The Automated Derivatisation and Extraction of Fatty Acids using the Gerstel MPS Autosampler and Agilent 7890 / 7200 GC/Q-TOF

Significance of the Topic

Reliable measurement of fatty acids is fundamental in metabolomics, where small chemical changes can reflect significant biological effects. Automating derivatisation and extraction improves reproducibility and throughput in complex sample analysis.

Objectives and Overview

This study demonstrates a fully automated workflow for fatty acid methyl ester (FAME) preparation and analysis using a Gerstel Dual Head MultiPurpose Sampler (MPS) and an Agilent 7890B GC coupled to a 7200 Q-TOF mass spectrometer. The goal is to standardise sample handling, reduce manual effort, and enable high-resolution fingerprinting of non-polar metabolites in plant material.

Used Instrumentation

• Agilent 7890B Gas Chromatograph
• Agilent 7200 Q-TOF mass spectrometer
• Gerstel Dual Head MPS 2 XL-xt autosampler
• Gerstel Cooled Injection System (CIS4)
• Gerstel Thermal Desorption Unit (TDU)
• Gerstel Heated Agitator
• Agilent MassHunter software B07.00
• Gerstel Maestro software 1.4.18.25/3.5

Methodology

Freeze-dried oregano (5–6 mg) was placed in a 2 mL vial on the MPS rack. A 500 μL aliquot of methanolic HCl was added using a 1 mL syringe and the vial agitated at 70 °C, 500 rpm for 15 minutes to complete methylation. After cooling, 500 μL hexane and 500 μL water were added. A 10 μL sample from the upper hexane layer containing FAMEs was withdrawn with a 10 μL syringe on the second head and injected via large volume injection (LVI) using the CIS4 for solvent venting and analyte focusing.

Results and Discussion

The automated workflow yielded consistent hexane extracts, as shown by total ion chromatograms (TIC) containing both FAMEs and other hexane-soluble components. Extracted ion chromatograms (EIC) at m/z 74.0366 (McLafferty rearrangement of the ester group) enabled rapid fatty acid screening. Replicate extractions gave a mean %CV of 8.2% for FAMEs present at ≥1% of the most abundant methyl hexadecanoate. Individual straight-chain FAMEs from C14 to C22 showed %CV values between 3.9% and 6.9%, demonstrating high reproducibility.

Benefits and Practical Applications

• Reduced solvent consumption and manual handling
• Lower labour and faster sample-to-sample time via prep-ahead feature
• High reproducibility suited for comparative metabolomic and fingerprinting studies
• High-resolution full scan Q-TOF data enables both target ion extraction and retrospective non-target screening
• Confirmation of analyte identity and structural elucidation from accurate mass spectra

Future Trends and Applications

The platform can be extended to other derivatisation and extraction protocols, such as Folch lipid extraction and Fiehn metabolite derivatisation, by integrating vortexing and centrifugation steps. Expanding to different sample types and coupling with data-mining tools will further enhance non-polar metabolic fingerprinting and novel metabolite discovery.

Conclusion

This work highlights the value of automating FAME derivatisation and extraction using a Gerstel MPS system in tandem with Agilent GC/Q-TOF. The approach streamlines workflows, ensures reproducibility, and provides rich analytical data for targeted and non-targeted metabolomics applications.

Reference

Sean O'Connor and Nathan Hawkins. Chromatography Technical Note No AS136. Anatune Ltd., 2014.