Thomas Andreas, Krombholz Sophia, Wolf Carina, Thevis Mario
Institute of Biochemistry/Center for Preventive Doping Research, German Sport University Cologne, Cologne, Germany.
European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany.
Drug Test Anal. 2021 Nov;13(11-12):1862-1870. doi: 10.1002/dta.3176. Epub 2021 Oct 26.
The hunger hormone ghrelin (G) is classified as prohibited substance in professional sport by the World Anti-Doping Agency (WADA), due to its known growth hormone releasing properties. The endogenous bioactive peptide consists of 28 amino acids with a caprylic acid attached to serine at position 3. Within this study, it was aimed to develop methods to determine G and desacyl ghrelin (DAG) in plasma and urine by means of LC-MS/MS. Two strategies were applied with a bottom-up approach for plasma and top-down analyses for urine. Both sample preparation procedures were based on solid-phase extraction for enrichment and sample clean-up. Method validation showed good results for plasma and urine with limits of detection (LODs) for G and DAG between 30 and 50 pg/ml, recoveries between 45-50%, and imprecisions (intra- and inter-day) between 3% and 24%. Plasma analysis was also valid for quantification with accuracies determined with ~100% for G and ~106% for DAG. The minimum required performance level for doping control laboratories is set to 2 ng/ml in urine, and the herein established method yielded acceptable results even at 5% of this level. As proof-of-concept, plasma levels (G and DAG) of healthy volunteers were determined and ranged between 30 and 100 pg/ml for G and 100-1200 pg/ml for DAG. In contrast to earlier reported studies using ligand binding assays for urinary G and DAG, in this mass spectrometry-based study, no endogenous urinary G and DAG were found, although the LODs should enable this.
饥饿激素胃饥饿素（G）因其已知的生长激素释放特性，被世界反兴奋剂机构（WADA）列为职业体育中的违禁物质。这种内源性生物活性肽由28个氨基酸组成，在第3位丝氨酸上连接有辛酸。在本研究中，旨在开发通过液相色谱-串联质谱法测定血浆和尿液中G和去酰基胃饥饿素（DAG）的方法。采用了两种策略，血浆分析采用自下而上的方法，尿液分析采用自上而下的方法。两种样品制备程序均基于固相萃取进行富集和样品净化。方法验证显示血浆和尿液的结果良好，G和DAG的检测限（LOD）在30至50 pg/ml之间，回收率在45%-50%之间，不精密度（日内和日间）在3%至24%之间。血浆分析的定量也有效，G的准确度约为100%，DAG的准确度约为106%。兴奋剂检测实验室的最低要求性能水平设定为尿液中2 ng/ml，本文建立的方法即使在该水平的5%时也能产生可接受的结果。作为概念验证，测定了健康志愿者的血浆水平（G和DAG），G的范围在30至100 pg/ml之间，DAG的范围在100至1200 pg/ml之间。与早期使用配体结合测定法检测尿液中G和DAG的研究不同，在这项基于质谱的研究中，未发现内源性尿液G和DAG，尽管检测限应能检测到它们。
