A rapid in-house validated GC-FID method for simultaneous determination of lipophilic bioactives in olive oil: Squalene, alpha-tocopherol, and beta-sitosterol

The analytical methodologies utilizing HPLC or GC techniques for the determination of predominant lipophilic bioactives in olive oil (beta-sitosterol, squalene, and alpha-tocopherol) may include a number of sample pre-treatment steps (such as clean up (using TLC and SPE), saponification, and derivatization) making them rather lengthy and impractical. The objective of this study was to develop a rapid and in-house validated GC-FID method for the simultaneous determination of these bioactives using response surface methodology for optimization of critical parameters (time and temperature of saponification and derivatization). In-house method validation based on method performance parameters (linearity, limit of detection (LOD), limit of quantification (LOQ), repeatability, and recovery) was carried out using optimized method parameters (10 min derivatization time for 30 degrees C derivatization temperature, 56 degrees C saponification temperature, and 10 min saponification time). The linear range of beta-sitosterol, squalene, and alpha-tocopherol was determined to be between LOQ value (8.4; 16.0; 12.5 mg/100 g, respectively) and upper end (500; 700 mg/100 g for beta-sitosterol; squalene, respectively) with acceptable accuracy, recovery (92-97%; 93-95%; 93-110%) and repeatability.