DETERMINATION OF THE ANTI-PLATELET-ACTIVATING FACTOR BN-50727 AND METABOLITES IN HUMAN URINE BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY USING SOLID-PHASE EXTRACTION

A sensitive and selective HPLC solid-phase extraction procedure was de veloped for the determination of platelet-activating factor antagonist BN-50727 and its metabolites in human urine. The procedure consisted in a double solid-phase extraction of the urine samples on cyanopropyl and silica cartridges, followed by an automated solid-phase extractio n of the drug and metabolites on CBA cartridges and posterior elution on-line to the chromatographic system for its separation. The method a llowed quantitation in the concentration range 10-2400 ng/ml urine for both BN-50727 and the main metabolite, the O-demethylated BN-50727 pr oduct. The limit of quantitation for both compounds was 10 ng/ml. The inter-assay precision of the method, expressed as relative standard de viation, ranged from 1.9 to 4.5% for BN-50727 and from 2.5 to 9.0% for the metabolite, The accuracy, expressed as relative error, ranged fro m -2.4 to 4.2% and from 0.2 to 6.2%, respectively. This paper describe s the validation of the analytical methodology for the determination o f BN-50727 in human urine and also for its metabolites. The method has been used to follow the time course of BN-50727 and its metabolites i n human urine after single-dose administration.