【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0.999 5，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。更多还原

【Abstract】 A method was developed for high throughput and high sensitive detection of free triiodothyronine(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 150 μL serum samples. First of all,the thyroid hormones bound to binding proteins were removed by washing with tri(hydroxymethyl) aminomethane hydrochloride(Tris-HCl) buffer solution(0. 1 mol/L,pH 7. 4). Following that,the free thyroid hormones were eluted using methanol. The established LC-MS/MS analysis method was employed for detection and quantification. The results of the methodological validation showed that the correlation coefficient(r2) was not less than 0. 999 5. 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 applied to the detection of 95 serum samples and compared with the equilibrium dialysis(ED),to evaluate 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 detection 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 approach for the detection of other free hormones such as cortisol and testosterone.更多还原