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Gradient reversed-phase liquid chromatography coupled on-line to receptor-affinity detection based on the urokinase
A J Oosterkamp1, R van der Hoeven, W Glässgen
1Leiden/Amsterdam Center for Drug Research, Division of Analytical Chemistry, University of Leiden, The Netherlands.
Abstract:
A postcolumn receptor-affinity detection (RAD) was developed for the detection of urokinase and cross-reactive compounds. The analytical method consisted of gradient reversed-phase HPLC coupled on-line to a RAD system based on fluorescein-labelled urokinase receptor (fluorescein-uPAR) as reagent. Fluorescein-uPAR was added continuously to the HPLC effluent to react with analytes eluting from the LC column. Unreacted fluorescein-uPAR was removed by a short affinity column packed with an immobilised urokinase support. The analyte-bound fluorescein-uPAR fraction passes the affinity column unretained and was detected downstream by means of a fluorescence detector. An absolute detection limit of 40 fmol urokinase was obtained in the flow injection mode. In the gradient HPLC-RAD system a detection limit of 40 nM (20-microl injection, absolute amount, 800 fmol) was obtained. The present method allowed the identification of active breakdown products of urokinase both in standard samples and biological matrices.