Stable isotope dilution analysis of N-acetylaspartic acid in urine by liquid chromatography electrospray ionization tandem mass spectrometry.

[canavan disease]

N-acetylaspartic acid (NAA ) is a specific urinary marker for Canavan disease , an autosomal recessive leukodystrophy . We developed a 'dilute and shoot ' stable isotope dilution liquid chromatography tandem mass spectrometry (LC -MS /MS ) method for determination of NAA in urine . Deuterated internal standard d (3 )-NAA was added to untreated urine and the mixture was injected into the LC -MS /MS system operated in the negative ion mode . Chromatography was carried out on a C (8 ) minibore column using 50 % acetonitrile solution containing 0 .05 % formic acid at a flow rate of 0 .25 mL /min . The retention time was 1 .6 min and the turnaround time was 2 .2 min . NAA and d (3 )-NAA were analyzed in multiple reaction monitoring mode . Calibrators and quality control samples were prepared in pooled control urine . The assay was linear up to 2000 micromol /L with limit of quantification at 1 micromol /L (S /N = 12 ). Interassay and intraassay coefficients of variation were less than 7 % and recovery at three different concentrations was 98 .9 -102 .5 %. The LC -MS /MS method for NAA as described involves no extraction and no derivatization , showed no interference and gave excellent recovery with low variability and short analytical time . The method was successfully applied for the retrospective analysis of urine from 21 Canavan disease cases .