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An integrated approach based on ultra high-performance liquid chromatography/high resolution mass spectrometry coupled with various data-mining methods was developed for metabolite profiling of piperine.Reverse molecular assembly strategy based on paired diagnostic product ions and neutral loss fragments was constructed to achieve rapid structural elucidation.A total of 148 metabolites as well as parent compound were positively or tentatively identified.Piperine, one of the major bioactive constituents isolated from natural flavorings and medicinal-culinary herbs, possesses various biological activities. In the present study, an integrated strategy based on ultra high-performance liquid chromatography/high resolution mass spectrometry was established to reveal piperine metabolism in rats. First of all, post-acquisition data-mining methods, including high resolution extracted ion chromatograms (HREICs) and multiple mass defect filtering (MMDF), were used to screen piperine metabolite candidates in a full-scan HRMS1 level. Then, parent ion list-dynamic exclusion coupled with data-dependent data-acquisition method was utilized to acquire MSn datasets. In addition, the established reverse molecular assembly (RMA) approach based on paired diagnostic product ions (pDPIs) coupled with neutral loss fragments (NLFs) was used to ascertain and identify the major-to-trace piperine metabolites efficiently. And then, the calculated ClogP values were utilized to distinguish the positional isomers. As a result, a total of 148 piperine metabolites were detected and characterized tentatively. The results demonstrated that piperine mainly underwent hydrogenation, dehydrogenation, hydroxylation, glucuronide conjugation, sulfate conjugation, ring-cleavage, and their composite reactions. Our results not only provided novel and useful data to better understand the safety, toxicity and efficacy of this potential therapeutic agent, but also indicated that the proposed strategy was reliable for a rapid discovery and identification drug-related constituents in vivo.