MSACL 2019 EU Abstract

Development and Comparison of Two High-throughput LC-MS Methods for the Accurate Quantitation of IGF1 in Human Serum

Pegah R. Jalili (1), Judy Cao(1), Yue Lu(1), Nicolas Caffarelli(1), Jeff Turner(1), Uma Sreenivasan(2), Kevin Ray (1) and Anders Fridstrom (3)
1) MilliporeSigma, St. Louis, MO; 2) MilliporeSigma, Round Rock, TX; 3) Merck Sweden AB

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Anders Fridström (Presenter) Merck Sweden AB >> POSTER (PDF)

Relevant Financial Disclosures (within past 24 months) Salary Merck Conflict mitigation NOT REQUIRED.

Introduction:
To achieve a high level of analytical quality for IGF1, we developed a Certified Reference Material (CRM) standard which is traceable to the SI and a full-length stable-isotope labeled (SIL) internal standard. Early introduction of SIL-IGF1 mitigates any source of variation throughout the analytical workflow. Subsequent to reagent development, two high-throughput plate-based LC-MRM formats for accurate quantification of IGF1 were developed. The first workflow incorporates protein precipitation followed by the analysis of intact IGF1. The second workflow utilizes immunoaffinity enrichment followed by rapid, in situ trypsin digestion and analysis of a surrogate peptide.
Method:
Two types of sample preparations methods, protein precipitation, and immunoaffinity enrichment were developed for LC-MS-based quantification of IGF1 in human serum. For calibration, recombinant human serum albumin was fortified with varying levels of IGF1 and spiked with SIL-IGF1. For the protein precipitation workflow, 100 µL of each calibrator was precipitated using acetonitrile with 6% acetic acid. The plate was centrifuged and supernatants were transferred to autosampler vials and intact IGF1 was analyzed by LC-MRM. For the immunoaffinity workflow, 20 µL of calibration standards were enriched using anti-human IGF1 capture antibody bound to a Protein A/G plate and digested with trypsin. A resulting tryptic peptide was analyzed by LC-MRM. MultiQuant software was used for data analysis.
Results:
For precipitation method, using 100 µL of serum, a linear calibration curve in a range of 40 ng/mL to 5 µg/mL with CV values of < 20% and accuracies of 90-116% was obtained. For immunoaffinity method, using 20 µL of serum, a linear calibration curve in a range of 20 ng/mL to 5 µg/mL with CV values of < 15% and accuracies of 85-115% was obtained. All samples were prepared in triplicate in 6% rHSA.
Conclusion:
A plate-based immunoaffinity enrichment kit and a protein precipitation workflow were developed to enable high throughput quantification of IGF1 by LC-MRM MS in less than five hours. The 15N SIL-IGF1 Certified Reference Material is an effective internal standard for both methods. Further experiments will be performed using human serum spiked with known amounts of IGF1 as test samples to check for matrix equivalency between 6% rHSA and human serum.