Identification of N1-methyl-2-pyridone-5-carboxamide and N1-methyl-4-pyridone-5-carboxamide as components in urine extracts of individuals consuming coffee

Caffeine metabolites were extracted from urine samples collected 4 h after consumption of a cup of coffee and were separated by high-performance liquid chromatography (HPLC) on a C18 (5 microm) reverse-phase column using an acetonitrile (5%), acetic acid (0.05%) solution as the mobile phase. The elution profiles indicated the constant presence of a major and a minor components eluting between the caffeine metabolites 5-acetamido-6-formyl-3-methyluracil (AFMU) and 7-methylxanthine (7X) in an approximate nine. A procedure was developed for the isolation of the major component in an apparent pure form, and the yield was 10-20 mg from 400 ml of urine. The minor component was isolated in an apparent pure form by this procedure as well, and the yield was 0.5 mg from 200 ml of urine. The average ratio of the two components in urine, UV absorption and 1H-NMR spectra of the two components, and 13C-NMR spectrum, mass spectrum and elemental analysis of the major component identified the major and minor components as N(1)-methyl-2-pyridone-5-carboxamide and N(1)-methyl-4-pyridone-5-carboxamide, respectively, two major metabolites of the vitamin niacin present in a significant amount in coffee beans. The two metabolites were present in the same average amount in urine extracts of individuals irregardless of coffee consumption. The findings are briefly discussed in relation to the nutritional sources of niacin and to current procedures for measuring amounts of the two metabolites in urine samples.