同位素稀释液相色谱-串联质谱法测定血清皮质醇候选参考方法的建立及评价  

Development and Evaluation of a Candidate Reference Method for Cortisol in Human Serum by Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry

作　　者：黄昌发 林丹 付晓雅 翁旭琦 孙雅玲 李伟 Huang Changfa;Lin Dan;Fu Xiaoya;Weng Xuqi;Sun Yaling;Li Wei(Guangzhou Wondfo Biotech Co.,Ltd.,Guangzhou 510663,China)

机构地区：[1]广州万孚生物技术股份有限公司,广州510663

出　　处：《中华检验医学杂志》2024年第8期927-935,共9页Chinese Journal of Laboratory Medicine

基　　金：广东省药品监督管理局科技创新项目(2024ZDZ16)。

摘　　要：目的建立同位素稀释液相色谱-串联质谱法(ID-LC/MS/MS)测定血清中皮质醇浓度的候选参考方法。方法方法学建立。将样本与皮质醇内标混匀后加入碳酸钠溶液碱化处理, 正己烷/乙酸乙酯进行液液萃取, 流动相采用甲醇水溶液等度洗脱, 质谱采用电喷雾电离源正离子模式(ESI+)及多反应监测(MRM)分析方法, 标准溶液及内标的配制、样本与内标的混合均采用重量法进行控制, 括号法计算皮质醇浓度。参照美国临床实验室标准化协会C62-A、C50-A及国内专家共识文件《液相色谱-质谱临床应用建议》《液相色谱串联质谱临床检测方法的开发与验证》等对方法正确度、精密度、特异性、线性、检测限和定量下限等性能进行系统评价。数据经Excel2019进行处理, 最小二乘法进行线性回归分析。结果方法准确精密, 测定有证标准物质ERM-DA192、ERM-DA193以及2021—2023年RELA样本的相对偏倚均<2%, 加标回收率98.2%~101.1%, 批内及批间不精密度均<2%, 检测结果不受其他结构类似物干扰, 在25~1 600 nmol/L范围内呈现出良好线性, 方法检测限和定量下限分别为0.5和1.0 nmol/L。经溯源后的皮质醇测定试剂盒(化学发光免疫分析法)与该候选参考方法同时测定46例临床血清样本的相关性良好。结论建立了一种灵敏、准确且操作简便的血清皮质醇浓度测定候选参考方法, 可为临床常规测定方法的量值溯源及正确度评价提供参考。Objective:To develop a candidate reference method for cortisol in human serum by isotope dilution liquid chromatography-tandem mass spectrometry(ID-LC/MS/MS).Methods:An isotope-labeled internal standard was added to samples,followed by alkalizing with sodium carbonate solution and liquid-liquid extraction with n-hexane/ethyl acetate.Isoelution with a methanol aqueous solution was employed for liquid chromatographic separation,while ESI in the positive ion and multiple reaction monitoring mode were used for mass spectrometry.The volume of sample,standard,and internal standard solutions were all controlled by weighing,and the results were calculated by bracketing method.The accuracy,precision,specificity,linearity,LOD and LOQ of this method were evaluated referring to the CLSI C62-A and C50-A guidelines and the domestic expert consensus documents.Results:The method demonstrated excellent accuracy and precision with the bias of ERM-DA192,ERM-DA193 and RELA 2021-2023 all being less than 2%.The recovery of added cortisol ranged from 98.2%to 101.1%.Both intra-and inter-assay imprecisions was<2%.The method was free from interference by structural analogue and showed a good linearity in the range of 25-1600 nmol/L.The LOD and LOQ were 0.5 nmol/L and 1.0 nmol/L,respectively.A cortisol assay kit(chemiluminescence immunoassay)traced to this candidate reference method was used to determine 46 clinical serum samples concurrently,and the two methods exhibited good correlation.Conclusions:A candidate reference method for the determination of cortisol in serum was established,demonstrating high sensitivity,good repeatability and accuracy.This method can serve as a reference for the measurement traceability and accuracy evaluation of routine methods.

关 键 词：氢化可的松 同位素稀释液相色谱-串联质谱 候选参考方法 

分 类 号：R446.11[医药卫生—诊断学]