Institute of Biochemistry/Center for Preventive Doping Research, German Sport University Cologne, Cologne, Germany.
Department of Diabetes, Endocrinology, Nutritional Medicine, and Metabolism, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland.
Drug Test Anal. 2020 Mar;12(3):382-390. doi: 10.1002/dta.2765. Epub 2020 Feb 5.
The quantification of peptide hormones by means of liquid chromatography (LC) coupled to mass spectrometry (MS) or other techniques (e.g. immunoassays) has been a challenging task in modern analytical chemistry. Especially for insulin, its synthetic analogs, and C-peptide, reliable determinations are urgently needed due to their diagnostic value in the management of diabetes and insulin resistance and because of the illicit use of insulin as a performance-enhancing agent in professional sports or as an effective toxin in forensic toxicology. The concomitant measurement of C-peptide and insulin offers an established tool for the diagnostic workup of hypoglycemia (endogenous vs. exogenous hyperinsulinemia), characterizing hepatic insulin clearance, and the assessment of beta-cell function (insulin secretion). Thus, the present approach offers the possibility to determine human insulin and its synthetic analogs (lispro, glulisine, aspart, glargine metabolite, degludec, detemir, porcine, and bovine) and C-peptide simultaneously after sample preparation utilizing protein precipitation and a mixed-mode cation-exchange solid-phase extraction, and subsequent detection by LC-high resolution MS. The method was fully validated regarding the following parameters: specificity, limit of detection (0.2 ng/mL), limit of quantification (0.6 ng/mL), recovery (40-90%), accuracy (78-128%), linearity, precision (< 21%), carry over, robustness, and matrix effects. The proof-of-concept was shown by analyzing authentic plasma samples from adults with class II obesity and prediabetes collected in the course of an oral glucose tolerance test. All sample preparation steps were controlled by two stable isotope-labeled internal standards, namely [[ H ] Leu ]-insulin, and [[ C ] Leu ] C-peptide.
利用液相色谱（LC）与质谱（MS）或其他技术（如免疫测定）对肽类激素进行定量分析一直是现代分析化学中的一项具有挑战性的任务。特别是对于胰岛素、其合成类似物和 C 肽，由于其在糖尿病和胰岛素抵抗管理中的诊断价值，以及由于胰岛素被非法用作职业运动中的兴奋剂或法医毒理学中的有效毒素，因此迫切需要进行可靠的测定。同时测量 C 肽和胰岛素为低血糖症（内源性与外源性高胰岛素血症）的诊断提供了一种既定的工具，可用于表征肝胰岛素清除率和评估β细胞功能（胰岛素分泌）。因此，本方法提供了一种可能，即在样品制备后利用蛋白质沉淀和混合模式阳离子交换固相萃取，同时检测人胰岛素及其合成类似物（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、地特胰岛素、猪胰岛素和牛胰岛素）和 C 肽，并通过 LC-高分辨 MS 进行检测。该方法在以下参数方面进行了全面验证：特异性、检测限（0.2ng/mL）、定量限（0.6ng/mL）、回收率（40-90%）、准确度（78-128%）、线性、精密度（<21%）、交叉污染、稳健性和基质效应。通过分析在口服葡萄糖耐量试验过程中收集的来自 II 类肥胖和糖尿病前期成年人的真实血浆样本，证明了该方法的概念验证。所有样品制备步骤均由两种稳定同位素标记的内标控制，即 [[ H ] Leu ]-胰岛素和 [[ C ] Leu ] C 肽。
