Development of bioanalytical method for determination of intact human insulin from plasma using LC/MS/MS

Significance of the Topic

The quantification of intact human insulin in plasma plays a pivotal role in pharmacokinetic and pharmacodynamic studies of diabetic therapies. Accurate measurement of this peptide hormone informs dosage regimens, improves therapeutic monitoring, and aids in the development of novel insulin analogues. Transitioning from traditional ligand binding assays (ELISA, RIA) to LC/MS/MS approaches addresses limitations such as cross-reactivity, lengthy assay optimization, and narrow dynamic ranges.

Objectives and Study Overview

This study aimed to establish a robust, high-sensitivity LC/MS/MS method for direct measurement of intact human insulin in plasma. Key objectives included:

• Developing a streamlined sample preparation protocol.
• Optimizing chromatographic and mass spectrometric conditions to achieve low picomolar detection limits.
• Validating linearity, accuracy, and precision over a 15–500 pM/L range.

Materials, Methodology and Instrumentation

• Sample Preparation: Inspection of charcoal-stripped human plasma spiked with insulin standards (15–500 pM/L) and bovine insulin internal standard. Solid phase extraction (SPE) using HLB cartridges minimized matrix interferences.
• Chromatography: UHPLC Nexera system with C18 column at 40 °C. Mobile phases comprised 0.1 % formic acid in water (A) and acetonitrile (B) under a gradient elution.
• Mass Spectrometry: Shimadzu LCMS-8060 triple quadrupole with heated ESI probe and fast polarity switching. MRM transitions: m/z 1162.30>1158.90 for insulin and 956.40>136.30 for the internal standard.
• Ionization Enhancement: Use of high-temperature gas in the heated ESI probe improved droplet desolvation and signal intensity.

Key Results and Discussion

• Linearity: Excellent correlation (r2 = 0.9968) across 15–500 pM/L.
• Sensitivity: Achieved a lower limit of quantitation (LLOQ) of 15 pM/L, satisfying precision (%RSD <20) and accuracy (80–120%) criteria.
• Precision and Accuracy: QC samples at low (15 pM/L), mid (50 pM/L), and high (300 pM/L) levels demonstrated acceptable variability (<18% RSD) and biases within ±20%.
• Specificity: No detectable interference at the insulin retention time in blank plasma.

Practical Benefits and Applications

The developed method offers:

• Rapid assay turnaround with minimal reagent dependency.
• Multiplexing capability for simultaneous quantitation of related peptides.
• A broad dynamic range compatible with clinical and preclinical studies.
• Reduced development time and cost compared to immunoassays.

Future Trends and Applications

Anticipated advancements include:

• Integration with automated sample processing platforms to increase throughput.
• Expansion to other peptide hormones and biotherapeutics using similar MRM strategies.
• Coupling with high-resolution mass spectrometry for detailed structural analysis of insulin analogues.
• Application in personalized medicine workflows leveraging precision pharmacokinetics.

Conclusion

A straightforward SPE-LC/MS/MS protocol was established for intact human insulin quantitation in plasma. The high sensitivity and reliability of the Shimadzu LCMS-8060 enabled precise measurement down to 15 pM/L, facilitating robust pharmacokinetic investigations and supporting therapeutic monitoring in diabetes research.

References

• Li Y-X et al. J Anal Bioanal Tech, S5 (2013) 004.
• Taylor SW et al. Clin Chim Acta, 455 (2016) 202–208.