SPE and LC-MS/MS Method for the Determination of 25-Hydroxyvitamin D2 and 25-Hydroxyvitamin D3 from Human Plasma

Importance of the Topic

The accurate measurement of 25-hydroxyvitamin D2 and D3 in human plasma is fundamental to evaluating vitamin D status, which influences bone health, immune function, and disease risk. Routine monitoring of these metabolites is preferred over the active hormone due to their higher abundance and longer half-life, enabling reliable assessment in clinical diagnostics and population health studies.

Objectives and Study Overview

This study aimed to develop and validate a rapid, sensitive, and reproducible SPE-LC-MS/MS workflow for quantifying 25-hydroxyvitamin D2 and D3 in human plasma. The method integrates Thermo Scientific SOLA HRP solid phase extraction with Syncronis C18 UHPLC separation and triple quadrupole MS detection to achieve high throughput and robust performance across a wide dynamic range.

Methodology and Sample Preparation

The sample preparation protocol disrupts vitamin D binding proteins with acetonitrile and isolates analytes on SOLA HRP reversed-phase SPE plates. Key steps include:

• Protein precipitation: 200 µL plasma mixed with 180 µL acetonitrile.
• Spiking: Addition of standard or internal standard solutions.
• Centrifugation: 5 min at 5,000 rpm.
• SPE cleanup: Conditioning with methanol and water, sample loading, wash with 40% methanol, and elution with 100% methanol.
• Drying and reconstitution: Nitrogen drying and reconstitution in 30:70 water/acetonitrile.

Instrumentation Used

The analytical setup comprised:

• UHPLC: Thermo Scientific UltiMate 3000 with Syncronis C18 column (1.7 µm, 50 × 2.1 mm).
• MS/MS: Thermo Scientific TSQ Vantage triple quadrupole with APCI source in positive mode.
• Data processing: Thermo Scientific LCQUAN 2.6 software.

Main Results and Discussion

The method demonstrated:

• Linearity over 5–1000 ng/mL for both analytes, with R² ≥ 0.9958.
• Limit of quantitation of 5 ng/mL, as evidenced by clear chromatographic peaks and signal-to-noise ratios.
• Precision below 4.2% RSD at QC levels (15, 250, 600 ng/mL).
• High recovery: 94.4% for D2 and 96.3% for D3 across QC concentrations.

The robust SIL-corrected quantification and rapid 2-minute cycle time support high-throughput laboratory operations.

Benefits and Practical Applications

This workflow offers:

• Fast turnaround with minimal sample and solvent use.
• High analytical sensitivity and specificity, minimizing matrix interferences.
• Reproducible performance suitable for clinical diagnostics, nutritional studies, and pharmaceutical research.

Future Trends and Potential Applications

Advancements may include:

• Automation of SPE and UHPLC steps to further increase throughput.
• Integration with high-resolution MS for untargeted metabolomics alongside vitamin D profiling.
• Development of dried blood spot protocols to expand accessibility in remote or resource-limited settings.

Conclusion

The combined use of SOLA HRP SPE, Syncronis C18 UHPLC, and triple quadrupole MS yields a streamlined, sensitive, and accurate method for quantifying 25-hydroxyvitamin D2 and D3 in plasma. This approach meets the demands of modern laboratories for speed, reliability, and data quality.

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.