Skip to main content
SpringerLink
Log in

Accurate analytical method for human plasma glucagon levels using liquid chromatography-high resolution mass spectrometry: comparison with commercially available immunoassays

  • Rapid Communication
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Accurate quantification of plasma glucagon levels in humans is necessary for understanding the physiological and pathological importance of glucagon. Although several immunoassays for glucagon are available, they provide inconsistent glucagon values owing to cross-reactivity of the antibodies with peptides other than glucagon. To overcome this limitation, we developed a novel method to measure glucagon levels by a liquid chromatography (LC)-high-resolution mass spectrometry (HRMS) assay via parallel reaction monitoring (PRM) without immunoaffinity enrichment. Using stable isotope-labeled glucagon as an internal standard and 200 μL of plasma, the lower limit of quantification was 0.5 pM. This method was applied to measure plasma glucagon levels during the oral glucose tolerance test (OGTT) and meal tolerance test (MTT) in healthy volunteers, and its results were compared with those of sandwich enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). During the OGTT, this method showed significant suppression of plasma glucagon levels, and similar patterns were observed with sandwich ELISA and RIA. In contrast, during the MTT, plasma glucagon levels were slightly elevated according to the LC-MS/MS and sandwich ELISA results and were reduced according to RIA results. Our newly developed LC-MS/MS method overcomes a lack of specificity among currently available immunoassays for glucagon and may contribute to a better understanding of the importance of glucagon.

Flowchart for the extraction and quantification of glucagon in human plasma, and plasma glucagon responses in healthy volunteers quantified by the present LC-MS/MS, sandwich ELISA, and RIA during OGTT and MTT

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Abbreviations

CV:

Coefficient of variation

ESI:

Electrospray ionization

HRMS:

High-resolution mass spectrometry

IS:

Internal standard

LLOQ:

Lower limit of quantification

MTT:

Meal tolerance test

OGTT:

Oral glucose tolerance test

PP:

Protein precipitation

PRM:

Parallel reaction monitoring

RE:

Relative error

SPE:

Solid-phase extraction

References

  1. Mighiu PI, Yue JTY, Filippi BM, Abraham MA, Chari M, Lam CK, et al. Hypothalamic glucagon signaling inhibits hepatic glucose production. Nat Med. 2013;19(6):766–72.

    Article  CAS  Google Scholar 

  2. Unger RH, Cherrington AD. Glucagonocentric restructuring of diabetes: a pathophysiologic and therapeutic makeover. J Clin Invest. 2012;122(1):4–12.

    Article  CAS  Google Scholar 

  3. Unger RH, Eisentraut AM, McCall MS, Keller S. Glucagon antibodies and their use for immunoassay for glucagon. Proc Soc Exp Biol Med. 1959;102:621–3.

    Article  CAS  Google Scholar 

  4. Bataille D, Dalle S. The forgotten members of the glucagon family. Diabetes Res Clin Pract. 2014;106(1):1–10.

    Article  CAS  Google Scholar 

  5. Bak MJ, Albrechtsen NW, Pedersen J, Hartmann B, Christensen M, Vilsbøll T, et al. Specificity and sensitivity of commercially available assays for glucagon and oxyntomodulin measurement in humans. Eur J Endocrinol. 2014;170(4):529–38.

    Article  CAS  Google Scholar 

  6. Holst JJ, Christensen M, Lund A, de Heer J, Svendsen B, Kielgast U, et al. Regulation of glucagon secretion by incretins. Diabetes Obes Metab. 2011;13(Suppl 1):89–94.

    Article  CAS  Google Scholar 

  7. Albrechtsen NJW, Hartmann B, Veedfald S, Windeløv JA, Plamboeck A, Bojsen-Møller KN, et al. Hyperglucagonaemia analysed by glucagon sandwich ELISA: nonspecific interference or truly elevated levels? Diabetologia. 2014;57(9):1919–26.

    Article  Google Scholar 

  8. Matsuo T, Miyagawa J, Kusunoki Y, Miuchi M, Ikawa T, Akagami T, et al. Postabsorptive hyperglucagonemia in patients with type 2 diabetes mellitus analyzed with a novel enzyme-linked immunosorbent assay. J Diabetes Invest. 2016;7(3):324–31.

    Article  CAS  Google Scholar 

  9. Chambers EE, Lame ME, Rainville PD, Murphy J, Johnson J, Fountain KJ, et al. Practical applications of integrated microfluidics for peptide quantification. Bioanalysis. 2015;7(7):857–67.

    Article  CAS  Google Scholar 

  10. Chappell DL, Lee AYH, Castro-Perez J, Zhou H, Roddy TP, Lassman ME, et al. An ultrasensitive method for the quantitation of active and inactive GLP-1 in human plasma via immunoaffinity LC-MS/MS. Bioanalysis. 2014;6(1):33–42.

  11. Lee AYH, Chappell DL, Bak MJ, Judo M, Liang L, Churakova T, et al. Multiplexed quantification of proglucagon-derived peptides by immunoaffinity enrichment and tandem mass spectrometry after a meal tolerance test. Clin Chem. 2016;62(1):227–35.

    Article  CAS  Google Scholar 

  12. Sano S, Tagami S, Hashimoto Y, Yoshizawa-Kumagaye K, Tsunemi M, Okochi M, et al. Absolute quantitation of low abundance plasma APL1β peptides at sub-fmol/mL level by SRM/MRM without immunoaffinity enrichment. J Proteome Res. 2014;13(2):1012–20.

    Article  CAS  Google Scholar 

  13. Grund B, Marvin L, Rochat B. Quantitative performance of a quadrupole-orbitrap-MS in targeted LC-MS determinations of small molecules. J Pharm Biomed Anal. 2016;124:48–56.

    Article  CAS  Google Scholar 

  14. Miyachi A, Murase T, Yamada Y, Osonoi T, Harada K. Quantitative analytical method for determining the levels of gastric inhibitory polypeptides GIP1–42 and GIP3–42 in human plasma using LC-MS/MS/MS. J Proteome Res. 2013;12(6):2690–9.

    Article  CAS  Google Scholar 

  15. Auld CA. White paper “Glucagon measurement—addressing long-standing analytical challenges.” 2015. https://www.mercodia.se. Accessed 24 Oct 2016.

  16. Lange V, Picotti P, Domon B, Aebersold R. Selected reaction monitoring for quantitative proteomics: a tutorial. Mol Syst Biol. 2008;4(222):1–14.

    Google Scholar 

  17. Bagger JI, Knop FK, Lund A, Holst JJ, Vilsbøll T. Glucagon responses to increasing oral loads of glucose and corresponding isoglycaemic intravenous glucose infusions in patients with type 2 diabetes and healthy individuals. Diabetologia. 2014;57(8):1720–5.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to the members of Thermo Fisher Scientific and to T. Murase, S. Yamashita, and H. Hashimoto for their fruitful discussions and technical support. We also thank the members of the Kitamura Laboratory for the discussion of the data.

Author information

Authors and Affiliations

  1. Radioisotope and Chemical Analysis Center, Sanwa Kagaku Kenkyusho Co., Ltd., 363 Shiosaki, Hokusei-cho, Inabe, Mie, 511-0406, Japan

    Atsushi Miyachi & Eri Mieno

  2. Metabolic Signal Research Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma, 371-8512, Japan

    Masaki Kobayashi & Tadahiro Kitamura

  3. Pharmaceutical Research Laboratories, Sanwa Kagaku Kenkyusho Co., Ltd., Inabe, Mie, 511-0406, Japan

    Moritaka Goto

  4. Medical Affairs Department, Sanwa Kagaku Kenkyusho Co., Ltd., Nagoya, Aichi, 461-8631, Japan

    Kenichi Furusawa

  5. Quality Assurance Sec., Cosmic Corporation, Tokyo, 112-0002, Japan

    Takashi Inagaki

Authors
  1. Atsushi Miyachi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Masaki Kobayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eri Mieno
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Moritaka Goto
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kenichi Furusawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Takashi Inagaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tadahiro Kitamura
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Atsushi Miyachi or Tadahiro Kitamura.

Ethics declarations

All participants gave oral and written consent. The study was approved by the Ethics Committee of Gunma University and was conducted in accordance with the Declaration of Helsinki.

Conflict of interest

The authors declare that they have no competing interests.

Electronic supplementary material

ESM 1

(PDF 261 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Miyachi, A., Kobayashi, M., Mieno, E. et al. Accurate analytical method for human plasma glucagon levels using liquid chromatography-high resolution mass spectrometry: comparison with commercially available immunoassays. Anal Bioanal Chem 409, 5911–5918 (2017). https://doi.org/10.1007/s00216-017-0534-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-017-0534-0

Keywords

Search

Navigation