The goal of this grant renewal application is the development of novel mass spectrometric (MS) techniques for detailed analysis of the vitamin D metabolome. Vitamin D comprises a group of secosteroidal compounds, of which vitamin D3 is the most important bioactive variant. Vitamin D plays a crucial role in bone health but has also been linked to many other diseases such as cancer and chronic liver diseases. To determine vitamin D status in humans, several automated clinical assays are available. Unfortunately, these assays lack specificity and accuracy. As a result, LC-MS/MS techniques are preferred today because of their ability to distinguish between vitamin D variants, their improved sensitivity and selectivity. Development of LC-MS/MS assays for vitamin D requires significant expertise to overcome the various inherent limitations, however. LC-MS/MS also exhibits significant potential for interferences. During the first funding phase, we established several novel analytical techniques addressing these limitations and interferences, allowing us to circumvent or remove some of the major weaknesses. We also developed powerful chemical derivatization tools that increased both sensitivity and specificity of analysis, thus permitting quantitative measurement of multiple vitamin D metabolites from serum of patients. In addition, we implemented an entirely novel calibration technique for analysis of vitamin D from dried blood spots and successfully applied MALDI-MS to analysis of the vitamin D status marker for the first time. In this grant renewal application, novel MS techniques will be developed that will allow much deeper access into the Vitamin D metabolome than presently possible. The proposed research will specifically target very low abundant, dynamic metabolites of vitamin D, many of which are currently not detectable in human serum because of insufficiently sensitive analytical techniques. Some of them have been seen in microsomal experiments, however, or have been predicted from the enzymes that can act on the major vitamin D compounds. Furthermore, we will investigate for the first time the parallel epimerization pathway of vitamin D via LC-MS/MS and develop entirely new methods for the determination of free vitamin D and its protein-bound species. To achieve this goal, we are suggesting the application of membrane introduction MS as well as separation by turbulent flow chromatography. A final objective is to systematically examine the spectrum and dynamic changes of a large number of vitamin D metabolites in sera of patient samples and to evaluate the diagnostic potential of these fingerprints. For example, we are planning to develop a statistical model from the measured metabolite concentrations that predicts response to supplementation after shortterm single dose of cholecalciferol and then suggests the required dose to reach a target level of the status marker in the patient’s blood.