Multidimensional LC-MS/MS Method for the Quantification of Intact Human Insulin

Importance of the Topic

Accurate measurement of human insulin in serum is critical for diabetes research, pharmacokinetics, and therapeutic monitoring. Traditional immunoassays can suffer from cross reactivity and lack of standardization. A robust LC-MS/MS assay enhances specificity and reliability in quantifying intact insulin, addressing unmet needs in clinical and analytical settings.

Objectives and Study Overview

This study aimed to develop and validate a multidimensional LC-MS/MS method using Shimadzu Nexera X2 LC×LC coupled to LCMS-8060 for the direct quantitation of intact human insulin in serum. Validation followed FDA bioanalytical method guidelines covering selectivity, accuracy, precision, recovery, calibration, sensitivity, reproducibility, and stability.

Methodology and Instrumentation

Sample preparation involved protein precipitation with ice-cold methanol (1:1 v/v) and addition of stable isotope-labelled insulin-d40 as internal standard. Chromatographic separation used a heart-cut 2D approach: a Kinetex C18 loading column (50×2.1 mm, 2.6 μm) and an analytical column (50×2.1 mm, 1.3 μm), both employing a 7.5-minute gradient. Detection was achieved via MRM under positive electrospray ionization on a Shimadzu LCMS-8060 triple quadrupole. Key MS parameters included interface temperature 350 °C, heat block 500 °C, desolvation line 250 °C, argon CID gas, and nitrogen nebulizing and drying gases.

Results and Discussion

Method optimization yielded two MRM transitions per analyte for quantitation and confirmation with a retention time of 5.8 minutes. Calibration curves were linear (R2>0.99) from 8.6 to 1720 pmol/L. Intra- and inter-day accuracy ranged 89–108% with precision CV<6.8%. Extraction recovery was 87–100% and matrix effects between 93–111%. The LLOQ of 103.3 pmol/L demonstrated acceptable sensitivity. Stability tests under freeze-thaw, room temperature, cold room, and long-term storage showed deviations within ±15% except at the lowest concentration. The method exhibited good reproducibility, including reinjection consistency. Comparison with immunoassay data across ten clinical samples showed differences mostly within ±6.5%, highlighting concordance. A pharmacokinetic profile was successfully generated over a 2-hour clamp study.

Benefits and Practical Applications

• Enhanced specificity and selectivity in intact insulin measurement
• Rapid throughput with a 7.5-minute run time
• Robust quantitation across a broad dynamic range
• Applicable to pharmacokinetic studies, clinical research, and quality control

Future Trends and Opportunities

Integration of high-resolution mass spectrometry for multiplexed peptide assays, automation of sample prep for higher throughput, miniaturized microflow LC systems to reduce solvent usage, and expansion to other peptide biomarkers in personalized medicine are promising avenues.

Conclusion

A validated 2D-LC-MS/MS assay for intact human insulin in serum delivers high specificity, accuracy, and robustness. It meets regulatory guidance and offers significant benefits for clinical and research applications.

Reference

• Daryl Kim Hor Hee, Jun Liang Ong, Lawrence Soon-U Lee et al. Multidimensional LC-MS/MS Method for the Quantification of Intact Human Insulin. Shimadzu Application Note AD-0182, 2019
• FDA Guidance for Industry, Bioanalytical Method Validation, U.S. Department of Health and Human Services