A Multi-Class Cyanobacterial Toxin Screening Method using Hydrophilic Interaction Liquid Chromatography with Tandem Mass Spectrometry (HILIC-MS/MS)

Cyanobacteria can produce diverse classes of toxins including microcystins (MCs), anatoxins (ATXs), cylindrospermopsins (CYNs), and saxitoxins (STXs). These range in polarities and molecular weights, generally requiring multiple preparation and chromatographic techniques for evaluation. Here we present the development and validation of a HILIC-MS/MS screening method for the detection and quantitation of all the aforementioned toxin classes. Various solvents and sample-to-solvent ratios were investigated with an in-house blend of toxic cyanobacteria to develop a universal extraction method using 75% acetonitrile-water (0.1% formic acid). HILIC-MS/MS was used with gradient elution (35 min run time) with selected reaction monitoring (SRM) settings optimized on a triple quadrupole MS using positive/negative polarity switching. Retention time matching with standards and product ion ratios were assessed for identification. Validation included evaluation of the calibration models, precision, and detection limits (between 0.2 (ATX) and 7 (GTX4) ng/mL). Excellent recoveries (\u3e 99%) were obtained using spiked algal extracts and in‑house reference materials. This method was applied to cultures, cyanobacterial dietary supplements, Canadian bloom and benthic algal samples, water, and shellfish samples, demonstrating suitability for screening and quantitation of all major cyanobacterial toxin classes in a range of sample types. Future applications include the characterization of cyanotoxin matrix reference materials