A Multiple Assay for Vitamin D Metabolites without High-Performance Liquid Chromatography

We describe a multiple assay of the three main vitamin D metabolites, 25OHD, 24,25(OH) 2D, and 1,25(OH) 2D, in 0.5 ml of serum, which does not require high-performance liquid chromatography. The assay involves extracting the serum with acetonitrile, separation and purification on a C-18/OH cartridge and a Sep-Pak silica cartridge, and quantitation using 1,25(OH) 2D receptors from calf mammary gland for 1,25(OH) 2D, and vitamin D binding protein for 25OHD and 24,25(OH) 2D. For 25OHD, 24,25(OH) 2D, and 1,25(OH) 2D, the method is sensitive to 0.125 ng/tube, 0.025 ng/tube, and 0.5 pg/tube, with the B 50 occurring at 1 ng/tube, 0.2 ng/tube, and 8 pg/tube, respectively. The coefficients of variation (SLD/mean × 100%) intraassay ( n = 8) were 5.4, 12.8, and 6.6% and interassay ( n = 6) 12.3, 10.8, and 8.6%. The overall recovery was 80.4 ± 5.5, 58.0 ± 6.3, and 77.4 ± 5.6% (mean ± SD, n = 40). The validity of the assay was confirmed by dilution test, analytical recovery of added vitamin D metabolites, and comparison with a standard assay using HPLC. This assay offers a simple, rapid, and precise method with which to determine the three main vitamin D metabolites in small serum samples, so it should be particularly useful in studies of pediatrics or small animals.