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 deter- mined in medium chain triacylglycerols were >0.012 and >0.039 ng/mL, respectively; whereas the recoveries ranged between 82% and 98%. The suit- ability of the analytical method was evaluated on palm oil (PO), palm olein (POL), and high oleic sunflower oil (HOSO) during the whole refining proces- sing (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 chro- matographic system coupled to the LC-Orbitrap-HRMS method demonstrated to be a useful and valid tool for a robust, precise, accurate, and sensitive deter- mination of POPs in refined and stored vegetable oils.