使用整体柱和二极管阵列检测同时测定人血清中维生素A和E的高效液相色谱法的优化与验证
Optimization and validation of a high performance liquid chromatography method for the simultaneous determination of vitamins A and E in human serum using monolithic column and diode-array detection.
作者信息
Urbánek Lubor, Solichová Dagmar, Melichar Bohuslav, Dvorák Josef, Svobodová Iveta, Solich Petr
机构信息
Charles University in Prague, Department of Analytical Chemistry, Faculty of Pharmacy, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic.
出版信息
Anal Chim Acta. 2006 Jul 28;573-574:267-72. doi: 10.1016/j.aca.2006.02.032. Epub 2006 Mar 6.
In this study a novel, simple and rapid reversed-phase high performance liquid chromatography (HPLC) procedure for simultaneous determination of vitamins A and E (retinol and alpha-tocopherol) in blood serum has been developed and validated using monolithic column and diode-array detection (DAD). The monolithic column Chromolith Performance RP-18e (100 mm x 4.6 mm) was operated at ambient temperature. One hundred percent methanol at flow rate 2.5 ml min(-1) was used as a mobile phase. Detection of both compounds was performed with diode-array detector, retinol was monitored at 325 nm and alpha-tocopherol at 295 nm. The linear dependence between peak area and concentration ranged from 0.25 to 10.00 micromol l(-1) for retinol and 0.5-50.0 micromol l(-1) for alpha-tocopherol. The limit of detection (LOD) for retinol was 0.02 micromol l(-1) and limit of quantification (LOQ) was 0.07 micromol l(-1). The limit of detection (LOD) for alpha-tocopherol was 0.1 micromol l(-1) and limit of quantification (LOQ) was 0.3 micromol l(-1). Retinol was eluted in 0.8 min and alpha-tocopherol in 1.4 min. The simultaneous analysis of vitamin A and E can be achieved in less than 2 min. The implementation of monolithic column Chromolith Performance shortens the time of analysis of both vitamins four times in comparison with using traditional particulate column Pecosphere C18 (150 mm x 4.6 mm), 5 microm. This fact may play an important role for routine clinical analysis of biological samples.
在本研究中，已开发并验证了一种新颖、简单且快速的反相高效液相色谱（HPLC）方法，用于同时测定血清中的维生素A和E（视黄醇和α-生育酚），该方法采用整体柱和二极管阵列检测（DAD）。整体柱Chromolith Performance RP - 18e（100 mm×4.6 mm）在室温下运行。以流速2.5 ml min⁻¹的100%甲醇作为流动相。两种化合物均用二极管阵列检测器进行检测，视黄醇在325 nm处监测，α-生育酚在295 nm处监测。视黄醇的峰面积与浓度之间的线性关系范围为0.25至10.00 μmol l⁻¹，α-生育酚为0.5 - 50.0 μmol l⁻¹。视黄醇的检测限（LOD）为0.02 μmol l⁻¹，定量限（LOQ）为0.07 μmol l⁻¹。α-生育酚的检测限（LOD）为0.1 μmol l⁻¹，定量限（LOQ）为0.3 μmol l⁻¹。视黄醇在0.8分钟内洗脱，α-生育酚在1.4分钟内洗脱。维生素A和E的同时分析可在不到2分钟内完成。与使用传统的5 μm粒径的Pecosphere C18（150 mm×4.6 mm）颗粒柱相比，整体柱Chromolith Performance的使用将两种维生素的分析时间缩短了四倍。这一事实可能在生物样品的常规临床分析中发挥重要作用。
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