Simultaneous Multiclass Analysis of Cyanotoxins in Cyanobacterial Samples Using Hydrophilic Interaction Liquid Chromatography‐Tandem Mass Spectrometry

The proliferation of cyanobacteria can result in algal blooms, which may cause environmental and biological harm due to the production and release of secondary metabolites, or cyanotoxins, into the affected waterway. Cyanobacteria can produce multiple classes of cyanotoxins; therefore, to understand the full toxic load of algal blooms, it is necessary to perform analyses that quantify each class. These classes are generally monitored individually due to the challenges associated with the differing physicochemical properties of the cyanotoxins. Hydrophilic interaction liquid chromatography (HILIC) is a form of chromatography capable of retaining multiple classes of cyanotoxins that differ in physicochemical properties. Here an HILIC-MS/MS method was developed and validated to detect 3 microcystins, 11 saxitoxins, and 2 anatoxins. The chromatographic conditions were optimized to allow for the separation of multiple pairs of saxitoxin epimers, and in-source fragmentation in the MS interface was used to develop unique MRMs between the pairs. The method was validated and had low limits of detection (LODs, between 0.00770 and 9.75 µg L^-1) and limits of quantification (LOQs, between 0.0257 and 32.5 µg L^-1) for all compounds. All analytes exhibited good linearity (R² values ≥ 0.991) and low percentage relative standard deviations for retention time (0%-1.74%) and peak area (4.54%-27.6%), with spiked recoveries ranging from 75.6% to 117% for all compounds. A multiclass sample preparation method to extract the three classes of analytes from cyanobacterial samples was developed and validated, with 80:20 acetonitrile:water and 0.1% formic acid as the optimal extraction solvent. The newly developed sample preparation and analysis methods were applied to cultured cyanobacteria and field samples, with microcystins and saxitoxins detected. The multiclass sample preparation and analysis methods developed here improve on individual methods as they reduce the complexity and time of sample preparation and analysis and will assist ecotoxicologists in assessing the full toxic risk of cyanobacterial blooms.