气相色谱-质谱联用法测定尿液中N-乙酰天门冬氨酸

目的 检测利用气相色谱-质谱(GC-MS)联用分析技术建立定量检测尿液中N-乙酰天门冬氨酸(NAA)的方法.方法 采用尿素酶法进行样本前处理,应用GC-MS技术分析,先通过全扫描离子扫描(SCAN)模式确定NAA经衍生化后生成物色谱峰的保留时间及其特征离子质荷比(m/z),再通过选择离子扫描(SIM)模式进行定量分析,并对其进行方法学评价.结果 NAA经衍生化后分别生成2个和3个活性位点硅烷化的产物,保留时间分别为17.008 min和17.249 min,采用SIM模式进行定量分析.NAA在1～1 000 μg/mL 内线性相关系数是0.998 4,在10、100和1 000 μg/mL 3种浓度其批内精密度分别为2.49%、7.61%和7.96%,批间精密度分别为4.28%、8.79%和8.1%,回收率分别为110%、90.03%和108%,偏差分别为10%、-9.97%和8.6%.用该方法检测正常健康对照组的尿液NAA浓度范围是(8.67±7.88)μmol/mmolCr(mean±2SD);检测Canavan病(CD)室间质控尿液NAA浓度均在95%的置信区间内.结论 通过GC-MS成功建立了一种能快速、敏感、准确地检测尿液中NAA的方法,为筛查及诊断CD提供实验室检测的参考依据.

Determination of urine N-acetyl aspartic acid using gas chromatography-mass spectrometry

Objective To evaluate the gas chromatography-mass spectrometry(GC-MS) in quantitative detection of N-acetyl aspartic acid(NAA).Methods The urine samples were pretreated by urease methods and assessed by GC-MS.Two modes of GC-MS,SCAN and SIM were applied.SCAN was applied for assessment of retention time of chromatographic peak of resultants and their characteristic ion mass charge ratio,while SIM was selected for quantitative analysis of NAA.Results There were two activated silylanize products after derivatization of NAA,NAAdi-TMS and NAAtri-TMS,with retention time of 17.008 min and 17.249 min,respectively.According to SIM,the linear correlation coefficient of NAA(1~1 000 μg/mL) was 0.998 4.At the levels of 10 μg/mL,100 μg/mL and 1 000 μg/mL,intra-assay precisions in NAA detection were 2.49%,7.61% and 7.96%,respectively;while inter-assay precisions were 4.28%,8.79% and 8.1%,respectively;and recovery rates were 110%,90.03% and 108%,respectively;and deviations were 10%,-9.97% and 8.6%,respectively.The concentration of NAA in healthy control group was 8.67±7.88 μmol/mmol(mean±2SD) using GC-MS.Furthermore,the NAA in urine sample of Canavan disease(CD) offered by ERNDIM(2009—2010) were in 95% confidence interval.Conclusion GC-MS quantitative analysis is a rapid,sensitive and accurate method in urine NAA detection,providing laboratory evidence for screening and diagnosing CD.