Analysis of phyto-oestrogens by gas chromatography-mass spectrometry

The metabolites of oestrogenic substances of plant origin, phyto-oestrogens, have been proposed as cancer-protective agents in Asian and vegetarian populations. The two principle classes of these weak oestrogens are isoflavonoids and mammalian lignans. The former is derived from soya-based foods and the latter from oilseeds, cereals and whole grains. Asian populations such as the Japanese have high plasma concentrations of the isoflavones, daidzein and genistein, whereas vegetarians excrete large quantities of the lignan enterolactone, in their urine. The concentrations of these compounds in biological samples and foods are usually determined by GC-MS, although other techniques such as HPLC and LC-MS have also been used. A simple, robust method employing isotope dilution GC-MS will be described which could be applied to the determination of phyto-oestrogens in biological samples and food matrices. Briefly, samples are hydrolysed with β-glucuronidase, the aglycones extracted and the phyto-oestrogen fraction isolated by chromatography on Sephadex LH20. This fraction is then derivatised for GC-MS by reaction with N,O-bistrimethylsilyl-triflouroacetatamide to form trimethylsilyl derivatives. Using this technique we have determined the concentrations of the lignans, enterodiol and enterolactone, and the isoflavonoids, equol, daidzein and genistein, in the serum of men from Japan (n=42). The mean levels of daidzein and genistein in these men were 82.5 ng/ml (range 1.9-577) and 158.6 ng/ml (range 5.3-852), respectively. The majority of these men (57%) produced equol concentrations of >5 ng/ml, with a mean value of 26.7 ng/ml. Mean Levels of enterodiol and enterolactone were 0.6 and 9.4 ng/ml, respectively. The levels of daidzein and genistein produced from the hydrolysis of soya bean hulls, soya bean hypocotyl, dehulled soya beans, soya flour, soya grit and soya concentrates have also been determined by this method. Soya bean hypocotyl, for example, produces 2.1 mg/g of daidzein and 1.2 mg/g genistein, whereas some concentrates yield as much as 40% isoflavones.