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Determination of human insulin and its analogues in human blood using liquid chromatography coupled to ion mobility mass spectrometry (LC‐IM‐MS)
Author(s) -
Thomas Andreas,
Schänzer Wilhelm,
Thevis Mario
Publication year - 2014
Publication title -
drug testing and analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.065
H-Index - 54
eISSN - 1942-7611
pISSN - 1942-7603
DOI - 10.1002/dta.1710
Subject(s) - chromatography , chemistry , mass spectrometry , insulin , insulin aspart , detection limit , ion suppression in liquid chromatography–mass spectrometry , liquid chromatography–mass spectrometry , human insulin , high performance liquid chromatography , medicine , postprandial
The qualitative and quantitative determination of insulin from human blood samples is an emerging topic in doping controls as well as in other related disciplines (e.g. forensics). Beside the therapeutic use, insulin represents a prohibited, performance enhancing substance in sports drug testing. In both cases accurate, sensitive, specific, and unambiguous determination of the target peptide is of the utmost importance. The challenges concerning identifying insulins in blood by liquid chromatography coupled to ion mobility mass spectrometry (LC‐IM‐MS) are detecting the basal concentrations of approximately 0.2 ng/mL and covering the hyperinsulinaemic clamps at > 3 ng/mL simultaneously using up to 200 μL of plasma or serum. This is achieved by immunoaffinity purification of the insulins with magnetic beads and subsequent separation by micro‐scale liquid chromatography coupled to ion mobility / high resolution mass spectrometry. The method includes human insulin as well as the synthetic or animal analogues insulin aspart, glulisine, glargine, detemir, lispro, bovine, and porcine insulin. The method validation shows reliable results considering specificity, limit of detection (0.2 ng/mL except for detemir: 0.8 ng/mL), limit of quantification (0.5 ng/mL for human insulin), precision (CV < 20%), linearity (r > 0.99), recovery, accuracy (>90%), robustness (plasma/serum), and ion suppression. For quantification of human insulin a labelled internal standard ([[ 2 H 10 ]‐Leu B6,B11,B15,B17 ] ‒ human Insulin) is introduced. By means of the additional ion mobility separation of the different analogues, the chromatographic run time is shortened to 8 min without losing specificity. As proof‐of‐concept, the procedure was successfully applied to different blood specimens from diabetic patients receiving recombinant synthetic analogues Copyright © 2014 John Wiley & Sons, Ltd.