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首页> 外文会议>American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics >A Quantitative UPLC-MS/MS Method for Metabolites of the Isothiocyanates Erucin and Sulforaphane
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A Quantitative UPLC-MS/MS Method for Metabolites of the Isothiocyanates Erucin and Sulforaphane

机译：用于异硫氰酸异素蛋白和亚磺酸盐代谢物的定量UPLC-MS / MS方法

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Isothiocyanates (ITCs) are potent inducers of human phase II enzymes and occur primarily as glucosinolates in cruciferous vegetables and are released by the action of myrosinase upon disruption of plant tissue (e.g. eating). Sulforaphane (SFN) and erucin (ERN) are ITCs with particular efficacy in many models of chemoprevention and both occur in cruciferous vegetables. ITCs are rapidly conjugated in vivo to glutathione and processed by the mercapturic acid pathway to cysteinyl-glycine, cysteine and N-acetyl cysteine derivatives for elimination in urine. We developed a UPLC-MS/MS method for determining the metabolites quantitatively in urine. UPLC-MS/MS was by electrospray positive mode on a triple quadrupole instrument (Quattro Ultima) with SRMs for each of nine metabolites (four each ERN and SFN plus free SFN). Reversed phase chromatography involved a C18 column (2.1X100mm, 1.7(mu)m) with a 0.1percent aqueous formic acid/acetonitrile gradient with 6min run time. Source parameters included 3.2kV capillary, 450 deg C desolvation, 800L/hr nitrogen flow and 35V cone. Dwell times ranged from 80-150ms. ERN metabolites and one SFN metabolite were synthesized in-house while other SFN metabolites were purchased. Urine was diluted ten-fold with 0.1percent aqueous formic acid and centrifuged before UPLC. Serum samples were protein-precipitated with acetonitrile and tissues were treated the same after probe sonication in 1percent formic acid. The UPLC-MS/MS method was applied to human urines as well as mouse serum and tissues from trials investigating the use of broccoli products for dietary intervention studies. We found that ITCs were well absorbed with recoveries in 24-hr urine as conjugates in excess of 80percent of glucosinolate dose. Surprisingly, some ERN recoveries were greater than 100percent suggesting that SFN metabolites may have interconverted post-absorption to ERN metabolites. The ratio of SFN to ERN metabolites varied greatly between human subjects but was quite consistent for a given person. In serum samples from a mouse bladder xenograft study where ERN or SFN were administered individually, SFN and ERN metabolites respectively were detected in serum clearly demonstrating interconversion. Similar metabolite profiles were observed in bladder xenograft tumors from the same mice as well as in the bladder tissue itself.
机译：异硫氰酸酯（ITC）是人II期酶的有效诱导剂，主要是芥菜蔬菜中的氨基葡萄糖酸盐，并通过近霉酶的作用释放到植物组织破坏时（例如吃）。亚磺酸盐（SFN）和芥酸素（EREN）是在许多化学预防型号中具有特殊功效的ITC，并且两者都发生在十字花果蔬菜中。 ITCs在体内迅速缀合至谷胱甘肽，并由巯基酸通路加工到半胱氨酸甘氨酸 - 甘氨酸，半胱氨酸和N-乙酰半胱氨酸衍生物以消除尿液。我们开发了一种用于定量在尿液中定量测定代谢物的UPLC-MS / MS方法。 UPLC-MS / MS在三重四极仪器（Quattro Ultima）上的电喷雾正模式，具有九个代谢物中的每一个的SRMS（每个插座和SFN加上免费SFN）。反相色谱涉及C18柱（2.1×100mm，1.7（亩），0.1％含水甲酸/乙腈梯度，具有6min运行时间。源参数包括3.2KV毛细管，450℃DESOLVATION，800L / HR氮气流量和35V锥。停留时间范围从80-150ms。在房屋内合成了代谢物和一个SFN代谢物，而购买其他SFN代谢物。用0.1分甲酸水溶液稀释10倍，并在UPLC之前离心。血清样品蛋白质沉淀，用乙腈沉淀，在1甲酸的探针超声处理后处理组织。 UPLC-MS / MS方法应用于人类尿以及来自试验的小鼠血清和组织，研究使用西兰花产品进行膳食干预研究。我们发现ITCS在24-HR尿液中的回收率很好地吸收，因为葡糖苷剂量过量的缀合物过量。令人惊讶的是，一些ERN回收率大于100％，表明SFN代谢物可能与ERN代谢物相互互连。人类受试者之间的SFN与塞代谢物的比率大大变化，但对于给定的人来说非常一致。在来自小鼠膀胱异种移植研究的血清样品中，分别在血清中单独施用ERN或SFN，分别检测SFN和塞代谢物，清楚地证明互联。在来自同一小鼠以及膀胱组织本身中，在膀胱异种移植肿瘤中观察到类似的代谢物型材。

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