Modifying AOAC Method 996.06 for FAME Analysis in Foods: Faster Throughput Using Hydrogen Carrier Gas

Significance of the Topic

Gas chromatography analysis of fatty acid methyl esters (FAMEs) plays a critical role in food quality assessment, nutritional labeling, and consumer safety. With regulatory bodies mandating accurate fat profiles—including total, saturated, and unsaturated fractions—laboratories must balance analytical rigor with operational efficiency. Rising helium costs and limited supply have driven the search for alternative carrier gases and faster methods.

Study Objectives and Overview

The primary goal was to adapt AOAC Method 996.06 for FAME analysis by replacing helium with hydrogen carrier gas and optimizing oven temperature programming. Specific objectives included:

• Reducing total analysis time by at least 50%
• Maintaining or improving baseline resolution of all 37 standard FAME components
• Demonstrating high calibration linearity and reproducibility
• Lowering operating costs through hydrogen use

Methodology and Instrumentation

A Shimadzu Nexis GC-2030 equipped with split/splitless inlet, flame ionization detector (FID), and automated Gas Selector was employed. Key modifications included:

• Carrier gas switched from helium (18 cm/s linear velocity) to hydrogen (35 cm/s)
• Oven program adjusted from 100 °C (4 min) → 3 °C/min to 240 °C (15 min) to 150 °C (2 min) → 4 °C/min to 220 °C → 2 °C/min to 240 °C (8 min)
• Shimadzu hydrogen sensor and gas filters ensured safety and purity

Calibration used a four-level quadratic fit (not forced through zero) on a 37-component FAME mix. Each standard level was analyzed in triplicate.

Results and Discussion

Compared to the original 65+ minute AOAC run, the hydrogen-based method completed all 37 analytes in under 32 minutes, achieving over 50% time reduction. Key findings:

• Baseline resolution of coeluting peaks (e.g., C20:3 and C20:4) improved, with all resolutions >1.5 (USP criteria)
• Calibration curves showed R2 >0.9995 for every analyte
• Area %RSD values remained below 2.1% across the concentration range, demonstrating excellent reproducibility
• Elimination of high split ratios reduced hydrogen consumption to negligible operating cost compared to helium

Practical Benefits and Applications

The modified method delivers substantial advantages for food testing laboratories:

• Increased sample throughput enables higher daily capacity
• Lower operating expenses by using in-house generated hydrogen instead of bottled helium
• Enhanced method robustness and reproducibility support QA/QC and regulatory compliance
• Flexibility to adopt higher linear velocities without sacrificing chromatographic efficiency

Future Trends and Potential Applications

Looking ahead, hydrogen carrier gas adoption is poised to grow, driven by sustainability and cost factors. Opportunities include:

• Integration with automated sample preparation and data processing workflows
• Extension to other high-polarity or thermally labile analytes
• Development of portable hydrogen generators and safety modules for decentralized testing
• Application of advanced column chemistries to further boost speed and resolution

Conclusion

By leveraging hydrogen’s favorable Golay curve characteristics and revising the oven program, the AOAC Method 996.06 was transformed into a fast, cost-effective protocol without compromising analytical performance. This approach supports high-throughput, low-cost FAME analysis, aiding food laboratories in meeting regulatory and commercial demands.

Reference

Official Methods of Analysis, AOAC INTERNATIONAL, Method 996.06: Fat (Total, Saturated, and Monounsaturated) in Foods.