Colorful, but hazardous: forbidden dyes

Importance of the Topic

Many synthetic azo dyes such as Sudan Red I–IV, Butter Yellow and Para Red are illegally used to color food products despite health hazards. Sensitive and reliable analytical methods are crucial for routine screening and regulatory control to protect consumers and ensure compliance with food safety standards.

Objectives and Study Overview

This study aimed to develop a rapid, robust and selective single-quadrupole LC/MS method for the determination of Sudan Red I–IV, Butter Yellow and Para Red in various food matrices, including spices and meat products. Key goals were to achieve low detection limits, minimal sample preparation, and short analysis times suitable for high-throughput testing.

Methodology and Instrumentation

Sample Preparation:

• Weigh 1 g of food sample (e.g., spice or sausage).
• Extract with acetonitrile for 30 minutes.
• Filter aliquot directly into LC vial; dilute if analyte concentrations are high.

Chromatographic Separation and MS Detection:

• Column: Shimadzu Pathfinder® AS Silica 100, 50 × 2.1 mm, 3.5 µm.
• Mobile phase: Gradient of water (A) and methanol (B), starting at 65% B, rising to 100% B within 3 min, total run time 7 min; flow 200 µL/min; column temperature 55 °C.
• Detector: Shimadzu LCMS-2010 single-quadrupole mass spectrometer.
• Ionization: Atmospheric pressure chemical ionization (APCI) in positive mode; optional negative mode for confirmatory analysis.
• Scan and SIM masses: Sudan I (249 m/z), II (277 m/z), III (353 m/z), IV (381 m/z), Butter Yellow (226 m/z), Para Red (294 m/z).

Main Results and Discussion

The optimized method achieved baseline separation of all six dyes within seven minutes. Matrix-matched calibration demonstrated linear responses from 0.1 to 2.0 mg/L. Extrapolated detection limits in spice mixtures were 5–20 µg/kg for most dyes, sufficient for regulatory screening. No significant ion suppression was observed in spiked samples. Larger injection volumes or modified extraction protocols can further lower detection limits.

Benefits and Practical Applications

• Rapid throughput: under 10 minutes per sample.
• Simple sample preparation: single-step extraction and filtration.
• High selectivity and sensitivity: APCI-MS detection minimizes interferences.
• Broad applicability: suitable for spices, chili products, meat matrices and other colored foods.

Future Trends and Potential Uses

Future developments may include integration of high-resolution mass spectrometry for simultaneous screening of additional contaminants, miniaturized extraction techniques for enhanced sensitivity, and automated sample handling to further increase laboratory productivity. Negative-ion APCI can serve as a confirmatory tool for ambiguous positive-mode findings.

Conclusion

The presented single-quadrupole LC/MS method combining semi-micro HPLC and APCI offers a fast, reliable and cost-effective tool for routine monitoring of forbidden azo dyes in food products. Its robustness and low detection limits meet current regulatory requirements and support quality assurance in food safety laboratories.