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

Abstract

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-d₂/glucose ratio in human serum and plasma following the intra-venous infusion of 6,6-d₂-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 µL) 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 (100×2 mm) using a mobile phase containing 85% acetonitrile with 20 mmol/L of ammonium acetate at a flow rate of 400 µL/min and an oven temperature of 40°C. Results: A linear response was obtained for the glucose-d₂/glucose ratio over the relative concentration range of 0–10%, r²>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-d₂-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-d₂/glucose ratios in clinical studies.

Key Words::

• Glucose
• glucose turnover
• deuterium glucose
• liquid chromatography-mass spectrometry
• human serum

Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the report.