Reverse Phase Liquid Chromatographic Analysis of the Impurities in Nicotinamide Adenine Dinucleotides

Abstract

Commercial preparations of several different nicotine adenine dinucleotides were examined by liquid chromatography on an octadecylsilane column (Margolis, S., et al., Clin. Chem., 22, 1322, 1976). Seven impurity peaks were detected in NADP⁺, eight in NADPH, and five in NAD⁺. The estimated purity of NADP⁺ from different commercial suppliers varied from 89 to 95 percent. For NADPH the purity ranged from 77.5 to 96 percent and for NAD⁺ from 90 to 93.5 percent. Preparations of NAD⁺ contained AMP, ADPR, nicotinamide, and two unidentified impurities. The impurities found in NADP⁺ and NADPH preparations did not correspond to compounds that we could identify. Four of the impurity peaks found in NADPH form under acidic storage conditions. Five of the impurity peaks observed in NADP⁺ and three of the impurity peaks in NAD⁺ form as products of alkali-catalyzed rearrangements.