Rapid high-performance liquid chromatography of 3-methylhistidine in human urine

Abstract An internally standardized method for the determination of 3-methylhistidine in human urine is presented. This methylated amino acid and chemically analogous internal standard 3-ethylhistidine were isolated from human urine specimens using small columns of cation-exchange resin. Quantification was accomplished by high-performance liquid chromatography using post-column derivatization with o -phthalicdicarboxaldehyde-2-mercaptoethanol followed by fluorometric detection. Sample-to-sample and day-to-day reproducibility were shown to have respective relative standard deviations of 2 and 5% for a human urine specimen containing 250 nmol/ml 3-methylhistidine when using 250 μl urine per analysis. The chromatographic separation was evaluated in terms of various peak descriptors (capacity factor and retention time) and “Chromatographic Figures of Merit” (peak symmetry and chromatographic efficiency). The utility of the method was demonstrated by its successful application to 1000 human urine specimens.