Quantitation of Acyl Chain Oxidation Products Formed upon Thermo-oxidation of Phytosteryl/-stanyl Oleates and Linoleates.

A GC-based approach involving preseparation via solid-phase extraction was established for the quantitation of acyl chain oxidation products (ACOPs) formed upon thermo-oxidation (180 °C, 40 min) of oleates and linoleates of phytostanols and phytosterols. The concentrations of ACOPs resulting from initially formed 9-hydroperoxides (octanoates, 8-hydroxyoctanoates, 9-oxononanoates) were higher than those from 8-hydroperoxides (heptanoates, 7-hydroxy- and 7-oxoheptanoates, 8-oxooctanoates) in both oleates and linoleates. Significantly higher amounts of ACOPs were found in heat-treated linoleates compared to oleates. However, despite lower thermally induced losses of stanyl oleates and linoleates compared to the respective steryl esters, higher concentrations of ACOPs (approximately 9 and 10% of the ester losses, respectively) were observed in the heat-treated stanyl esters. In contrast, in the heated steryl oleates and linoleates the contribution of the ACOPs to the ester losses was lower (approximately 3 and 5%, respectively), and there was a more pronounced formation of oxidation products of the sterol moieties (approximately 26 and 18% of the ester losses, respectively).

Identification of Acyl Chain Oxidation Products upon Thermal Treatment of a Mixture of Phytosteryl/-stanyl Linoleates.

A mixture of phytosterols/-stanols, consisting of 75% ß-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.

On-line liquid chromatography-gas chromatography: A novel approach for the analysis of phytosterol oxidation products in enriched foods.

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.