Quantitative analysis of illegal dyes in eggs using LC/MS/MS

Importance of the Topic

Egg yolk color is a key indicator of quality, freshness and consumer acceptance. To achieve the preferred yellow-orange hue, some producers add synthetic dyes to poultry feed. These unauthorized additives can remain as residues, posing potential health hazards. Sensitive and reliable detection of illegal dyes in eggs is crucial for food safety, consumer protection and regulatory enforcement.

Objectives and Study Overview

This study aimed to develop and validate a streamlined analytical method for the simultaneous quantitation of four Sudan dyes and Para Red in egg yolk. By coupling a rapid sample preparation protocol with ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS), the authors sought to achieve low parts-per-billion (ppb) detection levels, high specificity against complex egg matrices and robust repeatability.

Methodology and Instrumentation

Sample Preparation:

• Weighed 1 g egg yolk and mixed with 9 mL water and 10 mL acetonitrile.
• Added QuEChERS extraction salts (EN 15662 packet), vortexed, then centrifuged at 4000 rpm, –10 °C.
• Collected supernatant, performed cleanup on C-18 SPE cartridge, eluted with acetonitrile.
• Prepared matrix-matched calibration standards (1–100 ppb).

Instrumental Analysis:

• UHPLC Nexera with Shim-pack XR-C8 column (75×3 mm, 2.2 μm), flow 0.4 mL/min, 40 °C.
• Mobile phases: 0.1% formic acid in water (A) and in acetonitrile (B) with a 10-min gradient ramping B from 50% to 100%.
• LCMS-8040 triple quadrupole MS with electrospray ionization, rapid polarity switching and MRM transitions optimized for each dye.

Main Results and Discussion

The method achieved a limit of quantitation of 1 ppb for all five dyes in both aqueous and egg yolk matrices. Calibration curves exhibited excellent linearity (r2 > 0.99) and accuracy ranged from 90% to 110% across the concentration range. Background interference from the egg matrix was effectively minimized, enabling high sensitivity and specificity in multi-residue analysis.

Benefits and Practical Applications

By integrating a fast QuEChERS-based extraction with UHPLC-MS/MS, the method offers laboratories a cost-effective, high-throughput solution for routine surveillance of illegal dyes in eggs. Its low detection limits and strong repeatability support regulatory compliance, quality control in egg production and protection of public health.

Future Trends and Possibilities

Potential extensions include adapting the workflow to other food matrices (e.g., dairy, spices), expanding the target list to additional lipophilic contaminants and leveraging high-resolution MS for non-target screening. Automation of sample prep and incorporation of data processing software can further increase throughput and robustness.

Conclusion

A highly sensitive, reliable UHPLC-MS/MS method was established for simultaneous quantitation of Sudan I–IV and Para Red in egg yolk at low ppb levels. The approach demonstrates strong linearity, accuracy and minimal matrix interference, providing an effective tool for food safety laboratories.

References

• Marta Piatkowska, Piotr Jedziniak, Jan Zmudzki, "Residues of illegal dyes in eggs," Bull. Vet. Inst. Pulawy, vol. 58, pp. 247–253, 2014.
• Javier Dominguez-Alvarez et al., "Advanced QuEChERS cleanup for food analysis," J. Chromatogr. A, vol. 1278, pp. 166–174, 2013.