人体血浆中氧化三甲胺超高效液相色谱-串联质谱测定法

Determination of trimethylamine N-oxide in human plasma by ultra performance liquid chromatography-tandem mass spectrometry

目的建立超高效液相色谱三重四级串联质谱联用测定法测定人体血液中氧化三甲胺的含量。方法人体血浆样品中按要求加入适量内标,采用甲醇提取快速沉淀蛋白质,高速离心,取上清液再次高速离心后上机测定。选择亲水作用BEH HILIC(100 mm×2.1 mm,1.7μm)色谱柱,采用10 mmol/L的乙酸铵-乙腈流动相进行梯度洗脱,选用超高效液相色谱三重四级杆串联质谱进行测定,选用电喷雾正离子源和多反应监测扫描模式,定量离子对(m/z):76.10/58.10,内标法定量。结果甲醇溶液中氧化三甲胺含量在4~500μg/L的范围内(换算到血浆中浓度0.266~33.280μmol/L)线性关系良好,线性回归方程为y=0.07744x+0.00589,相关系数r为0.9998。目标化合物在2.663、6.656、13.314μmol/L 3个不同浓度添加水平下,回收率为89.5%~93.6%,相对标准偏差(relative standard deviation,RSD)为3.10%~5.25%。方法检出限为0.0266μmol/L。结论所建立的方法前处理简单、快速,准确度和精密度高,线性范围宽,适用于人体血浆中氧化三甲胺的检测。

Objective To establish an ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method for determining the content of trimethylamine N-oxidein in human blood.Methods Add an appropriate amount of internal standard to human plasma samples as required,extract and rapidly precipitate proteins using methanol,centrifuge at high speed,take the supernatant and centrifuge at high speed again before measuring on the machine.The hydrophilic BEH HILIC(100 mm×2.1 mm,1.7μm)column was selected for gradient elution with 10 mmol/L ammonium acetate acetonitrile mobile phase,UPLC-MS/MS was used for determination,electrospray ionization and multiple reaction monitoring mode were selected,the quantitative ion pair(m/z)was 76.10/58.10,and the internal standard method was used for quantification.Result The linear relationship between the content of trimethylamine N-oxide in methanol solution within the range of 4-500μg/L(converted to plasma concentration of 0.266-33.28μmol/L)was good,the linear regression equation was y=0.07744x+0.00589,with a correlation coefficient of 0.9998.At 2.663,6.656,13.314μmol/L of the three additive level,the recovery rate of the sample was 89.5%-93.6%.The relative standard deviation(RSD)was 3.10%-5.25%,and the detection limits was 0.0266μmol/L.Conclusion The established method has simple and fast preprocessing,high accuracy and precision,wide linear range,and is suitable for the detection of trimethylamine N-oxidein in human plasma.