title: Analysis of diarrhetic shellfish poisoning toxins and pectenotoxin-2 in the bottlenose dolphin (Tursiops truncatus) by liquid chromatography-tandem mass spectrometry.
abstract: Toxins produced by harmful algae are associated with detrimental health effects and mass mortalities of marine mammals. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is generally used to confirm the presence of algal toxins in marine mammals. Sample preparation and LC-MS/MS methods for the determination of three diarrhetic shellfish poisoning (DSP) toxins (okadaic acid, OA; dinophysistoxin-1, DTX1; dinophysistoxin-2, DTX2) and pectenotoxin-2 (PTX2) in bottlenose dolphin (Tursiops truncatus) urine and tissue samples were evaluated using spike-and-recovery tests. Sample clean-up with either reversed-phase silica or polymeric solid-phase extraction (SPE) reduced interference of sample matrices and improved toxin recoveries, with polymeric SPE showing higher sample loading capacity. LC separation on Xbridge C18 columns using acetonitrile/water gradient elutions with ammonia as the additive was chosen for its high detectivity and sensitivity in the MS detection of DSP toxins in negative ion mode. The retention times of OA, DTX1, and DTX2, separated as negative ions, increased with LC column temperature while the retention time of PTX2, separated as the neutral molecule, was weakly affected. At the same column temperature, retention times of OA, DTX1, and DTX2 gradually increased as the mobile phases aged while the retention time of PTX2 remained unchanged; higher column temperatures resulted in a greater increase in the retention time of each DSP toxin with mobile phase aging. Average recoveries of the 4 toxins in bottlenose dolphin samples ranged from 80% to 130% with relative standard deviations of less than 15% using the LC mobile phases prepared within one week at a column temperature of 30°C or 40°C. The preferred column temperature was 30°C, as the retention times of DSP toxins were less affected by mobile phase aging at this temperature. The limit of detection of each toxin analyzed in bottlenose dolphin samples was 2.8 ng/g or less in tissue samples and 0.7 ng/ml or less in urine.
authors.affiliation: Marine Biotoxins Program, Center for Coastal Environmental Health and Biomolecular Research, NOAA/National Ocean Service/National Centers for Coastal Ocean Science, 219 Fort Johnson Road, Charleston, SC 29412, USA. Electronic address: zhihong.wang@noaa.gov.
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Marine Biotoxins Program, Center for Coastal Environmental Health and Biomolecular Research, NOAA/National Ocean Service/National Centers for Coastal Ocean Science, 219 Fort Johnson Road, Charleston, SC 29412, USA.
authors.forename: Margaret H
authors.initials: MH
authors.lastname: Broadwater
authors.affiliation: Marine Biotoxins Program, Center for Coastal Environmental Health and Biomolecular Research, NOAA/National Ocean Service/National Centers for Coastal Ocean Science, 219 Fort Johnson Road, Charleston, SC 29412, USA.
authors.forename: John S
authors.initials: JS
authors.lastname: Ramsdell
mesh_terms: D000818:Animals; D050681:Bottle-Nosed Dolphin / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods*; D003967:Diarrhea / Q000139:chemically induced*; D005663:Furans / Q000032:analysis*; D018942:Macrolides; D008387:Marine Toxins / Q000032:analysis*; D019319:Okadaic Acid / Q000032:analysis / Q000031:analogs & derivatives; D011714:Pyrans / Q000032:analysis*; D057096:Shellfish Poisoning / Q000378:metabolism*; D052616:Solid Phase Extraction / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*; D000095702:Polyether Toxins
chemical_list: D005663:Furans; D018942:Macrolides; D008387:Marine Toxins; D019319:Okadaic Acid; D011714:Pyrans; C051904:dinophysistoxin 1; C098380:pectenotoxin 2; D000095702:Polyether Toxins; C076221:dinophysistoxin 2
keywords: Bottlenose dolphins; Diarrhetic shellfish poisoning toxins; LC–MS/MS; Mobile phase aging; Pectenotoxin; Solid-phase extraction