Bioanalytical Studies of the Assessment of Vitamin D Status

The most widely used and clinically accepted biochemical marker for assessing vitamin D status is the total serum 25-hydroxyvitamin D (25-OHD) concentration. In high-throughput clinical diagnostic laboratories, there is a trend towards the use of fully-automated clinical analysers for such assays. This means that immunoassays are commonly used, despite significant inter-assay variability due to varying concentrations of other related vitamin D metabolites and sample-to-sample matrix differences. It is important for clinicians requesting 25-OHD analyses to understand these issues and limitations, and where necessary to confront laboratories for details of analytical methods used. The availability of reference measurement procedures for 25-OHD based on liquid chromatography and tandem mass spectrometry (LC-MS/MS), whilst not intended for routine clinical sample analysis, should be utilised to improve assay harmonisation and reduce interlaboratory variability. The development of higher-throughput, semi-automated LC-MS/MS methods for routine application is also increasing due to recognition of the pitfalls of immunoassay-based methods for 25-OHD analysis, as well as the opportunity to individually measure multiple vitamin D metabolites. This thesis will discuss the reasons for ongoing 25-OHD assay variability, but will also discuss the novel application of LC-MS/MS to larger molecules related to the assessment vitamin D status. These include the analysis of parathyroid hormone (PTH) and PTH variants, and the three major isoforms of vitamin D binding protein.