Abstract
The role of the coenzyme flavin adenine dinucleotide (FAD) in the catalytic oxidation of glucose was elucidated by MS using a new extraction and ionization method. By a multiphase flow of liquid-gas, extractive electrospray ionization was achieved, and this technique (MF-EESI) decreased the salt-matrix interference effectively, avoided salt crystallizations at the capillary tip and increased ionization efficiency by a concentric-sprayed solvent. Notably, two intermediate complexes of FAD-glucose have been observed and differentiated for the first time using this MF-EESI technique. These intermediate complexes were demonstrated to be responsible for the hydride abstraction from glucose, as well as the cyclic coenzyme conversion of FAD during glucose oxidation. Online monitoring was also employed in MF-EESI, thereby providing a potential and informative tool to scrutinize enzymatic catalytic reactions.