Removal of Lipids for the Analysis of Toxicological Compounds in Plasma by LC/MS/MS

Significance of the Topic

In toxicological analysis of plasma, removal of lipids is essential to prevent chromatographic interferences and MS ion suppression.
The Enhanced Matrix Removal—Lipid (EMR—Lipid) dispersive SPE technique offers an efficient and streamlined approach for lipid cleanup in LC/MS/MS workflows.

Objectives and Overview of the Study

This application note presents a simplified protocol combining protein precipitation with EMR—Lipid dispersive SPE to quantify 25 toxicological compounds in human plasma.
The goals were to achieve >97% lipid removal, high analyte recovery (>95%), low RSDs (<6%), and LOQs of ≤1 ng/mL across diverse analytes.

Methodology and Process

• Protein precipitation of 500 µL plasma with acidified acetonitrile (0.2% formic acid), followed by centrifugation.
• Dispersive SPE cleanup using 200 mg EMR—Lipid sorbent, water conditioning, extract addition, and phase separation with MgSO4.
• Polishing and drying steps with MgSO4 to remove residual moisture and fine particulates.
• Final extract dilution (200 µL sample + 800 µL water) and analysis by LC/MS/MS in dynamic MRM mode.
• Calibration in matrix from 0.5 to 100 ng/mL with deuterated internal standards for quantitation.

Used Instrumentation

• Agilent 1290 Infinity LC with diode array detector.
• Agilent 6490 Triple Quadrupole LC/MS with Jet Stream and iFunnel technology.
• Bond Elut EMR—Lipid dispersive SPE tubes and ancillary centrifuge and vortex equipment.

Main Results and Discussion

• EMR—Lipid removed >97% of phospholipids, outperforming Oasis Prime HLB, Phenomenex Phree, and HybridSPE sorbents.
• Cleaner extracts prevented late-eluting lipid peaks and source contamination, as shown by precursor ion m/z 184 scans over 30 min.
• Calibration curves were highly linear (R²>0.97) with LOQs ranging from 0.5 to 5 ng/mL depending on compound.
• Average recoveries at 5 ng/mL exceeded 95% for most analytes (heroin ~65%), with RSDs below 6% across QC levels.

Benefits and Practical Applications

• Rapid, user-friendly workflow requiring only vortexing and centrifugation.
• Cleaner extracts reduce column fouling, source contamination, and instrument downtime.
• Applicable to high-throughput toxicology, clinical research, QA/QC, and forensic laboratories.

Future Trends and Potential Applications

The EMR—Lipid dispersive SPE approach can be extended to other biological matrices (whole blood, urine) and integrated with automated platforms to increase throughput and reproducibility.
Development of tailored sorbents targeting specific lipid classes may further refine cleanup for emerging analytes.

Conclusion

The combination of protein precipitation and EMR—Lipid dispersive SPE provides a robust, efficient, and cost-effective sample preparation strategy for LC/MS/MS quantification of toxicological compounds in plasma, delivering high lipid removal, excellent analytical performance, and ease of implementation.

Reference

• Stevens J. Removal of Lipids for the Analysis of Toxicological Compounds in Plasma by LC/MS/MS using EMR—Lipid. Agilent Application Note, 2016.
• Plössl F., et al. Multiresidue analytical method using dispersive SPE and GC/ion trap MS to determine pharmaceuticals in whole blood. J. Chromatogr. A. 2006;1135:19–26.