Fluorescence polarization immunoassay IV. Determination of phenytoin and phenobarbital in human serum and plasma.

Fluorescence polarization immunoassays for phenytoin and phenobarbital in human serum or plasma are described and shown to be clinically useful. No sample pretreatment, extraction, or phase separation is required. A determination can be made with less than 20 microL of sample in 15 min, with incubation at ambient temperature. Abnormal concentrations of protein, lipid, hemoglobin, or bilirubin do not interfere. In addition, drug metabolites and commonly co-administered drugs do not affect the assay results at clinically significant concentrations. Analytical recoveries of each of the two anticonvulsant drugs from serum averaged 101%. Between-assay CVs were less than 6.5%, with sensitivities of 0.5 mg/L. Comparison of this method with "high-performance" liquid chromatography, homogeneous enzyme immunoassay, and radioimmunoassay for phenytoin determinations yielded correlations of 0.99, 0.98, and 0.99, respectively. Similar comparison studies for phenobarbital yielded correlation coefficients of 0.99, 0.98, and 0.99, respectively.