Analysis of free and conjugated phytosterols in tobacco by an improved method using gas chromatography–flame ionization detection

An improved and simple method using capillary gas chromatography coupled with a flame ionization detection system (GC–FID) has been developed for quantitative analysis of free and conjugated phytosterols in tobacco. Direct acid and alkaline hydrolysis were first introduced into tobacco analysis to liberate free phytosterols from conjugates, followed by extraction with hexane, derivatization to trimethylsilyl ether derivatives and finally GC quantitative determination. The generality and applicability of this improved method for analyzing free and conjugated phytosterols in tobacco were validated after a series of optimization and comparison were done. Compared with traditional methods, this improved method not only simplified procedures, but also saved time and solvent. The limits of detection (LODs) of phytosterols varied from 0.35 to 0.10 μg mL−1, the relative standard deviations (RSD) were from 2.3% to 3.3% and recovery ranged from 87% to 99%. The analysis results showed that total phytosterols’ content in tobacco ranged from 1.0 to 2.5 mg g−1, and most phytosterols existed as conjugates, only approximately 15–25% phytosterols existed in free-form. Ergosterol was only found in mildewy flued-cured tobacco and the level was approximately 0.2–0.25 mg g−1.