An antibody-free LC-MS/MS method for the quantification of intact insulin-like growth factors 1 and 2 in human plasma

Abstract

AbstractInsulin-like growth factors 1 and 2 (IGF-1 and IGF-2) are important biomarkers in research and diagnosis of growth disorders. Quantitative analysis is performed using various ligand-binding assays or enzymatic digestion LC-MS/MS methods, whose widespread adoption is hampered by time-consuming sample preparation procedures. We present a simple and fast antibody-free LC-MS/MS method for the quantification of intact IGF-1 and IGF-2 in human plasma. The method requires 50 μL of plasma and uses fully 15N-labelled IGF-1 as internal standard. It features trifluoroethanol (TFE)-based IGF/IGF-binding protein complex dissociation and a two-step selective protein precipitation workflow, using 5% acetic acid in 80/20 acetone/acetonitrile (precipitation 1) and ice-cold ethanol (precipitation 2). Detection of intact IGF-1 and IGF-2 is performed by means of a Waters XEVO TQ-S triple quadrupole mass spectrometer in positive electrospray ionisation (ESI+) mode. Lower limits of quantification were 5.9 ng/mL for IGF-1 and 8.4 ng/mL for IGF-2. Intra-assay imprecision was below 4.5% and inter-assay imprecision was below 5.8% for both analytes. An excellent correlation was found between nominal and measured concentrations of the WHO reference standard for IGF-1. Comparison with the IDS-iSYS IGF-1 immunoassay showed good correlation (R2 > 0.97), although a significant bias was observed with the immunoassay giving substantially higher concentrations. The LC-MS/MS method described here allows for reliable and simultaneous quantification of IGF-1 and IGF-2 in plasma, without the need for enzymatic digestion. The method can be readily implemented in clinical mass spectrometry laboratories and has the potential to be adapted for the analysis of different similarly sized peptide hormones. Graphical abstract