title: Investigation of extraction and analysis techniques for Lyngbya wollei derived Paralytic Shellfish Toxins.
abstract: Paralytic Shellfish Toxins (PSTs) are highly toxic metabolic by-products of cyanobacteria and dinoflagellates. The filamentous cyanobacterium Lyngbya wollei produces a unique set of PSTs, including L. wollei toxins (LWT) 1-6. The accurate identification and quantification of PSTs from Lyngbya filaments is challenging, but critical for understanding toxin production and associated risk, as well as for providing baseline information regarding the potential for trophic transfer. This study evaluated several approaches for the extraction and analysis of PSTs from field-collected L. wollei dominated algal mats. Extraction of PSTs from lyophilized Lyngbya biomass was assessed utilizing hydrochloric acid and acetic acid at concentrations of 0.001-0.1 M. Toxin profiles were then compared utilizing two analysis techniques: pre-column oxidation (peroxide and periodate) High Performance Liquid Chromatography (HPLC) with Fluorescence (FL) detection and LC coupled with Mass Spectrometry (MS). While both acid approaches efficiently extracted PSTs, hydrochloric acid was found to convert the less toxic LWT into the more toxic decarbamoylgonyautoxins 2&3 (dcGTX2&3) and decarbamoylsaxitoxin (dcSTX). In comparison, extraction with 0.1 M acetic acid preserved the original toxin profile and limited the presence of interfering co-extractants. Although pre-chromatographic oxidation with HPLC/FL was relatively easy to setup and utilize, the method did not resolve the individual constituents of the L. wollei derived PST profile. The LC/MS method allowed characterization of the PSTs derived from L. wollei, but without commercially available LWT 1-6 standards, quantitation was not possible for the LWT. In future work, evaluation of the risk associated with L. wollei derived PSTs will require commercially available standards of LWT 1-6 for accurate determinations of total PST content and potency.
authors.affiliation: The Fisheries and Aquatic Sciences Program in the School of Forest Resources and Conservation, Institute of Food and Agricultural Sciences-IFAS, University of Florida, 7922 NW 71st Street, Gainesville, FL 32653, USA. amandafoss@greenwaterlab.com
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Foss
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Phlips
authors.forename: Mark T
authors.initials: MT
authors.lastname: Aubel
authors.forename: Nancy J
authors.initials: NJ
authors.lastname: Szabo
mesh_terms: D001681:Biological Assay / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D000458:Cyanobacteria / Q000378:metabolism*; D004141:Dinoflagellida / Q000378:metabolism*; D005453:Fluorescence; D008387:Marine Toxins / Q000302:isolation & purification*; D013058:Mass Spectrometry / Q000379:methods; D012530:Saxitoxin / Q000031:analogs & derivatives / Q000302:isolation & purification; D012758:Shellfish
chemical_list: D008387:Marine Toxins; D012530:Saxitoxin; C034903:decarbamylsaxitoxin