Quantification of 25-hydroxyvitamin D2 and D3 in human plasma by liquid chromatography coupled to high-resolution Orbitrap mass spectrometry for clinical research

Significance of the Topic

Vitamin D status is critical for bone health, calcium and phosphate homeostasis, and modulation of immune and cardiovascular functions. Precise measurement of its main circulating forms, 25-hydroxyvitamin D2 and D3, is essential in clinical research to diagnose deficiencies, monitor supplementation, and investigate associations with various diseases. High selectivity and sensitivity are required to accurately quantify low serum concentrations and to minimize interference from matrix components.

Objectives and Study Overview

The primary goal was to develop and validate a robust LC–HRMS method for the quantification of 25-OH-vitamin D2 and D3 in human plasma. This included implementing an offline sample preparation workflow using a ClinMass® kit, achieving rapid chromatographic separation with online SPE, and comparing full-scan and parallel reaction monitoring (PRM) acquisition modes on an Orbitrap platform.

Methodology and Instrumentation

The sample preparation involved:

• Addition of d6-25-hydroxyvitamin D3 internal standard to 50 µL plasma
• Protein precipitation with organic solvent, vortex mixing, cooling, centrifugation
• Transfer of supernatant for LC–MS analysis

Chromatographic setup on a Vanquish Duo UHPLC system:

• Online SPE cartridge for sample clean-up
• Analytical column separation with a 3.0-min total runtime
• Binary gradient to enrich and elute analytes

Mass spectrometry on a Q Exactive Plus quadrupole-Orbitrap:

• Atmospheric pressure chemical ionization (APCI) in positive mode
• FullMS and PRM acquisition to compare sensitivity and specificity
• TraceFinder software for data processing

Main Results and Discussion

Linearity and sensitivity:

• Calibration ranges: 9.8–81.0 ng/mL for D2, 9.0–78.9 ng/mL for D3
• Linear response with 1/x weighting across full range and down to LLOQ
• Lower limits of quantification (LLOQ): 4.92 ng/mL (D2) and 1.81 ng/mL (D3) in FullMS; PRM yielded 4.52 ng/mL for D3

Accuracy and precision:

• Bias within ±15% (±20% at LLOQ)
• Intra-assay CVs below 11.3%; inter-assay CVs below 9.3%
• Carryover below 0.4%

Comparison of acquisition modes:

• FullMS offered slightly lower LLOQ for D3
• PRM enhanced selectivity with comparable precision

Benefits and Practical Applications

The developed method provides:

• High selectivity and minimal matrix interference
• Rapid turnaround compatible with clinical research throughput
• Reliable quantification across the physiologically relevant range

It is readily adoptable in research laboratories investigating vitamin D status, disease associations, or supplementation efficacy.

Future Trends and Potential Applications

Emerging opportunities include:

• Automation of sample preparation to further increase throughput
• Expansion to multiplexed panels of vitamin D metabolites
• Integration with AI-driven data analysis for pattern recognition in large cohorts
• Application in pharmacokinetic and epidemiological studies

Conclusion

A precise, fast, and reproducible LC–HRMS method for 25-OH-vitamin D2 and D3 quantification was established using offline sample prep, online SPE, and Orbitrap detection. Both FullMS and PRM modes met stringent criteria for sensitivity, linearity, accuracy, and precision, supporting its use in clinical research settings.

Reference

De Nardi C., Kern K., Peters S. Quantification of 25-hydroxyvitamin D2 and D3 in human plasma by liquid chromatography coupled to high-resolution Orbitrap mass spectrometry for clinical research. Technical Note No. 73234, Thermo Fisher Scientific, 2019.