Liquid Chromatography with a Fluorimetric Detection Method for Analysis of Paralytic Shellfish Toxins and Tetrodotoxin Based on a Porous Graphitic Carbon Column

Abstract

Paralytic shellfish toxins (PST) traditionally have been analyzed by liquid chromatography with either pre- or post-column derivatization and always with a silica-based stationary phase. This technique resulted in different methods that need more than one run to analyze the toxins. Furthermore, tetrodotoxin (TTX) was recently found in bivalves of northward locations in Europe due to climate change, so it is important to analyze it along with PST because their signs of toxicity are similar in the bioassay. The methods described here detail a new approach to eliminate different runs, by using a new porous graphitic carbon stationary phase. Firstly we describe the separation of 13 PST that belong to different groups, taking into account the side-chains of substituents, in one single run of less than 30 min with good reproducibility. The method was assayed in four shellfish matrices: mussel (Mytillus galloprovincialis), clam (Pecten maximus), scallop (Ruditapes decussatus) and oyster (Ostrea edulis). The results for all of the parameters studied are provided, and the detection limits for the majority of toxins were improved with regard to previous liquid chromatography methods: the lowest values were those for decarbamoyl-gonyautoxin 2 (dcGTX2) and gonyautoxin 2 (GTX2) in mussel (0.0001 mg saxitoxin (STX)·diHCl kg−1 for each toxin), decarbamoyl-saxitoxin (dcSTX) in clam (0.0003 mg STX·diHCl kg−1), N-sulfocarbamoyl-gonyautoxins 2 and 3 (C1 and C2) in scallop (0.0001 mg STX·diHCl kg−1 for each toxin) and dcSTX (0.0003 mg STX·diHCl kg−1 ) in oyster; gonyautoxin 2 (GTX2) showed the highest limit of detection in oyster (0.0366 mg STX·diHCl kg−1). Secondly, we propose a modification of the method for the simultaneous analysis of PST and TTX, with some minor changes in the solvent gradient, although the detection limit for TTX does not allow its use nowadays for regulatory purposes.

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

“…However, it has the complication of the different fluorescence of other analogues (6-epi TTX is 20-fold more fluorescent than TTX, and 11-deoxy TTX is 100-fold less fluorescent than TTX (Yotsu et al, 1989;Pires et al, 2005). Although the simultaneous analysis of STX and analogues (paralytic shellfish poisoning (PSP) group) and TTX has been reported by one single LC-FL method (Rey et al, 2016), the differences in fluorescence between PSP and TTX is still a problem to solve.…”

Salinity Affects Saxitoxins (STXs) Toxicity in the Dinoflagellate Alexandrium pacificum, with Low Transcription of SXT-Biosynthesis Genes sxtA4 and sxtG

“…After boiling at 95 °C for 5 min, the samples were filtered using a 0.2 µm GVS syringe filter (GVS, Bologna, Italy). Toxin analysis was performed via high-pressure liquid chromatography-fluorescence detection (HPLC-FLD) (Waters, Milford, MA, USA) using the post-column reaction method following Rey et al [ 77 ], with slight modifications. In detail, the flow rate of binary pump (Waters, Milford, MA, USA) was 0.3 mL/min with solvent A: 0.075% ( v / v ) trifluoroacetic acid, and solvent B: 0.025% ( v / v ) trifluoroacetic acid.…”

Change in Paralytic Shellfish Toxins in the Mussel Mytilus galloprovincialis Depending on Dynamics of Harmful Alexandrium catenella (Group I) in the Geoje Coast (South Korea) during Bloom Season

“…PSTs were analyzed using HPLC (High-performance liquid chromatography) with postcolumn derivatization. [ 31 , 32 ]. HPLC analysis was performed using an Alliance 2690 Separations Module (Waters) fitted with porous graphitic carbon Hypercarb ® column (4.6 mm i.d.…”