Casilli Alessandro, Piper Thomas, de Oliveira Fábio Azamor, Padilha Monica Costa, Pereira Henrique Marcelo, Thevis Mario, de Aquino Neto Francisco Radler
Brazilian Laboratory Doping Control, LBCD-LADETEC/IQ-UFRJ, Rio de Janeiro, RJ, Brazil.
German Sport University Cologne, Center for Preventive Doping Research - Institute of Biochemistry, Köln, Germany.
Drug Test Anal. 2016 Nov;8(11-12):1204-1211. doi: 10.1002/dta.2119. Epub 2016 Nov 11.
Measuring carbon isotope ratios (CIRs) of urinary analytes represents a cornerstone of doping control analysis and has been particularly optimized for the detection of the misuse of endogenous steroids. Isotope ratio mass spectrometry (IRMS) of appropriate quality, however, necessitates adequate purities of the investigated steroids, which requires extensive pre-analytical sample clean-up steps due to both the natural presence of the target analytes and the high complexity of the matrix. In order to accelerate the sample preparation and increase the automation of the process, the use of multidimensional gas chromatography (MDGC) prior to IRMS experiments, was investigated. A well-established instrumental configuration based on two independent GC ovens and one heart-cutting device was optimized. The first dimension (1D) separation was obtained by a non-polar column which assured high efficiency and good loading capacity, while the second dimension (2D), based on a mid-polar stationary phase, provided good selectivity. A flame ionization detector monitored the 1D, and the 2D was simultaneously recorded by isotope ratio and quadrupole mass spectrometry. The assembled MDGC set-up was applied for measuring testosterone, 5α- and 5β-androstanediol, androsterone, and etiocholanolone as target compounds and pregnanediol as endogenous reference compound. The urine sample were pretreated by conventional sample preparation steps comprising solid-phase extraction, hydrolysis, and liquid-liquid extraction. The extract obtained was acetylated and different aliquots were injected into the MDGC system. Two high performance liquid chromatography steps, conventionally adopted prior to CIR measurements, were replaced by the MDGC approach. The obtained values were consistent with the conventional ones. Copyright © 2016 John Wiley & Sons, Ltd.
测量尿液分析物的碳同位素比率（CIR）是兴奋剂检测分析的基石，并且已针对检测内源性类固醇的滥用进行了特别优化。然而，质量合适的同位素比率质谱法（IRMS）需要所研究类固醇具有足够的纯度，由于目标分析物的天然存在以及基质的高度复杂性，这需要广泛的分析前样品净化步骤。为了加速样品制备并提高过程的自动化程度，研究了在IRMS实验之前使用多维气相色谱法（MDGC）。基于两个独立的气相色谱炉和一个中心切割装置的成熟仪器配置得到了优化。第一维（1D）分离通过非极性柱实现，该柱确保了高效率和良好的负载能力，而基于中极性固定相的第二维（2D）提供了良好的选择性。火焰离子化检测器监测1D，2D则通过同位素比率和四极杆质谱法同时记录。组装好的MDGC装置用于测量睾酮、5α-和5β-雄甾二醇、雄甾酮和本胆烷醇酮作为目标化合物，以及孕二醇作为内源性参考化合物。尿液样品通过包括固相萃取、水解和液-液萃取在内的常规样品制备步骤进行预处理。得到的提取物进行乙酰化处理，并将不同的等分试样注入MDGC系统。CIR测量之前常规采用的两个高效液相色谱步骤被MDGC方法所取代。获得的值与传统值一致。版权所有© 2016约翰威立父子有限公司。
