Center for Preventive Doping Research/Institute of Biochemistry, German Sport University, Cologne, Germany.
Anal Chem. 2012 Dec 4;84(23):10252-9. doi: 10.1021/ac302034w. Epub 2012 Nov 13.
New, potentially performance enhancing compounds have frequently been introduced to licit and illicit markets and rapidly distributed via worldwide operating Internet platforms. Developing fast analytical strategies to follow these new trends is one the most challenging issues for modern doping control analysis. Even if reference compounds for the active drugs are readily obtained, their unknown metabolism complicates effective testing strategies. Recently, a new class of small C-terminally amidated peptides comprising four to seven amino acid residues received considerable attention of sports drug testing authorities due to their ability to stimulate growth hormone release from the pituitary. The most promising candidates are the growth hormone releasing peptide (GHRP)-1, -2, -4, -5, -6, hexarelin, alexamorelin, and ipamorelin. With the exemption of GHRP-2, the entity of these peptides represents nonapproved pharmaceuticals; however, via Internet providers, all compounds are readily available. To date, only limited information on the metabolism of these substances is available and merely one metabolite for GHRP-2 is established. Therefore, a comprehensive in vivo (po and iv administration in rats) and in vitro (with human serum and recombinant amidase) study was performed in order to generate information on urinary metabolites potentially useful for routine doping controls. The urine samples from the in vivo experiments were purified by mixed-mode cation-exchange solid-phase extraction and analyzed by ultrahigh-performance liquid chromatography (UHPLC) separation followed by high-resolution/high-accuracy mass spectrometry. Combining the high resolution power of a benchtop Orbitrap mass analyzer for the first metabolite screening and the speed of a quadrupole/time-of-flight (Q-TOF) instrument for identification, urinary metabolites were screened by means of a sensitive full scan analysis and subsequently confirmed by high-accuracy product ion scan experiments. Two deuterium-labeled internal standards (triply deuterated GHRP-4 and GHRP-2 metabolite) were used to optimize the extraction and analysis procedure. Overall, 28 metabolites (at least three for each GHRP) were identified from the in vivo samples and main metabolites were confirmed by the human in vitro model. All identified metabolites were formed due to exopeptidase- (amino- or carboxy-), amidase-, or endopeptidase activity.
新的、潜在的具有增强性能的化合物经常被引入合法和非法市场，并通过全球运营的互联网平台迅速传播。开发快速分析策略来跟踪这些新趋势是现代兴奋剂控制分析面临的最具挑战性的问题之一。即使可以获得用于活性药物的参考化合物，它们未知的代谢也会使有效的测试策略复杂化。最近，一类新的小 C 末端酰胺化肽引起了运动药物检测机构的极大关注，因为它们能够刺激垂体释放生长激素。最有前途的候选物是生长激素释放肽（GHRP）-1、-2、-4、-5、-6、hexarelin、alexamorelin 和 ipamorelin。除了 GHRP-2 之外，这些肽的实体代表未经批准的药物；然而，通过互联网提供商，所有化合物都很容易获得。迄今为止，关于这些物质代谢的信息有限，仅建立了 GHRP-2 的一种代谢物。因此，进行了全面的体内（大鼠口服和静脉注射给药）和体外（用人血清和重组酰胺酶）研究，以生成可能对常规兴奋剂控制有用的尿代谢物信息。体内实验的尿样通过混合模式阳离子交换固相萃取进行纯化，并用超高效液相色谱（UHPLC）分离，然后用高分辨率/高精度质谱分析。将台式轨道阱质谱分析仪的高分辨率能力用于首次代谢物筛选，并将四极杆/飞行时间（Q-TOF）仪器的速度用于鉴定，通过灵敏的全扫描分析筛选尿代谢物，然后通过高精度产物离子扫描实验进行确认。使用两个氘标记的内标（三重氘标记的 GHRP-4 和 GHRP-2 代谢物）来优化提取和分析程序。总共从体内样本中鉴定出 28 种代谢物（每种 GHRP 至少三种），并通过人体体外模型确认主要代谢物。所有鉴定出的代谢物都是由于外肽酶（氨基或羧基）、酰胺酶或内肽酶活性形成的。
