Rapid measurement of free anticonvulsant drugs by direct liquid chromatography of serum ultrafiltrates.

In this simultaneous assay of unbound phenobarbital, phenytoin, primidone, and carbamazepine, serum ultrafiltrate is prepared by centrifugation in disposable filter units (Centrifree Micropartition System, Amicon Corp.) and injected directly onto a reversed-phase liquid-chromatography column. Drugs in the effluent are detected by absorbance at 210 nm. The measured concentrations were not critically dependent on the exact conditions of ultrafiltration (rotor angle, speed or time of centrifugation, amount of sample filtered). Pooled serum containing all four drugs gave reproducible results in repeated analyses during several days. For comparison we assayed 113 clinical specimens for one or more of these drugs by this method, and for the total (bound plus unbound) concentration of each drug by immunoassay. For each drug there was a nearly linear relationship between the two types of measurements, except for six specimens that had unusually high fractions of unbound drug. This assay appears highly suitable for routine determination of the free, biologically active fraction of anticonvulsant drugs, possibly as an alternative to measurements of total drug concentrations.