Determination of Triphenylmethane Dyes and Their Metabolites in Shrimp Using QuEChERS Extraction and the ACQUITY UPLC H-Class System with Xevo TQD

Significance of the topic

The widespread use of triphenylmethane dyes such as malachite green, crystal violet and brilliant green in aquaculture poses potential health risks when residues accumulate in seafood. Although banned in many countries, these compounds and their colorless leuco-metabolites persist in market products, creating a need for sensitive monitoring methods to ensure food safety and regulatory compliance.

Objectives and study overview

This study aims to develop a rapid, efficient and highly sensitive method for simultaneous determination of three triphenylmethane dyes and two of their metabolites in shrimp using a modified QuEChERS extraction combined with UPLC-MS/MS analysis.

Methodology and instrumentation used

The sample preparation employed a modified QuEChERS protocol: 10 g homogenized shrimp extracted with acidified acetonitrile, salt partitioning using magnesium sulfate and sodium acetate, followed by dispersive SPE cleanup with PSA and C18 sorbents. Chromatographic separation used a Waters ACQUITY UPLC H-Class system equipped with a BEH C18 column (2.1 × 100 mm, 1.7 μm) at 40 °C, a water/ammonium acetate mobile phase (pH 4) and acetonitrile gradient at 0.3 mL/min over 6 min. Mass spectrometric detection utilized a Xevo TQD in positive electrospray mode with multiple reaction monitoring, acquiring two transitions per analyte. Deuterated leuco-standards (LMG-D5 and LCV-D6) were evaluated as internal standards.

Main results and discussion

All five analytes demonstrated excellent linearity (R2 > 0.998) over 0.05–40 ppb, with limits of quantification well below regulatory thresholds (1–2 μg/kg). Matrix effects in shrimp were minimal (slope ratios 0.95–1.19), indicating effective cleanup. Recovery experiments using internal standard correction yielded 104–106% for LMG and LCV. Parent dyes without specific internal standards showed lower recoveries (33–83%) but remained detectable at regulatory limits. The high sensitivity of the Xevo TQD eliminated the need for additional concentration steps, streamlining sample throughput and lab efficiency.

Benefits and practical applications

• Rapid sample preparation with simple QuEChERS extraction and cleanup.
• Simultaneous quantification of multiple dyes and metabolites in under 6 minutes.
• Detection limits below FDA and EU requirements without concentration steps.
• Reduced matrix interferences and high reproducibility using deuterated internal standards.

Future trends and potential applications

Extending this approach to other seafood matrices and environmental samples can broaden monitoring capabilities. Integration of high-resolution mass spectrometry or automation in sample handling may further improve selectivity and throughput. Development of green extraction solvents and on-site screening tools could support regulatory enforcement and public health surveillance.

Conclusion

The combination of modified QuEChERS extraction with UPLC-MS/MS on the Waters ACQUITY H-Class and Xevo TQD provides a robust, fast and sensitive workflow for monitoring triphenylmethane dyes in shrimp. The method meets regulatory performance criteria with minimal matrix effects and high recoveries, offering a practical solution for routine seafood safety testing.

References

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