Automated high-pressure liquid chromatographic assay for antiepileptic drugs and their major metabolites by direct injection of serum samples.

: We present a fully automated high-pressure liquid chromatographic (HPLC) assay for simultaneous quantitation of five antiepileptic drugs [ethosuximide (ES), primidone, phenobarbital, phenytoin, and carbamazepine] and their major metabolites [phenylethylmalondiamide (PEMA), carbamazepine-10,11-dihydro-10,11-diol (CBZD), and carbamazepine-10,11-epoxide (CBZE)] in serum samples without sample pretreatment. Two other metabolites, p-hydroxyphenobarbital (HPB) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH), can be separated as well. Interference with serum constituents prevents a quantitative determination. Serum samples are injected onto a pre-column, the biological matrix is eluted with water, and the compounds of interest are subsequently separated on an analytical column. The anticonvulsants are eluted at 60 degrees C with a mobile phase containing acetonitrile:water (21:80 by vol) at a flow rate of 1.0-2.0 ml/min (flow-step gradient). The eluted compounds are monitored at 200 nm. Each analysis requires about 30 min. Analytical recoveries varied from 90 to 98%. Within-day coefficients of variation (CVs) ranged from 0.9 to 4.9%, between-day CVs from 2.8 to 7.6%. The lower limit of detection for all drugs is 0.2 microgram/ml serum, with a sample size of 100 microliters. At therapeutic concentrations we achieved baseline separation for all eight compounds, and there was no interference from other drugs.