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 (unlabelled phenobarbital) and the fluorescent-labelled phenobarbital to anti-phenobarbital antibodies. Mixing of the reagents and the immunoreaction takes place in a flow system. The products are separated on-line on a short gel chromatographic column and the fluorescence intensity of the marker is measured. The calibration curve shows good linearity in the range 5-80 microg/ml, corresponding to therapeutically relevant serum levels. Intra-day precision values are between 7.32 and 9.48%; the accuracy is between 0.97 and 9.43%. Inter-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 the proposed method show a good correlation with those of other methods (radioimmunoassay and fluorescence polarisation immunoassay) already established in clinical laboratories.
Bereczki
A
A
Institute of General and Analytical Chemistry, Budapest University of Technology and Economics, Hungary.
Horváth
V
V
Horvai
G
G
D000927:Anticonvulsants / Q000032:analysis* / Q000097:blood; D002850:Chromatography, Gel / Q000379:methods*; D016231:Fluorescence Polarization Immunoassay / Q000379:methods; D006801:Humans; D010634:Phenobarbital / Q000032:analysis* / Q000097:blood; D015203:Reproducibility of Results; D013997:Time Factors
D000927:Anticonvulsants; D010634:Phenobarbital