Novel analytical approaches to quantify low-abundant dynamic metabolites in the vitamin D metabolic cascade

• Applicant: Professor Dr. Dietrich Volmer
• Subject Area: Analytical Chemistry ; Public Health, Healthcare Research, Social and Occupational Medicine
• Term: from 2014 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 242744369

Vitamin D comprises a group of secosteroid compounds, of which vitamin D2 and D3 are the most important bioactive variants. Vitamin D plays a crucial role in bone health but has also been linked to many other diseases such as cancer and chronic liver disease. To determine vitamin D status in humans, several automated clinical assays are available, which measure the most important vitamin D marker, 25-hydroxyvitamin D. Unfortunately, these assays often 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, and the potential for interferences. This proposal addresses some of these limitations and suggests improved techniques for measurement of vitamin D metabolites. The approach comprises two areas of technical advancements; (1) chemical labeling techniques for increased sensitivity and selectivity, and (2) combined differential ion mobility (DMS)/mass spectrometry for separating isobaric/isomeric species, thus reducing potential systematic errors. Even though it is not the primary aim of this project, we are hoping that measurement of vitamin D metabolic distributions of well-defined patient samples will give us preliminary evidence for correlations of metabolite fingerprints with disease phenotypes. The first theme addresses important limitations of vitamin D analysis; that is limited detection sensitivity and issues of quantification, which currently restrict analysis to the main vitamin D metabolite in most assays. This precludes more elaborate diagnostic applications using low abundant metabolites. We intend to develop a differential quantification technique using new isotope-coded tags for LC-MS/MS, enabling comparative measurement of longitudinal up- and down-regulation of vitamin D metabolite levels, e.g. over the course of a supplementation study or for comparing stages of disease. We are also suggesting a metal-coded tag incorporating a lanthanide-chelating moiety, which will permit analysis at much lower concentration levels than currently possible, using analysis by ICP-MS.The second theme addresses the limited selectivity of LC-MS/MS from interfering endogenous components. In this proposal, we suggest a gas-phase separation step using DMS prior to mass spectrometry. DMS can often separate isobars and isomers because differential mobilities are largely independent of mass-to-charge-ratios but rather depend on conformation, charge distribution etc. Here we propose the use of DMS-MS for removing isobaric species and elimination of co-eluting interferences during vitamin D analysis, to lower detection limits and avoid systematic errors. We also want to examine whether DMS-MS is suited for separation of the C-3 epimers, to distinguish contributions of these species.

• DFG Programme: Research Grants