Automated and manual determination of N1-methylnicotinamide in urine

An automated AutoAnalyzer method and a manual adaptation of the automated method are described for determining N1-methylnicotinamide (NMN) in urine. The new methods incorporate improvements presented by Pelletier and Campbell (Anal. Biochem. 3:60, 1962) for the fluorometric determination of NMN after condensation with methyl ethyl ketone (MEK). The new methods are simpler and not limited by the usage of a single brand of MEK. Various brands of MEK into which hydrogen peroxide had been added in order to catalyze the formation of the condensation product were used and found to give comparable NMN values with different urine specimens. Results of the analysis of 21 urine specimens by the new procedures agreed well with those by the method of Carpenter and Kodicek (Biochem. J. 46: 421, 1950) for which interpretation guidelines exist. Furthermore, it was found that eluates from the chromatographic purification of thiamine in urine with Decalso retained the NMN present in the original specimens, gave complete recoveries of added NMN and were free from nearly all interfering fluorescing substances; this purification would be advantageous for determining with accuracy and precision NMN levels in the urine of deficient subjects and could be used routinely when thiamine analysis has to be performed.