Precise determination of glucose-d₂/glucose ratio in human serum and plasma by APCI LC-MS/MS

BACKGROUND: Determinations of glucose turnover based on infusion of deuterium-labelled glucose are frequently undertaken in studies involving the pathophysiology of diabetes or obesity or the metabolic response to physical exercise. METHODS: A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for determination of the glucose-d(2)/glucose ratio in human serum and plasma following the intra-venous infusion of 6,6-d(2)-glucose. Atmospheric pressure chemical ionization measuring negative ions in selected reaction monitoring mode (product ions at m/z 179-->89 and m/z 181-->89) was used. The serum or plasma samples (50 microL) were prepared by protein precipitation with acetonitrile and the resulting supernatant was directly used for the LC-MS/MS analysis. The chromatography was performed on a Luna 3 micron NH2 100A column (100 x 2 mm) using a mobile phase containing 85% acetonitrile with 20 mmol/L of ammonium acetate at a flow rate of 400 microL/min and an oven temperature of 40 degrees C. RESULTS: A linear response was obtained for the glucose-d(2)/glucose ratio over the relative concentration range of 0-10%, r(2)>0.999. The peak area ratio was determined with an imprecision of 1.20-8.19% (coefficient of variation). The ion suppression from matrix compared to water was in the order of 55%. Chromatographic retention time was between 4 and 5 min and the total analysis time was 10 min. The validated method was successfully applied for the analysis of human serum samples from a clinical study involving infusion of 6,6-d(2)-glucose and evaluation of endogenous glucose production. CONCLUSIONS: It is concluded that the described method provides an easy and precise technique for the determination of serum and plasma glucose-d(2)/glucose ratios in clinical studies.