24,25-dihydroxyvitamin D3 in serum: sample purification with Sep-Pak C-18 cartridges and liquid chromatography before protein-binding assay.
We describe a precise, specific method for measuring 24,25-dihydroxyvitamin 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 "high-performance" 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 micrograms/L (SD 1.9, range 6.3-0.42 microgram/L). The intra-assay CV was 7.7%, the interassay CV 11.2%. Purification of the sample with Sep-Pak C-18 and liquid chromatography on normal plus reversed-phase columns leads to a mean value of 3.4 micrograms/L (SD 1.6 micrograms/L, n = 12), not significantly different from results with our method.
D015650:24,25-Dihydroxyvitamin D 3; D000328:Adult; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004100:Dihydroxycholecalciferols / Q000097:blood / Q000302:isolation & purification*; D006801:Humans; D011485:Protein Binding; D012621:Seasons
D004100:Dihydroxycholecalciferols; D015650:24,25-Dihydroxyvitamin D 3