A UHPLC method for the rapid separation and quantification of phytosterols using tandem UV/Charged aerosol detection – A comparison of both detection techniques

Abstract

The presented work describes the development and validation of a rapid UHPLC-UV/CAD method using a core–shell particle column for the separation and quantitative analysis of seven plant sterols and stanols. The phytosterols (ergosterol, brassicasterol, campesterol, fucosterol, stigmasterol, and β-sitosterol) and the phytostanol stigmastanol were separated and analyzed in 8.5min. The sample pre-treatment procedure was optimized to be less time-consuming than any other published method, especially due to no need of derivatization, evaporation and even reconstitution step. The chromatographic separation was performed on the Kinetex 1.7μ Phenyl-hexyl column (100×2.1mm) with a mobile phase acetonitrile/water according to the gradient program at a flow rate of 0.9mLmin−1 and a temperature of 60°C. A tandem connection of PDA and CAD (Corona Charged Aerosol Detector) was used and both detection techniques were compared. The method was validated using saponification as a first step in sample pre-treatment and an universal CAD as the detector. Recoveries for all analyzed compounds were between 95.4% and 103.4% and relative standard deviation ranged from 1.0% to 5.8% for within-day and from 1.4% to 6.7% for between-day repeatability. The limits of detection were in the range of 0.4–0.6μgmL−1 for standard solutions and 0.3–1.2μgmL−1 for phytosterols in real samples. Although several gradient programs and different stationary phases were tested, two compounds, campesterol and campestanol, were not separated. Their peak was quantified as a sum of both analytes.