UPLC-MS/MS Determination of GLP-1 Analogue, Liraglutide A Bioactive Peptide in Human Plasma

Significance of the Topic

Accurate quantification of peptide therapeutics in biological matrices is critical for drug development and clinical monitoring. GLP-1 analogues such as liraglutide play a key role in type 2 diabetes management but pose analytical challenges due to low endogenous concentrations, complex matrices, and peptide instability. A robust LC-MS/MS assay enhances sensitivity, specificity and throughput in bioanalysis.

Objectives and Study Overview

The study aimed to develop a high-sensitivity, high-selectivity UPLC-MS/MS method for quantifying liraglutide in human plasma with a target lower limit of quantification of 0.5 ng/mL. The workflow integrates simplified sample preparation, optimized chromatographic separation and tandem quadrupole detection to support pharmacokinetic and biosimilar research.

Methodology and Instrumentation Used

Sample preparation employed protein precipitation with acetonitrile and water, followed by mixed-mode solid phase extraction using an Oasis WAX µElution 96-well plate to minimize losses and remove interferences. Chromatography utilized an ACQUITY UPLC I-Class FTN System with a Peptide BEH C18 column (300Å, 1.7 µm, 2.1×150 mm) at 80°C. A linear gradient of 0.3% formic acid in water and acetonitrile:methanol (50:50) at 0.3 mL/min delivered narrow peaks (<8 s). Mass spectrometric detection was performed on a Xevo TQ-XS tandem quadrupole in positive ESI mode, monitoring the 4+ precursor at m/z 938.68 and y-ion fragment at m/z 1064.08 under optimized cone and collision energies.

Main Results and Discussion

The assay achieved a linear calibration range of 0.5–200 ng/mL (r2>0.995) with accuracy between 85–115% and precision CVs ≤15%. The LLOQ of 0.5 ng/mL in extracted plasma provided a signal-to-noise ratio of 126. Quality control samples at low, mid and high concentrations met guideline criteria with accuracies of 93–106% and CVs ≤9%. The combined use of µElution SPE and UPLC separation resolved liraglutide from matrix interferences, ensured minimal carryover and enabled an 8-minute analytical cycle time.

Benefits and Practical Applications

This streamlined workflow delivers high throughput, reduced sample handling and enhanced peptide recovery without evaporation steps. It supports pharmacokinetic studies, biosimilar development and routine bioanalysis in research and industry laboratories. The method’s robustness and reproducibility facilitate reliable monitoring of liraglutide in clinical and preclinical settings.

Future Trends and Opportunities

Advances may include integration with microflow or nanoflow LC for further sensitivity gains, automation of SPE procedures and expansion to multiplexed peptide panels. High-resolution mass spectrometry and alternative ionization strategies could enhance selectivity. The approach can be adapted for next-generation GLP-1 analogues and other peptide therapeutics.

Conclusion

A sensitive, selective and reproducible UPLC-MS/MS method for liraglutide in human plasma was established. The combination of protein precipitation, µElution SPE and optimized UPLC and MS conditions achieved an LLOQ of 0.5 ng/mL with quantitative performance meeting bioanalytical guidelines. This protocol is fit for purpose in drug development and clinical research.

Reference

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