LC-MS/MS method for quantitation of seven biomarkers in human plasma for the assessment of insulin resistance and impaired glucose tolerance

Early detection of insulin resistance (IR) and/or impaired glucose tolerance (IGT) is crucial for delaying and preventing the progression toward type 2 diabetes. We recently developed and validated a straightforward metabolite-based test for the assessment of IR and IGT in a single LC-MS/MS method. Plasma samples were diluted with isotopically-labeled internal standards and extracted by simple protein precipitation. The extracts were analyzed by LC-MS/MS for the quantitation of 2-hydroxybutyric acid (0.500-40.0μg/mL), 3-hydroxybutyric acid (1.00-80.0μg/mL), 4-methyl-2-oxopentanoic acid (0.500-20.0μg/mL), 1-linoleoyl-2-hydroxy-sn-glycero-3-phosphocholine (2.50-100μg/mL), oleic acid (10.0-400μg/mL), pantothenic acid (0.0100-0.800μg/mL), and serine (2.50-100μg/mL). Liquid chromatography was carried out on a reversed phase column with a run time of 3.1min and the mass spectrometer operated in negative MRM mode. Method validation was performed on three identical LC-MS/MS systems with five runs each. Sufficient linearity (R²>0.99) was observed for all the analytes over the ranges. The imprecision (CVs) was found to be less than 5.5% for intra-run and less than 5.8% for inter-run for the seven analytes. The analytical recovery was determined to be between 96.3 and 103% for the seven analytes. This fast and robust method has subsequently been used for patient sample analysis for the assessment of IR and IGT.