Specific detection of nicotinamide coenzymes by liquid chromatography and amperometric detection with immobilized glucose-6-phosphate dehydrogenase

A liquid chromatographic system for the specific and simultaneous detection of nicotinamide coenzymes is constructed by combining an immobilized glucose-6-phosphate dehydrogenase reactor with an amperometric system based on a phenazine methosulphate-mediated reaction, after separation on a reversed-phase column. The calibration graphs are linear from 0.05 to 20 nmol for all four coenzymes. The detection limits are 3.2, 5.2, 7.9 and 9.4 pmol for NADP⁺, NADPH, NAD⁺ and NADH, respectively. The enzyme reactor retains most of its original activity after repeated use for 2 months.