Quantification of 25-hydroxyvitamin D2 and D3 in human plasma by liquid chromatography-tandem mass spectrometry for clinical research

Importance of the topic

Adequate measurement of 25-hydroxyvitamin D2 and D3 in human plasma is essential for assessing vitamin D status in clinical and research settings. Vitamin D regulates calcium and phosphate homeostasis, supports bone health, and influences a variety of conditions including rickets, osteomalacia, cardiovascular disease, cancer, and autoimmune disorders. Reliable quantification of 25(OH)D metabolites with high sensitivity and reproducibility is critical for accurate diagnosis, patient monitoring, and epidemiological studies.

Objectives and study overview

This study describes the implementation and validation of a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for quantifying 25-hydroxyvitamin D2 and D3 in human plasma. The method employs a Thermo Scientific™ TSQ Quantis™ triple quadrupole mass spectrometer coupled with a Thermo Scientific™ Transcend™ II TLX-2 system for online solid-phase extraction (SPE). Key goals included achieving rapid analysis, robust performance, and accurate quantitation across clinically relevant concentration ranges.

Methodology and instrumentation

• Sample preparation: Fifty microliters of plasma were spiked with d6-25-hydroxyvitamin D3 internal standard, protein-precipitated with organic solvent, vortexed, cooled at 4 °C, centrifuged, and the supernatant collected.
• Online SPE and chromatography: Extracts were processed on a Transcend II TLX-2 system using a dedicated SPE cartridge and analytical column. A fast gradient delivered total runtime of 3.0 minutes per injection.
• Mass spectrometry: APCI in positive mode was used for ionization. Two selected-reaction monitoring (SRM) transitions per analyte ensured quantification and confirmation. Calibration employed a linear 1/x-weighted model over 9–81 ng/mL ranges.

Used instrumentation

• Thermo Scientific Transcend II TLX-2 online SPE/LC system
• Thermo Scientific TSQ Quantis triple quadrupole mass spectrometer
• APCI source (vaporizer 400 °C, capillary 300 °C, 4 µA spray current)
• TraceFinder 4.1 software for data acquisition and processing

Main results and discussion

• Linearity: Calibration bias within ±10% across all concentration levels.
• Carryover: No detectable carryover observed in blank injections following highest calibrator.
• Accuracy: Percentage biases between nominal and measured control samples ranged from –5.5% to –1.7%.
• Precision: Intra-assay CVs ≤2.4%, inter-assay CVs ≤2.8% for both analytes.

Benefits and practical applications

• Rapid throughput: 3-minute runtime supports high sample volumes.
• Robustness: Online SPE reduces manual steps and variability.
• Sensitivity: Reliable detection across clinically relevant concentration ranges.
• Applicability: Suitable for clinical research, epidemiological studies, and quality control in diagnostic laboratories.

Future trends and applications

• Automation of sample handling to increase throughput and consistency.
• Expansion to additional vitamin D metabolites and related biomarkers.
• Integration with high-resolution mass spectrometry for improved specificity.
• Adaptation for alternative sample types such as dried blood spots.

Conclusion

The validated LC-MS/MS method using online SPE and APCI quantifies 25-hydroxyvitamin D2 and D3 in human plasma with high accuracy, precision, and speed. Implementation on the TSQ Quantis platform provides a robust workflow suitable for clinical research, supporting reliable vitamin D status assessment in diverse patient populations.