Automated and high-throughput derivatization for FAMEs analysis in vegetable oils and animal fats

Significance of FAME Derivatization in Edible Oil Analysis

Accurate profiling of fatty acid methyl esters (FAMEs) is essential for assessing nutritional quality and regulatory compliance of edible oils and animal fats. FAME analysis underpins nutritional labeling regulations, such as the U.S. FDA Nutrition Labeling and Education Act and trans fat labeling requirements, and international standards from ISO and AOAC. The derivatization of fatty acids into volatile methyl esters is a critical sample preparation step that directly influences data accuracy, reproducibility, and laboratory throughput.

Study Objectives and Overview

The study demonstrates a fully automated derivatization workflow using the Thermo Scientific™ TriPlus™ RSH SMART Robotic Sampler Handler to prepare FAMEs in vegetable oils and animal fats. It benchmarks three official methods (EU Regulation 2015/1833 Annex X, ISO 12966-2 “rapid”, and AOAC 996.01) and evaluates analytical performance in terms of speed, repeatability, and compliance with codified compositional limits.

Methodology and Instrumentation

The automated workflow integrates hydrolysis or trans-esterification steps followed by methylation and extraction, all controlled through Thermo Scientific™ Chromeleon™ CDS software. Key instrumentation:

• TriPlus RSH SMART autosampler configured with incubator, vortex mixer, solvent stations, large wash station, and automatic tool change for 10 mL reagent syringe and 10 µL injection syringe.
• Thermo Scientific™ TRACE™ 1610 gas chromatograph equipped with two iConnect™ split/splitless injectors and two iConnect™ flame ionization detectors serving dual GC channels for higher throughput.
• TRACE TR-FAME capillary column (10 m × 0.10 mm × 0.20 µm) operating with hydrogen carrier gas for fast, high-resolution separations (< 10 min run time).

Key Results and Discussion

• Chromatographic resolution of 37 FAME components achieved in under 10 minutes with critical pair Rs ≥ 1.0 and average peak asymmetry of 1.0.
• Automated derivatization throughput: up to 48 samples in 8 hours via ISO 12966-2 “rapid” method and 24 samples in ~4.5 hours by AOAC 996.01 procedure.
• High reproducibility demonstrated over six days: absolute peak area RSD < 9 %, relative peak area RSD < 3.3 %, and retention time deviations of 0.005 min.
• Compositional results for various oils (olive, peanut, soybean, sunflower, rapeseed) and animal fat matched quality ranges from UNI standards, FAO-WHO Codex Alimentarius, and EU regulations, confirming method suitability.

Benefits and Practical Applications

The automated protocol minimizes manual handling and exposure to toxic reagents (e.g., BF₃), reduces risk of cross-contamination, and enables unattended operation (including overnight runs). Dual-GC injection capability doubles sample throughput per autosampler, while Chromeleon CDS ensures secure data integrity in compliance with FDA 21 CFR Part 11 and EU Annex 11. Laboratories engaged in contract testing, QA/QC, and research benefit from faster turnaround, consistent data quality, and streamlined workflows.

Future Trends and Opportunities

Advances in autosampler robotics and integration with AI-driven data processing will further accelerate fatty acid analysis. Integration of mass spectrometric detectors could enhance specificity, while cloud-based CDS platforms will support real-time monitoring and remote operation. Continued development of ultra-fast columns and greener derivatization reagents will contribute to sustainable, high-throughput workflows.

Conclusion

The TriPlus RSH SMART–TRACE 1610 GC-FID configuration delivers a robust, high-throughput automated solution for FAME derivatization in edible oils and animal fats. It meets regulatory requirements, offers significant improvements in speed and reproducibility, and enhances laboratory safety and efficiency.

References

1. U.S. Food and Drug Administration. Nutrition Labeling and Education Act.
2. U.S. Food and Drug Administration. Small entity compliance guide: trans fatty acids in nutrition labeling, August 2003.
3. AOAC Official Method 996.01. Fat in Cereal Products by GC.
4. ISO 12966-2:2017. Animal and vegetable fats and oils — GC of FAMEs — Part 2: Preparation.
5. ISO 12966-4:2015. Animal and vegetable fats and oils — GC of FAMEs — Part 4: Determination.
6. Thermo Fisher Scientific. TriPlus RSH SMART Robotic Sampling System, BR52235-EN 0921C.
7. Thermo Fisher Scientific. TriPlus RSH SMART autosampler product specifications, PS000158.
8. Christie, W. W. Preparation of Ester Derivatives of Fatty Acids for Chromatographic Analysis. AOAC lipid library.
9. EU Commission Implementing Regulation 2015/1833, Annex X, October 2015.
10. Thermo Fisher Scientific. Technical Note TN001218: HeSaver-H2Safer for GC carrier gas.
11. UNI Standards. Composition and quality ranges for edible oils.
12. FAO-WHO Codex Alimentarius. General standards for edible fats and oils.
13. EU Commission Implementing Regulation 2019/1604 of 27 September 2019.