血浆1,5-脱水葡萄糖醇的UPLC-TQMS定量方法研究

Study on UPLC-TQMS quantification of plasma 1,5-anhydroglucitol

目的建立超高效液相色谱串联三重四级杆质谱(ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry,UPLC-TQMS)定量血浆中1,5-脱水葡萄糖醇(1,5-anhydroglucitol,1,5-AG)的方法.方法采用乙腈:甲醇(8:2,v:v)对样本进行除蛋白处理后直接进行色谱分离,在负离子的多反应监测模式下,对1,5-AG进行定量.结果在6 min内即可实现定量检测.1,5-AG在1~40μg/mL浓度范围内呈良好的线性关系,回归相关系数为0.9988,检出限和定量限分别为0.0025μg/mL和0.005μg/mL,重复性为3.85%~6.08%;日内与日间精密度的相对标准偏差(relative standard deviation,RSD)分别为3.77%和7.76%;1,5-AG在4℃环境下保存1周内的稳定性的RSD为2.51%~4.24%,准确度为97.28%~99.98%,RSD为2.72%~6.53%;平均加样回收率为95.54%~101.53%,RSD为6.52%~9.82%.结论UPLC-TQMS定量血浆中1,5-AG检测方法简单灵敏,准确稳定,可为临床大样本的筛查、定量提供一种快速有效的方法.

Objective To develope a method for quantification of plasma 1,5-anhydroglucitol(1,5-AG)by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry(UPLC-TQMS).Methods Acetonitrile:methanol(8∶2,v∶v)was chosen as the extraction solvent.After removed of protein,the supernatant was conducted to the chromatograph for separation and quantification using multiple reaction monitoring under negative mode.Results The whole separation and quantification could be completed within 6 min with a good linearity in the concentration range of 1~40μg/mL,and the regression correlation coefficient was 0.9988.The limits of detection and quantification were 0.0025μg/mL and 0.005μg/mL,respectively.The repeatability was 3.85%~6.08%,the relative standard deviation(RSD)of intra-day and inter-day precision was 3.77%and 7.76%.The RSD of the stability of 1,5-AG stored at 4℃for a week was 2.51%~4.24%.The accuracy was 97.28%~99.98%with RSD of 2.72%~6.53%.The average of added recovery was 95.54%~101.53%with RSD of 6.52%~9.82%.Conclusion A simple and sensitive,accurate and stable method which can be applied to screen and quantify clinical samples is achieved.