title: 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-1and 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-1for standard solutions and 0.3-1.2μgmL-1for 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.
authors.affiliation: The Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovského 1203, Hradec Králové 500 05, Czech Republic.
authors.forename: Jakub
authors.initials: J
authors.lastname: Fibigr
authors.affiliation: The Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovského 1203, Hradec Králové 500 05, Czech Republic. Electronic address: satinsky@faf.cuni.cz.
authors.forename: Dalibor
authors.initials: D
authors.lastname: Šatínský
authors.affiliation: The Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovského 1203, Hradec Králové 500 05, Czech Republic.
authors.forename: Petr
authors.initials: P
authors.lastname: Solich
mesh_terms: D000336:Aerosols; D002851:Chromatography, High Pressure Liquid; D010840:Phytosterols; D012855:Sitosterols; D013261:Sterols
chemical_list: D000336:Aerosols; D010840:Phytosterols; D012855:Sitosterols; D013261:Sterols; C025473:gamma-sitosterol
keywords: Core–shell column; Corona-charged aerosol detector (CAD); Plant extracts; Plant sterols; Stanols; UHPLC