凝胶过滤层析结合液相色谱-串联质谱法测定血清中游离甲状腺激素  

Determination of Free Thyroid Hormones in Serum by Gel Filtration Chromatography Combined with Liquid Chromatography-Tandem Mass Spectrometry

作　　者：邹海飞 李敏[2,3] 邓顺艳 夏兵 周燕[1] ZOU Hai-fei;LI Min;DENG Shun-yan;XIA Bing;ZHOU Yan(Chengdu Institute of Biology,Chinese Academy of Sciences,Chengdu 610041,China;Chengdu Institute of Organic Chemistry,Chinese Academy of Sciences,Chengdu 610041,China;University of Chinese Academy of Sciences,Beijing 101408,China)

机构地区：[1]中国科学院成都生物研究所,四川成都610041 [2]中国科学院成都有机化学研究所,四川成都610041 [3]中国科学院大学,北京101408

出　　处：《分析测试学报》2023年第12期1639-1644,1651,共7页Journal of Instrumental Analysis

基　　金：中国科学院仪器功能开发与技术创新项目(E1C3030001)。

摘　　要：基于凝胶过滤层析(GFC)进行前处理,结合液相色谱-串联质谱(LC-MS/MS)建立了血清中游离三碘甲腺原氨酸(FT3)和游离甲状腺素(FT4)的高通量、高灵敏检测新方法。选用Sephadex LH-20凝胶小柱对150μL血清样本进行过滤分离,先用Tris-HCl缓冲液(0.1 mol/L,pH 7.4)淋洗去除结合蛋白的甲状腺激素,然后用甲醇对游离的甲状腺激素进行洗脱,采用LC-MS/MS法进行检测和定量,并进行方法学验证。结果显示:FT3和FT4的线性相关系数(r^(2))均不小于0.9995,批内及批间相对标准偏差为1.8%~10%,准确度为85.4%~110%,基质效应为85.8%~114%,回收率为85.8%~107%。采用该方法与平衡透析法(ED)同时检测了95份血清样本,使用IBM SPSS Statistics 26和MedCalc统计软件(v.20.0.0)对检测结果进行配对t检验分析、PassingBablok回归分析和Bland-Altman一致性分析。结果表明,GFC/LC-MS/MS与ED/LC-MS/MS法对血清中FT3、FT4的测定结果无统计学差异(P>0.05)。所建立的方法快速、简便,受干扰因素相对较少,便于临床推广,可为临床实验室游离甲状腺激素的快速、高效、准确检测提供参考方法,同时为其它游离激素的检测提供了一种新的思路。A method was developed for high throughput and high sensitive detection of free triiodothy⁃ronine(FT3)and free thyroxine(FT4)in serum using gel filtration chromatography(GFC)and liquid chromatography-tandem mass spectrometry(LC-MS/MS).The hydroxypropyl cellulose gel Sephadex LH-20 column was used to filter and separate 150μL serum samples.First of all,the thyroid hor⁃mones bound to binding proteins were removed by washing with tri(hydroxymethyl)aminomethane hy⁃drochloride(Tris-HCl)buffer solution(0.1 mol/L,pH 7.4).Following that,the free thyroid hor⁃mones were eluted using methanol.The established LC-MS/MS analysis method was employed for de⁃tection and quantification.The results of the methodological validation showed that the correlation co⁃efficient(r^(2))was not less than 0.9995.The precision ranges from 1.8%to 10%,accuracy ranges from 85.4%to 110%,matrix effect ranges from 85.8%to 114%,and recoveries from 85.8%to 107%.According to the CLSI EP09-A3 method comparison guidelines,this method was further ap⁃plied to the detection of 95 serum samples and compared with the equilibrium dialysis(ED),to evalu⁃ate the accuracy and applicability of the established GFC/LC-MS/MS method.The statistical analysis was performed using IBM SPSS Statistics 26 and MedCalc statistical software(v.20.0.0)for paired ttest analysis,Passing-Bablok regression analysis,and Bland-Altman agreement analysis.The statistical analysis results indicated that there was no significant difference(P>0.05)between the detec⁃tion results of FT3 and FT4 in the serum measured by the developed GFC/LC-MS/MS method and the ED/LC-MS/MS method.The developed method can provide a reference method for rapid,efficient,and accurate detection of free thyroid hormones in clinical laboratories,as well as offering a new ap⁃proach for the detection of other free hormones such as cortisol and testosterone.

关 键 词：液相色谱-串联质谱法 游离甲状腺激素 凝胶过滤层析 血清 

分 类 号：O657.7[理学—分析化学] O657.63[理学—化学]