Quantification of N‐acetylaspartic acid in urine by LC‐MS/MS for the diagnosis of Canavan disease

• Published in: Journal of inherited metabolic disease 2007-08, Vol.30 (4), p.612-612
• Main Authors: Al‐Dirbashi, O. Y., Rashed, M. S., Al‐Qahtani, K, Al‐Mokhadab, M. A., Kurdi, W., Al‐Sayed, M. A. A.
• Format: Article
• Language: English
• Subjects: Aspartic Acid - analogs & derivatives; Aspartic Acid - urine; Canavan Disease - blood; Canavan Disease - diagnosis; Child; Child, Preschool; Chromatography, Liquid - methods; Female; Humans; Hydrolysis; Infant; Infant, Newborn; Male; Mass Spectrometry - methods; Models, Chemical; Reference Values; Urinalysis - methods
• ISSN: 0141-8955; 1573-2665
• DOI: 10.1007/s10545-007-0635-6

Summary Canavan disease is an autosomal recessive leukodystrophy characterized by excessive excretion of N‐acetylaspartic acid (NAA) in urine. The disease is caused by deficiency of aspartoacylase, the enzyme responsible for the hydrolysis of NAA into acetate and l‐aspartate. Patients, who are often asymptomatic in their early months, show a wide spectrum of clinical presentation thereafter that includes macrocephaly, poor head control, seizures, abnormal muscle tone, optic atrophy, significant developmental delay and death. In this work, we describe a simple liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for the determination of NAA in urine. The internal standard d3‐NAA was added to untreated urine and the mixture was injected into the LC‐MS/MS system operated in the negative ion mode. Detection was achieved in multiple reaction monitoring (MRM) mode by monitoring m/z 174 → 88, 174 → 130 and 174 → 58 for NAA and 177 → 89 for the internal standard. Separation was carried out on a C8 column (2.1 × 150 mm) using a mixture of acetonitrile and water (1:1 v/v) containing 0.05% formic acid at a flow rate of 0.25 ml/min. NAA was eluted at 1.6 min and the run time was approximately 2 min. Using spiked urine, the assay was linear up to 2 mmol/L with limit of quantification at 1 μmol/L (S/N = 12). NAA in patients’ urine (n = 17) ranged between 366 and 21235 mmol/mol creatinine compared to controls of