Quantitation of Human Insulin-Like Growth Factor-1 and 2 by MRM LC-MS/MS with Scheduled Ionization

Significance of the Topic

Insulin-like growth factors 1 (IGF-1) and 2 (IGF-2) play a pivotal role in human growth disorders assessment. Their low molecular weight and tight binding to specific proteins and receptors in serum require reliable dissociation and quantitation strategies to support clinical and research applications.

Objectives and Study Overview

This study aimed to establish a robust MRM LC-MS/MS workflow for direct quantitation of intact IGF-1 and IGF-2 in serum. It focused on optimized sample preparation to dissociate IGFs from binding partners and the use of scheduled ionization to minimize contamination risks in mass spectrometric analysis.

Methodology and Instrumentation

Sample Preparation:

• Serum samples spiked with IGF standards and internal standards (LR3 IGF-1 for IGF-1; bovine insulin for IGF-2).
• Use of 5% CHAPS detergent for efficient dissociation of IGFs from binding proteins.
• Protein precipitation followed by solid-phase extraction on a polymeric strong anion 96-well plate.

LC-MS/MS Analysis:

• SCIEX ExionLC AD system coupled to QTRAP 6500+ mass spectrometer (also compatible with Triple Quad 6500+).
• Phenomenex Kinetic C18 column for chromatographic separation.
• Gradient elution with 0.1% formic acid in water (buffer A) and acetonitrile (buffer B).
• Scheduled ionization in Analyst software version 1.7 to suppress electrospray voltage during CHAPS elution, preventing detergent entry into the mass analyzer.
• Data processing using MultiQuant software version 3.0.

Key Results and Discussion

The method achieved baseline separation of CHAPS from analytes, ensuring signal clarity. Recovery studies demonstrated that 5% CHAPS doubled IGF-1 recovery compared to 1% concentration. Quantitation in a surrogate mouse serum matrix yielded:

• Linear dynamic range: 2–1000 ng/mL for both IGF-1 and IGF-2.
• Limit of quantitation (LOQ): 2 ng/mL; limit of detection (LOD): 1 ng/mL.
• Accuracy within 92–110% and precision (%CV) below 15% across the concentration range.
• Endogenous levels in pooled human serum measured at 119.4 ng/mL (IGF-1) and 397.6 ng/mL (IGF-2).

Benefits and Practical Applications

This workflow offers high sensitivity, reproducibility, and speed suitable for challenging biological matrices. Scheduled ionization enhances instrument uptime by preventing contamination from sample preparation detergents. The simplicity and robustness support large molecule bioanalysis in clinical research and quality control environments.

Future Trends and Potential Applications

Further developments may include multiplexed quantitation of additional peptide hormones, automation of sample preparation, integration with immunocapture techniques for increased specificity, and deployment in high-throughput clinical laboratories. Advances in software-driven acquisition strategies will continue to enhance sensitivity and reduce analysis time.

Conclusion

An LC-MS/MS method featuring CHAPS-based sample preparation and scheduled ionization on the SCIEX QTRAP 6500+ platform enables quantitation of intact IGF-1 and IGF-2 at low nanogram-per-milliliter levels with high accuracy and precision. The approach balances simplicity, speed, and instrument protection, meeting the needs of modern large molecule bioanalysis.

Reference

1. Cote L, McCann K, Chu C. Determination of insulin-like growth factor-1 in serum by HRAM LC/MS for clinical research, 2014 MSACL.
2. Tanna N, Lame M, Wrona M, Chambers E. LC-MS/MS quantification of intact insulin-like growth factor 1 in serum for clinical research, 2017.
3. Luo J, Guo L, Jin W, Xiong L. Quantitation analysis of human insulin-like growth factor 1 in serum by SPE-LC-MS/MS workflow, SCIEX Technical Note, 2019.