Ultrahigh performance liquid chromatography–triple quadrupole mass spectrometry inhibitors fishing assay: A novel method for simultaneously screening of xanthine oxidase inhibitor and superoxide anion scavenger in a single analysis

Xanthine oxidase (XOD) inhibitors and superoxide anion scavengers play an important role in the treatment of gout and the inhibition of many diseases related to superoxide anion. The respective quantitation of uric acid and superoxide anion by traditional spectroscopic methods is routine in XOD inhibitors and superoxide anion scavengers screening at laboratories worldwide. In the present study, we established an ultrahigh performance liquid chromatography and triple quadrupole mass spectrometry (UHPLC–TQ-MS) method of higher accuracy and speed that combines screening of superoxide anion scavenger and XOD inhibitor in a single analysis by adding WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt) to the enzymatic reaction. We applied the established method to determine the XOD inhibitory activities and superoxide scavenging activities of some herbal extracts and compounds from natural products, which could be classified into six groups based on the results of the assay. Our innovative protocol is fast, accurate and robust. Moreover, it can eliminate false positive and false negative results which may occur in the traditional spectroscopic methods.

Graphical abstractIt is a novel method which combines screening of superoxide anion scavenger and xanthine oxidase inhibitor in a single analysis by adding WST-1 to the enzymatic reaction. This method can accurately quantify the amount of analytes by using multiple reaction monitoring (MRM) function which is through monitoring the ratio of mass and charge (m/z) of parent ions and diagnostic fragment ions, therefore, it can eliminate the false positive and false negative results.Figure optionsDownload full-size imageDownload as PowerPoint slideHighlights► A novel method to screen superoxide anion scavenger and xanthine oxidase inhibitor. ► This method can be used to detect and quantify superoxide anion. ► It is a fast, accurate and robust method. ► It can eliminate false positive and negative results.