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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

preprint
submitted on 26.02.2019 and posted on 27.02.2019 by Ashley Williams, Deborah Muoio, Guofang Zhang
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.

Funding

DK076169

Mitochondrial Protein Acetylation and Energy Metabolism in Muscle

National Institute of Diabetes and Digestive and Kidney Diseases

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Mechanisms of lipid-induced bioenergetic stress in muscle

National Institute of Diabetes and Digestive and Kidney Diseases

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History

Email Address of Submitting Author

ashley.s.williams@duke.edu

Institution

Duke Molecular Physiology Institute, Duke University

Country

USA

ORCID For Submitting Author

0000-0003-2298-2298

Declaration of Conflict of Interest

None

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