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Screening of xanthine oxidase inhibitors in complex mixtures using online HPLC coupled with postcolumn fluorescence‐based biochemical detection
Author(s) -
Li Deqiang,
Li Shaoping,
Zhao Jing
Publication year - 2014
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201301207
Subject(s) - xanthine oxidase , chemistry , hypoxanthine , allopurinol , xanthine oxidase inhibitor , chromatography , high performance liquid chromatography , xanthine , metabolite , uric acid , hyperuricemia , biochemistry , gout , probenecid , theobromine , enzyme , pharmacology , theophylline , biology , medicine , pathology
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.