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.

Elimination profile of methylthiouracil in cows after oral administration.

In addition to methods for the determination of residues, there is an important need for knowledge of the fate and excretion of growth promoting substances in fattening animals. In court, often simple questions are asked, such as, over what period of time can the drug be detected?, is it possible to find residues after 2 months, etc. These questions can only be answered by conducting animal experiments. Data on excretion and distribution of thiouracils in cows are rather scarce. At the beginning of the 1980s, animal experiments with methylthiouracil (MTU) were carried out in the Laboratory of Chemical Analysis. These experiments showed that treatment of cows with MTU results in the rapid appearance of the drug in plasma, urine and milk, whereas MTU selectively accumulates in the thyroid gland. The results of these experiments were only published in media with limited access (thesis, abstracts) and also there has been a considerable improvement in data handling with computer programs in the last 15 years. This investigation reinterprets the 'old' analytical data from animal experiments using a pharmacokinetic software package.