A Comprehensive HPLC-DAD-ESI-MS Validated Method for the Quantification of 16 Phytoestrogens in Food, Serum and Urine

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 (r(2) >= 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).