Analysis of Malachite Green Using a Triple Quadrupole LC/MS/MS [LCMS-8030]

Significance of the Topic

The synthetic dye malachite green and its metabolite leucomalachite green have been widely used in the textile industry and as an antimicrobial agent in aquaculture, despite concerns about their carcinogenicity, genotoxicity, and persistence in food products. Regulatory bans across major markets demand sensitive and reliable analytical methods to monitor these compounds in fish and seafood. The development of robust LC–MS/MS protocols is therefore critical for ensuring food safety and regulatory compliance.

Objectives and Study Overview

This work aimed to establish and validate a quantitative multiple-reaction monitoring (MRM) method using the Shimadzu LCMS-8030 triple quadrupole system for simultaneous determination of malachite green and leucomalachite green in salmon extracts. Spiked-recovery experiments were performed at 10 ng/mL to assess method performance under realistic matrix conditions.

Methodology

Optimization focused on selecting product ions and collision energies for the analytes and their stable isotope-labelled internal standards (malachite green-d5 and leucomalachite green-d6). Calibration curves were constructed over 0.5–50 ng/mL, demonstrating excellent linearity. Salmon extracts were prepared following the Japanese Ministry of Health, Labour, and Welfare protocol and spiked at 10 ng/mL prior to LC–MS/MS analysis.

Instrumentation

• Mass spectrometer: Shimadzu LCMS-8030 (triple quadrupole, ESI-positive mode)
• Column: Shim-pack XR-ODS II (75 mm L × 2.0 mm I.D., 2.2 µm)
• Mobile phases: (A) 10 mmol/L ammonium acetate in water; (B) acetonitrile; gradient from 10 % B to 100 % B over 5 min
• Flow rate: 0.2 mL/min; injection volume: 2 µL; column temperature: 40 °C
• Ion source: probe voltage +4.5 kV; nebulizing gas 3 L/min; drying gas 10 L/min; DL temp. 250 °C; heating block temp. 400 °C

Results and Discussion

Product ion spectra and calibration curves yielded correlation coefficients of 0.9984 for malachite green and 0.9996 for leucomalachite green. Chromatographic runs (8 min total) showed baseline resolution and minimal matrix interference. Spiked-recovery trials at 10 ng/mL delivered mean recoveries of 115.9 % (RSD 1.41 %) for malachite green and 112.5 % (RSD 1.39 %) for leucomalachite green, confirming method accuracy and precision.

Practical Benefits and Applications

• Sensitive detection limits down to 0.5 ng/mL
• Rapid analysis suitable for high-throughput monitoring
• Reliable quantitation in complex food matrices
• Compliance with international regulatory standards

Future Trends and Applications

Emerging directions include incorporation of high-resolution mass spectrometry for screening non-targeted dye residues, further automation of sample preparation workflows, and expansion to additional aquaculture species and environmental samples. Sustainable and green analytical practices may also drive solvent and reagent optimization.

Conclusion

The developed LC–MS/MS method on the LCMS-8030 provides a robust, accurate, and high-throughput solution for monitoring malachite green and its metabolite in fish matrices, supporting food safety and regulatory surveillance efforts.

References

• Ministry of Health, Labour and Welfare (Japan). Malachite Green Analytical Method.