| Objective1. To establish a quantitative method to detect N-Acetyl Aspartic acid (NAA) in urine, with Gas chromatography-Mass spectrometry(GC-MS), and to investigate its value in screening and diagnosing Canavan disease(CD).2. Two modes of GC-MS, SCAN and SIM, were compared in order to find out their differences and value in quantitative detection of NAA in urine.Methods1. To quantify NAA in urinary, which is key diagnostic metabolites by GC-MS.2. 100ul urine was taken as the quantitative basis, and the heptadecanoic acid was chosen as internal standard substance. Urease methods were used to pretreat the samples.3. Internal standard method of GC-MS was applied to establish the quantitative standard curve of NAA in urine.4. Validation data about quantitative analysis of NAA in urine, including linear range, correlation coefficient, precision, recovery and detection limit had been studied.5. Concentration range of NAA in urine from healthy enfants was tested as a control group.6. CD urine samples from ERNDIM(European Research Network Inhenited Disorders of Metabolism)external quality assurance(EQA)schemes(2009-2010) were mensurated. 7. Two modes of GC-MS method, SCAN and SIM were used respectively to analyse the formulation of the standard curve, evaluation of methodology, concentration range of NAA in normal urineand and concentration of NAA from ERNDIM.8. SCAN and SIM modes of GC-MS were compared based on linear range, correlation coefficient, precision, recovery and detection limit of NAA in urine.Results1. There were two substances, NAAdi- TMS and NAAtri-TMS, after derivatization of NAA. And their retention time were 17.008min and 17.249min, respectively. Under the SCAN mode of GC-MS analysis method, NAA's linear correlation coefficient was 0.9956 in 10～1000μg/ml, and its intra-assay imprecisions ranged from 5.4% to 12.15%, inter-assay imprecisions ranged from 5.82% to 10.43%, which obtained an excellent stability and repeatability. When coming to the concentration of 100ug/ml and 1000ug/ml, the recoveries were 87% and 95%, respectively, which obtained a highly accuracy. However, the recovery rate was 180% in 10ug/ml. The bias was too apparent, so that lower limit of quantification would be 10ug/ml. Concentration range of NAA from control group was 48.88±17.2umol/mmolCr (mean±2SD). Nevertheless, when testing the CD urine samples offered by ERNDIM (2009-2010), all the concentrations were in 95% confidence interval.2. The differences and significance of SCAN and SIM were compared in the quantitative analysis of NAA in urine. When in application of SIM mode, NAA's linear correlation coefficient was 0.9984 in 1～1000μg/ml.Intra-assay imprecisions ranged from 2.49% to 7.96%, and inter-assay imprecisions ranged from 4.28%～8.79%. The stability and repeatability were superior to SCAN mode. The recovery rate were 110%,90.03% and 108% when at the levels of 10ug/ml,100ug/ml and 1000ug/ml, respectively. When NAA was at a low level, SIM was more accurate than SCAN. Concentration range of NAA from healthy control group was 8.67±7.88umol/mmolCr(mean±2SD). When testing the CD urine samples offered by ERNDIM (2009-2010), all the concentrations were in 95% confidence interval. Conclusions1. GC-MS quantitative analysis is a rapid, sensitive and accurate method to detect NAA in urine, and it is in line with European quality control standards,which could provide evidence for screening and diagnosing CD.2. When two modes of GC-MS, SCAN and SIM are compared in the quantitative analysis of NAA in urine, SIM mode is more stabile, repeatable and accurate than SCAN, especially when NAA in urine is at a low concentration. |
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