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Gymnodimines (GYMs) are fast-acting toxins that belong to the cyclic imine group, a subclass of lipophilic marine toxins. GYMs are considered to be emerging toxins but have not yet been linked to incidents of human poisoning, Limited knowledge on the metabolism of GYMs means that a proper risk assessment has not been possible and caution must be taken when establishing the relevance of GYMs in terms of food safety of marine products.

The combination of liquid chromatography and mass spectrometry LC-MS) is a powerful tool for the detection and quantitation of phycotoxins in plankton and shellfish at trace levels, the identification of new toxins, the investigation of toxin production by plankton, and the study of toxin metabolism in shellfish. Progress in development of LC-MS as a “universal” toxin analysis method for the comprehensive analysis of a wide range of toxins is now described. Two basic methods have been developed: lipophilic toxins are separated on a reversed phase column using gradient elution, while the paralytic shellfish poisoning (PSP) toxins are separated on a hydrophilic interaction LC column using isocratic elution., The 10th International IUPAC Symposium on Mycotoxins and Phycotoxins, May 21-25, 2000 Guaruja, Brazil

The cyclic imines group includes gymnodimine, spirolides, pinnatoxins, prorocentrolide and spirocentrimine. The presence of this group of compounds in shellfish was discovered because of their very high acute toxicity in mice upon i.p. injections of lipophilic extracts. When present at elevated levels, they rapidly kill mice, and their presence may interfere with the MBA for OA, BTXs, and AZA groups. At sublethal doses, the mice recover rapidly. The toxic potential of the cyclic imines is much lower via the oral route. The regulatory significance of the cyclic imine toxins is still unclear. Although gymnodimine and spirolides are now known to commonly occur in microalgae and/or bivalve molluscs from several parts of the world (Canada, Denmark, New Zealand, Norway, Tunisia, the United Kingdom of Great Britain and Northern Ireland, and the United States of America), there have been no reports of adverse effects in humans., Series: FAO Fisheries and Aquaculture Technical Paper; no. 551

Sixteen laboratories participated in a collaborative study to evaluate method performance parameters of a liquid chromatographic method of analysis for paralytic shellfish toxins (PST) in blue mussels (Mytilus edulis), soft shell clams (Mya arenaria), sea scallops (Placopectin magellanicus), and American oysters (Crassostrea virginicus). The specific analogs tested included saxitoxin, neosaxitoxin, gonyautoxins-1 to -5, decarbamoyl-gonyautoxins-2 and -3, decarbamoyl-saxitoxin, and N-sulfocarbamoylgonyautoxin- 2 and -3. This instrumental technique has been developed as a replacement for the current AOAC biological method (AOAC Official MethodSM 959.08) and an alternative to the pre-column oxidation LC method (AOAC Official MethodSM 2005.06). The method is based on reversed-phase liquid chromatography with post-column oxidation and fluorescence detection (excitation 330 nm and emission 390 nm). The shellfish samples used in the study were prepared from the edible tissues of clams, mussels, oysters, and scallops to contain concentrations of PST representative of low, medium, and high toxicities and with varying profiles of individual toxins. These concentrations are approximately equivalent to = maximum level (ML), ML, or 2×ML established by regulatory authorities (0.40, 0.80, and 1.60 mg STẊdiHCl eq/kg, respectively). Recovery for the individual toxins ranged from 104 to 127%, and recovery of total toxin averaged 116%. Horwitz Ratio (HorRat) values for individual toxins in the materials included in the study were generally within the desired range of 0.3 to 2.0. For the estimation of total toxicity in the test materials, the reproducibility relative standard deviation ranged from 4.6 to 20%. A bridging study comparing the results from the study participants using the post-column oxidation (PCOX) method with the results obtained in the study director's laboratory on the same test materials using the accepted reference method, the mouse bioassay (MBA; AOAC Official MethodSM 959.08), showed that the average ratio of results obtained from the two methods was 1.0. A good match of values was also achieved with a new certified reference material. The results from this study demonstrated that the PCOX method is a suitable method of analysis for PST in shellfish tissue and provides both an estimate of total toxicity, equivalent to that determined using the MBA AOAC Official MethodSM 959.08, and a detailed profile of the individual toxin present in the sample.

IMSC 10: 10th International Mass Spectrometry Conference, September 9-13, 1985, Swansea, Wales, UK

In recent years the number of known marine algal toxins has increased tremendously, mainly by discovery of new analogues and metabolites within known toxin groups. This is due in particular to the use of LC-MS/MS. In this presentation, we will outline some new strategies based on various MS/MS scanning techniques that can be used for comprehensive detection and identification of new structural analogues of toxins. One example is the detection of assorted new analogues of the cyclic imine toxins, spirolides (SPX). Targeting key collision-induced product ions (m/z 150 or 164) in a precursor ion scanning mode and presenting the ion intensity data in a 2-D contour plot (m/z vs. retention time) allowed the detection of new toxins as individual 'spots' and also determines their masses. Product ion scans of those masses then allow detailed structure information to be generated. Thus, several new SPX analogues including fatty acid acyl ester metabolites have been identified. Examples will also be shown for the yessotoxin (YTX), okadaic acid (OA) and pectenotoxin (PTX) groups. The discovery of all these new toxin analogues raises some important questions such as their toxicological significance and whether we will be able to monitor for the toxins without standards., 12th International Conference on Harmful Algae, 4-8 September 2006, Copenhagen, Denmark

Toxins responsible for incidents of shellfish poisoning, including okadaic acid (OA), dinophysistoxins-1 and -2 (DTX1, DTX2), pectenotoxins, brevetoxins and spirolides can occur as acyl esters in plankton and shellfish creating complications during routine analysis. Alkaline hydrolysis is usually used to release free OA/DTX and two analyses are conducted, before and after hydrolysis, to determine the amounts of unesterified and esterified toxin. However, alkaline hydrolysis is not suitable for all acylated compounds such as pectenotoxins or spirolides. Harsh alkaline conditions destroy the parent toxins. An alternative approach to alkaline hydrolysis is the use of enzymes. Two enzymatic methods were developed for the hydrolysis of 7-O-acyl fatty acid esters (“DTX3”) and the carboxylate esters of OA (“diol-esters”). Lipase (EC 3.1.1.3) was found to be specific for complete conversion of DTX3 compounds to OA, DTX1 and DTX2. OAdiol was hydrolyzed completely to OA by an esterase (EC 3.1.1.1). It has also been determined that the lipase is able to hydrolyze acyl esters of spirolides and the esterase is able to convert the acyl esters of pectenotoxins, without any degradation of parent compounds. Applications to contaminated Norwegian shellfish samples containing acyl esters of OA and DTX1 have shown complete hydrolysis within 4 hours. Analytes, including the acyl esters, the released toxins and even the released fatty acids, were monitored using LC-MS. Enzymatic hydrolysis has been shown to have potential as an alternative to the conventional alkaline hydrolysis procedure in preparation of shellfish samples for analysis of toxins.

54th ASMS Conference on Mass Spectrometry, May 28–June 1, 2006, Seattle, Washington, USA

During 2002 and 2003, poisoning incidents caused by the consumption of snail Nassarius spp.were reported in Fujian Province and Jiangsu Province, China. After observation of the symptoms in the victims it was suspected that PSP toxins were involved, and the toxicity screening of the snail samples with mouse bioassay method for monitoring of PSP toxins gave numbers exceeding 10,000MU/100g tissue (wet weight). However, no PSP toxins were detected after analysis of snail samples using high performance liquid chromatography (HPLC) with post-column derivatization. The toxins in Nassarius spp. were finally identified as tetrodotoxin (TTX), after re-analysis of the samples using HPLC coupled with a mass detector. The derivatives of TTX in toxic snail samples were also analyzed, using HPLC coupled with a triple quadrupole mass detector (API 4000), in the mode of precursor ion scan and product ion scan. Two isomers of trideoxyTTX, two isomers of TTX, two isomers of 11-oxoTTX, anhydroTTX and trace amounts of deoxyTTX were identified from the toxic snail samples. Analytical methods using select ion monitoring and select reaction monitoring targeted on these compounds were then developed, 12th International Conference on Harmful Algae, September 4-8, 2006, Copenhagen, Denmark

A novel hydrophilic interaction liquid chromatography mass spectrometry (HILIC-MS) method was developed for the separation of excitatory amino acids (EAA) including kainic acid, domoic acid, glutamic acid, aspartic acid, ß-oxalylamino-Lalanine and L-ß-methylaminoalanine in a variety of marine algae. Several HILIC stationary phases were assessed during this study including TSK-gel amide-80 (Tosoh), Atlantis HILIC silica (Waters) and the highly polar, zwitterionic ZIC HILIC (SeQuant). In each case, a variety of parameters were optimised including buffer concentration, organic modifiers, ion-pairing agents, pH, column temperature and flow rate. Single ion monitoring (SIM) of the positive and negative parent ions was performed using an API III, API 165 and API 4000 mass spectrometer. Positive and negative MS/MS spectra were obtained for each EAA examined with the API4000 triple quadrupole MS which facilitated optimization of collision energy and declustering potentials of major fragment ions for single and multiple reaction monitoring. All aspects of quantification will be discussed including linearity assessment, level of detection, level of quantification and effects of sample matrix for each EAA under investigation. These studies were applied to purity assessment of algal derived products such as kainic and domoic acid, and excitotoxin identification in seaweeds, diatoms and cyanobacteria, 12th International Conference on Harmful Algae,4-8 September 2006, Copenhagen, Denmark

Reference materials play a very important role in analytical laboratories. They are used for calibration of instruments, development and validation of measurement methods, and routine assessment of method performance. Certified reference materials (CRMs) are essential for laboratories maintaining a quality system such as ISO-17025 because they ensure accuracy and traceability to international standards. The NRC-IMB Certified Reference Materials Program (CRMP) produces CRMs for shellfish toxins and distributes them on a not-for-profit basis. Diarrhetic shellfish poisoning (DSP) is a gastrointestinal illness caused by ingestion of shellfish contaminated with toxins produced by certain species of dinoflagellates. The principal toxins associated with DSP are okadaic acid (OA) and the dinophysistoxins, DTX1 and DTX2. The most common methods of analysis for these toxins are based upon liquid chromatography with either mass spectrometric (LC-MS) or fluorescence detection (LC-FLD). Accurate calibration is an essential step in such analyses, particularly for regulatory work. Two CRMs for DSP toxins are currently available from CRMP: (a) CRM-OA-b, a certified calibration solution for OA; and (b) CRM-DSP-Mus-b, a certified mussel tissue CRM for OA and dinophysistoxin-1. Two other calibration solution CRMs are in preparation for DTX1 and DTX2. OA, isolated from Prorocentrum lima, was carefully assessed for its purity by LC-MS and nuclear magnetic resonance (NMR). An exhaustive study of OA stability was performed in various solvents and under different conditions. Based on this work, methanol was selected as the best solvent. NMR was then used to measure the absolute concentration of OA in a CD3OH stock solution, which was then diluted very accurately in degassed, high purity methanol to a concentration of 20 µmol L-1. Aliquots were dispensed into argon-filled glass ampoules, which were immediately flame-sealed. The mussel tissue CRM was produced by blending blue mussel (Mytilus edulis) digestive glands with a small amount of P. lima. The CRM (~4 grams of homogenate) is distributed in polypropylene bottles that were heat sealed, thermally sterilized and individually packed in trilaminate pouches. The toxin levels in this CRM were determined to be 10.1 mg/kg for OA and 1.3 mg/kg for DTX1 using LC-MS and LC-FLD after derivatization with 9-anthryldiazomethane (ADAM).