Natsume K, Suzumura E, Suzuki T, Watanabe Y
Kaku Igaku. 1995 Jan;32(1):99-104.
In this study, the three major vitamin D metabolites: 25-hydroxyvitamin D [25(OH)D], 24,25-dihydroxy-vitamin D [24,25(OH)2D], 1,25-dihydroxyvitamin D [1,25(OH)2D] were clearly separated using a NH2 cartridge after acetonitrile and C18 cartridge extraction. In the NH2 cartridge purification procedure, 25(OH)D was eluted with hexane/dichloromethane (50:50), 24,25(OH)2D was eluted with hexane/dichloromethane (20:80) and 1,25(OH)2D was eluted with hexane/isopropanol (75:25). Contamination of each fraction with two other metabolites were less than 1.4%. Recoveries of added 3H-25(OH)D, 3H-24,25-(OH)2D and 3H-1,25(OH)2D were 73.2 +/- 2.45%, 60.0 +/- 2.98% and 63.5 +/- 3.37%, respectively. Using the 25(OH)D fraction after the NH2 cartridge procedure, we measured 25(OH)D using a competitive protein binding assay. The intra- (n = 10) and interassay (n = 8) coefficients of variation were 4.60-8.41% and 6.62-16.4%, respectively. Analytical recovery of added 25(OH)D was in the range of 81.2-130%. The 25(OH)D values were 17.4 +/- 6.02 ng/ml (mean +/- SD) in serum from 110 healthy volunteer collected in May. The correlation of 25(OH)D values was good between cartridge purification and high performance liquid chromatography (HPLC) purification. (gamma = -0.38 + 1.03x, r = 0.953, n = 36). This purification using a simple cartridge procedure was suitable for the measurement of 25(OH)D, and preferable to the time-consuming HPLC purification.
在本研究中，使用氨基柱在乙腈和C18柱萃取后，三种主要维生素D代谢物：25-羟基维生素D [25(OH)D]、24,25-二羟基维生素D [24,25(OH)₂D]、1,25-二羟基维生素D [1,25(OH)₂D] 得到了清晰分离。在氨基柱纯化过程中，25(OH)D用己烷/二氯甲烷（50:50）洗脱，24,25(OH)₂D用己烷/二氯甲烷（20:80）洗脱，1,25(OH)₂D用己烷/异丙醇（75:25）洗脱。每个馏分中其他两种代谢物的污染率均低于1.4%。添加的³H-25(OH)D、³H-24,25-(OH)₂D和³H-1,25(OH)₂D的回收率分别为73.2±2.45%、60.0±2.98%和63.5±3.37%。使用氨基柱纯化后的25(OH)D馏分，我们采用竞争性蛋白结合法测定25(OH)D。批内（n = 10）和批间（n = 8）变异系数分别为4.60 - 8.41%和6.62 - 16.4%。添加的25(OH)D的分析回收率在81.2 - 130%范围内。5月份采集的110名健康志愿者血清中的25(OH)D值为17.4±6.02 ng/ml（平均值±标准差）。柱纯化和高效液相色谱（HPLC）纯化之间的25(OH)D值相关性良好。（γ = -0.38 + 1.03x，r = 0.953，n = 36）。这种使用简单柱程序的纯化方法适用于25(OH)D的测定，且优于耗时的HPLC纯化方法。
