Method Development and Validation of Vitamin D2 and Vitamin D3 using Mass Spectrometry

Thesis (Master's)--University of Washington, 2016-06

Vitamin D has long been known to maintain bone health by regulating calcium and phosphorous homeostasis. In recent years, scientists have discovered additional physiological roles for vitamin D. The complex interaction between the active vitamin D hormone and its metabolic precursors continues to be a rich area of research. Fundamental to this research is the availability of accurate and precise assays. Few published assays for vitamins D2 and D3 have contained sufficient details on method validation or performance characteristics. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay developed for this thesis has undergone a rigorous validation and proven to yield a sensitive and specific method that exceeds the capabilities of all previously published methods. Developing and validating a novel assay is often complicated by the lack of established acceptability standards. This thesis explores this challenge, specifically for establishing meaningful interpretations and qualification standards of the lower limit of the measuring interval. Altogether, future research focused on vitamins D2, D3 and the Vitamin D pathway can benefit from this robust LC-MS/MS assay and the associated quality parameters outlined in this thesis.