Development of 2D-LC/MS/MS Bioanalytical Method for Quantitative Determination of Insulin Glargine in Human Plasma

Significance of the Topic

Insulin glargine is a long-acting recombinant insulin analog widely used in diabetes management. Accurate quantification at low plasma concentrations is essential for pharmacokinetic profiling and therapeutic monitoring. Two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D-LC/MS/MS) offers enhanced sensitivity and selectivity by effectively reducing matrix interferences.

Objectives and Study Overview

This study aimed to develop and validate a high-sensitivity 2D-LC/MS/MS assay on the Shimadzu LCMS-8060 platform for quantifying insulin glargine in human plasma, while eliminating solid-phase extraction from the sample preparation workflow.

Methodology and Instrumentation

Sample preparation involved protein precipitation by adding acetonitrile/methanol (3:1 v/v) to plasma, vortexing, centrifugation, filtration, evaporation under nitrogen, and reconstitution in water/methanol (80:20) with 0.1% acetic acid. Chromatographic separation employed a Nexera X2 UHPLC system with automated 6-way valve switching: first dimension on a Kinetex C18 column (2.6 µm, 50×2.1 mm) to trap the 1.50–1.73 min fraction in a 200 µL loop, followed by transfer to a second Kinetex C18 column (1.3 µm, 50×2.1 mm) for final separation. MS/MS detection used a Shimadzu LCMS-8060 triple quadrupole with positive-mode ESI, monitoring MRM transitions m/z 867→984 for quantitation and 1011.5→1179 for confirmation, with optimized collision energies and bias voltages.

Instrumentation Used

• Shimadzu Nexera X2 UHPLC with binary pumps, column oven, and automated switching valve
• Shimadzu LCMS-8060 triple quadrupole mass spectrometer with ESI source
• LabSolutions software for system control, MRM optimization, and data analysis

Main Results and Discussion

• A seven-point calibration from 0.05 to 10 ng/mL achieved linearity (R2 > 0.9996) using 1/conc weighting.
• Accuracy ranged from 98% to 105% across calibration levels; the limit of quantitation was 0.018 ng/mL in treated plasma (≈70 pg/mL pre-treatment).
• Precision (RSD) was 11.1% at 0.05 ng/mL, improving to 2.3–7.8% at higher levels.
• Recovery after protein precipitation ranged from 63% to 87% without SPE cleanup.
• Method specificity was confirmed by dual MRM transitions, consistent transition ratios, and retention time matching.

Benefits and Practical Applications of the Method

• High analytical sensitivity without laborious solid-phase extraction.
• Effective removal of matrix components via 2D separation, reducing instrument contamination.
• Automated valve switching and simplified workflow support high-throughput bioanalysis.

Future Trends and Potential Applications

The described 2D-LC/MS/MS approach can be extended to other peptide therapeutics and their metabolites. Integration with robotic sample handling and ongoing advances in UHPLC and MS hardware are expected to further lower detection limits, enhance throughput, and broaden applications in clinical pharmacokinetic and bioequivalence studies.

Conclusion

An automated 2D-LC/MS/MS assay on the Shimadzu LCMS-8060 was successfully validated for insulin glargine in human plasma, achieving sub-100 pg/mL sensitivity. The streamlined, SPE-free workflow enhances robustness and is well-suited for pharmacokinetic and clinical bioanalysis.

References

1. Blackburn M, Ewles M, Gray N, McDougall S, Stanta J. Presented at EBF, Barcelona, June 2014.
2. Chambers EE, Fountain KJ, Smith N, Ashraf L, Karalliedde J, Cowan D, Legido-Quigley C. Anal Chem. 2014;86:694–702.
3. Li YX, Ke Y, Li J, Chen X, Mollah S, Wang X. J Anal Bioanal Tech. 2013;S5.