Purification and analysis of the purity of NADH

We developed an analytical reverse-phase high-performance liquid chromatographic procedure for rapid assessment of the purity of NADH. The method completely separates adenosine monophosphate and adenosine diphosphoribose from NADH. By use of this analytical technique we found that preparative chromatography on DEAE-cellulose gives NADH that is free from adenine nucleotides as well as other impurities that commonly are present in NADH. The absorbance ratio at 260 and 340 nm of the purified NADH in 1.8 mmol/liter ammonium carbonate is 2.261 +/- 0.002 (+/- 1 SD).