Determination of creatinine and ultraviolet-absorbing amino acids and organic acids in urine by reversed-phase high-performance liquid chromatography.

A simple and reliable method for the determination of urinary creatinine, amino acids and organic acids was developed. A urine sample was preliminarily separated into an organic acid fraction (including neutral species) and an amino acid fraction by cation-exchange chromatography. Both fractions were analysed by reversed-phase high-performance liquid chromatography, with a phosphoric acid-methanol gradient elution system and ultraviolet detection at 210 nm. Relationships between concentrations and peak heights were linear from 2 to 500 microM for the analytes. Overall recoveries were ca. 100%. The concentrations of creatinine for 37 urine samples, from 20 healthy newborns and from 17 patients with several inherited metabolic disorders, were 2.35 +/- 2.29 mM (ranging between 0.27 and 10.15 mM). The method was applied to the determination of several diagnostically useful metabolites in urine. The concentrations of phenylalanine and phenylacetic acid for five urine samples from patients with phenylketonuria were 347 +/- 177 and 282 +/- 224 microM/mM creatinine, respectively. The concentrations of tyrosine and 4-hydroxyphenyllactic acid in the urine of a patient with tyrosinemia were 112 and 1871 microM/mM creatinine, respectively.