Simultaneous measurement of 25-hydroxy, 24,25-dihydroxy-, and 1,25-dihydroxyvitamin D without use of HPLC.
A procedure is described for measuring the concentration of three major vitamin D metabolites: 25(OH)D, 24,25(OH)2D and 1,25(OH)2D, in 0.5 ml serum. The analytes are extracted using C18, and separated using aminopropyl solid phase extraction cartridges. 25(OH)D is separated completely; less than or equal to 10% overlap is observed between the 24,25(OH)2D and the 1,25(OH)2D fractions, and this overlap did not interfere in subsequent competitive radioligand assay. Coefficient of variation (SEM/mean x 100%) is intra-assay (n = 10) 5.8, 3.1, 5.2%, and inter-assay (n = 5) 10.1, 8.7 and 6.4%, respectively. Recoveries of the three analytes added to a single specimen are 103, 95 and 111%, respectively. One technician can extract and fractionate up to 24 specimens in one day, ready for HPLC or direct estimation.
McGraw
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CA
Division of Enzymology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-2899.
Hug
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D015650:24,25-Dihydroxyvitamin D 3 / Q000097:blood; D015652:25-Hydroxyvitamin D 2 / Q000097:blood; D002117:Calcitriol / Q000097:blood; D005591:Chemical Fractionation / Q000379:methods; D006801:Humans; D006887:Hydroxycholecalciferols / Q000097:blood*; D011869:Radioligand Assay
D006887:Hydroxycholecalciferols; D015652:25-Hydroxyvitamin D 2; D015650:24,25-Dihydroxyvitamin D 3; D002117:Calcitriol