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

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.

Metabolomics Technologies for the Identification and Quantification of Dietary Phenolic Compound Metabolites: An Overview

“…This technique has been used for the extraction of PCM from the main dietary sources, such as extra virgin olive oil [27,107], orange juice [44], tea [38], yerba mate [42], puree of five (poly)phenol-rich berry fruits [66], cocoa products [72], and specific compounds, such as resveratrol [90] and daidzein and genistein [78].…”

“…QTrap technology has been extensively utilized to develop a targeted metabolomic approach for specific PC groups, being applied to plasma and human urine containing phenolic acid metabolites [119] or specific compounds such as chlorogenic acid [33]. Regarding flavonoids, phase-I, phase-II, or microbial metabolites were detected in plasma [97] and urine [46,78,105]. As shown in Table 3, acetic and formic acids are the major additives used to improve compound ionization.…”

“…The most common mobile phases for urine are both ammonium acetate and water (with acid), together with ACN/methanol, and for plasma, water, and ACN. Flavonoids and phenolic acids [43] Daidzein and genistein [78] Urine…”

Effects of Pasture Type on Metabolism, Liver and Kidney Function, Antioxidant Status, and Plant Secondary Compounds in Plasma of Grazing, Jersey Dairy Cattle During Mid-lactation

“…The collision gas pressure was 230 kPa, and the SRM dwell time for each transition was 15 ms. Data acquisition and integration were carried out using Shimadzu Lab Solutions software version 5.7. The analysis of equol was carried out according to a prior published method (Saha and Kroon, 2020), except that the HPLC separation was shortened from 15 to 6 min.…”

A rapid ultra high performance liquid chromatography–tandem mass spectrometry method for the quantification of daidzein, its valine carbamate prodrug, and glucuronide in rat plasma samples: Comparison of the pharmacokinetic behavior of daidzine valine carbamate prodrugs

“…Currently, several analytical methods for DAN quantification have been developed in biological matrices [18][19][20][21][22][23][24], but only a few allow simultaneous determination of its intact main phase II metabolites, such as DAN-7-G. Hosoda et al developed an HPLC-DAD method to analyze soy isoflavones and their conjugated metabolites with a sensitivity of 8 ng/mL for DAN and DAN-7-G [25]. To quantify different metabolites of DAN, the SPE method, which is expensive and requires complicated procedures for sample preparation and a run time of 35 min, was used.…”