High performance liquid chromatography tandem mass spectrometry dual extraction method for identification of green tea catechin metabolites excreted in human urine
High performance liquid chromatography tandem mass spectrometry dual extraction method for identification of green tea catechin metabolites excreted in human urine
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DOI：
10.1016/j.jchromb.2014.09.035
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发表时间：
2014-12-01
影响因子：
3
通讯作者：
Williamson, Gary
中科院分区：
文献类型：
--
作者：
Clarke, Kayleigh A.;Dew, Tristan P.;Williamson, Gary
在同一样品中同时分析游离形式和共轭类黄酮是困难的，但有必要正确估计其生物利用度。开发了一种方法，以优化提取的游离和共轭形式的儿茶素和代谢物的生物样品消费后的绿色茶。进行了一项双盲随机对照试验，其中26名志愿者每天服用绿色茶和维生素C补充剂，24名志愿者服用安慰剂，为期3个月。在4个不同的时间点（服用前和服用后）收集尿液24小时，以确认对补充剂的依从性，并区分安慰剂和补充剂的服用。在组合提取方法后，使用LC-MS 2分析评估尿液中绿色茶的游离和结合代谢物，该方法包括乙酸乙酯提取，然后乙腈蛋白沉淀。组合方法可获得EC-O-硫酸盐（91 +/- 7%）、EGC-O-葡糖苷酸（94 +/- 6%）、EC（95 +/- 6%）、EGC（111 +/- 5%）和没食子酸乙酯（74 +/- 3%）的良好回收率。共研究了55种潜在的儿茶素代谢物，其中，在绿色茶摄入后的尿液中鉴定了26种结合（与甲基、葡糖苷酸或硫酸酯基团结合）和3种游离形式（非结合）化合物。与基线（补充前）样品相比，大多数EC和EGC缀合物显著增加了绿色茶的消费后。与最高峰面积相关的结合代谢物为O-甲基-EC-O-硫酸盐和戊内酯M6/M6 '-O-硫酸盐。与先前的研究一致，EC和EGC仅被鉴定为缀合衍生物，并且未发现EGCG和ECG为单缀合或游离形式。总之，本文报道的方法提供了儿茶素化合物的良好回收，并且适用于评估类黄酮生物利用度，特别是对于可能含有内源性解缀合酶的生物组织。(C)2014爱思唯尔有限公司版权所有。
The simultaneous analysis of free-form and conjugated flavonoids in the same sample is difficult but necessary to properly estimate their bioavailability. A method was developed to optimise the extraction of both free and conjugated forms of catechins and metabolites in a biological sample following the consumption of green tea. A double-blind randomised controlled trial was performed in which 26 volunteers consumed daily green tea and vitamin C supplements and 24 consumed a placebo for 3 months. Urine was collected for 24 h at 4 separate time points (pre- and post-consumption) to confirm compliance to the supplementation and to distinguish between placebo and supplementation consumption. The urine was assessed for both free and conjugated metabolites of green tea using LC-MS2 analysis, after a combination extraction method, which involved an ethyl acetate extraction followed by an acetonitrile protein precipitation. The combination method resulted in a good recovery of EC-O-sulphate (91 +/- 7%), EGC-O-glucuronide (94 +/- 6%), EC (95 +/- 6%), EGC (111 +/- 5%) and ethyl gallate (74 +/- 3%). A potential total of 55 catechin metabolites were investigated, and of these, 26 conjugated (with methyl, glucuronide or sulphate groups) and 3 free-form (unconjugated) compounds were identified in urine following green tea consumption. The majority of EC and EGC conjugates significantly increased post-consumption of green tea in comparison to baseline (pre-supplementation) samples. The conjugated metabolites associated with the highest peak areas were O-methyl-EC-O-sulphate and the valerolactones M6/M6'-O-sulphate. In line with previous studies, EC and EGC were only identified as conjugated derivatives, and EGCG and ECG were not found as mono-conjugated or free-forms. In summary, the method reported here provides a good recovery of catechin compounds and is appropriate for use in the assessment of flavonoid bioavailability, particularly for biological tissues that may contain endogenous deconjugating enzymes. (C) 2014 Elsevier B.V. All rights reserved.