Journal of Pharmaceutical and Biomedical Analysis 146 (2017) 387–401 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis j o ur na l ho mepage: www.elsevier.com/locate/jpba An integrated strategy for rapid discovery and identification of the sequential piperine metabolites in rats using ultra high-performance liquid chromatography/high resolution mass spectrometery Zhanpeng Shang a , Wei Cai b , Yanfeng Cao a , Fei Wang a , Zhibin Wang c , Jianqiu Lu d,∗ , Jiayu Zhang c,∗ a School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China b Dong Pharmaceutical Research of Hunan Key Laboratory, Hunan University of Medicine, Huaihua, Hunan 418000, China c Beijing Research Institute of Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China d Library of Beijing University of Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China a r t i c l e i n f o Article history: Received 5 July 2017 Received in revised form 6 September 2017 Accepted 8 September 2017 Available online 9 September 2017 Keywords: Piperine Ultra high-performance liquid chromatography/high resolution mass spectrometry Metabolic profile Multiple data processing methods Reverse molecular assembly strategy a b s t r a c t 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 HRMS 1 level. Then, parent ion list-dynamic exclusion cou- pled with data-dependent data-acquisition method was utilized to acquire MS n 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, hydroxy- lation, 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. © 2017 Published by Elsevier B.V. 1. Introduction Piperine is an active natural cinnamamide-type alkaloid found in many medicinal-culinary herbs, such as Piper nigrum L. (Black pepper and White pepper), Zingiber officinale (ginger) (Jiang in Chinese), Piper longum L. (Biba, mongolian medicine), Piper sar- mentosum Roxb, and Dangzuo (Tibetan medicine) [1–5]. It has been reported to have anti-carcinogenic, anti-hyperlipidemia, immunomodulatory, anti-depressant, anti-inflammatory, anti- diarrheal, anti-ulcer, anti-ischemic and anti-oxidative activities [6–14]. Meanwhile, piperine could also enhance the in vivo absorp- tion of other drugs such as aulin, amoxicillin, and phenytoin due to ∗ Corresponding authors. E-mail addresses: lujq@vip.sina.com (J. Lu), zhangjiayu0615@163.com (J. Zhang). its potential effects on regulating microsomal metabolic enzymes [15]. However, although 12 metabolites had been tentatively iden- tified in SD rats recently, the in vivo metabolic pathways of piperine might not be holistically proposed due to the absence of scientific analytical strategy [16]. And thus, it still plays a towing role to com- prehensively profile piperine metabolism which will benefit the researches for its further therapeutic applications [17,18]. Xenobiotics are metabolized into various forms via different metabolic pathways. The metabolites are often found in small amount and hence their signals are usually masked by background noise produced by endogenous substances. In past decades, with the rapid developing of liquid chromatography/mass spectrome- try (LC/MS) sensitivity, to establish a comprehensive and efficient analytical method to obtain useful information from complex back- grounds is made feasible and become a major project [19–21]. Thus, various post-acquisition data-mining methods emerged to acquire http://dx.doi.org/10.1016/j.jpba.2017.09.012 0731-7085/© 2017 Published by Elsevier B.V.