LC-MS/MS QUANTIFICATION OF INTACT INSULIN-LIKE GROWTH FACTOR-1 FOR CLINICAL RESEARCH

Importance of the Topic

The accurate quantification of intact Insulin-like Growth Factor-1 (IGF-1) is crucial for clinical research and endocrinology, as IGF-1 mediates many of the physiological actions of growth hormone and serves as a biomarker in metabolic disorders, growth deficiencies, and doping control.

Objectives and Study Overview

This study aimed to develop and validate a streamlined LC-MS/MS workflow for direct measurement of intact IGF-1 in human serum, achieving low ng/mL sensitivity without requiring protein digestion or complex affinity enrichment.

Methodology and Sample Preparation

The workflow combined protein precipitation, denaturation, and mixed-mode solid-phase extraction (SPE) to isolate intact IGF-1 from biological matrices. Key steps included:

• Protein binding dissociation using sodium dodecyl sulfate (SDS) at optimized concentrations to disrupt IGF-1/IGFBP complexes.
• Solvent evaluation for protein precipitation, identifying acetonitrile as the most effective.
• Optimization of acid concentration (5% acetic acid) for maximal complex disruption and recovery.

Used Instrumentation

• UHPLC system: Waters ACQUITY UPLC I-Class with CORTECS C18+ column (2.1×50 mm, 1.6 µm), 60 °C.
• Tandem quadrupole MS: Waters Xevo TQ-XS operated in multiple reaction monitoring (MRM) mode.
• Surrogate matrix: Mouse plasma for calibration, human serum for quality controls.

Main Results and Discussion

The method demonstrated:

• Linear dynamic range from 5 to 1000 ng/mL (r²>0.99) with LLOQ at 5 ng/mL.
• High recovery (>90%) using 0.6% SDS for protein binding disruption.
• Precision and accuracy within ±10% bias and CV<10% across low, medium, and high QC levels.
• No significant interference from IGF-II spiked during extraction.

Representative calibration curves and chromatograms confirmed method robustness and reproducibility.

Benefits and Practical Applications

The direct quantification approach offers:

• Simplified sample preparation without enzymatic digestion or immunoaffinity steps.
• High throughput suitable for clinical laboratories and doping control.
• Sensitivity and selectivity required for low-level endogenous biomarker measurement.

Future Trends and Potential Applications

Emerging directions include:

• Automation of sample preparation to increase throughput and consistency.
• Extension to other small proteins or peptide hormones using similar intact-protein workflows.
• Integration with high-resolution MS for broader biomarker panels and structural variant analysis.

Conclusion

This study presents a robust, reproducible LC-MS/MS method for intact IGF-1 quantification in human serum, combining simple SPE cleanup and MRM detection to achieve single-digit ng/mL sensitivity. The approach streamlines workflow and meets bioanalytical validation criteria, making it highly applicable to clinical research and diagnostic laboratories.

Reference

1. Niederkofler EE et al. (2013) Targeted Selected Reaction Monitoring Mass Spectrometric Immunoassay for Insulin-like Growth Factor 1. PLoS ONE 8(11): e81125.
2. Lopes F et al. (2014) Quantification of intact human insulin-like growth factor-I in serum by nano-UHPLC/MS. Rapid Commun. Mass Spectrom. 28:1426–1432.