Simultaneous Quantitation and Stability Evaluation of Cholecalciferol and α-Tocopherol in Vitamin-Fortified Nanoemulsions by UHPLC-MS/MS Coupled with QuEChERS Extraction

A novel QuEChERS-UHPLC-MS/MS method was developed for the simultaneous quantification of cholecalciferol and α-tocopherol in vitamin-fortified nanoemulsions. Critical extraction parameters, including solvent volume, extraction time, agitation type, and QuEChERS salt composition, were optimized using recovery tests and a Box-Behnken design. The method demonstrated excellent linearity (R² > 0.9999), low detection limits, high repeatability (0.12-1.96% RSD), reproducibility (0.58-1.97% RSD), and accuracy (97.18-106.21%), with negligible matrix effects (97.66-105.67%). The thermal stability of both vitamins was evaluated under different temperature treatments: low-temperature long-time (65 °C for 30 min), high-temperature short-time (95 °C for 15 s), and autoclave treatment (121 °C for 15 min). Significant degradation was observed only after autoclave treatment, while higher soy lecithin content in the nanoemulsions enhanced vitamin retention under heat stress. This method offers a reliable tool for the routine analysis and stability evaluation of vitamin-fortified nanoemulsions, contributing to their quality control and development.