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

Abstract

Recently, liquid chromatography-mass spectrometry (LC-MS) has been recognized as a useful method to analyze tetrodotoxin (TTX), the primary toxin for puffer fish poisoning. TTX usually exists with its chemically interchangeable analogs, such as 4-epiTTX and 4, 9-anhydroTTX. TTX and 4-epiTTX have the same molecular weight and the same fragmentation pattern by tandem mass spectrometry (MS-MS). In this study, we optimized conditions for hydrophilic interaction liquid chromatography (HILC)-MS-MS to quantitate TTX, 4-epiTTX, 4,9-anhydroTTX, and 5,6,11-trideoxyTTX with good separation. The relationships between the applied amounts of each TTX analog to the HILC-MS-MS instrument and the peak areas showed good linearity in the ranges examined. The lower limits of detection (signal-to-noise ratio = 3) were 64 pg on-column for TTX, 128 pg for both 4-epiTTX and 4,9-anhydroTTX, and 180 pg for 5,6,11-trideoxyTTX using an API 2000 mass spectrometer.

First Identification of 5,11-Dideoxytetrodotoxin in Marine Animals, and Characterization of Major Fragment Ions of Tetrodotoxin and Its Analogs by High Resolution ESI-MS/MS

“…In this mode, both the Q1 scan mass chromatograms and the fragment ion spectra from the specific precursor ions ([M + H] + m/z 288.1190 ± 0.1 for dideoxyTTX) are simultaneously obtained. In this experiment, TTXs were separated in HILIC mode as we reported previously [29,41,43,44]. The extract ion chromatograms (EICs) for [M + H] + of these dideoxy analogs are shown in Figure 2A, their fragment ion spectra with the high resolution masses and predicted molecular formulas are shown in Figure 2B, and the fragment ion spectra of TTX and 5,6,11-trideoxyTTX are also shown in Figure 3 for comparison.…”

“…Liquid chromatography was performed on a 2.0 i.d. × 150 mm (5 µm) TSK-GEL Amide-80 column (Tosoh, Tokyo, Japan) using 16 mM ammonium formate in water/acetonitrile/formic acid (30:70:0.002, v/v) as a mobile phase at the flow rate of 0.2 mL/min at 25 °C [44]. The liquid chromatography system was connected to a Q-TOF MS spectrometer, MicrOTOFQII (Bruker Daltonics, Bremen, Germany), equipped with an ESI source.…”

“…We also reported that TTX and its analogs commonly give the major fragment ion at m/z 162 using ESI-triple quadrupole mass spectrometer [42]. This fragment ion has been widely used to detect TTX and its analogs in MRM (multiple reaction monitoring) mode by detecting m/z 320/162 transition in tandem mass spectrometry [40,43,44]. However, the structures of the major fragment ions of TTX and its analogs, including this m/z 162 ion, have not been determined using a high resolution mass spectrometer.…”

Risks for public health related to the presence of tetrodotoxin (TTX) and TTX analogues in marine bivalves and gastropods

“…The typical positive ionisation provides a TTX ion of 320 ( m / z ) (McNabb et al., ). Although the main product ion for TTX is 162 ( m / z ), other TTX analogues can generate this same ion, and hence the MRM mode is commonly used to identify different analogues (Shoji et al., ; Jang et al., ; McNabb et al., , ; Yotsu‐Yamashita et al., , ).…”

Study of the matrix effects of tetrodotoxin and its content in cooked seafood by liquid chromatography with triple quadrupole mass spectrometry

“…The quantitative ion pair m/z 320→302 is attributed to the transition [M+H] + →[M+H-H 2 O] + and the qualitative ion pair m/z 320→162 is attributed to the product ion of 2-aminohydroquinazoline in tandem MS [49]. Prior reports showed that almost all TTX analogs were associated with these two transitions in LC-MS/MS measurement [18,31,49]. Rodríguez et al and Pires et al reported possible TTX analogs [18,31] in pufferfish and + , respectively).…”