Food Chemistry 136 (2013) 1584–1589

Contents lists available at SciVerse ScienceDirect

Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Comparison of sample preparation methods, validation of an UPLC–MS/MSprocedure for the quantification of tetrodotoxin present in marine gastropodsand analysis of pufferfish

Judith Kouassi Nzoughet a,⇑, Katrina Campbell a, Paul Barnes b, Kevin M. Cooper a, Olivier P. Chevallier a,Christopher T. Elliott a

a Institute of Agri-Food and Land Use, School of Biological Sciences, Queen’s University Belfast, Belfast BT9 5AG, UKb Agri-Food and Biosciences Institute, Belfast BT4 3SD, UK

a r t i c l e i n f o a b s t r a c t

Article history:Available online 8 February 2012

Keywords:Emerging biotoxinsTetrodotoxinUPLC–MS/MSValidationAccelerated solvent extraction

0308-8146/$ - see front matter � 2012 Elsevier Ltd. Adoi:10.1016/j.foodchem.2012.01.109

⇑ Corresponding author. Address: Institute of Agri-Biological Sciences, Queen’s University Belfast, StranNorthern Ireland, UK. Tel.: +44 28 9097 5564; fax: +4

E-mail address: jk.nzoughet@qub.ac.uk (J.K. Nzoug

Tetrodotoxin (TTX) is one of the most potent marine neurotoxins reported. The global distribution of thistoxin is spreading with the European Atlantic coastline now being affected. Climate change and increas-ing pollution have been suggested as underlying causes for this. In the present study, two different sam-ple preparation techniques were used to extract TTX from Trumpet shells and pufferfish samples. Bothextraction procedures (accelerated solvent extraction (ASE) and a simple solvent extraction) were shownto provide good recoveries (80–92%). A UPLC–MS/MS method was developed for the analysis of TTX andvalidated following the guidelines contained in the Commission Decision 2002/657/EC for chemical con-taminant analysis. The performance of this procedure was demonstrated to be fit for purpose. This studyis the first report on the use of ASE as a mean for TTX extraction, the use of UPLC–MS/MS for TTX analysis,and the validation of this method for TTX in gastropods.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Seafood has always been considered as nutritionally healthyand an important part of a balanced diet. However more attentionis now being shown to seafood safety due adverse changes in themarine environment believed to be caused by an increase in pollu-tion and global changes in climate. Over the past few decades, var-ious types of poisoning incidents have been reported (Blanco,Moroño, & Fernández, 2005; Botana, 2000; Daranas, Manuel, &Fernandez, 2001; McMahon & Silke, 1996; Munday, 2008) and thishas led to growing concerns with regard to the consumption ofseafood. Both public health and aquaculture industries have beenaffected by such marine toxin associated poisoning.

The occurrence of TTX has been mainly reported in Asian coun-tries and more specifically in Japan (Gessner & McLaughlin, 2008),with reports of many cases of TTX-food poisoning related to con-suming pufferfish, served as a delicacy called ’fugu’ (Raybouldet al., 1992). However, TTX distribution is spreading, with theUnited States (Nunez-Vazquez, Yotsu-Yamashita, Sierra-Beltran,Yasumoto, & Ochoa, 2000) and the European Atlantic coast being

ll rights reserved.

food and Land Use, School ofmillis Road, Belfast BT9 5AG,4 28 9097 6513.het).

now affected (Katikou, Georgantelis, Sinouris, Petsi, & Fotaras,2009; Rodriguez et al., 2008). Indeed, TTX-contaminated pufferfish(Katikou et al., 2009; Nunez-Vazquez et al., 2000) and Trumpetshells (Rodriguez et al., 2008) have been reported in these areas.

TTX is one of the most potent marine biotoxins reported. Thetoxin acts by blocking site 1 of the voltage-gated sodium channel,resulting in respiratory paralysis and often death in human. TTXhas an LD50 in mammals of 2–10 lg/kg intravenously and10–14 lg/kg subcutaneously (Alcaraz, Whipple, Gregg, Andressen,& Grant, 1999). Unlike many other marine biotoxins which are ofalgal origin, bacteria were suggested as probable primary sourceof TTX; though the ultimate origin of TTX is still a matter of debate(Williams, 2010). TTX is believed to be produced by bacteriaassociated with TTX-bearing animals and their food (Simidu,Kita-Tsukamoto, Yasumoto, & Yotsu, 1990). Symbiotic bacteria(Vibrio, Pseudomonas, Shewanella or Alteromonas) isolated fromvarious sources (starfish, xanthid crab, pufferfish, and red alga)have been confirmed as TTX-producers (Noguchi & Arakawa,2008; Noguchi, Arakawa, & Takatani, 2006; Yotsu-Yamashita,Mebs, Kwet, & Schneider, 2007). The distribution of TTX in puffer-fish bodies appears to be species-specific. In marine species, theliver and ovary generally have the highest toxicity, followed byintestines and skin. Muscles and/or testis are non-toxic or weaklytoxic, except for in Lagocephalus lunaris and Chelonodon patoca(Noguchi & Arakawa, 2008). Currently, there are no EU regulatory

J.K. Nzoughet et al. / Food Chemistry 136 (2013) 1584–1589 1585

limits specifically set for TTX but for muscle paralysing toxins,(referred to as Paralytic Shellfish Poisons (PSP) toxins) with whichthey share the same mechanism of action, symptoms and physicalsigns of intoxication; the legal limit is stated as 800 lg of saxitoxinequivalents per kg of shellfish meat. In the EU, there are severe lim-itations in the legislation in relation to TTX contamination in sea-food. Regulation (EC) no 854/2004 stipulates that: ‘‘Fisheryproducts derived from poisonous fish of the following familiesmust not be placed on the market: Tetraodontidae, Molidae, Dio-dontidae and Canthigasteridae. Fishery products containing bio-toxins such as ciguatoxin or muscle-paralysing toxins must notbe placed on the market. However fishery products such as gastro-pods complying with Regulation 853/2004, may be placed on themarket’’. In Japan, the government has set regulatory limits forTTX in food of 2000 lg/kg TTX equivalents (Hungerford, 2006)whereas the United States has a zero tolerance level due to the factthat no product sold legally in the United States is expected to con-tain this toxin (Yakes, Deeds, White, & Degrasse, 2011). With therecently observed occurrence of TTX contaminated seafood in theAtlantic area, which can be life threatening upon consumption,reliable analytical methods to perform TTX analysis are now essen-tial. In the present study, two different sample preparation tech-niques were used to extract TTX from gastropods (Trumpet shellsCharonia lampas lampas) and pufferfish (Lagocephalus sceleratus)harvested from the European Atlantic coastal area. An acceleratedsolvent extraction (ASE) system operating at elevated temperatureand pressure, and a simple solvent extraction procedure notinvolving heat treatment, were applied as sample preparation. AnUPLC–MS/MS method was developed for TTX and the overall pro-cedure was validated using the simple solvent extraction andTrumpet shells, following the guidelines contained in the Commis-sion Decision 2002/657/EC.

2. Material and methods

2.1. TTX standard, solvents and reagents

Tetrodotoxin standard (min. 96% purity by HPLC, IR, NMR), puri-fied from fugu fish, was purchased from Latoxan (Valence, France).Acetonitrile, water and acetic acid were of HPLC grade and wereobtained from Sigma Aldrich (Poole, UK).

2.2. Test materials

Trumpet shells (Charonia lampas lampas) were collected in 2010on the North coast of Portugal, and screened using a surface plas-mon resonance biosensor assay to be tetrodotoxin negative (man-uscript in preparation). Naturally contaminated Trumpet shells(Charonia lampas lampas) with TTX were harvested in October2009 from Angeiras Coast, on the Portuguese littoral. TTX incurredpufferfish (Lagocephalus sceleratus) materials were harvested fromGreece in 2007. Pufferfish sample 1 was collected in the NorthWest part of Aegean Sea in the area of Horeyto, in June 2007 andpufferfish sample 2 was collected in November 2007 from theSoutheast Aegean Sea, near the island of Rhodes. The levels previ-ously detected by MBA for these samples were: less than 1100 and1690 lg/kg for samples 1 and 2, respectively (Katikou et al., 2009).In addition, 27 Trumpet shells (Charonia lampas lampas) samples(meat, viscera and whole animal homogenate) collected in 2010on the North coast of Portugal were analysed. Samples were storedfrozen prior to use.

2.3. Preparation of solvent and matrix-matched calibration standards

A stock TTX standard solution (1 mg/mL) was serially dilutedwith 0.03 M acetic acid solution to provide seven calibration stan-

dards (5, 10, 25, 50, 100, 200, and 500 ng/mL). These solutions wereused for method development and optimisation. Matrix-matchedstandards were used for quantification during the validation study.These were prepared daily by adding the appropriate amount ofTTX to 2 g of negative Trumpet shell sample (whole animal homog-enate) to provide seven levels matrix-matched calibration stan-dards (50, 100, 200, 500, 1000, 2000, and 3000 lg/kg). Thecalibration range was chosen in order to include concentrationsof TTX currently found in various aquatic species (Deeds, White,Etheridge, & Landsberg, 2008; Huang, Lin, & Lin, 2008; Jang, Lee,& Yotsu-Yamashita, 2010; Katikou et al., 2009; Rodriguez et al.,2008) and also to encompass the EU regulatory limit (800 lg/kg)for PSP toxins (Regulation (EC) no 853/2004). Matrix-matched cal-ibration standards were extracted with 0.03 M acetic acid solutionfollowing the procedures described below (Section 2.4.1).

2.4. Sample preparation

Trumpet shells (Charonia lampas lampas) were excised fromtheir shells. Trumpet shell and pufferfish samples (20–100 g) werehomogenised for 3 min at 13500 rpm, using a high speed homoge-nizer (Ultra-Turrax� T25 basic IKA�). Aliquots (2 g wet weight) ofhomogenised tests materials, i.e., negative Trumpet shells (wholeanimal homogenate), pre-fortified Trumpet shells (whole animalhomogenate), 27 additional Trumpet shells (meat, viscera, andwhole animal homogenate), naturally contaminated Trumpet shell(viscera and muscle) and pufferfish (muscle) samples, were ex-tracted using two different procedures, one involving heat treat-ment (see Section 2.4.1).

2.4.1. Accelerated solvent extraction (ASE) and solvent extractionprocedures

Accelerated solvent extraction (ASE) is a technique which hasbeen seldom applied to marine biotoxins analysis and to the bestof our knowledge, not previously applied as a method for samplepreparation for TTX analysis. The ASE system used was ASE 350�

Accelerated solvent extractor, an automated system for extractingorganic compounds from a variety of solid samples (ASE 350 oper-ator’s manual), thus there is the necessity to lyophilise the samplesbefore application to the system. It has the advantage of being anautomated technique which operates at elevated temperatureand pressure, thereby increasing the efficiency of the extractionprocess. In the present study test materials were first lyophilised,before being extracted with 0.03 M acetic acid solution at 75 �C,1500 psi, in one cycle. Each sample was extracted for 15 min andprovided a 10 ml extract volume.

For the solvent extraction procedure, test materials were alsoextracted with 0.03 M acetic acid solution using a ratio weight tovolume of 1:5. The mixture was vortex-mixed for one minute, son-icated for 10 min and finally centrifuged at 4500 rpm at 4 �C for10 min.

Both resultant extracts were spin-filtered through 3000 molec-ular weight cut-off Amicon Ultracel filters, at 10000g for 30 min,prior to UPLC–MS/MS analysis.

2.4.2. Assessment of recoveryNegative Trumpet shells (whole animal homogenate) were used

to assess the recovery. The absolute recovery of both extractionprocedures was determined by comparing UPLC–MS/MS peakareas of TTX in pre-fortified test material extract (fortified beforeextraction procedure) with post-fortified extract (fortified afterextraction procedure). The absolute recovery was assessed at 400and 800 lg/kg fortified TTX; six individual replicates were usedfor each concentration level. Statistical analysis was carried outusing SPSS Statistics 18.0 package. The Student’s (two-tailed) t-testwas performed to compare absolute recoveries from ASE and

1586 J.K. Nzoughet et al. / Food Chemistry 136 (2013) 1584–1589

manual solvent extraction procedures. The level of significancewas selected at p < 0.05.

2.4.3. Method validationNegative Trumpet shell materials (i.e. free from TTX) were for-

tified at three concentrations, i.e. 400, 800 and 1200 lg/kg, equiv-alent to 0.5, 1 and 1.5 times the regulatory limit for PSP toxins forthe purpose of evaluating the method performance. Fortified mate-rials were extracted following the solvent extraction procedure(see Section 2.4.1) with seven independent replicates for each con-centration. This procedure was performed over three analyticalruns on three different days. The method was validated accordingto guidelines contained in Commission Decision 2002/657/EC.One-way analysis of variance (ANOVA) was applied to the 21 val-idation data in order to generate repeatability precision coeffi-cients of variation. The mean method recovery, precision underrepeatability conditions, decision limit (CCa), detection capability(CCb) and calibration curves linearity were assessed. Limit ofdetection (LOD) and limit of quantification (LOQ) based on signalto noise ratios were also assessed.

2.5. UPLC–MS/MS (ultra performance liquid chromatography coupledto tandem mass spectrometry)

An Acquity UPLC system coupled to a Quattro Premier XE massspectrometer (Waters, Manchester, UK) equipped with a Z-SprayESI source was used for sample analysis. The UPLC system wasequipped with an Acquity UPLC BEH HILIC column (2.1 �100 mm, 1.7 lm) maintained at 30 �C. The mobile phase consistedof 5% acetonitrile (ACN) in channel A, and 95% ACN containing 1%acetic acid (pH 3.5) in channel B. The system was programmedto hold initial condition (95% B) for 3.0 min and then to performgradient elution from 95% to 50% B over a 5.5 min period, held at50% B for 2.5 min, return to initial conditions over 3 min and thenheld at these conditions for a further 4 min. The flow rate was0.4 mL/min and the injection volume was 3.5 lL (partial loop withneedle overfill mode). The mass spectrometer operated in electro-spray positive mode with the capillary voltage set at 3.7 kV. Thesource temperature was set at 120 �C, desolvation temperature at350 �C, cone gas flow at 80 L nitrogen/h with desolvation gas flowat 850 L nitrogen/h. Data acquisition was in multiple reaction mon-itoring mode (MRM). The cone voltage and collision energy wereoptimised for TTX by standard infusion. The precursor/product ionsmonitored were 319.92 > 161.80 (cone voltage 40 V, collision en-ergy 35 eV) and 319.92 > 302.00 (cone voltage 40 V, collision en-ergy 25 eV). MassLynx 4.1 with QuanLynx software was used fordata processing.

Fig. 1. Chemical structure of tetrodotoxin.

3. Results and discussion

3.1. Extraction procedures recoveries

The extraction procedure using the ASE resulted in a meanrecovery of 88% for TTX at 400 lg/kg compared to the solventextraction procedure which gave a mean value of 80% TTX recoveryat the same spiking level. At 800 lg/kg spiked TTX, the absoluterecovery using both procedure was 92%. A t-test was performedto assess the TTX recoveries obtained by the two different extrac-tion procedures, i.e., ASE and solvent extraction. The results of thetest indicated that there is no significant difference (p > 0.05) be-tween the two procedures toxin recovery at 800 lg/kg whilst thisdifference was significant (p < 0.05) at 400 lg/kg.

The ASE technique was successfully applied for the extraction ofTTX from Trumpet shell samples and was found to provide aslightly better toxin recovery. However, the simpler solvent extrac-

tion procedure was selected to undertake the full method valida-tion because it was found to be overall faster compared to theASE procedure which required a day long freeze drying step. Previ-ously reported sample preparation techniques for TTX contami-nated materials involving manual extraction procedures in somecases used a boiling bath (Huang et al., 2008; Jang et al., 2010)whereas in other cases no heat treatment was applied (Chenet al., 2011; Fong, Tam, Tsui, & Leung, 2011; Jen et al., 2008; Rodri-guez et al., 2008). The appliance of heat as part of the extractionprocedure was investigated in the present study. As explainedabove, the ASE extraction has led to a better recovery at lower le-vel, 400 lg/kg spiked TTX.

3.2. UPLC–MS/MS method development and optimisation

Due to the nature of TTX (Fig. 1) a HILIC (hydrophobic interac-tion chromatography) column was selected to overcome the chal-lenge of retaining and separating polar, basic analytes as TTX is ahydrophilic toxin, polar, weak base with a pKa of 8.76. Toxin stan-dard solutions prepared as described previously (Section 2.3.)showed good linearity over the calibration ranges: 5–500 ng/mlfor solvent standards and 50–3000 lg/kg for matrix-matched stan-dards; correlation coefficients (r2) were 0.9973 and 0.9851, respec-tively. Matrix effect was observed during this study; to compensatefor that, matrix-matched standard was used for TTX quantification.Fig. 2 presents a chromatogram obtained for (A) an extracted ma-trix calibration standard and (B) a naturally contaminated puffer-fish sample, with the monitored MRM transitions 319.92 >302.00 (base peak) and 319.92 > 161.80 (confirmatory peak). Thesecond transition was monitored to confirm the analyte identity(TTX). The limit of detection (LOD) and limit of quantification(LOQ) were respectively 0.074 and 0.123 ng/ml for the solventstandards, and 7.3 and 24.5 lg/kg for the matrix matched stan-dards. LOD and LOQ were determined at signal to noise ratios equalto 3 and 10, respectively. The method was further validated underoptimized conditions and using a matrix-matched calibrationcurve around the 800 lg/kg legal limit for muscle paralysing tox-ins. This study also presents the first use of UPLC chromatographyfor TTX analysis since the previous published methods have mainlyutilised HPLC equipment. Comparing the present method to otherswho also assessed their detection limit in gastropod extract(100 ng/ml) (Huang et al., 2008), the method presented here showsa better sensitivity, i.e., 7.3 lg/kg (matrix matched standard), cor-responding to 1.46 ng/ml. UPLC offers significant advantages com-pared to HPLC, including the reduction of the mobile phase flowrate and injection volume. It also uses columns with smaller parti-cle sizes (1.7 lm) which provide increased efficiency and resolu-tion, while reducing run times. The particle technology has alsothe advantage of being mechanically strong to operate at elevatedpressures, up to 15000 psi compared to pressure of 6000 psi forconventional HPLC.

Fig. 2. UPLC–MS/MS chromatograms of tetrodotoxin MRM transitions 319.92 > 302.00 (base peak) and 319.92 > 161.80 (confirmatory peak) in (A) extracted matrixcalibration standard (1000 lg/kg), and (B) naturally contaminated Pufferfish sample.

J.K. Nzoughet et al. / Food Chemistry 136 (2013) 1584–1589 1587

3.3. Method validation

The performance parameters assessed for the validation of themethod were as follows: recovery (accuracy), precision underrepeatability conditions, decision limit (CCa) and detection capa-bility (CCb). The method validation was performed followingguidelines in Commission Decision 2002/657/EC. Table 1 summa-rises the data obtained. Extracts were injected onto the UPLC–MS/MS system described above (Section 2.5.) and TTX concentra-tion was calculated by comparing peak area of the analyte basepeak with the peak areas in the matrix-matched calibration curve.The ion ratio between peak areas of the base and confirmatoryMRM transitions was monitored throughout the study as an addi-tional tool to confirm the presence of TTX in the samples. Ion ratiosin samples consistently fell within the ±20% tolerance of the mean

Table 1Mean recovery, repeatability precision parameters expressed as CV (%), decision limit(CCa) and detection capability (CCb) for Trumpet shell fortified at three tetrodotoxinconcentrations (n = 7 independent replicates).

TTX fortification level 400 lg/kg 800 lg/kg 1200 lg/kg

Mean TTX detected (lg/kg) 435.6 898.5 1232.6Mean recovery (%) 109 112 103Within day CV (%) 14.6 19.2 9.2Between day CV (%) 3.1 4.0 3.2Intermediate precision CV (%) 14.9 19.6 9.7CCa 1025.2 lg/kgCCb 1250.3 lg/kg

ratio in the calibration standards as laid down in Commission Deci-sion 2002/657/EC.

Under Commission Decision 2002/657, for compounds with apermitted MRL (Maximum residue limit), a non-compliant sampleis defined as one containing the compound at a concentration abovethe MRL with at least 95% certainty. The decision limit (CCa) wascalculated on the basis of the 800 lg/kg regulatory limit for PSP tox-ins, to define the observed concentration at which samples could beconsidered as non-compliant. Based on the fortified validation sam-ples, the concentration at the regulatory limit plus 1.64 times thestandard deviation of the within laboratory reproducibility equalsthe decision limit (a error = 5%). The decision limit obtained was1025.2 lg/kg. Ideally this CCa should be lower, but its calculationwas adversely affected by the method’s within day precision. Theavailability of an isotopically labelled internal standard for TTXwould greatly improve assay precision and lower the CCa. The meanTTX recovery during validation was good, 109%, 112% and 103% at400, 800 and 1200 lg/kg, respectively (Table 1). Reported validated

Table 2Tetrodotoxin detected in naturally contaminated Trumpet shell (Charonia lampaslampas) and immigrant Pufferfish (Lagocephalus sceleratus) harvested from EU waters.

Sampledetails

Trumpetshellviscera

Trumpetshellmuscle

Pufferfishmuscle sample1

Pufferfishmuscle sample2

TTXdetected(lg/kg)

22.4 66.6 1199 2957

1588 J.K. Nzoughet et al. / Food Chemistry 136 (2013) 1584–1589

methods for TTX extraction and analysis by LC–MS/MS have beenperformed using human urine and plasma (Fong et al., 2011) andTTX solvent standards (Chen et al., 2011). Methods recovery, linear-ity, precision, accuracy, LOD and LOQ have been assessed, but notthe CCa and CCb. Rodriguez et al. (2008) were first to report theoccurrence of TTX in European Trumpet shell (Charonia lampas lam-pas). They examined their extracts by confocal microscopy, mousebioassay and LC–MS, but these methods had not been validatedon this matrix.

3.4. Application of the method to incurred TTX materials and 27Trumpet shel samples

Naturally contaminated Trumpet shell (Charonia lampas lampas)and Puffer fish (Lagocephalus sceleratus) were extracted followingthe solvent extraction procedure (Section 2.4.1.) and analysed byUPLC–MS/MS (Section 2.5) in order to determine their TTX content.Incurred Trumpet shell samples (viscera and muscle) were foundto contain substantially less TTX as compared to the incurred Puf-fer fish samples (muscle). The results are illustrated in Table 2 andemphasise the potential threat of TTX for seafood and fish har-vested in European waters. The levels of TTX found using theprocedure reported here (1199 and 2957 lg/kg for samples 1 and2, respectively) are higher than the level reported by Katikouet al. (2009) using the mouse bio assay (less than 1100 and1690 lg/kg for samples 1 and 2, respectively). TTX contentsassessed by UPLC–MS/MS and mouse bioassay do not correlate,though both methods agree on the high amount of toxin presentin the pufferfish samples analysed. The 27 Trumpet shell materials(meat, viscera and whole animal homogenate) were also analysedand tested TTX negative.

4. Conclusions

Two different sample preparation approaches were used to ex-tract TTX from seafood and both were shown to provide goodrecoveries. This study is the first report on the use of ASE as a meanfor TTX extraction and the method was found to be highly satisfac-tory in recovering TTX from food matrices. This automated tech-nique though reducing hands on time during the extractionprocess was found to be overall longer compared to the solventextraction procedure used because of the one day freeze dryingstep required. Thus a simple and rapid solvent based extractionmethod for TTX extraction was trialled and found to be fit for pur-pose. This was combined with a UPLC–MS/MS system and vali-dated following the guidelines contained in the CommissionDecision 2002/657/EC. The performance of the method was foundto be acceptable but could be improved with the incorporation of adeuterated internal standard. Analytical methods such as UPLC–MS/MS are very useful for detecting emerging toxins such asTTX, which have been shown to occur in European waters (Katikouet al., 2009; Rodriguez et al., 2008). This is the first published re-port of a validated method for the detection and quantification ofTTX in gastropods. In the opinion of the authors, Commission Deci-sion 2002/657/EC should be applied for the validation of all meth-ods developed for the detection and quantification of smallmolecular weight contaminants present in foods, to bring about abetter degree of harmonisation to the field of food analysis andto allow all stakeholders to compare methods and make selectionsof procedures for inclusion in control and monitoring programmes.

Acknowledgements

This research was funded by The Interreg Programme throughthe project ‘Atlantox: Advanced Tests about New Toxins appeared

in the Atlantic Area’. The authors would like to thank Professor Vi-tor Vasconcelos, Joana Azevedo, Cowan Higgins, and Panagiota Kat-ikou for providing the test materials used during this study and toBrett Greer for assistance with mass spectrometry analysis.

References

Alcaraz, A., Whipple, R. E., Gregg, H. R., Andresen, B. D., & Grant, P. M. (1999).Analysis of tetrodotoxin. Forensic Science International, 99, 35–45.

ASE 350 operator’s manual, Dionex Corporation, Document N� 065220, revision 02,September 2008.

Blanco, J., Moroño, A., Fernández, M.L. (2005). Toxic episodes in shellfish, producedby lipophilic phycotoxins: An overview. Revista Galega de Recursos Mariños(Monog.): 1, 1–70. Xunta de Galicia.

Botana, L. M. (2000). Seafood and Freshwater toxins: pharmacology, physiology, anddetection. New York: Marcel Dekker.

Chen, X. W., Liu, X. H., Jin, Y. B., Li, S. F., Bi, X., Chung, S., et al. (2011). Separation,identification and quantification of tetrodotoxin and its analogs by LC–MSwithout calibration of individual analogs. Toxicon, 57, 938–943.

Commission Decision 2002/657/EC, implementing Council Directive 96/23/ECconcerning the performance of analytical methods and the interpretation ofresults. Official Journal of the European Communities, L221, 8–36.

Daranas, A. H., Manuel, N., & Fernandez, J. J. (2001). Review Toxic marinemicroalgae. Toxicon, 39, 1101–1132.

Deeds, J. R., White, K. D., Etheridge, S. M., & Landsberg, J. H. (2008). Concentrationsof saxitoxin and tetrodotoxin in three species of puffers from the Indian riverlagoon, Florida, the location for multiple cases of Saxitoxin Puffer Poisoningfrom 2002 to 2004. Transactions of the American Fisheries Society, 137,1317–1326.

Fong, B. M. W., Tam, S., Tsui, S. H., & Leung, K. S. Y. (2011). Development andvalidation of a high-throughput double solid phase extraction–liquidchromatography–tandem mass spectrometry method for the determination oftetrodotoxin in human urine and plasma. Talanta, 83, 1030–1036.

Gessner, B. D., & McLaughlin, J. B. (2008). Seafood and freshwater toxins.Pharmocology, Physiology and Detection (2nd ed.). CRC Press: Boca Raton, FL.

Huang, H. N., Lin, J., & Lin, H. L. (2008). Identification and quantification oftetrodotoxin in the marine gastropod Nassarius by LC–MS. Toxicon, 51,774–779.

Hungerford, J. M. (2006). Committee on natural toxins and food allergens – Marineand freshwater toxins. The Journal of AOAC International, 89(1), 248–269.

Jang, J. H., Lee, J. S., & Yotsu-Yamashita, M. (2010). LC/MS analysis of tetrodotoxinand its deoxy analogs in the marine PufferFish Fugu niphobles from the SouthernCoast of Korea, and in the Brackishwater PufferFishes Tetraodon nigroviridis andTetraodon biocellatus from Southeast Asia. Marine Drugs, 8, 1049–1058.

Jen, H. C., Lin, S. J., Tsai, Y. H., Chen, C. H., Lin, Z. C., & Hwang, D. F. (2008).Tetrodotoxin poisoning evidenced by solid-phase extraction combining withliquid chromatography–tandem mass spectrometry. Journal of ChromatographyB, 871(2008), 95–100.

Katikou, P., Georgantelis, D., Sinouris, N., Petsi, A., & Fotaras, T. (2009). First reporton toxicity assessment of the Lessepsian migrant pufferfish Lagocephalussceleratus (Gmelin, 1789) from European waters (Aegean Sea, Greece). Toxicon,54, 50–55.

McMahon, T., & Silke, J. (1996). Winter toxicity of unknown aetiology in mussels.Harmful Algae News, 14, 2.

Munday, R. (2008). Occurrence and Toxicology of Palytoxins. In: Botana, L. M. (2nded). Seafood and Freshwater toxins: pharmacology, physiology, and detection(pp. 693–709). CRC Press (Taylor and Francis Group): Boca Raton., FL.

Noguchi, T., & Arakawa, O. (2008). Tetrodotoxin-distribution and accumulation inaquatic organisms, and cases of human intoxication. Marine Drugs, 6, 220–242.

Noguchi, T., Arakawa, O., & Takatani, T. (2006). TTX accumulation in pufferfish.Comparative Biochemistry and Physiology – Part D: Genomics and Proteomics, 1,145–152.

Nunez-Vazquez, E. J., Yotsu-Yamashita, M., Sierra-Beltran, A. P., Yasumoto, T., &Ochoa, J. L. (2000). Toxicities and distribution of tetrodotoxin in the tissues ofpuffer fish found in the coast of the Baja California Peninsula. Mexico, Toxicon,38, 729–734.

Raybould, T. J. G., Bignami, G. S., Inouye, L. K., Simpson, S. B., Byrnes, J. B., Grothaus,P. G., et al. (1992). A monoclonal antibody-based immunoassay for detectingtetrodotoxin in biological samples. Journal of Clinical Laboratory Analysis, 6,65–72.

Regulation (EC) no 853/2004 of the European parliament and of the council of 29April 2004 laying down specific hygiene rules for on the hygiene of foodstuffs.Chapter v: health standards for live bivalve molluscs. Official Journal of theEuropean, Communities, L139.

Regulation (EC) no 854/2004 of the European parliament and of the council Of 29April 2004 Laying down specific rules for the organisation of official controls onproducts of animal origin Intended for human consumption. Official Journal ofthe European, Communities, L226.

Rodriguez, P., Alfonso, A., Vale, C., Alfonso, C., Vale, P., Tellez, A., et al. (2008). FirstToxicity report of tetrodotoxin and 5,6,11-trideoxyttx in the Trumpet shellCharonia lampas lampas in Europe. Analytical Chemistry, 80, 5622–5629.

Simidu, U., Kita-Tsukamoto, K., Yasumoto, T., & Yotsu, M. (1990). Taxonomy of fourmarine bacterial strains that produce tetrodotoxin. International Journal ofSystematic Bacteriology, 40(4), 331–336.

J.K. Nzoughet et al. / Food Chemistry 136 (2013) 1584–1589 1589

Williams, B. L. (2010). Behavioral and chemical ecology of marine organisms withrespect to tetrodotoxin. Marine Drugs, 8, 381–398.

Yakes, B. J., Deeds, J., White, K., & Degrasse, S. L. (2011). Evaluation of surfaceplasmon resonance biosensors for detection of tetrodotoxin in food matrices

and comparison to analytical methods. Journal of Agricultural and FoodChemistry, 59, 839–846.

Yotsu-Yamashita, M., Mebs, D., Kwet, A., & Schneider, M. (2007). Tetrodotoxin andits analogue 6-epitetrodotoxin in newts (Triturus spp.; Urodela, Salamandridae)from southern Germany. Toxicon, 50, 306–309.