A Simultaneous High-Performance Liquid Chromatographic Analysis of the Most Common Anticonvulsants and Their Metabolites in the Serum

Abstract

We present a method for the simultaneous analysis of some common anticonvulsants and their metabolites (phenylethyl malonamide, primidone, ethosuximide, 5-ethyl-5-phenylhydantoin, phenobarbital, α-methyl-α-phenylsuccinimide, mephenytoin, methsuximide, mephobarbital, phenytoin, and carbamazepine) in as little as 25 µl of serum. Serum proteins are precipitated with an acetonitrile solution containing 5-(4-methylphanyl)-5-phenylhydantoin, the internal standard. The drugs are eluted from a reversed-phase column with a mobile phase consisting of an acetonitrile/phosphate buffer, at a flow rate of 3.0 ml/min. The eluted drugs are detected by their absorption at 195 nm; their quantities are estimated from their peak heights. Each analysis requires no longer than 30 minutes at the optimum column temperature of 50°C. The lower limit of detection for all these drugs is less than 10 ng/sample for drug standards. A sensitivity of 1.0 mg/liter of serum is attained routinely for each of the drugs. Analytical recoveries for the eleven drugs varied from 88 to 102%, with good day-to-day precision (CV, between 2.0 and 8.0%). Of more than 35 drugs tested for possible interference, only ethotoin and pentobarbital interfere with the analysis of phenobarbital and mephobarbital, respectively.