Quantification of soy isoflavones and their conjugative metabolites in plasma and urine: an automated and validated UHPLC-MS/MS method for use in large-scale studies
Quantification of soy isoflavones and their conjugative metabolites in plasma and urine: an automated and validated UHPLC-MS/MS method for use in large-scale studies
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DOI：
10.1007/s00216-014-8034-y
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发表时间：
2014-09-01
影响因子：
4.3
通讯作者：
Kulling, Sabine E.
中科院分区：
文献类型：
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作者：
Soukup, Sebastian T.;Al-Maharik, Nawaf;Kulling, Sabine E.
通过肠道微生物群和药物代谢酶的生物转化对了解其潜在的健康促进作用起着至关重要的作用。我们的工作的目的是开发一种同时，灵敏，和强大的自动化超高效液相色谱串联质谱（UHPLC-MS/MS）方法来定量大豆黄酮大豆苷元和染料木素，其共轭代谢产物，以及其主要的微生物降解产物，以提供一种方法用于大型临床试验或动物研究。使用自动化的96孔固相萃取方法从血浆和尿液中提取β-内酰胺酶分析物。分离染料木素，大豆苷元，和19个代谢产物，包括5个葡萄糖醛酸苷，7个硫酸盐，和两个磺基葡萄糖醛酸苷，以及5个微生物代谢产物，在不到25分钟内实现使用亚2 μ m的颗粒柱和梯度洗脱，乙腈/甲醇/水作为移动的相。在负离子电喷雾质谱下通过多反应监测（MRM）进行分析。根据美国食品药品监督管理局（FDA）和人用药品注册技术要求国际协调会议（ICH）的分析方法验证指导原则进行验证，包括选择性、准确度、精密度、线性、检测限、回收率、基质效应和耐用性。所有验证参数基本符合FDA和ICH要求。该方法在绝经后妇女中的药代动力学研究的应用表明，阿托伐他汀酮在体内被广泛代谢。开发了一种稳健的自动化分析方法，该方法允许处理大样本量，但仍提供了关于药物代谢物谱的详细信息，从而更好地理解和解释临床和动物研究。
The biotransformation of isoflavones by gut microbiota and by drug metabolizing enzymes plays a crucial role in the understanding of their potential health-promoting effects. The purpose of our work was to develop a simultaneous, sensitive, and robust automated ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method to quantify the soy isoflavones daidzein and genistein, their conjugative metabolites, as well as their major microbial degradation products in order to provide a method for use in large clinical trials or animal studies. An automated, 96-well solid-phase extraction method was used to extract the isoflavone analytes from plasma and urine. Separation of genistein, daidzein, and 19 of its metabolites, including five glucuronides, seven sulfates, and two sulfoglucuronides, as well as five microbial metabolites, was achieved in less than 25 min using a sub-2 mu m particle column and a gradient elution with acetonitrile/methanol/water as mobile phases. Analysis was performed under negative ionization electrospray MS via the multiple reaction monitoring (MRM). Validation was performed according to the analytical method validation guidelines of Food and Drug Administration (FDA) and International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) consisting of selectivity, accuracy, precision, linearity, limit of detection, recovery, matrix effect, and robustness. All validated parameters essentially matched the FDA and ICH requirements. The application of this method to a pharmacokinetic study in postmenopausal women showed that isoflavones are extensively metabolized in vivo. A robust automated analytical approach was developed, which allows the handling of large sample sizes but nevertheless provides detailed information on the isoflavone metabolite profile leading to a better understanding and interpretation of clinical and animal studies.