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

• Published in: Clinical biochemistry Vol. 38; no. 11; pp. 997 - 1008
• Main Authors: Tavazzi, Barbara, Lazzarino, Giuseppe, Leone, Paola, Amorini, Angela Maria, Bellia, Francesco, Janson, Christopher G., Di Pietro, Valentina, Ceccarelli, Lia, Donzelli, Sonia, Francis, Jeremy S., Giardina, Bruno
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.11.2005
• Subjects: Adolescent; Adult; Amino Acids - isolation & purification; Amniotic Fluid - chemistry; Aspartic Acid - analogs & derivatives; Aspartic Acid - blood; Aspartic Acid - urine; Biological fluids; Canavan disease; Canavan Disease - diagnosis; Chemical diagnosis; Child; Child, Preschool; Chromatography, High Pressure Liquid - methods; Dicarboxylic acids; Dicarboxylic Acids - isolation & purification; HPLC; Humans; Inborn errors of metabolism; Infant; Mass Screening - methods; Metabolism, Inborn Errors - diagnosis; Middle Aged; N-acetylated amino acids; Prenatal and neonatal screening; Prenatal Diagnosis - methods; Purines; Purines - isolation & purification; Pyrimidines; Pyrimidines - isolation & purification; Reproducibility of Results; Sensitivity and Specificity; Spectrophotometry, Ultraviolet; Canavan disease; Purines; N-acetylated amino acids; Pyrimidines; Inborn errors of metabolism; Biological fluids; Dicarboxylic acids; Prenatal and neonatal screening; Chemical diagnosis; HPLC
• ISSN: 0009-9120; 1873-2933
• DOI: 10.1016/j.clinbiochem.2005.08.002

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). The separation was set up using a Hypersil C-18, 5-μm particle size, 250 × 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. 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 μmol/L plasma; not detectable in healthy adults) and dramatically high urinary N-acetylaspartate concentrations (1872.03 ± 631.86 μmol/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. The results demonstrate the suitability of the present ion-pairing HPLC separation with UV detection of cytosine, cytidine, creatinine, uracil, uridine, β-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.