Single-shot multi-reaction monitoring of intact marker conjugates for quantitative profiling of human major microsomal glucuronidations and its utility to screen inhibitors from medicinal herbs.
UDP-glucuronosyltransferase (UGT) is a polymorphic family of conjugating enzymes responsible for the elimination of a myriad of xenobiotics and endogenous compounds. The precise reaction phenotyping of this multi-isoform superfamily is hampered by a lack of fast generic methods for directly measuring the diverse glucuronoconjugate metabolites for comprehensive profiling of UGT isoform-specific glucuronidations. We report here a single-shot liquid chromatography-tandem mass spectrometry (LC-MS/MS) method enabling the simultaneous direct measurement of nine intact glucuronides from hepatic microsomal glucuronidations mediated by a battery of isoforms (1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 2B10, 2B15, and 2B17), which represent the majority of human UGTs in drug metabolism. This new method is based on post-incubation pooling of the individual probe reaction samples for nine-in-one cassette analysis with polarity switching multiple reaction monitoring (MRM) of all the marker glucuronides within a single LC-MS/MS injection. The pooled sample strategy overcomes the cross-interferences among the cocktail substrates and also increases the throughput. The periodic polarity switching of the LC-MRM acquisition expands the glucuronide profiling coverage using a generic single-run analysis. The source-induced dissociation of the glucuronoconjugates was evaluated as a generic alternative for their quantitation as their free aglycones, but a significant bias occurs against the traditional assumption that the parent substrates could be used as the surrogates for quantifying their glucuronide metabolites without authentic standards. After collective validations for analyte quantitation and enzyme kinetics, this single-shot cassette quantitative profiling approach may prove useful in large-scale phenotyping of human glucuronidations and rapid screening for UGT inhibitors in natural products. Graphical abstract Multi-reaction monitoring of intact conjugate metabolites for quantitative profiling of human major glucuronidations.
Key Laboratory of Phytochemical R&D of Hunan Province, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha, Hunan, 410081, China.
Yuqing
Y
Cheng
Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), Hunan Normal University, Changsha, Hunan, 410081, China.
Jing
J
Zhou
College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, 410083, China.
Meiling
M
Wang
Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), Hunan Normal University, Changsha, Hunan, 410081, China.
Yanyan
Y
Liu
Key Laboratory of Phytochemical R&D of Hunan Province, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha, Hunan, 410081, China. binguo@hunnu.edu.cn.
Bin
B
Guo
Key Laboratory of Phytochemical R&D of Hunan Province, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha, Hunan, 410081, China.
Bo
B
Chen
D015415:Biomarkers / Q000032:analysis; D002853:Chromatography, Liquid; D016903:Drug Monitoring / Q000379:methods*; D004365:Drugs, Chinese Herbal / Q000493:pharmacokinetics / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000493:pharmacokinetics / Q000494:pharmacology*; D020719:Glucuronides / Q000032:analysis*; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors* / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008862:Microsomes, Liver / Q000201:enzymology; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
D015415:Biomarkers; D004365:Drugs, Chinese Herbal; D004791:Enzyme Inhibitors; D020719:Glucuronides; C070600:yin zhi huang; D014453:Glucuronosyltransferase
Glucuronides; Inhibition screening; Liquid chromatography-tandem mass spectrometry (LC-MS/MS); Polarity switching; Source-induced dissociation; UDP-glucuronosyltransferase (UGT)