Purpose: Insulin-like growth factor 1 (IGF-1) is a hormone of 70 amino acids that plays key roles in childhood growth and anabolic effects in adults. IGF1 is routinely measured in clinical laboratories to monitor for abnormal levels of the hormone which may indicate a developmental disorder or dysfunctional pituitary gland. The role of IGF1 associated with cancer, aging, neuropathy, and stroke also leads to significant interest for academic and industry research. Historically, IGF1 has been measured by immunological assays; however, recent studies have raised concerns over inter-assay variability of commercially available immunoassays. These studies have demonstrated the use of affinity enrichment of IGF1 coupled with various mass spectrometry detectors. Here, we show an alternative sample preparative method to the affinity-based enrichment method by using traditional solid phase resin chemistries on automated liquid handling system without any offline sample process (i.e. centrifugation) coupled with low resolution LC-MS/MS for monitoring IGF-1 isoforms, variants, and analogs.
Methods: The pooled human plasma samples processed by Na EDTA and Heparin were purchased from Fisher Scientific and used for the initial method development. Liquichek™ tumor quality control from Bio-rad was used for precision and accuracy measurements. The enrichment and clean-up from plasma samples were designed for Microlab STAR from Hamilton Robotics with dispersive pipette extraction totaling less than 1 hour of total processing time for a batch of 96 samples. To measure the intact IGF-1 quantity without endopeptidase cleavage, we optimized the selected reaction monitoring using UPLC and TSQ Endura triple quadrupole mass spectrometry (Thermo Fisher Scientific).
Results: The automated workflow required no centrifugation step and implemented a two step solid phase extraction process using dispersive pipette extraction on the robotic liquid handling system. To enrich polypeptide hormones from plasma samples for liquid chromatography and mass spectrometry analyses using automated liquid handling system, we utilized strong-cation exchange and reverse-phase dispersive pipette extraction for a robust plasma sample analysis on the LC-MS/MS system.
We developed a selected reaction monitoring method to measure intact human IGF-1 and to test efficiency of the enrichment period using mouse IGF-1, an internal standard. We achieved R2 > 0.9692 for the linear regression of the calibration curve over three orders of magnitude, 0.5 ng - 500 ng, on column with serial dilutions of certified reference IGF-1 standard. The coefficient of variation (CV) percentage of intra-day assay was < 13.7 % and the CV of inter-week assay was < 14.2 %.
Using a mouse IGF1 standard, we achieved over 65% recovery after the two step SPE enrichment. Based on the relative abundance to the certified reference material standard, human IGF1, we calculated the concentration of endogenous level of IGF1 from plasma samples. The median level of endogenous human IGF1 was 120.5 ng/mL, range 84-211 ng/mL (n=12).
Conclusion: These results demonstrate a robust automatic plasma sample preparation of IGF-1 enrichment and clean-up for a targeted LC-MS/MS quantification. This application will be useful to improve accuracy of clinical quantification of IGF-1 isoforms compared to conventional immunological assays.