Liquid chromatographic determination of six antiepileptic drugs and two metabolites in microsamples of human plasma

A simple, rapid, sensitive, and reproducible high-performance liquid chroma tographic (HPLC) method for simultaneous determination of the antiepileptic lugs (ethosuximide, primidone, lamotrigine, phenobarbital, phenytoin, and carbamazepine) and two metabolites (carbamazepine-diol and carbamazepine-ep oxide) in human plasma is described. The procedure involves extraction of t he drugs from human plasma (100 mu L) with ether using 9-hydroxymethyl-10-c arbamyl acridan as an internal standard. The extract was evaporated and rec onstituted with mobile phase and then injected onto the chromatograph. The drugs and the internal standard were eluted from a Supelcosil LC-18 stainle ss steel column at ambient temperature with a mobile phase consisting of a 0.01 M phosphate buffer/methanol/acetonitrile (65/18/17, v/v/v) adjusted to a pH of 7.5 with phosphoric acid and a flow rate of 1 mL/min. The effluent was monitored at 220 nm. Quantitation was achieved by using peak area rati o of each drug to the internal standard. The intraassay and interassay coef ficients of variation (CV) ranged from 2.43% to 6.25% and from 3.02% to 5.8 5%, respectively. The absolute (extraction) and relative (analytical) recov eries for the drugs ranged from 70.7% to 104.4% and from 88.3% to 106.1%, r espectively. Stability tests showed that the drugs were stable in plasma fo r at least 4 weeks when stored at -20 degrees C. The method was applied cli nically for monitoring the AEDs in epileptic patients.