Comprehensive steroid screening in bovine and porcine urine by GC-HRMS

Steroids are a large and diverse group of molecules containing both natural hormones and synthetic derivatives. Steroid administration can have a growth-promoting effect in food-producing livestock. However, the exogenic use of steroids to promote growth has adverse health effects in livestock and consumers, and, consequently, is forbidden in the European Union (EU). Therefore, broad monitoring of steroids and other growth-promoting compounds in animal matrices is desirable. Commission Implementing Regulation (EU) 2021/808 was recently implemented to provide explicit room and quality guidance for analyzing food residues, including steroids and growth-promoting compounds, using the technique of high-resolution mass spectrometry (HRMS). This manuscript presents a method for broad steroid monitoring in animal urine by gas chromatography (GC) Q-Orbitrap HRMS, using a minimal cleanup of liquid–liquid extractions and a 96-well plate solid-phase extraction (SPE). An in-house library was built to detect 104 steroid and steroid-like molecules from subclasses of androgens, estrogens, progestogens, stilbenes, and resorcylic acid lactones (RALs) in the HRMS datafiles. The method was fully validated according to Regulation (EU) 2021/808 as a qualitative screening method for bovine and porcine urine, and used for analysis of bovine urine samples originating from previously performed animal studies and from the Dutch National Residue Control Plan. Analyses of samples from previous animal studies show performance of the developed method for monitoring synthetic steroid misuse, as all incurred samples were flagged non-compliant. To our knowledge, it is the first time a method was developed and validated with such a broad scope of growth promoters in cattle with GC-HRMS. Since the sample preparation is generic for multiple steroid classes, and the HRMS data acquisition is untargeted, the generated data can be used for further expansion of the scope with other steroids, for investigating unexpected or new steroids, and for retrospective (trend-)analysis, thus facilitating risk-based monitoring of steroids and other growth promoting compounds.