Highly selective isolation and separation of 25-hydroxyvitamin D and 3-epi-25-hydroxyvitamin D metabolites from serum

Increasing health concerns related to vitamin D deficiency including Alzheimer's and immune diseases, along with various cancers, have heightened awareness of the nutrient. As the associated health concerns grow, so does the need for fast and accurate analytical testing for diagnostics and treatment. Established immunoassay methods have been challenged for accuracy caused largely by endogenous interferences. This has driven the interest in more specific LC-MS/MS methodology where specificity is gained through chromatographic and MS resolution.

Herein, a pentafluorophenyl stationary phase is shown to provide superior selectivity for the separation of the closely related 25-hydroxyvitamin D(3) and 3-epi-25-hydroxyvitamin D(3) as compared with many methods reported in the literature. To increase robustness and reliability, a novel protein precipitation/phospholipid removal device is also utilized. The novel approach was applied to human samples with a comparison with established clinical LC-MS/MS services for measuring 25-hydroxyvitamin D in adults and for infants (<1 year old).

The data showed a good correlation between the routine service for adults and infant patient samples and illustrated the need to resolve the epimers. The unique selectivity of the pentafluorophenyl phase combined with the selective protein depletion and phospholipid enable a fast, accurate and robust analysis of 25-hydroxyvitamin D and related forms, which are otherwise unattainable with commonly used sample preparation and reversed-phase HPLC approaches.