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The preanalytical stability of glucagon as measured by liquid chromatography tandem mass spectrometry and two commercially available immunoassays

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posted on 24.08.2017 by Jaimini Cegla, Ben Jones, James W. Howard, Richard G. Kay, Colin Creaser, Steve Bloom, Tricia Tan
© 2016, © The Author(s) 2016. Background: One of the main challenges in the measurement of glucagon is the premise that it is unstable in human plasma. Traditionally, protease inhibitors have been used to prevent its degradation; however, their use is controversial. Here, we investigated the optimal method of sample collection for glucagon, with measurement by liquid chromatography tandem mass spectrometry (LC-MS/MS) and two commercially available immunoassays. Methods: Blood from healthy fasting volunteers (n = 10) was processed under a variety of preanalytical conditions including collection in EDTA vs. lithium heparin tubes and the addition of aprotinin and/or a dipeptidyl-peptidase IV (DPPIV) inhibitor. Additionally, the effect of freeze thaw was assessed. Plasma glucagon concentrations were measured by LC-MS/MS and two commercially available immunoassays (HTRF® sandwich immunoassay, Cisbio and Milliplex MAP Human Metabolic Hormone Panel, Merck Millipore). Results: A systematic bias of Milliplex > LC-MS/MS > HTRF was noted and plasma glucagon concentrations were significantly different between methods (Milliplex vs. LC-MS/MS P < 0.01; Milliplex vs. HTRF P < 0.0001; LC-MS/MS vs. HTRF P < 0.001). The addition of aprotinin, DPPIV inhibitor or a combination of aprotinin and DPPIV inhibitor had no effect on plasma glucagon concentrations when compared to ‘non-stabilized’ samples or each other. Whether samples were taken in EDTA tubes or lithium heparin tubes made no difference to plasma glucagon concentrations. These findings were consistent for all three methods. Plasma glucagon concentrations were not significantly different after two freeze–thaw cycles (performed on samples in EDTA tubes containing aprotinin and DPPIV inhibitor). Conclusions: This study demonstrates that glucagon is stable in both EDTA and lithium heparin tubes when stored at −80℃. Furthermore, the addition of aprotinin and DPPIV inhibitors is unnecessary.

Funding

The Section of Endocrinology and Investigative Medicine is funded by grants from the MRC, BBSRC, NIHR, an Integrative Mammalian Biology (IMB) Capacity Building Award, an FP7‐HEALTH‐2009‐241592 EuroCHIP grant and is supported by the NIHR Imperial Biomedical Research Centre Funding Scheme.

History

School

  • Science

Department

  • Chemistry

Published in

Annals of Clinical Biochemistry

Volume

54

Issue

2

Pages

293 - 296

Citation

CEGLA, J....et al., 2017. The preanalytical stability of glucagon as measured by liquid chromatography tandem mass spectrometry and two commercially available immunoassays. Annals of Clinical Biochemistry, 54(2), pp. 293-296.

Publisher

© The authors. Published by SAGE Publications Ltd

Version

AM (Accepted Manuscript)

Publisher statement

This work is made available according to the conditions of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) licence. Full details of this licence are available at: https://creativecommons.org/licenses/by-nc-nd/4.0/

Acceptance date

29/09/2016

Publication date

2016-10-24

Notes

This paper was accepted for publication in the journal Annals of Clinical Biochemistry and the definitive published version is available at http://dx.doi.org/10.1177/0004563216675648

ISSN

0004-5632

eISSN

1758-1001

Language

en

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