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

J Tekeł, E Daeseleire, A Heeremans, C Van Peteghem

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    A simple method was developed for the determination of free and/or total isoflavones 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 ChemElut 1010 column connected on-line to a Florisil cartridge by a Teflon stopcock 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 using GC/MS-SIM after separation on an HP-5MS fused silica column. TMS ethers were obtained by using BSTFA containing 1% of TMCS. For the determination of free isoflavones 6-hydroxyflavone was used as internal standard, whereas robigenin was used in the case of total isoflavone determination. Recoveries 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. Analytical curves were linear between 5 and 25 ng mL(-)(1). Detection limits varied from 1 ng mL(-)(1) for formononetin to 2.3 ng mL(-)(1) for daidzein. Recoveries for total isoflavone determination after enzymatic hydrolysis with glucuronidase from Helix pomatia ranged from 56.5 to 77.1% at the 25 ng mL(-1) level.
    Original languageEnglish
    JournalJournal of Agricultural and Food Chemistry
    Issue number9
    Pages (from-to)3489-94
    Number of pages6
    Publication statusPublished - 1999


    • Anticarcinogenic Agents
    • Chromatography
    • Estrogens, Non-Steroidal
    • Gas Chromatography-Mass Spectrometry
    • Genistein
    • Humans
    • Indicators and Reagents
    • Isoflavones
    • Spectrometry, Mass, Secondary Ion


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