Swiss Laboratory for Doping Analyses, University Center of Legal Medicine Lausanne-Geneva, Lausanne University Hospital and University of Lausanne, Switzerland; University Center of Legal Medicine Lausanne-Geneva, Lausanne University Hospital, University of Geneva, Switzerland.
Swiss Laboratory for Doping Analyses, University Center of Legal Medicine Lausanne-Geneva, Lausanne University Hospital and University of Lausanne, Switzerland.
J Chromatogr A. 2024 Oct 11;1734:465224. doi: 10.1016/j.chroma.2024.465224. Epub 2024 Aug 8.
The first step in the detection of testosterone (T) doping is to measure the urinary steroid profile for the athlete biological passport (ABP). To harmonise the analysis between anti-doping laboratories, urinary steroid profiling is parametrised in deep detail and shall be performed by gas chromatography hyphenated to mass spectrometry (GC-MS). However, due to its requirement for extensive sample preparation, alternatives to GC-MS are being actively pursued. The aim of this study was the evaluation of Ultra-High-Performance Supercritical Fluid Chromatography hyphenated to tandem Mass Spectrometry (UHPSFC-MS/MS) as an alternative for the quantification of endogenous urinary steroids. In this context, we developed a high throughput sample extraction method, followed by a novel UHPSFC-MS/MS method for the analysis of 10 endogenous urinary steroids which are relevant for doping control analysis. Depending on the steroid, the herein presented method is capable of quantification from 0.5 ng/mL up to 10 µg/mL. After validation, the applicability of the method was evaluated by analysing 132 authentic urine samples, which demonstrated results similar to classical GC-MS analysis. Steroid concentrations determined by UHPSFC-MS/MS were slightly overestimated in comparison with GC-MS, but the ratios had <10 % difference between the two methods. As the ABP considers the steroid ratios for passport evaluation, the herein presented method could be used for steroid profiling without reducing the sensitivity of the ABP. Thus, we would propose to consider UHPSFC-MS/MS as an alternative to GC-MS after more tests would have been performed to support our findings. Furthermore, we have also investigated the potential of this technology for sample purification prior to Isotope Ratio Mass Spectrometry (IRMS) for the differentiation between exogenous and endogenous origin of T and its metabolites. While the achieved separation was sufficient to purify urine samples for IRMS analysis in our proof-of-concept study, the instrumental parameters should be further refined for future use.
检测睾酮（T）兴奋剂的第一步是测量运动员生物护照（ABP）中的尿甾体谱。为了使反兴奋剂实验室之间的分析保持一致，尿甾体谱被详细参数化，并通过气相色谱-质谱联用（GC-MS）进行分析。然而，由于其需要广泛的样品制备，因此正在积极寻求 GC-MS 的替代品。本研究的目的是评估超高效超临界流体色谱-串联质谱（UHPSFC-MS/MS）作为替代定量内源性尿甾体的方法。在这方面，我们开发了一种高通量样品提取方法，随后开发了一种新颖的 UHPSFC-MS/MS 方法，用于分析 10 种与兴奋剂控制分析相关的内源性尿甾体。根据类固醇的不同，本文提出的方法能够从 0.5ng/mL 到 10μg/mL 进行定量。经过验证，通过分析 132 个真实尿液样本评估了该方法的适用性，结果与经典 GC-MS 分析相似。与 GC-MS 相比，UHPSFC-MS/MS 测定的类固醇浓度略有高估，但两种方法的比值差异<10%。由于 ABP 考虑了护照评估中的类固醇比值，因此本文提出的方法可用于类固醇谱分析，而不会降低 ABP 的灵敏度。因此，我们建议在进行更多测试以支持我们的发现后，将 UHPSFC-MS/MS 视为 GC-MS 的替代品。此外，我们还研究了这项技术在同位素比质谱（IRMS）之前用于区分 T 及其代谢物的外源性和内源性来源的样品净化的潜力。虽然在我们的概念验证研究中，实现的分离足以净化用于 IRMS 分析的尿液样本，但仪器参数应进一步细化，以用于未来。
