Open AccessDevelopment of an UPLC/MS–MS method for quantification of intact IGF-I from human serum
Author(s) -
Nikunj Tanna,
Mary E. Lame,
Mark Wrona
Publication year - 2020
Publication title -
bioanalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.566
H-Index - 58
eISSN - 1757-6199
pISSN - 1757-6180
DOI - 10.4155/bio-2019-0234
Subject(s) - bioanalysis , chromatography , protein precipitation , chemistry , sample preparation , selected reaction monitoring , reproducibility , immunoassay , triple quadrupole mass spectrometer , tandem mass spectrometry , high performance liquid chromatography , mass spectrometry , antibody , immunology , biology
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 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.