Clinical Chemistry
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Clinical Chemistry 29: 1806-1807, 1983;
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Clinical Chemistry, Vol 29, 1806-1807, Copyright © 1983 by American Association for Clinical Chemistry

24,25-dihydroxyvitamin D3 in serum: sample purification with Sep-Pak C- 18 cartridges and liquid chromatography before protein-binding assay

ML Traba, M Babe, C de la Piedra and A Marin

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.




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Copyright © 1983 by the American Association for Clinical Chemistry.