Simultaneous high performance liquid chromatographic separation of purines, pyrimidines, N-acetylated amino acids, and dicarboxylic acids for the chemical diagnosis of inborn errors of metabolism

Clin Biochem. 2005 Nov;38(11):997-1008. doi: 10.1016/j.clinbiochem.2005.08.002. Epub 2005 Sep 1.


Objectives: To set up a novel simple, sensitive, and reliable ion-pairing HPLC method for the synchronous separation of several purines, pyrimidines, N-acetylated amino acids, and dicarboxylic acids for the chemical diagnosis and screening of inborn errors of metabolism (IEM).

Design and methods: The separation was set up using a Hypersil C-18, 5-microm particle size, 250 x 4.6 mm column, and a step gradient using two buffers and tetrabutylammonium hydroxide as the pairing reagent. A highly sensitive diode array UV detector was set up at a wavelength between 200 and 300 nm that revealed purines and pyrimidines at 260 nm and other compounds at 206 nm.

Results: Compounds were determined in the plasma of 15 healthy adults, in the urine of 50 healthy subjects (1-3 years, 4-6 years, 8-10 years, 12-18 years, 25-35 years), and in 10 non-pathological amniotic fluid samples. To assess the validity of the chemical diagnosis of IEM, plasma and urine samples were analyzed in patients affected by Canavan disease (n = 10; mean age 4.6 +/- 2.3). Low plasma levels of N-acetylaspartate (16.96 +/- 19.57 micromol/L plasma; not detectable in healthy adults) and dramatically high urinary N-acetylaspartate concentrations (1872.03 +/- 631.86 micromol/mmol creatinine; 450 times higher than that which was observed in age-matched controls) were recorded. Neither N-acetylglutamate nor N-acetylaspartylglutamate could be detected in the plasma or urine of controls or patients with Canavan disease.

Conclusions: The results demonstrate the suitability of the present ion-pairing HPLC separation with UV detection of cytosine, cytidine, creatinine, uracil, uridine, beta-pseudouridine, adenine, 3-methyladenine, hypoxanthine, xanthine, xanthosine, inosine, guanosine, ascorbic acid, thymine, thymidine, uric acid, 1-methyluric acid, orotic acid, N-acetylaspartate, N-acetylglutamate, N-acetylaspartylglutamate, malonic acid, methylmalonic acid, GSH, and GSSG as a reliable method for the prenatal and neonatal chemical diagnosis and screening of IEM using biological fluids.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Adolescent
  • Adult
  • Amino Acids / isolation & purification*
  • Amniotic Fluid / chemistry
  • Aspartic Acid / analogs & derivatives
  • Aspartic Acid / blood
  • Aspartic Acid / urine
  • Canavan Disease / diagnosis
  • Child
  • Child, Preschool
  • Chromatography, High Pressure Liquid / methods*
  • Dicarboxylic Acids / isolation & purification*
  • Humans
  • Infant
  • Mass Screening / methods
  • Metabolism, Inborn Errors / diagnosis*
  • Middle Aged
  • Prenatal Diagnosis / methods
  • Purines / isolation & purification*
  • Pyrimidines / isolation & purification*
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Spectrophotometry, Ultraviolet


  • Amino Acids
  • Dicarboxylic Acids
  • Purines
  • Pyrimidines
  • Aspartic Acid
  • N-acetylaspartate