An LC-MS/MS Clinical Research Method for the Measurement of 25-OH Vitamin D2and D3 Metabolites

Importance of Topic

Vitamin D status assessment is critical due to the widespread prevalence of deficiency and its association with bone disorders, cancer risk, and various chronic diseases. Accurate quantification of the circulating metabolites 25-hydroxyvitamin D2 and D3 in plasma is essential for both clinical research and quality-controlled testing.

Objectives and Study Overview

The primary aim was to develop and validate a rapid, sensitive, and reproducible LC-MS/MS method using the ACQUITY UPLC Online SPE Manager (OSM) for simultaneous measurement of 25-OH D2 and 25-OH D3 in human plasma. The study compared the new workflow against an established online SPE-LC-MS/MS assay.

Methodology and Instrumentation

The method integrates automated online solid-phase extraction (SPE) with UPLC-MS/MS:

• Sample preparation: 150 µL plasma, addition of isotopically labeled internal standards, protein precipitation with 0.4 M ZnSO₄, centrifugation.
• Online SPE: MassTrak C8 cartridge, conditioning with organic solvents, wash steps with gradient methanol/ammonia solutions, elution in 0.75 min.
• Chromatography: ACQUITY UPLC CSH Phenyl-Hexyl column (2.1×100 mm, 1.7 µm); mobile phases A (10% methanol, ammonium acetate, formic acid) and B (methanol, ammonium acetate, formic acid); flow 0.4 mL/min; 6.25-min total runtime at 35 °C.
• Mass spectrometry: Xevo TQ-S; multiple reaction monitoring transitions optimized for 25-OH D2, 25-OH D3, and their deuterated analogs (e.g., m/z 383.4→257.3 for D3).

Main Results and Discussion

Chromatographic separation yielded sharp, well-resolved peaks for both metabolites at physiologically relevant concentrations (e.g., 55 nmol/L D3, 14 nmol/L D2).

Reproducibility:

• Intra-assay CVs: 1.5–3.8% for D3, 3.1–5.3% for D2 across low to high QC levels.
• Inter-assay CVs: 3.3–3.9% for D3, 3.7–5.0% for D2 over six days.

Method comparison with an existing online SPE LC-MS/MS assay showed excellent agreement (Deming fit slope 1.08), while delivering a more streamlined workflow.

Benefits and Practical Applications

• Simultaneous quantification of 25-OH D2 and 25-OH D3 with high sensitivity at low nmol/L levels.
• Fully automated online SPE reduces manual handling and operator time.
• Rapid analysis (~6 min per sample) and small plasma volume requirement (150 µL).
• Robust performance suitable for high-throughput clinical research and QA/QC laboratories.

Future Trends and Applications

• Expansion to additional vitamin D metabolites or related biomarkers using tailored MRM transitions.
• Integration into large-scale epidemiological studies and routine clinical monitoring platforms.
• Coupling with data processing software and laboratory information systems for seamless workflow.
• Adaptation of the online SPE-UPLC approach to other bioanalytical targets requiring high sensitivity.

Conclusion

The developed ACQUITY UPLC OSM-based LC-MS/MS method delivers a rapid, sensitive, and reproducible workflow for measuring 25-OH vitamin D2 and D3 in plasma. Its high automation level, low sample volume requirement, and strong agreement with established assays make it a valuable tool for clinical research laboratories.