LC-MS Method Using Cloud Point Extraction for the Determination of Permitted and Banned Azo Colors in Liquid, Semi-liquid and Solid Food Matrices

Importance of the Topic

The accurate determination of food colorants is crucial for ensuring compliance with regulatory limits and for protecting public health. Both permitted water-soluble dyes such as sunset yellow and allura red, and banned fat-soluble azo dyes like Sudan I–IV can pose carcinogenic risks when used illegally. Developing a single, robust method that can detect multiple dye classes across diverse food matrices addresses industry and regulatory demand for efficient monitoring tools.

Objectives and Overview of the Study

This study introduces a cloud point extraction coupled with single quadrupole LC–MS method for simultaneous analysis of 14 regulated and banned azo colors in liquid, semi-liquid, and solid food samples. The main goals were to optimize extraction, achieve low detection limits, and validate the procedure for matrices such as wine, paprika sauce, and chili powder.

Methodology and Instrumentation

The procedure combines sample homogenization with non-ionic surfactant–based extraction and LC–MS detection. Key steps include:

• Fortification of 0.5 g homogenized sample with d6-Sudan IV internal standard.
• Addition of 3 mL 10% Triton X-114 and 1 mL 1 M ammonium acetate; volume adjusted to 10 mL with water.
• Shaking for 10 min followed by heating at 50 °C for 30 min to induce phase separation.
• Removal of the upper aqueous layer and reconstitution of the surfactant-rich phase in 10 mL acetone.
• Filtration through a 0.2 μm PTFE filter and injection into the LC–MS system.

Chromatographic separation was achieved on a Hypersil GOLD 150 × 2.1 mm column using a gradient of 0.1% formic acid in water and acetonitrile. Detection employed multi-ion monitoring in positive and negative electrospray ionization modes.

Instrumentation Used

• Thermo Fisher MSQ Plus single quadrupole LC–MS
• Accela 1250 pump and autosampler
• Hypersil GOLD 1.9 μm column
• Ultra-Turrax homogenizer and vortex shaker
• Büchi heater bath and centrifuge

Main Results and Discussion

The method demonstrated high specificity with retention time deviations within ±2.5% and confirmatory fragment ions for each dye. Calibration curves were linear over 0.3–25 mg/kg (fat-soluble) and 20–300 mg/kg (water-soluble) with R² > 0.985. Recoveries ranged between 60%–120% for most analytes, and intra-day and inter-day RSDs were below 20%. Limits of detection in all matrices were between 0.1 and 0.5 mg/kg, meeting regulatory action limits. Robustness testing confirmed method stability against variations in surfactant source, shaking time, extraction time, and temperature.

Benefits and Practical Applications

• Green extraction using biodegradable surfactant instead of organic solvents.
• Simultaneous analysis of multiple water- and fat-soluble dye classes.
• Applicable to diverse food types: beverages, sauces, and spices.
• Low detection limits enabling compliance with EU action limits.
• High-throughput compatible with routine laboratory workflows.

Future Trends and Potential Applications

Emerging directions include integration of cloud point extraction with high-resolution mass spectrometry for improved selectivity, miniaturized and automated extraction platforms for on-site testing, and incorporation of data-driven algorithms for rapid screening. Broadening the method scope to new synthetic dyes and adapting to real-time monitoring systems can further enhance food safety surveillance.

Conclusion

The presented cloud point extraction LC–MS method offers a fast, cost-effective, and environmentally friendly approach for simultaneous detection of permitted and banned azo colors across liquid, semi-liquid, and solid foods. Its validation performance supports its use in regulatory and quality control laboratories.

References

• Commission Decision 1994/36/EC on colors for foodstuffs. Official Journal L237, 1994.
• Title 21 CFR Part 73 and Part 74, Food and Drug Administration, USA.
• GB 2760-2007 Hygienic standards for uses of food additives. People’s Republic of China.
• IARC Monographs on the evaluation of carcinogenic risk of chemicals to man, Vol. 8, IARC, Lyon, 1975.
• Rapid Alert System for Food and Feed (RASFF) Annual Report 2009.
• Commission Decision 2005/402/EC on emergency measures regarding chili products. Official Journal L135, 2005.
• Commission Decision 657/2002/EC implementing Council Directive 96/23/EC on analytical method performance. Official Journal L221, 2002.