Brunetto M R, Obando M A, Gallignani M, Alarcón O M, Nieto E, Salinas R, Burguera J L, Burguera M
IVAIQUIM, Venezuelan Andean Institute for Chemical Research, Faculty of Sciences, Los Andes University, Ipostel La Hechicera, P.O. Box 3, Mérida 5101-A, Venezuela.
Talanta. 2004 Dec 15;64(5):1364-70. doi: 10.1016/j.talanta.2004.04.035.
A HPLC method with automated column switching and UV-diode array detection is described for the simultaneous determination of Vitamin D(3) and 25-hydroxyvitamin D(3) (25-OH-D(3)) in a sample of human plasma. The system uses a BioTrap precolumn for the on-line sample cleanup. A sample of 1ml of human plasma was treated with 2ml of a mixture of ethanol-acetonitrile (2:1 (v/v)). Following centrifugation, the supernatant was evaporated to dryness under a stream of dry and pure nitrogen. The residue was reconstituted in 250muL of a solution of methanol 5mmoll(-1) phosphate buffer, pH 6.5 (4:1 (v/v)), and a 200mul aliquot of this solution was injected onto the BioTrap precolumn. After washing during 5min with a mobile phase constituted by a solution of 6% acetonitrile in 5mmoll(-1) phosphate buffer, pH 6.5 (extraction mobile phase), the retained analytes were then transferred to the analytical column in the backflush mode. The analytical separation was then performed by reverse-phase chromatography in the gradient elution mode with the solvents A and B (Solvent A: acetonitrile-phosphate buffer 5mmoll(-1), pH 6.5; 20:80 (v/v); solvent B: methanol-acetonitrile-tetrahydrofuran, 65:20:15 (v/v)). The compounds of interest were detected at 265nm. The method was linear in the range 3.0-32.0ngml(-1) with a limit of quantification of 3.0ngml(-1). Quantitative recoveries from spiked plasma samples were between 91.0 and 98.0%. In all cases, the coefficient of variation (CV) of the intra-day and inter-day-assay precision was </=2.80%. The proposed method permitted the simultaneous determination of Vitamin D(3) and 25-OH-D(3) in 16min, with an adequate precision and sensitivity. However, the overlap of the sample cleanup step with the analysis increases the sampling frequency to five samplesh(-1). The method was successfully applied for the determination of Vitamin D(3) and 25-OH-D(3) in plasma from 46 female volunteers, ranging from 50 to 94 years old. Vitamin D(3) and 25-OH-D(3) concentrations in plasma were found from 4.30-40.70ngml(-1) (19.74 +/- 9.48ngml(-1)) and 3.1-36.52ngml(-1) (7.13 +/- 7.80ngml(-1)), respectively. These results were in good agreement with data published by other authors.
描述了一种采用自动柱切换和紫外二极管阵列检测的高效液相色谱法，用于同时测定人血浆样品中的维生素D(3)和25-羟基维生素D(3)（25-OH-D(3)）。该系统使用BioTrap预柱进行在线样品净化。取1ml人血浆样品，加入2ml乙醇-乙腈混合液（2:1（v/v））。离心后，上清液在干燥纯氮气流下蒸发至干。残渣用250μL 5mmol/L磷酸盐缓冲液（pH 6.5）与甲醇的混合溶液（4:1（v/v））复溶，取200μl该溶液注入BioTrap预柱。用由6%乙腈的5mmol/L磷酸盐缓冲液（pH 6.5）组成的流动相（萃取流动相）洗涤5分钟后，保留的分析物以反冲模式转移至分析柱。然后采用反相色谱梯度洗脱模式进行分析分离，使用溶剂A和B（溶剂A：乙腈-5mmol/L磷酸盐缓冲液，pH 6.5；20:80（v/v）；溶剂B：甲醇-乙腈-四氢呋喃，65:20:15（v/v））。在265nm处检测目标化合物。该方法在3.0 - 32.0ng/ml范围内呈线性，定量限为3.0ng/ml。加标血浆样品的定量回收率在91.0%至98.0%之间。在所有情况下，日内和日间测定精密度的变异系数（CV）≤2.80%。所提出的方法能够在16分钟内同时测定维生素D(3)和25-OH-D(3)，具有足够的精密度和灵敏度。然而，样品净化步骤与分析步骤的重叠将采样频率提高到了每小时5个样品。该方法成功应用于46名年龄在50至94岁之间的女性志愿者血浆中维生素D(3)和25-OH-D(3)的测定。血浆中维生素D(3)和25-OH-D(3)的浓度分别为4.30 - 40.70ng/ml（19.74±9.48ng/ml）和3.1 - 36.52ng/ml（7.13±7.80ng/ml）。这些结果与其他作者发表的数据高度一致。
