Highly Sensitive Quantitation of Microcystins and Nodularin in Water using the Triple Quad LCMS-8060NX in Accordance with EPA Method 544

Importance of the Topic

Cyanobacterial toxins such as microcystins and nodularin are potent hepatotoxins that threaten drinking water sources and public health worldwide. The prevalence of harmful algal blooms has increased due to nutrient enrichment, making sensitive and rapid monitoring essential for environmental safety.

Study Objectives and Overview

This study demonstrates the application of the Shimadzu LCMS-8060NX triple quadrupole mass spectrometer for the quantification of six microcystin variants and nodularin in water. The work follows EPA Method 544 guidelines, aiming to achieve lower limits of quantitation and faster analysis without the need for solid-phase extraction preconcentration.

Methodology

• Standards preparation: Stock solutions of six microcystins and nodularin were diluted to establish calibration curves over relevant concentration ranges.
• Chromatography: Separation was performed on a Shim-pack Velox SP-C18 column (2.1×100 mm, 2.7 μm) using a 0.1% formic acid water/acetonitrile gradient at 0.3 mL/min, 40 °C, and 10 μL injection volume.
• Detection: Electrospray ionization in positive mode with multiple reaction monitoring (MRM) transitions was employed for quantitation and confirmation.
• Calibration and LOQ determination: Calibration curves (0.5–500 μg/L) achieved R² > 0.99; LOQs were 0.5 μg/L (1.0 μg/L for MC-LF) based on S/N ≥ 10 and accuracy criteria.

Instrumentation Used

Shimadzu LCMS-8060NX triple quadrupole mass spectrometer coupled with a Shim-pack Velox SP-C18 column. LabSolutions Insight software was used for data acquisition and processing.

Main Results and Discussion

The optimized method achieved baseline chromatographic separation of seven cyanotoxins in an 8 min run time. Calibration curves displayed excellent linearity (R² > 0.99) across broad ranges. LOQs were more than ten times lower than those specified in EPA Method 544, enabling direct injection analysis. Spike recovery experiments at 8, 25, and 80 μg/L in water samples yielded accuracy within 88–108% and precision (%RSD) below 10%, meeting regulatory requirements.

Benefits and Practical Applications

• Significantly reduced sample preparation by eliminating SPE preconcentration.
• Short analysis time (8 min) enhances throughput for routine monitoring.
• Improved sensitivity supports detection of trace levels in drinking water.
• Robustness and reproducibility suitable for environmental QA/QC laboratories.

Future Trends and Opportunities

Advancements may include integration with automated sampling systems, expansion to additional cyanotoxin variants, and coupling with high-resolution MS for non-targeted screening. Method transfer to field-deployable platforms and further miniaturization could support real-time water quality monitoring.

Conclusion

The Shimadzu LCMS-8060NX method offers a rapid, sensitive, and reliable approach for quantifying microcystins and nodularin in water, exceeding EPA Method 544 performance without the need for preconcentration. This workflow enhances environmental monitoring capabilities and ensures compliance with health advisory limits.

References

• US EPA Method 544: Determination of Microcystins and Nodularin in Drinking Water by SPE and LC-MS/MS.
• WHO Guidelines for Drinking-Water Quality: Cyanobacterial toxins—Microcystins.
• EPA Drinking Water Health Advisories for Cyanotoxins.