LC/MS/MS Analysis of Cylindrospermopsin and Anatoxin-a in Drinking Water Using US EPA Method 545

Significance of the topic

Monitoring cyanobacterial toxins in drinking water is essential to protect public health and comply with regulatory requirements. Cylindrospermopsin and anatoxin-a present acute and chronic risks, demanding sensitive, accurate analytical methods. EPA Method 545 provides a standardized protocol for quantifying these toxins. Implementing a robust LC/MS/MS workflow enhances data quality and supports regulatory monitoring programs.

Objectives and Study Overview

The study aimed to demonstrate the performance of an Agilent 1290 Infinity LC coupled to an Agilent 6460 Triple Quadrupole LC/MS system with Jet Stream technology for EPA Method 545.
Key objectives:

• Achieve required precision and accuracy for anatoxin-a and cylindrospermopsin.
• Determine Lowest Calculated Minimum Reporting Limits (LCMRLs) equal to or below method targets.
• Minimize sample injection volume to reduce maintenance.

Methodology

Sample preparation involved collecting 10 mL drinking water with preservatives (ascorbic acid and sodium bisulfate), spiking internal standards, and filtering 1 mL through 0.2 µm PVDF filters.
Chromatographic separation used a C18-Ether column (3 × 150 mm, 3 µm) at 30 °C with a gradient of 0.15 % acetic acid in water and methanol (0.3 mL/min). Multiple reaction monitoring (MRM) transitions were optimized for each analyte and internal standard.

Instrumentation used

• Agilent 1290 Infinity Binary Pump, Autosampler, Column Compartment
• Agilent 6460A Triple Quadrupole LC/MS with Jet Stream ESI in positive mode
• Agilent Polaris C18-Ether column

Main Results and Discussion

Calibration was assessed over two ranges (0.030–0.300 µg/L and 0.005–0.050 µg/L) with R² ≥ 0.996. Recoveries for spiked tap and reagent water at 0.100 µg/L ranged 90–106 % with RSDs ≤ 10 %. No carryover was observed below the reporting limits following highest-level injections. LCMRLs calculated at 0.018 µg/L for anatoxin-a and 0.038 µg/L for cylindrospermopsin met or exceeded EPA Method 545 requirements.

Benefits and Practical Applications

• High sensitivity with only 20 µL injection volume reduces source fouling and maintenance.
• Consistent precision and accuracy simplify compliance with regulatory monitoring.
• Rapid run times and straightforward sample prep support routine laboratory workflows.

Future Trends and Potential Applications

Advances may include expanding the method to additional cyanotoxins, integrating automated sample handling, and leveraging high-resolution MS for broader screening. Real-time monitoring in field-deployable platforms and data integration for early warning systems represent emerging opportunities.

Conclusion

The Agilent LC/MS/MS configuration reliably meets EPA Method 545 performance criteria for anatoxin-a and cylindrospermopsin in drinking water. The workflow delivers low reporting limits, robust quantitation, and reduced maintenance, making it suitable for regulatory and research laboratories.

References

1. US EPA, Cyanobacterial Harmful Algal Blooms (CyanoHABs), 2014.
2. EPA Method 545: Cylindrospermopsin and Anatoxin-a in Drinking Water by LC/ESI-MS/MS, 2013.
3. S. D. Winslow et al., Environ. Sci. Technol. 40, 281–288, 2006.