ONE-STEP MICROWAVE-ASSISTED EXTRACTION AND DERIVATIZATION FOLLOWED BY COMPREHENSIVE TWO-DIMENSIONAL CHROMATOGRAPHY COUPLED WITH FLAME IONIZATION DETECTOR TO FAMEs ANALYSIS IN COMPLEX FOOD MATRICES

Importance of the Topic

Analysis of fatty acid methyl esters (FAMEs) in complex food matrices is critical for nutritional labeling, quality control, and understanding health impacts of dietary fats. Traditional extraction and derivatization methods are often time-consuming, solvent-intensive, and may suffer from incomplete extraction or co-elution of isomers. Implementing microwave-assisted extraction and derivatization (MAED) coupled with comprehensive two-dimensional gas chromatography and flame ionization detection (GC×GC-FID) offers a rapid, greener, and more sensitive alternative.

Objectives and Study Overview

This study aimed to:(ol)

Evaluate MAED as an alternative to official AOCS methods Ce 2b-11 and Ce 2c-11 for FAME preparation.

Compare the reliability and quantitative performance of one-dimensional (1D) GC against two-dimensional (2D) GC×GC for FAME analysis in complex food samples.

Methodology and Instrumentation

Sample Preparation and MAED:

• Sample: 0.5 g of homogenized food matrix (e.g., mussel tissue).
• Reagents: 10 mL of HCl in methanol and 25 mL of cyclohexane in a closed microwave vessel.
• Conditions: 120 °C for 15 minutes, single-step extraction and derivatization.

Official AOCS Methods:

• Ce 2c-11: Sequential acid–alkali hydrolysis (NaOH, BF₃) and hexane extraction.
• Ce 2b-11: Alkaline hydrolysis (NaOH), followed by BF₃ derivatization and hexane extraction.

GC×GC-FID Analysis:

• Flow modulation system for comprehensive 2D separation without cryogens.
• Column set: SepSolve 1D-FAMEs (20 m × 0.18 mm × 0.1 μm) as the non-polar first dimension, SepSolve 2D-FAMEs (5 m × 0.25 mm × 0.1 μm) as the polar second dimension.
• Detector: Flame ionization detector (FID) for quantification of FAME classes.

Main Results and Discussion

• MAED performance matched AOCS Ce 2b-11 and Ce 2c-11 for total saturated (SFA), monounsaturated (MUFA), and polyunsaturated fatty acids (PUFA) with deviations below 2%.
• Precision: Intra-day RSD <1.2% and inter-day RSD <3.1% for standard FAME mix.
• GC×GC provided: structured chromatograms, enhanced resolution of co-eluting isomers, and 5- to 10-fold sensitivity gains over 1D GC.
• Quantitative results for 82 identified peaks and over 100 detected components in mussel samples were equivalent between 1D and 2D approaches, confirming reliability of 2D quantification.
• Greenness assessment: MAED reduced solvent use, energy consumption, and processing time (12 samples per 15 minutes), outperforming conventional methods in sustainability metrics.

Benefits and Practical Applications

• High throughput: Up to 48 samples per hour with minimal manual handling.
• Green chemistry: Reduced solvent volumes and energy requirements, elimination of cryogenic cooling in GC×GC.
• Automation potential: Compatible with robotic sample loaders and batch processing.
• Robust separation: Flow modulation and GC×GC avoid the need for mass spectrometry, lowering instrument costs and complexity.

Future Trends and Applications

Further developments may include integration of MAED-GC×GC workflows with automated data processing and machine learning to accelerate fatty acid profiling in diverse matrices. Miniaturized reactors and alternative green solvents could enhance sustainability. Expansion to targeted lipidomics and coupling with high-resolution mass spectrometry will broaden analytical capabilities in food science and biomedical research.

Conclusion

The one-step MAED protocol combined with GC×GC-FID offers a reliable, rapid, and environmentally friendly alternative to official AOCS methods for FAME analysis. Comprehensive 2D chromatography ensures accurate quantification and superior resolution of complex lipid profiles without requiring cryogenic modulation or MS detection.

References

• Ferrara D., Beccaria M., Cordero C. E. I., Purcaro G., Advances in Sample Preparation, 4 (2022) 100039.
• Ferrara D., Beccaria M., Cordero C. E. I., Purcaro G., Comprehensive comparison of fatty acid methyl ester profiles in food matrices using MAED and GC×GC-FID, Sample Prep. 2024, 100124.