液相色谱串联质谱法测定血清维生素A、E含量

Determination of Vitamin A and E in Serum by Liquid Chromatography Tandem-mass Spectrometry

目的建立液相色谱串联质谱法(LC-MS/MS法)测定血清维生素A和维生素E含量的方法。方法采用硫酸锌沉淀蛋白,96孔蛋白沉淀板过滤。采用LCMS/MS[正离子电喷雾离子化(ESI+)的多反应监测模式(MRM)]氘代同位素内标法检测血清维生素A、E含量并进行相关方法学验证。结果 LC-MS/MS检测血清维生素A的批内精密度变异系数为3.2%(2.4%~3.9%),批间变异为5.5%(5.0%~6.0%);维生素E的批内精密度变异系数为4.1%(3.0%~5.1%),批间变异为8.0%(7.6%~8.3%)。维生素A在0.06~2μg/m L、维生素E在1.2~40μg/m L范围内线性良好,线性相关系数分别为0.9987、0.9909。维生素A、维生素E的定量限分别为0.03μg/m L、0.8μg/m L;检测限分别为0.02μg/m L and 0.01μg/m L。维生素A低浓度回收率为107.5%,高浓度为106.9%;维生素E的低浓度回收率为91.9%,高浓度为88.4%。与液相色谱方法进行比对,两种方法的结果差异无统计学意义(P值均大于0.30)说明两种方法具有一致性。结论 LC-MS/MS检测血清维生素A及维生素E的灵敏度高,结果准确、稳定,可应用于临床分析。

Objective To establish a method for the determinations of serum vitamin A and vitamin E contents by liquid chromatography tandem-mass spectrometry（ LC-MS/MS）. Methods After serum levels of proteins were precipitated by zinc sulfate and filtrated by 96 pore protein-precipitate plate,t hen analyzed by LC-MS/MS in the positive electrospray ionization（ ESI ＋） mode and multiple reaction monitor（ MRM） mode. The quantitative analysis for vitamin A and vitamin E contents was carried out by deuterium isotope as internal standard,and the methodology validation was performed. Results The within-run precision of vitamin A was3. 2%（ 2. 4%-3. 9%）,and between-run precision of vitamin A was 5. 5%（ 5. 0%-6. 0%）. The withinrun precision of vitamin E was 4. 1%（ 3. 0%-5. 1%）,and between-run precision of vitamin E was 8. 0%（ 7. 6%-8. 3%）. The good correlation coefficients were 0. 9987 and 0. 9909 at the concentration of 0. 06-2μg/mL vitamin A and 1. 2-40μg/mL vitamin E,respectively. The detection limits of vitamin A and vitamin E were 0. 03 g/mL and 0. 8 g/mL respectively. The quantitation limits of vitamin A and vitamin E were 0. 02 ug/mL and 0. 01 ug/mL respectively. Low and high concentration of vitamin A recovery rate were107. 5% and 106. 9% respectively; low and high concentration of vitamin E recovery rate were 91. 9% and88. 4% respectively. Compared with the liquid chromatography method,the results of the two methods were not statistically significant（ P〉 0. 30）,indicating the consistency of the two methods. Conclusion This method is sensitive,accurate and stable,satisfying the quantification requirements of vitamin A and vitamin E in serum. So it can be used in clinical analysis.