Direct determination of phenobarbital in serum or plasma by polarization fluoroimmunoassay

A polarization fluoroimmunoassay for the direct determination of phenobarbital in serum or plasma was developed, optimized, and validated. The drug was derivatized by the introduction of a p-amino function in the phenyl group to enable coupling to carrier protein (used to raise antiserum in sheep) and to fluorescein label. The assay required simply the mixing of prediluted sample, labeled drug, and antiserum, followed by a short incubation and measurement of fluorescence polarization. Normal reliability criteria were satisfied and there was good correlation with established chromatographic and radioimmunoassay methods. Nonseparation, nonisotopic immunoassays are inherently susceptible to sample interferences, but it was shown that the polarization fluoroimmunoassay was subject to inaccuracy only in the presence of markedly elevated levels of bilirubin (above 500 mumol/L), or with gross hemolysis or lipemia.