Selective and sensitive quantification of glucagon and glucagon-related peptide hormones in human plasma using conventional LC/MS/MS

Importance of the Topic

Glucagon and related peptides derived from proglucagon are key regulators of glucose metabolism and are implicated in the pathophysiology of diabetes and other metabolic disorders. Conventional immunoassays face cross-reactivity challenges when measuring closely related peptide hormones. A robust, selective, and sensitive LC/MS/MS approach addresses these limitations and provides accurate quantification of glucagon and its analogues in human plasma.

Goals and Study Overview

This study aimed to develop and validate a conventional flow LC/MS/MS method for the simultaneous quantification of intact glucagon, GLP-1 isoforms, insulin, and therapeutic GLP-1 analogues in human plasma. The key objectives were to achieve low picomolar detection limits, resolve structurally similar peptides, and demonstrate applicability to endogenous samples.

Methodology and Instrumentation

Sample Preparation

• Human plasma (500 µL) collected with protease inhibitors
• Acidification with 40% acetic acid followed by alkalization with ammonium hydroxide
• SPE cleanup using EVOLUTE EXPRESS AX cartridges
• Elution with acetonitrile water acetic acid mixture and dilution prior to analysis

LC/MS/MS Setup

• Shimadzu Nexera X2 UHPLC with Shim-Pack ODS II or Phenomenex Kinetex C18 columns (2.0–2.1 mm × 100–150 mm)
• Binary gradient of 0.1% formic acid in water and acetonitrile
• Shimadzu LCMS-8060 triple quadrupole mass spectrometer with ESI positive mode
• MRM transitions optimized for each peptide charge state (3+ to 6+)

Main Results and Discussion

The method achieved baseline separation of intact hormones and analogues within 10–13 minutes. Calibration curves for glucagon and insulin showed linearity over four orders of magnitude (r2 > 0.9989). The lower limit of detection for glucagon was 2.5 pM, well below physiological levels (10–50 pM). Recovery experiments in spiked plasma yielded accuracies near 100%. Analysis of healthy volunteer samples revealed a twofold increase in fasting versus casual glucagon levels, consistent with physiological expectations.

Benefits and Practical Applications

This LC/MS/MS protocol offers highly selective quantification of proglucagon-derived peptides without antibody cross-reactivity. Practical applications include:

• Clinical research on diabetes and metabolic syndrome
• Therapeutic monitoring of GLP-1 analogues
• Pharmacokinetic and pharmacodynamic studies
• Quality control in peptide drug development

Future Trends and Potential Uses

Advances may focus on multiplexed panels of peptide hormones, microflow LC for higher throughput, integration with automated sample preparation systems, and extension to novel biomarkers. Coupling with high-resolution MS or ion mobility could further enhance selectivity and expand the method to complex clinical matrices.

Conclusion

The developed conventional flow LC/MS/MS approach enables reliable, sensitive, and selective quantification of glucagon and related peptide hormones in human plasma. The method overcomes immunoassay limitations, demonstrates robust performance in endogenous samples, and offers broad utility for clinical and pharmaceutical research.

Reference(s)

• Matsubara T, Yokoi N, Hoshikawa R, Hirano I, Seino S. Selective and sensitive quantification of glucagon and glucagon-related peptide hormones in human plasma using conventional LC/MS/MS. ASMS 2016; ThP-494.