COMPARISON OF SPE PROTOCOLS FOR PHOSPHOLIPID REMOVAL IN BASIC ANALYTE BIOANALYTICAL QUANTITATION

Significance of the topic

Phospholipids (PLs) in biological matrices pose a major analytical challenge in LC–MS bioanalysis due to their high concentrations and structural diversity, leading to significant ion suppression or enhancement effects. Efficient removal of PLs prior to analysis is essential for accurate quantitation of small molecules in plasma and other complex samples.

Objectives and study overview

This study directly compares two mixed-mode reversed-phase/cation-exchange solid-phase extraction (SPE) protocols—Oasis MCX and Oasis PRiME MCX—for the removal of major endogenous PL classes (phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, lysophosphatidylcholine, sphingomyelin) and cholesterol. Both protocols were evaluated using human blood plasma and individual PL standards to assess their efficiency at each SPE step (load, aqueous wash, organic wash, elution).

Methodology and instrumentation

Sample preparation and SPE protocols were performed as follows:

• Human plasma (200 µL) diluted 1:1 with 4 % phosphoric acid.
• Oasis MCX: 96-well plate packed with 30 mg sorbent per well.
• Oasis PRiME MCX: 96-well plate cartridge format.
• Washes: aqueous (2 % formic acid or 100 mM ammonium formate with 2 % formic acid), organic (methanol), two sequential steps.
• Elution: 5 % NH4OH in methanol.

LC–MS/MS conditions:

• Instrument: ACQUITY UPLC I-Class coupled to XEVO TQ-S triple quadrupole mass spectrometer.
• Column: ACQUITY BEH C18, 1.7 µm, 2.1 × 100 mm, 50 °C.
• Mobile phases: A—0.1 % formic acid in water; B—0.1 % formic acid in acetonitrile. Gradient from 2 % B to 95 % B.
• ESI positive mode; capillary 3 kV; desolvation 500 °C; MRM transitions for each PL and cholesterol.
• Data acquired and processed in MassLynx v4.1.

Additional LC–PDA analysis of standards used ACQUITY UPLC with BEH HILIC column and Empower 3 software.

Main results and discussion

Removal efficiencies for each SPE step demonstrated:

• Oasis PRiME MCX achieved > 95 % removal of all PL classes and cholesterol during the combined aqueous and organic wash steps, with minimal analyte loss in the elution step.
• Oasis MCX effectively removed phosphatidylglycerol, phosphatidylinositol, and cholesterol (> 95 %) in wash steps but retained significant fractions of phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and phosphatidylserine, which required strong-base elution.

Differences were attributed to sorbent chemistry, analyte pKa values, charge states at pH < 1.0, and hydrophobic versus ion-exchange interactions. The higher pH and ionic strength in PRiME MCX washes partially zwitterionize amine-containing PLs, weakening cation-exchange binding and promoting early removal.

Advantages and practical applications

The Oasis PRiME MCX protocol offers:

• Cleaner extracts with broad phospholipid removal prior to LC–MS analysis.
• Reduced ion suppression and enhanced method robustness.
• Simplified workflows with fewer elution steps and lower solvent usage.

These benefits support high-throughput bioanalytical laboratories, QA/QC environments, and pharmacokinetic studies requiring reliable quantitation in complex matrices.

Future trends and application potential

Emerging directions include:

• Automated SPE platforms integrating PRiME sorbents for increased throughput.
• Development of mixed-mode materials optimized for specific lipid subclasses.
• Coupling with high-resolution mass spectrometry for comprehensive lipidomic profiling.
• Miniaturized protocols to reduce solvent consumption and waste.

Conclusion

Both Oasis MCX and PRiME MCX protocols effectively remove key PLs and cholesterol from plasma samples. The PRiME MCX method demonstrates superior performance in broad phospholipid depletion during wash steps, yielding cleaner samples and simplifying downstream LC–MS workflows.

References

1. Journal of Lipid Research, Volume 51, 2010, 3299.
2. Analytical and Bioanalytical Chemistry, 2015, Volume 407, 5227.
3. Journal of Lipid Research, Volume 51, 2010, 3299.
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5. Clinical Methods: The History, Physical, and Laboratory Examination, 3rd Ed., Chapter 31.