Efficient dual-mode liquid chromatography-tandem mass spectrometry methods for quantifying major cyanotoxins in...

Cyanobacterial blooms in freshwater resources frequently originate from toxin-producing strains capable of generating chemically diverse cyanotoxins. These blooms not only disrupt aquatic ecosystems but also pose significant risks to public health due to the production of hepatotoxins (such as microcystins (MCs) and nodularins (NODs)) and neurotoxins (such as anatoxins (ATXs), saxitoxins (STXs), and cylindrospermopsins (CYNs)). It is therefore essential to accurately detect and quantify cyanotoxins in order to assess exposure risks, inform water management decisions, and guide public health interventions. However, this task remains analytically challenging due to the wide polarity range of these compounds (-5.1 < XLogP < 5.2), their structural diversity, and their presence at trace levels (ng L⁻¹). In this study, we developed and validated two high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) methods for the quantification of 24 priority cyanotoxins across diverse families. The panel included eleven MC variants, one NOD, three ATXs, three CYNs, and six STX analogs. Peptides (MCs and the NOD) were analyzed using reversed-phase online solid-phase extraction (SPE)-HPLC-MS/MS, whereas more polar toxins (ATXs, STXs, and CYNs) were analyzed using direct injection in hydrophilic interaction liquid chromatography mode (DI-HPLC-MS/MS) after minimal sample preparation. Quantification was performed using isotope-labeled surrogate internal standards, with one representative labeled analogue assigned to each toxin class to improve correction for matrix effects and analytical variability. Both methods were optimized and validated following SANTE/EURL guidelines, using representative water matrices. Calibration curves exhibited close quadratic fits (R² > 0.99). Mean recoveries ranged from 77 to 120% with relative standard deviations below 15% and maximum measurement uncertainties under 40% for most analytes. Quantification limits reached 20 ng L⁻¹ for MCs and the NOD, and ranged from 50 to 250 ng L⁻¹ for ATXs, CYNs, and STXs. Application of these methods to the summer monitoring campaign for 2025 in the Grand-Est region of northeastern France confirmed their robustness and successfully quantified six MC variants, two STX analogs, and ATX in bathing waters.