title: 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.
abstract: OBJECTIVE
  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).
  
  
  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.
authors.affiliation: Institute of Biochemistry and Clinical Biochemistry, Catholic University of Rome Sacro Cuore, Largo F. Vito 1, 00168 Rome, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Tavazzi
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Lazzarino
authors.forename: Paola
authors.initials: P
authors.lastname: Leone
authors.forename: Angela Maria
authors.initials: AM
authors.lastname: Amorini
authors.forename: Francesco
authors.initials: F
authors.lastname: Bellia
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Janson
authors.forename: Valentina
authors.initials: V
authors.lastname: Di Pietro
authors.forename: Lia
authors.initials: L
authors.lastname: Ceccarelli
authors.forename: Sonia
authors.initials: S
authors.lastname: Donzelli
authors.forename: Jeremy S
authors.initials: JS
authors.lastname: Francis
authors.forename: Bruno
authors.initials: B
authors.lastname: Giardina
mesh_terms: D000293:Adolescent; D000328:Adult; D000596:Amino Acids / Q000302:isolation & purification*; D000653:Amniotic Fluid / Q000737:chemistry; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000097:blood / Q000652:urine; D017825:Canavan Disease / Q000175:diagnosis; D002648:Child; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003998:Dicarboxylic Acids / Q000302:isolation & purification*; D006801:Humans; D007223:Infant; D008403:Mass Screening / Q000379:methods; D008661:Metabolism, Inborn Errors / Q000175:diagnosis*; D008875:Middle Aged; D011296:Prenatal Diagnosis / Q000379:methods; D011687:Purines / Q000302:isolation & purification*; D011743:Pyrimidines / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000596:Amino Acids; D003998:Dicarboxylic Acids; D011687:Purines; D011743:Pyrimidines; D001224:Aspartic Acid; C000179:N-acetylaspartate