Saha Shikha, Kroon Paul A
Quadram Institute Bioscience, Norwich Research Park, Norwich NR4 7UQ, UK.
J Anal Methods Chem. 2020 Jan 20;2020:2359397. doi: 10.1155/2020/2359397. eCollection 2020.
Isoflavones and isoflavandiols have shown many health benefits, such as reducing cardiovascular disease, cancer, age-related disease, and osteoporosis. However, to investigate the relationships between consumption of isoflavones and their health benefits, it is important to be able to accurately quantify exposure in the large numbers of samples typically produced in association studies (i.e., several thousands). Current methods rely on solid-phase extraction protocols for sample cleanup, resulting in protracted extraction and analysis times. Here, we describe a fast and easy sample preparation method of human urine samples for subsequent quantification of daidzein, genistein (isoflavones), and equol (isoflavandiol) using LC-MS/MS. Sample preparation involves only the addition of dimethylformamide (DMF) and formic acid (FA) after enzymatic hydrolysis of their metabolites by a -glucuronidase and sulfatase mixture. The method was validated by precision, linearity, accuracy, recoveries, limit of detection (LOD), and limit of quantification (LOQ). Linear calibration curves have been shown by daidzein, genistein, and equol. The correlation coefficients values are > 0.99 for daidzein, genistein, and equol. LOD for daidzein and genistein was 1 ng/ml and equol was 2 ng/ml. Recoveries were >90%, and the relative standard deviation for intraday (<10%) and interday (≤20% over 10 days) was good. This method is suitable for quantification of isoflavones and the microbial metabolite equol in human urine and is particularly useful where large numbers of samples require analysis.
异黄酮和异黄酮二醇已显示出许多健康益处，如降低心血管疾病、癌症、与年龄相关的疾病以及骨质疏松症的风险。然而，为了研究异黄酮摄入量与其健康益处之间的关系，能够准确量化关联研究（即数千例）中通常产生的大量样本中的暴露量非常重要。目前的方法依赖于固相萃取方案进行样品净化，导致提取和分析时间延长。在此，我们描述了一种快速简便的人尿液样本制备方法，用于随后使用液相色谱-串联质谱法（LC-MS/MS）对大豆苷元、染料木黄酮（异黄酮）和雌马酚（异黄酮二醇）进行定量分析。样品制备仅需在使用β-葡萄糖醛酸酶和硫酸酯酶混合物对其代谢物进行酶解后加入二甲基甲酰胺（DMF）和甲酸（FA）。该方法通过精密度、线性、准确度、回收率、检测限（LOD）和定量限（LOQ）进行了验证。大豆苷元、染料木黄酮和雌马酚均呈现线性校准曲线。大豆苷元、染料木黄酮和雌马酚的相关系数值均>0.99。大豆苷元和染料木黄酮的检测限为1 ng/ml，雌马酚的检测限为2 ng/ml。回收率>90%，日内相对标准偏差（<10%）和日间相对标准偏差（10天内≤20%）良好。该方法适用于人尿液中异黄酮和微生物代谢产物雌马酚的定量分析，在需要分析大量样本的情况下特别有用。
