Thomas Andreas, Benzenberg Lukas, Bally Lia, Thevis Mario
Institute of Biochemistry/Center for Preventive Doping Research, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany.
Department of Diabetes: Endocrinology, Nutritional Medicine, and Metabolism, Inselspital, Bern University Hospital, University of Bern, 3010 Bern, Switzerland.
Metabolites. 2021 May 12;11(5):309. doi: 10.3390/metabo11050309.
The increasing importance to determine bioactive peptide hormones such as insulin, its synthetic analogs, and C-peptide in urine samples represents an analytical challenge. The physiological concentrations of insulin in urine are commonly found at sub-ng/mL levels and thus represent a complex analytical task. C-peptide concentrations, on the other hand, tend to be in the moderate ng/mL range and are hence much easier to determine. Insulin and C-peptide are important in the diagnostics and management of metabolic disorders such as diabetes mellitus and are also particularly relevant target analytes in professional sports and forensics. All insulins are classified on the World Anti-Doping Agency's (WADA) list of prohibited substances and methods in sports with a minimum required performance level (MRPL) of 50 pg/mL. Until now, methods combining immunoextraction and subsequent mass spectrometric detection have mostly been used for this purpose. With the method developed here, sample preparation has been simplified considerably and does not require an antibody-based sample purification. This was achieved by a sophisticated mixed-mode solid-phase extraction and subsequent separation with liquid chromatography coupled to high-resolution mass spectrometry. Included target insulins were human, lispro, glulisine, aspart, glargine metabolite, degludec, and additionally, human C-peptide. The method was validated for the synthetic insulin analogs considering WADA requirements including specificity, limit of detection (10-25 pg/mL), limit of identification, recovery (25-100%), robustness, carry over (<2%), and matrix effects. All sample preparation steps were controlled by two stable isotope-labeled internal standards, namely, [[2H10] LeuB6, B11, B15, B17]-insulin and [[13C6] Leu26, 30] C-peptide. Finally, the method was applied to samples from patients with treated with synthetic insulins.
测定尿液样本中生物活性肽激素（如胰岛素、其合成类似物和C肽）的重要性日益增加，这是一项分析挑战。尿液中胰岛素的生理浓度通常处于亚纳克/毫升水平，因此是一项复杂的分析任务。另一方面，C肽浓度往往处于中等纳克/毫升范围，因此更容易测定。胰岛素和C肽在糖尿病等代谢紊乱的诊断和管理中很重要，也是职业体育和法医学中特别相关的目标分析物。所有胰岛素都被列入世界反兴奋剂机构（WADA）的体育禁用物质和方法清单，最低所需性能水平（MRPL）为50皮克/毫升。到目前为止，结合免疫萃取和后续质谱检测的方法大多用于此目的。利用此处开发的方法，样品制备已大大简化，并且不需要基于抗体的样品纯化。这是通过复杂的混合模式固相萃取以及随后与液相色谱联用高分辨率质谱进行分离来实现的。包括的目标胰岛素有人胰岛素、赖脯胰岛素、谷赖胰岛素、门冬胰岛素、甘精胰岛素代谢物、德谷胰岛素，此外还有人C肽。该方法针对合成胰岛素类似物进行了验证，符合WADA要求，包括特异性、检测限（10 - 25皮克/毫升）、鉴定限、回收率（25 - 100%）、稳健性、残留（<2%）和基质效应。所有样品制备步骤均由两种稳定同位素标记的内标物控制，即[[2H10]亮氨酸B6、B11、B15、B17] - 胰岛素和[[13C6]亮氨酸26、30] - C肽。最后，该方法应用于接受合成胰岛素治疗的患者的样本。
