A novel specific assay of 25-hydroxy vitamin D

The synthesis of 25-hydroxy-[26-2H3]vitamin D3 is described. A fixed amount of this compound (usually 250 ng) is added to a fixed amount of serum (usually 2.5 ml) and the mixture is extracted with a chloroform/methanol mixture. The extract is chromatographed on a Sephadex LH-20 column together with a trace amount of 25-hydroxy-[263H3]vitamin D3. The chromatographic fraction corresponding to 25-hydroxy vitamin D3 is converted into trimethylsilyl ether and the amount of unlabeled 25hydroxy vitamin D3 is determined from the ratio between the mass fragmentographic recording at m/e 131 (base peak of unlabeled 25-hydroxy vitamin D3) and m/e 134 (base peak 25-hydroxy-[26-2H3]vitamin D3). The relative standard deviation of the method was about 5%.