title: A revisited hemolytic assay for palytoxin detection: Limitations for its quantitation in mussels.
abstract: Palytoxin (PLTX) and its analogues have been detected as seafood contaminants associated with a series of human foodborne poisonings. Due to a number of fatalities ascribed to the ingestion of PLTX-contaminated marine organisms, the development of methods for its detection in seafood has been recommended by the European Food Safety Authority (EFSA). Due to its feasibility, the spectrophotometric hemolytic assay is widely used to detect PLTX in different matrices, even though a standardized protocol is still lacking. Thus, on the basis of available assay procedures, a new standardized protocol was set up using purified human erythrocytes exposed to PLTX (working range: 3.9 × 10(-10)-2.5 × 10(-8) M) in a K(+)-free phosphate buffered saline solution, employing a 5 h incubation at 41 °C. An intra-laboratory characterization demonstrated its sensitivity (limit of detection, LOD = 1.4 × 10(-10) M and quantitation, LOQ = 3.4 × 10(-10) M), accuracy (bias = -0.8%), repeatability (RSDr = 15% and 6% for intra- and inter-day repeatability, respectively) and specificity. However, the standardized method seems not to be suitable for PLTX quantitation in complex matrices, such as mussel (Mytilus galloprovincialis) extracts, at least below the limit suggested by EFSA (30 μg PLTXs/Kg shellfish meat). Thus, the hemolytic assay for PLTX quantitation in seafood should be used only after a careful evaluation of the specific matrix effects.
authors.affiliation: Department of Life Sciences, University of Trieste, Via A. Valerio 6, 34127, Trieste, Italy. Electronic address: valentina.brovedani@phd.units.it.
authors.forename: Valentina
authors.initials: V
authors.lastname: Brovedani
authors.affiliation: Department of Life Sciences, University of Trieste, Via A. Valerio 6, 34127, Trieste, Italy. Electronic address: ssosa@units.it.
authors.forename: Silvio
authors.initials: S
authors.lastname: Sosa
authors.affiliation: U.S. Army Medical Research Institute of Infectious Diseases, Ft Detrick, MD, United States. Electronic address: mark.a.poli.civ@mail.mil.
authors.forename: Mark
authors.initials: M
authors.lastname: Poli
authors.affiliation: Department of Pharmacy, University of Napoli Federico II, 80131, Napoli, Italy. Electronic address: forino@unina.it.
authors.forename: Martino
authors.initials: M
authors.lastname: Forino
authors.affiliation: Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Via Bologna 148, 10154, Torino, Italy. Electronic address: katia.varello@izsto.it.
authors.forename: Katia
authors.initials: K
authors.lastname: Varello
authors.affiliation: Department of Life Sciences, University of Trieste, Via A. Valerio 6, 34127, Trieste, Italy. Electronic address: tubaro@units.it.
authors.forename: Aurelia
authors.initials: A
authors.lastname: Tubaro
authors.affiliation: Department of Life Sciences, University of Trieste, Via A. Valerio 6, 34127, Trieste, Italy. Electronic address: mpelin@units.it.
authors.forename: Marco
authors.initials: M
authors.lastname: Pelin
mesh_terms: D000178:Acrylamides / Q000032:analysis* / Q000633:toxicity; D000818:Animals; D049872:Bivalvia / Q000737:chemistry*; D003064:Cnidarian Venoms; D003429:Cross Reactions; D004912:Erythrocytes / Q000187:drug effects; D006461:Hemolysis / Q000187:drug effects*; D006801:Humans; D057230:Limit of Detection; D015203:Reproducibility of Results; D000095702:Polyether Toxins
chemical_list: D000178:Acrylamides; D003064:Cnidarian Venoms; C010272:palytoxin; D000095702:Polyether Toxins
keywords: Hemolytic assay; Matrix effect; Mytilus galloprovincialis; Palytoxin