HPLC-MS/MS method for the measurement of seven phytoestrogens in human serum and urine

The elevated exposure of children to hormonally active dietary phytoestrogens has led to the need for rapid, sensitive, and precise assays for phytoestrogen metabolites in physiological matrices. Here we report the development of a high-performance liquid chromatography (HPLC) MS/MS method for the quantitative detection of seven phytoestrogens in human serum and urine. The method uses enzymatic deconjugation of the phytoestrogen metabolites followed by solid phase extraction (SPE) and reverse-phase HPLC. The phytoestrogens are detected using a Sciex API III heated nebulizer atmospheric pressure chemical ionization (HN-APCI) interface coupled with tandem mass spectrometry. This method allows the detection of the primary dietary phytoestrogens (isoflavones and lignans) in human serum and urine with limits of detection (LODs) in the low parts per billion range. The combination of tandem mass spectrometry and chromatographic separation of the analytes helps ensure the selectivity of the method. Stable isotope-labeled internal standards for all seven analytes improve the precision of the assay, resulting in interday CV values of < 10% for most compounds studied. The accuracy and precision of the method were monitored over time using quality control (QC) samples containing known amounts of phytoestrogens. The majority of phytoestrogens in human sera and urine are present as their glucuronide and sulfate conjugates. Therefore, the thoroughness of deconjugation for each sample was monitored by the addition of a conjugated internal standard and subsequent detection of deconjugated compound. This method proves to be efficacious for measuring baseline urinary phytoestrogen levels in the American population and should prove useful for assessing the modulatory effects of dietary phytoestrogens on endocrine disrupter action in children.