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

Abstract

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.

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

“…Cholesteryl octanoate and cholesteryl 9-oxononanoate were synthesized via esterification with the respective acid, and cholesteryl 9hydroxynonanoate was obtained by reduction of cholesteryl 9oxononanoate with sodium borohydride as described in a previous publication for the corresponding sitosteryl esters. 10 The NMR spectra of cholesteryl octanoate and cholesteryl 9-oxononanoate corresponded to previously published data. 12,13 The identity of cholesteryl 9hydroxynonanoate was confirmed by two-dimensional NMR of the fatty acid part ( Thermal Treatment of Phytosteryl/-stanyl Oleates and Linoleates.…”

“…This sample was directly subjected to GC-FID/MS analysis for the determination of nonpolar oxidation products. Their identification was based on retention times and mass spectra as previously described; 10 quantitation was performed relative to the internal standard cholesteryl octanoate using a response factor (R f ) of 1.0 as default value.…”

“…After removal of the solvent under a gentle stream of nitrogen, the polar oxidation products were separated from nonoxidized esters by silica solid-phase extraction as described recently. 10 Briefly, nonoxidized esters and nonpolar oxidation products were eluted from a silica SPE cartridge with cyclohexane (fraction 1), followed by the elution of the polar oxidation products with MTBE (fraction 2). The residue of the polar MTBE fraction was silylated with 100 μL of pyridine and 100 μL of BSTFA/TMCS for 20 min at 80 °C.…”

“…The silylation reagent was evaporated under a gentle nitrogen stream; the residue was dissolved in 100−400 μL of hexane/MTBE (3:2, v/v) and subjected to GC-FID/MS analysis. Identification of polar oxidation products was based on retention times and mass spectra, 10 and quantitation was done relative to the internal standards using a response factor (R f ) of 1.0 as default value.…”

“…The oxidation products covered by the employed GC-based methodology comprised three classes: phytosteryl/stanyl alkanoates (heptanoates, octanoates), hydroxyalkanoates (7-hydroxyheptanoates, 8-hydroxyoctanoates), and oxoalkanoates (7-oxoheptanoates, 8-oxooctanoates, 9-oxonona-noates). 10 Representative structures are shown in Figure 1.…”