ANALYSIS OF PLASMA ISOFLAVONES BY REVERSED-PHASE HPLC-MULTIPLE REACTION ION MONITORING-MASS SPECTROMETRY

A HPLC-MS procedure for the rapid, sensitive and specific measurement of the isoflavones, daidzein, dihydrodaidzein, O-desmethylangolensin a nd genistein, in human plasma has been developed. Synthetic radiolabel ed genistein conjugates were used for evaluation of optimum conditions for solid phase extraction. Biochanin A was added to plasma as a reco very marker for isoflavones and phenolphthalein glucuronide and 4-meth ylumbelliferone sulfate were added to ensure completeness of hydrolysi s with beta-glucuronidase/sulfatase. Isoflavones in plasma extracts we re separated using an isocratic HPLC method and analyzed by negative i on multiple reaction ion monitoring-mass spectrometry using a heated n ebulizer-atmospheric pressure chemical ionization interface. Using pla sma samples from four subjects consuming two servings a day of an isol ated soy protein beverage for 14 days, the mean plasma genistein and d aidzein concentrations were 556 and 345 nM, respectively. Within assay and between assay coefficients of variation for measurement of daidze in and genistein in five aliquots of the same plasma sample were 8.51% and 7.76%, and 5.98% and 6.12%, respectively.