Rapid micromethod for measuring anticonvulsant drugs in serum by high-performance liquid chromatography

We describe an assay system for measuring phenobarbital, diphenylhydantoin, primidone, ethosuximide, and carbamazepine in 25 mul of serum. The procedure involves precipitation of proteins with an acetonitrile solution containing cyheptamide as an internal standard, and reverse-phase chromatography on a 4 mm X 30 cm column containing "muBondapak C18." The anticonvulsants are eluted with an equivolume mixture of potassium phosphate buffer (10 mmol/liter, pH 8.0) and acetonitrile at a flow rate of 0.8 ml/min, detected by their absorbance at 200 nm, and quantitated by measuring peak areas. When measurement of primidone is not required, a 254-nm detector may be used. Each analysis requires 10 min. Optimum column temperature has been found to decrease with use. Analytical recoveries for the five drugs varied from 92% to 102% with good precision (coefficients of variation between 2.8% and 9.2% for therapeutic and toxic concentrations). The results obtained with this method compare favorably with results obtained by gas-liquid chromatography (correlation coefficients between 0.77 and 0.98). In over 1300 patients' samples analyzed to date, the only drugs known to have interfered with the assay are gentamicin, diazoxide, and mephobarbital.