Some problems in development of a high-performance liquid chromatographic assay to measure 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 simultaneously in human serum

Abstract

A high performance liquid chromatographic technique was examined that may allow simultaneous measurement of 25-hydroxyvitamin D3 in a single sample of human serum. Concentrations of 4 μg/liter or greater are accurately measured. However, the prepurification procedure allows partial separation of the two metabolites, and so tritiated 25-hydroxyvitamin D3 can be used as an internal standard for accurately monitoring the analytical recovery of 25-hydroxyvitamin D3, but not that of 25-hydroxyvitamin D2. The ability of most prepurification procedures to partly separate the 25-hydroxy metabolites of vitamin D2 and D3 mandates the need for individual internal standard to accurately monitor the recovery of each compound. Values obtained for 25 hydroxyvitmin D2 or composite 25-hydroxyvitamin D when only tritiated 25-hydroxyvitamin D3 is used as a means of monitoring recovery are probably in error.