Development of a rapid and detailed structural identification system with an on-line immobilized enzyme reactor integrated into LC-NMR

Abstract

Immobilized enzyme reactors (IMERs) integrated into an LC-NMR system were developed for rapid and detailed structural identification of enzymatic reaction products. An on-column enzymatic reaction was achieved for immobilized cytochrome-c and dog microsomes. After the reaction, these products were analyzed by LC-NMR without any work-up processes. The immobilized cytochrome-c column integrated into LC-NMR was used to characterize the reaction product formed on N-demethylation by measurement of (1)H-NMR. In the case of reaction taking place on the microsome column, a glucuronidation product was identified by (1)H-NMR and (1)H-(1)H correlated spectroscopy. The chemical structures of the enzymatic reaction products could be elucidated by IMER-LC-NMR without the need for authentic samples or isolation processes.