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Simultaneous determination and validated quantification of human insulin and its synthetic analogues in human blood serum by immunoaffinity purification and liquid chromatography-mass spectrometry

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Abstract

Possible fatal complications of human insulin and its synthetic analogues like hypoglycemia require precise classification and quantitative determination of these drugs both for clinical purposes as well as for forensic toxicologists. A procedure was developed for the identification and quantification of human insulin and different long-acting as well as short-acting synthetic insulins in human blood serum specimens. After an immunoaffinity purification step and separation by liquid chromatography, the insulins were characterized by their five- or sixfold protonated molecule ions and diagnostic product ions. Clinical samples of 207 diabetic and 50 non-diabetic patients after the administration of human insulin or oral antidiabetics and forensic samples were analyzed for human/synthetic insulin concentrations. The method was validated according to international guidelines. Limits of detection of the insulins ranged between 1.3 and 2.8 μU/ml. Recoveries ranged between 33.2 % and 51.7 %. Precision data was in accordance with international guidelines. Clinical samples showed concentrations of human insulin lower than 301 μU/ml. Our liquid chromatography tandem mass spectrometry procedure allows unambiguous identification and quantification of the intact human insulin and its intact synthetic analogues Humalog®, Novolog®, Apidra®, Lantus®, and Levemir® in human blood serum in clinical and overdose cases. The assay could be successfully tested in patients with diabetes mellitus on therapy with insulins or oral antidiabetics.

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Acknowledgments

We would like to thank Pia Aymans and Yvonne Mattern for their help in the laboratory and Julia Brünig and Charlotte Meister for sample collection.

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Authors and Affiliations

  1. Institute of Forensic Medicine, University Hospital Bonn, Stiftsplatz 12, 53111, Bonn, Germany

    Cornelius Hess, Burkhard Madea & Frank Musshoff

  2. Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany

    Andreas Thomas & Mario Thevis

  3. Heart and Diabetes Center Northrhine-Westphalia, Ruhr-University of Bochum, Georgstrasse 11, 32545, Bad Oeynhausen, Germany

    Bernd Stratmann, Wulf Quester & Diethelm Tschoepe

Authors
  1. Cornelius Hess
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  2. Andreas Thomas
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  3. Mario Thevis
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  4. Bernd Stratmann
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  5. Wulf Quester
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  6. Diethelm Tschoepe
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  7. Burkhard Madea
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  8. Frank Musshoff
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Correspondence to Cornelius Hess.

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Hess, C., Thomas, A., Thevis, M. et al. 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 (2012). https://doi.org/10.1007/s00216-012-6271-5

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  • DOI: https://doi.org/10.1007/s00216-012-6271-5

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