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.