Simultaneous determination of daidzein, equol, genistein and bisphenol A in human urine by a fast and simple method using SPE and GC-MS

摘要

Human diet contains weakly estrogenic compounds such as daidzein (DAI) and genistein (GEN), phytoestrogens present in soy and many vegetables as well as bisphenol A (BPA), a contaminant from packing materials and plastic containers for foods and beverages. In light of concerns about hormonally active agents, biomonitoring methods are needed to assess human exposure to such compounds. A method for simultaneous determination of DAI, its metabolite equol (EQ), GEN, and BPA by GC-MS analysis was established, validated and applied to measure concentrations in human urine. Sample preparation involves enzymatic conjugate cleavage, SPE and derivatization by silylation. For GC/MS analysis, deuterated DAI and GEN and( 13)C-BPA are used as internal standards. LOD are 4, 4, 5 and 3 ng/mL urine for DAI, EQ, GEN and BPA, respectively. Interassay variations were 9% for DAI, 15% for EQ, 18% for GEN and 10% for BPA. Simple workup and accuracy of the method are suited for biomonitoring. An analysis of urine samples from 15 adults consuming typical German food revealed dietary exposure to phytoestrogens in all samples: GEN concentrations ranged between 13 and 238 ng/mL, those for DAI ranged from 12 to 356 ng/mL. More than half of the individuals excreted also the more estrogenic metabolite EQ, at levels of 8-128 ng/mL. Higher concentrations (GEN: 820, DAI: 960 and EQ: 1740 ng/mL) were measured in a 24 h urine sample upon ingestion of soy protein (50 g with 12.9 mg DAI and 25.2 mg GEN). Only urine collected after some days on strict phytoestrogen-free diet had undetectable isoflavone levels. BPA was detected in 9 of 15 urine samples ranging from 3 to 11 ng/mL, and at 55 ng/mL in one sample. In conclusion, a reliable method to determine BPA and isoflavones in urine was established and applied in a pilot study: Biomonitoring results show much higher dietary exposure to phytoestrogens than to BPA in German adults.