气-质联用技术测定尿有机酸方法的建立及在遗传代谢病诊断中的应用

目的 利用气-质联用技术测定尿液中有机酸种类及含量的变化,为先天性遗传代谢病,特别是有机酸代谢紊乱提供新的诊断依据.方法 收集临床高度怀疑为有机酸代谢紊乱的195例患儿的尿液标本,根据肌酐含量取相应体积的尿样和内标,加盐酸羟胺对带羟基的有机酸进行圬化反应生成酮体后,用乙酸乙酯和乙醚萃取有机酸,三甲基硅烷衍生;测定采用Agilent GC/MS 6890/5973i气相质谱仪,选择扫描模式检测,测定质荷比(m/z)范围在50～550 m/z的所有有机酸,数据处理采用Agilent GCMSD ChemStationGl701DA软件.用健康对照样本中加人的内标和阳性对照样本中加入的阳性有机酸进行分析方法的线性范围、精密度、准确性、样本回收率和残留分析.结果 该方法可测定尿液中100余种有机酸,以健康对照者尿样中加入的二甲基丙二酸(MMA)和2-苯基丁酸(2-PA)内标为参考,最低检测极限为2.5～2.8μmol/L;批内和批间变异系数均<10%,样本前处理批间变异系数为11.4%;样品回收率为95%～105%,残留分析<1%,所有参数均符合临床检测的要求.用该方法检测的临床患儿尿样中,发现诊断阳性病例12例,包括6例甲基丙二酸血症、1例丙酸血症、3例酪氨酸血症Ⅰ型、1例枫糖尿病和1例Ketosis病(酮症病).结论本研究建立了气-质联用技术分析测定尿液中有机酸的方法,可用于先天性遗传代谢性疾病的筛查诊断.

The determination of organic acids in urine with gas chromatography-mass spectrometry and its application in clinical diagnosis

Objectivo To analyze urine organic acids in the urine using gas chromatography-mass spectrometry(GC/MS)for diagnosis of inherited metabolic diseases,especially for organic acids metabolic disorders.Methods 195 clinical urine samples from the patients with suspected organic acids metabolic disorders and 5 normal urine from adults were collected.After mixing some urine with intemal standards according to the concentration creatinine and adding hydroxylamine hydrochloride to mixture,the organic acids with hydroxyl group were oximated to the ketobodies.Organic acids were extracted twice with ethyl acetate and ethyl ether and derivatized with BSTFA-TMCS.An the organic acids were determined with Agilent GC/MS 6890/5973i with scan model.the mass-to-charge ratio range is 50-550 m/z,all data were nalyzed with Agilent GCMSD ChemStationG1701DA.We also investigated the linearity, accurate,precision.recovery and Carry-over by determining the internal standards in normal samples and positive organic acids in spiked control samples.Results More than one hundred kinds of organic acids in urine samples can be analyzed with this method.According to the two internal standards in normal urine samples,the minimal detection limit MMA and 2.PA was 2.5-2.8 μmol/L.Intra-and interassay coefficient of variation for MMA and 2-PA are both less than 10%.Pre-processing Interassay coefficient by sequential preparations of the same sample was 14%.The recoveries of the spiked samples were 95%-105%.Carryover analysis was less than 1%.All the parameters meet the requirement for clinical diagnosis.12 samples demonstrated positive including 6 cases of methylmalonic acidemia,1 case of propionic acidemia,3 cases of tyrosinemia Ⅰ,1 case of maple syrup urine disease and 1 cases of ketosis.Conclusions The method for the determination of organic acids in urine by GC/MS has been successfully established.It can be used for clinical screening and diagnosis for inherited genetic metabolic diseases.