Laboratorio Antidoping FMSI, Largo Giulio Onesti 1, Rome, Italy.
Laboratorio Antidoping FMSI, Largo Giulio Onesti 1, Rome, Italy.
J Pharm Biomed Anal. 2021 Feb 5;194:113807. doi: 10.1016/j.jpba.2020.113807. Epub 2020 Nov 30.
The detection of the abuse of pseudo-endogenous steroids in sport is articulated in two different levels: an initial testing procedure, based on the longitudinal evaluation of the urinary androgenic steroid profile by gas-chromatography mass spectrometry (GC-MS), and a confirmation analysis, based on the differentiation between the endogenous and exogenous origin of the pseudo-endogenous steroids by gas-chromatography coupled to isotopic ratio mass spectrometry (GC/C/IRMS). The abuse of pharmaceutical preparations displaying a carbon isotopic composition values within a range similar to those reported for endogenous urinary steroids makes more difficult the application of GC/C/IRMS technique. To overcome this limitation, the direct detection of an intact synthetic anabolic steroid ester in blood matrices (plasma and/or serum) could supply the unequivocal proof of exogenous administration of pseudo-endogenous steroids. Here we are presenting a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the analysis of 14 testosterone (T) esters and 2 nandrolone (Nand) esters in human serum. Sample pre-treatment consisted of protein precipitation, liquid-liquid extraction and derivatization. The formation of three different derivatives (oxime derivatives, Girard P and Girard T hydrazones) is considered, in order to guarantee an improvement in the detection capability of the assay with respect to underivatized compounds. Once the most suitable derivative was selected, the method was validated, according to the World Anti-Doping Agency (WADA) criteria, in terms of specificity, linearity, limit of detection (LOD), extraction recovery, matrix effect (ion suppression/enhancement), carry over and autosampler stability. The formation of Girard P hydrazones of T and Nand esters provides the best results compared to the underivatized compounds, oxime and Girard T derivatives, respectively. The presented analytical method is specific for all considered compounds and linear in the range of concentrations investigated (0.25-10 ng/mL). The LODs are between 0.03 and 0.30 ng/mL, the extraction recovery higher than 70 % for all esters and no remarkable matrix effect, expressed in terms of ion enhancement and ion suppression, was observed. Finally, the developed and validate method was applied in the analysis of serum samples collected after the administration of a single dose (40 mg, 1 capsule) of testosterone undecanoate (Andriol ®) demonstrating its applicability.
一是基于气相色谱-质谱联用（GC-MS）对尿雄激素谱进行纵向评估的初始检测程序；二是基于 GC/同位素质谱联用（GC/C/IRMS）区分内源性和外源性伪内源性激素的确认分析。药物制剂的滥用会导致其碳同位素组成值与内源性尿类固醇相似，这使得 GC/C/IRMS 技术的应用变得更加困难。为了克服这一限制，可以直接检测血液基质（血浆和/或血清）中完整的合成合成代谢类固醇酯，从而提供外源性给予伪内源性激素的明确证据。本文介绍了一种液相色谱串联质谱（LC-MS/MS）法，用于分析人血清中的 14 种睾酮（T）酯和 2 种诺龙（Nand）酯。样品预处理包括蛋白质沉淀、液-液萃取和衍生化。考虑形成三种不同的衍生物（肟衍生物、Girard P 和 Girard T 腙），以保证与未衍生化合物相比，该测定的检测能力得到提高。选择最合适的衍生物后，根据世界反兴奋剂机构（WADA）的标准，对方法的特异性、线性、检测限（LOD）、提取回收率、基质效应（离子抑制/增强）、交叉污染和自动进样器稳定性进行了验证。与未衍生化合物、肟和 Girard T 衍生物相比，T 和 Nand 酯的 Girard P 腙的形成提供了最佳的结果。所提出的分析方法对所有考虑的化合物均具有特异性，在研究浓度范围内（0.25-10ng/mL）呈线性。LOD 在 0.03-0.30ng/mL 之间，所有酯的提取回收率均高于 70%，未观察到显著的基质效应，表现为离子增强和离子抑制。最后，该方法应用于单次剂量（40mg，1 粒）十一酸睾酮（Andriol®）给药后血清样本的分析，证明了其适用性。
