Thomas Andreas, Brinkkötter Paul, Schänzer Wilhelm, Thevis Mario
Center for Preventive Doping Research and Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany; Department of Geriatric Medicine, St. Marien-Hospital, Cologne, Germany.
Anal Chim Acta. 2015 Oct 15;897:53-61. doi: 10.1016/j.aca.2015.09.036. Epub 2015 Oct 9.
The misuse of insulin for performance enhancement in sport or as toxic agent has frequently been reported in the past. In contrast to synthetic insulin analogues, the administration of recombinant human insulin is hardly recognized by mass spectrometry. The present study was designed to uncover the misuse of recombinant human insulin for doping control purposes as well as for forensic applications. It is hypothesized that an altered metabolite profile of circulating insulin prevails after subcutaneous administration due to exposure of insulin to epidermal proteases. In vitro experiments with skin tissue lysates (S9 fraction and microsomes), different biological fluids (urine, serum, plasma) and recombinant human insulin were performed and the deriving metabolites were characterized by liquid chromatography coupled to high resolution mass spectrometry (HRMS). Afterwards, authentic blood samples of patients suffering from diabetes mellitus and a control group of healthy humans were analysed. Therefore, a method using protein precipitation, ultrafiltration and antibody-coated magnetic beads for purification with subsequent separation by nano-scale liquid chromatography coupled a Q Exactive mass spectrometer was applied. Several metabolites of insulin with C-terminally truncated sequences of the B-chain (and A-chain in minor extent) were identified within this study. Here, the DesB30 human insulin represents the major metabolite in all experiments. This metabolite is frequently found in urine samples due to degradation processes and, thus, disqualifies this matrix for the intended purposes. In contrast, blood samples do commonly not contain DesB30 insulin, which was corroborated by data obtained from the control group. In post-administration blood samples, minute but distinct amounts (approx. 50 pg mL(-1)) of DesB30 insulin were found and suggest the use of this analyte as potential marker for subcutaneous human insulin administration, supporting the attempts to uncover illicit recombinant human insulin administrations.
过去经常有关于在体育运动中滥用胰岛素以提高成绩或将其用作有毒物质的报道。与合成胰岛素类似物不同，重组人胰岛素的给药很难通过质谱法识别。本研究旨在揭示重组人胰岛素在兴奋剂检测以及法医应用中的滥用情况。据推测，皮下给药后，由于胰岛素暴露于表皮蛋白酶，循环胰岛素的代谢产物谱会发生改变。对皮肤组织裂解物（S9组分和微粒体）、不同生物体液（尿液、血清、血浆）和重组人胰岛素进行了体外实验，并通过液相色谱与高分辨率质谱联用（HRMS）对衍生代谢产物进行了表征。之后，对糖尿病患者的真实血样和健康人类对照组进行了分析。因此，采用了一种使用蛋白质沉淀、超滤和抗体包被磁珠进行纯化，随后通过纳米级液相色谱与Q Exactive质谱仪联用进行分离的方法。在本研究中鉴定出了几种胰岛素的代谢产物，其B链（在较小程度上还有A链）的C末端序列被截断。在此，DesB30人胰岛素是所有实验中的主要代谢产物。由于降解过程，这种代谢产物经常在尿液样本中发现，因此不符合预期用途的基质要求。相比之下，血样中通常不含DesB30胰岛素，这一点得到了对照组数据的证实。在给药后的血样中，发现了微量但明显的DesB30胰岛素（约50 pg mL(-1)），这表明将这种分析物用作皮下注射人胰岛素的潜在标志物，支持了揭露非法重组人胰岛素给药行为的尝试。
