串联质谱检测干血滤纸片氨基酸方法的建立

Analysis of amino acid in dry blood filter paper with tandem mass spectrometry

目的 建立可靠的串联质谱技术检测干血滤纸片中氨基酸方法 ,以便用于氨基酸代谢紊乱疾病的筛查和诊断.方法 取含不同浓度氨基酸(苯丙氨酸、酪氨酸、亮氨酸、蛋氨酸、瓜氨酸和缬氨酸)的血滤纸片,用含已知量相应氨基酸内标的甲醇萃取,盐酸正丁醇衍生后,用串联质谱仪检测血片中氨基酸谱及其浓度.分析该方法 的精确性、样品回收率及浓度相关性.结果 氨基酸批内和批间变异系数分别为6.3%～9.7%、9.9%～14.3%.低浓度回收率为92%～100%,中等浓度回收率为94%～104%,高浓度回收率为98%～107%.不同浓度的氨基酸实测浓度与加入浓度之间的相关系数为0.999 0～0.999 9.结论 串联质谱技术检测干血滤纸片中氨基酸能够达到较高的精确性、准确性和回收率,为临床进行氨基酸分析提供了一项新技术,可用于新生儿遗传代谢病筛查、高危儿童遗传代谢病的诊断及其他与氨基酸代谢有关的疾病诊治.

Objective To establish a method for amino acid analysis in dry blood filter paper by tandem mass spectrometry for newborn screening and diagnosis of amino acid disorders in children. Methods Dry blood spots of 3 mm diameter with different concentration of Phe, Tyr, Leu, Met, Val, Cit and stable isotope labeled internal standards were punched and extracted in methanol solution then derivatized with butanolic-HCl. After ward, the samples were analysed by tandem mass spectrometry (API 2000). Precision (coefficient of variation, CV), recovery, and linearity were analyzed. Results The intraassay CV and interassay CV were 6.3%-9.7% and 9.9%-14.3%.The recoveries of low level, middling level and high level of amino acids were 92%-100%, 94%-104% and 98%-107% respectively. The linearities of different concentration of amino acids were 0.999 0-0.999 9. Conclusions The precision, recovery, and linearity of the tandem mass spectrometry method are high enough for amino acids analysis, and it could be applied on newborn screening and diagnosis for high risk children with inborn error of metabolism.