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 and genistein, in human plasma has been developed. Synthetic radiolabeled genistein conjugates were used for evaluation of optimum conditions for solid phase extraction. Biochanin A was added to plasma as a recovery marker for isoflavones and phenolphthalein glucuronide and 4-methylumbelliferone sulfate were added to ensure completeness of hydrolysis with beta-glucuronidase/sulfatase. Isoflavones in plasma extracts were separated using an isocratic HPLC method and analyzed by negative ion multiple reaction ion monitoring-mass spectrometry using a heated nebulizer-atmospheric pressure chemical ionization interface. Using plasma samples from four subjects consuming two servings a day of an isolated soy protein beverage for 14 days, the mean plasma genistein and daidzein concentrations were 556 and 345 nM, respectively. Within assay and between assay coefficients of variation for measurement of daidzein and genistein in five aliquots of the same plasma sample were 8.51% and 7.76%, and 5.98% and 6.12%, respectively.