Selective and sensitive quantification of glucagon in human plasma using microflow LC/Q-TOF MS

Significance of the topic

Accurate measurement of glucagon, a key hormone in blood glucose regulation, is critical for understanding and managing diabetes. Conventional immunoassays often suffer from cross-reactivity with structurally related peptides derived from proglucagon, limiting specificity. A sensitive, interference-free analytical method would enable reliable quantification of endogenous glucagon levels in clinical and research settings.

Objectives and study overview

The study aimed to develop a streamlined sample-preparation protocol and a high-sensitivity microflow liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (microflow LC-Q-TOF MS) method to selectively quantify intact glucagon in human plasma. Key goals included minimizing peptide degradation, reducing matrix effects, and achieving detection limits below physiological concentration ranges.

Methodology

Samples of human plasma (500 μL) were collected into tubes containing a protease inhibitor cocktail to prevent peptide degradation. After acidification with acetic acid, samples were subjected to solid-phase extraction on an anion-exchange cartridge (EVOLUTE EXPRESS AX). The eluate was concentrated and reconstituted for microflow LC analysis.

Instrumentation used

• Liquid chromatograph: Nexera Mikros microflow system
• Analytical column: HALO C18, 2.7 μm, 0.2 mm ID × 100 mm
• Trap column: L-Column2 C8, 5 μm, 0.2 mm ID × 100 mm
• Mass spectrometer: LCMS-9030 Q-TOF with microflow electrospray ionization
• Flow rates: 3 μL/min for analysis; 20 μL/min trap loading
• Gradient: binary 0.1% formic acid in water and acetonitrile

Main results and discussion

Intact glucagon was detected as multiply charged ions (3+ to 5+). The microflow LC-ESI configuration delivered a ten-fold sensitivity improvement over semi-microflow setups. The lower limit of detection for glucagon was estimated at 10 pM, well below normal plasma concentrations (10–50 pM). Calibration was linear from 10 to 10 000 pM (r2 > 0.999) with accuracy within 93–104% and relative standard deviations below 5%. Endogenous glucagon in pooled human plasma was quantified reliably within physiological ranges.

Benefits and practical applications

• Highly selective quantification of glucagon without cross-interference from related peptides
• Sensitivity sufficient to measure low-picomolar endogenous levels
• Streamlined sample preparation compatible with routine laboratory workflows
• Potential use in clinical research, drug development, and QA/QC of peptide therapeutics

Future trends and potential applications

Future developments may include automated on-line sample cleanup, multiplexed analysis of multiple peptide hormones in a single run, and miniaturized platforms for point-of-care testing. Integration with data-independent acquisition modes and further enhancements in mass spectrometer speed and resolution could expand applications to broader biomarker panels.

Conclusion

The described microflow LC-Q-TOF MS method, combined with a simplified solid-phase extraction protocol, enables sensitive and selective quantification of intact glucagon in human plasma. The approach overcomes limitations of immunoassays and offers a robust tool for diabetes research and clinical studies.