Speciated Isotope Dilution for the Determination of Methylmercury in Tuna Fish by GC-MS

Significance of the Topic

Methylmercury is a highly toxic and bioaccumulative form of mercury that poses significant health risks through the food chain. Reliable trace-level detection in complex matrices like fish is essential for food safety, environmental monitoring, and regulatory compliance. Speciated isotope dilution offers enhanced accuracy and precision, addressing challenges in analyte recovery and matrix effects.

Objectives and Scope of the Study

This study aimed to develop and validate a gas chromatography–mass spectrometry (GC-MS) method employing speciated isotope dilution for the quantitation of methylmercury in tuna samples. The goal was to achieve a single-injection workflow that eliminates lengthy calibrations, standard additions, and recovery corrections while maintaining high reliability.

Methodology

• Sample Preparation: 0.4 g of homogenized tuna was spiked with 201Hg-enriched methylmercury. Extraction was carried out with saturated NaCl and HCl under mechanical shaking for 5 hours.
• Derivatization: Ethylation of methylmercury was performed in situ by adding sodium tetraethyl borate in a pH 5.3 acetate buffer.
• Headspace SPME: A DVB/CAR/PDMS fiber was exposed to the sample headspace for 15 minutes with stirring to extract the volatile derivative.
• Isotope Ratio Correction: Molecular ions corresponding to Hg isotopes 198–202 were monitored. Contributions from 13C to m+1 clusters were corrected using established equations to ensure accurate isotope ratio measurements.

Used Instrumentation

• Gas Chromatograph: Agilent 6890N with split/splitless injector and HP-5MS capillary column (30 m × 0.25 mm id × 0.25 µm).
• Mass Spectrometer: Agilent 5973 MSD quadrupole with electron impact ionization at 70 eV; transfer line at 280 °C and ion source at 230 °C; helium carrier gas at 1.2 mL/min.
• SPME Device: Manual holder and replaceable DVB/CAR/PDMS fibers (50/30 µm) for headspace extraction.

Main Results and Discussion

The method was validated with the certified reference material BCR-464 (tuna). Three independent spikes and triplicate GC-MS injections yielded an average methylmercury concentration of 5.05 ± 0.04 µg/g (RSD 0.71 %), in excellent agreement with the certified value of 5.12 ± 0.16 µg/g. Isotope dilution effectively compensated for potential losses and matrix interferences, enabling accurate quantitation with a single injection.

Benefits and Practical Applications

• High accuracy and precision through isotope dilution calibration.
• Rapid single-injection analysis without external calibration or recovery corrections.
• Reduced sample handling and simplified workflow suitable for routine laboratories.
• Applicable to food safety monitoring, environmental assessments, and regulatory testing.

Future Trends and Potential Applications

• Automation of SPME sampling for increased throughput.
• Extension of the approach to other organomercury species and complex matrices.
• Integration with high-resolution mass spectrometry for enhanced selectivity.
• Development of portable GC-MS or field-deployable systems for on-site screening.

Conclusion

The developed GC-MS method with speciated isotope dilution provides a robust, accurate, and streamlined approach for methylmercury analysis in fish. Its simplicity and precision make it an excellent choice for routine applications in food safety and environmental laboratories.

References

• Centineo G., García Alonso J. I. Speciated isotope dilution for the determination of methylmercury in tuna fish by GC-MS. Agilent Application Note, 2008.