Enzyme Immunoassay in Combination with Liquid Chromatography for Sensitive and Selective Determination of Drugs in Biosamples

Abstract: Two different sensitive microtitre-plate based enzyme immunoassays were developed and evaluated for determination of the model drug compound, budesonide, in blood plasma. In the first approach, high sensitivity was obtained by using amperometric detection in a flow injection system for determination of the enzyme product p-aminophenol. The high sensitivity in the second approach was obtained using an enzyme amplified ELISA, based on the substrate recycling technique. Both immunoassays allowed for determination of the steroid, budesonide, in blood plasma samples at low picomolar concentrations, when combined with solid phase extraction and column liquid chromatography. Flow injection enzyme immunoassays based on three different formats; namely competitive with post-column reaction technique, non-competitive for haptens and the displacement format, were developed and evaluated for potential bioanalytical applications with emphasis on sensitivity, precision and throughput. Alkaline phosphatase was used as enzyme label and fluorometric or amperomtric detection was used for determination of the enzyme product. Micellar liquid chromatography was evaluated as a biocompatible sample pretreatment procedure preceding immunoassay. Blood plasma samples were injected directly into a pre-column venting system where the analyte, cortisol, was separated from cross-reacting steroids in the sample. A biocompatible mobile phase, consisting of only buffer solution and the surfactant Tween 20, was used in the chromatographic system. This allowed for direct transfer of collected fractions to the immunoassay for quantification of the analyte.