[Extraction and purification of the three major vitamin D metabolites using C18 and NH2 cartridges and measurement of 25-hydroxyvitamin D].

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.