Confident quantitation of 25-hydroxyvitamin D2 and D3 in human plasma for clinical research by liquid chromatography-tandem mass spectrometry with simple, robust sample preparation

Significance of the Topic

Vitamin D status is critically linked to bone health and mineral homeostasis. Accurate quantitation of 25-hydroxyvitamin D2 and D3 in human plasma supports clinical research and patient care by providing reliable biomarkers of vitamin D nutrition.

Objectives and Study Overview

The study aimed to develop and validate a fast, sensitive, and robust LC-MS/MS method for quantifying 25-hydroxyvitamin D2 and D3 in human plasma. The workflow integrates a multichannel UHPLC system with a triple quadrupole mass spectrometer to meet productivity and performance requirements for clinical research laboratories.

Methodology and Instrumentation

Sample Preparation

• Plasma volume 50 µL was protein precipitated with 150 µL acetonitrile containing d6-25-hydroxyvitamin D3 as internal standard
• Vortex mixing and centrifugation yielded supernatant for injection

Liquid Chromatography

• System – two-channel Thermo Scientific Transcend LX-2 in multichannel mode
• Column – Hypersil GOLD 50 × 2.1 mm, 1.9 µm, at 40 °C
• Mobile phases – water (A) and methanol (B), each with 10 mM ammonium formate and 0.1% formic acid
• Gradient – 3.5 min runtime, flow rate 0.5 mL/min, rapid transition from 20% to 100% organic

Mass Spectrometry

• Instrument – Thermo Scientific TSQ Quantis triple quadrupole
• Ionization – APCI in positive mode
• Acquisition – selected reaction monitoring with two transitions per analyte and internal standard
• Key settings – vaporizer 400 °C, capillary 300 °C, spray current 4 µA, collision gas 1.5 mTorr, resolution 0.7 FWHM

Main Results and Discussion

Calibration and Linearity

• Calibration ranges covered 9.84–81.0 ng/mL for D2 and 9.04–78.9 ng/mL for D3
• Quadratic curves with 1/x weighting achieved bias within ±10% across all levels

Accuracy and Precision

• Analytical accuracy bias for control samples remained between -2.5% and 2.6%
• Intra-assay precision CV ≤3.6% and inter-assay precision CV ≤4.1%

Carryover was negligible with no detectable signal in blanks following high calibrators. Chromatographic separation delivered sharp peaks in under 1 min retention time per analyte, enabling 3.5 min total cycle time per injection.

Benefits and Practical Applications

• High throughput via multichannel LC doubles effective sample analysis compared to single-channel setups
• Simple offline protein precipitation reduces preparation time and increases reproducibility
• Mid-range triple quadrupole MS delivers best-in-class sensitivity suitable for low-level vitamin D metabolites
• Method meets regulatory expectations for clinical research quantitation and can support large cohort studies

Future Trends and Opportunities

Further automation of sample preparation and integration of online extraction could streamline workflows. Expansion of analyte panels to include additional vitamin D metabolites and related biomarkers may enhance clinical insights. Advances in high-resolution MS and ion mobility may offer even greater specificity for complex biological matrices. Application of multiplexed LC-MS platforms will continue to drive efficiency in biomonitoring studies.

Conclusion

A rapid, robust LC-MS/MS method was established for 25-hydroxyvitamin D2 and D3 quantification in human plasma. The combination of Transcend LX-2 multichannel LC with TSQ Quantis triple quadrupole MS achieved high sensitivity, throughput, and reproducibility. This approach fulfils the stringent demands of clinical research laboratories for reliable vitamin D status assessment.

References

No formal literature references were provided in the source document.