The Analysis of Sudan and other Azo Dyes in Spices Using Liquid Chromatography - Tandem Quadrupole Mass Spectrometry

Significance of the Topic

Azo dyes have been illicitly added to spices to intensify their color, posing health risks and regulatory challenges. Reliable analytical approaches are essential to detect these banned colorants and ensure food safety.

Objectives and Study Overview

This study presents a single-run method for the simultaneous quantitation of 11 azo dyes, including seven Sudan dyes, Rhodamine, Dimethyl Yellow, Para Red, and Orange II. The method enables rapid screening in spice matrices, focusing on paprika, with a total run time of 14 minutes.

Methodology

Sample preparation involves direct extraction of spices with acetonitrile after spiking stable isotope-labelled internal standards. No extensive cleanup is required. Chromatography is performed on an ACQUITY UPLC I-Class system using a BEH C18 column (2.1 x 100 mm, 1.7 µm) at 40 °C. The mobile phase gradient employs 0.1% formic acid/5 mM ammonium formate in water (A) and 0.1% formic acid in methanol (B), ramping from 25% to 95% B over 14 minutes. Mass spectrometry is conducted on a Xevo TQ-S micro in positive ESI mode, using two MRM transitions per compound and automatic dwell time optimization.

Used Instrumentation

• ACQUITY UPLC I-Class with FTN Sample Manager
• ACQUITY UPLC BEH C18 column (2.1 x 100 mm, 1.7 µm)
• Xevo TQ-S micro Triple Quadrupole Mass Spectrometer
• MassLynx and TargetLynx software

Main Results and Discussion

• Chromatography achieved baseline separation of isobaric dyes, e.g., Sudan Red B and Sudan IV, with sharp peak shapes.
• Sensitivity allowed detection at matrix-matched levels down to 0.125 mg/kg, below regulatory action limits.
• Specificity and selectivity were confirmed with no interfering peaks, except trace Rhodamine below 0.5% of the lowest calibrator.
• Calibration curves displayed excellent linearity (R2 > 0.996) with residuals mostly under 10%.
• Method trueness showed recoveries between 93.8% and 115.2% across concentration levels.
• Precision was robust, with repeatability (RSDr) and reproducibility (RSDRL) both under 7.7%.

Benefits and Practical Applications

• Simultaneous analysis of a broad panel of azo dyes streamlines workflows.
• High sensitivity and selectivity support reliable quantitation below action limits.
• Simplified sample preparation without lengthy clean-up accelerates throughput.
• Applicable to regulatory compliance and routine quality control in spice industries.

Future Trends and Potential Applications

Advancements may include:

1. Extension to additional food matrices such as oils and sauces.
2. Integration with high-throughput platforms and robotic sample handling.
3. Development of wider multiresidue methods combining dyes with other contaminants.
4. Adoption of portable or field-deployable MS systems for on-site screening.

Conclusion

The described UPLC-MS/MS method offers a fast, reliable, and sensitive solution for detecting illegal azo dyes in spices. Its high throughput and robust performance make it suitable for routine monitoring and regulatory enforcement well below established action thresholds.

References

1. Regulation (EC) 1333/2008 of the European Parliament and of the Council on food additives, OJ L 354, 31.12.2008, p.16–33.
2. Galvin-King P et al. Herb and spice fraud: the drivers, challenges and detection. Food Control 2018;88:85–97.
3. Oplatowska-Stachowiak M, Elliott C. Food colors: existing and emerging food safety concerns. Crit Rev Food Sci Nutr 2017;57(3):524–548.
4. Standing Committee on the Food Chain and Animal Health (SCoFCAH) Summary Record SANCO–D.1(06)D/411990, 23 June 2006.
5. European Commission Decision 2002/657/EC implementing Council Directive 96/23/EC, OJ L 221, 17.8.2002, p.8–36.
6. EFSA Guidance Document SANTE/12682/2019 on analytical quality control and method validation procedures for pesticide residues in food and feed.