Competitive immunoassay of phenobarbital by microchip electrophoresis with laser induced fluorescence detection.

A microchip electrophoresis method with laser induced fluorescence detection was developed for the immunoassay of phenobarbital. The detection was based on the competitive immunoreaction between analyte phenobarbital and fluorescein isothiocyanate (FITC) labeled phenobarbital with a limited amount of antibody. The assay was developed by varying the borate concentration, buffer pH, separation voltage, and incubation time. A running buffer system containing 35mM borate and 15mM sodium dodecyl sulfate (pH 9.5), and 2800V separation voltage provided analysis conditions for a high-resolution, sensitive, and repeatable assay of phenobarbital. Free FITC-labeled phenobarbital and immunocomplex were separated within 30s. The calibration curve for phenobarbital had a detection limit of 3.4nM and a range of 8.6–860.0nM. The assay could be used to determine the phenobarbital plasma concentration in clinical plasma sample.