滤纸干血片6种标志物UPLC-MS/MS方法的建立和性能评价  

Establishment and performance evaluation of UPLC-MS/MS for 6 markers in dried blood spots

作　　者：李西就 周健伟[1] 张芹 龙允麟 吴英松[1] LI Xijiu;ZHOU Jianwei;ZHANG Qin;LONG Yunlin;WU Yingsong(Institute of Antibody Engineering,Southern Medical University,Guangzhou 510515,Guangdong,China)

机构地区：[1]南方医科大学抗体工程研究所,广东广州510515

出　　处：《检验医学》2023年第5期446-453,共8页Laboratory Medicine

基　　金：广州市科技计划项目(201704020230)。

摘　　要：目的建立可同时检测新生儿苯丙酮尿症(PKU)、先天性肾上腺皮质增生症(CAH)和先天性甲状腺功能减退症(CH)6种标志物的超高效液相色谱串联质谱(UPLC-MS/MS)方法。方法对滤纸干血片的处理条件进行优化,建立可同时检出17-羟孕酮(17-OHP)、雄烯二酮(ASD)、皮质醇(COR)、苯丙氨酸(PHE)、酪氨酸(TYR)和甲状腺素(T4)的UPLC-MS/MS方法并进行方法学评价(线性范围、准确度、灵敏度、精密度、参考物质分析)。收集正常新生儿滤纸干血片样本1000份、CH患儿滤纸干血片样本30份,PKU患儿滤纸干血片样本30份、CAH患儿滤纸干血片样本30份。对建立的UPLC-MS/MS方法进行验证,并初步建立6种标志物筛查的参考区间。结果UPLC-MS/MS检测PHE、TYR、17-OHP、ASD、COR、T4的线性范围分别为12.1~980.7μmol/L、3.7~298.0μmol/L、6.1~490.2 nmol/L、0.7~56.6 nmol/L、13.8~1117.3 nmol/L、32.2~2606.6 nmol/L,最低检测限分别为0.5μmol/L、0.7μmol/L、1.6 nmol/L、0.2 nmol/L、2.6 nmol/L、3.2 nmol/L。3个浓度水平质控品的日内精密度[变异系数(CV)]为2.2%~7.5%,日间精密度(CV)为2.3%~7.3%,加标回收率为92.5%~115.4%。国际新生儿筛查学会(ISNS)PHE和17-OHP标准物质5个浓度的检测值与标示值的相对偏差分别为1.78%~7.01%、0.70%~10.78%。相对于正常新生儿,CAH患儿17-OHP水平和(17-OHP+ASD)/COR比值升高(P<0.05),PKU患儿PHE水平和PHE/TYR比值均升高(P<0.05),CH患儿T4水平降低(P<0.05)。17-OHP和17-OHP联合(17-OHP+ASD)/COR比值筛查CAH的阳性率均为100%。PHE和PHE联合PHE/TYR比值筛查PKU的阳性率均为100%。T4筛查CH的阳性率为40%。结论建立了可同时检测PKU、CAH和CH 6种标志物的UPLC-MS/MS方法,该方法特异性强、快速、灵敏,缩短了样本分析时间,可作为新生儿疾病筛查的新方法。Objective To establish a method for the determination of 6 markers in neonatal phenylketonuria(PKU),congenital adrenal hyperplasia(CAH)and congenital hypothyroidism(CH)by ultra-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS).Methods The processing conditions of dried blood spots were optimized to establish a UPLC-MS/MS that can simultaneously determine 17-hydroxyprogesterone(17-OHP),androstenedione(ASD),hydrocortisone(COR),phenylalanine(PHE),tyrosine(TYR)and thyroxine(T4)and carry out methodological validation(linear range,accuracy,sensitivity,precision,reference material determination).Totally,1000 normal neonatal dried blood samples,30 CH dried blood samples,30 PKU dried blood samples and 30 CAH dried blood samples for clinical validation were collected,and a reference interval for 6 analytes was preliminarily established.Results The linear ranges for determining PHE,TYR,17-OHP,ASD,COR and T4 by UPLC-MS/MS were 12.1-980.7μmol/L,3.7-298.0μmol/L,6.1-490.2 nmol/L,0.7-56.6 nmol/L,13.8-1117.3 nmol/L and 32.2-2606.6 nmol/L,respectively.The minimum determination limits were 0.5μmol/L,0.7μmol/L,1.6 nmol/L,0.2 nmol/L,2.6 nmol/L and 3.2 nmol/L,respectively.The intraday precision[coefficient of variation(CV)]of the quality control samples at 3 concentration levels was 2.2%-7.5%,the interday precision(CV)was 2.3%-7.3%,and the spiked recovery rate was 92.5%-115.4%.The relative deviations between the determined values and standard values of the 5 concentrations of the International Society for Neonatal Screening(ISNS)PHE and 17-OHP reference materials were 1.78%-7.01%and 0.70%-10.78%,respectively.Compared to normal newborns,the 17-OHP and(17-OHP+ASD)/COR ratio in CAH newborns were increased(P<0.05).The PHE level and PHE/TYR ratio of PKU newborns were increased(P<0.05).The T4 level in CH newborns was decreased(P<0.05).The positive rates of 17-OHP and 17-OHP combined with(17-OHP+ASD)/COR screening for CAH were 100%and 100%,respectively.The positive rates of PHE and PHE combined with PHE/TYR screening

关 键 词：苯丙酮尿症 先天性肾上腺皮质增生症 先天性甲状腺功能减退症 超高效液相色谱串联质谱 新生儿 滤纸干血片 

分 类 号：R446.1[医药卫生—诊断学]