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Determination of Tetrodotoxin and Its Analogs in the Puffer Fish Takifugu Oblongus from Bangladesh by Hydrophilic Interaction Chromatography and Mass-spectrometric Detection

Marc Diener
Institute of Nutrition, University of Jena, Dornburger Street 25, 07743 Jena, Germany

Bernd Christian
Institute of Nutrition, University of Jena, Dornburger Street 25, 07743 Jena, Germany

M. Sagir Ahmed
Laboratory of Aquatic Resource Management, Department of Zoology, University of Dhaka, Dhaka 1000, Bangladesh

Bernd Luckas
Institute of Nutrition, University of Jena, Dornburger Street 25, 07743 Jena, Germany

Abstract/ Executive Summary:

Tetrodotoxin (TTX) and its analogs (TTXs), widely distributed among marine as well as terrestrial animals, induce dangerous intoxications. These highly potential toxins are also known as the causative agent of pufferfish poisoning. A newly developed highly sensitive method for the determination of TTXs based on hydrophilic interaction chromatography and mass-spectrometric detection is presented. TTX, anhydrotetrodotoxin, 11-deoxytetrodotoxin and trideoxytetrodotoxin were determined in separated tissues of Bangladeshi marine puffers, Takifugu oblongus. TTX was predominant in skin, muscle and liver, whereas trideoxytetrodotoxin preponderated in the ovary. The toxicity of the various tissues was determined by a mouse bioassay.

Keywords: Hydrophilic interaction chromatography, Liquid chromatography–tandem mass spectrometry, Takifugu oblongus,Tetrodotoxin, Zwitterionic hydrophilic interaction chromatography column

Location:

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Puffer fish

Objectives:

Here we report an approach that facilitates the quantification of TTXs in pufferfish by the enhanced sensitivity and accuracy of a reliable LC-MS method. The LC-MS method applied a zwitterionic (ZIC) hydrophilic interaction chromatography (HILIC) column and decreased the LODs 3 times compared with previous LC-MS methods. Therefore, the method allowed the affirmation of the toxicity results obtained by a mouse bioassay. Moreover, a similar ZIC-HILIC column allows the detection of PSP toxins which could appear as major toxins in pufferfish or combined with TTX.

Methodology:

Pufferfish specimens Five specimens of T. oblongus (body weight from 240 to 625 g) were collected from the area adjacent to the poisoning incident of 2002, in the coastal district of Khulna on 25 March 2006. They were caught concomitantly with other commercially important fish by seine nets from the offshore area of the Bay of Bengal. All the samples were immediately frozen at −20°C, transported to the laboratory at the Department of Zoology, University of Dhaka, and subsequently kept frozen at below −30°C until use. Assay of toxicity The puffer specimens were partially thawed and dissected into skin, muscle, liver, testis/ovary and viscera. Each tissue was minced and a small portion (3–5 g) was extracted with an equal volume of 0.1% acetic acid by heating in a boiling water bath for 5 min and the extractant was centrifuged at 11,000 g for 10 min. The supernatant was examined for toxicity by the standard method for TTX, since the toxin was suspected to be TTX. Lethal potency was expressed in mouse units (MU), where 1 MU is defined as the amount of toxin that kills an 18–20 g male mouse in 30 min after intraperitoneal injection. Representative parts of the remaining tissues (except viscera) were sun-dried and transported to the Institute of Nutrition, University of Jena, Germany, for determination of toxic components. Extraction The dried T. oblongus samples (0.2 g) were homogenized with 2 mL acetic acid (0.03 M) using an ultrasonic probe. Afterward, the samples were centrifuged (Z 200 MH, Hermle Labortechnik, Wehingen, Germany) for 10 min (16,000 g), and subsequently, the supernatant was passed through a 0.45 μm nylon filter. Toxin standards The TTX standard (part number 630–002–M001) was purchased from Axxora, Grünberg, Germany. This solution also contained anhydro TTX and 11-deoxyTTX as minor compounds. Reference material for trideoxyTTX was not available; therefore, the concentration of trideoxyTTX was calculated with the calibration equation for TTX. Chemicals All solvents used were of analytical grade; acetonitrile, ammonium formate and formic acid were purchased from Acros Organics (Geel, Belgium). Water (high-performance LC grade) was taken from a Millipore-Q RG ultrapure water system (Millipore, Milford, USA). Liquid chromatography-tandem mass spectrometry LC-MS/MS was performed using the following system: an SIL-10ADvp intelligent autosampler, LC-10AT pumps, an SCL-10Avp system controller, a CTO 10ASvp column oven (Shimadzu, Duisburg, Germany) and a triple-quadrupole API 365 PE Sciex mass spectrometer equipped with a turbo ion spray interface (PE Sciex, Halifax, Canada). HILIC was operated with eluent A consisting of 10 mM ammonium formate and 10 mM formic acid in water. Eluent B contained 80% acetonitrile and 20% water with a final concentration of 5 mM ammonium formate and 2 mM formic acid. Gradient formation applied for the chromatographic separation of TTX is presented in Table 1.A5−μm ZIC-HILIC column, 150 mm×2.1−mm inner diameters and a corresponding guard column 20 mm×2.1−mm inner diameter (SeQuant, Haltern, Germany) were used. The mobile phase, flow 0.25 mL min−1, was transferred directly into the ion spray interface and the mass-spectrometric detection was carried out in positive mode. Multiple reaction monitoring (MRM) transitions and the selected ion monitoring (SIM) parameters for HILIC-MS detection of TTX are presented in Table 2. The turbo ion spray source was operated with nitrogen (8 L min−1 ) as the nebulizer gas at 500°C.

Source of Information: Anal Bioanal Chem (2007) 389:1997–2002

Article Link (Online Journal): DOI 10.1007/s00216-007-1602-7

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Achievement/Findings:

The new HILIC-MS/MS method allows the selective and sensitive detection of the major TTXs in a single 30 min analysis. The composition of the mobile phase, low buffer concentration and a high percentage of organic solvent results in a high ionization efficiency and guarantees low LODs for TTXs. The results demonstrate the successful application of this method for qualitative analysis of different organs of T. oblongus. It was demonstrated that the occurrence of TTXs in Bangladeshi marine puffers caused the intoxication of consumers.

Knowledge Management: Journal