基于非衍生化串联质谱技术分析新生儿滤纸干血片中的乳清酸  

Analysis of Orotic Acid in Dry Blood Spots from Newborns by Non-derivatized Flow Injection Analysis Coupled to Tandem Mass Spectrometry

作　　者：张潇分 樊静静 陈武炼 王燕敏 纪伟 田国力 ZHANG Xiao-Fen;FAN Jing-Jing;CHEN Wu-Lian;WANG Yan-Min;JI Wei;TIAN Guo-Li(Department of Neonatal Screening Center,Shanghai Children's Hospital,School of Medicine,Shanghai Jiao Tong University,Shanghai 200040,China;Anpel Laboratory Technologies(Shanghai)Inc.,Shanghai 201600,China)

机构地区：[1]上海市儿童医院,上海交通大学医学院附属儿童医院新生儿筛查中心,上海200040 [2]上海安谱实验科技股份有限公司,上海201600

出　　处：《分析化学》2024年第3期373-379,共7页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(No.21803009);上海市科学技术委员会科研项目(No.18441905100);上海交通大学“交大之星”医工交叉项目(No.YG2022QN097);上海市儿童医院院级课题国家自然科学基金培育专项项目(No.2021YGZQ09)资助。

摘　　要：建立了一种基于非衍生化的流动注射串联质谱技术检测新生儿滤纸干血片中乳清酸(ORA)的方法。本方法采用负离子多反应监测模式(MRM)进行检测,通过同位素内标定量,单次仪器分析时间仅需2 min,检测ORA的线性范围为0.25~40μmol/L,线性关系良好(R^(2)>0.99),检出限(S/N=3)为0.12μmol/L。混合实验中,混合基质样本与滤纸干血片(DBS)基质样本和溶剂基质样本的均值,差值百分比低于20%,表明本方法的基质效应不影响ORA的准确定量分析。将本方法用于实际DBS样本中ORA的检测,加标回收率为101.4%~122.2%,相对标准偏差(RSD)为0.8%~13.3%,采用高低浓度转换进样实验评估了携带污染效应。采用本方法对900例正常新生儿DBS临床实际样本进行了检测,分别以99.00%和1.00%百分位数的浓度值作为参考区间的上限值和下限值,初步获得了基于本实验室新生儿人群DBS中ORA浓度的临床诊断参考区间(1.10~1.52μmol/L),为未来新生儿遗传代谢疾病筛查诊断提供了基础数据。Orotic acid(ORA)is a key targeted biomarker in inherited metabolic diseases,especifically ornithine transcarboxylase deficiency.However,ORA is not included into the current first-tier newborn screening program due to the absence of a suitable assay method.Therefore,some false positives and error results need to be corrected by a second-tier assay,which requires additional time and urine of newborns.The aim of this study was to develop a convenient method for analysis of ORA in dry blood spots(DBS)by non-derivatized flow injection analysis coupled to tandem mass spectrometry.ORA was extracted from DBS with 78%methanol aqueous solution containing ORA-15N2 as internal standard.The samples were analyzed in MRM mode with negative-mode ionization from a single injection in 2 min acquisition time.A linear response range was found in the ORA concentration range from 0.25 to 40.0μmol/L,and the correlation coefficient was good(R^(2)>0.99).The limit of detection of ORA(S/N=3)was 0.12μmol/L.Matrix effects of the method was carefully evaluated and determined,and the difference of their response values was within 20%of the target,indicating that the matrix effects would not affect the accurate quantification of ORA.Then,the proposed method was successfully applied to analyze ORA in actual DBS samples.The recoveries of ORA were in the range of 101.4%-122.2%,and the relative standard deviation(RSD)was 0.8%-13.3%.Meanwhile,the residual contamination of the method was evaluated,and the results showed that the residual contamination was acceptable.Based on the method established in this study,900 clinical DBS samples from healthy newborns was analyzed.Herein,the lower limit cut-offs were set at below 1.0%,and the upper limit cut-offs were set at above 99.0%.Thus,the reference intervals of ORA(1.10-1.52μmol/L)in DBS of this laboratory was preliminarily obtained,which provided a preliminary foundation for the promotion of NBS program in China in the near future.

关 键 词：非衍生化流动注射串联质谱 乳清酸 滤纸干血片 新生儿 遗传代谢疾病 筛查 

分 类 号：R446.11[医药卫生—诊断学] R722.1[医药卫生—临床医学] O657.63[理学—分析化学]