Flow injection assays for NADH and ethanol using photosensitized rose bengal and luminol-copper (II) chemiluminescence system.

The online photoreaction of the rose bengal photosensitized luminol-copper (II) chemiluminescence (CL) system was used for the determination of ß-nicotinamide adenine dinucleotide (NADH) and ethanol (EtOH) in pharmaceutical formulations combined with a flow injection technique. NADH can significantly enhance the CL emission of the reaction. For EtOH, alcohol dehydrogenase in soluble form was utilized in the presence of nicotinamide adenine dinucleotide resulting in NADH production. The limit of detection (3σ blank, 𝑛 = 3) of 4.0 × 10-8 and 2.17 × 10-5  M, and linear range 1.3 × 10-7 to 2.5 × 10-5  M (R2  = 0.9998, n = 6) and 0.11-2.17 × 10-3  M (R2  = 0.9996, n = 6) were obtained for NADH and EtOH respectively. The injection rate was 100 h-1 with a relative standard deviation (n = 3) of 1.5-4.8% in the range studied for both analytes. The procedure was satisfactorily applied to pharmaceutical formulations with recoveries in the range 91.6 ± 3.0% to 110 ± 2.0% for NADH and 88 ± 3.0% to 95.4 ± 4.0% for EtOH. The results obtained were very consistent and did not differ considerably from the reported approaches at a 95% confidence limit. The possible mechanism of the CL reaction is also explained briefly.