Simplified quantification of insulin, its synthetic analogs and C-peptide in human plasma by means of LC-HRMS.

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