Immunoassay-based determination of phenobarbital using size-exclusion chromatography

The measurement of the anti-epileptic drug phenobarbital from serum samples combining immunoassay and size-exclusion chromatography is presented. The immunoreaction is based on the competitive binding of the analyte (unlabell ed phenobarbital) and the fluorescent-labelled phenobarbital to anti-phenob arbital antibodies. Mixing of the reagents and the immunoreaction takes pla ce in a Row system. The products are separated on-line on a short gel chrom atographic column and the fluorescence intensity of the marker is measured. The calibration curve shows good linearity in the range 5-80 mug/ml, corre sponding to therapeutically relevant serum levels. Intra-day precision valu es are between 7.32 and 9.48%; the accuracy is between 0.97 and 9.43%. Inte r-day precision and accuracy measured on 6 different days fall between 5.38 and 10.05% and -8.27 and -4.97%, respectively. The results obtained with t he proposed method show a good correlation with those of other methods (rad ioimmunoassay and fluorescence polarisation immunoassay) already establishe d in clinical laboratories. (C) 2000 Elsevier Science B.V. All rights reser ved.