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Simplified quantification of insulin, its synthetic analogs and C-peptide in human plasma by means of LC-HRMS.
Drug Testing and Analysis ( IF 2.9 ) Pub Date : 2020-02-05 , DOI: 10.1002/dta.2765
Andreas Thomas 1 , Rouxue Yang 1 , Simon Petring 1 , Lia Bally 2 , Mario Thevis 1, 3
Affiliation  

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 [[2H10] Leu B6, B11, B15, B17]‐insulin, and [[13C6] Leu 26, 30] C‐peptide.

中文翻译:

通过LC-HRMS简化了人血浆中胰岛素,其合成类似物和C肽的定量。

通过液相色谱(LC)结合质谱(MS)或其他技术(例如免疫测定)对肽类激素进行定量分析已成为现代分析化学中一项艰巨的任务。尤其是对于胰岛素,其合成类似物和C肽,迫切需要可靠的测定方法,因为它们在管理糖尿病和胰岛素抵抗方面具有诊断价值,并且由于在职业体育活动中或在体育运动中非法使用胰岛素作为性能增强剂法医毒理学中的有效毒素。C肽和胰岛素的同时测量为诊断低血糖(内源性与外源性高胰岛素血症),表征肝胰岛素清除率以及评估β细胞功能(胰岛素分泌)提供了一个成熟的工具。从而,本方法提供了在使用蛋白质沉淀和混合模式阳离子制备样品后同时测定人胰岛素及其合成类似物(赖脯,甘氨酸,天冬氨酸,甘精代谢产物,地格列克,德特米尔,猪和牛)和C肽的可能性。交换固相萃取,并随后通过LC高分辨率MS进行检测。该方法已针对以下参数进行了充分验证:特异性,检测限(0.2 ng / mL),定量限(0.6 ng / mL),回收率(40–90%),准确性(78–128%),线性,精度(<21%),残留,鲁棒性和矩阵效应。通过分析从口服糖耐量测试过程中收集的II类肥胖和糖尿病前期成年人的真实血浆样本,可以显示出概念验证。2 ħ 10 ]亮氨酸B6,B11,B15,B17 ] -胰岛素,和[ 13 C ^ 6 ]亮氨酸26,30 ] C-肽。
更新日期:2020-02-05
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