Universität zu Köln

Thomas, Andreas, Benzenberg, Lukas, Bally, Lia ORCID: 0000-0003-1993-7672 and Thevis, Mario (2021). Facilitated Qualitative Determination of Insulin, Its Synthetic Analogs, and C-Peptide in Human Urine by Means of LC-HRMS. Metabolites, 11 (5). BASEL: MDPI. ISSN 2218-1989

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Abstract

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 diabetes mellitus treated with synthetic insulins.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Thomas, Andreas UNSPECIFIED UNSPECIFIED UNSPECIFIED Benzenberg, Lukas UNSPECIFIED UNSPECIFIED UNSPECIFIED Bally, Lia UNSPECIFIED orcid.org/0000-0003-1993-7672 UNSPECIFIED Thevis, Mario UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-581485
DOI:	10.3390/metabo11050309
Journal or Publication Title:	Metabolites
Volume:	11
Number:	5
Date:	2021
Publisher:	MDPI
Place of Publication:	BASEL
ISSN:	2218-1989
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language MASS-SPECTROMETRY Multiple languages Biochemistry & Molecular Biology Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/58148