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Williams et al_2DG_2019.v2.pdf (1.64 MB)
A Fast and Sensitive Method Combining Reversed-Phase Chromatography with High Resolution Mass Spectrometry to Quantify 2-Fluoro-2-Deoxyglucose and Its Phosphorylated Metabolite for Determining Glucose Uptake
Preprints are manuscripts made publicly available before they have been submitted for formal peer review and publication. They might contain new research findings or data. Preprints can be a draft or final version of an author's research but must not have been accepted for publication at the time of submission.
Quantative measurements of the glucose analogue, 2-deoxyglucose (2DG), and its phosphorylated metabolite (2-deoxyglucose-6-phosphate (2DG-6-P)) are critical for the measurement of glucose uptake. While the field has long identified the need for sensitive and reliable assays that deploy non-radiolabled glucose analogues to assess glucose uptake, no analytical MS-based methods exist to detect trace amounts in complex biological samples. In the present work, we show that 2DG is poorly suited for MS-based methods due to interfering metabolites. We therefore developed and validated an alternative C18-based LC-Q-Exactive-Orbitrap-MS method using 2-fluoro-2-deoxyglucose (2FDG) to quantify both 2FDG and 2FDG-6-P by measuring the sodium adduct of 2FDG in the positive mode and deprotonation of 2FDG-6-P in the negative mode. The low detection limit of this method can reach 81.4 and 48.8 fmol for both 2FDG and 2FDG-6-P, respectively. The newly developed method was fully validated via calibration curves in the presence and absence of biological matrix. The present work is the first successful LC-MS method that can quantify trace amounts of a nonradiolabeled glucose analogue and its phosphorylated metabolite and is a promising analytical method to determine glucose uptake in biological samples.