22种氨基酸衍生化串联质谱高通量检测方法的建立及其在遗传代谢病中的应用

Establishment of a high throughput method for the determination of 22 amino acids by derivatization tandem mass spectrometry and its application in genetic metabolic disease

目的建立高通量快速检测全血中22种氨基酸的衍生化串联质谱法,考察该方法的影响因素及其在遗传代谢病筛查中的应用和意义。方法新生儿采足后跟血、儿童采末梢血于空白采血滤纸上,完全渗透,晾干。用已知浓度的氨基酸同位素内标液萃取干血滤纸片中的22种氨基酸,衍生化20 min,建立衍生化串联质谱法,高通量快速检测全血中22种氨基酸,采用方差分析优化其影响因素,分析2869例2014年5月至2018年1月西部5省部队在空军军医大学第一附属医院自愿选择衍生化串联质谱法筛查遗传代谢病的3 d至1岁新生儿及儿童的干血滤纸片。结果建立衍生化串联质谱法高通量快速检测全血中22种氨基酸并优化了该方法的影响因素:使用新鲜末稍血,标本4℃保存不超过2周,内标液4℃保存有效期为1个月左右,氮气最佳吹干时间为20 min。衍生化串联质谱法定量检出限为0.3~10.0μmol/L。批内变异系数为5.3%~7.9%,批间变异系数为7.1%~8.6%。经统计学分析男、女氨基酸浓度差异无统计学意义(P>0.5)。加标回收率为96.1%~98.1%,偏倚<10.0%。结论衍生化串联质谱法高通量快速检测全血中22种氨基酸有较高的回收率、精确性和准确性,可以灵敏、特异地检测全血中22种氨基酸,满足临床对遗传代谢病筛查及诊断的需要。

Objective To establish a high-throughput derivatization tandem mass spectrometry method for rapid detection of 22 amino acids in whole blood,and to investigate the influencing factors of this method and its application and significance in screening of genetic and metabolic diseases.Methods The heel blood of newborn and the peripheral blood of children were collected on the blank blood filter paper,completely penetrated and dried.Twenty-two kinds of amino acids in dry blood filter paper were extracted with known concentration of amino acid isotope internal standard solution and derivatized for 20 min.A derivatization tandem mass spectrometry method was established for rapid detection of 22 kinds of amino acids in whole blood.The influencing factors were optimized by analysis of variance,and 2869 cases of dry blood filter paper of infants and children aged 3 days to 1 year old in the First Affiliated Hospital of Air Force Medical University from May 2014 to Jan 2018 were analyzed.Results The high flux rapid detection of 22 amino acids in the whole blood by derivatization tandem mass spectrometry was established and the influencing factors of the method were optimized:the whole blood without anticoagulant was used,the sample was kept at 4℃for no more than 2 weeks,the internal standard solution was kept for about 1 month at 4℃,and the best blowing dry time of nitrogen was 20 min.The quantitative detection limit of this method was 0.3-10.0μmol/L.The variation coefficient in batch was 5.3%-7.9%,and the coefficient of variation between batches was 7.1%-8.6%,and there was no statistical significance in amino acid content between men and women(P>0.5).The recovery rate of the standard addition is 96.1%-98.1%,the bias was less than 10.0%.Conclusion Derivatization tandem mass spectrometry has high recovery,accuracy and accuracy in rapid detection of 22 amino acids in whole blood.It can be used to determine the concentration of 22 amino acids in whole blood sensitively and specifically,and meet the needs of clinical screening and diagnosis of genetic and metabolic diseases.