Preparation and storage of isotopically labeled reduced nicotinamide adenine dinucleotide

A method for obtaining highly purified NADH in a dry, solid, and stable form is described. The method involves improvements of the ion-exchange and reversed-phase chromatographic procedures of C. J. Newton and S. M. Faynor, and D. B. Northrop (Anal. Biochem., 1983, 132, 50-53). The necessary time to prepare pure NADH has been reduced to a few hours. The final product, obtained by drying the nucleotide from absolute ethanol, shows no detectable decomposition either during the drying procedure or during storage under nitrogen gas at -20 degrees C for several months. Using dry product prepared from fixed volumes of ethanolic solution, standardized solutions of known amounts of the highly purified and stored NADH can be obtained in a few seconds.