Gradient reversed-phase liquid chromatography coupled on-line to receptor-affinity detection based on the urokinase receptor.
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.
Oosterkamp
A J
AJ
Leiden/Amsterdam Center for Drug Research, Division of Analytical Chemistry, University of Leiden, The Netherlands.
van der Hoeven
R
R
Glässgen
W
W
König
B
B
Tjaden
U R
UR
van der Greef
J
J
Irth
H
H
D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017022:Flow Injection Analysis; D019793:Fluorescein / Q000737:chemistry; D011956:Receptors, Cell Surface / Q000737:chemistry*; D055293:Receptors, Urokinase Plasminogen Activator; D013057:Spectrum Analysis
D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D019793:Fluorescein