Analysis of Total-Forms of Cyanotoxins Microcystins in Biological Matrices: A Methodological Review.

Microcystins (MCs) are cyclic heptapeptidic toxins produced by many cyanobacteria. Microcystins can be accumulated in various matrices in two forms: a free cellular fraction and a covalently protein-bound form. To detect and quantify the concentration of microcystins, a panel of techniques on various matrices (water, sediments, and animal tissues) is available. The analysis of MCs can concern the free or the total (free plus covalently bound) fractions. Free-form analyses of MCs are the most common and easiest to detect, whereas total-form analyses are much less frequent and more complex to achieve. The objective of this review is to summarize the different methods of extraction and analysis that have been developed for total forms. Four extraction methods were identified: MMPB (2-methyl-3-methoxy-4-phenylbutyric acid) method, deconjugation at basic pH, ozonolysis, and laser irradiation desorption. The study of the bibliography on the methods of extraction and analysis of the total forms of MCs showed that the reference method for the subject remains the MMPB method even if alternative methods and, in particular, deconjugation at basic pH, showed results encouraging the continuation of the methodological development on different matrices and on naturally-contaminated samples.

Multilaboratory Collaborative Study of a Nontarget Data Acquisition for Target Analysis (nDATA) Workflow Using Liquid Chromatography-High-Resolution Accurate Mass Spectrometry for Pesticide Screening in Fruits and Vegetables.

Nontarget data acquisition for target analysis (nDATA) workflows using liquid chromatography-high-resolution accurate mass (LC-HRAM) spectrometry, spectral screening software, and a compound database have generated interest because of their potential for screening of pesticides in foods. However, these procedures and particularly the instrument processing software need to be thoroughly evaluated before implementation in routine analysis. In this work, 25 laboratories participated in a collaborative study to evaluate an nDATA workflow on high moisture produce (apple, banana, broccoli, carrot, grape, lettuce, orange, potato, strawberry, and tomato). Samples were extracted in each laboratory by quick, easy, cheap, effective, rugged, and safe (QuEChERS), and data were acquired by ultrahigh-performance liquid chromatography (UHPLC) coupled to a high-resolution quadrupole Orbitrap (QOrbitrap) or quadrupole time-of-flight (QTOF) mass spectrometer operating in full-scan mass spectrometry (MS) data-independent tandem mass spectrometry (LC-FS MS/DIA MS/MS) acquisition mode. The nDATA workflow was evaluated using a restricted compound database with 51 pesticides and vendor processing software. Pesticide identifications were determined by retention time (tR, ±0.5 min relative to the reference retention times used in the compound database) and mass errors (δM) of the precursor (RTP, δM ≤ ±5 ppm) and product ions (RTPI, δM ≤ ±10 ppm). The elution profiles of all 51 pesticides were within ±0.5 min among 24 of the participating laboratories. Successful screening was determined by false positive and false negative rates of <5% in unfortified (pesticide-free) and fortified (10 and 100 µg/kg) produce matrices. Pesticide responses were dependent on the pesticide, matrix, and instrument. The false negative rates were 0.7 and 0.1% at 10 and 100 µg/kg, respectively, and the false positive rate was 1.1% from results of the participating LC-HRAM platforms. Further evaluation was achieved by providing produce samples spiked with pesticides at concentrations blinded to the laboratories. Twenty-two of the 25 laboratories were successful in identifying all fortified pesticides (0-7 pesticides ranging from 5 to 50 µg/kg) for each produce sample (99.7% detection rate). These studies provide convincing evidence that the nDATA comprehensive approach broadens the screening capabilities of pesticide analyses and provide a platform with the potential to be easily extended to a larger number of other chemical residues and contaminants in foods.

First Evidence of the Presence of Anatoxin-A in Sea Figs Associated with Human Food Poisonings in France.

From January 2011 to March 2018, 26 patients aged from 20 to 80 years old reported being sick in France after eating sea figs of the genus Microcosmus. The patients had symptoms evoking a cerebellar syndrome: blurred or double vision, ataxia and dizziness, asthenia, headache, muscle cramps, paresthesia and digestive disorders (nausea, vomiting and diarrhea). Three of the 18 food poisoning events recorded by the Poison Control Center in Marseille and involving four patients were further investigated as the meal leftovers were collected and analyzed. A previous study ruled out the presence of the regulated lipophilic marine toxins after high-resolution mass spectrometry, but further analyses were required to look for hydrophilic cyanotoxins. The sea fig leftovers from food poisoning case Numbers 1 (January 2011), 6 (December 2012) and 17 (March 2018) of this published case series were analyzed by hydrophilic interaction liquid chromatography coupled to low- and high-resolution mass spectrometry to investigate the presence of hydrophilic cyanotoxins. The sea fig samples showed anatoxin-a (ATX-a) concentrations ranging from 193.7 to 1240.2 µg/kg. The sea fig control sample analyzed was also contaminated with ATX-a but in a much smaller concentration (22.5 µg/kg). To the best of our knowledge, this is the first report of human food poisoning involving ATX-a as the possible causative toxin where the cyanotoxin could be unequivocally identified.

Extended Targeted and Non-Targeted Strategies for the Analysis of Marine Toxins in Mussels and Oysters by (LC-HRMS).

When considering the geographical expansion of marine toxins, the emergence of new toxins and the associated risk for human health, there is urgent need for versatile and efficient analytical methods that are able to detect a range, as wide as possible, of known or emerging toxins. Current detection methods for marine toxins rely on a priori defined target lists of toxins and are generally inappropriate for the detection and identification of emerging compounds. The authors describe the implementation of a recent approach for the non-targeted analysis of marine toxins in shellfish with a focus on a comprehensive workflow for the acquisition and treatment of the data generated after liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) analysis. First, the study was carried out in targeted mode to assess the performance of the method for known toxins with an extended range of polarities, including lipophilic toxins (okadaic acid, dinophysistoxins, azaspiracids, pectenotoxins, yessotoxins, cyclic imines, brevetoxins) and domoic acid. The targeted method, assessed for 14 toxins, shows good performance both in mussel and oyster extracts. The non-target potential of the method was then challenged via suspects and without a priori screening by blind analyzing mussel and oyster samples spiked with marine toxins. The data processing was optimized and successfully identified the toxins that were spiked in the blind samples.

Mixtures of Lipophilic Phycotoxins: Exposure Data and Toxicological Assessment.

Lipophilic phycotoxins are secondary metabolites produced by phytoplanktonic species. They accumulate in filter-feeding shellfish and can cause human intoxication. Regulatory limits have been set for individual toxins, and the toxicological features are well characterized for some of them. However, phycotoxin contamination is often a co-exposure phenomenon, and toxicological data regarding mixtures effects are very scarce. Moreover, the type and occurrence of phycotoxins can greatly vary from one region to another. This review aims at summarizing the knowledge on (i) multi-toxin occurrence by a comprehensive literature review and (ii) the toxicological assessment of mixture effects. A total of 79 publications was selected for co-exposure evaluation, and 44 of them were suitable for toxin ratio calculations. The main toxin mixtures featured okadaic acid in combination with pectenotoxin-2 or yessotoxin. Only a few toxicity studies dealing with co-exposure were published. In vivo studies did not report particular mixture effects, whereas in vitro studies showed synergistic or antagonistic effects. Based on the combinations that are the most reported, further investigations on mixture effects must be carried out.

Occurrence of ß-N-methylamino-l-alanine (BMAA) and Isomers in Aquatic Environments and Aquatic Food Sources for Humans.

The neurotoxin ß-N-methylamino-l-alanine (BMAA), a non-protein amino acid produced by terrestrial and aquatic cyanobacteria and by micro-algae, has been suggested to play a role as an environmental factor in the neurodegenerative disease Amyotrophic Lateral Sclerosis-Parkinsonism-Dementia complex (ALS-PDC). The ubiquitous presence of BMAA in aquatic environments and organisms along the food chain potentially makes it public health concerns. However, the BMAA-associated human health risk remains difficult to rigorously assess due to analytical challenges associated with the detection and quantification of BMAA and its natural isomers, 2,4-diamino butyric acid (DAB), ß-amino-N-methyl-alanine (BAMA) and N-(2-aminoethyl) glycine (AEG). This systematic review, reporting the current knowledge on the presence of BMAA and isomers in aquatic environments and human food sources, was based on a selection and a score numbering of the scientific literature according to various qualitative and quantitative criteria concerning the chemical analytical methods used. Results from the best-graded studies show that marine bivalves are to date the matrix containing the higher amount of BMAA, far more than most fish muscles, but with an exception for shark cartilage. This review discusses the available data in terms of their use for human health risk assessment and identifies knowledge gaps requiring further investigations.

Hunt for Palytoxins in a Wide Variety of Marine Organisms Harvested in 2010 on the French Mediterranean Coast.

During the summer of 2010, 31 species including fish, echinoderms, gastropods, crustaceans, cephalopods and sponges were sampled in the Bay of Villefranche on the French Mediterranean coast and screened for the presence of PLTX-group toxins using the haemolytic assay. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for confirmatory purposes and to determine the toxin profile. The mean toxin concentration in the whole flesh of all sampled marine organisms, determined using the lower- (LB) and upper-bound (UB) approach was 4.3 and 5.1 µg·kg(-1), respectively, with less than 1% of the results exceeding the European Food Safety Authority (EFSA) threshold of 30 µg·kg(-1)and the highest values being reported for sea urchins (107.6 and 108.0 µg·kg(-1)). Toxins accumulated almost exclusively in the digestive tube of the tested species, with the exception of octopus, in which there were detectable toxin amounts in the remaining tissues (RT). The mean toxin concentration in the RT of the sampled organisms (fishes, echinoderms and cephalopods) was 0.7 and 1.7 µg·kg(-1) (LB and UB, respectively), with a maximum value of 19.9 µg·kg(-1) for octopus RT. The herbivorous and omnivorous organisms were the most contaminated species, indicating that diet influences the contamination process, and the LC-MS/MS revealed that ovatoxin-a was the only toxin detected.

Collaborative study for the detection of toxic compounds in shellfish extracts using cell-based assays. Part I: screening strategy and pre-validation study with lipophilic marine toxins.

Human poisoning due to consumption of seafood contaminated with phycotoxins is a worldwide problem, and routine monitoring programs have been implemented in various countries to protect human consumers. Following successive episodes of unexplained shellfish toxicity since 2005 in the Arcachon Bay on the French Atlantic coast, a national research program was set up to investigate these atypical toxic events. Part of this program was devoted to fit-for-purpose cell-based assays (CBA) as complementary tools to collect toxicity data on atypical positive-mouse bioassay shellfish extracts. A collaborative study involving five laboratories was conducted. The responses of human hepatic (HepG2), human intestinal (Caco2), and mouse neuronal (Neuro2a) cell lines exposed to three known lipophilic phycotoxins-okadaic acid (OA), azaspiracid-1 (AZA1), and pectenotoxin-2 (PTX2)-were investigated. A screening strategy composed of standard operating procedures and a decision tree for dose-response modeling and assay validation were designed after a round of "trial-and-error" process. For each toxin, the shape of the concentration-response curves and the IC(50) values were determined on the three cell lines. Whereas OA induced a similar response irrespective of the cell line (complete sigmoid), PTX2 was shown to be less toxic. AZA1 induced cytotoxicity only on HepG2 and Neuro2a, but not on Caco2. Intra- and inter-laboratory coefficients of variation of cell responses were large, with mean values ranging from 35 to 54 % and from 37 to 48 %, respectively. Investigating the responses of the selected cell lines to well-known toxins is the first step supporting the use of CBA among the panel of methods for characterizing atypical shellfish toxicity. Considering these successful results, the CBA strategy will be further applied to extracts of negative, spiked, and naturally contaminated shellfish tissues.

Collaborative study for the detection of toxic compounds in shellfish extracts using cell-based assays. Part II: application to shellfish extracts spiked with lipophilic marine toxins.

Successive unexplained shellfish toxicity events have been observed in Arcachon Bay (Atlantic coast, France) since 2005. The positive mouse bioassay (MBA) revealing atypical toxicity did not match the phytoplankton observations or the liquid chromatography-tandem mass spectrometry (LC-MS/MS) investigations used to detect some known lipophilic toxins in shellfish. The use of the three cell lines (Caco2, HepG2, and Neuro2a) allows detection of azaspiracid-1 (AZA1), okadaic acid (OA), or pectenotoxin-2 (PTX2). In this study, we proposed the cell-based assays (CBA) as complementary tools for collecting toxicity data about atypical positive MBA shellfish extracts and tracking their chromatographic fractionation in order to identify toxic compound(s). The present study was intended to investigate the responses of these cell lines to shellfish extracts, which were either control or spiked with AZA1, OA, or PTX2 used as positive controls. Digestive glands of control shellfish were extracted using the procedure of the standard MBA for lipophilic toxins and then tested for their cytotoxic effects in CBA. The same screening strategy previously used with pure lipophilic toxins was conducted for determining the intra- and inter-laboratory variabilities of the responses. Cytotoxicity was induced by control shellfish extracts whatever the cell line used and regardless of the geographical origin of the extracts. Even though the control shellfish extracts demonstrated some toxic effects on the selected cell lines, the extracts spiked with the selected lipophilic toxins were significantly more toxic than the control ones. This study is a crucial step for supporting that cell-based assays can contribute to the detection of the toxic compound(s) responsible for the atypical toxicity observed in Arcachon Bay, and which could also occur at other coastal areas.

Solid phase extraction for removal of matrix effects in lipophilic marine toxin analysis by liquid chromatography-tandem mass spectrometry.

The potential of solid phase extraction (SPE) clean-up has been assessed to reduce matrix effects (signal suppression or enhancement) in the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of lipophilic marine toxins. A large array of ion-exchange, silica-based, and mixed-function SPE sorbents was tested. Polymeric sorbents were found to retain most of the toxins. Optimization experiments were carried out to maximize recoveries and the effectiveness of the clean-up. In LC-MS/MS analysis, the observed matrix effects can depend on the chromatographic conditions used, therefore, two different HPLC methods were tested, using either an acidic or an alkaline mobile phase. The recovery of the optimized SPE protocol was around 90% for all toxins studied and no break-through was observed. The matrix effects were determined by comparing signal response from toxins spiked in crude and SPE-cleaned extracts with those derived from toxins prepared in methanol. In crude extracts, all toxins suffered from matrix effects, although in varying amounts. The most serious effects were observed for okadaic acid (OA) and pectenotoxin-2 (PTX2) in the positive electrospray ionization mode (ESI(+)). SPE clean-up on polymeric sorbents in combination with the alkaline LC method resulted in a substantial reduction of matrix effects to less than 15% (apparent recovery between 85 and 115%) for OA, yessotoxin (YTX) in ESI(-) and azaspiracid-1 (AZA1), PTX2, 13-desmethyl spirolides C (SPX1), and gymnodimine (GYM) in ESI(+). In combination with the acidic LC method, the matrix effects after SPE were also reduced but nevertheless approximately 30% of the matrix effects remained for PTX2, SPX1, and GYM in ESI(+). It was concluded that SPE of methanolic shellfish extracts can be very useful for reduction of matrix effects. However, the type of LC and MS methods used is also of great importance. SPE on polymeric sorbents in combination with LC under alkaline conditions was found the most effective method.

Confirmation by LC-MS/MS of azaspiracids in shellfish from the Portuguese north-western coast.

The search for azaspiracids (AZAs) in shellfish on the Portuguese coast started in 2002, but the presence of these toxins could not be demonstrated until the summer of 2006. Analysis by liquid-chromatography-tandem mass spectrometry (LC-MS/MS) allowed the confirmation of AZA2 as a dominant compound, followed by AZA1, in blue mussel (Mytilus galloprovincialis), common cockle (Cerastoderma edule), clams (Venerupis senegalensis, Ruditapes decussatus), razor clam (Solen marginatus) and oyster (Crassostrea spp). Traces of AZA3 were found only in blue mussel. Total levels of AZA1-3 determined in the whole flesh by LC-MS/MS ranged from 1.6 to 6.1 microg/kg. The finding of low levels of AZAs since 2002 suggests a low risk level when compared with the highest risks posed by diarrhetic shellfish poisoning (DSP) and paralytic shellfish poisoning (PSP) toxins. However, the limited number of years studied might generate a misleading conclusion. The contamination with PSP is an example, as no contamination occurred for an extended period of time between 1996 and 2004, despite high levels having occurred outside this period. Thus, there appears overall a moderate likelihood of occurrence of AZAs in the range that may be relevant to consumers.

Development of an ultra-performance liquid chromatography-mass spectrometry method for the detection of lipophilic marine toxins.

A rapid method for the detection of marine toxins was developed using an ultra-performance liquid chromatography (UPLC) system coupled to a latest generation mass spectrometry (MS) system. The analysis of 21 lipophilic marine toxins was achieved on an Acquity C18 column using a water-acetonitrile gradient with a cycle time of 6.6 min, reducing analysis time by more than a factor two compared to HPLC while maintaining peak resolution. Linear ranges, limits of detection and limits of quantification were established for okadaic acid (OA), pectenotoxin-2, azaspiracid-1 (AZA1), yessotoxin, gymnodimine and 13-desmethylspirolide C. The method was found to be accurate when using a triplicate methanolic extraction. Matrix effects were assessed by standard addition of OA and AZA1 in extracts of raw and heat-treated flesh of mussels and oysters. For the analysis of AZA1, the UPLC-MS method was always prone to signal suppression, while for OA analysis signal suppression was observed in extracts of raw shellfish flesh and signal enhancement in extracts of heat-treated flesh. Matrix effects occurring in the method presented are diminished compared to previous studies.

Improved solid-phase extraction procedure in the analysis of paralytic shellfish poisoning toxins by liquid chromatography with fluorescence detection.

The analysis of shellfish extracts for the determination of paralytic shellfish poisoning (PSP) toxins by liquid chromatography with fluorescence detection repeatedly showed the presence of a compound suspected to interfere with gonyautoxin 4. The first aim of this study was to confirm by liquid chromatography coupled to tandem mass spectrometry that this compound was not gonyautoxin 4. The second part of this work was to improve a nonvolumetric C(18) solid-phase extraction (SPE) procedure to evaluate the removal of the interference associated with the recovery of PSP toxins. The cleanup procedure was modified into a volumetric SPE procedure and proved to efficiently and totally remove the interference while recovering from 78 to 85% of the PSP toxins available as commercial standards (saxitoxin, neosaxitoxin, gonyautoxins 1-4) and considered as major PSP toxins in human intoxication, with 85% recovery for gonyautoxin 4. The efficiency of this cleanup procedure was checked on shellfish extracts containing this interference and originating from France and Turkey.

First evidence on occurrence of gymnodimine in clams from Tunisia.

Among several batches of clams harvested in Tunisia and imported to France, a small number of them were found to be neurotoxic to mice (intraperitoneal injection) as determined by the diarrhetic shellfish poisoning (DSP) bioassay developed by Yasumoto et al. (1978). The present study was conducted to confirm the nature of the toxic agent, suspected to be gymnodimine. Liquid chromatography tandem mass spectrometry analyses unequivocally revealed the presence of gymnodimine in the shellfish, making Tunisia the second country, after New Zealand, where shellfish contamination with this phycotoxin is reported. Gymnodimine B and C analogues were not detected in the clam samples. Gymnodimine preferentially accumulates in the digestive gland of the Tunisian clams, although substantial amounts are also found in the meat.