[45] Mass fragmentographic assay of 25-hydroxyvitamin D3

Publisher Summary This chapter discusses mass fragmentographic assay of 25-hydroxyvitamin D 3 . The problems with respect to instability and lack of specificity can be overcome with the use of mass fragmentography and deuterium-labeled 25-hydroxyvitamin D 3 as internal standard. The internal standard is added to the biological fluid prior to purification and analysis, and it can be assumed that this standard is degraded or decomposed to the same extent as unlabeled 25-hydroxyvitamin D 3 . In the last step, the amount of unlabeled 25-hydroxyvitamin D 3 is determined by combined gas chromatography mass spectrometry with a multiple ion detector (MID). With the latter detector, a specific ion corresponding to the labeled and unlabeled derivative of 25-hydroxyvitamin D 3 , respectively, can be followed through a gas chromatogram, and the ratio between the two tracings can be used for the calculation of unlabeled 25-hydroxy vitamin D 3 . The specificity of the present assay is based on the fact that, to influence the results, a contaminating compound must have the same retention time in gas-liquid chromatography and contain the same ions in its mass spectrum as the derivative of 25-hydroxyvitamin D 3 . The very specific derivative used in the assay is expected to further increase the specificity.