DETERMINATION OF DRUGS IN BIOSAMPLES AT PICOMOLAR CONCENTRATIONS USING COMPETITIVE ELISA WITH ELECTROCHEMICAL DETECTION - APPLICATION TO STEROIDS

A competitive ELISA with electrochemical detection in a flow injection system (FIA) has been developed for determinations of the steroid drug budesonide in biological samples. Plasma samples were cleaned from interfering and cross-reacting compounds by two pretreatment steps consisting of a solid-phase extraction and a liquid chromatography fractionation. The enzyme label was alkaline phosphatase, which was used with p-aminophenyl phosphate (PAPP) as a substrate. The product, p-aminophenol, was detected electrochemically at a glassy carbon electrode at 250 mV (vs Ag/AgCl). The limited stability of both the substrate and the product influenced the performance of the method and had to be taken into account in the procedure by a normalization with time. Budesonide could be quantified in plasma samples down to 10 pM. The major sensitivity-limiting factor was the amperometric background response, probably due to spontaneous hydrolysis of PAPP to p-aminophenol.