同位素稀释液相色谱串联质谱法测定人血清中5-甲基四氢叶酸

摘要

目的 建立测定人血清中5-甲基四氢叶酸的同位素稀释液相色谱串联质谱法(ID-LC/MS/MS).方法 对5-甲基四氢叶酸纯品定性分析后,用两种不同原理(质量平衡法和定量核磁共振法)的定值方法测定纯品纯度,再以5-甲基四氢叶酸同位素标记物(13C5-5-甲基四氢叶酸)为内标物,用电喷雾三重四级杆串联质谱多重反应监测模式对6例相同水平浓度的血清进行定值及不确定度评定,并对方法的精密度、正确度、检测限、定量限进行考察.结果 定性分析确认样品主成分后,质量平衡法与定量核磁共振法测定5-甲基四氢叶酸纯品的纯度为76.65％;人血清5-甲基四氢叶酸定值结果为8.22 ng/mL,扩展不确定度为0.54 ng/mL;方法检测限为0.05 ng/mL,定量限为0.50 ng/mL,重复性与期间精密度的变异系数(CV)均在5.0％以内,用国际标准物质NIST SRM 1955进行正确度验证,3个水平测量结果均在认定值±扩展不确定度范围内.结论 建立了测定人血清中5-甲基四氢叶酸的ID-LC/MS/MS法,该法特异性好,灵敏度高,可准确测定人血清中5-甲基四氢叶酸的含量.%Objective To develop an isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) method for determining the concentration of 5-methyltertrahydrofolate (5MT) in human serum.Methods After qualitative analysis of 5MT reference,its purity was identified by two methods with different principle,i.e.,mass balance and quantitative nuclear magnetic resonance.The definite value and uncertainty of 5MT concentrations in 6 serum samples containing same level of 5MT were assessed by ultra performance liquid chromatography-triple quadrupole tandem mass spectrometer equipped with electrospray ionization in monitoring mode of multiple reactions,in which 13C5-5MT was used as the internal standard.The precision,accuracy,limit of detection (LOD) and limit of quantification (LOQ) of the method were also evaluated.Results After qualitative analysis for confirming the principal component in the sample,the purity of 5MT reference was measured as 76.65％ by mass balance and quantitative nuclear magnetic resonance method.The definite value of 5MT in the serum sample was 8.22 ng/mL,the extended uncertainty was 0.54 ng/mL,LOD was 0.05 ng/mL,LOQ was 0.50 ng/mL and the repeatability and inter-batch precision CV were within 5.0％.The accuracy of measured results was verified by using NIST SRM 1955 since all the values of 3 levels were within the range of certified value with the extended uncertainty.Conclusion A accurately quantitative determination for the concentration of 5 MT in human serum with high specificity and sensitivity was established by using ID-LC-MS/MS method.