抄録
A simultaneous micro-determination of nicotinamide, N^1-methyl-2-pyridone-5-carboxamide (2-Py) and N^1-methyl-4-pyridone-3-carboxamide (4-Py), as well as an ultramicro-determination of N^1-methylnicotinamide (MNA) by HPLC are described. The method for measuring nicotinarnide, 2-Py and 4-Py employs a 7-ODS-L (4.6×250mm, particle size 7μm) column eluted with 10mM potassium dihydrogenphosphate-acetonitrile (96:4, v/v, pH adjusted to 3.0 by the addition of concentrated phosphoric acid) at a flow-rate of 1.0 ml/min. The UV detector was set at 260nm. The detection limits for nicotinamide, 2-Py and 4-Py were 10pmol (1.22ng), 2pmol (304pg) and 2pmol (304pg), respectively, at a signal-to-noise ratio 5:1. Isonicotinamide was used as an internal standard. MNA reacted with acetophenone in a strong alkali medium at 0℃ in the presence of a large amount of isonicotinamide. After being kept for 10min, formic acid was added and the mixture was kept for further 15min at 0℃. Then, the mixture was heated for 5 min. The reaction product, 1-methyl-7-phenyl-1, 5-dihydro-5-oxo-1; 6-naphthyridine was adsorbed on a 300-SCX (4.6×150mm, particle size 7μm) column, eluted with 25mM potassium dihydrogenphosphate containing 20% acetonitrile (pH 4.5) at a flow-rate of 1.0ml/min, and estimated at excitation wavelength of 382nm as well as emission wavelength of 440nm. The detection limit was 0.01pmol (1.36pg in terms of MNA). These techniques were applied to the analysis of biological materials.
