Determination of 60 steroid hormones in sports nutrition foods by ultra performance liquid chromatography-tandem mass spectrometry

<p id="p00005">Recent societal developments and improving living standards have witnessed a growing enthusiasm for sport and fitness along with a boom in sports nutrition foods. However, rapid industrial development has led to product quality and safety issues becoming increasingly important. Steroid hormones exhibit physiological effects that include promoting protein synthesis, increasing muscle mass, and reducing body fat. Driven by vested interests, some unscrupulous traders illegally add steroid hormones to sports nutrition foods to improve sports performance and relieve muscle pain, which poses a potential threat to human health. Therefore, establishing a method for determining steroid hormones in sports nutrition foods is of great practical importance. <p id="p00010">In this study, an analytical method for the simultaneous determination of 60 steroid hormones in sports nutrition foods was developed using QuEChERS combined with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The MS parameters and chromatographic conditions were optimized for the 60 steroid hormones, and the extraction solvents and purification methods were systematically optimized to facilitate rapid screening and accurate quantitative analysis of these hormones in sports nutrition foods. Samples were extracted with 1.0% formic acid in methanol, partitioned with sodium chloride, and purified using 150 mg of PSA and 150 mg of C₁₈ powder. The clean-up solution was evaporated to near dryness under a stream of nitrogen and the residue was reconstituted with 50% methanol aqueous solution. The 60 target compounds were separated on a Waters Acquity UPLC HSS T₃ column (100 mm×2.1 mm, 1.8 μm) with 0.1% (v/v) formic acid aqueous solution (containing 10 mmol/L ammonium acetate) and methanol as the mobile phases. The analytes were determined by multiple reaction monitoring (MRM) in positive electrospray ionization mode (ESI⁺) and quantified using the external standard method. The method was validated under the optimized conditions, with the 60 steroid hormones found to exhibit good linearities in the 1.0-100 ng/mL range with correlation coefficients (<italic>r</italic>) greater than 0.99. The limits of detection (LODs) and limits of quantification (LOQs) were 0.2-0.8 μg/kg and 0.6-2.4 μg/kg, respectively. At three spiked levels on whey protein powder (solid), pectin protein peptide (semi-solid) and energy drink (liquid) as blank matrix, the mean recoveries of the 60 steroid hormones were 73.7%-112.7% with relative standard deviations (RSDs) of 3.2%-10.1% (<italic>n</italic>=6). Thirteen batches of commercially available sports nutrition products were screened using the newly developed method, with five positive samples detected. Progesterone (an endogenous hormone) was detected at levels of 12.6-25.4 μg/kg in four samples of whey protein powder, and boldenone (which is a prohibited drug for athletes) was detected at 14.6 μg/kg in one sports nutrition liquid, confirming that illegal steroid hormones are indeed added to sports nutrition foods; consequently, relevant national departments need to strengthen their monitoring of such substances in such foods. The developed method is simple, efficient, sensitive, reproducible, and suitable for the rapid screening and quantitative analysis of steroid hormones in sports nutrition foods, thereby providing technical support for the daily supervision of steroid hormones in these foods.