Highly Sensitive Quantitation of Cylindrospermopsin and Anatoxin-a in Water using the Triple Quad LCMS-8060NX in Accordance with EPA Method 545

Importance of the Topic

Cyanobacterial toxins such as cylindrospermopsin and anatoxin-a pose serious risks to drinking and recreational water due to harmful algal blooms, necessitating sensitive and reliable methods for their detection and quantitation.

Objectives and Study Overview

This application note describes the development of a rapid LC-MS/MS workflow using the Shimadzu LCMS-8060NX triple quadrupole system to quantify cylindrospermopsin and anatoxin-a in water, in line with EPA Method 545, aiming to achieve lower limits of quantitation (LOQs), an extended dynamic range, and high throughput with minimal sample preparation.

Methodology

Standard solutions of cylindrospermopsin and anatoxin-a were prepared in methanol/water (1:1) and diluted to final calibration ranges of 0.005–10 ng/mL and 0.02–20 ng/mL, respectively, using water with sodium bisulfate and ascorbic acid. Water samples were filtered (0.2 µm PVDF) and spiked at two concentration levels. Chromatographic separation was achieved on a Shim-pack GIST C18 column (100×2.1 mm, 2.0 µm) using a gradient of 0.2% acetic acid in water (solvent A) and methanol (solvent B) at 0.3 mL/min, 40 °C, with an 8-minute runtime. The LCMS-8060NX operated in positive electrospray ionization with multiple reaction monitoring (MRM) and a divert valve to waste during the first minute. Data processing and QC were performed with LabSolutions Insight software.

Used Instrumentation

• Shimadzu LCMS-8060NX triple quadrupole mass spectrometer
• Shim-pack GIST C18 column (100×2.1 mm, 2.0 µm)
• LabSolutions Insight data analysis software
• HPLC autosampler with 20 µL injection volume

Main Results and Discussion

The method provided baseline separation of analytes and internal standards in under 8 minutes. LOQs were 0.005 ng/mL for cylindrospermopsin and 0.02 ng/mL for anatoxin-a (S/N≥10), with accuracy 80–120% and %RSD<7%. Calibration curves exhibited linearity (R2>0.99) over the tested ranges with no carryover. Spiked water samples (LC-MS grade, tap, filtered tap, stream, pond) showed recoveries within 80–125% and RSD<10% across both spiking levels, demonstrating robustness across diverse matrices.

Benefits and Practical Applications

• Compliance with EPA Method 545 requirements with improved sensitivity
• Extended dynamic range allowing analysis of variable concentrations without re-preparation
• High throughput with an 8-minute run time
• Simplified sample preparation suitable for routine monitoring

Future Trends and Applications

Potential developments include extending this workflow to additional cyanotoxins, integrating automated online sample preparation, coupling with high-resolution mass spectrometry for non-target screening, deploying portable LC-MS platforms for in-field analysis, and leveraging advanced data analytics for trend monitoring.

Conclusion

This study demonstrates that the Shimadzu LCMS-8060NX system delivers a rapid, sensitive, and reproducible LC-MS/MS method for monitoring cylindrospermopsin and anatoxin-a in water, fulfilling regulatory QA/QC criteria and offering broad applicability for environmental laboratories.

Reference

1. US EPA. Health Effects Support Document for the Cyanobacterial Toxin Cylindrospermopsin. 2015.
2. US EPA. EPA Drinking Water Health Advisories for Cyanotoxins. 2019.
3. US EPA. Health Effects Support Document for the Cyanobacterial Toxin Anatoxin-A. 2015.
4. US EPA. Method 545: Determination of Cylindrospermopsin and Anatoxin-a in Drinking Water by LC/ESI-MS/MS. 2015.