A fluorometric method for the determination of the 6-pyridone of N1-methylnicotinamide in urine.

Since Knox and Grossman demonstrated the excretion by man of the 6-pyridone of N’-methylnicotinamide (hereafter referred to as the pyridone) in appreciable amounts after the ingestion of nicotinamide (2), it became necessary to devise a method for its quantitative estimation in urine. Knox and Grossman isolated the compound from human urine and described its properties, including its ultraviolet absorption spectrum. They found that this metabolite did not yield a fluorescent derivative under the conditions employed for the determination of N1-methylnicotinamide (3); nor could it be determined microbiologically with the use of the customary organisms. The above observation by Knox and Grossman was confirmed that the condensation reaction with acetone in an aqueous alkaline medium which yields a fluorescent product with N1-methylnicotinamide and the pyridone nucleotides (3) did not yield a fluorescent compound with the pyridone. It was soon found, however, that a fluorescent compound could be obtained by agitating the pyridone in acetone solution with KOH in the presence of a minimal amount of water. With the applicability of this reaction to analytical purposes in pure solutions of the pyridone established, it was then necessary to find a method of separation and purification of relatively small amounts of the pyridone present in normal human urine which would yield reasonable recovery and blank values. This proved to be extremely difficult. The usual adsorption and elution techniques either were altogether inapplicable or gave variably low recovery values. The final resolution of this difficulty was found in two steps: the removal of pigments and of other interfering substances in urine with lead