Highly sensitive and rapid analysis of synthetic dyes in sea food by LC/MS/MS

Importance of the Topic

Rapid and sensitive detection of banned synthetic dyes such as malachite green and crystal violet in seafood is critical for food safety and regulatory compliance. These compounds, widely used for their antimicrobial properties in aquaculture, pose health risks to consumers and are subject to zero-tolerance policies in major markets. Effective analytical methods ensure consumer protection and support quality control in the seafood industry.

Objectives and Study Overview

This study aimed to develop and validate an ultra-sensitive LC/MS/MS method for simultaneous quantitation of malachite green, leucomalachite green, crystal violet and leucocrystal violet in seafood samples. The approach integrates streamlined sample preparation, solid-phase extraction cleanup and high-throughput analysis using Shimadzu’s UHPLC-MS/MS instrumentation.

Methodology and Instrumentation

Sample Preparation

• Shrimp tissue was homogenized and extracted with acidified acetonitrile containing ascorbic acid to stabilize redox-sensitive leuco dyes.
• Extracts were centrifuged, diluted with McIlvaine’s buffer and cleaned up using strong cation-exchange SPE cartridges.
• Dyes were eluted with methanol enriched with triethylamine and formic acid, then injected into the LC/MS/MS system.

Instrumental Setup

• UHPLC System: Shimadzu Nexera with a Shim-pack GISS column (75×3.0 mm, 3 µm).
• Mass Spectrometer: Shimadzu LCMS-8045 triple quadrupole with heated ESI probe for enhanced desolvation.
• Chromatographic Conditions: 0.4 mL/min flow, gradient from 30% to 95% organic phase over 3 minutes, 40 °C column temperature.
• MRM Transitions: Optimized precursor-to-product ion pairs for each dye with collision energies determined via auto-optimization.

Main Results and Discussion

The method achieved baseline separation of all four target dyes within a 6.5 minute run time. Calibration curves prepared in solvent and matrix-matched standards exhibited excellent linearity (r2 > 0.99) over 0.05–0.5 ppb. Method detection limits reached 0.05 ppb, with recoveries between 80% and 112% at the lowest fortification level. Overlay of blank, control and spiked samples demonstrated minimal matrix interference, confirming the effectiveness of SPE cleanup and the sensitivity of the heated ESI source.

Benefits and Practical Applications

This streamlined protocol offers:

• High throughput analysis with rapid chromatography and fast polarity switching.
• Robust stabilization of unstable leuco dye metabolites through antioxidant addition.
• Enhanced sensitivity and reproducibility for trace-level quantitation in complex seafood matrices.

It supports regulatory monitoring laboratories, aquaculture quality control and research applications requiring reliable pesticide and dye residue testing.

Future Trends and Possibilities

Advances in ion source design and high-resolution instrumentation may further lower detection limits and expand the range of detectable contaminants. Automation of sample preparation workflows and incorporation of high-capacity SPE formats could increase analytical throughput. Emerging ambient ionization techniques may also enable direct analysis of seafood surfaces without extensive extraction.

Conclusion

The presented LC/MS/MS method enables rapid, sensitive and reliable determination of banned synthetic dyes in seafood at sub-ppb levels. The combination of ascorbic acid stabilization, SPE cleanup and a high-performance UHPLC-MS/MS system yields a practical solution for regulatory compliance and food safety monitoring.

References

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• [2] Lopez-Gutierrez et al. Anal. Methods. 5:3434–3449, 2013.
• [3] J.C. Hashimoto et al. J. AOAC Int. 95:913–922, 2012.