Multi-Toxin Quantitative Analysis of Paralytic Shellfish Toxins and Tetrodotoxins in Bivalve Mollusks with Ultra-Performance Hydrophilic Interaction LC-MS/MS—An In-House Validation Study

Abstract

Ultra-performance hydrophilic interaction liquid chromatography tandem mass spectrometry system (UP-HILIC–MS/MS) was used in multi-toxin analysis of paralytic shellfish toxins (PSTs) and tetrodotoxins (TTXs) in sample matrices from bivalve molluscan species commercially produced for human consumption in Sweden. The method validation includes 17 toxins of which GTX6 and two TTX analogues, TTX and 4,9-anhydroTTX, were previously not analyzed together with hydrophilic PSTs. 11-deoxyTTX was monitored qualitatively with a non-certified reference standard. The performance of the method was evaluated for selectivity, repeatability, and linearity by analyzing spiked samples which generated linear calibration curves across the concentration ranges used (R2 > 0.99). The in-house reproducibility (RSD) was satisfactory including the LOD and LOQ for both PST and TTX toxins being far below their regulatory action limits. The major advantage of the method is that it allows direct confirmation of the toxin identity and specific toxin quantification using a derivatization-free approach. Unlike the PST-chemical methods used in routine regulatory monitoring until now for food control, the UP-HILIC-MS/MS approach enables the calibration set-up for each of the toxin analogs separately, thereby providing the essential flexibility and specificity in analysis of this challenging group of toxins. The method is suitable to implement in food monitoring for PSTs and TTXs in bivalves, and can serve as a fast and cost-efficient screening method. However, positive samples would, for regulatory reasons still need to be confirmed using the AOAC official method (2005.06).

Electrophysiological Evaluation of Pacific Oyster (Crassostrea gigas) Sensitivity to Saxitoxin and Tetrodotoxin

“…It is likely that other variables, such as filtration activity and toxin assimilation, are involved in PST bio-accumulation in bivalve molluscs. Additionally, the present results show that C. gigas nerves are resistant to TTX, whereas recent studies point out controversial results concerning this toxin and analogues bio-accumulation in oysters, even using optimized and in-house validated methods [20][21][22]. This further supports that the relationship between toxin load in bivalve molluscs and toxin-sensitivity of nerves is more complex than expected.…”

Tetrodotoxins in French Bivalve Mollusks—Analytical Methodology, Environmental Dynamics and Screening of Bacterial Strain Collections

“…This latter condition was still considered appropriate for our screening purpose but will deserve more optimization in the future. Overall, matrix effects for TTXs appear to depend on the analog (−55 vs. +82% for TTX and 11-deoxyTTX in Patria et al [38], +41 for TTX vs. −29/43% for the other three analogs in oyster here), the matrix (from −45 to +23% in the different organs of oyster [37], −61% in scallop but +29% in mussel in Rey et al [40], +355% in bacteria here), the analyte concentration (−1 up to +183% at high vs. low concentration in Bordin et al [35]) and is possibly also chromatography/mass spectrometry-dependent (e.g., −17% in Turner et al [21] vs. −55% in Patria et al [38], based on comparison of published data with the EURLMB-protocol). Therefore, matrix effects should be determined and corrected, especially if matrix-matched calibration curves are not used.…”

“…Many of the published methods about the analysis of TTXs in bivalves by HILIC-MS/MS are based on the valuable work of Turner et al [21] that led to the protocol adopted by the European Union Reference Laboratory for Marine Biotoxins [33] (e.g., [26,28,[34][35][36]). This method of reference uses an extraction in acetic acid 1%, a boiling step followed by a graphitized carbon SPE clean-up and a dilution in ACN before analysis, yielding recoveries between 66 ± 2% [37] and 84 ± 18% [21] for the oyster whole flesh matrix and 68 ± 11/20% up to 77 ± 11% for mussel matrix [21,35,38]. In the present study, similar recoveries were obtained after a graphitized carbon SPE step only when using the GCB cartridge (65 ± 11%), while they were unexpectedly poor with the ENVI-carb cartridge despite an identical protocol (<5%).…”

“…Unfortunately, they were not always assessed in previous studies. Despite the use of an SPE clean-up, the reported matrix effects can still be as high as 61 or 82% for TTX analogs in bivalves (e.g., [38,40]), while a significant dilution of the extract before analysis may be a good alternative (e.g., matrix effects of <9% in mussel and oyster matrices after a 10-fold dilution [39] compared to the 70-80% ion suppression after a two-fold dilution only [27]). These observations confirmed that our sample preparation led to satisfactory matrix effects (ca.…”

Revisiting the HPLC-FLD Method to Quantify Paralytic Shellfish Toxins: C3,4 Quantification and the First Steps towards Validation

“…Over the years, several methods [ 4 , 5 , 6 , 7 , 8 ] have been developed and/or optimized, aiming to improve analytical parameters (e.g., sensitivity and specificity). Most of them require mass spectrometry techniques, implying higher costs than the methods currently in use (e.g., AOAC Official Method 2005.06 [ 9 , 10 ]).…”