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

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 x 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 micromol/L (S/N = 12). NAA in patients' urine (n = 17) ranged between 366 and 21,235 mmol/mol creatinine compared to controls of <39 mmol/mol creatinine (n = 159). This LC-MS/MS method for NAA as described involved no extraction and no derivatization, showed no interference, and gave excellent recovery with low variability and short analytical time.