Reduced injection volume applied to the quantitation of cylindrospermopsin and anatoxin-a in drinking water according to EPA Method 545

Significance of the topic

Cyanotoxins produced by harmful algal blooms are a growing concern for drinking water safety due to their acute neurotoxic and hepatotoxic effects. Regular monitoring of anatoxin-a and cylindrospermopsin is essential to protect public health and comply with regulatory programs such as the UCMR 4 under the US Safe Drinking Water Act.

Objectives and study overview

This application note evaluates a liquid chromatography tandem mass spectrometry workflow for quantifying anatoxin-a and cylindrospermopsin in drinking water according to EPA Method 545. The study focuses on using reduced injection volumes (5 µL, 10 µL, and 25 µL) to enhance method robustness while preserving sensitivity.

Methodology and instrumentation

Sample preparation follows EPA Method 545 with triple freeze-thaw cycles, preservative addition (0.1% sodium bisulfate and 0.01% ascorbic acid), and filtration through 0.2 µm PVDF. Direct injection of 1 mL sample spiked with phenylalanine-d5 and uracil-d4 internal standards is performed on a Thermo Scientific UltiMate 3000 LC system coupled to a TSQ Quantis triple quadrupole MS with heated electrospray ionization. Mobile phases are 100 mM acetic acid in water and methanol. Key MS source parameters include 3500 V spray voltage, 45 Arb sheath gas, 10 Arb auxiliary gas, and vaporizer and transfer tube temperatures of 275 °C and 325 °C, respectively. SRM transitions are optimized for each analyte and internal standard.

Key results and discussion

• Calibration linearity was achieved for both toxins at all injection volumes, meeting ±50% accuracy at or below the MRL and ±30% at higher levels.
• System blanks showed no carryover or background contamination above one-third of the MRL for any analyte.
• Precision and accuracy demonstrated <20% RSD and recoveries within ±30% at 10× MRL across four replicates.
• MRL confirmation with seven replicates at the target reporting limit yielded prediction intervals within 50–150% recovery criteria.
• Analysis of a real tap water sample confirmed method applicability, with recoveries of 91–125% and RSD below 6%.

Benefits and practical applications of the method

By reducing injection volumes up to tenfold relative to EPA recommendations, this workflow decreases matrix load, minimizes system maintenance, and extends instrument uptime without compromising sensitivity. The robust performance makes it well suited for routine monitoring of unregulated cyanotoxins in drinking water laboratories.

Future trends and potential applications

• Expansion of analyte panels to include additional cyanotoxins or emerging contaminants.
• Integration of automated sample preparation and online SPE for higher throughput.
• Advances in high-resolution and portable mass spectrometers for field-deployable near real-time monitoring.
• Data analytics and AI-driven workflows to enhance detection confidence and trend analysis.

Conclusion

The Thermo Scientific TSQ Quantis triple quadrupole MS platform delivers sensitive, accurate, and reproducible quantitation of anatoxin-a and cylindrospermopsin in drinking water per EPA Method 545. Reduced injection volumes maintain performance while improving method robustness and reducing maintenance burdens.

Reference

1. U.S. EPA Method 545 Determination of Cylindrospermopsin and Anatoxin-A in Drinking Water by LC/ESI-MS/MS Version 1.0 April 2015
2. U.S. EPA Monitoring Unregulated Drinking Water Contaminants Fourth Unregulated Contaminant Monitoring Rule Accessed April 23, 2017