Simultaneous very fast liquid-chromatographic analysis of ethosuximide, primidone, phenobarbital, phenytoin, and carbamazepine in serum.

Abstract

Abstract We describe a sensitive, specific, and very fast liquid-chromatographic assay for simultaneously determining five anticonvulsants (ethosuximide, primidone, phenobarbital, phenytoin, and carbamazepine) by using commercially available 5- or 3-microns particle size reversed-phase columns and a microflow-cell-equipped ultraviolet detector. The anticonvulsant drugs are extracted from 200 microL of serum containing 50 mg of cyclopal per liter as an internal standard, by elution from a Bond-Elut (Analytichem International, Harbor City, CA 90710) column with 300 microL of methanol. A 5-microL aliquot of the eluate is applied to an analytical column and eluted with a mobile phase of acetonitrile/methanol/phosphate buffer, 20 mmol/L, pH 3.7 (13.5/35/51.5 by vol), at a flow rate of 3.0 mL/min and at 50 degrees C. Detection is at 210 or 195 nm. The chromatography is complete in less than 2.5 min with the 5-microns-particle column, and in less than 1.4 min with the 3-microns-particle column. The sensitivity of the method for all drugs is less than 1 mg/L. Analytical recovery of drugs added to serum ranged from 92 to 109% for concentrations up to 200 mg/L. Between-run precision (CV) ranged from 1.3 to 4.1%.