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A mixture of phytosterols/-stanols, consisting of 75% β-sitosterol, 12% sitostanol, 10% campesterol, 2% campestanol, and 1% others, was esterified with linoleic acid. The resulting mixture of phytosteryl/-stanyl linoleates was subjected to thermal oxidation at 180 °C for 40 min. A silica solid-phase extraction was applied to separate a fraction containing the nonoxidized linoleates and nonpolar degradation products (heptanoates, octanoates) from polar oxidation products (oxo- and hydroxyalkanoates). In total, 15 sitosteryl, sitostanyl, and campesteryl esters, resulting from oxidation of the acyl chain, could be identified by GC-FID/MS. Synthetic routes were described for authentic reference compounds of phytosteryl/-stanyl 7-hydroxyheptanoates, 8-hydroxyoctanoates, 7-oxoheptanoates, 8-oxooctanoates, and 9-oxononanoates, which were characterized by GC-MS and two-dimensional NMR spectroscopy. The study provides data on the formation and identities of previously unreported classes of acyl chain oxidation products upon thermal treatment of phytosteryl/-stanyl fatty acid esters.

A novel methodology for the automated qualitative and quantitative determination of phytosterol oxidation products in enriched foods via on-line liquid chromatography-gas chromatography (LC-GC) was established. The approach is based on the LC pre-separation of acetylated phytosterols and their corresponding oxides using silica as stationary phase and a mixture of n-hexane/methyl tert-butyl ether/isopropanol as eluent. Two LC-fractions containing (i) 5,6-epoxy- and 7-hydroxyphytosterols, and (ii) 7-ketophytosterols are transferred on-line to the GC for the analysis of their individual compositions on a medium polar trifluoropropylmethyl polysiloxane capillary column. Thus, conventionally employed laborious off-line purification and enrichment steps can be avoided. Validation data, including recovery, repeatability, and reproducibility of the method, were elaborated using an enriched margarine as example. The margarine was subjected to a heating procedure in order to exemplarily monitor the formation of phytosterol oxidation products. Quantification was performed using on-line LC-GC-FID, identification of the analytes was based on on-line LC-GC-MS. The developed approach offers a new possibility for the reliable and fast analysis of phytosterol oxidation products in enriched foods.

An increasing number of nutritional supplements contain phytoestrogens, in particular isoflavones, which potentially alleviate climacteric complaints. Intention of the present study was the qualitative and quantitative determination of isoflavones in soy based nutritional supplements in order to compare the actual content with the labeling of these products. For the analysis high-performance liquid chromatography (HPLC) with photo diode array (PDA) detection was used. The amount of isoflavones was determined via external calibration functions. Our analyses revealed certain variations in the isoflavone content of soy based nutritional supplements, with regard to the individual and the total amount. Furthermore, the total amount of isoflavones per serving unit in some products was higher than declared on the labeling, although only the major isoflavones were taken into account. This study shows that there is the necessity to clearly specify the isoflavone composition on the labeling and - due to safety aspects - to standardize and control the isoflavone content.

A method for the analysis of both individual phytosteryl and phytostanyl fatty acid esters in complex mixtures was established. The approach was based on a previously not described combination of three elements: (i) the formation of [M-FA+H](+) fragment ions via APCI (atmospheric pressure chemical ionization), (ii) a highly efficient UHPLC-based separation on a 1.7 μ C8 column, previously established for phytostanyl fatty acid esters, allowing the distinction of individual fatty acid esters sharing the same sterol/stanol nucleus and of isotope peaks of phytosteryl fatty acid esters and corresponding phytostanyl fatty acid esters based on these [M-FA+H](+) fragment ions, and (iii) the adjustment of the APCI conditions allowing the differential APCI-MS-SIM (single ion monitoring) detection of phytostanyl esters of linoleic and linolenic acid based on their distinct formation of a [M+H](+) ion. The usefulness of the methodology was demonstrated by the analysis of a commercially available enriched margarine. Two runs per sample allowed the quantification of 35 target analytes; the total amounts of esters were between 124.7 and 125.3g/kg, being in good agreement with the labelled 125 g/kg. Validation data were elaborated for 35 individual fatty acid esters of sitosterol, campesterol, brassicasterol, stigmasterol, sitostanol and campestanol. Recovery rates ranged from 95 to 106%; the coefficients of variation were consistently

The stability of α- (1) and β-asarone (2) in ethanolic solutions (30 %; 100 %; pH 7; pH3) was studied under daylight conditions over a period of six months. After rapid initial light-induced isomerization of 1 to 2, a number of (i) oxidation products, i.e. 2,4,5-trimethoxybenzaldehyde (4); l-(2,4,5-trimethoxyphenyl) propan-2-one (5); 2,4,5-trimethoxypropiophenone (6); and (ii) addition products, i.e. l-ethoxy-l-(2,4,5-trimethoxyphenyl) propane (3); (1R, 2R; 1S, 2S)-l-ethoxy-l-(2,4,5-trimethoxyphenyl) propan-2-ol (7A); (1R, 2S; 1S, 2R)-l-ethoxy-l-(2,4,5-trimethoxy-phenyl propan-2-ol (7B); (1R, 2R; 1S, 2S)-l-(2,4,5-trimethoxyphenyl) propan-1, 2-diol (8A); (1R, 2S; 1S 2R)-1-(2,4,5-trimethoxyphenyl) propan-1,2-diol (8B); as well as (iii) the dimers l-(2′,4′,5′-trimethoxyphenyl)-2-methyl-3-(2,4,5-trimethoxyphenyl)-lE-pentene (9) and l-(2′,4′,5′-trimethoxyphenyl)-2-methyl-3-ethyl-lα, 2β, 3α (H)-4,6,7-trimethoxyindane (13) were detected after extractive sample preparation. In addition, three dimers, l-(2′,4′,5′-trimethoxyphenyl)-2-methyl-3(2,4,5-trimethoxyphenyl)-penta-1E, 4-diene (10); 1 -(2′,4′,5′-trimethoxyphenyl)-2-(2,4,5-trimethoxyphenyl)-3,4-dimethylcyclobutane (11) and l-(2′,4′,5′-trimethoxyphenyl)-2-rnethyl-3-(2,4,5-trimethoxyphenyl) prop-l-en-3-one (12) were tentatively identified. Identifications were carried out by capillary gas chromatography (HRGC) and on-line HRGC techniques, i.e. HRGC-mass spectrometry (HRGC-MS) and HRGC-Fourier transform infrared spectroscopy (HRGC-FTIR) as well as 1H-NMR spectroscopy.

Numerous studies have demonstrated the potential of isoflavones occurring in soy (Glycine max L.) and red clover (Trifolium pratense L.) to alleviate climacteric complaints. They have also shown beneficial effects on the cardiovascular system and in the prevention of osteoporosis. As a result, several companies offer nutraceuticals based on soy or red clover extracts. The aim of the present study was the isolation of pure isoflavones on a preparative scale, in order to obtain standards for biological tests and for the quantification of isoflavones in nutraceuticals and foods. High-speed countercurrent chromatography, a special type of liquid-liquid partition chromatography, was applied to the preparative isolation of isoflavones. By using this technique the major monoglucosylated and acetylated isoflavones from soy extracts were obtained after a cleaning-up step on Amberlite XAD-7 material. Furthermore, it was possible to isolate isoflavone aglycones as well as glycosides from a red clover extract. Purity and identity of the isolated isoflavones were confirmed by HPLC with DAD, HPLC-ESI multiple-MS, and NMR spectroscopy.