Simultaneous determination of 11 phytoestrogens in human serum using a 2 min liquid chromatography/tandem mass spectrometry method

Abstract

A rapid 2 min liquid chromatography-tandem mass spectrometry (LC-MS/MS) method operating in multiple reaction ion monitoring mode was developed and validated that allows for the characterization and simultaneous quantification of 11 phytoestrogen metabolites with mass transitions m/z 241/119 (equol), 253/132 (daidzein), 255/149 (dihydrodaidzein), 257/108 (O-desmethylangolesin), 269/133 (genistein), 283/184 (glycitein), 267/191 (formononetin), 289/109 (biochanin A), 267/91 (coumestrol), enterodiol (301/253), and enterolactone (297/253). The method was demonstrated to be specific and sensitive, and a linear response for each phytoestrogen was observed over a range of 1-5000 ng/mL in human serum with the exception of dihydrodaidzein, whose lower limit of quantification was 2 ng/mL. The separation was carried out on a Synergi Polar-RP 2.5 micron (50 mm x 2.0 mm i.d.) column at 50 degrees C with water and acetonitrile (both containing 10 mM ammonium acetate) as the mobile phase under gradient conditions at a flow rate of 0.75 mL/min. This LC-MS/MS method is very useful for high-throughput analysis of phytoestrogens and proved to be simple, sensitive, reproducible, and reliable.

Fig. 1

Chemical structures of standard phytoestrogens.

Fig. 2

MRM chromatogram showing separation of eleven phytoestrogens using a 2 min run time.

Fig. 3

Representative MRM chromatograms of serum spiked with phytoestrogens (50 ng/mL) (A); internal standards 4-MU, phenolpthalein, and chrysin (B).

Fig. 4

Representative MRM chromatograms for phytoestrogens in plasma (5000 ng/mL) (A); 10 µL methanol blank (B).

Fig. 5

MRM chromatograms from a human serum sample showing daidzein (27.94 ng/mL), genistein (27.54 ng/mL), equol (24.68 ng/mL), DHD (32.51 ng/mL), O-DMA (19.60 ng/mL) and IS (200 ng/mL).