Enabling Automated, Low-Volume Plasma Metabolite Extraction with the Agilent Bravo Platform

Significance of Topic

Efficient and reproducible extraction of metabolites from small volumes of plasma is critical in both basic and translational research, particularly when sample quantities are limited as in pediatric or animal studies. Automating low-volume workflows reduces operator variability, enhances throughput, and supports consistent quality in LC-MS metabolomics assays.

Objectives and Study Overview

This study describes modifications to the Agilent Bravo Metabolomics Sample Prep Platform that enable automated extraction from just 25 µL of plasma instead of the standard 100 µL. The goals were to assess metabolite recovery using an Agilent 6546 LC/Q-TOF system and compare reproducibility against manual processing by multiple technicians.

Methodology and Instrumentation

The protocol preserves a 225:225:100 ratio of methanol:ethanol:plasma to precipitate proteins and quenche enzymatic activity. Captiva EMR–Lipid plates are used for protein filtration and lipid depletion. Key steps include:

• Automated addition of organic solvents and water with 10-minute incubations after quenching and water addition
• Filtrate collection of all eluant and wash fractions to minimize loss
• Vacuum drying and reconstitution in 100 µL of LC-MS solvent (80 µL acetonitrile then 20 µL water)

Instrumentation:

• Agilent Bravo Metabolomics Sample Prep Platform
• Agilent PlateLoc thermal microplate sealer
• Agilent 1260 Infinity II Prime LC with HILIC-Z column (2.1×150 mm, 2.7 µm) and quaternary pump
• Agilent 6546 LC/Q-TOF with Dual Agilent Jet Stream ion source

Chromatography uses a gradient from 96% to 65% acetonitrile with 10 mM ammonium acetate, pH 9. Mass spectrometry in negative mode acquires from m/z 50–1600 at 1.5 spectra/s.

Key Results and Discussion

Recovery experiments spiked U-13C yeast extract before and after extraction. Of 31 representative metabolites covering amino acids, nucleotides, organic acids, sugars, and vitamins, 26 exhibited recoveries above 80% (average 85.9% with 7.7% RSD). D-fructose 1,6-bisphosphate showed lower recovery at 38.7%, attributed to its low endogenous presence in plasma.
Reproducibility testing compared 60 automated preparations to 60 manual preparations by three users. Automated extraction yielded an average RSD of 4.8% across metabolites, outperforming the combined manual RSD of 10.3%.

Benefits and Practical Applications

The low-volume automated method delivers outstanding recovery and superior reproducibility compared with manual processing. Reducing plasma input to 25 µL expands applicability to scarce samples and high-throughput studies, while minimizing human error and labor demands in metabolomics workflows.

Future Trends and Opportunities

Further miniaturization and integration of sample preparation with on-line LC-MS systems may drive even higher throughput and sensitivity. Advances in robotic platforms, microfluidics, and data-driven process control will support personalized medicine, small cohort studies, and single-cell metabolomics.

Conclusion

The modified Agilent Bravo protocol enables reliable, automated extraction of metabolites from low-volume plasma samples with excellent recovery and enhanced reproducibility. This workflow is well suited to demanding clinical and research applications where sample volume and consistency are critical.

Reference

1. Automated Metabolite Extraction for Plasma using the Agilent Bravo Platform. Agilent Technologies Technical Overview, publication number 5994-0685, 2019.
2. Discovery Metabolomics LC/MS Methods Optimized for Polar Metabolites. Agilent Technologies Application Note, publication number 5994-1492, 2019.