Gabapentin in Horses: Validation of an Analytical Method for Gabapentin Quantitation

Abstract

Gabapentin [1-(aminomethyl)cyclohexaneacetic acid, Neurontin], is a new anticonvulsant used as adjunctive therapy in the treatment of partial seizures in humans not controlled with standard antiseizure drugs, and it has also been used in veterinary medicine. In performance horses, gabapentin is listed as a class 3 performance-enhancing substance by the Association of Racing Commissioners International, and thus is considered to have the potential to influence the outcome of races. Therefore, we developed and validated a sensitive gas chromatographic-mass spectrometric (GC-MS) method for gabapentin detection. Gamma-aminobutyric acid-d(2) (GABA-d(2)) was used as an internal standard during solid-phase extraction; lacking the cyclohexyl ring of gabapentin, GABA-d(2) formed a lactam structure to only a minor extent. Gabapentin, on the other hand, readily formed a lactam on thermal exposure during trimethylsilyl-derivatization and/or GC analysis; electrospray-ionization MS was employed to verify that the original compound was present as the expected 171 m.w. compound. Extraction efficiency for the assay was about 60%, and a curvilinear standard curve ranging from 50 ng/mL to 3000 ng/mL provided excellent within-run and between-run coefficients of variation and accuracies over a range of low, medium, and high values. The limit of detection, defined as the concentration calculated from the mean response at zero concentration plus two times the standard deviation, was calculated at 7.6 ng/mL; the limit of quantitation, defined as the concentration calculated from the mean of the zero responses plus five times the standard deviation, was calculated at 17 ng/mL. This method will enable accurate quantification of gabapentin in equine biological fluids for use in both pharmacokinetic and forensic studies.

Pharmacokinetic profile and behavioral effects of gabapentin in the horse

“…Although Dirikolu et al. (2008) applied a GC‐MS/MS technique with similar LOQ (17 ng/mL; Lehner et al. , 2007), the authors could not include any time point beyond 20 h in their pharmacokinetic analysis because in all study animals the plasma concentrations of gabapentin at 24 h had decreased below the lowest standard concentration (50 ng/mL).…”

“…Plasma gabapentin concentrations were measured using LC‐MS/MS in positive electrospray ionization mode, following previously described techniques (Carlsson & Reubsaet, 2004; Lehner et al. , 2007).…”

“…, 2006; Gilron, 2007). In performance horses, gabapentin is listed as a class 3 performance‐enhancing substance by the Association of Racing Commissioners International (Lehner et al. , 2007).…”

Analysis of Gabapentin in Equine Plasma with Measurement Uncertainty Estimation by Liquid Chromatography-Tandem Mass Spectrometry

“…The risk and danger posed to other horses, jockeys, and drivers that are competing in the same race with a horse under masked pain cannot be underestimated. GPT is listed as a class 3 performance-enhancing substance by the Association of Racing Commissioners International (6), and thus, it is a prohibited substance and should not be present in the horse at the time of competition. GPT has been reported to be effective in humans for a broad range of acute and chronic conditions (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26).…”

“…The need to demonstrate the "chemical fingerprint" of the suspect substance in a test sample cannot be overemphasized. A method for quantification of GPT in horses using GC-MS had been reported; however, the drawback in the method is the use of time-consuming derivatization steps (6).…”