Facilitated Qualitative Determination of Insulin, Its Synthetic Analogs, and C-Peptide in Human Urine by Means of LC-HRMS.

Andreas Thomas,Lukas Benzenberg,Lia Bally,Mario Thevis

doi:10.3390/metabo11050309

Andreas Thomas, Lukas Benzenberg + Show 2 more

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https://doi.org/10.3390/metabo11050309

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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.

Highlights

While insulin is an important, endogenous peptide hormone that regulates blood glucose homeostasis and other aspects of metabolism, the cosecreted C-peptide owns merely limited or unknown physiological effects
All synthetic insulin analogs have their individual and specific pharmacokinetic profile, which facilitates their usage as therapeutic agents to treat diabetes mellitus (DM) [3]
The World Anti-Doping Agency (WADA) has set the minimum required performance level (MRPL) for all insulins to 50 pg/mL, and this concentration represents by far the lowest MRPL of all prohibited substances, emphasizing the analytical challenge for these bioactive peptides [5]

Summary

Introduction

While insulin is an important, endogenous peptide hormone that regulates blood glucose homeostasis and other aspects of metabolism, the cosecreted C-peptide owns merely limited (if any) or unknown physiological effects. All published methods for the determination of insulin and C-peptide in urine by means of liquid chromatography/mass spectrometry mainly use immunoaffinity extraction of the target peptides from the matrix [3,6,7,8,9,10,11,12]. These immunoaffinity-enriched sample aliquots are of highly purified quality and allow for nanoscale liquid chromatography

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