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

• On-line LC–GC is a suitable approach for the analysis of phytosterol oxides.
• Acetylated phytosterols and their oxides are pre-separated via LC.
• Phytosterol oxides are on-line transferred to the GC for subsequent analysis.
• The methodology is fully automated and analyses are performed in a closed system.

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.