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

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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 8.5 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 μ Phenyl-hexyl column (100 × 2.1 mm) with a mobile phase acetonitrile/water according to the gradient program at a flow rate of 0.9 mL min−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 0.4–0.6 μg mL−1 for standard solutions and 0.3–1.2 μg 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.

Highlights

Corona-charged aerosol detector for the first time in the analysis of phytosterols and phytostanols was used.The fast chromatography method for separation of 7 phytosterols was developed.The method shows short time (8 min) of analysis using phenyl-hexyl stationary phase.Method shows novel and alternative approach compared to C-18 reversed phase separation and UV detection.

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