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 (0.500–40.0 μg/mL), 3-hydroxybutyric acid (1.00–80.0 μg/mL), 4-methyl-2-oxopentanoic acid (0.500–20.0 μg/mL), 1-linoleoyl-2-hydroxy-sn-glycero-3-phosphocholine (2.50–100 μg/mL), oleic acid (10.0–400 μg/mL), pantothenic acid (0.0100–0.800 μg/mL), and serine (2.50–100 μg/mL). Liquid chromatography was carried out on a reversed phase column with a run time of 3.1 min 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.