Efficiency of Biological Fluid Matrix Removal Using Agilent Captiva EMR— Lipid Cleanup

Significance of the Topic

Phospholipids are abundant in biological fluids and are major contributors to matrix effects in LC-MS/MS bioanalysis, leading to ion suppression, decreased sensitivity, and contamination of chromatographic columns and mass spectrometer sources. Efficient removal of these lipids prior to analysis is essential to protect instruments, ensure data integrity, and achieve reliable quantification.

Study Objectives and Overview

This study evaluates the performance of Agilent Captiva Enhanced Matrix Removal—Lipid (Captiva EMR—Lipid) cartridges and 96-well plates for selective removal of phospholipids from various human and animal biological fluids. Key objectives include:

• Quantifying phospholipid removal efficiency across matrices (plasma with different anticoagulants, serum, CSF, and animal plasmas).
• Comparing Captiva EMR—Lipid to five commercially available cleanup products in terms of removal performance, eluent clarity, ease of use, and clogging.

Methodology

Sample preparation combined in situ protein precipitation (PPT) and pass-through cleanup:

• Add 600 µL acetonitrile with 1 % formic acid to each cartridge well or plate well, then 200 µL sample; mix by pipetting.
• Apply controlled vacuum (~1 drop per 3–5 s) to elute the supernatant through Captiva EMR—Lipid sorbent; dry eluents and reconstitute for analysis.
• Evaluate cleanup by precursor ion scan for m/z 184 to monitor phospholipids, gravimetric residue measurement, eluent clarity assessment, and flow observations.

Used Instrumentation

• Agilent 1290 Infinity UHPLC system (binary pump, autosampler, column compartment).
• Agilent G6490 Triple Quadrupole LC/MS with JetStream iFunnel ESI source; MassHunter software.
• Laboratory equipment: Centra CL3R and Eppendorf centrifuges, vortexers, pipettes, ViaFlo 96 liquid handler, Captiva vacuum manifold, CentriVap and TurboVap concentrators.

Main Results and Discussion

• Ease of Use and Eluent Clarity: Captiva EMR—Lipid demonstrated smooth, clog-free flow under low vacuum for all biological fluids, yielding clear eluents. Competing products often required higher vacuum, experienced clogging, and produced cloudy extracts.
• Gravimetric Residue: Cleanup with Captiva EMR—Lipid resulted in the lowest residual mass compared to other methods, indicating superior removal of co-extractives.
• Phospholipid Removal: Captiva EMR—Lipid achieved >99 % removal of phospholipids in all tested matrices and anticoagulant conditions. Competitor products varied widely, with removal efficiencies ranging from 82 % to just under 100 %.

Benefits and Practical Applications

• Enhanced analytical sensitivity and reproducibility by eliminating phospholipid interferences.
• Protection of LC-MS instrumentation and extended column and source lifetimes.
• Streamlined, in situ PPT workflow compatible with high-throughput automation in 96-well plate format.

Future Trends and Opportunities

• Extension of EMR cleanup approaches to other lipid-rich matrices such as tissue homogenates, food, and environmental samples.
• Integration with fully automated platforms and microfluidic systems to increase throughput and reduce solvent consumption.
• Development of multimodal sorbents capable of simultaneous removal of diverse matrix components and further miniaturization for point-of-care applications.

Conclusion

Agilent Captiva EMR—Lipid cartridges and 96-well plates offer robust and selective phospholipid removal (>99 %) from a wide range of biological fluids. The combination of in situ protein precipitation and pass-through cleanup yields clear eluents, minimal instrument contamination, and reliable, high-throughput workflows.

References

1. Chambers, E.; et al. Systematic and comprehensive strategy for reducing matrix effects in LC/MS/MS analyses. J. Chromatogr. B, Analytical technologies in the biomedical and life sciences 2007, 852, 22–34.
2. Matuszewski, B. K.; Constanzer, M. L.; Chavez-Eng, C. M. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal. Chem. 2003, 75, 3019–3030.
3. Little, J. L.; Wempe, M. F.; Buchanan, C. M. Liquid chromatography-mass spectrometry/mass spectrometry method development for drug metabolism studies: Examining lipid matrix ionization effects in plasma. J. Chromatogr. B, Analytical technologies in the biomedical and life sciences 2006, 833, 219–230.
4. Lehotay, S. J.; Mastovská, K.; Lightfield, A. R. Use of buffering and other means to improve results of problematic pesticides in a fast and easy method for residue analysis of fruits and vegetables. J. AOAC Int. 2005, 88, 615–629.
5. Janusch, F.; et al. Evaluation and subsequent minimization of matrix effects caused by phospholipids in LC-MS analysis of biological samples. Bioanalysis 2013, 5, 2101–2114.
6. Korfmacher, W. A. Using Mass Spectrometry for Drug Metabolism Studies. 2004.