Assessment of a Metabolomics Automated Sample Prep Platform for Low Volume Plasma Samples

Significance of the Topic

In metabolomics analysis of plasma samples, preparing low volume samples poses challenges in reproducibility and completeness of metabolite recovery. This is critical in both basic and translational research where sample availability from infants, small animal models, or precious clinical specimens may be limited.

Objectives and Study Overview

This study aimed to adapt an automated platform for metabolomics sample preparation to accommodate 25 μL plasma inputs and to evaluate its performance in terms of recovery and reproducibility relative to manual processing by multiple lab personnel.

Methodology and Instrumentation

An Agilent Bravo Metabolomics Sample Prep Platform protocol was modified to reduce plasma volume from 100 μL to 25 μL. Plasma proteins were precipitated using ethanol/methanol, lipids were removed via Captiva EMR-Lipid plates, and metabolites were eluted, dried, and reconstituted for analysis. A custom PCDL based on Agilent METLIN, and Agilent MassHunter Quantitative Analysis software (Ver 10.1) was used for data processing.
Used instrumentation:

• Agilent Bravo 96-well automation
• Captiva EMR-Lipid 96-well plates
• Agilent 1260 Infinity II Prime LC system with Poroshell 120 HILIC-Z column
• Agilent 6546 LC/Q-TOF with Jet Stream ionization

Main Results and Discussion

• Recovery assessment using 13C-labeled yeast spike-in demonstrated excellent results: 28 of 32 measured compounds showed recoveries above 80%, with an average recovery of 86%; one nonendogenous sugar phosphate showed poor recovery.
• Reproducibility testing across 60 samples showed automated preparation yielded average %RSD of 4.8% for metabolite peak areas, outperforming combined manual processing (%RSD 10.3%) and most individual operators.

Benefits and Practical Applications

• Significantly reduces required plasma volume to 25 μL, facilitating studies with limited sample availability.
• Automated workflow improves reproducibility and throughput, supporting high-quality quantitative metabolomics in clinical and research laboratories.

Future Trends and Applications

Emerging metabolomics workflows may integrate further sample diversity, multiplexed automation, and advanced data analysis tools. Continued miniaturization and coupling to high-resolution MS will expand capabilities in biomarker discovery and personalized medicine.

Conclusion

The modified automated protocol for low volume plasma sample preparation delivers robust metabolite recovery and superior reproducibility compared to manual methods, enabling reliable high-throughput metabolomics with limited sample volumes.

References

• Agilent Technologies. Automated Metabolite Extraction for Plasma using the Agilent Bravo Platform. Technical Overview no. 5994-0685, 2019.
• Agilent Technologies. Discovery Metabolomics LC/MS Methods Optimized for Polar Metabolites. Application Note no. 5994-1492, 2019.