Optimization and validation of an HPLC-HRMS method through semipreparative HPLC system for determining phytosterol oxidation products during refining processing and storage of vegetable oils

A semi-preparative liquid chromatographic system (LC-Prep) has been used to isolate and collect the main oxidation products generated from β-sitosterol, campesterol, and stigmasterol, which represent the main sterols detected in vegetable oils. In less than 50 min, 15 different phytosterol oxidation products (POPs) were separated with a satisfactory resolution (R >1) and collected. From each pure phytosterol standard the 5,6α-epoxy (α-E), 5,6β-epoxy (β-E), 7-keto (7-K), 7α-hydroxy (7α-H), and 7β-hydroxy (7β-H) isomers were obtained. The purity of POPs was >90%. Then, the obtained pure POPs were used to validate an HPLC-Orbitrap-HRMS analytical method. The LOD and LOQ determined in medium chain triacylglycerols were >0.012 and >0.039 ng/mL, respectively; whereas the recoveries ranged between 82% and 98%. The suitability of the analytical method was evaluated on palm oil (PO), palm olein (POL), and high oleic sunflower oil (HOSO) during the whole refining processing (crude oil, neutralization and degumming, bleaching and deodorization) and under storage conditions (45°C, 16 days). In the refining steps, the total POPs content significantly increased (p < 0.05) by 29%, 20%, and 13% in PO, POL, and HOSO, respectively. The highest amount was found in HOSO (15.046 mg/kg), followed by POL (1.067 mg/kg) and PO (0.538 mg/kg). Under storage conditions, the content of POPs did not significantly (p > 0.05) change and was lower than 8.965 mg/kg. The developed semi-preparative liquid chromatographic system coupled to the LC-Orbitrap-HRMS method demonstrated to be a useful and valid tool for a robust, precise, accurate, and sensitive determination of POPs in refined and stored vegetable oils.