Quantification of 25-hydroxyvitamin D2 and D3 in human plasma by LC-HRAM-MS for clinical research

Significance of the Topic

Measurement of 25-hydroxyvitamin D2 and D3 informs vitamin D status impacting bone health, immune function, and chronic disease risk. High-resolution LC-MS overcomes limitations of protein binding bias and cross-reactivity in immunoassays and provides selective, sensitive quantification for clinical research.

Study Objectives and Overview

The aim was to develop and validate a rapid, robust LC-HRAM-MS method using a Thermo Scientific Orbitrap Exploris 120 to quantify 25-OHD2 and 25-OHD3 in human plasma. Method performance was assessed for linearity, sensitivity, accuracy, precision, and carryover.

Methodology and Sample Preparation

Plasma (50 µL) was spiked with a d6-25-OHD3 internal standard and precipitated with acetonitrile. After centrifugation the supernatant was directly injected.
Chromatographic separation used a Vanquish Flex UHPLC system with a Hypersil GOLD 2.1×50 mm column at 40 °C. Gradient elution with 5 mM ammonium formate/0.1% formic acid in water (A) and methanol (B) at 0.5 mL/min yielded a 3.5 min run time.
Mass spectrometry on Orbitrap Exploris 120 with an APCI positive ion source acquired full scan (100–500 m/z at 60 000 resolution) and targeted MS2 (30 000 resolution) with HCD at 40% collision energy.

Used Instrumentation

• Thermo Scientific Vanquish Flex UHPLC
• Hypersil GOLD 2.1×50 mm, 1.9 µm column
• Thermo Scientific Orbitrap Exploris 120 MS with APCI source
• TraceFinder 5.1 data processing software

Main Results and Discussion

The calibration curve used quadratic fitting with 1/x weighting; bias remained within ±10% across all calibrators. LLOQs were 1.97 µg/L (25-OHD2) and 4.52 µg/L (25-OHD3) for both acquisition modes. Accuracy bias ranged 1.9–5.8% (full scan) and 1.8–6.7% (MS2). Intra-assay precision (%CV) was ≤7.5% (full scan) and ≤8.4% (MS2); inter-assay precision was ≤5.0% and ≤5.9%, respectively. No significant carryover was observed.

Benefits and Practical Applications

• High selectivity and sensitivity enable reliable quantification of vitamin D metabolites.
• Rapid sample preparation and short run times support high throughput.
• Robust performance suits clinical research and biomarker studies.

Future Trends and Opportunities

Advances may include automation of sample preparation, multiplexed assays for additional metabolites, integration into high-throughput workflows, and application in large-scale epidemiological studies. Emerging high-resolution platforms and enhanced ionization techniques will further improve sensitivity and selectivity.

Conclusion

A streamlined LC-HRAM-MS method was validated for precise, accurate quantification of 25-OHD2 and 25-OHD3 in plasma. The technique offers robust performance suitable for clinical research quantifying vitamin D status.

Reference

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