Aim: Developing LC-MS methods for biomolecules is often challenging due to issues with molecular size and complexity, nonspecific binding, protein binding, solubility and sensitivity. As a result, complex sample preparation workflows, including immune-affinity and/or protein digestion and lengthy analysis potentially using nano-flow LC, may be needed to achieve the required sensitivity. This work aims to provide a simple, sensitive, fast and robust method for quantification of intact IGF-I from human serum using UPLC-MS/MS. Methods: IGF-I serum samples were denatured with sodium dodecyl sulfate, followed by organic protein precipitation to effectively disrupt protein binding and subsequent SPE of the resulting supernatant for sample cleanup and enrichment prior to LC-MS/MS analysis. Separation was performed on an analytical scale LC using a reversed-phase column containing <2 μm solid core particle followed by detection on a tandem quadrupole MS in multiple reaction monitoring mode. Results: Intact IGF-I was quantified from serum using the method described above at a LLOQ of 5 ng/ml with a dynamic range 5-1000 ng/ml (r2>0.99) and mean accuracy of 101.76%. Accuracies for quality control samples were between 93.9-107.7% with RSD <7%. Conclusion: The analytical sensitivity, linear dynamic range and excellent reproducibility of this method reliably measures endogenous and elevated serum IGF-I levels, demonstrating its utility in discovery, bioanalysis and clinical research.
Keywords: IGF-I; LC–MS/MS; bioanalysis; biomarker quantification; insulin-like growth factor 1; intact protein quantitation.