同位素稀释液相色谱串联质谱法测定血清17α-羟孕酮候选参考测量程序的研究

Analytical performance of a candidate reference measurement procedure for serum 17α-hydroxyprogesterone based on liquid chromatography tandem mass spectrometry

目的研究1种基于液相色谱串联质谱(LC-MS/MS)技术的血清17α-羟孕酮(17α-OHP)候选参考测量程序并对其进行评价。方法2019年12月,取上海市临床检验中心员工体检留存的血清标本,采用正己烷乙酸乙酯混合液(体积比3∶2)提取加入同位素内标的血清样本,C18反向色谱柱分离,正离子电喷雾质谱仪检测,参照美国临床实验室标准化协会(CLSI)C62-A文件对建立的候选参考方法进行校准曲线、检测限与定量限、基质效应、不精密度、正确度、特异性、携带污染、线性范围等基本分析性能验证。结果17α-OHP的线性范围为0.21~119.67μg/L。检测限和定量限分别为5.218 ng/L、0.116μg/L。3种不同比例(50∶50、80∶20、20∶80)的血清与溶液混合物的相对基质效应分别为-0.02%、-0.40%、-0.90%。批内变异系数(CV)在0.164、14.81、81.63μg/L浓度处分别为1.73%~2.11%、0.98%~1.71%、0.47%~0.87%,批间CV在上述3个浓度点处分别为1.82%、1.03%、0.80%。加标样本平均回收率在0.5、20、100μg/L处分别为100.4%、101.7%、102.8%;检测中国计量科学研究院标准参考物质GBW09829,结果均在规定的不确定度范围内。无携带污染和特异性干扰。结论成功建立了基于同位素稀释LC-MS/MS技术的血清17α-OHP候选参考测量程序。该候选参考测量程序有着良好的精密度和准确度,能用于常规临床检验方法的量值溯源。

Objective To determine the analytical performance of a candidate reference measurement procedure for 17α-hydroxyprogesterone in human serum by liquid chromatography-tandem mass spectrometry(LC-MS/MS).Methods The serum spiked with a deuterium-labeled internal standard was extracted from serum from individual undergoing physical examination by liquid-liquid extraction with n-hexane/ethyl acetate(3∶2,v/v),separated by C18 reversed-phase chromatography and detected by positive electrospray ionization mass spectrometry.According to the Clinical and Laboratory Standards Institute(CLSI)C62-A documents,the analytical performance including linearity,limit of detection,limit of quantitation,relative matrix effect,precision and trueness,carry-over and specificity was evaluated.Results The linear range of 17α-hydroxyprogesterone by LC-MS/MS was 0.21-119.67μg/L.The limit of detection and limit of quantitation were 5.218 ng/L and 0.116μg/L.The relative matrix effects were-0.02%,-0.40%and-0.90%for sera and solution mixtures in 3 different ratios(50∶50,80:20 and 20∶80).The coefficients of variation(CVs)of intra-assay were 1.73%-2.11%,0.98%-1.71%,0.47%-0.87%at 0.164μg/L,14.81μg/L,81.63μg/L and the CVs of inter-assay were 1.82%,1.03%,0.80%at above three concentrations.The average recovery rates of 3 levels(0.5,20 and 100μg/L)were 100.4%,101.7%,102.8%,respectively.The measured values of GBW09829 of National Institute of Metrology were within the specified uncertainty range.Conclusion The candidate reference measurement procedure for 17α-hydroxyprogesterone in human by LC-MS/MS is established with good accuracy and precision,which can be clinically used for measurement traceability.