Simultaneous quantification of labeled ²H₅-glycerol, ¹³C₆-glucose, and endogenous D-glucose in mouse plasma using liquid chromatography tandem mass spectrometry

Abstract

Monitoring the level of glucose and glycerol or their labeled derivatives in biological fluid for kinetic studies has always been challenging, especially in mice, because of the limited volume in addition to the complexity of plasma. For such application, we developed a simple, fast, and sensitive method for the simultaneous measurement of absolute concentrations of labeled ²H₅-glycerol and ¹³C₆-glucose as well as endogenous D-glucose using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In our study, 15.0 μL of mouse plasma was processed by a one-step protein precipitation, followed by LC-MS/MS analysis. The quantification of the analytes was carried out by monitoring the product ion scan of their corresponding deprotonated molecular ions and constructing the extracted ion fragmentogram by choosing a specific product ion for each analyte (equivalent to precursor ion to product ion transitions). The limit of detection (LOD) was evaluated to be 1.0 μM for both ²H₅-glycerol and ¹³C₆-glucose, and the limit of quantitation (LOQ) was observed to be 5.0 μM for both ²H₅-glycerol and ¹³C₆-glucose in diluted mice plasma that corresponds to 50 μM in plasma or 4.60 and 9.01 mg/dL of glycerol and glucose in plasma, respectively. The extraction recoveries are 81.9 % (CV = 8.1 %) for ²H₅-glycerol and 26.2 % (CV = 13.6 %) for ¹³C₆-glucose.