Agilent Captiva EMR–Lipid Manual Fractionation of Low‑Volume Plasma Samples for LC/MS Multi‑Omics

Significance of the topic

Manual fractionation of low-volume plasma samples for LC/MS multi-omics provides a reliable foundation for high-throughput metabolomics, lipidomics, and proteomics analyses. Developing a consistent, easy-to-implement protocol that can be readily automated meets the growing demand for reproducibility, throughput, and flexibility in omics workflows.

Objectives and study overview

This method guide presents a configurable protocol for processing 20 µL plasma aliquots in 96-well format. Key goals are to enable selective collection of metabolite, lipid, and protein fractions, test compatibility with the Agilent Bravo Metabolomics Sample Prep Platform, and demonstrate suitability for downstream LC/MS analysis on instruments such as LC/TQ and LC/Q-TOF.

Methodology and instrumentation

The protocol comprises the following steps:

• Sample aliquoting: Transfer 20 µL plasma to a 96-well glass-lined or polypropylene plate.
• Protein precipitation: Add 112.5 µL 1:1 methanol:ethanol, mix, and optionally pellet proteins by centrifugation or sonicating.
• Metabolite fractionation: Pass sample through preconditioned Agilent Captiva EMR–Lipid plate under positive pressure; collect filtrate containing polar metabolites.
• Lipid fractionation: Wash EMR–Lipid plate with 2:1:1 water:methanol:ethanol, then elute lipids with two sequential 900 µL aliquots of 2:1 methanol:DCM into glass-lined plates.
• Protein pellet wash: If proteins are collected, wash pellets with 2:1 methanol:DCM, re-sonicate and centrifuge to recover residual lipids.
• Sample drying and reconstitution: Dry metabolite and lipid fractions under nitrogen or vacuum without heat; reconstitute in 50–200 µL LC/MS solvent before analysis.

Used instrumentation:

• Agilent Captiva EMR–Lipid plates and collection plates
• Positive pressure manifold processor (Agilent PPM-96)
• Agilent Bravo Metabolomics Sample Prep Platform
• PlateLoc thermal microplate sealer and pierceable seals
• Centrifuge capable of low-speed plate centrifugation

Key results and discussion

Testing demonstrated robust recovery of polar metabolites, lipids, and proteins with minimal cross-contamination. The decision tree allows users to prioritize metabolite versus hydrophobic lipid recovery and choose whether to isolate proteins. Slow elution through EMR–Lipid sorbent maximizes lipid yield. The protocol scaled effectively from manual processing to automated workflows on the Bravo platform, maintaining reproducibility across 96-well plates.

Benefits and practical applications

• Flexibility: Configurable fractionation paths support targeted metabolomics, lipidomics, proteomics, or combined multi-omics.
• Low sample volume: Efficient processing of 20 µL plasma conserves precious clinical and experimental samples.
• Automation readiness: Compatible with standard robotic platforms for high-throughput labs.
• Streamlined workflow: Single-plate, 96-well format reduces handling time and risk of error.
• LC/MS compatibility: All fractions are directly suitable for Agilent LC/MS methods (metabolite, lipid, peptide).

Future trends and potential applications

Integration with high-resolution mass spectrometers and data-independent acquisition will expand coverage in lipidomics and metabolomics. Combining this protocol with advanced sample multiplexing (e.g., TMT labelling for proteomics) can further increase throughput. Emerging microfluidic platforms may reduce sample and solvent consumption even further. Adaptation to other biofluids and single-cell analysis could broaden applications in clinical and translational research.

Conclusion

The Agilent Captiva EMR–Lipid manual fractionation method offers a robust, flexible, and automation-ready solution for multi-omics analysis of low-volume plasma. By enabling selective recovery of metabolites, lipids, and proteins in a streamlined 96-well workflow, it supports high-throughput studies requiring reproducible and scalable sample preparation for LC/MS.

References

1. Van de Bittner, G. C. et al. Automated Fractionation of Low-Volume Plasma Samples for LC/MS Multi-Omics. Agilent Technologies Tech. Overview 5994-7357EN (2024).
2. Sartain, M. et al. Enabling Automated, Low-Volume Plasma Metabolite Extraction with the Agilent Bravo Platform. Agilent Tech. App. Note 5994-2156EN (2020).
3. Van de Bittner, G. C. et al. An Automated Dual Metabolite + Lipid Sample Preparation Workflow for Mammalian Cell Samples. Agilent Tech. Overview 5994-5065EN (2022).
4. Agilent AssayMAP Bravo. Agilent Technologies Citation Index 5994-4198EN (2024).
5. Yannell, K. E. et al. An End-to-End Targeted Metabolomics Workflow. Agilent Tech. App. Note 5994-5628EN (2023).
6. Huynh, K. et al. A Comprehensive, Curated, High-Throughput Method for the Detailed Analysis of the Plasma Lipidome. Agilent Tech. App. Note 5994-3747EN (2021).
7. Wu, L. et al. In-Depth Peptide Mapping with Iterative MS/MS Acquisition on the Agilent 6545XT AdvanceBio LC/Q-TOF. App. Note 5991-8633EN (2020).
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