A Simple and Rapid LC-MS/MS Method for Quantification of Total Daidzein, Genistein, and Equol in Human Urine.

Shikha Saha,Paul A Kroon

doi:10.1155/2020/2359397

Abstract

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. 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 r2 > 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.

Highlights

Isoflavones belong to the polyphenols family of plant secondary metabolites [1] and are common components of the human diet
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 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
Human Study. e human intervention study rationale, design, and other details were reported previously [30] and was approved by a National Research Ethics Committee. is trial sought to assess the effects of dietary intervention with soy isoflavones and cocoa flavanols on biomarkers of cardiometabolic risk in postmenopausal women with type-2 diabetes, which is a group of the population at high risk of cardiovascular disease

Summary

Introduction

Isoflavones belong to the polyphenols family of plant secondary 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]. Previous studies showed that glucuronide and sulfate conjugates of daidzein, genistein, and equol are the main circulating metabolites in humans [8]. 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 quantified against authentic standards [9]. Sample preparation and sample cleanup before analysis by mass spectroscopy can be very complicated and time

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