Ultra Fast Liquid Chromatography/Tandem Mass Spectrometry for Simultaneous Quantification of Ten Phytoestrogens in Plasma

Phytoestrogens have been the subject of intense study owing to their beneficial effects on human health, and identification of such compounds at the physiological concentration in biological systems has immense implication for many areas of research [1,2]. It is therefore, necessary to have analytical methods which are sensitive, reproducible, and high throughput. A rapid 2 min UFLC-MS/MS method operating in MRM mode was developed that allows for the characterization and simultaneous quantification of 10 phytoestrogen metabolites and chrysin (internal standard) 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), 272/65 (6-hydroxy-O-desmethylangolesin), 289/109 (biochanin A), 253/143 (chrysin), 267/91 (coumestrol). This method allowed to achieve ultra fast analysis without sacrificing chromatographic resolution with a lower limit of quantification (LLQ, 5 nM), except for equol. The separation was carried out on a Synergi 2.5 micron (50 × 2.0 mm, i.d.) column 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 ultra fast method is very useful for high throughput analysis of phytoestrogens and proved to be reproducible, reliable and the results were comparable to those obtained by regular flow LC/MS/MS analysis. Acknowledgements: These studies were supported in part by grants from the National Center for Complementary and Alternative Medicine and the National Institutes of Health Office of Dietary Supplements (5P50 AT-00477 – to the Purdue University-UAB Botanicals Center for Age-Related Disease, Connie Weaver, PI). The mass spectrometer was purchased by funds from a NIH/NCRR Shared Instrumentation Grant (S10 RR19231) and from this institution. Operation of the UAB Comprehensive Cancer Center Mass Spectrometry Shared Facility has been supported in part by a NCI Core Research Support Grant to the UAB Comprehensive Cancer (P30 CA13148). References: [1] Meezan E, et al. (2005) J Agric Food Chem 53: 8760–8767. [2] Prasain Jet al. (2007) Biomed Chromatogr 21: 410–414.