Quantification of intact human insulin-like growth factor-I in serum by nano-ultrahigh-performance liquid chromatography/tandem mass spectrometry

RATIONALEInsulin-like growth factor-I is one of the biomarkers used to detect growth hormone administration prohibited in human sport. Current testing approaches for IGF-I rely on commercial immunoassays, which may change from time to time requiring complex revalidation. Mass spectrometry (MS)-based approaches often rely on enzymatically digesting the protein and measuring specific peptide concentrations. In order to reinforce the current available methodology for IGF-I testing, a reliable and equally sensitive MS method is required for the analysis of intact protein using small sample volumes (<25 L). METHODSIGF-I was extracted from human serum samples by a simple protein precipitation procedure. Separation was achieved via nano-ultrahigh-performance liquid chromatography and MS analysis was conducted by nano-electrospray ionisation triple-quadrupole mass spectrometry in the selected reaction monitoring mode using a stable-isotope-labelled internal standard. RESULTSA six-point calibration curve ranging from 50 to 1000ng/mL of human IGF-I in rat serum was used to establish instrument response. The method provided a limit of quantification of 50ng/mL, with intra- and inter-day precision 5% and intra- and inter-day accuracy 95%. CONCLUSIONSA quantitative method was developed for the quantification of intact IGF-I in human serum samples. The data generated provided important information for the development of a new reference method for the growth hormone biomarker test and helped create a reliable system for monitoring peptide hormones in individual athletes, a possible extension to the athlete biological passport system. Nano-electrospray has here been shown to be sufficiently robust for routine use in an analytical laboratory, allowing for the analysis of minute sample volumes. Copyright (c) 2014 John Wiley & Sons, Ltd.