Development of a high throughput 96-well plate sample preparation method for the determination of trileptal (oxcarbazepine) and its metabolites in human plasma

A high throughput preparation method for the determination of trileptal (ox carbazepine, OXC) and its mono (MHD) and dihydroxy (DHD) metabolites in hum an plasma, using 96-well plate technology, has been developed and validated according to international regulatory requirements. Preparation of plasma samples (50 mul) containing the compounds to be analysed involved solid-pha se extraction (SPE) on Empore C-18 96-well SPE plates. Eluates from the pla te were injected onto a reversed-phase column (Hypersil C-18, 3 mum) with U V detection at 210 nm. Detector response was linear over the ranges 0.2-10, 0.1-200 and 0.1-20 mu mol/l, for OXC, MHD and DHD, respectively, with rela tive standard deviations from I to 10% and mean accuracies within 4% of the nominal values (number of standard curves=3 in duplicate). The limits of q uantitation were 0.2, 0.1 and 0.1 mu mol/l, respectively. The overall mean accuracies ranged from 96 to 106% and precision was in the range 4 to 11%. Cross validation indicated no significant difference between plasma concent rations obtained using the 96-well method and the previous method using a t raditional SPE method with a 50 mg C-18 cartridge. About a threefold increa se in sample throughput and a twofold decrease of plasma volume required fo r the assays, were the main advantages obtained from the previous method. T he method was applied for the determination of 3000 plasma samples from cli nical studies. (C) 2001 Elsevier Science B.V. All rights reserved.