尹逸丛;禹松林;于佳磊;王丹晨;马超超;邹雨桐;谢少伟;程倩;邱玲;
YIN Yicong;YU Songlin;YU Jialei;WANG Danchen;MA Chaochao;ZOU Yutong;XIE Shaowei;CHENG Qian;QIU Ling;Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College;Graduate School, Chinese Academy of Medical Sciences & Peking Union Medical College;

• 1:中国医学科学院北京协和医学院北京协和医院检验科疑难重症及罕见病国家重点实验室
• 2:中国医学科学院北京协和医学院研究生院

摘要(Abstract)：

目的建立一种同位素稀释超高效液相色谱串联质谱法(isotope dilution ultra performance liquid chromatography tandem mass spectrometry, ID-UPLC-MS/MS)检测血浆18-羟皮质酮(18-Hydroxycorticosterone, 18-OHB)的方法。方法取血浆标本或标准溶液200μL置于离心管中,然后加入同位素氚标记的18-OHB为内标,用甲醇沉淀血浆蛋白,离心后取上清液,用Prime HLB Elution 96孔SPE板进行萃取,收集洗脱液,在正离子电喷雾离子化的多离子反应监测模式下检测18-OHB。评价该方法的精密度、加标回收率、定量检测下限、线性和基质效应。2019年11月起招募表观健康志愿者,采用ID-UPLC-MS/MS检测其血浆18-OHB水平,并验证梅奥医学实验中心提供的18-OHB参考区间。结果该方法检测血浆18-OHB的分析时间约为3.0 min,检测低、中、高3个水平18-OHB的重复性变异系数和实验室内不精密度分别为2.2%～3.5%和3.7%～5.0%,平均加标回收率为98.1%～101.7%,定量检测下限为0.01μg/L,在0.1～10μg/L范围内线性良好(r>0.990),血浆基质效应为86.83%～119.00%。基于本研究建立的ID-UPLC-MS/MS方法,73名表观健康人群血浆18-OHB第2.5、97.5百分位数分别为0.01、0.60μg/L,其中66%(48/73)血浆18-OHB水平处于梅奥医学实验中心提供的参考范围外。结论本研究建立了一种ID-UPLC-MS/MS测定血浆18-OHB的方法,该方法检测快速、结果准确可靠,性能满足临床需求。
Objective To establish a method for quantification of 18-Hydroxycorticosterone(18-OHB)in plasma by isotope dilution ultra performance liquid chromatography tandem mass spectrometry(ID-UPLC-MS/MS). Methods This study was a methodology-validation on the evaluation of plasma 18-OHB with LC-MS/MS. Two hundreds microliter(μL) of serum samples or standard solution were mixed with 18-OHB-d4(internal standard) and treated with methanol solution to precipitate protein and anion-exchange solID-phase extraction(SPE). After SPE, the eluates were detected in the positive electro-spray ionization mode and multiple reaction-monitor mode. The precision, recovery, lower limits of quantification, linearity and the matrix-effect of LC-MS/MS have been evaluated. Since November 2019, healthy participants were recruited continuously to the study to validate the reference intervals of Mayo Clinical Laboratory. Results The analyzing time was 3.0 min. The repeatability coefficient of variation and laboratory imprecision for detecting low, medium and high levels of 18-OHB were 2.2%-3.5% and 3.7%-5.0%, respectively. The average recovery of 18-OHB ranged between 98.1% and 101.7%.The lower limit of quantification was 0.1 μg/L. The matrix of plasma had no significant effect on the measurement of 18-OHB. The 2.5 th to 97.5 th percentiles of 18-OHB measured by ID-UPLC-MS/MS in apparently healthy population was 0.01 and 0.60 μg/L, The plasma level of 18-OHB is outside the reference range provided by the Mayo Medical Laboratory Center in 66%(48/73) of the population. Conclusions A reliable and specific LC-MS/MS method for evaluating plasma 18-OHB was established in the clinical laboratory. The method was simple, rapid and suitable for the diagnosis and classification of primary aldosteronism.

关键词(KeyWords)： 18-羟皮质酮;超高效液相色谱串联质谱法;同位素稀释;原发性醛固酮增多症
18-Hydroxycorticosterone;ultra performance liquid chromatography tandem mass spectrometry;isotope dilution;primary aldosteronism

Abstract:

Keywords:

基金项目(Foundation): 北京市临床重点专科医学检验科卓越项目(ZK201000)~~

作者(Authors): 尹逸丛;禹松林;于佳磊;王丹晨;马超超;邹雨桐;谢少伟;程倩;邱玲;
YIN Yicong;YU Songlin;YU Jialei;WANG Danchen;MA Chaochao;ZOU Yutong;XIE Shaowei;CHENG Qian;QIU Ling;Department of Clinical Laboratory, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College;Graduate School, Chinese Academy of Medical Sciences & Peking Union Medical College;

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