Quantitation of 25-Hydroxyvitamin D2 and 25-Hydroxyvitamin D3 in Plasma for Clinical Research Using the Prelude SPLC and TSQ Endura Mass Spectrometer

Significance of the Topic

Accurate quantification of 25-hydroxyvitamin D2 and D3 is essential for understanding vitamin D status and its role in human health. Clinical laboratories benefit from methods that balance precision, speed, and cost-efficiency to support large-scale studies and routine analysis.

Objectives and Study Overview

This study evaluated the performance of a coupled sample preparation and LC-MS/MS platform—Thermo Scientific Prelude SPLC with TurboFlow technology and TSQ Endura mass spectrometer—for quantifying 25OH D2 and D3 in human plasma. The goal was to assess sensitivity, accuracy, precision, and throughput under conditions relevant to clinical research.

Methodology and Used Instrumentation

Sample preparation involved protein precipitation of plasma with methanol containing a deuterated internal standard, followed by direct online cleanup. Key instrumentation included:

• Thermo Scientific Prelude SPLC system with dual-channel TurboFlow online cleanup
• Thermo Scientific Cyclone-P column (0.5×50 mm) for initial extraction
• Thermo Scientific Accucore aQ column (2.1×100 mm) for chromatographic separation
• Thermo Scientific TSQ Endura triple quadrupole MS with APCI source in SRM mode
• Thermo Scientific TraceFinder software for data acquisition and processing

Main Results and Discussion

The method achieved limits of quantitation of 4 ng/mL for both 25OH D2 and D3, with a linear range up to 200 ng/mL. Inter-assay precision was below 7% RSD, and accuracy ranged between 93% and 107% when benchmarked against NIST reference materials. Recovery experiments yielded 102–132% for 25OH D2 and 100–118% for 25OH D3. Matrix effects showed ion suppression (absolute values of 61–79%) but were largely mitigated by internal standard correction (relative effects above 75%). The dual-channel operation facilitated a sample throughput of up to 24 injections per hour.

Benefits and Practical Applications

• Automated online cleanup reduces manual handling and sample prep time
• High throughput supports large clinical studies and routine testing
• Robust performance with minimal carryover and maintenance
• Cost-effective operation through optimized mass spectrometer utilization

Future Trends and Possibilities

• Integration of multiplexed assays for broader vitamin D metabolite panels
• Advancements in microflow and nanoflow LC to reduce solvent and sample consumption
• Implementation of artificial intelligence for data analysis and quality control
• Expansion of high-throughput platforms for personalized medicine applications

Conclusion

The described LC-MS/MS approach combining Prelude SPLC and TSQ Endura offers a streamlined, high-performance solution for quantifying 25-hydroxyvitamin D metabolites in plasma. It meets key criteria for clinical research, delivering sensitivity, accuracy, and throughput while reducing operational complexity.

References

No external literature citations were provided in the original document.