A novel high-performance liquid chromatographic assay for vitamin D metabolites using a coulometric electrochemical detector.
A novel high-performance liquid chromatographic assay for vitamin D metabolites using a coulometric electrochemical detector.
复制标题
使用库仑电化学检测器对维生素 D 代谢物进行新型高效液相色谱测定。
DOI：
10.1016/s0731-7085(96)02005-5
复制
发表时间：
1997
影响因子：
3.4
通讯作者：
Tadashi Kobayashi
中科院分区：
文献类型：
--
作者：
S. Masuda;Toshio Okano;M. Kamao;Yasuhiro Kaneda;Tadashi Kobayashi
本文报道了一种新的、高灵敏度的高效液相色谱电化学检测器（ECD）测定维生素D代谢物的方法。该测定包括用二氯甲烷和甲醇从血浆中提取脂质，在Zorbax SIL柱上用5.5%（v/v）异丙醇的己烷溶液纯化，并通过HPLC-ECD定量。由双电极分析池和保护池组成的库仑系统用于洗脱化合物的ECD。将施加到双电极分析池中的检测器1和2的电势分别调节到+0.20 V和+0.60 V。该方法对20 μ g 25-羟基维生素D3[25（OH）D3]和24 R，25-二羟基维生素D3[24，25（OH）2D 3]敏感。25（OH）D3和24，25（OH）2D 3的校准曲线在20-1000 pg范围内呈线性。血浆中25（OH）D3和24，25（OH）2D 3的检测限约为50 pg ml− 1。该灵敏度结合25（OH）D3的总体回收率（81.5 ± 2.6%，平均值± S.E.）仅需20 μl血浆即可测定痕量25（OH）D3。25（OH）D3的批内和批间RSD值分别为5.3%和9.7%，24，25（OH）2D 3分别为6.3%和9.7%。正常成人血浆25（OH）D3和24，25（OH）2D 3水平分别为15.9 ± 2.8 ng ml−1（n = 10）和1.4 ± 0.5 ng ml−1（n = 10）。该方法可用于25（OH）D2和25（OH）D3的测定，以评价其营养和临床状况。从这些结果中，可以得出结论，建议的HPLC-ECD测定系统是一种高灵敏度的物理化学方法，用于测定生物体液中的维生素D代谢产物。
A new, highly sensitive HPLC assay method using an electrochemical detector (ECD) for multiple assay of vitamin D metabolites is reported. The assay involves extracting lipids from plasma with methylene chloride and methanol, purification on Zorbax SIL column with 5.5% (v/v) iso-propanol in hexane and quantification by HPLC-ECD. A coulometric system, composed of the dual electrode analytical cell and a guard cell, was used for ECD of the eluting compounds. The potentials applied to detectors 1 and 2 in a dual electrode analytical cell were adjusted to +0.20 V and +0.60 V, respectively. This method is sensitive to 20 pg of 25-hydroxyvitamin D3[25(OH)D3] and of 24R,25-dihydroxyvitamin D3[24,25(OH)2D3]. Calibration curves gave linearity from 20–1000 pg for 25(OH)D3and 24,25(OH)2D3. The detection limit was approximately 50 pg ml−1for 25(OH)D3and 24,25(OH)2D3in plasma. This sensitivity combined with an overall recovery of 25(OH)D3(81.5 ± 2.6%, mean ± S.E.) allows the measurement of trace amount of 25(OH)D3with only 20 μl of plasma. Intra- and interassay RSD values were 5.3 and 9.7% for 25(OH)D3and 6.3 and 9.7% for 24,25(OH)2D3, respectively. Plasma levels of 25(OH)D3and 24,25(OH)2D3in normal adults were 15.9 ± 2.8 ng ml−1(n = 10) and 1.4 ± 0.5 ng ml−1(n = 10), respectively. This method allows the determination of 25(OH)D2and 25(OH)D3for evaluating their nutritional and clinical status. From these results, it is concluded that the proposed HPLC-ECD assay system is useful for the determination of vitamin D metabolites in biological fluids as a highly sensitive physicochemical method.