Research Article A Simple and Rapid LC-MS/MS Method for Quantification of Total Daidzein, Genistein, and Equol in Human Urine Shikha Saha and Paul A Kroon Quadram Institute Bioscience, Norwich Research Park, Norwich NR4 7UQ, UK Correspondence should be addressed to Paul A Kroon; paul.kroon@quadram.ac.uk Received 14 October 2019; Revised 20 December 2019; Accepted 26 December 2019; Published 20 January 2020 Academic Editor: Krishna K. Verma Copyright © 2020 Shikha Saha and Paul A Kroon. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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 β-glucuronidase and sulfatase mixture. e 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. e correlation coefficients values are r 2 > 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. is 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. 1. Introduction Isoflavones belong to the polyphenols family of plant sec- ondary metabolites [1] and are common components of the human diet. Daidzein and genistein are isoflavones, and equol is the end metabolite of daidzein produced by the metabolic action of a particular intestinal bacteria [2]. ere is much supporting evidence that isoflavones have health- promoting effects and alleviate many diseases [3]; its role in the reduction of hormone-related diseases, cancers, meno- pausal problems, osteoporosis, and cardiovascular diseases has all been reported [4–7]. Previous studies showed that glucuronide and sulfate conjugates of daidzein, genistein, and equol are the main circulating metabolites in humans [8]. To quantify daidzein, genistein, and equol in human bodily fluids, it is common practice to first hydrolyse samples with β-glucuronidase and/or sulfatase enzymes. e hydrolysed products are the aglycons, daidzein, genistein, and equol, which are then quantified against authentic standards [9]. e hydrolysis reaction is affected by pH, temperature, hydrolysis time, concentration, the source of the enzyme, and the kind of sample matrix. After hydrolysis, samples are cleaned up using either a solid-phase extraction (SPE) or a liquid-liquid extraction (LLE) method. A variety of chromatography and detection techniques, for example, HPLC-UV, LC-UV/PDA, fluorescence, mass spectrometry (MS) or coularray electrode array detection [10–12], and gas chromatography with mass spectroscopy detection (GC-MS) [13] have been used for quantification of the aglycone forms. Nowadays, mass spectroscopy is a common detection technique because mass spectroscopy is very sensitive and specific [14–20]. However, sample preparation and sample cleanup before analysis by mass spectroscopy can be very complicated and time Hindawi Journal of Analytical Methods in Chemistry Volume 2020, Article ID 2359397, 9 pages https://doi.org/10.1155/2020/2359397