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 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.
Tekeł
J
J
Faculty of Pharmacy, Comenius University, Odbojarov 10, SK 832 32 Bratislava, Slovak Republic.
Daeseleire
E
E
Heeremans
A
A
van Peteghem
C
C
D016588:Anticarcinogenic Agents / Q000652:urine*; D002845:Chromatography / Q000379:methods; D004968:Estrogens, Non-Steroidal / Q000652:urine; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D019833:Genistein / Q000652:urine*; D006801:Humans; D007202:Indicators and Reagents; D007529:Isoflavones / Q000652:urine*; D018629:Spectrometry, Mass, Secondary Ion / Q000379:methods
D016588:Anticarcinogenic Agents; D004968:Estrogens, Non-Steroidal; D007202:Indicators and Reagents; D007529:Isoflavones; C007768:formononetin; C004742:daidzein; D019833:Genistein; C004541:biochanin A