2.3. Solid phase extraction (SPE)

Due to the variety of physiochemical properties that cyanotoxins possess, isolation of target analytes from water was split into two SPE extractions methods, corresponding to the HILIC or RPLC separation methods. SPE extraction methodology and optimization were developed from a previously reported study [15], with modifications for individual SPE extraction and the incorporation of SAX. Water samples were filtered through two filters: a glass fiber prefilter (1.0-μm pore size, 47 mm, Pall Corporation, Port Washington, NY, USA) and a nitrocellulose filter (0.45-μm pore size, 47 mm, GE Healthcare, Little Chalfont, BUX, UK). 2 Ls were separated into 2 – 1 L volumetric flasks for extraction. ANA, CLD, and SAX were extracted on a Supelclean ENVI-carb (6cc, 500mg, Supelco Inc., Bellefonte, PA, USA). MCs and NOD were extracted using an Oasis HLB (6 cc, 200 mg, Waters Corporation, Milford, MA, USA). Prior to extraction all samples were spiked with 100 μg L⁻¹ of corresponding Iss. HLB cartridges were pretreated with 6 mL of MeOH and 6 mL of nanopure water. ENVI-carb cartridges were pretreated with 6 mL of DCM, 6 mL of MeOH, and 6 mL of nanopure H₂O (Barnstead™ Nanopure™). 1 L of sample was loaded on each cartridge separately at ~10 mL/min (visible dripping) via a 24 port Visiprep™ vacuum manifold (Supelco Inc., Bellefonte, PA, USA). Cartridges were then blown dry under nitrogen. HLB cartridges were eluted with 10 mL of MeOH (0.5% formic acid) into 20 mL test tubes. ENVI-carb cartridges were eluted with 10 mL 60:40 (v v⁻¹) MeOH:DCM (0.5% formic acid) into 20 mL test tubes. Eluates were blown to dryness under a gentle stream of nitrogen in a Turbovap (Zynmark, Hopkinton, MA, USA) set to 55°C. HLB samples were reconstituted with 1 mL 90:10 (v v⁻¹) H₂O:MeCN buffered with 5mM NH₄OOCH and 3.6 mM HCOOH (pH 3.7). ENVI-carb samples were reconstituted with 1 mL 10:90 (v v⁻¹) H₂O:MeCN buffered with 5mM NH₄OOCH and 3.6 mM HCOOH (pH 3.7). All reconstituted samples were syringe filtered using a BD 1 mL TB syringe (BD, Franklin Lakes, NJ, USA) and Acrodisc® hydrophobic Teflon Supor membrane syringe filters (13-mm diameter; 0.2-μm pore size, Pall Corporation, Port Washington, NY, USA) and placed in 2 mL analytical vials (Agilent Technologies, Santa Clara, CA, USA) for analysis.