Gross and separative determination of 1 alpha,25-dihydroxyvitamin D2 and D3 in plasma using calf thymus receptor

Determination of 1 alpha,25-dihydroxyvitamin D [1,25(OH)2D, gross amounts of 1,25(OH)2D2 and 1,25(OH)2D3] and separative determination of 1,25(OH)2D2 and 1,25(OH)2D3 in plasma using calf thymus receptor have been investigated. A lipid extract from 1 ml of plasma is applied to a Bond Elut C18OH column and an eluate corresponding to 1,25-(OH)2D including both 1,25(OH)2D2 and 1,25(OH)2D3 is applied to calf thymus receptor to assay a gross amount of the two compounds. On the other hand, when separative assay of the two compounds is performed, the 1,25(OH)2D eluate obtained from the Bond Elut C18OH column is further applied to HPLC using a Zorbax SIL column with 5% isopropanol in methylene chloride as a developing solvent to separate the two compounds from one another. The separated eluates are independently applied to the receptor to assay the two compounds. Since less amounts of unknown components non-specifically bound to interfering concomitants besides 1,25(OH)2D exist in the calf thymus receptor, complicated purification steps to eliminate the concomitants are unnecessary. The detection limit by this method is 1.25 pg/tube which is sensitive enough for a routine method to assay 1,25(OH)2D in plasma.