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 beta-sitosterol) and the phytostanol stigmastanol were separated and analyzed in 8.5 min.
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 1.7 mu Phenyl-hexyl column (100 x 2.1 mm) with a mobile phase acetoni-trile/water according to the gradient program at a flow rate of 0.9 mL min(-1) and a temperature of 60 degrees 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 pretreatment 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 0.4-0.6 mu g mL(-1) for standard solutions and 0.3-1.2 mu g mL(-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.