Careful GC optimisation gives phyto-oestrogen separation

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  • Published: May 15, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: Gas Chromatography
thumbnail image: Careful GC optimisation gives phyto-oestrogen separation

Improved methods are needed for the detection of phyto-oestrogens in foods

Phyto-oestrogens are compounds occurring in plants that mimic, to some extent, the effect of female hormones such as oestradiol. Sources include tea, soya beans, parsley, broccoli, berries and peppers. Flavones and isoflavones are two of the most common groups of phyto-oestrogens, most of which contain a number of phenolic OH groups.

Functional foods are foods that are used for a secondary purpose, usually medical, in addition to providing nourishment. Those that are believed to have medical benefits are sometimes referred to as health functional foods (HFF). Foods containing phyto-oestrogens are marketed in many countries for the alleviation of menopausal symptoms. Although phyto-oestrogens are believed to be beneficial for a variety of conditions, there is a concern that too high a level could be harmful. It is therefore important to be able to accurately monitor a wide range of these compounds in food matrices. Existing methods tend to be limited to only a few phyto-oestrogens, or else lack accuracy. The Korean researchers aimed to devise a workable GC method that could detect a wide range of phyto-oestrogens in functional foods. Derivatisation is needed for the GC of these compounds, since they are mostly too polar to readily undergo GC without it.

Hydrolysis, derivatisation and GC-MS used for phyto-oestrogen determination

Food samples were dissolved in 80% aqueous methanol with sonication. The internal standard, a mixture of three deuterated phyto-oestrogens, daidzein, genistein and catechin, was added, and the mixture was evaporated to dryness. Enzymatic hydrolysis was then carried out; this was necessary since many of the compounds were present as non-volatile derivatives, such as glucosides. The residues were dissolved in aqueous ascorbic acid (used as an antioxidant) and hydrolysed in sodium acetate buffer with β-glucuronidase and arylsulfatase enzymes at 55 °C for 3 hours. The hydrolysed compounds were extracted with MTBE (methyl tert-butyl ether), a little trimethylamine was added in order to remove any acid present and to inhibit volatilisation, and the samples were evaporated to dryness. The hydrolysis and extraction methods were carefully optimised.

Derivatisation was carried out with BSTFA/1% TMCS (N,O-bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane) at 80 °C, giving the trimethylsilyl derivatives. This combination was shown to be superior to other derivatisation methods. GC was done using an Agilent 6890 Plus instrument with an Agilent Ultra-2 polysiloxane column. The oven temperature was ramped from 160 to 270 °C at 20 °C/min, held at 270 °C for 10 minutes, ramped to 300 °C at 5 °C/min and finally held at 300 °C for 4 minutes. The GC conditions were optimised to give good separation for 21 phyto-oestrogens using standard samples. Mass spectrometry was carried out using an Agilent 5975 mass selective detector (MSD) with electron impact ionisation. Characteristic ions were identified for each of the 21 compounds and the three internal standards.

The method was validated, showing good sensitivity and linearity, and was then applied successfully to the analysis of 59 functional food samples. The foods showed a wide variety of phyto-oestrogens, although all contained at least some of the flavone kaempferol, which is found in fruit, green tea and broccoli. It has been suggested that this compound helps prevent ovarian cancer in post-menopausal women.

New GC-MS method detects a wide range of phyto-oestrogens

This new method was carefully optimised in order to detect a wide range of phyto-oestrogens with a single GC-MS run. It could usefully be applied to foodstuffs from countries other than Korea. The growing market for functional foods means that analysis of the many active compounds in them will become increasingly important in the future.

Related Links

Bulletin of the Korean Chemical Society, 2017, Early View Paper. Kim et al. GC-MS analysis of various phytoestrogens in health functional foods.

British Nutrition Foundation, Functional Foods

Wikipedia, Phytoestrogens

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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