Optimization of phytosterol extraction and analysis from animal feeds by D-Optimal design and UHPLC-MS/MS

Phytosterols are omnipresent in plants, as they play an important role in cell membrane stability and as signal transducers. Over the last few decades, the scientific interest in phytosterols has peaked. Most of the interest has focused on the cholesterol-lowering properties of phytosterols, but they may also interfere with the endogenous steroid hormone synthesis. This possible involvement in the synthesis, excretion and thus detection of steroids in sports or farm animals is currently under discussion as the conversion of phytosterols to steroids has been reported in different biological systems, mainly involving a variety of microorganisms. Additionally, recent work indicated that a novel mechanism of endogenous steroid-synthesis is to be considered: non-toxic feed-borne fungi, naturally present on animal feed, were shown capable of converting phytosterols into C19-steroids, which include AED, ADD, precursors of the androgenic-anabolic steroids boldenone and testosterone. Despite this dual interest in phytosterols, accurate and fully validated methods for the quantification of phytosterols are scarce, for food and especially for feed samples. During this study a new extraction and detection method was optimised and fully validated according to EC, AORC and EURC guidelines. A fractional factorial design was used to optimize the extraction procedure. This resulted in the discrimination of five influential factors (amount of sample, solvent, drying temperature, washing and hydrolysis), for which the most optimal conditions were perfected by Surface Response Modeling (SRM) (D-Optimal). Detection was carried out on a UHPLC-MS/MS equipment. The newly developed extraction and UHPLC-MS/MS detection method reached all evaluated performance parameters. The individual recoveries ranged between 90 and 107 %. Good results for repeatability and intra-laboratory reproducibility (RSD%) were observed (<10%). Excellent linearity was proven based on determination coefficient (R2 < 0.99) and lack-of-fit test (F test, alpha = 0.05). With this method we were able to quantify all main phytosterols in different grains and it was shown that the extraction and detection method can be used on other, both liquid and solid, feed and food samples as well. Multiple feed samples, including grass, hay, straw, yellow peas, grains and apples, were analyzed, and all main phytosterols could be detected and quantified. Grass for example showed to contain 28 ± 2 mg β-sitosterol, 4.1 ± 0.3 mg campesterol and 0.41 ± 0.02 mg stigmasterol per 100 g fresh weight (25% dry weight)