TY - JOUR
T1 - 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
AU - Tavazzi, Barbara
AU - Lazzarino, Giuseppe
AU - Leone, Paola
AU - Amorini, Angela Maria
AU - Bellia, Francesco
AU - Janson, Christopher G.
AU - Di Pietro, Valentina
AU - Ceccarelli, Lia
AU - Donzelli, Sonia
AU - Francis, Jeremy S.
AU - Giardina, Bruno
N1 - Funding Information:
This work has been supported in part by research funds from the University of Catania and Catholic University of Rome (Grant D1-2004). Patent no. RM2005A000085, covering the HPLC method and the respective analytical kit, has been filed by the Italian Ministry for Productive Activities, Italian Office of Patents and Trademarks.
Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2005/11
Y1 - 2005/11
N2 - 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-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. 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 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. Conclusions: 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.
AB - 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-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. 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 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. Conclusions: 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.
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U2 - 10.1016/j.clinbiochem.2005.08.002
DO - 10.1016/j.clinbiochem.2005.08.002
M3 - Article
C2 - 16139832
AN - SCOPUS:27744482480
SN - 0009-9120
VL - 38
SP - 997
EP - 1008
JO - Clinical Biochemistry
JF - Clinical Biochemistry
IS - 11
ER -