Gas-chromatography coupled mass spectrometry for rapid and sensitive quantitation of glucose and glucose phosphates derived from glycogen

Glycogen is the primary storage carbohydrate in mammals and it is synthesized in most tissues. Glycogen contains covalently attached phosphate groups at C2-, C3-, and C6-hydroxyls of the glucose units. The addition of phosphate modulates branching pattern, granular size, and crystallinity of a glycogen molecule, which all impact its accessibility to glycogen interacting enzymes. As glycogen architecture modulates its role in metabolism, it is essential to accurately evaluate and quantify each parameter. Simultaneous quantitation of glucose and its phosphate esters is challenging and requires an assay with high sensitivity and a robust dynamic range. Currently, this method is lacking in the field. Herein, we describe a highly sensitive method for the detection of both glycogen-derived glucose and glucose-phosphate esters utilizing gas-chromatography coupled mass spectrometry. Using this method, we observed higher glycogen levels in the liver compared to skeletal muscle, but skeletal muscle contained much more phosphate esters. These results confirm previous findings and establish the validity of the method. Importantly, this method can detect femtomole levels of glucose and glucose phosphate esters within an extremely robust dynamic range with excellent accuracy and reproducibility. The method can also be easily adapted for the quantification of glucose from plant starch, amylopectin or other biopolymers as well as covalently attached phosphate within them.