There has been substantial interest in phytoestrogens, because of their potential effect in reducing cancer and heart disease risk. Measuring concentrations of phytoestrogens in urine is an alternative method for conducting epidemiological studies. Our objective was to evaluate the urinary excretion of phytoestrogens as biomarkers for dietary phytoestrogen intake in Mexican women. Participants were 100 healthy women from 25 to 80 years of age. A food frequency questionnaire (FFQ) and a 24 h recall were used to estimate habitual and recent intakes of isoflavones, lignans, flavonols, coumestrol, resveratrol, naringenin, and luteolin. Urinary concentrations were measured by liquid chromatography (HPLC) coupled to mass spectrometry (MS) using the electrospray ionization interface (ESI) and diode array detector (DAD) (HPLC-DAD-ESI-MS). Spearman correlation coefficients were used to evaluate associations between dietary intake and urine concentrations. The habitual consumption (FFQ) of total phytoestrogens was 37.56 mg/day. In urine, the higher compounds were naringenin (60.1 µg/L) and enterolactone (41.7 µg/L). Recent intakes (24 h recall) of isoflavones (r = 0.460, p < 0.001), lignans (r = 0.550, p < 0.0001), flavonoids (r = 0.240, p < 0.05), and total phytoestrogens (r = 0.410, p < 0.001) were correlated to their urinary levels. Total phytoestrogen intakes estimated by the FFQ showed higher correlations to urinary levels (r = 0.730, p < 0.0001). Urinary phytoestrogens may be useful as biomarkers of phytoestrogen intake, and as a tool for evaluating the relationship of intake and disease risk in Mexican women.

There has been increased interest in phytoestrogens due to their potential effect in reducing the risk of developing cancer and cardiovascular disease. To evaluate phytoestrogens’ exposure, sensitive and accurate methods should be developed for their quantification in food and human matrices. The present study aimed to validate a comprehensive liquid chromatography-mass spectrometry (LC-MS) method for the quantification of 16 phytoestrogens: Biochanin A, secoisolariciresinol, matairesinol, enterodiol, enterolactone, equol, quercetin, genistein, glycitein, luteolin, naringenin, kaempferol, formononetin, daidzein, resveratrol and coumestrol, in food, serum and urine. Phytoestrogen extraction was performed by solid-phase extraction (food and serum) and liquid-liquid extraction (urine), and analyzed by LC diode-array detector (DAD) coupled with a single quadrupole MS with electrospray ionization (ESI) in negative mode. Validation included selectivity, sensibility, recovery, accuracy and precision. The method was proved to be specific, with a linear response (r² ≥ 0.97). Limits of quantification were 0.008–3.541 ng/mL for food, 0.01–1.77 ng/mL for serum and 0.003–0.251 ng/mL for urine. Recoveries were 66–113% for food, 63–104% for serum and 76–111% for urine. Accuracy and precision were below 15% (except for enterodiol in food with 18% and resveratrol in urine with 15.71%). The method is suitable for the quantification of a wide number of phytoestrogens in food, serum and urine. The method was successfully applied in highly consumed food items (n = 6) from North Mexico and biofluids from healthy women (n = 10).