title: Determination of diarrheic shellfish toxins in mussels by microliquid chromatography-tandem mass spectrometry.
abstract: A fast, sensitive, and specific procedure for determining toxins that cause diarrheic shellfish poisoning (DSP) using microliquid chromatography coupled with tandem mass spectrometry (micro-LC-MS-MS) is reported. The lipophylic polyether acidic toxins okadaic acid (OA), its isomer dinophysistoxin-2 (DTX-2), the 35-methylokadaic acid dinophysistoxin-1 (DTX-1), and the novel toxin dinophysistoxin-1B (DTX-2B; recently isolated from Irish mussels) were extracted from shellfish tissues with acetone and chromatographed by isocratic elution at 10 microL/min with CH3 CN-H2O, 80 + 20 (v/v), containing 0.1% trifluoroacetic acid, through a C18 reversed-phase column (1.0 mm id). The chromatograph is coupled via an ion spray interface to an atmospheric pressure ionization source. Collision-induced-dissociation (CID) ion mass spectra of the protonated molecule, [M + H]+, at m/z 805 for OA, DTX-2, and DTX-2B and at m/z 819 for DTX-1, were obtained in MS-MS experiments to identify 2 diagnostic fragment ions for each analyte that could be used for selected-reaction-monitoring (SRM) micro-LC-MS-MS analysis. The CID spectrum of DTX-2B confirmed it to be a new OA isomer, like DTX-2. Standard curves obtained by SRM micro-LC-MS-MS were linear (r2 > or = 0.9992) over the range 0.05-1.00 micrograms/mL (i.e., 0.10-2.00 micrograms toxin/g hepatopancreas), and a detection limit of 15 pg/injection was obtained for each DSP toxin. Average recoveries ranged from 95 to 101%, and coefficients of variation ranged from 1.8 to 3.4%. This novel SRM micro-LC-MS-MS method was used to confirm acidic DSP toxins in Irish and Italian toxic mussels. It offers a high degree of specificity because analyte confirmation is based on retention time, molecular weight, structural information obtained from the presence of 2 diagnostic fragments for each analyte, and ion ratios. OA was found in both Irish (< or = 0.7 micrograms/g hepatopancreas) and Italian (< or = 1.5 micrograms/g hepatopancreas) mussels. DTX-1 was found only in Italian mussels (< or = 0.3 micrograms/g hepatopancreas). DTX-2 (< or = 6.1 micrograms/g hepatopancreas) and DTX-2B (< or = 0.08 micrograms/hepatopancreas) were unique to Irish shellfish.
authors.affiliation: Istituto Superiore di Sanità, Laboratorio Medicina Veterinaria, Rome, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Draisci
authors.forename: L
authors.initials: L
authors.lastname: Lucentini
authors.forename: L
authors.initials: L
authors.lastname: Giannetti
authors.forename: P
authors.initials: P
authors.lastname: Boria
authors.forename: K J
authors.initials: KJ
authors.lastname: James
authors.forename: A
authors.initials: A
authors.lastname: Furey
authors.forename: M
authors.initials: M
authors.lastname: Gillman
authors.forename: S S
authors.initials: SS
authors.lastname: Kelly
mesh_terms: D000818:Animals; D049872:Bivalvia / Q000737:chemistry*; D002853:Chromatography, Liquid; D003967:Diarrhea / Q000139:chemically induced*; D004064:Digestive System / Q000737:chemistry; D008387:Marine Toxins / Q000032:analysis* / Q000633:toxicity; D013058:Mass Spectrometry; D019319:Okadaic Acid / Q000032:analysis / Q000031:analogs & derivatives; D011714:Pyrans / Q000032:analysis; D012758:Shellfish / Q000032:analysis*; D012997:Solvents
chemical_list: D008387:Marine Toxins; D011714:Pyrans; D012997:Solvents; D019319:Okadaic Acid; C076221:dinophysistoxin 2