A Semi-Automated Lipid Extraction Protocol Using the Agilent Bravo Automated Liquid Handling Platform

Significance of the topic

Lipidomics is a vital branch of metabolomics that profiles hundreds of lipid species to understand their biological roles. Traditional extraction methods are laborious, error-prone, and time-consuming, especially when processing large cohorts. Semi-automation addresses these challenges by improving throughput, reproducibility, and operator safety, while reducing sample preparation time and cost.

Objectives and overview

This study presents a semi-automated workflow for extracting phospholipids and sphingolipids from human plasma using the Agilent Bravo Automated Liquid Handling Platform. The protocol transfers plasma aliquots into 96-well plates, adds a single-phase butanol:methanol extraction solvent with internal standards, and integrates manual sonication and centrifugation steps. Extracts are analyzed by UHPLC–MS/MS to assess method performance versus conventional manual extraction.

Methodology and instrumentation

This semi-automated procedure uses:

• Agilent Bravo Automated Liquid Handling Platform with 96-channel tip head and VWorks software
• Butanol:methanol (1:1) extraction solvent containing ammonium formate and a mix of deuterated/internal lipid standards
• CPAC Ultra-flat cooling plates to maintain samples at 4 °C during transfers
• Multi-well reservoirs, PCR tube caps, and dedicated tip waste containers
• Branson ultrasonic bath and Sorvall centrifuge for protein precipitation and phase consolidation
• Agilent 1290 Infinity LC coupled to a 6460 Triple Quadrupole MS using reverse-phase C18 chromatography and dynamic MRM in positive/negative modes

Critical method development steps included calibrating low-volume solvent handling to correct for butanol:methanol vapor pressure and surface tension effects, and optimizing deck layout for sample, solvent, and tip positions.

Main results and discussion

Comparison of 12 replicates each of manual and semi-automated extractions revealed:

• Correlation of endogenous lipid concentrations: R² = 0.9964 across 115 molecular species
• Reduced relative standard deviations for lipid standards: semi-automated RSDs ranged from ~3.8 % to 7.3 % versus 4.96 % to 11.97 % manually
• Throughput improvement: sample preparation time decreased from ~8 hours to ~2 hours per 96-well plate

The semi-automated protocol delivered consistent recovery and repeatability, matching or exceeding traditional manual methods while significantly reducing hands-on time and potential for human error.

Benefits and practical applications

The adoption of this semi-automated extraction workflow offers:

• Higher throughput for large clinical or epidemiological lipidomics studies
• Enhanced reproducibility and lower inter-operator variability
• Reduced exposure to toxic organic solvents and minimized safety risks
• Lower per-sample labor costs and optimized laboratory resource allocation

Future trends and applications

Advances may include full integration of sonication and centrifugation modules into the robotic platform, expansion to multi-phase extraction protocols, and adaptation to other biofluids or tissue matrices. Coupling with high-resolution mass spectrometry and AI-driven automation workflows will further streamline lipidomics in clinical diagnostics and drug discovery.

Conclusion

The Agilent Bravo platform enables semi-automated single-phase lipid extraction from plasma with high precision, reproducibility, and a four-fold reduction in sample preparation time. This protocol supports scalable lipidomics pipelines in research and quality-control laboratories.

References

1. Wenk MR. Lipidomics: New Tools and Applications. Cell. 2010;143(6):888–895.
2. Bligh EG, Dyer WJ. A Rapid Method of Total Lipid Extraction and Purification. Can J Biochem Physiol. 1959;37(8):911–917.
3. Albert KJ. Optimizing Accuracy Performance on an Agilent Bravo Platform using the Artel MVS. Artel Application Note. 2013.