Selectivity Comparison of Agilent Poroshell 120 Phases in the Separation of Butter Antioxidants

Significance of the Topic

Unsaturated fats in foods are prone to oxidation leading to rancidity and nutritional loss. Synthetic antioxidants such as ascorbyl palmitate and phenolic compounds are added to inhibit oxidation but can pose health risks at high levels. Regulatory limits necessitate robust analytical methods for quantifying food antioxidants, particularly in complex matrices like butter.

Objectives and Study Overview

This study compared selectivity of five Agilent Poroshell 120 superficially porous LC stationary phases (EC-C18, SB-C18, SB-Aq, Bonus-RP, Phenyl-Hexyl) for separating nine common synthetic antioxidants. The goal was to optimize a fast, accurate HPLC method on the Poroshell 120 SB-Aq phase to quantify these compounds in butter samples.

Methodology and Instrumentation

• Instrumentation: Agilent 1290 Infinity LC system with binary pump, autosampler, thermostatted column compartment and diode-array detector.
• Columns: Agilent Poroshell 120, 3.0×100 mm, 2.7 µm, phases EC-C18, SB-C18, SB-Aq, Bonus-RP, Phenyl-Hexyl.
• Sample preparation: Butter extracted with n-hexane/saturated acetonitrile, filtered, concentrated to 2 mL with isopropanol.
• Mobile phase: Water with 1.5% acetic acid and acetonitrile, gradient optimized on SB-Aq.
• Detection: UV at 280 nm, injection volume 2 µL.

Main Results and Discussion

• Selectivity tests revealed SB-Aq provided baseline separation of nine antioxidants in ~8.5 min using acetonitrile/1.5% acetic acid, outperforming other phases in resolution and peak shape.
• Method exhibited excellent linearity (R2>0.9992) over 0.2–10 ppm and LODs ≤ 0.1 ppm (≈0.2 mg/kg in butter).
• Retention time reproducibility was high (RSD<0.1% for eight consecutive injections).
• Analysis of five commercial butter samples and 1 ppm spiked controls demonstrated minimal matrix interferences; critical peaks were well resolved.

Benefits and Practical Applications

The method reduces analysis time to 15 min, one-third that of traditional 5 µm columns, while retaining high resolution at lower backpressure. It is suitable for QC laboratories in the food industry for routine monitoring of antioxidant additives in dairy fats.

Future Trends and Opportunities

• Integration with tandem mass spectrometry could lower detection limits and enhance selectivity in complex matrices.
• Expansion to other lipid-based foods and antioxidants via tailored Poroshell phases.
• Automation of sample preparation for high-throughput screening in industrial QA/QC settings.

Conclusion

Agilent Poroshell 120 SB-Aq provides an efficient, robust, and rapid HPLC platform to separate and quantify nine synthetic antioxidants in butter. The method meets regulatory requirements with high sensitivity, reproducibility, and throughput.

References

1. Li Tao et al. (2003) Food Research and Development (Japan), 24, 23.
2. Vanhoenacker G, David F, Sandra P. (2009) Ultrafast analysis of synthetic antioxidants in vegetable oils using the Agilent 1290 Infinity LC system. Application Note 5990-4378EN. Agilent Technologies.
3. Anonymous. (2002) Method for the determination of antioxidants in oils and fats for import and export – liquid chromatography. SN/T 1050-2002. China Standard.