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A simple, reversed-phase high performance liquid chromatography method with mass spectrometric detection (HPLC-MS/MS) was developed for determination of an antiepileptic drug, phenytoin, in human plasma. The procedure involves a simple extraction step by mixing 0.2 ml plasma with 0.6 ml methanol. After centrifugation, 1 µl of the supernatant was injected onto a Zorbax SB-C18 100 mm x 3 mm, 3.5 µm column, and eluted with a mobile phase consisting in a mixture of water containing 2 mM ammonium acetate and methanol 50:50 (v/v). Detection was in MRM mode, using an electrospray positive ionization. The ion transition monitored was 253.1→(182.1+225.1). The method was evaluated in terms of linearity (between 2.0 µg/ml to 80.0 µg/ml), accuracy, precision, recovery, sensitivity. The lower limit of quanti-fication was established at 2.0 µg/ml. The simple extraction procedure and short chromatographic runtime make the method suitable for therapeutic drug monitoring studies.

Keywords: phenytoin, HPLC-MS/MS, human plasma