DOI 10.1515/revac-2012-0020 Rev Anal Chem 2013; 32(1): 15–34

Ana M. Botana , Paz Otero , Paula Rodr í guez , Amparo Alfonso and Luis M. Botana * Current situation on analysis of marine toxins

Abstract: Marine toxins are a food safety concern world- economic losses. The microalgae are usually present in wide. This review discusses current analytical methods the plankton in low concentrations, but under some envi- for those toxins that are legally regulated in Europe, ronmental conditions they can rapidly multiply in dense namely domoic acid, saxitoxin, okadaic acid, yessotoxin, blooms. These microalgae produce phycotoxins, which pectenotoxin and azaspiracids, and all their analogues, accumulate in the digestive organs of filter-feeding shell- and those that are not regularly monitored but are a threat fish, zooplankton and herbivorous fishes and through to humans, such as tetrodotoxin, ciguatoxins and cyclic the trophic chain can produce human intoxications. The imines. Because of legislative changes that were imple- toxins are generally considered to be secondary metabo- mented in 2011, most legally required methods are chro- lites and therefore not essential to the basic metabolism matographic. Saxitoxin and domoic acid are monitored and growth of the microorganism (Van Dolah 2000 ). These by high-performance liquid chromatography with fluo- episodes previously regarded as sporadic and localised rescent and ultraviolet detection, respectively. All other phenomena and restricted to certain geographic areas toxins are monitored nowadays by means of chromato- have started to show up as recurrent and ubiquitous in graphic separation and mass spectrometry detection. any coast of the world and sometimes cause great damage to the local fishing industry and also to the natural com- Keywords: analytical standards; chromatography; marine munities of marine organisms (Vieites and Cabado 2008 ). toxins; mass spectrometry. Different countries have established standards about the total amount of phycotoxins that can be present in seafood products and also about the testing methods *Corresponding author: Luis M. Botana, Department of for detecting these molecules. Legal requirements for Pharmacology, Veterinary School , Faculty Veterinaria, Universidad de Santiago de Compostela, Campus de Lugo, Lugo 27002, Spain , paralytic shellfish toxins [usually named paralytic shell- e-mail: [email protected] fish poisoning (PSP)], amnesic shellfish toxins [amnesic Ana M. Botana: Department of Analytical Chemistry, Universidad de shellfish poisoning (ASP)], lipophilic toxins, ciguatoxins Santiago de Compostela, Campus de Lugo, Lugo 27002, Spain (CTXs), brevetoxins or tetrodotoxins (TTXs) have been set Paz Otero: Department of Pharmacology, Veterinary School, Faculty around the world (Rodrí guez-Velasco 2008). The Euro- Veterinaria, Universidad de Santiago de Compostela, Campus de Lugo, Lugo 27002 , Spain pean legislation establishes a maximum level of toxins Paula Rodr í guez: Department of Pharmacology, Veterinary School , in bivalve molluscs, echinoderms, tunicates and marine Faculty Veterinaria, Universidad de Santiago de Compostela, gastropods (the whole body or any part edible separately) Campus de Lugo, Lugo 27002 , Spain of 800 μ g of PSP equivalent/kg, 20 mg of domoic acid Amparo Alfonso: Department of Pharmacology, Veterinary School , (DA)/kg, 160 μ g of okadaic acid (OA) equivalents/kg, 160 Faculty Veterinaria, Universidad de Santiago de Compostela, μ g of azaspiracids (AZAs) equivalents/kg and 1 mg of yes- Campus de Lugo, Lugo 27002 , Spain sotoxin equivalent/kg, and the total absence of TTX and CTX (European Commission 2004a ) (see structures in Figure 1 ). To check compliance with the limits laid down, several methods including mouse bioassay (MBA), high- Introduction performance liquid chromatography (HPLC) with fluo- rescence (FL), ultraviolet (UV) or mass spectrometry (MS) Phycotoxins are an acute worldwide seafood safety detection, immune assays or functional assays have been concern; hence, their detection and quantification in food proposed. However, not all these methods are officially with reliable methods is important. The methods must be accepted as a validation process is necessary (European validated according to international guidelines to ensure Commission 2007 ). MBA has been for many years consid- adequate results and to protect public health and reduce ered as the reference monitoring method for phycotoxins, their economic impact. although the European Commission established that to be Harmful algal blooms is the term used to describe in accordance with animal health protection, all possible a proliferation of marine microalgae (dinoflagellates, replacement, refinement and reduction of animals must be diatoms) that carries a toxic effect and, in many cases, taken into account when laboratory animal methods are 16 A.M. Botana et al.: Analysis of marine toxins

Palytoxin

Azaspiracid1

Saxitoxin

Ciguatoxin3C Okadaic acid

Yessotoxin

Pectenotoxin OH HO R1 10 9 H O 2 + NH 4 2 N R O H H 2 5 1 N 7 HO R4 H H Tetrodotoxin H H R3

Brevetoxin

Figure 1 Structures of representative toxins. A.M. Botana et al.: Analysis of marine toxins 17 used (Hess et al. 2006 ), and therefore alternative methods calculated (Commission Regulation 2011 ). In this sense, should be applied. Any method proposed as an alternative the toxicity equivalent factor (TEF) of each compound to the biological assay should not be less effective than compared with the potency of the reference compound the biological method, and the implementation should must be applied to calculate the total toxicity of a sample provide an equivalent level of public health protection. (Botana et al. 2010 ). The Working Group on marine bio- Following these premises, several methods were validated toxins, as part of the Contaminants Panel at the European as alternatives to the MBA (Botana et al. 2009 , 2010, Camp- Food Safety Agency (EFSA), has defined, on the basis of bell et al. 2011 ). In the case of PSP toxins, the total content current knowledge, the TEF that should be used for the of toxins in the edible parts of molluscs must be officially conversion of analytical results into toxic concentrations detected by MBA (European Commission 2005 ) and also by in each toxin group (European Commission Panel 2009b ). the HPLC Lawrence method with pre-column oxidation as Another essential item to implement and to validate any published in the Association of Official Analytical Chemi- chemical detection method is the availability of standards sts Official Method2205.06 (European Commission 2006 ). as the identification of one toxin by using other analogues If the results are challenged, the reference method shall be can lead to gross errors (Otero et al. 2011a ). In addition, the biological method. To detect PSP toxins, other analyti- pure toxins in high amount are necessary to calculate the cal non-validated methods are available, such as an HPLC toxicity and TEF of each molecule to achieve results to method with post-column oxidation (Oshima et al. 1976 , protect human health. It can be concluded that analytical Rodriguez et al. 2010 ), plasmon resonance biosensors methods are useful tools to quantify the amount of known (Fonfria et al. 2007 ) or enzyme immunoassays [enzyme- marine toxins in seafood samples, but several important linked immunoassay (ELISA)] (Huang et al. 1996 , Garet details such as toxicity, calibrants and new toxins should et al. 2010 ), based on both the use of antibodies and several be considered. functional assays based on the mechanism of action of the toxins (Vieytes et al. 1993 , Louzao et al. 2001 ). In the case of ASP, the total content of toxins in edible parts of molluscs may be detected by using either an HPLC method with UV Analysis of PSP toxins detection (HPLC-UV) or an ELISA method (European Com- mission 2007 ). In this case, if results are challenged, the PSP is a fatal human syndrome produced by a naturally reference method shall be the HPLC method. For the detec- occurring group of neurotoxic alkaloids present in several tion of lipophilic toxins, a series of MBA procedures, dif- species of dinoflagellates. This group of toxins constitutes fering in the test portion of flesh and the solvents used for the most spread worldwide affecting the whole of Europe, extraction, were the official method for many years (Euro- South Africa, India, Morocco, eastern Asian coast and pean Commission 2005 ); however, after July 2011 a liquid North and South America as well. chromatography (LC) tandem MS (LC-MS/MS) method was Saxitoxin (STX) is the first described and most studied approved to be applied as the reference method for the compound of the group (it is the representative com- detection of these toxins (Commission Regulation 2011 ). In pound), and 57 other analogues also have been researched addition to this, several methods based on the mechanism (Wiese et al. 2010 ). Traditionally, STX analogues have been of action of each lipophilic toxin group have been devel- composed only of hydrophilic compounds and divided oped (Vieytes et al. 1997 , Alfonso et al. 2004 , Vilarino et al. into subgroups on the basis of substituent side chains: 2009 , Otero et al. 2011b ). There are no official methods to carbamate [STX, neo-STX and gonyautoxins (GTX1 – 4)], detect the presence of TTX or CTX even though several decarbamoyl (dc-STX, dc-neo-STX and dc-GTX1 – 4) and N - LC-MS/MS methods can be used to detect these toxins sulfocarbamoyl (GTX5– 6 and C1– 4). These groups present (Otero et al. 2010 , Rodr í guez et al. 2012 ). different toxicities, with the carbamoyl analogues being As was mentioned earlier, the presence of phycotox- the most toxic, followed by decarbamoyl analogues with ins is a dynamic and ubiquitous process in any coast of intermediate toxicity, and N -sulfocarbamoyl analogues the world and therefore the presence of new analogues, the least toxic (Vale et al. 2008a,b ). Figure 1 shows the new toxins or toxins from different locations are issues chemical structure of the more often found compounds in that must be taken into account when any analytical toxic dynoflagellates and poisoned seafood. method is used. However, the legislation also established More recently, analogues from a novel family of PSP that in addition to the amount of toxin, the total toxicity, toxins containing a hydrophobic side chain were isolated which is calculated by using conversion factors based and structurally characterised from Australian strains of on the available toxicity data of each toxin, should be Gymnodinium catenatum and designated GC1-GC3 (Negri 18 A.M. Botana et al.: Analysis of marine toxins et al. 2003 ). Other hydrophilic analogues of STX with an Following this, the method was modified by chang- acetate R4 side chain were reported in the freshwater fila- ing chromatographic conditions to reduce analysis time mentous cyanobacterium Lyngbya wollei but not in the and improve performance (Lawrence et al. 1995 ). In 2005, marine environment so far (Onodera et al. 1997 ). the Lawrence method was adopted as the official method PSP toxins specifically block the excitation current in to detect PSP toxins and then approved by the European nerve and muscle cells by means of site one of the sodium Union (EU) for monitoring these toxins (Association of channel (Messner and Catterall 1986 ); therefore, the accu- Official Analytical Chemists 2005, European Commis- mulation of PSP toxins in shellfish creates a serious public sion 2006 ). It is based on the pre-column oxidation of PSP health problem and affects the fisheries industry. Sommer toxins with hydrogen peroxide and sodium periodate fol- and Meyer (1937) were the first to develop a method to lowed by fluorimetric detection. It was validated for the determine PSP toxins: their MBA method established the determination of STX, NEO, GTX2,3, GTX1,4, dc-STX, GTX5 basis for that. This is the reference method internationally (B1), C1,2 and C3,4 in molluscs (mussels, clams, oysters accepted to quantify PSP toxicity, and it is used worldwide and scallops). In 2009 a validation study was under- in monitoring programmes. The chemicals methods used taken for the extension of this method to two more toxins: to determine PSP toxins are fluorimetric assays, HPLC dc-NEO and dc-GTX2,3 (Turner et al. 2009). with fluorimetric detection (either pre-column or post- Pre- and post-column HPLC methods, despite the column oxidation), LC/MS and capillary electrophoresis many benefits of each, which includes an increased sen- (CE) methods. sitivity to low concentrations of toxins and less variability The HPLC methods are widely used to quantify PSP in results, also present some drawbacks that should be toxins present in seafood samples, and they are also resolved. In the case of hydrophobic analogues, they are useful to provide the PSP profile because chromatographic retained by C18 resins (Negri et al. 2003 ), because of which methods are identification methods as well. These toxins the use of HPLC methods in monitoring programmes will have only a weak chromophore group, and it must be miss the presence of hydrophobic analogues (Vale 2008 , modified before detection: when they are oxidised in alka- Vale et al. 2009 ). LC-MS methods are also being developed line solution, a purine is formed that becomes fluorescent to obtain a good characterisation of these compounds: at acidic pH. This reaction can be either a pre-column one Electrospray ionisation MS (ESI-MS) is effective for the or a post-column one, and obtained purines are monito- detection of polar PSP toxins (Quilliam 2003 , Dell’ Aversano rised with an FL detector. et al. 2005 ); however, variable retention times for PSP Bates and Rapoport (1975) first studied the oxidative toxins in different seafood matrixes were observed. At alkaline conditions of PSP toxins to get fluorescent com- present, it is not possible to achieve a complete separa- pounds; then, Buckley et al. (1978) added them to their tion of all PSP toxins, and the sensitivity of MS detection is method and established the basis of the HPLC method for not good enough to control seafood toxin at the regulatory these compounds with the post-column reaction. limit established (Diener et al. 2007 ). In 1984, Oshima et al. (1984) proposed a method Although the analysis of PSP toxins provides a profile based on alkaline oxidation to produce highly fluores- of the composition of a given toxic episode, the use of cent derivatives, but with problems in separating toxins surface plasmon resonance biosensors has reached a con- such as GTX1, GTX4 and GTX3, GTX5. Afterwards, they siderable level of development (Campbell et al. 2007 ). The described a method that was able to separate almost all high sensitivity of this approach allows fast throughput, the PSP toxins with three different eluents according to as sample extraction and detection is rather simple, plus the basicity of the three groups of toxins (group I: C1 – C4; the chips can be used many times (Fonfria et al. 2007 ). group II: GTX1,4, GTX5 (B1) and GTX6 (B2), dc-GTX1,4; For this techonology, an antibody-based method has been group III: NEO, dc-STX and STX) (Oshima et al. 1987 ). described and validated both at the single laboratory level In a later work, Oshima (1995) improved the system and (Campbell et al. 2010 ) and in an interlabotoratory study included an extract cleaning procedure. The first dis- (van den Top et al. 2011b ). advantage is that it is a time-consuming method for the analysis of PSP toxins. In the group of pre-column methods, the method developed by Lawrence and Menard (1991) needs to be Analysis of DA highlighted. One of the biggest disadvantages was the ina- bility to easily distinguish between different toxin groups DA, belonging to the kainoid class of compounds, is the with similar toxicity. chemical responsible for ASP. It was originally isolated as A.M. Botana et al.: Analysis of marine toxins 19 a product from the red algae Chondria armata and subse- because of interferences from crude extracts (Hess et al. quently isolated from other red algae and several species of 2001 ). Mainly, tryptophan and its derivatives, normally diatoms, mainly of the genus Pseudonitzschia (Bates et al. contained in shellfish and finfish tissues, may be eluted 1989 , Lundholm et al. 1994 ). DA has also been detected close to DA with some columns and chromatographic con- in several species of molluscs worldwide including the ditions. Thus, there are other alternatives, such as FL (James United States, New Zealand, Mexico, several European et al. 2000 , Chan et al. 2007 ) and chemiluminescence countries such as France, Portugal, Ireland and Spain and detection (Kodamatani et al. 2004 ). As DA concentra- the Croatian coast (Horner et al. 1993 , M í guez et al. 1996 , tions in Pseudonitzschia cultures and phytoplankton field Vale, 2001, Ujevic et al. 2010 ). Although the DA is the main samples are often much lower, a more sensitive method toxin found in a variety of shellfish species, other minor of detection is required. The most common technique for analogues, about 10 isomers of DA (isodomoic acids A – H the analysis of DA in seawater samples uses a derivatising and DA 5′ diasteriomer), have been identified in marine agent, which results in low LODs (Pocklington et al. 1990 ), samples (Wright et al. 1990 , Zaman et al. 1997). but this procedure involves some problems. Among them, The first method developed to detect the compound a poor selectivity because many compounds in the sample, was LC-UV (Lawrence et al. 1989 ), and a protocol involv- such as amino acids, can be derivatised and interfere with ing aqueous methanol extraction and strong anion- DA detection. Also, the handling during the derivatisation exchange (SAX) clean-up has been applied extensively to step is time consuming for the use of LC-FL on a large scale. the analysis of DA and its known isomers in shellfish and In addition to the LC-UV and LC-FL, various extraction and fish tissues on a regulatory basis. Nowadays, the LC-UV screening techniques, such as thin-layer chromatography remains the most widely used method, and it has been (Quilliam et al. 1998 ), CE (Gago-Martinez et al. 2003), gas validated and standardised as the reference method for chromatography coupled to MS (GC-MS) (Pleasance et al. DA quantification (International 2000 , European Com- 1990 ), LC-MS/MS (Wang et al. 2007 , Mafra et al. 2009 ) and mittee for Standardization 2003 ). Furthermore, ELISA ultraperformance liquid chromatography with tandem MS has also been validated (Kleivdal et al. 2007 ) and is used detection (UPLC-MS/MS) (de la Iglesia et al. 2008 ), have officially in the EU for screening purposes. These methods allowed the detection of novel compounds inside of the DA have limits of detection (LODs) low enough to detect DA at group in new coastal areas of the world. a concentration of 4.5 mg/kg of shellfish meat. This dose As the seafood matrix can interfere in the detection was established as the maximum amount that a portion of of trace levels, pre-treatment steps to concentrate and 400 g of meat should contain for not exceeding the acute purify DA are needed. Solid-phase extraction (SPE) is the reference dose of 30 mg DA/kg body weight (European most common method used for the clean-up of shellfish Commission Panel 2009a ). samples before using a chromatography or other analyti- Several other biological methods have been deve- cal technique to quantify the toxin amount (Zhao et al. loped to detect and quantify DA in seawater and tissue 1997 , Quilliam et al. 1998 , James et al. 2000 , Pardo et al. samples, such as electrochemical ELISA (Micheli et al. 2007 , He et al. 2010 ]. So, several methods have been based 2004 ), radioimmunoassay (Lawrence et al. 1994 ), recep- on SAX and strong cation exchange (Pleasance et al. 1990 , tor binging assay (Lefebvre et al. 2010 ), cytotoxicity assay Furey et al. 2001 , Gago -Martinez et al. 2003 , Ciminiello (Beani et al. 2000 , Leira et al. 2003 ) and surface plasmon et al. 2005 ). The purification with SPE cartridges results in resonance (Le Berre and Kane 2006 , Traynor et al. 2006 , a valid approach for the routine monitoring of DA in shell- Stevens et al. 2007 ). Some of these techniques, such as fish to prevent the matrix effect and signal suppression. enzyme immunoassay and ELISA, sometimes may require However, this is a manual time-consuming procedure; little clean-up of shellfish samples and have the advan- even some authors concluded that an additional step tages of being easy and fast (Yu et al. 2004 ). However, with an SPE cartridge did not significantly improve the the cross-reactivity with similar toxins will result in false recovery of DA from shellfish samples (Powell et al. 2002 ). positives and limit the demonstration of toxicity (He et al. Therefore, there are many analytical methods that do use 2010 ). a sample clean-up and could be avoided when the pH of DA contains a characteristic conjugated diene chromo- the mobile phase in the LC methods is properly adjusted phore with strong absorbance at 242 nm, which permits (He et al. 2010 ). Table 1 summarises several chemical its detection by LC-UV at concentrations as low as 4– 80 methods, specifying the sample clean-up procedure in ng/ml, depending on the sensitivity of the detector (Mafra different matrices and the LOD obtained in each method. et al. 2009 ). This is suitable for regulatory purposes; MS techniques are being increasingly used in some however, false positives are frequently encountered laboratories, and today, LC-MS/MS is perhaps the most 20 A.M. Botana et al.: Analysis of marine toxins

Method Matrices Treatment sample (clean-up) LODs References

LC-UV Mussels Molecularly imprinted polymer- 0.1 mg/ml (Ciminiello et al. 2005) solid-phase extraction (MISPE) LC-UV Fur seals (fluidics) SPE 8 ng/ml (European Commission Panel 2009a ) UPLC-MS/ < 1 ng/ml MS LC-UV Phytoplankton, seawater Without sample pre- 42 pg/ml (Traynor et al. 2006 ) samples concentration and derivatisation (on column) LC-ESI-MS 15 pg/ml LC-FL ∼ 15 pg/ml LC-FL Mussels SPE and post-column 25 ng/ml (Powell et al. 2002 ) derivatisation LC-FL Shellfish and phytoplankton SPE and pre-column ≤ 1 ng/ml (He et al. 2010 ) derivatisation (shellfish) Without SPE (phytoplankton) UPLC-MS/ Seawater SPE-C18 (with Empore disks) 0.02 ng/ml (Gago -Martinez et al. 2003 ) MS UPLC-MS/ Crude extracts Without clean-up 0.05 – 0.09 mg/kg (McNabb et al. 2005 ) MS LC-MS/MS Seawater and phytoplankton SPE-C18 cartridges 30 pg/ml (Quilliam et al. 1998 ) LC-MS/MS Mussels, clams, cockles Pressurised liquid extraction 0.2 μ g/ml (Wang et al. 2007 ) (PLC) with purification inside the extraction cell LC-UV Mussels and scallops Without SPE clean-up 25 ng/ml (Doucette et al. 2009 ) LC-ESI-MS 0.008 μ g/ml LC-UV-CLD Mussels Without any pre-concentration or 8 pg/ml (Hess et al. 2001 ) derivatisation steps LC-MS/MS Seawater, phytoplankton, SPE-C18 cartridges without pre- 1 – 4 pg/ml (Mosher et al. 1965 ) mammalian fluids and tissues concentration (on column) TLC Mussels SAX clean-up∼ 10 μ g/g (Miyazawa and Noguchi 2001 ) GC-MS Mussels SPE clean-up: reversed phase 1 μ g/ml (Pocklington et al. 1990 ) and SCX cartridges LC-UV Clams and scallops SAX cartridges 0.47 μg/ml (Kodamatani et al. 2004 ) CE-EIA Mussels, oysters, clams and – 0.02 ng/ml (Noguchi and Arakawa 2008 ) scallops CE Mussels, razor clams and SAX clean-up 0.15 μ g/g (Pleasance et al. 1990 ) anchovy

Table 1 Some representative analytical chemical methods for the detection of DA in a range of matrices. CE, capillary electrophoresis; CE-EIA, capillary electrophoresis-enzyme immunoassay; DA, domoic acid; EIA, enzyme immunoassay; GC-MS, gas chromatography-mass spectrometry; LC-ESI-MS, liquid chromatography-enzyme immunoassay-tandem mass spectrometry; LC-FL, liquid chromatography with fluorescence detection; LC-MS/MS, liquid chromatography with tandem mass spectrometry; LC-UV, liquid chromatography with ultraviolet detection; LC-UV-CLD, liquid chromatography-ultraviolet-chemiluminescence detection; LODs, limits of detection; SAX, strong anion exchange; SCX, strong cation exchange; UPLC-MS/MS, ultraperformance liquid chromatography with tandem mass spectrometry; TLC, thin layer chromatography.

important and legally accepted confirmatory tool, provid- the peak’ s identity at low DA levels ( < 3 ng/ml), when a ing a high sensitivity, accuracy and selectivity for DA and sensible UV spectrum cannot be obtained (Mafra et al. its isomers in crude extracts (McNabb et al. 2005 ). Nev- 2009 ). In this case, confirmation by LC-MS is sometimes ertheless, LC-UV is often the only analytical instrument necessary and should also be required whenever DA is available in many research institutes and regulatory agen- reported in a new geographical area. Some studies on cies responsible for monitoring the occurrence of marine algal production of DA to track the toxin in seawater, as an toxins. Although the LOD of DA can be improved by using early alert for toxin accumulation in marine organisms, different injection volumes in the LC-UV, the matrix com- are available for remote, subsurface detection (Doucette plexity may result in some degree of uncertainty about et al. 2009 ). A.M. Botana et al.: Analysis of marine toxins 21

Overall, an improvement in techniques based on are regulations for TTX in countries such as Japan and LC and/or MS to achieve reliable, simple and low-cost Korea, where official control does not regulate the amount methods is essential for DA detection in any sample of toxin in fish that can be placed on the market, but res- matrix. taurants that want to serve species containing TTX require special licenses. EFSA has not published any documents related to the risks involving TTX, but some authors claim that the minimum lethal dose in humans is 2 mg, though Analysis of TTX it may vary with factors such as age, health and sensitivity to the toxin (Noguchi and Ebesu 2001 , Cohen et al. 2009 ). TTX, a well-known potent neurotoxin, was first isolated Currently, there is no official method for TTX detection, from puffer fish (Mosher et al. 1965 ) and has also been but the MBA has been used in many cases to determine found in a wide variety of animals including arthropods, TTX toxicity in food matrices (Kawabata 1978 , Yasumoto echinoderms, mollusks, worms, newts or frogs (Miyazawa 1991 ). However, the MBA is not completely satisfactory, and Noguchi 2001 , Noguchi and Arakawa 2008 ). Neither due to its low sensibility. MBA is not suitable for meas- its biochemical path nor its true origin is fully clarified, as uring TTX in biological samples as it cannot distinguish three hypotheses point to its origin: endogenous (Lehman between TTX and STX or TTX analogues. Other biological et al. 2004 ), through food chain (Lin and Hwang 2001 , methods have been developed for TTX detection, such as Kono et al. 2008 ) or through symbionts (Narita et al. 1987 , ELISA, methods that use antibodies specific to the toxin Wang et al. 2008 ). (Raybould et al. 1992 , Neagu et al. 2006 , Zhou et al. 2007 ), Although TTX exists mainly in tropical waters tests with cultured neuroblastoma, hemolytic assays worldwide, this toxin has appeared on European coasts (Hamasaki et al. 1996 ) and also techniques using biosen- (Katikou et al. 2009 ). It is possibly due to the migration of sors (Kreuzer et al. 2002 , Neagu et al. 2006 , Yakes et al. toxic species from the Red Sea to the Mediterranean Sea 2011 ). Nevertheless, to obtain specific information of a through the Suez Canal, a phenomenon known as Lessep- sample, as the toxic profile or the amount of an individual sian migration (Golani 1998 , Galil and Zenetos 2002 ). toxin, chemical methods have been developed, including This migration may happen because of the opening of immunoaffinity chromatography (Kawatsu et al. 1999 ), new corridors allied to the increase in water temperature GC-MS (Man et al. 2010 ), HPLC with FL detection (HPLC- as a result of climate change. So, marine species typical FL) ( O ’ Leary et al. 2004 , Jen et al. 2007 ) and HPLC-UV of tropical and subtropical waters are adapted to waters (Yu et al. 2010 ). Although these techniques ensure low less warm, and changes in environmental conditions LODs and high sensitivity to detect TTX, they have some lead to poisoning incidents in new geographical areas. In limitations. In the case of immunoaffinity or ELISA, both fact, recent intoxication cases caused by the consumption methods require a costly monoclonal antibody or reagent of TTX bearers have been described in the Israeli coast (Leung et al. 2011 ), and the GC-MS requires a complex (puffer fish: Lagocephalus sceleratus ) and in Spain (gastro- extraction procedure (Jen et al. 2008 ). However, HPLC-FL pod: Charonia lampas ) (Zaki and Mossa 2005 , Bentur et al. shows differences in the FL intensities of some TTX ana- 2008 , Rodriguez et al. 2008 ). This last episode was the logues compared with TTX itself, which causes problems first report of TTX occurrence in autochthonous species in in quantifying (Shoji et al. 2001 ). For these reasons, ana- Atlantic and Mediterranean waters. It triggered an investi- lytical methods based on LC-MS and LC-MS/MS have been gation monitoring different invertebrate species in several developed (Jang et al. 2010 , Chen et al. 2011 , Rodrí guez sites of Portuguese coast, where low amounts of TTXs were et al. 2012). These LC methods, together with appropri- found in two intertidal gastropod species (Silva et al. 2012 ). ate extraction procedures, have been able to determine As a consequence of the migration of TTX bearers, interna- the presence of TTX, not only in the remains of fish and tional fisheries agreements in the EU could be modified. other animals but also in the blood and urine of poisoned At the moment, the importation of puffer fish and other patients (Tsai et al. 2006 , Rodriguez et al. 2008 ). More- toxic species is not permitted in some countries, including over, these methods provide better LODs when compared the United States and Europe, and no regulatory limits for with HPLC-FL or UV (Jen et al. 2008 ). Most of the LC-MS TTX and its analogues have been established yet (Gessner instruments are equipped with an atmospheric pressured and McLaughlin 2008 ). With regard to current EU legis- ionisation (API) fitted with an ESI source based on colli- lation, toxic fish belonging to the family Tetraodontidae sion-induced dissociation (CID) of a triple quadrupole or their products should not be placed on the European mass analyser. For the detection of TTXs, the ESI interface markets (European Commission 2004b ). However, there operates in the positive ionisation mode. So, TTX and its 22 A.M. Botana et al.: Analysis of marine toxins analogues are analysed by MS, in which positive ionisa- a HILIC column and detected under the MRM mode were tion produces a typical molecular ion of [M+ H] + for each the following: 5,6,11-trideoxy-TTX (m/z 272 > 254/162), one. Because of the continuous emergence of new TTX 5-deoxy-TTX, and 11-deoxy-TTX (m/z 304 > 286/176), analogues, the most recent 8-epi-type TTX analogues 4,9-anhydro-TTX (m/z 302 > 256/162), 11-nor-TTX-6(R)-ol found in newts (Kudo et al. 2012 ), some authors prefer to and 11-nor-TTX-6(S)-ol (m/z 290 > 272/162),4-epi-TTX and operate with the MS in the multiple reaction monitoring TTX (m/z 320 > 302/162). (MRM) mode to quantify the individual toxins. However, In summary, the LC-MS/MS technology is the most others use the MS operating in the single ion monitoring appropriate and chosen by some laboratories world- mode or the single ion recording mode, such that only a wide for detecting TTXs. One problem is the difficulty in selected m/z value is detected in the analysis. On the con- obtaining purified TTX analogues for calibration. TTX is trary, the MRM mode not only allows detecting the precur- frequently used as an internal standard for their quantifi- sor molecule but also tracks multiple product ions from cation, and this could lead to errors in the toxin amount. the fragmentation pattern in the same run. In this case, Nevertheless, because of the timely appearance of TTX in only one product ion is used in the quantification and the new geographical areas, monitoring programmes coupled others to confirm the toxin. to a suitable and standardised analytical method should Today, the investigation is focused in developing be developed and for this, certified standards of TTXs LC-MS methods that lead to a good separation and iden- would be necessary. tification of all TTX analogues in any sample with low LODs. So, different analytical columns and LC condi- tions are tested by researchers to improve the detection Detection of lipophilic marine of TTX. Table 2 summarises several LC-MS/MS methods to detect TTX and its analogues. The toxin separation is toxins typically achieved in reverse-phase columns and with solvents containing an ion pair reagent (i.e., ammonium Marine lipophilic toxins are a heterogeneous group of heptafluorobutyrate). But this ion pair sometimes tends toxins with different molecular weights and specific to remain in the MS, causing spectral noise (Shoji et al. chemical characteristics (Table 3 ). This group includes 2001 , Rodrí guez et al. 2012). Also, because of the pola- yessotoxins (YTXs), AZAs, pectenotoxins, gymnodimines rity of TTXs, some of them are not well retained in these (GYMs), spirolides (SPXs), CTXs and diarrhetic shellfish columns. To solve all these problems, the hydrophilic poisoning (DSP). Members of the DSP toxin group are OA interaction liquid chromatography (HILIC) is becoming and its derivatives dinophysistoxin-1 (DTX-1), dinophysis- more useful in the analysis of TTXs (Chen et al. 2011 , toxin-2 (DTX-2) and dinophysistoxin-3. To detect them, a Chulanetra et al. 2011 , Cho et al. 2012 , Kudo et al. 2012 , number of analytical methods have been developed in the Rodr í guez et al. 2012 , Silva et al. 2012 ). The HILIC tech- last decades, comprising MBAs, in vitro assays and chemi- nique employs low aqueous/high polar organic solvents cal assays (Botana et al. 2012 ). Each method has its merits without ion pair reagents, resulting in increased sensitiv- and drawbacks, and ultimately it is the responsibility of ity compared with the reverse phase. So, TTX, which is regulators and analysts to decide upon the most appropri- the more polar compound between its analogues, elutes ate technology for their particular situation. The regula- with the higher proportion of water in a gradient elution. tion of lipophilic marine toxins worldwide differs widely. Also, problems with noise peaks are reduced in HILIC In Japan, Canada and South America, MBA is the method columns. Almost all TTX analogues show a common used for the official control of these toxins, while in New major product ion, which is usually 162 (m/z) formed Zealand, chromatographic methods have been used for from the molecular ions in MS/MS, suggesting that this their monitoring programmes (McNabb 2008 , Gerssen product ion can be used in the quantification. However, et al. 2011 ). In Europe, chromatographic techniques there are analogues such as TTX and 4-epi-TTX that have coupled to MS were also the method of choice to replace the same molecular weight and the same fragmentation MBAs to detect lipophilic marine toxins (Commission Reg- pattern; therefore, a complete separation is needed to ulation 2011 ). MBA, which has been used for many years quantify them. As a result, the HILIC separation system as an exclusive reference method for lipohilic marine is very useful for the simultaneous quantification of toxin detection, was replaced by the LC-MS/MS approach TTXs by LC-MS/MS in the MRM mode. Figure 2 shows an with the new regulation. example of a chromatogram obtained by LC-ESI-CID-MS/ To date, more than 200 lipophilic marine toxins MS from puffer fish sample. The TTX analogues eluted in have been described in the literature (Gerssen et al. A.M. Botana et al.: Analysis of marine toxins 23

Separation Eluents Toxins [M+ H] + (m/z) Matrices LODs References

Reverse 30 m m ammonium heptafluorobutyrate in 320, 336, 302, 290, Amphibian – (Stobo et al. phase 1 m m ammonium acetate buffer (pH 5.0) 304 and unknown 2005) analogues (348, 330) ZIC-HILIC (A) 10 mm ammonium formate and 10 m m 320, 302, 304, 272 Puffer fish 0.09 – 0.2 (Villar -Gonzalez formic acid in water ng/ml et al. 2008 ) (B) acetronitrile:water (80:20) in 5 m m ammonium formate and 2 mm formic acid HILIC 16 m m ammonium formate/acetonitrile 256, 272, 288, 302, Puffer fish 0.9 pmol/ (Wang et al. (3:7 v/v) 304, 320, 336 injection 2008) HILIC (A) 0.1 % formic acid/water and (B) 100 % 320 Blood serum 0.1 ng/ml (Jang et al. 2010) methanol Reverse 1 % acetonitrile, 10 m m trimethylamine and 320, 272 Gastropods and human < 10 ng/ml (Gessner and phase 10 m m ammonia formate in water fluids (blood and urine) McLaughlin 2008) HILIC 16 m m ammonium formate/acetonitrile 320, 302, 304, 288, Puffer fish < 0.5 (Cho et al. 2012) (3:7 v/v) 272 nmol/g ZIC-HILIC (A) 10 mm ammonium formate and 10 m m 320, 302, 304 Puffer fish – (McNabb 2008) formic acid in water (B) acetronitrile:water (80:20) in 5 m m ammonium formate and 2 mm formic acid HILIC 10 mm ammonioum formate/acetonitrile 272, 288, 290, 302, Puffer fish 0.10 ng/ml (Kudo et al. 2012) (22:78 v/v) 304, 320, 336 HILIC 1 % acetic acid in acetonitrile/1 % acetic 320 Blood 0.32 ng/ml (Gerssen et al. acid in water (88:12 v/v, pH 4.1) 2011) Reverse (A) 1 % acetonitrile, 20 m m 320, 290, 304, 302 Puffer fish – (Rodr í guez et al. phase heptafluorobutyric acid, 20 m m ammonium 2012) hydroxide and 10 m m ammonium formate in water (B) and same mixture (A) with 5 % acetonitrile HILIC (A) 10 m m ammonium formate and 10 m m 320, 290, 304, 302, Puffer fish 16 ng/ml (Rodr í guez et al. formic acid in water 272 2012) (B) acetronitrile:water (95:5) in 5 m m ammonium formate and 2 mm formic acid HILIC Not clear 254, 270, 272, 286, Netws – (Botana et al. 288, 290, 302, 304, 2012) 320 HILIC (A) 10 m m ammonium formate and 10 m m 320, 272 Gastropods 1.7 ng/ml (Cohen et al. formic acid in water 2009) (B) acetronitrile:water (95:5) in 5 m m ammonium formate and 2 mm formic acid

Table 2 Summary of several LC-MS/MS methods to detect TTX and its analogues. HILIC, hydrophilic interaction liquid chromatography; LC-MS/MS, liquid chromatography with tandem mass spectrometry; LODs, limits of detection; TTX, tetrodotoxin; ZIC-HILIC, Highly polar Zwitterionic hydrophilic interaction liquid chromatography.

2011 ). However, most LC-MS/MS methods are focused for the monitoring of lipophilic marine toxin. If an acid on the analysis of the 13 toxins that are legislated in the chromatography is employed, the mobile phase is com- EU (Table 3). GYMs and SPXs are usually included in the posed of water and ACN/water (95:5), both containing multi-toxin detection methods despite the fact that they 50 mm formic acid and 2 mm ammonium formate (Alfonso are not legislated in the EU. Hence, LC-MS/MS methods et al. 2008 , Villar -Gonzalez et al. 2008 , Otero et al. 2010 , detect different marine toxin groups in a short period of Blay et al. 2011 ). Mobile phases for basic chromatogra- time (Stobo et al. 2005 , McNabb 2008 , Gerssen et al. 2011 ), phy are composed of water and ACN/water (90:10), both and they are classified according to the chromatographic containing 0.05% ammonia, 2 mm bicarbonate or 6.7 mm solvents used. Basic or acid mobile phases can be used ammonium hydroxide (pH 11) (Gerssen et al. 2009 , 2011). with an elution gradient. Table 4 summarises the LC char- MRM data are detailed in Table 4. The transition with acteristics of two types of chromatography developed the highest intensity is used for quantification, while the 24 A.M. Botana et al.: Analysis of marine toxins

100 TIC of +MRM 2 the positive mode and the other with the equipment oper- 5 Max. 4.5 x 10 ating in the negative mode. 1: 5-deoxyTTX 5,6,11-trideoxyTTX 2: 11-deoxyTTX This technique has been evaluated, and it is consid- 3: 11-norTTX-6(R)-ol 4: 4,9-anhydroTTX 5 ered to be successful by many laboratories (Gerssen et al. 5: 11-norTTX-6(S)-ol 6: 4-epiTTX 2009 , Blay et al. 2011 ). The short, narrow-bore column 7: TTX 3 7 packed with 3 μ m Hypersil-BDS-C8 phase and the X-Bridge C18 are two of the most widely used columns, which are 1 Relative intensity (%) capable of separating a wide range of toxins by using rapid 4 6 gradient (Villar-Gonzalez et al. 2007, Ciminiello et al. 2010 , 0 Gerssen et al. 2011 ). The method with both basic and acid 2 4 6 8 10 12 14 16 18 20 22 24 Time (min) mobile phases was also adjusted to the new technologies, UPLC-MS/MS (Fux et al. 2007 , Rundberget et al. 2011 ), and Figure 2 Mass chromatogram of the LC-ESI-CID-MS/MS obtained more than 20 analogues were separated in only 6.6 min under MRM operation from the puffer fish sample. Toxins eluted (Fux et al. 2007 ). One advantage of the acid against basic in an XBridge Amide column (i.d. 2.1 × 150 mm; 3.5 μ m) at 25° C, 0.2 ml/min. The LC was operated in gradient with eluent A consisting chromatography is that the first one facilitates good sepa- of 10 mm ammonium formate and 10 mm formic acid in water. Eluent ration of acidic OA analogues by suppressing the ionisa- B contained 95 % acetonitrile and 5 % water with a final concentration tion of the carboxyl groups and preventing deleterious ion of 5 mm ammonium formate and 2 mm formic acid. Minutes 16 to exchange interactions with residual silanol groups in the 22 represent a broadening of the chromatogram where the toxins stationary phase (Suzuki and Quilliam 2011 ). Neverthe- are eluted. CID, collision-induced dissociation; ESI, electrospray ionisation; i.d., inner diameter; LC, liquid chromatography; MRM, less, the chromatography of compounds included in the multiple reaction monitoring; MS, mass spectrometry; TIC, total ion YTX group can be problematic under acidic conditions chromatogram. (Gerssen et al. 2009 ). LODs for lipophilic toxins achieved by the LC-MS/MS approach are low (Table 4), and toxins can be detected at levels below the current regulatory limit. For both acid and basic chromatography conditions, transition with the lowest intensity is used for confirma- these LODs are lower for toxins ionised in the positive tory purposes. With the exception of OA and YTX toxins mode than in the negative one, specially for SPXs (Blay groups, which are ionised in the negative mode, the et al. 2011 , van den Top et al. 2011a ). remaining lipophilic toxins are preferably ionised in the Although analytical methods are by far the target of positive mode. If the instrument is able to simultaneously most of the international efforts for the detection of this operate in positive and negative ionisation modes, the MS group of compounds, it should be highlighted that enzyme- methods include transitions for the toxins that are ionised based assays are available for high-throughput detection in both ionisation modes. If it is not capable of detecting of these compounds (Vieytes et al. 1997 , Gonz á lez et al. all toxins in a single injection, compounds are detected in 2002 , Rubiolo et al. 2012 ), and biosensor methods that use two separated runs, one with the equipment operating in the surface plasmon resonance technology (Llamas et al.

Toxin groups Chemical class Main Formula Toxin analogues covered by the

compounds ( Mr ) EU legislation

Okadaic acid (OA) Polyether, spiro-keto OA (804) C 44 H68 O13 OA, DTX-1, DTX-2, DTX-3 assembly

Azaspiracid (AZA) Polyether, second amine, AZA-1 (841.5) C47 H71 NO12 AZA-1, AZA-2, AZA-3 3-spiro ring

Petecnotoxin (PTX) Polyeter, ester PTX-1 (874.5) C47 H70 O14 PTX-1, PTX-2 macrocycle

Yessotoxin (YTX) Ladder-shaped polyether YTX (1141) C55 H82 O21 S2 YTX, 45-OH-YTX, homo-YTX, 45-homo-YTX

Gymnodimines (GYMs) Macrocycle; cyclic imine GYM (507) C32 H45 NO4 None

Spirolides (SPXs) Macrocycle; cyclic imine SPX-1 (691.5) C41 H61 NO7 None

Table 3 Chemical characteristics of lipophilic marine toxins. EU, European Union. A.M. Botana et al.: Analysis of marine toxins 25 (van den Top et al. Top et (van den 2011) (Gerssen et al. (Gerssen et 2009) g/kg; μ g/kg Limits of detection (LODs) of several several of detection (LODs) Limits of m particle size a μ μ g/kg, AZA-3: g/kg 2010) (Kilcoyne, 213.2), PTX-2 (m/z 876.5/823.4, m/z 876.5/823.4, m/z (m/z 213.2), PTX-2 μ μ 77.7, m/z 1157.5/871.5), homo-YTX 1157.5/871.5), homo-YTX 77.7, m/z 3 mm, 5 g/kg; DTX-2: 3 g/kg; DTX-2: z 828.5/792.5). z × μ y under acidic conditions and (2) chromatography (2) chromatography conditions and acidic under y g/kg, YTX: 16 μ e; YTX, yessotoxin. e; YTX, yessotoxin. ng two transitions per toxin: OA and DTX-2 (m/z 803.5/255.0, (m/z DTX-2 and OA per toxin: ng two transitions g/kg; AZA-2: 2 μ g/kg, DTX-1: 5 g/kg, DTX-1: g/kg, AZA-1: 0.5 l μ μ μ g/kg, PTX-2: 2 g/kg, PTX-2: μ OA: 3 OA: AZA-1: 2 3 OA: 9.1 pg, YTX: 2.2 pg, AZA-1: 1.1 pg; PTX-2: 9.1 pg, YTX:OA: 2.2 pg, AZA-1: 1.1 pg; PTX-2: 3.7 pg 0.8 pg; GYM: SPX-1: 7.4 pg; 5–10 0.25–0.4 ml/min 25°C–40°C (Villar-Gonzalez (Villar-Gonzalez 2007) et al. (Gerssen et al. (Gerssen et 2009) OA, okadaic acid; PTX, petecnotoxin; SPX, spirolid PTX, petecnotoxin; acid; OA, okadaic for the detection of lipohilic marine toxins: (1) chromatograph toxins: marine lipohilic for the detection of m particle size C18, 150 mm X-Bridge μ was performed in the multiple reaction monitoring (MRM) mode by usi monitoring (MRM) mode by performed in the multiple reaction was g/kg 2010 #24) (Kilcoyne, 5 OA: g/kg, SPX-1: g/kg, SPX-1: 2 mm, 3 μ μ × g/kg, AZA-1: 0.3 g/kg, PTX-2: 10 g/kg, PTX-2: μ μ g/kg l μ μ B: ACN (95%)ACN B: 081090101090 acid) 50 mm formic and 1111.517 14.1 pg 1.9 pg; GYM: SPX-1: 6.9 pg; PTX-2: 70 10 70 70 B: ACN (90%) 30 (pH 11) 90 30 30 0–1 13 15 19 90 10 90 90 10 90 10 10 OA: 10 OA: OA: 15 OA: 4 Cromatography under acid conditions acid under Cromatography conditions basic under Cromatography Main characteristics of two types of liquid chromatography (LC) chromatography liquid two types of of Main characteristics Table 4 Table under basic conditions. under basic gymnodimine; GYM, DTX, dinophysistoxin; AZA,ACN, acetonitrile; azaspiracid; FlowInjection vol. TColumn Mobile phase 5 Gradient 0.2 ml/min Water A: 25°C Time (min)LODs formate 2 mm ammonium (both containing A (%) Mobile phase 22.1 pg, YTX: OA: 6.1 pg, AZA-1: 1.1 pg; Water A: B (%) Mobile phase Time (min) hydroxide) 6.7 mm ammonium (both containing A (%) Mobile phase B (%) Mobile phase lipophilic toxins are also indicated on it. MS/MS detection on it. MS/MS are also indicated toxins lipophilic 1157.5/10 (m/z 1141.5/855.0), 45-OH-YTX 1141.5/1061.7, m/z 817.5/113.0), YTX (m/z 817.5/255.0, m/z (m/z DTX-1 m/z 803.5/113.0), 892.5/ 892.5/821.0, m/z (m/z 1171.5/869.5), PTX-1 1171.5/1091.5, m/z (m/z 1155.5/869.5), 45-OH-homo-YTX 1155.5/1075.5, m/z (m/z 828.5/810.5, m/ AZA-3 (m/z 856.5/820.5) and 856.5/838.5, m/z 842.5/806.5), AZA-2 (m/z 842.5/824.5, m/z 876.5/213.2), AZA-1 (m/z Column C8, 50 mm BDS-Hypersil 26 A.M. Botana et al.: Analysis of marine toxins

2007 ), in combination with antibodies that match their Nowadays, the most used chemical method used for cross-reactivity to the relative toxicity of each analogue detecting CTXs in shellfish samples is LC-MS/MS. Methods (Stewart et al. 2009a,b ). based on HPLC-UV and HPLC-FL have been also used for In general, methods based on LC-MS/MS are spe- many years, but if they are compared with LC-MS/MS, it cific, have sufficient LODs and also have the possibility is obvious that both have disadvantages. As CTXs do not for multi-toxin group detection in a single run. However, have characteristic chromophore groups in their struc- one of the major problems found using this approach is tures (like DA), the use of HPLC-UV results in methods the standard availability. When toxins are not available, with poor sensitivity and selectivity (Caillaud et al. 2010 ). the calibration curve of one toxin standard is often used Despite this, several authors have used this technique as a to quantify other toxins from the same group, assum- strategy for the isolation and purification of CTXs prior to ing a given ionisation conversion factor. This important their characterisation in fish tissues and extracts of dino- issue was checked by using the DSP toxin group (OA, flagellates (Lewis and Jones 1997; Lewis and Vernoux 1998, DTX-1 and DTX-2) (Otero et al. 2011a ), and no compara- Satake et al. 1998 , Hamilton et al. 2002a,b ). However, the ble results were obtained. When each one of the three presence of primary hydroxy groups in some CTXs (P-CTX- analogues was quantified by using the calibration 1, P-CTX-2, P-CTX-3 and 2,3-dihydroxy-CTX-3C) suggested curve of the other ones, the amounts were increased that these toxins could be derivatised into fluorescent or decreased; that is, wrong results were obtained. The esters and thus become suitable for HPLC-FL (Yasumoto highest variability was obtained when DTX-2 was quanti- et al. 1995 ). These analyses are performed in a Develosil fied by using a DTX-1 calibration curve. In this case, the ODS-5 column (4.6 × 250 mm) using an isocratic elution overestimation was up to 204 % . When DTX-2 was quanti- gradient with around 90 % acetonitrile (Yasumoto et al. fied by using an OA calibration curve, the overestimation 1995 ). This method was successfully applied to samples was 88 % . This last quantification type is very usual in of Gambierdiscus toxicus and to different carnivorous fish many laboratories as the OA standard is more available species, including G. javanicus , Lutjanus bohar , Plectro- than DTX-2. Therefore, the use of quality standards or pomus leopardus and Epinephelus fuscoguttatus (Caillaud certified reference materials of each one of the marine et al. 2010 ). However, this approach lacks applicability toxins is essential for the exact quantification of these as many CTX analogues such as P-CTX-3C do not exhibit products by MS, and all compounds for which standards this primary hydroxy group, and therefore they cannot be are not available or unknown should not be monitored derivatised. in the samples. Later, the application of MS played a critical role in the structure elucidation of many CTX congeners (Lewis and Jones 1997 , Hamilton et al. 2002a,b , Pottier et al. 2002a ). Today, LC-MS/MS technology is widely used by many Analysis of CTX laboratories for their detection in contaminated samples. This technique is not the reference method, as happens in This toxin group is the only one that does accumulate the case of other lipophilic toxins. Each laboratory uses in fish of certain species, such as G. javanicus , Lutjanus LC conditions that are considered more properly and thus, bohar , Plectropomus leopardus and Epinephelus fuscogut- the bibliography describes different methods of analy- tatus (Caillaud et al. 2010 ), not in bivalves. CTXs are potent sis. Prior to the chromatographic analysis of CTXs in fish polyether toxins issued from Gambierdiscus species of samples, strict clean protocols are used (Hamilton et al. dinoflagellates. These toxins are usually found in areas 2002a,b , Pottier et al. 2002a , Otero et al. 2010 ). Although between 35° N and 35° S latitude, mainly Indo-Pacific and these steps improve the sensitivity and reduce the matrix Caribbean areas. However, in recent years, these toxins effects, it is certain that they do not remove metallic impu- are increasingly appearing in countries not expected for rities, which result in the formation of adducts (Hamilton their latitude such as waters close to the European and et al. 2002a , Otero et al. 2010 ). African continents (Perez -Arellano et al. 2005 , Bentur and LC systems comprise C18 columns, such as 5-μ m Spanier 2007 , Otero et al. 2010 ). Its distribution is expected Phenomenex Luna (Lewis et al. 2009 ) or 3.5-μ m Zorbax to shift towards other countries, and in fact this seems to 300SB (Hamilton et al. 2002a,b ) and C8 columns such have begun the trend. For this reason, some laboratories as 3- μ m Phenomenex Hyperclone (Roeder et al. 2010 ) that did not normally include these compounds in the or 3-μ m Phenomenex Luna (Dechraoui et al. 2005 ). toxin analysis are now in need of improving the methods Mobile phases are composed generally by acetonitrile for detecting them. and water, and the use of gradients of these organic A.M. Botana et al.: Analysis of marine toxins 27 solvents buffered with 1 mm ammonium acetate improve Another consideration to be taken into account in the the separation and detection of CTXs compared with analysis of CTXs by LC-MS/MS is the big number of ana- an acetonitrile:water gradient modified with 0.1 % tri- logues belonging to this toxin group and the few stand- fluorocetic acid (Lewis and Jones 1997 ). MS detection ards available. To date, more than 50 different congeners is usually performed in the positive MRM mode (Perez- have been identified (Litaker et al. 2010 ), and the pres- Arellano et al. 2005, Roeder et al. 2010 ) or in the Q1 mode ence of several CTXs with equal molecular weight in the selecting a range of mass (Hamilton et al. 2002a , Pottier same extract is very frequent. Up to six different analogues et al. 2002a ). In the last case, the MS spectrum will show for the compound 1111.6 m/z (CTX-1B) eluting in different the characteristic pattern of ion formation for CTX poly- retention times have been described (Lewis and Jones ethers (multiple losses of water and the formation of 1997 ). This issue makes the identification of CTX difficult. sodium and ammonium adducts) (Pottier et al. 2002a ). Moreover, because of the lack of certified standards and Each CTX gives rise to prominent ammonium, potassium reference materials and the limited amounts of contami- and sodium adducts and losses of waters. An example nated material available for method development, the vali- of a typical chromatogram and spectrum of a CTX stand- dation status of LC-MS/MS methods is very restricted and ard is shown in Figure 3 . The compound is the synthetic up to now no collaborative study has been undertaken.

51-hydroxy-CTX-3C with M r 1038.5, and the identification Recently, a method with 14 reference toxins prepared was performed in a UPLC system coupled to an Xevo TQ by either synthesis or isolation from natural sources was MS mass spectrometer from Waters (Manchester, UK). used to identify and quantify 16 toxins from the CTXs

The column used was a Waters Acquity UPLC BEH C18 group (Yogi et al. 2011 ). LC separation was performed in (100 × 2.1 mm; 1.7 μm), and the mobile phase consisted of a Zorbax Eclipse Plus C18 column in < 14 min by using a gradient of acetonitrile/water. As can be observed, the a linear gradient of mobile phases: 5 m m ammonium ion 1039.5 m/z due to the [M+ H] + is not the most promi- formate and 0.1% formic acid in water and in methanol. nent. However, the ions m/z 1077.5 [M+ K] + and m/z 1061.5 The mass spectrometer operates in the positive mode [M+ Na] + have a high intensity. In addition, the spectrum for the monitoring of sodium adduct ions [M+ Na] + . This shows two water losses, m/z 1021.5, which corresponds method has greatly reduced the time of analysis as up to to [M + H-H2 O], and m/z 1003.5, which corresponds to now almost all methods described use run times of around

[M+ H-H 2 O]. 50 min (Hamilton et al. 2002a,b, 2009, Pottier et al. 2002b ,

XEVO-TQMS A 51-OH-CTX-3C SIR of 1 channel ES+ 5.80 TIC 100 1039.5 6.36e4 %

0 2.00 4.00 6.00 8.00 10.00 12.00 Time (min)

B 100 + [M+H]+ MS2 ES+ [M+H - 2H2O] 1077.5 1003.5 1.09e5 [M+Na]+ 1061.5

[M+H - 2H O]+ % 2 1021.5 [M+H]+ 1039.5

0 1000 1020 1040 1060 1080 1100 1120 1140 1160 m/z

Figure 3 Chromatogram (A) and mass spectrum (B) of 51-OH-CTX-3C (M r 1038.5) standard at 1000 ng/ml, using selected ion recording (SIR) UPLC mode of 1039.5 m/z [M +H] + . Analysis was performed in an ACQUITY UPLC system coupled with Xevo TQ MS mass spectrometer. × μ Chromatographic identification was achieved in a Waters Acquity UPLC BEH C18 column (100 2.1 mm; 1.7 m). The mobile phase consisted of two components: acetonitrile/water (95:5) (A) and water (B), both containing 50 m m formic acid and 2 m m ammonium formate using a gradient elution. 51-OH-CTX-3C, 51-hydroxy-CTX-3C; UPLC, ultraperformance liquid chromatography. 28 A.M. Botana et al.: Analysis of marine toxins

Dechraoui et al. 2005 , Roeder et al. 2010 ). This method pre- Conclusions sents limits of 0.25 pg for CTX detection. This means that it can detect CTX-1B at 45 pg/g in fish flesh. However, an Although there are many choices for the analysis and obstacle to the widespread use and further validation of detection of marine toxins, the current legal requirement this method is the difficulty in obtaining standard toxins. demands the use of LC-MS for most of the groups, with In summary, the LC-MS/MS methodology has proven the exception of the DA and saxitoxin groups, which can to be extremely useful for identifying characteristic profiles be detected by HPLC. The chemical complexity of each of CTXs in different strains of Gambierdiscus spp. (Rhodes toxin group, with new analogues being added every year, et al. 2010 , Roeder et al. 2010 ) and fish (Pottier et al. 2002a , and their different profiles depending on the geographi- Dechraoui et al. 2005 , Otero et al. 2010 ) worldwide. The LC cal area and the climate conditions highlight the impor- and MS methods and instruments have greatly improved tance of robust analytical methods and a good supply of in recent years. Because of the high selectivity, accurate analytical standards. Along with this demand, it is very mass spectra are obtained, susceptible to being compared important to continue with the development of high- with spectra of known molecules or standard spectra in throughput methods that may provide quick results about the case they are available. These advances give rise to the the presence of these very toxic compounds in food, as confirmation of many suspected cases of ciguateric fish food safety will always be a concern for industries that by selecting the transitions object study. Techniques such deal with certain mollusks and certain types of fish. as the UPLC-MS/MS improve sensitivity; however, at the time of protecting the human health they are still far from being used as a routine method in the laboratories for the Received June 7, 2012; accepted October 5, 2012; previously detection of CTX. published online November 23, 2012

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Ana M. Botana professor of Analytical Chemistry; 60 international papers. Paula Rodr í guez and Paz Otero Postdoctoral researchers; 20 international papers.

Luis M. Botana full professor of Pharmacology at the University of Santiago de Compostela (USC), expert in marine toxin pharmacology; 200 international papers, editor of 8 books and author of 25 patents. Amparo Alfonso Professor of Pharmacology at the USC; 90 International papers, and 6 patents.