Quantitation Analysis of Human Insulin-like Growth Factor I (IGF1) in Serum by Using SPE-LC-MS/MS Workflow

Significance of the Topic

Human insulin-like growth factor 1 (IGF1) is a 70-amino acid polypeptide hormone produced mainly by the liver. It plays an essential role in mediating growth hormone effects in peripheral tissues and is implicated in conditions such as aging, cancers, metabolic disorders and developmental diseases. Accurate quantification of IGF1 in serum is critical for clinical research, therapeutic monitoring and biomarker discovery.

Objectives and Study Overview

This study aimed to develop and validate a robust SPE-LC-MS/MS workflow for quantifying IGF1 in serum over a biologically relevant concentration range (10–2430 ng/mL). The method was designed to offer high sensitivity, specificity and throughput as an orthogonal alternative to traditional ligand binding assays.

Methodology

Sample preparation involved protein precipitation and denaturation with CHAPS, followed by solid-phase extraction (SPE) on a 96-well PAX plate. Calibration standards were prepared in rat serum owing to endogenous IGF1 in human samples, with concentrations of 10, 30, 90, 279, 810 and 2430 ng/mL. Blank and spiked human serum samples (50 ng/mL IGF1) served to evaluate accuracy. After precipitation and centrifugation, samples were adjusted to alkaline conditions and loaded onto SPE wells. Eluates were combined, dried and reconstituted for LC-MS/MS analysis.

Used Instrumentation

• SCIEX Triple Quad 4500 or QTRAP® 4500 mass spectrometer with Turbo V™ ion source and Curtain Gas™ interface
• Shimadzu Prominence HPLC system
• Waters ACQUITY UPLC CSH C18 column (2.1×100 mm, 1.7 μm)
• MultiQuant™ Software 3.0.2 for data processing

Main Results and Discussion

The optimized assay achieved a lower limit of quantification (LLOQ) of 10 ng/mL for IGF1, with a calibration curve range of 10–2430 ng/mL and a correlation coefficient (r) of 0.999 (1/x weighting). Accuracy across calibration points ranged from 91 % to 106 %, and reproducibility at the LLOQ (10 ng/mL; n=6) yielded a CV of 5.6 %. Unknown human serum samples measured 81.9 ng/mL (baseline) and 149.3 ng/mL (50 ng/mL spike), corresponding to 113.2 % recovery for the quality control sample.

Benefits and Practical Applications

The developed SPE-LC-MS/MS method offers:

• High sensitivity and specificity without the need for antibodies
• Wide dynamic range covering physiological and pathological IGF1 levels
• Shorter development time and high throughput suitable for large-scale studies
• Robust performance with minimal instrument maintenance

This workflow can be applied in pharmacokinetic studies, biomarker validation and quality control laboratories.

Future Trends and Potential Applications

Advances in mass spectrometry, such as high-resolution instruments and automated sample handling, will further improve peptide quantification. Multiplexed assays for simultaneous measurement of IGF family proteins, integration with microfluidic SPE and data-driven calibrations are promising directions. Expansion to dried blood spots or other matrices could broaden clinical utility.

Conclusion

A validated SPE-LC-MS/MS assay on the SCIEX Triple Quad 4500/QTRAP 4500 platforms enables reliable quantification of IGF1 in serum at concentrations as low as 10 ng/mL. The method demonstrates excellent accuracy, precision and throughput, providing a robust tool for research and clinical applications.

References

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3. Chambers MC, et al. Anal Chem. 2014;86(2):694–702.