Development of Simple, Fast SPE Protocols for Basic Analyte Extraction with Phospholipid Removal Using Oasis PRiME MCX

Significance of the Topic

Effective removal of phospholipids from biological samples is critical to ensure reliable quantification of basic analytes by LC-MS/MS. Endogenous phospholipids cause significant matrix effects, suppressing or enhancing analyte signals, altering retention and selectivity, and leading to reduced column life and system fouling. Developing streamlined sample preparation protocols that minimize phospholipid carryover while preserving analyte recovery is essential for robust bioanalytical workflows.

Objectives and Study Overview

This study aimed to design and evaluate rapid solid-phase extraction (SPE) protocols using a new mixed-mode sorbent, Oasis PRiME MCX, to remove phospholipids from plasma samples containing basic drug analytes. Key goals included maximizing phospholipid removal, maintaining high and reproducible analyte recoveries, and simplifying workflows compared with conventional Oasis MCX 2×4 protocols.

Methodology and Instrumentation

Two sample types were tested: a phosphatidylcholine (PC) standard in organic solvent and rat plasma spiked with fifteen basic drugs. SPE was performed using Oasis MCX cartridges or 96-well plates (10 mg sorbent) and Oasis PRiME MCX formats. Protocols varied loading and wash solutions, introducing 100 mM ammonium formate in acidic washes to disrupt ionic and hydrophobic interactions of phospholipids. Eluates were analyzed by UPLC and tandem mass spectrometry.

Main Results and Discussion

• Using the conventional Oasis MCX 2×4 protocol, ~85% of PC eluted in the final step, indicating limited removal in washes.
• Incorporating 100 mM ammonium formate in the organic wash step of a new 4-step Oasis PRiME MCX protocol shifted ~89% of PC into the wash, reducing eluate PC by >100-fold.
• A simplified 3-step variant, combining ammonium formate in the load and skipping the aqueous wash, achieved comparable phospholipid reduction with only a slight increase in residual PC.
• In plasma extractions, the 4-step and 3-step Oasis PRiME MCX methods lowered phospholipid signals by approximately tenfold versus the traditional protocol while preserving >90% recovery for basic analytes with pKa >5.
• Diazepam (pKa 3.4) exhibited lower recovery due to partial loss during acidic washes, highlighting the importance of analyte pKa in method selection.

Benefits and Practical Applications

• Dramatic phospholipid removal reduces matrix effects, improving quantitation accuracy and reproducibility.
• Lower system fouling and extended column lifetime support high-throughput LC-MS/MS operations.
• High and consistent recoveries for a broad range of bases streamline method development and validation.
• The simplified 3-step workflow minimizes hands-on time and solvent consumption, ideal for 96-well plate automation.

Future Trends and Potential Applications

• Expansion to additional compound classes, including acidic and neutral analytes, through customized mixed-mode sorbent chemistries.
• Integration with high-resolution mass spectrometry for multi-residue screening and untargeted lipidomic studies.
• Development of universal SPE cartridges combining phospholipid removal with protein depletion for clinical and toxicological testing.
• Automated on-line SPE-LC-MS platforms to further reduce sample handling and enhance throughput.

Conclusion

The Oasis PRiME MCX 4-step and 3-step SPE protocols deliver rapid, robust phospholipid removal from plasma samples while maintaining high recovery of basic analytes. Compared to traditional methods, these workflows offer substantial reductions in matrix interferences, simplified operation, and improved LC-MS/MS system performance.

Instrumentation Used

• ACQUITY UPLC I-Class System with PDA Detector
• ACQUITY UPLC BEH C8 and C18 Columns
• Xevo TQ-S Tandem Quadrupole Mass Spectrometer
• Empower 3 CDS and MassLynx 4.1 Software

References

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