Tian Tingting, Jin Yiran, Ma Yinghua, Xie Weiwei, Xu Huijun, Zhang Kerong, Zhang Lantong, Du Yingfeng
Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University, Shijiazhuang, Hebei 050017, PR China.
The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei 050000, PR China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Dec 1;1006:80-92. doi: 10.1016/j.jchromb.2015.10.006. Epub 2015 Oct 21.
Oridonin (ORI) is an active natural ent-kaurane diterpenoid ingredient originating from well-known traditional Chinese herb medicine and is expected to be pursued as a new anticancer agent. In the present study, a novel and efficient approach was developed for in vivo screening and identification of ORI metabolites using ultra high performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UPLC-Triple-TOF-MS/MS). This analytical strategy was as follows: an effective on-line data acquisition method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS), was developed to trace all of potential metabolites of ORI. The MMDF and DBS method could trigger an information dependent acquisition scan, which could give the information of low-level metabolites masked by background noise and endogenous components in complex matrix. Moreover, the sensitive and specific multiple data-mining techniques including extracted ion chromatography, mass defect filtering, product ion filtering and neutral loss filtering were employed to identify the metabolites of ORI. Then, structures for the metabolites were successfully assigned based on accurate masses, the mass fragmentation of ORI and metabolic knowledge. Finally, an important parameter Clog P was used to estimate the retention time of isomers. Based on the proposed strategy, 16 phase I and 2 phase II metabolites were detected in rats after oral administration of ORI. The main biotransformation route of ORI was identified as reduction, oxidation, dehydroxylation and glucuronic acid conjugation. This is the first study of ORI metabolism in vivo. This study not only proposed a practical strategy for rapidly screening and identifying metabolites, but also provided useful information for further study of the pharmacology and mechanism of ORI in vivo. At the same time this methodology can be widely applied for the structural characterization of the metabolites of other ent-kaurane diterpenoid.
冬凌草甲素（ORI）是一种活性天然对映-贝壳杉烷二萜成分，源自著名的传统中草药，有望成为一种新型抗癌药物。在本研究中，开发了一种新颖且高效的方法，使用超高效液相色谱联用混合三重四极杆飞行时间质谱（UPLC-Triple-TOF-MS/MS）对ORI代谢物进行体内筛选和鉴定。该分析策略如下：开发了一种有效的在线数据采集方法——多重质量亏损过滤（MMDF）结合动态背景扣除（DBS），以追踪ORI的所有潜在代谢物。MMDF和DBS方法可触发信息依赖采集扫描，该扫描能够提供被复杂基质中的背景噪声和内源性成分掩盖的低水平代谢物的信息。此外，采用了包括提取离子色谱、质量亏损过滤、产物离子过滤和中性丢失过滤在内的灵敏且特异的多重数据挖掘技术来鉴定ORI的代谢物。然后，基于精确质量、ORI的质量碎片和代谢知识成功确定了代谢物的结构。最后，使用一个重要参数Clog P来估计异构体的保留时间。基于所提出的策略，口服给予ORI后在大鼠体内检测到16种I相和2种II相代谢物。确定ORI的主要生物转化途径为还原、氧化、脱羟基和葡萄糖醛酸结合。这是首次关于ORI体内代谢的研究。本研究不仅提出了一种快速筛选和鉴定代谢物的实用策略，还为进一步研究ORI在体内的药理学和作用机制提供了有用信息。同时，该方法可广泛应用于其他对映-贝壳杉烷二萜代谢物的结构表征。
