RESEARCH PAPER 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 Anneleen I. Decloedt 1,2 & Anita Van Landschoot 2 & Lynn Vanhaecke 1 Received: 30 May 2016 /Revised: 29 July 2016 /Accepted: 9 August 2016 # Springer-Verlag Berlin Heidelberg 2016 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 perfor- mance 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 coef- ficient (R 2 > 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 quanti- fication 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). Keywords Phytosterols . Ultra high-performance liquid chromatography . Tandem mass spectrometry . Foods/ beverages . Extraction . Bioanalytical methods Introduction Phytosterols are ubiquitous in plants, as they play an impor- tant role in cell membrane stability and fluidity. They can also participate in the control of membrane-associated metabolic processes, as signal transducers. Over the last few decades, the scientific interest in phytosterols increased significantly, based upon two very distinct properties related to their chemical structure: the cholesterol-lowering properties and the possible influence on endogenous steroid synthesis [1]. It is generally assumed that phytosterols reduce cholesterol absorption in the intestinal tract through the displacement of cholesterol from the micelles. Phytosterols are structurally very similar to cho- lesterol except that they contain a substitution at the C24 po- sition on the sterol side chain and, in the case of stigmasterol and brassicasterol, an additional double bound between C22 and C23 (Fig. 1)[2, 3]. The European Food Safety Authority (EFSA) and Food and Drug Administration (FDA) concluded that, relative to a placebo, blood low-density lipoprotein (LDL)-cholesterol Electronic supplementary material The online version of this article (doi:10.1007/s00216-016-9870-8) contains supplementary material, which is available to authorized users. * Lynn Vanhaecke Lynn.Vanhaecke@ugent.be 1 Faculty of Veterinary Medicine, Department of Veterinary Public Health and Food Safety, Laboratory of Chemical Analysis, Ghent University, 133 Salisburylaan, 9820 Merelbeke, Belgium 2 Faculty of Bioscience Engineering, Laboratory of Biochemistry and Brewing, Ghent University, Valentin Vaerwyckweg 1, 9000 Ghent, Belgium Anal Bioanal Chem DOI 10.1007/s00216-016-9870-8