Abstract
An ultramicro-determination of N^1-methylnicotinamide by high-performance liquid chromatography is elaborated. N^1-methylnicotinamide reacted with ace-tophenone in a strong alkali medium at 0℃ in the presence of a large amount of isonicotinamide. After being kept for 10 min, formic acid was added and the mixture was kept for another 15 min at 0℃. Then, the mixture was heated at above 93℃ for 5 min. The reaction product, 1-methyl-7-phenyl-1, 5-dihydro-5-oxo-1, 6-naphthyridine was analyzed by high-performance liquid chromatography. The method employs adsorption on a Chemcosorb 300-SCX (150 × 4.6mm, I.D., 7 μm) column, elution with 25 mM KH_2PO_4 containing 20% acetonitrile (pH 4.5) at a flow rate of 1.0 ml/min, and estimation at excitation wavelength of 382 nm as well as emission wavelength of 440 nm. The detection limit was 0.01 pmol (1.36 pg in terms of N^1-methylnicotinamide). The technique was applied to the analysis of niacin-deficient rat urine. The total HPLC analysis time is approximately 10 min.
