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Simultaneous determination of insulin, DesB30 insulin, proinsulin, and C-peptide in human plasma samples by liquid chromatography coupled to high resolution mass spectrometry

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Abstract

The endocrine pancreatic processes comprising the main actions of insulin, proinsulin, and C-peptide, represent a well-investigated system in the human organism because of the widespread epidemic of diabetes mellitus. Besides their medical relevance, insulin and its related compounds have further been of particular interest also to forensic science and doping controls, and in all disciplines, a specific and sensitive determination of the target analytes is critical for accurate result interpretation. In the present study, a method was developed allowing for the simultaneous determination of human insulin (and its synthetic analogues), the degradation product DesB30 human insulin, proinsulin, and C-peptide in plasma samples (100 µL) by liquid chromatography coupled to high resolution mass spectrometry. Immunoaffinity extraction employing magnetic nanoparticles and two monoclonal antibodies for insulin and C-peptide were used in combination with two internal standards. The assay was validated for quantitative result evaluation considering the parameters specificity, linearity (0–10 ng/mL), limit of detection (~0.05 ng/mL), precision at high and low levels (6–25%), accuracy at high and low levels (85–127%), recovery (33–44%), ion suppression, and stability. Furthermore, the method was applied to samples from healthy athletes, patients with diabetes mellitus (type II) with and without treatment with insulin, and patients being treated for diabetes mellitus (type I). The results enable a thorough interpretation of the data with respect to endocrine, forensic and doping control samples.

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References

  1. Fosgerau K, Hoffmann T (2015) Peptide therapeutics: current status and future directions. Drug Discov Today 20:122–128

    Article  CAS  PubMed  Google Scholar 

  2. Parfitt C, Church D, Armston A, Couchman L, Evans C, Wark G, McDonald TJ (2015) Commercial insulin immunoassays fail to detect commonly prescribed insulin analogues. Clin Biochem 48:1354–1357

    Article  CAS  PubMed  Google Scholar 

  3. Chevenne D, Trivin F, Porquet D (1999) Insulin assays and reference values. Diabetes Metab 25:459–476

    CAS  PubMed  Google Scholar 

  4. Rodriguez-Cabaleiro D, Van Uytfanghe K, Stove V, Fiers T, Thienpont LM (2007) Pilot study for the standardization of insulin immunoassays with isotope dilution liquid chromatography/tandem mass spectrometry. Clin Chem 53:1462–1469

    Article  CAS  PubMed  Google Scholar 

  5. Sonksen PH (2001) Insulin, growth hormone and sport. J Endocrinol 170:13–25

    Article  CAS  PubMed  Google Scholar 

  6. Green RP, Hollander AS, Thevis M, Thomas A, Dietzen DJ (2010) Detection of surreptitious administration of analog insulin to an 8-week-old infant. Pediatrics 125:e1236–e1240

    Article  PubMed  Google Scholar 

  7. Lebowitz MR, Blumenthal SA (1993) The molar ratio of insulin to C-peptide. An aid to the diagnosis of hypoglycemia due to surreptitious (or inadvertent) insulin administration. Arch Intern Med 153:650–655

    Article  CAS  PubMed  Google Scholar 

  8. Marks V (1999) Murder by insulin. Med Leg J 67:147–163

    Article  CAS  PubMed  Google Scholar 

  9. Marks V (2009) Murder by insulin: suspected, purported and proven–a review. Drug Test Anal 1:162–176

    Article  CAS  PubMed  Google Scholar 

  10. Marks V, Richmond C (2008) Kenneth Barlow: the first documented case of murder by insulin. J R Soc Med 101:19–21

    Article  PubMed  PubMed Central  Google Scholar 

  11. Ojanperä I, Sajantila A, Vinogradova L, Thomas A, Schanzer W, Thevis M (2013) Post-mortem vitreous humour as potential specimen for detection of insulin analogues by LC-MS/MS. Forensic Sci Int 233:328–332

    Article  PubMed  Google Scholar 

  12. Thanawala N, Cheney S, Wark G, Thevis M, Thomas A, Madira W, Tziaferi V, Greening J (2016) Factitious administration of analogue insulin to a 2-year-old child. Br J Diabetes 16:82–84

    Article  Google Scholar 

  13. Kippen AD, Cerini F, Vadas L, Stöcklin R, Vu L, Offord RE, Rose K (1997) Development of an isotope dilution assay for precise determination of insulin, C-peptide, and proinsulin levels in non-diabetic and type II diabetic individuals with comparison to immunoassay. J Biol Chem 272:12513–12522

    Article  CAS  PubMed  Google Scholar 

  14. Stöcklin R, Vu L, Vadas L, Cerini F, Kippen AD, Offord RE, Rose K (1997) A stable isotope dilution assay for the in vivo determination of insulin levels in humans by mass spectrometry. Diabetes 46:44–50

    Article  PubMed  Google Scholar 

  15. Darby SM, Miller ML, Allen RO, LeBeau M (2001) A mass spectrometric method for quantitation of intact insulin in blood samples. J Anal Toxicol 25:8–14

    Article  CAS  PubMed  Google Scholar 

  16. Van Uytfanghe K, Rodriguez-Cabaleiro D, Stockl D, Thienpont LM (2007) New liquid chromatography/electrospray ionisation tandem mass spectrometry measurement procedure for quantitative analysis of human insulin in serum. Rapid Commun Mass Spectrom 21:819–821

    Article  PubMed  Google Scholar 

  17. Hess C, Thomas A, Thevis M, Stratmann B, Quester W, Tschoepe D, Madea B, Musshoff F (2012) Simultaneous determination and validated quantification of human insulin and its synthetic analogues in human blood serum by immunoaffinity purification and liquid chromatography-mass spectrometry. Anal Bioanal Chem 404:1813–1822

    Article  CAS  PubMed  Google Scholar 

  18. Blackburn M (2013) Advances in the quantitation of therapeutic insulin analogues by LC–MS/MS. Bioanalysis 5:2933–2946

    Article  CAS  PubMed  Google Scholar 

  19. Chambers EE, Fountain KJ, Smith N, Ashraf L, Karalliedde J, Cowan D, Legido-Quigley C (2014) Multidimensional LC-MS/MS enables simultaneous quantification of intact human insulin and five recombinant analogs in human plasma. Anal Chem 86:694–702

    Article  CAS  PubMed  Google Scholar 

  20. Peterman S, Niederkofler EE, Phillips DA, Krastins B, Kiernan UA, Tubbs KA, Nedelkov D, Prakash A, Vogelsang MS, Schoeder T, Couchman L, Taylor DR, Moniz CF, Vadali G, Byram G, Lopez MF (2014) An automated, high-throughput method for targeted quantification of intact insulin and its therapeutic analogs in human serum or plasma coupling mass spectrometric immunoassay with high resolution and accurate mass detection (MSIA-HRAM). Proteomics 14:1445–1456

    Article  CAS  PubMed  Google Scholar 

  21. Thevis M, Thomas A, Delahaut P, Bosseloir A, Schänzer W (2005) Qualitative determination of synthetic analogues of insulin in human plasma by immunoaffinity purification and liquid chromatography-tandem mass spectrometry for doping control purposes. Anal Chem 77:3579–3585

    Article  CAS  PubMed  Google Scholar 

  22. Thomas A, Schänzer W, Thevis M (2014) Determination of human insulin and its analogues in human blood using liquid chromatography coupled to ion mobility mass spectrometry (LC-IM-MS). Drug Test Anal 6:1125–1132

    Article  CAS  PubMed  Google Scholar 

  23. Thomas A, Thevis M, Delahaut P, Bosseloir A, Schänzer W (2007) Mass spectrometric identification of degradation products of insulin and its long-acting analogues in human urine for doping control purposes. Anal Chem 79:2518–2524

    Article  CAS  PubMed  Google Scholar 

  24. Taylor SW, Clarke NJ, Chen Z, McPhaul MJ (2016) A high-throughput mass spectrometry assay to simultaneously measure intact insulin and C-peptide. Clin Chim Acta 455:202–208

    Article  CAS  PubMed  Google Scholar 

  25. Thomas A, Schänzer W, Delahaut P, Thevis M (2009) Sensitive and fast identification of urinary human, synthetic and animal insulin by means of nano-UPLC coupled with high-resolution/high-accuracy mass spectrometry. Drug Test Anal 1:219–227

    Article  CAS  PubMed  Google Scholar 

  26. Thomas A, Brinkkötter P, Schänzer W, Thevis M (2015) Metabolism of human insulin after subcutaneous administration: a possible means to uncover insulin misuse. Anal Chim Acta 897:53–61

    Article  CAS  PubMed  Google Scholar 

  27. Roepstorff P, Fohlman J (1984) Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed Mass Spectrom 11:601

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The study was carried out with support of the World Anti-Doping Agency (Grant #13D25MT), the Manfred Donike Institute for Doping Analysis, Cologne, and the Federal Ministry of the Interior of the Federal Republic of Germany (Bonn, Germany). The authors further acknowledge the valuable support by Dr. Philippe Delahaut from the CER Groupe, Département Santé, Marloie, Belgium.

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Authors and Affiliations

  1. Center for Preventive Doping Research and Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany

    Andreas Thomas, Wilhelm Schänzer & Mario Thevis

  2. Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany

    Paul T. Brinkkötter

Authors
  1. Andreas Thomas
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  2. Paul T. Brinkkötter
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  3. Wilhelm Schänzer
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  4. Mario Thevis
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Correspondence to Andreas Thomas.

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The authors declare that they have no conflicts of interest.

Ethical approval

No animal experiments were conducted within this study. For the blood sampling of the patients written consent and approval of the local ethical committee were obtained.

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Thomas, A., Brinkkötter, P.T., Schänzer, W. et al. Simultaneous determination of insulin, DesB30 insulin, proinsulin, and C-peptide in human plasma samples by liquid chromatography coupled to high resolution mass spectrometry. Forensic Toxicol 35, 106–113 (2017). https://doi.org/10.1007/s11419-016-0343-8

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  • DOI: https://doi.org/10.1007/s11419-016-0343-8

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