Paul McNabb

Complex toxin profiles in phytoplankton and Greenshell mussels (Perna canaliculus), revealed by LC–MS/MS analysis

Toxin profiles were determined in phytoplankton cell concentrates and Greenshell mussels (Perna canaliculus) exposed to a dinoflagellate bloom dominated by Dinophysis acuta and Protoceratium reticulatum. This was achieved by using a method for the simultaneous identification and quantification of a variety of micro-algal toxins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionisation (+/-) and monitoring of daughter ions in multiple reaction modes. Plankton concentrates and shellfish contained high levels of yessotoxins (YTXs) and pectenotoxins (PTXs) and low levels of okadaic acid (OA). A high proportion (>87%) of the OA in both plankton and shellfish was released by alkaline hydrolysis. An isomer of pectenotoxin 1 (PTX1i) was nearly as abundant as pectenotoxin 2 (PTX2) in the plankton and shellfish, and the latter contained high levels of their respective seco acids. DTX1, DTX2, and PTX6 were not detected. MS-MS experiments revealed that the shellfish contained several other oxygenated metabolites of YTX in addition to 45-hydroxy yessotoxin (45OH-YTX). Gymnodimine (GYM) was present in the shellfish but not plankton and it was probably the residue from a previous GYM contamination event. Unlike the other toxins, GYM was concentrated in tissues outside the digestive gland and levels did not decrease over 5 months. The depuration rates of YTX and PTXs from mussels were modelled.

Isolation, structural determination and acute toxicity of pinnatoxins E, F and G.

Pinnatoxins and pteriatoxins are a group of cyclic imine toxins that have hitherto only been isolated from Japanese shellfish. As with other cyclic imine shellfish toxins, pinnatoxins cause rapid death in the mouse bioassay for lipophilic shellfish toxins, but there is no evidence directly linking these compounds to human illness. We have identified the known pinnatoxins A (1) and D (6), and the novel pinnatoxins E (7), F (8) and G (5), in a range of shellfish and environmental samples from Australia and New Zealand using LC-MS. After isolation from the digestive glands of Pacific oysters, the structures of the novel pinnatoxins were determined by mass spectrometry and NMR spectroscopy, and their LD(50) values were evaluated by ip administration to mice. Examination of the toxin structures, together with analysis of environmental samples, suggests that pinnatoxins F and G are produced separately in different dinoflagellates. Furthermore, it appears probable that pinnatoxin F (8) is the progenitor of pinnatoxins D (6) and E (7), and that pinnatoxin G (6) is the progenitor of both pinnatoxins A-C (1 and 2) and pteriatoxins A-C (3 and 4), via metabolic and hydrolytic transformations in shellfish.

Development of a sensitive and selective liquid chromatography–mass spectrometry method for high throughput analysis of paralytic shellfish toxins using graphitised carbon solid phase extraction

Routine regulatory monitoring of paralytic shellfish toxins (PST) commonly employs oxidative derivitisation and complex liquid chromatography fluorescence detection methods (LC-FL). The pre-column oxidation LC-FL method is currently implemented in New Zealand and the United Kingdom. When using this method positive samples are fractionated and two different oxidations are required to confirm the identity and quantity of each PST analogue present. There is a need for alternative methods that are simpler, provide faster turnaround times and have improved detection limits. Hydrophilic interaction liquid chromatography (HILIC) HPLC-MS/MS analysis of PST has been used for research purposes, but high detection limits and substantial sample matrix issues have prevented it from becoming a viable alternative for routine monitoring purposes. We have developed a HILIC UPLC-MS/MS method for paralytic shellfish toxins with an optimised desalting clean-up procedure on inexpensive carbon solid phase extraction cartridges for reduction of matrix interferences. This represents a major technical breakthrough and allows sensitive, selective and rapid analysis of paralytic shellfish toxins from a variety of sample types, including many commercially produced bivalve molluscan shellfish species. Additionally, this analytical approach avoids the need for complex calculations to determine sample toxicity, as unlike other methods each PST analogue is able to be quantified as a single resolved peak. This article presents the method development and optimisation information. A thorough single laboratory validation study has subsequently been performed and this data will be presented elsewhere.

Detection of tetrodotoxin from the grey side-gilled sea slug - Pleurobranchaea maculata, and associated dog neurotoxicosis on beaches adjacent to the Hauraki Gulf, Auckland, New Zealand

Investigations into a series of dog poisonings on beaches in Auckland, North Island, New Zealand, resulted in the identification of tetrodotoxin (TTX) in the grey side-gilled sea slug, Pleurobranchaea maculata. The levels of TTX in P. maculata, assayed by liquid chromatography-mass spectrometry (LC-MS) ranged from 91 to 850 mg kg(-1) with a median level of 365 mg kg(-1) (n = 12). In two of the dog poisoning cases, vomit and gastrointestinal contents were found to contain TTX. Adult P. maculata were maintained in aquaria for several weeks. Levels of TTX decreased only slightly with time. While in the aquaria, P. maculata spawned, with each individual producing 2-4 egg masses. The egg masses and 2-week old larvae also contained TTX. Tests for other marine toxins were negative and no other organisms from the area contained TTX. This is the first time TTX has been identified in New Zealand and the first detection of TTX in an opisthobranch.

Toxic dinoflagellates (Dinophyceae) from Rarotonga, Cook Islands

Dinoflagellate species isolated from the green calcareous seaweed, Halimeda sp. J.V. Lamouroux, growing in Rarotongan lagoons, included Gambierdiscus australes Faust & Chinain, Coolia monotis Meunier, Amphidinium carterae Hulburth, Prorocentrum lima (Ehrenberg) Dodge, P. cf. maculosum Faust and species in the genus Ostreopsis Schmidt. Isolates were identified to species level by scanning electron microscopy and/or DNA sequence analysis. Culture extracts of G. australes isolate CAWD149 gave a response of 0.04 pg P-CTX-1 equiv. per cell by an N2A cytotoxicity assay (equivalent to ca 0.4 pg CTX-3C cell(-1)). However, ciguatoxins were not detected by LC-MS/MS. Partitioned fractions of the cell extracts potentially containing maitotoxin were found to be very toxic to mice after intraperitoneal (i.p.) injection. A. carterae was also of interest as extracts of mass cultures caused respiratory paralysis in mice at high doses, both by i.p. injection and by oral administration. The Rarotongan isolate fell into a different clade to New Zealand A. carterae isolates, based on DNA sequence analysis, and also had a different toxin profile. As A. carterae co-occurred with G. australes, it may contribute to human poisonings attributed to CTX and warrants further investigation. A crude extract of C. monotis was of low toxicity to mice by i.p. injection, and an extract of Ostreopsis sp. was negative in the palytoxin haemolysis neutralisation assay.

Dinoflagellate Vulcanodinium rugosum identified as the causative organism of pinnatoxins in Australia, New Zealand and Japan

Rhodes L., Smith K., Selwood A., McNabb P., Munday R., Suda S., Molenaar S. and Hallegraeff G. 2011. Dinoflagellate Vulcanodinium rugosum identified as the causative organism of pinnatoxins in Australia, New Zealand and Japan. Phycologia 50: 624–628. DOI: 10.2216/11-19.1 The producers of pinnatoxins E and F were first identified through systematic light microscopy-mass spectrometry (LC-MS) screening of New Zealand (NZ) dinoflagellate isolates. Pinnatoxins E, F and G were subsequently detected for South Australian (AUS) isolates, and a pinnatoxin G producer was recently identified from Okinawa, Japan. The NZ, AUS and Japanese isolates appear morphologically identical to Mediterranean field samples recently described as Vulcanodinium rugosum. Motile forms (24–32 µm long, 20–30 µm wide) have a wide, deeply incised cingulum displaced by 17% of the cell length and a deep sulcus. Photosynthetic motile cells have the same distinctive apical pore complex with raised collar and short canal plate. The reticulate thecal ornamentation resembles that of Bysmatrum because the three intercalary plates are connected. The posterior sulcal plate Sp does not touch the cingulum (as in Bysmatrum, Pentapharsodinium and Ensiculifera). The plate formula for the NZ, AUS and Japanese isolates is Po, X, 4′, 3a, 7″, 6c (5c + T), 5s, 5′″, 2″″. The dominant non-motile life-cycle stage comprises clusters of brown to black, spherical, unornamented, non-calcareous division cells (30–35 µm diameter). Genetically these isolates fall within the Peridiniales, closely related to the calciodinellid genera, Pentapharsodinium, Ensiculifera and Scrippsiella. The large subunit (LSU) rDNA and internal transcribed spacer (ITS) sequences exhibit sequence differences compared to each other and to the French field material (96–97% LSU and 84–88% ITS similarity, respectively). This variation and pinnatoxin profile differences suggest a possible species complex.

Single-Laboratory Validation of a Multitoxin Ultra-Performance LC-Hydrophilic Interaction LC-MS/MS Method for Quantitation of Paralytic Shellfish Toxins in Bivalve Shellfish.

A single-laboratory validation study was conducted for the hydrophilic interaction-LC-MS/MS analysis of paralytic shellfish toxins (PSTs) in bivalve shellfish. The method was developed as an alternative to the precolumn oxidation AOAC 2005.06 and postcolumn oxidation AOAC 2011.02 LC with fluorescence detection methods, receptor binding assay AOAC 2011.27, as well as the mouse bioassay AOAC 959.08. PSTs assessed were saxitoxin, neosaxitoxin, deoxydecarbamoylsaxitoxin, decarbamoylsaxitoxin, decarbamoylneosaxitoxin, gonyautoxins 1-6, decarbamoylgonyautoxins 2-3, and N-sulfocarbamoyl gonyautoxins 2&3. The method also included the determination of decarbamoylgonyautoxins 1&4, N-sulfocarbamoyl gonyautoxins 1&4, and M toxins. Twelve commercially produced bivalve species from both New Zealand and the United Kingdom were assessed, including mussels, oysters, scallops, and clams. Validation studies demonstrated acceptable method performance characteristics for specificity, linearity, recovery, repeatability, and within-laboratory reproducibility. LOD and LOQ were significantly improved in comparison to current fluorescence-based detection methods, and the method was shown to be rugged. The method performed well in comparison to AOAC 2005.06, with evidence obtained from both comparative analysis of 1141 PST-contaminated samples and successful participation in proficiency testing schemes. The method is suitable for use in regulatory testing and will be submitted for an AOAC collaborative study.

A sensitive assay for palytoxins, ovatoxins and ostreocins using LC-MS/ MS analysis of cleavage fragments from micro-scale oxidation

Palytoxin is a highly toxic non-proteinaceous marine natural product that can pass through the food chain and result in human illnesses. A recent review by the European Food Safety Authority concluded that palytoxin requires regulation in seafood and a limit of 30 μg kg⁻¹ for shellfish flesh was suggested. Current methods based on LC-MS detection of intact palytoxins do not have sufficient sensitivity to enforce this limit for palytoxin. To improve sensitivity for trace analysis, a novel screen approach has been developed that uses LC-MS/MS analysis of substructures generated by oxidative cleavage of vicinal diol groups present in the intact toxin. Oxidation of palytoxins, ovatoxins or ostreocins using periodic acid generates two nitrogen-containing aldehyde fragments; an amino aldehyde common to these toxins, and an amide aldehyde that may vary depending on toxin type. Conditions for micro-scale oxidation of palytoxin were optimised, which include a novel SPE cleanup and on-column oxidation step. Rapid analysis of cleavage fragments was established using LC-MS/MS. Linear calibrations were established for the amino aldehyde from a palytoxin reference standard, which is suitable for all known palytoxin-like compounds, and for the confirmatory amide aldehydes of palytoxin and ostreocin-D. Palytoxin recoveries (at 10 μg kg⁻¹) from shellfish and fish tissues were 114-119% (as amine aldehyde) and 90-115% (as amide aldehyde) with RSDs for both of ≤ 18% (all tissues, n = 12). The method LOD was determined to be approximately 1 ng mL⁻¹ and the LOQ 4 ng mL⁻¹, which corresponds to 10 μg kg⁻¹ in tissue (flesh of shellfish or fish). The method has potential for use in research and is sufficiently sensitive for regulatory testing, should it be required.

Production of pinnatoxins E, F and G by scrippsielloid dinoflagellates isolated from Franklin Harbour, South Australia

Abstract A pinnatoxin producing dinoflagellate was isolated in cyst form from sediments from Franklin Harbour, South Australia, December 2009. One isolate (CAWD180) produced pinnatoxin G, E, F and A (87, 10, 41 and 1.3 pg/cell respectively; liquid chromatograph–mass spectrometer, LC-MS, analysis) and another (CAWD183) produced pinnatoxin G only (13 pg/cell). Australian strains were identical to pinnatoxin E and F producers isolated from Northland, New Zealand (2008), based on large subunit (LSU) rDNA and ITS spacer region sequence data. Cysts were capable of division and produced more toxin per cell than the excysted motile form. Crude extracts of mass cultures were tested for toxicity in mice by intraperitoneal (i.p.) injection, gavage and voluntary consumption, and toxicity ratios were 1.0:1.8:4.5 (CAWD180) and 1.0:2.9:7.8 (CAWD183). This is similar to the ratios for New Zealand isolates, but differs from other cyclic imines for which oral toxicity can be 10–1000-fold less than i.p. administration.

Comparative toxicity to mice of domoic acid and isodomoic acids A, B and C

Seafood in many parts of the world may become contaminated with high levels of domoic acid and domoic acid isomers, and such seafood has been shown to cause toxic effects in humans and in marine animals. Domoic acid itself has been held responsible for the observed effects, although the possible contribution of the isomers to toxicity has not been investigated. In the present study, the acute intraperitoneal toxicity of isodomoic acid C in mice was found to be lower than that of domoic acid. Furthermore, the severities of the behavioural changes induced by isodomoic acids A, B and C were all much lower than that of domoic acid itself, suggesting that these substances pose relatively little risk to human or animal health.