Screening of xanthine oxidase inhibitors in complex mixtures using online HPLC coupled with postcolumn fluorescence-based biochemical detection

Xanthine oxidase (XO) catalyzes the metabolism of hypoxanthine and xanthine to uric acid, the overproduction and/or underexcretion of which could cause the incidence of hyperuricemia such as gout. Herein, the inhibition of XO is recognized as one of the therapeutic approaches to treat gout. In the present study, an off-line fluorescence-based microplate method was first developed for an XO assay in which the enzyme converted pterin to its fluorescent metabolite isoxanthopterin. Then, a postcolumn continuous XO assay as a means of bioactivity assessment was coupled to HPLC separation to establish the online HPLC with diode array detection, biochemical detection, and MS/MS system for the screening of XO inhibitors. The availability of the online system was first tested with a positive drug, allopurinol, a well-known XO inhibitor, and subsequent analysis of Scutellaria baicalensis extract showed that two main bioactive compounds with XO inhibitory activities were observed, indicating that the developed online system was applicable to complex mixtures.