Mass-fragmentographic determination of 25-hydroxy-vitamin D3 using deuterium labeled internal standard

25-[26-2H3] Hydroxy-vitamin D3 has been synthesized according to the following route: 3β-acetoxy-27-nor-cholest-5-en-25-one → 3β-acetoxy-27-nor-cholesta-5,7-dien-25-one → [26-2H3] cholesta-5,7-diene-3β,25-diol → 25-[26-2H3] hydroxy-vitamin D3. A fixed amount of 25-[26-2H3] hydroxy-vitamin D3, 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-[26-3H] hydroxy-vitamin D3. The chromatographic fraction corresponding to 25-hydroxy-vitamin D3 is collected and the amount of unlabeled 25-hydroxy-vitamin D3 is determined from the ratio between the mass fragmentographic recording of m/e 131 (base peak of unlabeled 25-hydroxy-vitamin D3) and m/e 134 (base peak of 25-[26-2H3] hydroxy-vitamin D3). The relative standard deviation of the method was about 5%. The mean value for 25-hydroxy-vitamin D3 obtained from 23 different serum samples from healthy Swedish men and women was 27 ng/ml with a standard deviation of 10 ng/ml