Simultaneous Analysis of Intact Human Insulin and Five Analogs in Human Plasma Using μElution SPE and a CORTECS UPLC Column

Significance of the Topic

This application addresses the critical need for accurate, sensitive, and specific quantification of intact human insulin and its therapeutic analogs in plasma. Reliable measurement of insulin species underpins diabetes management, pharmacokinetic profiling, and anti-doping controls. Mass spectrometry methods that directly detect intact peptides offer advantages over conventional immunoassays, including multiplexing capability and reduced development time.

Objectives and Study Overview

The primary goal was to establish a streamlined LC-MS/MS workflow for simultaneous determination of human insulin, lispro, glargine, aspart, glulisine, and detemir in human plasma without enzymatic digestion or affinity purification. The method integrates selective sample cleanup and high-efficiency chromatography to achieve low picogram-per-milliliter detection limits, robust quantitative performance, and clear differentiation of structurally similar analogs.

Methodology and Instrumentation

Sample Preparation:

• Protein precipitation: 1:1 methanol:acetonitrile with 1% acetic acid added to plasma containing bovine insulin IS; centrifugation to remove proteins.
• pH adjustment: Dilution with 5% NH₄OH to pH ~11 to impart negative charge on insulin species.
• Mixed-mode SPE: Oasis MAX µElution 96-well plate for combined anion exchange and reversed-phase cleanup; optimized wash and elution solvents (60% methanol/30% water/10% acetic acid).

Chromatography and Mass Spectrometry:

• LC system: ACQUITY UPLC I-Class with 2D at-column dilution, trap and back-elute configuration.
• Analytical column: CORTECS UPLC C18+ (1.6 µm, 2.1 × 50 mm) at 60 °C; flow 0.25 mL/min; 8.5 min run with gradient from 15% to 40% organic.
• Mass spectrometer: Xevo TQ-S, ESI positive mode; optimized MRM transitions for each analog with cone and collision energies tailored to maximize specificity.

Main Results and Discussion

The validated method achieved limits of detection between 50 and 200 pg/mL for all six analytes. Calibration curves (50–10 000 pg/mL) were linear (r²≥0.995), with mean curve-point accuracy of 99–100%. Inter- and intra-day accuracies for QCs ranged from 92% to 106%, and precisions (CV) were ≤7.5% and ≤5.3%, respectively. Matrix factor CVs across six plasma lots were below 15%, confirming negligible ion suppression. Critical specificity studies demonstrated that high concentrations of human insulin did not interfere with analog quantification. The combination of mixed-mode SPE and high-efficiency solid-core chromatography enabled clear separation of insulin and lispro, which differ by only two reversed amino acids.

Benefits and Practical Applications

This approach eliminates time-consuming immunoaffinity steps and nano-LC, leveraging existing analytical-scale LC-MS instrumentation. It supports high throughput in clinical and bioanalytical laboratories, facilitates multiplexed pharmacokinetic and clinical studies, and can be adapted for regulatory anti-doping assays.

Future Trends and Potential Applications

Emerging opportunities include integration with automated online SPE, miniaturized flow paths for further sensitivity gains, expansion to next-generation analogs, and coupling with high-resolution MS for broader peptide profiling. Advancements in microfluidic sample handling and machine-learning–guided chromatography optimization will further streamline bioanalysis of peptide therapeutics.

Conclusion

A robust, simple, and highly selective LC-MS/MS method for direct quantification of intact human insulin and five analogs in plasma has been developed. It delivers low-picogram sensitivity, excellent quantitative performance, and the ability to distinguish closely related analogs, meeting rigorous bioanalytical validation criteria and offering broad applicability in clinical, research, and regulatory settings.

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