Available online xxxKeywords:High-performance liquid chromatography coupled with fluorescence detection Liquid chromatographyetandem mass spectrometry Paralytic shellfish poison Tetrodotoxin a b s t r a c t Among marine toxins, tetrodotoxin (TTX) and paralytic shellfish poisons (PSPs) are known as notorious neurotoxins that induce serious food poisoning incidents in the Southeast Asia region. The aim of this study was to investigate whether TTX and PSP toxins are important issues of seafood safety. Paralytic toxicity was observed in mice exposed to 34 specimens from five species of gastropods using a PSP bioassay. Five species of gastropods, Natica vitellus, Natica tumidus, Oliva hirasei, Oliva lignaria, and Oliva annulata, were collected from the coastal seawaters in Nha Trang City, Vietnam, between August 2007 and October 2007. The average lethal potency of gastropod specimens was 90 AE 40 (mean AE standard deviation) mouse units (MU) for N. vitellus, 64 AE 19 MU for N. tumidus, 42 AE 28 MU for O. hirasei, 51 AE 17 MU for O. lignaria, and 39 AE 18 MU for O. annulata. All toxic extracts from the sample species were clarified using a C18 Sep-Pak solid-phase extraction column and a microcentrifuge filter prior to analysis. High-performance liquid chromatography coupled with fluorescence detection indicated that the toxins of the olive shell (O. hirasei, O. lignaria,and O. annulata) were mainly composed of saxitoxin (STX) (73e82%), gonyautoxin (GTX) 2, 3 (12e22%), and minor levels of TTX (5e6%). The toxins of N. vitellus and N. tumidus were mainly composed of STX (76e81%) and GTX 1, 4 (19e24%). Furthermore, liquid chromatographyetandem mass spectrometry analysis was used to verify the identity of the PSPs and TTX. Our evidence shows that these gastropods have novel toxin profiles.
The optical properties of tetrodotoxin (TTX), a scarce toxin with anesthetic properties, were studied using nanoparticle arrays-assisted surface-enhanced Raman scattering (SERS). The nanoparticles arrays were fabricated using nanosphere lithography and a metallic lift-off process to control the particle size, shape, and spacing in the arrays. Using density functional methods, the Raman spectrum of TTX was also calculated with Gaussian03 software. The main peaks of the spectrum are originated from the vibration of the NH 2 molecule group. In the SERS experiment, we were able to measure the Raman spectrum with a TTX concentration as less as 0.9 ng/mL. This sensitivity is comparable to that from high performance liquid chromatography.
To differentiate the species of processed eel products, the gene identification of four fresh eel species was first established and the species of eel products collected from markets were investigated. Polymerase Chain Reaction (PCR) and sequence analysis were used to determine the genetic variation in a 362-nucleotide region of the mitochondrial cytochrome b gene in four fresh eels including Anguilla japonica, Anguilla anguilla, Anguilla rostrata, and Muraenesox cinereus. It was found that each eel species had a unique genotype, which was no different among fresh, frozen, and sterilized meats. The restriction enzyme HinfI could differentiate the species of A. japonica and A. rostrata but could not differentiate A. anguilla and M. cinereus. Another restriction enzyme, Sau96 I, was valuable in the differentiation of M. cinereus from the other three species of Anguilla. By applying PCR and restriction enzymes, the species of 12 commercial eel products were identified as A. japonica (9 samples), A. anguilla (2), and A. rostrata (1). This indicated that the sequence and restriction enzyme cutting site analyses were very usable to authenticate species of different processed eel products.
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