SPE and LC-MS/MS method for the determination of 25-Hydroxyvitamin D2 and 25-Hydroxyvitamin D3 from human plasma

Importance of the Topic

Accurate quantification of 25-hydroxyvitamin D2 and D3 in human plasma is essential for assessing vitamin D status in clinical, research, and quality control laboratories. These metabolites serve as reliable markers due to their higher circulating levels and longer half-life compared to the active hormone form.

Objectives and Overview of the Study

This application note describes the development and validation of a rapid, robust solid-phase extraction (SPE) and LC-MS/MS method for simultaneous determination of 25-hydroxyvitamin D2 and D3 from human plasma. Key goals included high extraction recovery, excellent precision, and a fast two-minute chromatographic cycle.

Methodology

• Sample Pre-treatment: Human plasma samples (200 µL) were protein-precipitated with acetonitrile (final ratio ≤1:1) to release bound analytes.
• SPE Cleanup: SOLA HRP 10 mg/2 mL hydrophobic reversed-phase plate was conditioned with methanol and water, loaded with sample supernatant, washed with 40% methanol, and eluted with 100% methanol.
• Drying and Reconstitution: Eluates were evaporated under nitrogen and reconstituted in 100 µL water/acetonitrile (30:70 v/v).
• Chromatographic Separation: Syncronis C18 column (1.7 µm, 50 × 2.1 mm) at 40 °C using a gradient from 70% to 100% acetonitrile (+0.1% formic acid) over 2 minutes at 0.8 mL/min.
• Mass Spectrometry: APCI in positive mode on a TSQ Vantage triple quadrupole; optimized transitions monitored for D2, D3, and deuterated internal standard.

Used Instrumentation

• Dionex UltiMate 3000 HPLC system
• Syncronis C18, 1.7 µm, 50 × 2.1 mm column
• TSQ Vantage triple stage quadrupole mass spectrometer with APCI source
• Thermo Scientific SOLA HRP SPE plate and vacuum manifold
• UltraVap nitrogen evaporator

Main Results and Discussion

• Linearity: 5–1000 ng/mL for both analytes with r2 values of 0.9994 (D2) and 0.9958 (D3).
• Limit of Quantitation: 5 ng/mL demonstrated by clear chromatographic peaks.
• Precision: %RSD ≤4.2% at quality control levels of 15, 250, and 600 ng/mL.
• Recovery: 94.4% for D2 and 96.3% for D3 across QC levels.

Benefits and Practical Applications

The streamlined SPE workflow using SOLA HRP reduces solvent consumption and sample handling time while improving extract cleanliness. Fast UHPLC separation enhances throughput, making the method suitable for high-volume clinical and bioanalytical laboratories.

Future Trends and Opportunities

• Integration into automated platforms for fully automated sample preparation.
• Expansion to multiplex vitamin D metabolite panels including 1,25-dihydroxyvitamin D.
• Application to alternative matrices such as dried blood spots or saliva.
• Use of advanced sorbent chemistries to further reduce matrix effects.

Conclusion

The validated SPE and LC-MS/MS protocol delivers rapid, reliable quantification of 25-hydroxyvitamin D2 and D3 in human plasma with excellent recovery, precision, and throughput. This approach supports routine clinical monitoring and research requiring high sample throughput.

Reference

1. Holick MF, Binkley NC, Bischoff-Ferrari HA et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2011;96(7):1911–1930.
2. Institute of Medicine, Food and Nutrition Board. Dietary Reference Intakes for Calcium and Vitamin D. Washington, DC: National Academy Press; 2010.