Lipidomics Analysis of Human Plasma Using Agilent Bond Elut Lipid Extraction 96-Well Plates

Importance of the Topic

Lipidomics sample preparation is critical for large-scale clinical research, offering insights into lipid function, disease biomarkers and treatment monitoring. Conventional liquid–liquid extraction workflows are time-consuming, labor-intensive and difficult to automate, creating a bottleneck in high-throughput studies. The Agilent Bond Elut Lipid Extraction 96-well plate SPE method delivers a swift, reproducible and automation-friendly alternative for human plasma lipid profiling.

Objectives and Overview of the Study

This study compares the high-throughput 96-well plate SPE workflow against the established Agilent 1 mL cartridge method for processing human plasma. Key aims include assessing background cleanliness, recovery of isotopic internal standards, lipid profiling performance and operational efficiency in a side-by-side evaluation.

Methodology and Instrumentation

Human plasma samples underwent protein precipitation with acetonitrile/methanol, followed by loading onto Bond Elut Lipid Extraction plates. Washing with ACN:water and elution with dichloromethane/methanol isolated lipids, which were dried under nitrogen and reconstituted in n-butanol/methanol containing EquiSPLASH internal standards. Recovery was studied by pre- and post-spiking with UltimateSPLASH One standards across fifteen lipid classes.

Used Instrumentation

• Agilent 6545 LC/Q-TOF with Dual Jet Stream ESI source and 1290 Infinity II LC
• Agilent 6490 LC/TQ with Dual Jet Stream ESI source and 1290 Infinity II LC
• Agilent Bond Elut Lipid Extraction 96-well plate and related 2 mL square-well collection plates
• PlateLoc thermal microplate sealer, PPM-96 positive pressure manifold, 96-well plate evaporator and multiprobe vortexer
• Integra ViaFlo 96 liquid handler, Eppendorf pipettes and sonicator

Main Results and Discussion

Background blanks showed minimal contamination in both ionization modes. Total ion chromatograms revealed comparable lipid profiles for cartridge and 96-well plate formats, with the latter often providing higher signal intensity. Quantitative recovery of 63 deuterated lipids across 15 classes exceeded 70% for both methods, with most class-to-class differences below 5%. Untargeted LC/Q-TOF profiling identified 377 lipids for the cartridge and 381 for the 96-well plate, exhibiting similar class distributions.

Benefits and Practical Applications of the Method

The 96-well SPE workflow simplifies handling, reduces processing time and effort, and integrates readily with automation platforms. It supports high-throughput clinical and industrial lipidomics, offering robust reproducibility and increased productivity for large cohort studies.

Future Trends and Possibilities for Application

Advances will focus on deeper integration with automated liquid handlers, application to diverse biological matrices and coupling with advanced MS informatics. Standardized high-throughput workflows are poised to accelerate clinical diagnostics and biomarker discovery in lipidomics.

Conclusion

The Agilent Bond Elut Lipid Extraction 96-well plate method provides a reliable, high-throughput alternative to cartridge-based SPE for human plasma lipidomics. It matches or exceeds cartridge performance in cleanliness, recovery and profiling while streamlining workflow and enhancing automation compatibility.

Reference

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