Pfeffer Sandra, Gmeiner Guenter, Forsdahl Guro
Department of Pharmacy, UiT - The Arctic University of Norway, Tromsø, Norway.
Doping Control Laboratory, Seibersdorf Labor GmbH, Seibersdorf, Austria.
Drug Test Anal. 2025 Feb;17(2):205-215. doi: 10.1002/dta.3694. Epub 2024 Apr 21.
The study of intact phase II metabolites of endogenous anabolic androgenic steroids (EAAS) gives important information about metabolism and has the potential to improve the detection of doping with testosterone. For analysis with liquid chromatography-mass spectrometry (LC-MS), chemical derivatization at the steroid moiety is a technique to improve the positive ionization efficiency of glucuronidated/sulfated EAAS under collision-induced dissociation (CID) conditions. However, regarding the chromatographic performance, there are still challenges to address, for example, poor peak shape, which is mainly caused by nondefined adsorption in the chromatographic system. Here, we show a novel derivatization technique for the analysis of selected phase II metabolites of EAAS, where the acidic moiety of the glucuronide/sulfate is methylated with different methylation reagents to reduce nondefined adsorption. The methylation reagent trimethylsilyl-diazomethane (TMSD) was preferred over the other tested reagents methyl iodide (MeI) and dimethyl sulfate (DMS). Glucuronidated and sulfated testosterone and epitestosterone were methylated, and their chromatographic performance and CID ion mass spectra obtained in positive ionization mode were investigated. The peak width and peak height were significantly improved for all substances. Methylated testosterone sulfate showed the best results with a 3.5 times narrower peak and 14 times increased intensity compared with underivatized testosterone sulfate. Furthermore, CID ion mass spectra obtained in positive ionization mode showed product ions characteristically for the steroidal backbone for all substances. This preliminary study shows the potential of methylation as a supplementary derivatization technique, which can assist in the development of more sensitive methods due to the improvements in method performance.
对内源性合成代谢雄激素类固醇（EAAS）完整的II相代谢物进行研究，可为代谢提供重要信息，并有可能改进睾酮兴奋剂的检测方法。对于液相色谱-质谱联用（LC-MS）分析而言，在类固醇部分进行化学衍生化是一种在碰撞诱导解离（CID）条件下提高葡萄糖醛酸化/硫酸化EAAS正离子化效率的技术。然而，在色谱性能方面，仍存在一些挑战需要解决，例如峰形不佳，这主要是由色谱系统中不确定的吸附引起的。在此，我们展示了一种用于分析EAAS选定II相代谢物的新型衍生化技术，其中葡萄糖醛酸/硫酸盐的酸性部分用不同的甲基化试剂进行甲基化，以减少不确定的吸附。甲基化试剂三甲基硅基重氮甲烷（TMSD）优于其他测试试剂碘甲烷（MeI）和硫酸二甲酯（DMS）。对葡萄糖醛酸化和硫酸化的睾酮及表睾酮进行甲基化，并研究其在正离子化模式下的色谱性能和CID离子质谱。所有物质的峰宽和峰高均有显著改善。与未衍生化的硫酸睾酮相比，甲基化硫酸睾酮的峰宽窄3.5倍，强度增加14倍，效果最佳。此外，在正离子化模式下获得的CID离子质谱显示，所有物质的甾体骨架特征性产物离子均存在。这项初步研究表明甲基化作为一种辅助衍生化技术具有潜力，由于方法性能的改进，它可以帮助开发更灵敏的方法。
