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Abstract：
Anabolic androgenic steroids (AAS) have been the most commonly abused substances taken by not only professional sportsmen but also recreational bodybuilders. The detection of micro-dose testosterone (T) misuse is particularly challenging as it possesses pseudo-endogenous origin and is sometimes impossible to be identified in urine samples. Dried blood (DB) obtained by finger pricking has been proven to be an alternative matrix for better correlating to physiological responses. Moreover, the introduction of the volumetric absorptive microsampling (VAMS) technology allows overcoming some major limitations of spotting blood onto a filter paper card. In this work, a fast and sensitive GC-MS/MS method was developed and validated for the quantification of AAS in DB collected by means of VAMS. T and the eight top abused synthetic AAS, namely nandrolone, boldenone, mesterolone, drostanolone, metenolone, metandienone, oxandrolone, and dehydrochloromethyl T were selected as the target analytes. The method based on VAMS exhibited good precision, accuracy as well as stability, and superior extraction recoveries over the punched DB spots reported in the literature. The chromatographic separation was achieved within 6.4 min and the detection limit is as little as 50 fg (i.e. able to detect 0.10 ng mL-1 in 20 μL of DB). Confirmed by forty real blood samples, the Deming regression and Bland-Altman analysis revealed that the VAMS DB could be employed for quantifying blood T level in agreement with using the serum specimen. The feasibility of the method was then successfully proven by the analysis of samples collected from a three-arm T administration trial. Our results highlighted that DB total T was a sensitive indicator for identifying transdermal micro-dosing of T. In the groups of receiving T gel administration, T concentrations could rise up to ten times higher than the baseline at 9 h after the application. As a future step, this approach is being expanded to a large cohort screening of bodybuilders at gym and ultimately may allow universal applications on monitoring sports drug misuse.
摘要：
合成代谢雄激素类固醇（AAS）不仅是职业运动员，而且是休闲健美运动员最常滥用的物质。微剂量睾酮(T)误用的检测特别具有挑战性,因为其具有假内源性起源,并且有时不可能在尿液样品中被鉴定。通过刺破手指获得的干血（DB）已被证明是与生理反应更好相关的替代基质。此外，体积吸收微量取样(VAMS)技术的引入允许克服将血液点样到滤纸卡上的一些主要限制。在这项工作中,开发了一种快速灵敏的GC-MS/MS方法，并对通过VAMS收集的DB中的AAS进行了定量验证。T和八个滥用的合成AAS，即Nandrolone,boldenone,美酯酮,屈斯坦龙,美替诺酮,美坦酮,oxandrolone,选择了脱氢氯甲基T作为目标分析物。基于VAMS的方法具有良好的精度,准确性和稳定性，与文献中报道的穿孔DB点相比，提取回收率更高。色谱分离在6.4分钟内实现，检测极限低至50fg（即在20μLDB中能够检测到0.10ngmL-1）。由40份真实血液样本证实，Deming回归和Bland-Altman分析显示，VAMSDB可用于定量血液T水平，与使用血清样本一致。然后通过分析从三臂T给药试验收集的样品成功地证明了该方法的可行性。我们的结果强调，DBtotalT是识别T的透皮微量给药的敏感指标。在接受T凝胶给药的组中，在施用后9小时,T浓度可以上升到比基线高10倍。作为未来的一步，这种方法正在扩展到健身房健美运动员的大型队列筛查，最终可能允许监测运动药物滥用的普遍应用。
