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A LC-MS/MS method was developed for simultaneous determination of okadaic acid, dinophysistoxin-1 and dinophysistoxin-2 in shellfish. The development of such a method was necessary for specific determination of these toxins, which was not assured by available officical methods in Vietnam using mouse bioassay and HPLC with fluorescent detection. Chromatographic separation of toxins was done on a ultra performance C18 column (50 mm x 2.1 mm; 1.8 μm) and isocratic elution was carried out with a acetonitrile – water mixture (35 : 65, v/v) containing 0.1% formic acid and 0.01 M of ammonium formate as mobile phase. The detection of toxins was done with electrospray ionization in negative mode, with the ESI ion source set at 5500 V, using one precursor ion (m/z = 803 for okadaic acid and dinophysistoxin-2, m/z = 817 for dinophysistoxin-2) and two product ions (m/z = 563 and 255) for identification of each toxin and the intensity of product ion m/z = 255 for quantitative determination of each toxin. The method was fully validated and proved to be reliable for intended purpose. The method was applied to analyze 39 oyster samples collected in Thanh Hoa province (Vietnam), in which 6 samples contained trace amounts of okadaic acid (3.1 – 9.5 ppb), 2 other samples contained trace amounts of dinophysistoxin-2 (4.4 and 6.4 ppb). The toxin detection in oyster showed a seasonal variation with higher frequency in dry season. No oyster sample was detected with toxin level higher than accepted limit (160 ppb). 162 T.T.T. Vuong et al. such as P. lima and P. concavum1. The first DSP outbreak was recorded in Japan in 19784, following by other outbreaks in Europe5, North America6 and South America7. In South East Asia, although officially no DSP outbreak has been detected yet, but algae bloom of Dinophysis species accompanied by high levels of DSP toxins in green mussels has been recorded in Philippines8, while persistent low levels of DSP toxins were found in green mussels in Singapore9. Among the dinoflagellate species producing OA and DTXs, D. caudata has been detected in Vietnamese sea10, therefore, the bivalves shellfish in Vietnamese coastal area can be exposed to OA and DTXs and accumulate these toxins in their bodies. Currently, DSP toxins (principally OA and DTXs) were among natural toxins to be controlled in shellfish according to requirements of authority in many countries. In general, the accepted limit for DSP toxins in shellfish was 0.16 mg of okadaic acid equivalent per kg of shellfish meat11,12. In Vietnam, the same limit is applied, more from the need to conform with requirements of stringent import markets than from the knowledge of actual presence of OA and DTXs in shellfish at Vietnamese sea coast because the risk of their accumulation in shellfish has not yet been thoroughly studied. The content of OA and DTXs in shellfish can be estimated indirectly through the toxicity on testing animal (mouse bioassay method-MBA)13, or can be determined directly by physicochemical analytical technique, such as liquid chromatography with fluorescent detector14,15 or mass spectrometry detection16-18. In Europe, LCMS/MS was selected as preference technique to determine OA and DTXs in shellfish17 due to its high specificity and sensitivity, enabling direct analyze of toxin without the need of derivatization as with fluorescent detection. However, in Vietnam, no official LC-MS/MS was yet developed for the determination of these toxins in shellfish. Moreover, no study using LC-MS/MS for accurate assessment of OA and DTXs in shellfish was carried out in Vietnam, leaving an important gap on comprehensive knowledge about the actual presence of these toxins in seafood along Vietnamese seacoast. In order to contribute to a better understanding of DSP risk in Vietnam, in this paper, an UPLC-MS/MS was developed and applied to determine OA, DTX-1 and DTX-2 in oyster, a shellfish widely used for human consume in Vietnam. This method was rapid (running time was 7 minutes), simple (using isocratic elution instead of gradient elution for chromatographic separation of toxin, and a simple methanol extraction for sample preparation), and will be used as analytical tool for a more long-term and widespread study on the actual contamination rate and level of OA and DTXs in shellfish along the seacoast of Vietnam. 2.MATERIALS AND METHODS 2.1.Objects of study Oyster samples: 39 oyster samples were collected in Thanh Hoa province in the period from September 2016 to May 2017. All samples belonging to the species Crassostrea rivularis. 2.2. Standards, chemicals, reagents Toxin standards: OA (Batch No: 20141119, 8.4 ppm solution in methanol), DTX-1 (Batch No: 20151219, 8.5 ppm solution in methanol), DTX-2 (Batch No:20150819, 3.8 ppm solution in methanol): All standards obtained from National Research Council Canada (NRCC) (Ottawa, Ontario, Canada). HPLC grade solvents: Methanol, acetonitrile purchased from Merck (Darmstad, Germany). PA grade chemicals: ammonium format, formic acid, isopropyl acetate purchased from Merck (Darmstad, Germany). 2.3. Instrumentation UPLC column: Zorbax Eclipse Plus C18 (2.1 X 50 mm; 1.8mm) purchased from Agilent (Santa Clara, CA, USA). 163 Simultaneous detection of three biotoxins causing diarrhetic shellfish poisoning (okadaic acid, dinophysistoxin-1, dinophysistoxin-2) in oyster by LC-MS/MS UPLC-MS/MS system: UPLC apparatus LC20AD-XR of Shimadzu (Kyoto, Kyoto, Japan) connected to ABSciex Triple Quad 5500 of Sciex (USA).

Simultaneous detection of three biotoxins causing diarrhetic shellfish poisoning (okadaic acid, dinophysistoxin-1, dinophysistoxin-2) in oyster by LC-MS/MS