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Anal Bioanal Chem (2014) 406:1975–1984 DOI 10.1007/s00216-013-7612-8 RESEARCH PAPER Discovery of xanthine oxidase inhibitors from a complex mixture using an online, restricted-access material coupled with column-switching liquid chromatography with a diode-array detection system De-qiang Li & Jing Zhao & Shao-ping Li & Qing-wen Zhang Received: 22 October 2013 / Revised: 25 December 2013 / Accepted: 30 December 2013 / Published online: 9 February 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract To find potential lead compounds for antigout drug discovery, an automated online, restricted-access material coupled with column-switching liquid chromatography with a diode-array detection (RAM–LC–DAD) system was developed for screening of xanthine oxidase (XO) inhibitors and their affinity rankings in complex mixtures. The system was first evaluated by analyzing a mixture of six compounds with known inhibition of XO. Nonspecific binding to the denatured XO was investigated and used as the control for screening. Subsequently, the newly developed system was applied to screening of a natural product, Oroxylum indicum extract, and four compounds which could specifically interact with XO were found and identified as oroxin B, oroxin A, baicalin, and baicalein. The results were verified by a competitive binding test using the known competitive inhibitor allopurinol and were further validated by an inhibition assay in vitro. The online RAM–LC–DAD system developed was shown to be a simple and effective strategy for the rapid screening of bioactive compounds from a complex mixture. Keywords Xanthine oxidase inhibitors . Restricted-access material . Column switching . Liquid chromatography Abbreviations DAD Diode-array detection HPLC High-performance liquid chromatography IC50 Half-maximal inhibitory concentration LC Liquid chromatography MS Mass spectrometry D.<q. Li : J. Zhao (*) : S.<p. Li (*) : Q.<w. Zhang State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China e-mail: [email protected] e-mail: [email protected] RAM RSD SEC SPE TFC XO Restricted-access material Relative standard deviation Size-exclusion chromatography Solid-phase extraction Turbulent flow chromatography Xanthine oxidase Introduction Gout, a fairly common metabolic disorder characterized by chronic hyperuricemia, is markedly increasing in prevalence in the USA as well as other countries with established and emerging economies, and has become a health problem worldwide [1, 2]. The population with gout in the USA has been estimated at 6.1 million, and studies in the UK have reported a prevalence approaching 7 % [3]. Global studies have found an increase in mean serum urate levels in both genders during the past four decades [4]. Inhibition of xanthine oxidase (XO) is recognized as the major treatment of gout and its complications [5]. At present, allopurinol used as a primary XO inhibitor is being prescribed to prevent gout attacks, but it was claimed to have potentially serious side effects such as hypersensitivity syndrome [6]. Therefore, considerable efforts are being made to find alternative inhibitors from natural sources because of their chemical diversity and excellent efficacy as well as low side effects [7]. However, it is a great challenge to discover active compounds from complex matrices, commonly involving numerous repeated chemical analysis, sample fractionation, and biological screening. Therefore, there is an urgent need to develop rapid and simple methods for screening of XO inhibitors from natural products. At present, high-throughput screening of bioactive compounds from complex mixtures mainly relies on the binding