Streamlined Vitamin D Metabolite Fingerprinting Analysis Using Isotope-Coded Multiplexing MS with Cost-Effective...

Area of Science:

• Clinical Chemistry
• Analytical Chemistry
• Mass Spectrometry

Background:

• Accurate analysis of vitamin D and its metabolites is crucial for clinical diagnostics.
• Existing methods for vitamin D metabolite analysis face challenges in multiplexing and separating critical epimers.
• Conventional separation techniques often require specialized columns (e.g., PFP) with limited long-term stability.

Purpose of the Study:

• To develop the first routine isotope-coded multiplex derivatization method for vitamin D and its metabolites.
• To enable simultaneous analysis of two samples in a single liquid chromatography-tandem mass spectrometry (LC-MS/MS) run.
• To improve the chromatographic separation of 25-hydroxyvitamin D3 epimers on conventional C-18 columns.

Main Methods:

• Extended a one-pot double derivatization reaction using commercial reagents.
• Employed a Diels-Alder reaction with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) followed by acetylation with acetic anhydride (or its d3-isotopologue).
• Utilized C-18 column with methanol/water gradient elution for enhanced separation of vitamin D3 metabolites.

Main Results:

• Established a routine isotope-coded multiplex derivatization enabling simultaneous measurement of two samples.
• Achieved baseline separation of critical vitamin D3 metabolites (24,25(OH)2D3, 3α-25(OH)D3, 3β-25(OH)D3, and vitamin D3) on a C-18 column.
• Demonstrated method applicability by testing on 40 serum samples from patients with chronic liver diseases (CLD).

Conclusions:

• The developed duplex derivatization method offers a robust and routine approach for clinical vitamin D analysis.
• This method overcomes limitations of specialized columns and enhances the separation of challenging epimers.
• The isotope-coded multiplexing strategy facilitates higher throughput and efficiency in clinical laboratories.