A novel, simple and sensitive LC-MS/MS method for simultaneous quantification of insulin glargine and its metabolites (M1 and M2)

Significance of the topic

Insulin glargine is a long-acting insulin analog widely used in diabetes management. Accurate measurement of glargine and its active metabolites M1 and M2 at low plasma concentrations is critical for pharmacokinetic studies, therapeutic monitoring, and quality control. The structural similarity among insulin analogs and metabolites, combined with low circulating levels, presents analytical challenges.

Study objectives and overview

This study presents a novel, simple, and sensitive LC-MS/MS method for the simultaneous quantification of insulin glargine and its metabolites M1 and M2 in human plasma. The method development focused on overcoming issues of poor ionization, non-specific adsorption, and carryover, enabling reliable detection over a wide concentration range.

Methodology and instrumentation

The method integrates solid phase extraction (SPE) for sample cleanup and high-performance UHPLC separation followed by tandem mass spectrometry in multiple reaction monitoring mode.

• Sample preparation: Calibration standards and QC samples were spiked into human plasma at 75–10000 pg/mL for calibration and 100–2000 pg/mL for QC. Samples underwent protein precipitation, SPE conditioning (methanol and water), loading, washing, elution, evaporation, and reconstitution in 100 µL of solution.
• Chromatography: Separation was achieved on a Shim-pack velox biphenyl column (2.1 × 100 mm, 2.7 µm) using a Nexera X2 UHPLC system. A gradient of 0.1% formic acid in water and acetonitrile at 0.2 mL/min and 40 °C provided efficient resolution.
• Mass spectrometry: A Shimadzu LCMS-8060 with heated ESI interface (250 °C desolvation line, 400 °C block, 300 °C interface) and nitrogen nebulizing (3 L/min) and drying gas (10 L/min) was operated in MRM mode for selective detection of glargine, M1, and M2.

Main results and discussion

Calibration curves exhibited linearity from 75 to 10000 pg/mL with correlation coefficients above 0.98 for all analytes. Recovery rates were consistent and reproducible (glargine ~33%, M1 ~51%, M2 ~51%). Intra- and inter-day precision met acceptance criteria (<20% at LLOQ, <15% at other QC levels) and accuracy fell within 80–120% at LLOQ and 85–115% at higher QC levels.

Benefits and practical applications

The validated assay enables rapid, high-throughput analysis of insulin glargine and its metabolites at trace levels, making it suitable for bioequivalence studies, drug interaction assessments, and therapeutic drug monitoring in clinical research and QC laboratories.

Future trends and potential applications

Advancements may include automation of SPE workflows, adoption of high-resolution mass spectrometry for wider analog profiling, and integration with laboratory information management systems. The methodology can be extended to other insulin analogs and novel peptide therapeutics.

Conclusion

This fully validated LC-MS/MS method offers a straightforward, highly sensitive, and reproducible approach for simultaneous quantification of insulin glargine and its two primary metabolites in human plasma, supporting both research and potential clinical workflows.

Reference

• PubChem. Insulin glargine; accessed July 31, 2019.
• Bolli GB et al. Plasma exposure to insulin glargine and its metabolites M1 and M2 after subcutaneous injection of therapeutic and supratherapeutic doses in subjects with type 1 diabetes. Diabetes Care. 2012;35(12):2626–2630.