人血清哇巴因UPLC-MS/MS检测方法的建立和方法比对  

Determination of human serum ouabain by UPLC-MS/MS and methodology comparison

作　　者：赵银霞[1] 欧美贤[1] 曲毅[2] 陆优丽[1] 张美微[1] 李水军[1] ZHAO Yinxia;OU Meixian;QU Yi;LU Youli;ZHANG Meiwei;LI Shuijun(Central Laboratory,Shanghai Xuhui Central Hospital,Shanghai 200031,China;Department of Geriatrics,Shanghai Xuhui Central Hospital,Shanghai 200031,China)

机构地区：[1]上海市徐汇区中心医院中心实验室,上海200031 [2]上海市徐汇区中心医院老年科,上海200031

出　　处：《检验医学》2018年第11期1013-1017,共5页Laboratory Medicine

基　　金：上海市徐汇区中心医院科研资助项目(2012XHCH04)

摘　　要：目的建立超高效液相色谱串联质谱(UPLC-MS/MS)检测人血清哇巴因的方法。方法采用高特异性的UPLC-MS/MS,以氘标记的哇巴因-d3作为内标。样本采用固相萃取(SPE)前处理方法,以反相色谱柱负离子模式及电喷雾电离源(ESI)检测血清哇巴因水平。对建立的方法进行方法学(基质效应、回收率、准确度、批内精密度、批间精密度及稳定性)验证。采用建立的UPLC-MS/MS方法检测20名体检健康者及40例高血压患者血清哇巴因水平,并与酶联免疫吸附试验(ELISA)进行比较。结果 UPLC-MS/MS检测血清哇巴因的标准曲线范围为0.02～5.0 ng/mL,最低定量检测限(LLOQ)为0.02ng/mL。采用ABN固相萃取小柱进行样本前处理的基质效应较小,且回收率较高,达85%。LLOQ和低值(0.06 ng/mL)、中值(0.6 ng/mL)、高值(4 ng/mL)质控品的准确度分别为108.0%、89.2%、101.0%、103.0%。3个水平质控品的批内变异系数(CV)分别为2.87%、1.95%、0.56%,批间CV分别为5.98%、1.90%、0.75%。样本室温过夜放置16 h及样本前处理后室温放置自动进样器48 h的偏差均<15%。采用UPLC-MS/MS检测哇巴因,正常对照者及高血压患者血清中均未检测到哇巴因。采用ELISA测定血清哇巴因,高血压患者为0.096 ng/mL,正常对照者为0.062 ng/mL。UPLC-MS/MS检测5个水平(0.02、0.05、0.10、0.20、0.50 ng/mL)的哇巴因标准品,其测定结果与对应的哇巴因标准品浓度呈正相关,且线性较好(r2>0.99),准确度较高;而ELISA检测5个水平哇巴因标准品的结果均很接近(0.024 9～0.029 6 ng/mL)。结论建立了检测人血清哇巴因的UPLC-MS/MS方法,未检测到正常人及高血压患者的血清哇巴因。UPLC-MS/MS与ELISA检测血清哇巴因的结果存在较大差异。Objective To establish an ultra high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)for the determination of human serum ouabain.Methods High-specific UPLC-MS/MS was used.A deuterium-labeled ouabain-d3 was used as internal standard.Serum samples were prepared with solid phase extraction(SPE),and a reversed phase column was used for chromatography separation.The mass spectrometer was operated in negative-ion mode with electrospray ionization(ESI)source.Methodology validation included the evaluation of matrix effect,recovery,accuracy,within-run precision,between-run precision and stability.The established UPLC-MS/MS was used to determine serum ouabain in 40 hypertension patients and 20 healthy subjects(healthy control group).The results of UPLC-MS/MS were compared with those of enzyme-linked immunosorbent assay(ELISA).Results The UPLC-MS/MS had a linear range from 0.02 to 5.00 ng/mL.The lower limit of quantification(LLOQ)was 0.02 ng/mL.The matrix effect of pretreatment with ABN solid phase extraction column was the smallest.The recovery rate was high(85%).The accuracies of LLOQ,low-level(0.06 ng/mL),middle-level(0.6 ng/mL)and high-level(4 ng/mL)quality control materials were 108.0%,89.2%,101.0%and 103.0%,respectively.The within-run coefficients of variation(CV)were 2.87%,1.95%and 0.56%,and the between-run CV were 5.98%,1.90%and 0.75%.The samples placed at room temperature for 16 h were stable,and the processed samples placed in autosampler at room temperature for 48 h were stable.Both biases were<15%.The results showed that both healthy control and hypertension groups had undetectable serum ouabain level by the established UPLC-MS/MS.The levels were 0.096 ng/mL in hypertension group and 0.062 ng/mL in healthy control group when they were determined by ELISA.Ouabain standard materials at 5 different levels(0.02,0.05,0.10,0.20 and 0.50 ng/mL)were determined by UPLC-MS/MS.It showed a positive correlation between the results and the corresponding levels with good linearity(r2>0.99)and high accura

关 键 词：哇巴因 超高效液相色谱串联质谱 固相萃取 酶联免疫吸附试验 高血压 

分 类 号：R446.1[医药卫生—诊断学]