Identification of Unknown Microcystins in Alberta Lake Water

Importance of topic

Freshwater sources in Canada are regularly contaminated by cyanobacterial toxins known as microcystins, which pose serious risks to public health and livestock. These cyclic heptapeptides inhibit protein phosphatases, cause liver damage, and may promote tumor growth. Rapid, sensitive detection of both known and emerging microcystin variants is critical to meet regulatory guidelines for recreational and drinking water quality.

Objectives and overview

This study aimed to detect, characterize, and tentatively identify previously unknown microcystins in Alberta lake water in the absence of analytical standards. A two‐pronged approach was used: targeted quantitation by triple quadrupole LC/MS and structural confirmation by accurate‐mass LC/Q‐TOF MS. A Personal Compound Database (PCD) based on the World Health Organization list of microcystins supported the non‐targeted search for additional analogues.

Methodology and instrumentation

Two analytical workflows were established:

• Targeted MRM quantitation on an Agilent 1290 Infinity LC coupled to a 6460 Triple Quadrupole MS. Chromatography used a Poroshell SB‐C18 column with a gradient of ammonium fluoride in water and isopropanol/acetonitrile.
• Accurate‐mass confirmation on an Agilent 1290 Infinity LC coupled to a 6540 Q‐TOF MS with Jet Stream ESI source. Full‐scan and MS/MS data were collected for mass accuracy and fragment ion analysis.

Samples underwent freeze/thaw cycles, sonication, and filtration prior to injection. Data processing used Agilent MassHunter software and a custom PCD of 52 microcystin formulas for retrospective formula matching.

Main results and discussion

When screening lake samples with the triple quadrupole method, an unexpected peak sharing the YR transition (m/z 1045→135/213) appeared at a distinct retention time with an altered qualifier ratio. Calibration curves for eight target microcystins showed excellent linearity (R2>0.9978) and quantitation limits well below Canadian guidelines (1.5 µg/L).

Accurate‐mass Q‐TOF MS confirmed the unknown’s protonated mass of 1059.5500, consistent with a desmethyl‐HtyR structure (Δ5.0 ppm). MS/MS fragment analysis of characteristic Adda ions (m/z 135.0804, 135.1168), the Glu+Mdha cleavage product (m/z 199.0713), and diagnostic “f” and “h” ions established loss of a methyl group at position 7 (Mdha→Dha) while maintaining methylaspartate at position 3.

Using the PCD and MassHunter’s Find By Formula, seven additional microcystins were tentatively identified in two lake samples, demonstrating the value of retrospective screening for known and novel analogues.

Benefits and practical applications

• Detection of microcystins at 0.1 ng/mL, enabling early warning below regulatory thresholds.
• Identification of unknown analogues without reference standards through accurate‐mass and fragment‐based confirmation.
• Retrospective mining of stored LC/Q‐TOF data to discover emerging toxins as they are added to the database.
• Robust quantitation for QA/QC of drinking and recreational water monitoring programs.

Future trends and opportunities

• Expansion of PCD libraries with newly identified microcystin variants and their predicted masses.
• Integration of machine‐learning tools for exact‐mass prediction and automated annotation of non‐targeted toxins.
• Coupling high‐resolution MS with ion mobility or HRAM MS/MS to resolve isobaric variants and positional isomers.
• Development of rapid field‐deployable screening kits using portable MS platforms for real‐time water quality assessment.

Conclusion

An integrated LC/triple quadrupole and LC/Q‐TOF MS strategy successfully detected and confirmed an unknown desmethylated HtyR microcystin in Alberta lake water without a reference standard. The methodology delivers high sensitivity, excellent linearity, and structural insight through accurate‐mass fragmentation. Coupled with a customizable PCD, this approach enables comprehensive targeted and non‐targeted surveillance of microcystins in environmental waters.

Reference

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• Orihel DM et al. High microcystin concentrations occur only at low nitrogen-to-phosphorus ratios in nutrient-rich Canadian lakes. Can J Fish Aquat Sci. 2012;69:1457–1462.
• Mayumi T et al. Structural characterization of microcystins by LC/MS/MS under ion trap conditions. J Antibiot (Tokyo). 2006;59:710–719.
• Chorus I, Bartram J, editors. Toxic Cyanobacteria in Water—A Guide to Their Public Health Consequences, Monitoring, and Management. E&FN Spon on behalf of WHO; 1999.