QUANTITATIVE DETERMINATION OF FREE PHYTOSTEROLS IN TOBACCO LEAVES BY UPLC-MS/MS

A simple, sensitive, and rapid method for quantitative determination of free phytosterols (cholesterol, ergosterol, stigmasterol, and beta-sitosterol) in tobacco leaves by ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) has been developed. The separation of the four phytosterols was realized on a C18 reversed-phase column within 6.0min. A triple quadrupole mass analyzer API 4000 MS/MS System was evaluated for its performance in the study; the identification and quantitation of analytes were performed in the positive ion multiple reactions monitoring mode (MRM). Satisfactory recovery ranged from 86.3% to 106.4% at middle quality concentration (MQC) levels in tobacco leaves. The intra-day precision and inter-day precision accuracy of the method were between 0.43% and 2.42%. The limit of quantification (LOQ) was 4.8-9.7ng/mL. The applicability of this newly developed method was demonstrated by analysis of free phytosterols in tobacco leaves samples.