A practical and reliable method for determination of urinary 3-methylhistidine

A practical and reliable semiautomated method for analysis of urinary 3-methylhistidine (3-MH) was designed combining the isolation of 3-MH by ion-exchange chromatography with the color reaction given by ninhydrin-orthopthalaldehyde (ninhydrin-OPT) reagent after alkalinization. 2 ml of urine were passed through disposable columns packed with an ion-exchange resin (Dowex 50-X8, 200–400 mesh) and the acidic and neutral amino acids were eluted with 10 ml of 0.2 M pyridine solution. Then, the 3-MH was quantitatively eluted and separated from histidine with a volume of 9 ml of a 1.5 M pyridine solution. Standard Autoanalyzer equipment was used for the automation of spectrophotometry. The method permits the analysis of 40 samples in duplicate per day. The 3-MH color reaction was linear for concentrations from 0.015 to 0.24 μ mol/ml. The mean recoveries of 3-MH from standards and urine were 98.6 ± 1.3 and 99.0 ± 1.3%, respectively. Duplicate determinations of urine samples showed a variation coefficient of 1.88%. An excellent agreement was obtained between urine samples analyzed by the present method and by an amino acid analyzer. The need for the elimanation of the interfering amino acids was clearly demonstrated.