

Gabapentin [1-(aminomethyl) cyclohexaneacetic acid] is an antiepileptic drug structurally related to the neurotransmitter gammaaminobutyric acid (GABA). Gabapentin is indicated as adjunctive therapy in the treatment of partial seizures with and without secondary generalization in adults with epilepsy and for the management of postherpetic neuralgia [1]. Gabapentin can be actively transported across the brain–blood barrier and the gut via the L-system amino acid transporter, which recognizes L-isoleucine, L-leucine, Lphenylalanine and L-valine [2]. The exact mode of action of GB has not yet been clearly defined. Several cellular actions have been described but are likely to be related to multiple concentration-dependent actions resulting in seizure control [3].


Introduction
Gabapentin [1-(aminomethyl) cyclohexaneacetic acid] is an antiepileptic drug structurally related to the neurotransmitter gammaaminobutyric acid (GABA). Gabapentin is indicated as adjunctive therapy in the treatment of partial seizures with and without secondary generalization in adults with epilepsy and for the management of postherpetic neuralgia [1]. Gabapentin can be actively transported across the brain-blood barrier and the gut via the L-system amino acid transporter, which recognizes L-isoleucine, L-leucine, Lphenylalanine and L-valine [2]. The exact mode of action of GB has not yet been clearly defined. Several cellular actions have been described but are likely to be related to multiple concentration-dependent actions resulting in seizure control [3].
Maximum plasma gabapentin concentrations (mean) are attained 2 to 3 hours after a single oral 300 mg dose in healthy volunteers. Absorption kinetics of gabapentin are dose-dependent and not dose-proportional, Possibly due to a saturable transport system. Bioavailability of a single 300 mg oral dose of gabapentin is 60%, decreasing with Increasing dose. Gabapentin has some lipophilicity and readily crosses the blood-brain barrier. Its volume of distribution is large, estimated as 50 to 60 L in healthy volunteers. The drug is not bound to human plasma proteins. Elimination of gabapentin is wholly accountable by renal clearance. The elimination half-life of gabapentin is about 5 to 7 hours after a single oral dose of 200 to 400 mg. Renal impairment reduces drug clearance and augments plasma gabapentin concentrations in a linear fashion [4,5].
Mild adverse events, commonly fatigue, somnolence, ataxia and dizziness have been reported in about 75% of gabapentin recipients. Other events such as tremor, diplopia, nausea and vomiting were each experienced by < 10% of gabapentin recipients. The overall proportion of patients reporting adverse events during gabapentin administration has been calculated to be about 75%, versus 55% for placebo [4].
The objective of this study was to compare in healthy volunteers, the pharmacokinetics profiles and evaluate the bioequivalence of one test formulation of 400 mg capsule of gabapentin, elaborated by Arrow Farmacêutica S/A, Brazil (test formulation). The test formulation was compared to one commercial formulation of 400 mg of gabapentin (Neurontin ® ) by Pfizer, Brazil (reference formulation).

Study protocol
The study was performed in accordance with the Helsinki Declaration and Good Clinical Practice Guideline, and informed consent was obtained from participants prior to study commencement. The clinical part of the study was condueted at Scentryphar Clinical Research (Campinas City, São Paulo, Brazil) and the bioanalytical part at Nucleus of Bioequivalence and Clinical Research/NUBEC (São Paulo, Brazil).

Subjects
Twenty six healthy volunteers of both sexes (13 males and 13 females) who were between the ages of 20 and 45 (mean ± SEM: 28.7 ± 7.4 years), who had heights between 150.0 cm and 180.0 cm (170.0 ± 0.1 cm), and who weighed between 52.0 kg and 87.8 kg (65.7 ± 9.8 kg) and within 15% of their ideal body weight were enrolled in the study. Subjects were judged eligible for enrolment in this study if they were in compliance with all the inclusion and exclusion criteria described in the protocol.
All the subjects provided written informed consent to participate after explaining the nature and purpose of the study. The study protocol was approved by the M.M. Assert Serviços Médicos S/A Ltda with the ethical principles described in the Declaration of Helsinki, guidelines for International Conference on Harmonization-Good clinical practices (ICH-GCP).
All volunteers were healthy as assessed by physical examination, ECG, and the following laboratory tests: blood glucose, urea, creatinine, AST, ALT, alkaline phosphatase, Gamma GT, total bilirrubin, albumin and total protein, triglycerides, total cholesterol, hemoglobin, hematocrit, total and differential white cell counts and routine urine. All subjects were negative for HIV, HBV (except for serological scare) and HCV.

Drug products
The test formulation employed was Gabapentin 400 mg capsule (lot number AE87) and the reference formulation was: Neurontin ® 400 mg capsule (lot number 835F4C).

Study design
The study was performed to compare the bioavailability of two gabapentin 400 mg capsule formulation (Gabapentin from Arrow Farmacêutica S/A as test formulation and Neurontin ® from Pfizer, Brazil, as reference formulation) under fasting conditions.
The study was conducted in an open randomized 2 period crossover balanced design with a 1 week wash out period between the doses. During each period, the volunteers were hospitalized at 8:00 pm having already had a normal evening meal, and after an overnight fast they received at 7:00 am a single 400 mg capsule gabapentin dose of either formulation. Water (200 mL) was given immediately after drug administration. All volunteers were then fasted 05 hours following the drug administration, after which a standard lunch was consumed and an evening meal was provided 10 hours after dosing. No other food was permitted during the "in-house" period. Liquid consumption was permitted ad libitum after lunch but xanthine-containing drinks including tea, coffee and cola were avoided. Systolic and Diastolic arterial pressure, heart rate and temperature were recorded just before and hourly after drug administration.
Blood samples (08 mL) from a suitable antecubital vein were collected into heparin containing tubes before and 0. 20

Drug analysis
Blood samples were cooled in an bath and centrifuged at 3.000 rpm for at least 10 min at approximately 4°C. At least 3mL of plasma were dispensed into polypropylene tubes. Sample tubes were frozen at -20°C, and maintained to that temperature until analysis. All samples from a single volunteer were analyzed on the same day in order to avoid interassay variation.

Method validation
Quantitation was based on determination of relationship between gabapentin peaks areas and I.S. peaks areas. Selectivity was evaluated by extracting plasma samples of plasma from six different volunteers, including a lipemic and hemolysed plasma. Recoveries of gabapentin at the three QC concentrations and I.S. were determined by comparing peak areas of spiked plasma samples with the peak area in solutions prepared with the same nominal concentration. For precision (as relative standard deviation, R.S.D.) and accuracy (as relative error, R.E.) studies, samples were prepared at three QC and were analysed in the same day (intraday precision and accuracy), and analysed in 3 consecutive days (inter-day precision and accuracy).
The validated method show a Lower limit of quantification of 50 ng/mL and linearity > 0, 98.
The intra-day accuracy and precision of the quality control samples ranged from 91.63-100.67% and 1.14-4.73%. The inter-day accuracy and precision of the quality control samples ranged from 99.83-102.03% and 0.69-7.99%. Similar accuracy and precision values were observed during the study sample analysis.
The recovery of gabapentin was greater than 62.87%. No matrix effect on quantification was observed. The stability of gabapentin was also evaluated in post-extracted samples kept in the autosampler at room temperature (23°C) for 24 h, as well as in plasma samples kept at -20°C for 89 days and after being submitted to 3 freeze-thawing cycles (24 h each cycle). All samples described above were compared to freshly prepared gabapentin samples at the same concentration level. The method was validated according to ANVISA's (National Health Surveillance Agency of Brazilian Government) criteria and used to determine the concentration of gabapentin in volunteers' plasma.

Pharmacokinetic analysis and statistical analysis
The first-order terminal elimination rate constant (Ke) was estimated by linear regression from the points describing the elimination phase on a log-linear plot, using the software SAS ® Institute (Version 9.1.3). Elimination half-life (T ½ ) was derived from this rate constant (T ½ = ln (2)/Ke). The maximum observed plasma concentration (C max ) and the time taken to achieve this concentration (T max ) were obtained directly from the curves. The areas under the gabapentin metabolite plasma concentration versus time curves from 0 to 48 hours (AUC 0-48h ) were calculated by applying the linear trapezoidal rule. Extrapolation of these areas to infinity (AUC 0-∞ ) was done by adding the value C48/Ke to the calculated AUC 0-48h (where C48=plasma concentration calculated from the log-linear regression equation obtained for the estimation of Ke 48 hours after dose).
The bioequivalence between both formulations was assessed by calculating individual C max , AUC 0-48h , AUC 0-∞ and C max /AUC 0-48h ratios (test/reference) together with their mean and 90% confidence intervals (CI) after log transformation of the data. The inclusion of the 90% CI for the ratio in the 80% to 125% range was analyzed by nonparametric (SAS ® Institute Version 9.1.3) and parametric (ANOVA) methods.

Tolerability analysis
Gabapentin was well tolerated at the administered dose. All the biochemical parameters did not any clinical relevant alterations. No adverse effects serious were either reported or observed.

Pharmacokinetic and statistical analysis
The number of volunteers must always ensure enough statistical power to ensure the reliability of the results of the bioequivalence study. The number of volunteers in the study in question was calculated using the coefficient of variation and statistical power available in the literature. The study protocol established sufficient number of volunteers providing potential "dropouts".
The mean (± SD) plasma concentration time profile of the 2 formulations, shown in Figure 1, was similar and superimposable.
Central and dispersion measures for all pharmacokinetic parameters for both formulations are shown in Table 1, and Table 2. Table 3 presents the ratios and the respective confidence intervals for bioequivalence analysis.

Discussion
Several analytical methods have been reported for the determination of gabapentin in human plasma and biological samples. The methods are based on high-performance liquid chromatography (HPLC) with UV [6,7] or fluorescence detection [8][9][10][11][12][13], capillary electrophoresis (CE) [14,15], gas chromatography (GC) with flame ionization [16] or mass spectrometry (MS) [17][18][19], and liquid chromatography-tandem mass spectrometry (LC-MS-MS) [20][21][22][23][24][25]. The HPLC and CE methods are time-consuming because they require derivatization of gabapentin to produce a detectable chromophore. The GC methods also require derivatization of gabapentin to improve volatility and avoid column interactions. For routine analysis, derivatization step increases the time of sample preparation and cost. LC-MS-MS is currently considered the method of choice for determining gabapentin levels in biological samples because the drug can be directly detected without derivatization, thus making sample preparation time shorter.
The purpose of the present study was to develop and validate an LC-MS-MS method with simple sample preparation to determine gabapentin concentration in human plasma and to apply it to a bioequivalence study of 400 mg gabapentin capsule in 26 healthy volunteers. A rapid, sensitive and reliable LC/MS/MS method for the determination of gabapentin in human plasma has been successfully developed and validated using deproteinized extraction as sample preparation procedure. This assay method demonstrated acceptable sensitivity (LLOQ: 50 ng/mL), precision, accuracy, selectivity, recovery and stability, and less absolute and relative matrix effect. The validated method was successfully applied to assay human plasma samples from the bioequivalence study of gabapentin.   The bioavailability of a pharmaceutical form refers to the extent and speed of absorption of the active principle in contained it. Two pharmaceutical forms are said bioequivalent when, to be administered to the same individual, in the same experimental conditions and at the same dose, showed no significant differences in relation to bioavailability. In this study two formulations of gabapentin had been evaluated. The mean ratio of parameters C max and AUC 0-t and 90% confidence intervals of correspondents were calculated to determine the bioequivalence.
The AUC 0-t and AUC 0-∞ are both recognized as an uncontaminated measurement of the extent of absorption. The present study showed that 90% CI of mean AUC 0-t and AUC 0-∞ (after log-transformation of individual ratios) were included into the bioequivalence range (80-125%), consequently, the two formulations of gabapentin are equivalent for the extend of absorption.
The statistical comparison of C max , AUC 0-t and AUC 0-∞ clearly indicated no significant difference in the two formulations of gabapentin 400 mg capsule. 90% confidence intervals for the mean ratio (T/R) of C max , AUC 0-t and AUC 0-∞ were entirely within the US Food and Drug Administration acceptance range. Based on the pharmacokinetic and statistical results of this study, we can conclude that gabapentin 400 mg capsule (Arrow Farmacêutica S/A, Brazil) is bioequivalent to Neurontin ® 400 mg capsule (Pfizer, Brazil), and that then the test product can be considered interchangeable in medical practice.