New Use Agriculture & Natural Plant Products Program, Department of Plant Biology, Rutgers University, New Brunswick, NJ 08901, USA; Department of Medicinal Chemistry, Rutgers University, Piscataway, NJ 08854, USA.
New Use Agriculture & Natural Plant Products Program, Department of Plant Biology, Rutgers University, New Brunswick, NJ 08901, USA.
J Pharm Biomed Anal. 2018 Sep 10;159:374-383. doi: 10.1016/j.jpba.2018.06.034. Epub 2018 Jun 20.
Grape-derived products contain a wide array of bioactive phenolic compounds which are of significant interest to consumers and researchers for their multiple health benefits. The majority of bioavailable grape polyphenols, including the most abundant flavan-3-ols, i.e. (+)-catechin and (-)-epicatechin, undergo extensive microbial metabolism in the gut, forming metabolites that can be highly bioavailable and bioactive. To gain a better understanding in microbial metabolism of grape polyphenols and to identify bioactive metabolites, advanced analytical methods are needed to accurately quantitate microbial-derived metabolites, particularly at trace levels, in addition to their precursors. This work describes the development and validation of a high-throughput, sensitive and reproducible GC-QqQ/MS method operated under MRM mode that allowed the identification and quantification of 16 phenolic acid metabolites, along with (+)-catechin and (-)-epicatechin, in flavanol-enriched broth samples anaerobically fermented with human intestinal bacteria. Excellent sensitivity was achieved with low limits of detection and low limits of quantification in the range of 0.24-6.18 ng/mL and 0.480-12.37 ng/mL, respectively. With the exception of hippuric acid, recoveries of most analytes were greater than 85%. The percent accuracies for almost all analytes were within ±23% and precision results were all below 18%. Application of the developed method to in vitro samples fermented with different human gut microbiota revealed distinct variations in the extent of flavanol catabolism, as well as production of bioactive phenolic acid metabolites. These results support that intestinal microbiota have a significant impact on the production of flavanol metabolites. The successful application of the established method demonstrates its applicability and robustness for analysis of grape flavanols and their microbial metabolites in biological samples.
葡萄衍生产品含有广泛的生物活性酚类化合物，这些化合物因其多种健康益处而受到消费者和研究人员的关注。大多数可生物利用的葡萄多酚，包括最丰富的黄烷-3-醇，即（+）-儿茶素和（-）-表儿茶素，在肠道中会经历广泛的微生物代谢，形成具有高度生物利用度和生物活性的代谢物。为了更好地了解葡萄多酚的微生物代谢，并确定生物活性代谢物，需要先进的分析方法来准确定量微生物衍生的代谢物，特别是痕量水平的代谢物及其前体。本工作描述了一种高通量、灵敏和重现性好的 GC-QqQ/MS 方法的开发和验证，该方法采用 MRM 模式操作，允许在无氧条件下用人类肠道细菌发酵富含黄烷醇的肉汤样品中鉴定和定量 16 种酚酸代谢物，以及（+）-儿茶素和（-）-表儿茶素。该方法具有出色的灵敏度，检测限和定量限分别在 0.24-6.18ng/mL 和 0.480-12.37ng/mL 范围内。除了马尿酸外，大多数分析物的回收率均大于 85%。几乎所有分析物的准确度百分比都在±23%范围内，精密度结果均低于 18%。将该方法应用于用不同的人类肠道微生物群发酵的体外样品，揭示了黄烷醇代谢的程度以及生物活性酚酸代谢物的产生存在明显差异。这些结果表明肠道微生物群对黄烷醇代谢物的产生有重大影响。所建立方法的成功应用证明了其在生物样品中分析葡萄黄烷醇及其微生物代谢物的适用性和稳健性。
