GRADIENT REVERSED-PHASE LIQUID-CHROMATOGRAPHY COUPLED ONLINE TO RECEPTOR-AFFINITY DETECTION BASED ON THE UROKINASE RECEPTOR

A postcolumn receptor-affinity detection (RAD) was developed for the d etection of urokinase and cross-reactive compounds. The analytical met hod 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 fra ction passes the affinity column unretained and was detected downstrea m by means of a fluorescence detector. An absolute detection limit of 40 fmol urokinase was obtained in the flow injection mode. In the grad ient HPLC-RAD system a detection limit of 40 nM (20-mu l injection, ab solute amount, 800 fmol) was obtained. The present method allowed the identification of active breakdown products of urokinase both in stand ard samples and biological matrices. (C) 1998 Elsevier Science B.V. Al l rights reserved.