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

• Final Report Year: 2024

Final Report Abstract

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. The research conducted addressed many of these limitations and delivered improved techniques for measurement of vitamin D metabolites. Among them were chemical labeling techniques for increased sensitivity and selectivity, combined ion mobility spectrometry-mass spectrometry, high resolution MS and MALDI-MS. By applying these new techniques, we were able to remove or circumvent many of the existing limitations and strongly improve accuracy and precision of analysis. Furthermore, we gained much deeper access into the Vitamin D metabolome than was possible before and were able to measure and quantify very low abundant, dynamic metabolites of vitamin D. We developed rapid profiling techniques for quantitatively capturing the dynamic metabolic phenotypes of several vitamin D metabolites in human serum samples, which gave us preliminary evidence for correlations of metabolite fingerprints with disease phenotypes. In addition, we implemented an entirely novel calibration technique for analysis of vitamin D from dried blood spots and successfully applied MALDI-MS to high-speed analysis of the vitamin D status marker for the first time. Finally, we investigated in detail the parallel Vitamin D epimerization pathway and developed novel isotope-coded chemical labeling tools for multiplex-high throughput-LC-MS/MS.

Publications

• On the isobaric space of 25‐hydroxyvitamin D in human serum: potential for interferences in liquid chromatography/tandem mass spectrometry, systematic errors and accuracy issues. Rapid Communications in Mass Spectrometry, 29(1), 1-9.
  Qi, Yulin; Geib, Timon; Schorr, Pascal; Meier, Florian & Volmer, Dietrich A.
  
• Assessment of 3-epi-25-hydroxyvitamin D levels during cholecalciferol supplementation in adults with chronic liver diseases. Applied Physiology, Nutrition, and Metabolism, 41(12), 1311-1317.
  Stokes, Caroline S. & Volmer, Dietrich A.
  
• Chemotyping the distribution of vitamin D metabolites in human serum. Scientific Reports, 6(1).
  Müller, Miriam J.; Stokes, Caroline S.; Lammert, Frank & Volmer, Dietrich A.
  
• Triple Quadrupole Versus High Resolution Quadrupole-Time-of-Flight Mass Spectrometry for Quantitative LC-MS/MS Analysis of 25-Hydroxyvitamin D in Human Serum. Journal of the American Society for Mass Spectrometry, 27(8), 1404-1410.
  Geib, Timon; Sleno, Lekha; Hall, Rabea A.; Stokes, Caroline S. & Volmer, Dietrich A.
  
• Quantification of the 3α and 3β epimers of 25-hydroxyvitamin D3 in dried blood spots by LC-MS/MS using artificial whole blood calibration and chemical derivatization. Talanta, 165, 398-404.
  Müller, Miriam J.; Stokes, Caroline S. & Volmer, Dietrich A.
  
• Overestimation of 3α- over 3β-25-Hydroxyvitamin D3 Levels in Serum: A Mechanistic Rationale for the Different Mass Spectral Properties of the Vitamin D Epimers. Journal of the American Society for Mass Spectrometry, 32(4), 1116-1125.
  Schorr, Pascal; Kovačević, Borislav & Volmer, Dietrich A.
  
• Stability of sample extracts of vitamin D3 metabolites after chemical derivatization for LC–MS/MS analysis. Analytical and Bioanalytical Chemistry, 415(2), 327-333.
  Alexandridou, Anastasia & Volmer, Dietrich A.
  
• 2-fluoro-1-methylpyridinium p-toluene sulfonate: a new LC-MS/MS derivatization reagent for vitamin D metabolites. Journal of Lipid Research, 64(8), 100409.
  Alexandridou, Anastasia & Volmer, Dietrich A.
  
• Comparing derivatization reagents for quantitative LC–MS/MS analysis of a variety of vitamin D metabolites. Analytical and Bioanalytical Chemistry, 415(19), 4689-4701.
  Alexandridou, Anastasia; Schorr, Pascal & Volmer, Dietrich A.
  
• Improved quantitative LC-MS/MS analysis of vitamin D metabolites in serum after one-pot double derivatization. Journal of Pharmaceutical and Biomedical Analysis, 234, 115522.
  Schorr, Pascal; Stokes, Caroline S. & Volmer, Dietrich A.