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

A high-performance liquid chromatographic technique was examined that may allow simultaneous measurement of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in a single sample of human serum. Concentrations of 4 microgram/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 standards to accurately monitor the recovery of each compound. Values obtained for 25-hydroxyvitamin D2 or composite 25-hydroxyvitamin D when only tritiated 25-hydroxyvitamin D3 is used as a means of monitoring recovery are probably in error.