Development of a method of liquid chromatography coupled to mass spectrometry for determination of tetrodotoxins

Identifier:  TFG:122

Handle: https://hdl.handle.net/20.500.11797/TFG122

Authors:  Gil Pagà, Lourdes

Abstract:
A high-performance liquid chromatography (LC) coupled with mass spectrometric detection (-MS/MS) via an electrospray ion source has been developed for the determination of tetrodotoxins (TTXs) in puffer fish samples. Separation was based upon hydrophilic interaction chromatography (HILIC) on an XBridge Amide column (2.1mm × 100 mm, 3,5 μm particle size) and mobile phases consisted in 30 mM ammonium acetate at pH 5.8 and acetonitrile/30 mM ammonium acetate (90:10), for mobile phases A and B, respectively. The gradient elution was optimized using positive control samples from the puffer fish species Lagocephalus sceleratus, from 100% B to 50% A in a total run time cycle of 12 min including post-run equilibration. Compound-dependent mass spectrometry detection parameters were optimized by direct infusion of TTX standard to the mass spectrometer, among others, a declustering potential of 50 V and a collision energy of 29 and 53 V for the transitions precursor ion > product ion of m/z 320 > 302 and 320 > 162, respectively. The electrospray ion source temperature was adjusted to 600ºC after LC coupling to maximize the ionization yield. The triple-quadrupolo multi reaction monitoring (MRM) method allowed the determination of 10 different tetrodotoxins in puffer fish samples, including a fish individual of the species Sphoeroides pachygaster caught at Palamós, coast of Catalonia. Preliminary results on method validation have been obtained, showing good linearity in the range 25-1250 μg TTX/Kg (r² = 0.999) and a very low limit of detection of 1 μg TTX/Kg. A preliminary assessment of the puffer fish found in Catalonia revealed a much lower toxicity compared to that found in the highly toxic L. sceleratus, the former showing TTX > 25 μg /Kg only the skin and at relatively low level 300 μg TTX/Kg. The set up and optimization of this method will contribute in the next future to perform the risk assessment of the invasion of these Lessepsian migrants to the Mediterranean Sea.