Dihydroxyvitamin D 3 in Serum : Sample Purification with Sep - Pak C - i 8 Cartridges and Liquid Chromatography before Protein - Binding Assay

We describe a precise, specific method for measunng 24,25dihydroxyvitamin D3 in human serum. A 2-mL serum sample is extracted with acetonitrile and passed through a Sep-Pak C-18 cartridge. The sample is further purified by “highperformance’ liquid chromatography under isocratic conditions on a normal-phase column (Radial-Pak silica-gel cartridge), then subjected to a protein-binding assay. The mean concentration of 24,25-dihydroxyvitamin D3 in serum from 22 normal adults (measured during the spring) was 2.9 tg/L (SD 1.9, range 6.3-0.42 g/L). The intra-assay CV was 7.7%, the interassay CV 11.2%. Purification of the sample with SepPak C-i 8 and liquid chromatography on normal plus reversed-phase columns leads to a mean value of 3.4 /.Lg/L(SD 1.6 g/L, n = 12), not significantly different from results with our method.