Determination of total and free phenytoin in serum by non-isotopic immunoassays and gas chromatography.

Four different homogeneous non-isotopic immunoassays for the determination of total phenytoin in serum were evaluated and compared with a gaschromatographic method (GC) described by W. R. Külpmann & M. Oellerich [1981), J. Clin. Chem. Clin. Biochem. 19, 249-258) enzyme multiplied immunoassay technique (EMIT), fluorescence polarization immunoassay (FPIA), nephelometric inhibition immunoassay (NIIA) and substrate labeled fluorescent immunoassay (SLFIA). The between-days coefficients of variation in the medium therapeutic range were 4.0% (n = 29) with EMIT, 4.6% (n = 15) with FPIA, 7.8% (n = 10) with NIIA, 2.8% (n = 12) with SLFIA and 5.7% (n = 15) with GC. The recovery in spiked serum samples (phenytoin concentration: 11.9-99.1 mumol/l) was 98-101% with EMIT, 97-107% with FPIA, 102-110% with NIIA, 94-97% with SLFIA and 97-104% with GC. All of the tested immunoassays and GC yielded comparable results. The NIIA showed a somewhat lower correlation. In samples from an uraemic patient, however, great deviations from GC values were obtained with EMIT (bias: +22 to +85%) NIIA (+68 to +114%) and SLFIA (+48 to +52%). Only the results of FPIA were in good agreement with those of GC (bias: +1 to -7%). All the immunoassays showed a cross-reaction with 5-(4-hydroxyphenyl)-5-phenylhydantoin, which was most expressed with SLFIA and NIIA. The detectability of the immunoassays was adequate to allow precise measurements within the therapeutic range. After ultrafiltration of the serum, free phenytoin was measured by EMIT, FPIA and capillary gas chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)