Simplified Quantification of Insulin, Its Synthetic Analogues and C-peptide in Human Plasma by Means of LC-HRMS

Simplified Quantification of Insulin, Its Synthetic Analogues and C-peptide in Human Plasma by Means of LC-HRMS / Andreas Thomas, Rouxue Yang, Simon Petring, Lia Bally, Mario Thevis. - (Drug Testing and Analysis (2020) 12 January)

  • PMID: 31930697.
  • DOI: 10.1002/dta.2765


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 analogues and C-peptide, reliable determinations are in urgent demand due to their diagnostic value in the management of diabetes and insulin resistance and because of the illicit use of insulins as performance-enhancing agent in professional sports or as effective toxin in forensic toxicology. The concomitant measurement of C-peptide and insulin offers an established tool for the diagnostic workup of hypoglycaemia (endogenous vs. exogenous hyperinsulinemia), characterizing of 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 a 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.

Original document


Research / Study
21 January 2020
Bally, Lia
Petring, Simon
Thevis, Mario
Thomas, Andreas
Yang, Rouxue
Other organisations
European Monitoring Center for Emerging Doping Agents (EuMoCEDA)
Universität Bern - University of Bern
Universitätsspital Bern - University Hospital of Bern
Cologne, Germany: Institute of Biochemistry - German Sport University Cologne
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Mass spectrometry analysis
Testing method development
Doping classes
S4. Hormone And Metabolic Modulators
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Scientific article
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Date generated
23 January 2020
Date of last modification
7 September 2020
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