Development of a simple method for the determination of genistein, daidzein, biochanin A, and formononetin (Biochanin B) in human urine

A simple method was developed for the determination of free and/or total is oflavones daidzein, genistein, and their respective 4'-methoxy derivatives biochanin A and formononetin (biochanin B) at low levels in human urine. A solid-phase extraction on octadecyl silica (C-18) columns was used for the isolation of the phytoestrogens from the matrix. An extraction on a ChemElu t 1010 column connected on-line to a Florisil cartridge by a Teflon stopcoc k was used for effective eluate purification. A mixture of dichloromethane and ethyl acetate was used for elution of the isoflavones from the columns in tandem. The isoflavones were determined as trimethylsilyl (TMS) ethers u sing GC/MS-SIM after separation on an HP-SMS fused silica column. TMS ether s were obtained by using BSTFA containing 1% of TMCS. For the determination of free isoflavones 6-hydroxyflavone was used as internal standard, wherea s robigenin was used in the case of total isoflavone determination. Recover ies for free isoflavones under study varied from 63.5 to 89.6% at the 25 ng mL(-1) level and from 63.5 to 89.2% at the 5 ng mL(-1) level in urine. Ana lytical curves were linear between 5 and 25 ng mL(-1). Detection limits var ied from 1 ng mL(-1) for formononetin to 2.3 ng mL(-1) for daidzein. Recove ries for total isoflavone determination after enzymatic hydrolysis with glu curonidase from Helix pomatia ranged from 56.5 to 77.1% at the 25 ng mL(-1) level.