Determination of tetrodotoxin in puffer-fish by liquid chromatography-electrospray ionization mass spectrometry

Abstract

A simple and reliable method using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) for the determination of tetrodotoxin in the puffer-fish has been developed. The LC separation was performed on a Shodex RSpak NN-414 column (15 cm x 4.6 mm id) using 20 mM ammonium acetate-methanol (75 + 25) as the mobile phase at a flow rate of 0.5 ml min(-1). The positive ionization produced the typical [M + H]+ molecular ion of tetrodotoxin (m/z 320). The calibration graph for tetrodotoxin was rectilinear from 0.01 to 1 microg ml(-1) with selected ion monitoring (SIM). Tetrodotoxin was extracted with 0.1% acetic acid by heating in a boiling water-bath and the extracts were cleaned up on a Bond Elut C18 (500 mg) cartridge. The recoveries of the tetrodotoxin from the puffer-fish fortified at 1 microg g(-1) were 77.7-80.7% and the detection limit was 0.1 microg g(-1) (equivalent to ca. 0.5 mouse units per gram).

Screening of tetrodotoxin in puffers using gas chromatography–mass spectrometry

“…The lengthy evaporation steps as practiced by others [6,25,29] was omitted. In several studies, the recovered supernatant was subjected to defatting procedure using dichloromethane [6,11,25], chloroform [29,30] or diethyl ether [28] followed by further cleanup procedures before the sample could be analyzed by analytical instruments. For over 30 years, SPE and preparative column have been used to extract TTX.…”

“…Mass spectrometry (MS) coupled with LC or GC is a sensitive technique for identification of TTX. However, due to the complexity of sample matrices and insolubility of TTX in organic solvent, the LC-MS [8,9,11,15,18,[24][25][26][27] or LC-MS/MS [4,6,13,15,18,27] is a preferred method than GC-MS. The drawback for LC-MS and LC-MS/MS analysis is that it involves the use of expensive instruments that require higher maintenance compared to GC-MS.…”

“…Analytical assay of TTX generally involves pre-extraction of the compound from fish tissues using acetic acid (HOAc) accompanied by a 5-10 min heating [4,10,11,17,19,[25][26][27], or non-heating [6,7,18,20]. After centrifugation and/or evaporation, the recovered supernatant is subjected to sample cleanup using either solid-phase extraction (SPE) [6,7,25,26], preparative column [7,18], membrane or nylon filtration [2,4,11] or a combination of any of the two cleanup methods.…”

“…After centrifugation and/or evaporation, the recovered supernatant is subjected to sample cleanup using either solid-phase extraction (SPE) [6,7,25,26], preparative column [7,18], membrane or nylon filtration [2,4,11] or a combination of any of the two cleanup methods. In GC-MS analysis, structure modification of TTX is necessary as it is heat stable and not-volatile.…”

Optimization of simultaneous analysis of tetrodotoxin, 4-epitetrodotoxin, 4,9-anhydrotetrodotoxin, and 5,6,11-trideoxytetrodotoxin by hydrophilic interaction liquid chromatography–tandem mass spectrometry

“…Recently, liquid chromatography-mass spectrometry (LC-MS) has been recognized as a useful method to analyze TTX [8][9][10][11][12][13][14][15][16]. We previously reported two different systems to separate TTX analogs by LC-MS analysis.…”