Simultaneous extraction and determination of free and conjugated phytosterols in tobacco.

Acid hydrolysis and alkaline saponification were incorporated into a microwave-assisted extraction process for the simultaneous extraction of free and conjugated phytosterols from tobacco. The crude extract of the microwave-assisted extraction was purified by C18 solid-phase extraction and then determined by high-performance liquid chromatography. Phytosterols of cholesterol, ergosterol, stigmasterol, campesterol, and β-sitosterol were determined by chromatographic quantification. The multiple parameters of microwave-assisted extraction were optimized by a uniform design method. The optimal ratio of extraction ethanol solvent to tobacco mass was 30 mL/g. The microwave-assisted extraction acid hydrolysis was carried out in sulfuric acid medium by heating for 10 min at 55°C. The microwave-assisted extraction alkaline saponification was performed after adding excessive sodium hydroxide by heating another 10 min. The repeatability of the proposed method was acceptable with recoveries from 69.68 to 88.17% for the phytosterols. Five target phytosterols were all found in the tobacco samples, and the contents were significantly different in samples from different producing areas.