Advantages of Oasis PRiME HLB for the LC-MS/MS Analysis of Vitamin D Metabolites in Serum for Clinical Research

Importance of the Topic

Accurate quantification of vitamin D metabolites in serum is critical for clinical research and patient management. Low abundance and structural similarities between analytes and endogenous phospholipids can compromise sensitivity and specificity in LC-MS/MS assays. Adopting effective sample cleanup strategies enhances analytical performance and data reliability.

Objectives and Study Overview

The aim of this study was to establish a robust LC-MS/MS method for simultaneous measurement of four key vitamin D metabolites: 25OHD3, 25OHD2, 24,25(OH)2D3 and C3-epi-25OHD3. The approach focuses on minimizing phospholipid interference and maximizing sensitivity without derivatization.

Methodology and Instrumentation

Sample preparation involved protein precipitation of 100 µL serum with methanol/zinc sulfate, followed by microelution SPE using Waters Oasis PRiME HLB plates. Optimized wash (25% acetonitrile) and elution (100% acetonitrile) conditions were selected based on elution profiles of vitamin D metabolites and lysophosphatidylcholines (LysoPCs). Chromatographic separation was performed on an ACQUITY UPLC HSS PFP column with a water/methanol gradient containing ammonium acetate and formic acid. Detection employed multiple reaction monitoring on a Xevo TQ-S micro mass spectrometer.

Instrumentation Used

• Waters ACQUITY UPLC I-Class system with HSS PFP column
• Waters Xevo TQ-S micro mass spectrometer
• Oasis PRiME HLB µElution SPE plates

Key Results and Discussion

The optimized SPE conditions removed over 99% of targeted LysoPCs, boosting vitamin D metabolite signal by up to 10-fold compared to protein precipitation. Calibration curves for each analyte showed correlation coefficients above 0.99 across clinically relevant ranges. Chromatographic separation resolved isobaric compounds including C3-epi-25OHD3 and 25,26(OH)2D3 within an 8-minute runtime. Precision (total CV ≤ 8.2%) and accuracy demonstrated strong agreement with NIST SRM972a and DEQAS reference materials. Limits of quantification achieved were 1 nmol/L for 25OHD3 and 25OHD2, and 0.5 nmol/L for C3-epi-25OHD3 and 24,25(OH)2D3.

Benefits and Practical Applications

• Simplified workflow with efficient phospholipid removal
• High analytical precision and reproducibility
• Enhanced sensitivity without derivatization
• Rapid throughput suitable for clinical research laboratories

Future Trends and Potential Applications

Continued improvements in sorbent design and MS instrumentation may further reduce matrix effects and push detection limits. Integration with automated sample preparation platforms could streamline high-volume workflows. Expanding the method to additional vitamin D metabolites and other lipid-related biomarkers may extend its clinical and research utility.

Conclusion

This study demonstrates that Oasis PRiME HLB SPE coupled with UPLC-MS/MS provides a sensitive, precise and high-throughput method for vitamin D metabolite analysis in serum. The streamlined protocol effectively eliminates phospholipid interference, obviates derivatization and supports reliable clinical research applications.