Development of a Simple Method for the Determination of Genistein, Daidzein, Biochanin A, and Formononetin (Biochanin B) in Human Urine

Abstract

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.

The red clover isoflavone irilone is largely resistant to degradation by the human gut microbiota

“…The intact molecular ion of the TMS-ether of DHI was nearly absent. This was also true for the precursor irilone and has generally been observed for isoflavones with a hydroxy group in position C-5 [35,36]. The second metabolite peak observed in the GC chromatogram could be assigned to the TMS-ether of the enolic tautomer of DHI.…”

Determination of phytoestrogens in traditional medicinal herbs using gas chromatography–mass spectrometry

“…In previous reports, hexamethyldisilane and bis(trimethylsilyl)trifluoroacetatamide containing 1% TMCS in pyrimidine were used for derivatizing agents [14,20,23,24]. Those derivatization agents could form O-TMS functional groups from the reaction with hydroxyl groups in the phytoestrogens.…”

Determination of isoflavones in soybean food and human urine using liquid chromatography with electrochemical detection

“…A lot of chromatographic procedures for more or less efficient separation of isoflavones have been described in the literature [17,28,[45][46][47][48][49], but only part of them applied an HPLC-ED hyphenated technique [24,37,38,[50][51][52][53][54]. Recently, we have published highly efficient and sensitive methods for separation of particular isoflavones in several plant species [34,39,40].…”