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

The elevated exposure of children to hormonally active dietary phytoestroge ns has led to the need for rapid, sensitive, and precise assays for phytoes trogen metabolites in physiological matrices. Here we report the developmen t of a high-performance liquid chromatography (HPLC) MS/MS method for the q uantitative detection of seven phytoestrogens in human serum and urine. The method uses enzymatic deconjugation of the phytoestrogen metabolites follo wed by solid phase extraction (SPE) and reverse-phase HPLC. The phytoestrog ens are detected using a Sciex API III heated nebulizer atmospheric pressur e chemical ionization (HN-APCI) interface coupled with tandem mass spectrom etry. This method allows the detection of the primary dietary phytoestrogen s (isoflavones and lignans) in human serum and urine with limits of detecti on (LODs) in the low parts per billion range. The combination of tandem mas s spectrometry and chromatographic separation of the analytes helps ensure the selectivity of the method. Stable isotope-labeled internal standards fo r all seven analytes improve the precision of the assay, resulting in inter day CV values of <10% for most compounds studied. The accuracy and precisio n 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 conju gates. Therefore, the thoroughness of deconjugation for each sample was mon itored by the addition of a conjugated internal standard and subsequent det ection of deconjugated compound. This method proves to be efficacious for m easuring baseline urinary phytoestrogen levels in the American population a nd should prove useful for assessing the modulatory effects of dietary phyt oestrogens on endocrine disrupter action in children.