Stable isotope dilution analysis of N-acetylaspartic acid in urine by liquid chromatography electrospray ionization tandem mass spectrometry.
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.
Al-Dirbashi
Osama Y
OY
National Laboratory for Newborn Screening, King Faisal Specialist Hospital and Research Centre, PO Box 3354, Riyadh 11211, Saudi Arabia.
Rashed
Mohamed S
MS
Al-Mokhadab
Manhal A
MA
Al-Qahtani
Khalid
K
Al-Sayed
Moeen A A
MA
Kurdi
Wesam
W
D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000652:urine; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007554:Isotopes; D012015:Reference Standards; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D007554:Isotopes; D001224:Aspartic Acid; C000179:N-acetylaspartate