title: Fractional factorial design-based optimisation and application of an extraction and UPLC-MS/MS detection method for the quantification of phytosterols in food, feed and beverages low in phytosterols.
abstract: Phytosterols are ubiquitous in plants, as they play an important role in cell membrane stability and as signal transducers. Over the last few decades, scientific interest in phytosterols has significantly increased. Most of the interest has focused on the cholesterol-lowering properties of phytosterols, but they may also interfere with endogenous steroid hormone synthesis. Despite this dual interest in phytosterols, accurate and fully validated methods for the quantification of phytosterols in food and feed samples are scarce. During this study an extraction and detection method for the main free phytosterols (β-sitosterol, campesterol, stigmasterol and brassicasterol) was optimised using a fractional factorial design. Detection was carried out on a UPLC-MS/MS triple stage quadrupole apparatus. The extraction and UPLC-MS/MS detection method was fully validated according to EU Council Decision 2002/657 guidelines and Association of Analytical Chemists (AOAC) MS criteria, reaching all evaluated performance parameter requirements. The individual recoveries ranged between 95 and 104 %. Good results for repeatability and intralaboratory reproducibility (RSD %) were observed (<10 %). Excellent linearity was proven on the basis of determination coefficient (R2> 0.99) and lack-of-fit test (F test, alpha = 0.05). The limits of detection (LODs) and lower limits of quantification (LLOQs) in grain matrices were as low as 0.01-0.03 mg per 100 g and 0.02-0.10 mg per 100 g. This method allowed quantification of all main, free phytosterols in different grains (oats, barley, corn, malt) and it was shown that the method can be used for other solid food and feed samples as well, including new matrices such as straw, hay, mustard seeds, grass and yellow peas. Additionally, the method was shown to perform well in liquid samples low in phytosterols such as concentrate-based juices, soft drinks and beers (<5 μg per 100 mL). Graphical Abstract An extraction and detection method for the main free phytosterols (β-sitosterol, campesterol, stigmasterol and brassicasterol) was optimised using a fractional factorial design. Detection was carried out on a UPLC-MS/MS triple stage quadrupole apparatus. The extraction and UPLC-MS/MS detection method was fully validated according to EU Council Decision 2002/657 guidelines and Association of Analytical Chemists (AOAC) MS criteria and applied on different matrices including feed and beverages.
authors: Faculty of Veterinary Medicine, Department of Veterinary Public Health and Food Safety, Laboratory of Chemical Analysis, Ghent University, 133 Salisburylaan, 9820, Merelbeke, Belgium.|Faculty of Bioscience Engineering, Laboratory of Biochemistry and Brewing, Ghent University, Valentin Vaerwyckweg 1, 9000, Ghent, Belgium.
authors: Anneleen I
authors: AI
authors: Decloedt
authors: Faculty of Bioscience Engineering, Laboratory of Biochemistry and Brewing, Ghent University, Valentin Vaerwyckweg 1, 9000, Ghent, Belgium.
authors: Anita
authors: A
authors: Van Landschoot
authors: Faculty of Veterinary Medicine, Department of Veterinary Public Health and Food Safety, Laboratory of Chemical Analysis, Ghent University, 133 Salisburylaan, 9820, Merelbeke, Belgium. Lynn.Vanhaecke@ugent.be.
authors: Lynn
authors: L
authors: Vanhaecke
mesh_terms: D002775:Cholestadienols / Q000302:isolation & purification*; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002523:Edible Grain / Q000737:chemistry*; D005163:Factor Analysis, Statistical; D000067030:Fruit and Vegetable Juices / Q000032:analysis; D006801:Humans; D057230:Limit of Detection; D059627:Liquid Phase Microextraction / Q000379:methods; D018532:Pisum sativum / Q000737:chemistry; D010840:Phytosterols / Q000302:isolation & purification*; D006109:Poaceae / Q000737:chemistry; D015203:Reproducibility of Results; D012855:Sitosterols / Q000302:isolation & purification*; D013265:Stigmasterol / Q000302:isolation & purification*; D053719:Tandem Mass Spectrometry
chemical_list: D002775:Cholestadienols; D010840:Phytosterols; D012855:Sitosterols; C003532:brassicasterol; C021273:campesterol; C025473:gamma-sitosterol; D002784:Cholesterol; D013265:Stigmasterol
keywords: Bioanalytical methods; Extraction; Foods/beverages; Phytosterols; Tandem mass spectrometry; Ultra high-performance liquid chromatography