Analysis of oxidized and reduced pyridine dinucleotides in rat liver by high-performance liquid chromatography

NADP+, NAD+, NADPH, and NADH were assayed by selective extraction and isocratic reversephase HPLC. Sample preparation involves freeze clamping and powdering liver under liquid nitrogen, extraction of dinucleotides with basic (reduced species) or acidic (oxidized species) cold ethanol, and injection onto the HPLC for quantitation at 340 nm (reduced) and 254 nm (oxidized). The mobile phase for the oxidized species is pH 5.25, 0.2 M ammonium phosphate/methanol, and for the reduced species is pH 6.0, 0.2 M ammonium phosphate/methanol/tributylamine. The method is linear over the range 0.016 to 2.0 nmol for the reduced species, and from 0.005 to 0.8 nmol for the oxidized pyridine dinucleotides. The recoveries were from 94.5% for NAD+ to 99.3% for NADPH, with standard deviations of approximately 2.5% for all species other than NADP+, which had a standard deviation of 10.4%. The coefficients of variation for repeated determinations of standards over 3 months were less than 4%.