Journal of Chromatography B, 854 (2007) 63–67 Stereoselective determination of vigabatrin enantiomers in human plasma by high performance liquid chromatography using UV detection Valentina Franco a , Iolanda Mazzucchelli a , Cinzia Fattore a , Roberto Marchiselli a , Giuliana Gatti a , Emilio Perucca a,b,∗ a Clinical Pharmacology Unit, Department of Internal Medicine and Therapeutics, University of Pavia, Pavia, Italy b Institute of Neurology, IRCCS C. Mondino Foundation, Pavia, Italy Received 28 November 2006; accepted 30 March 2007 Available online 8 April 2007 Abstract A rapid and simple high-performance liquid chromatographic method for the determination of the R-(-)- and S-(+)-enantiomers of the antiepilep- tic drug vigabatrin in human plasma is described. After adding the internal standard (1-aminomethyl-cycloheptyl-acetic acid), plasma samples (200 L) are deproteinized with acetonitrile and the supernatant is derivatized with 2,4,6 trinitrobenzene sulfonic acid (TNBSA). Separation is achieved on a reversed-phase cellulose-based chiral column (Chiralcel-ODR, 250 mm × 4.6 mm i.d.) using 0.05 M potassium hexafluorophosphate (pH 4.5)/acetonitrile/ethanol (50:40:10 vol/vol/vol) as mobile phase at a flow-rate of 0.9 mL/min. Chromatographic selectivity is improved by concentrating the derivatives on High Performance Extraction Disk Cartridges prior to injection. Detection is at 340nm. Calibration curves are linear (r 2 ≥ 0.999) over the range of 0.5–40 g/mL for each enantiomer, with a limit of quantification of 0.5 g/mL for both analytes. The assay is suitable for therapeutic drug monitoring and for single-dose pharmacokinetic studies in man. © 2007 Elsevier B.V. All rights reserved. Keywords: Vigabatrin; Enantiomers; Enantioselective assay; HPLC; UV detection 1. Introduction Vigabatrin (4-amino-5-hexenoic acid; -vinyl-GABA) is an antiepileptic drug introduced in clinical practice in the early 90s [1]. It is a structural analogue of -aminobutyric acid (GABA) with a vinyl appendage (Fig. 1), and it exerts its pharmacologi- cal activity by increasing brain GABA levels through selective and irreversible inhibition of GABA-transaminase, the enzyme responsible for the degradation of GABA in the central nervous system [1,2]. Vigabatrin was widely used as adjunctive treat- ment of refractory partial seizures until the discovery, in 1997, of severe irreversible visual field constriction associated with its chronic use [3,4]. Today, vigabatrin is rarely used in the treat- ∗ Corresponding author at: Clinical Pharmacology Unit, Department of Inter- nal Medicine and Therapeutics, University of Pavia, Piazza Botta 10, 27100 Pavia, Italy. Tel.: +39 0382 986370; fax: +39 0382 22741. E-mail address: perucca@unipv.it (E. Perucca). ment of partial seizures, but it is regarded by many authorities as a drug of choice in infants with West syndrome (infantile spasms), particularly in cases associated with tuberous sclerosis [5–7]. Vigabatrin is a chiral molecule commercialized as the race- mate, but only the S-(+)-enantiomer is pharmacologically active. No chiral inversion was detected for the R-(-)-vigabatrin and its presence does not interfere with the action of S-(+)-vigabatrin [1]. Evidence has been provided that the two enantiomers differ not only in pharmacological activity, but also in pharmacokinetic properties [8,9]. This implies that the sum of the concentrations of the two enantiomers in the plasma of patients treated with vigabatrin does not provide a reliable estimate of the concentra- tion of the pharmacologically active S-(+)-enantiomer. Based on the evidence summarized above, meaningful phar- macokinetic studies with vigabatrin can only be performed by using assays which differentiate between the two enantiomers. Although few such assays have been described [10–16], they are all relatively complex and all involve the use of a mass 1570-0232/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2007.03.042