Liquid chromatography--multiple tandem mass spectrometry for the determination of ten azaspiracids, including hydroxyl analogues in shellfish

Azaspiracids (AZAs) are a group of polyether toxins that cause food poisoning in humans. These toxins, produced by marine dinoflagellates, accumulate in filter-feeding shellfish, especially mussels. Sensitive liquid chromatography-electrospray ionisation mass spectrometry (LC-ESI-MS(n)) methods have been developed for the determination of the major AZAs and their hydroxyl analogues. These methods, utilising both chromatographic and mass resolution, were applied for the determination of 10 AZAs in mussels (Mytilus edulis). An optimised isocratic reversed phase method (3 microm Luna-2 C18 column) separated 10 azaspiracids using acetonitrile/water (46:54, v/v) containing 0.05% trifluoroacetic acid (TFA) and 0.004% ammonium acetate in 55 min. Analyte determination using MS3 involved trapping and fragmentation of the [M + H]+ and [M + H - H2O]+ ions with detection of the [M + H - 2H2O]+ ion for each AZA. Linear calibrations were obtained for AZA1, using spiked shellfish extracts, in the range 0.05-1.00 microg/ml (r2 = 0.997) with a detection limit of 5 pg (signal : noise = 3). The major fragmentation pathways in hydroxylated azaspiracids were elucidated using hydrogen/deuterium (H/D) exchange experiments. An LC-MS3 method was developed using unique parent ions and product ions, [M + H - H2O - CgH10O2R1R3]+, that involved fragmentation of the A-ring. This facilitated the discrimination between 10 azapiracids, AZA1-10. Thus, this rapid LC-MS3 method did not require complete chromatographic resolution and the run-time of 7 min had detection limits better than 20 pg for each toxin.     

A mussel tissue certified reference material for multiple phycotoxins. Part 2: liquid chromatography-mass spectrometry, sample extraction and quantitation procedures

A freeze-dried mussel tissue certified reference material (CRM-FDMT1) containing multiple groups of shellfish toxins has been prepared. Toxin groups present in the material include okadaic acid and the dinophysistoxins, azaspiracids, yessotoxins, pectenotoxins, spirolides and domoic acid. In this work, analytical methods have been examined for the characterisation of the candidate CRM. A comprehensive extraction procedure was developed, which gave good recovery (>98%) for all lipophilic toxins studied. A fast liquid chromatography–mass spectrometry (LC-MS) method was developed that separates the major toxins according to the MS ionisation mode of optimum sensitivity. Matrix effects associated with analysis of these extracts using the developed LC-MS method were assessed. Standard addition and matrix-matched calibration procedures were evaluated to compensate for matrix effects. The methods and approaches will be used for the precise characterisation of the homogeneity and stability of the various toxins in CRM-FDMT1 and for the accurate assignment of certified values. The developed methods also have excellent potential for application in routine regulatory monitoring of shellfish toxins.     

Discovery of new analogs of the marine biotoxin azaspiracid in blue mussels (Mytilus edulis) by ultra-performance liquid chromatography/tandem mass spectrometry

Azaspiracids (AZAs) are a group of lipophilic marine biotoxins that were first discovered in blue mussels harvested in 1995 in Killary Harbour on the west coast of Ireland. At least eight people fell ill after the consumption of contaminated mussels and developed symptoms of nausea, stomach cramps, vomiting and severe diarrhoea. Until now, eleven different analogs of these toxins have been described, with a twelfth one theoretically postulated. This paper describes the detection and identification of twenty new analogs of azaspiracid, including dihydroxy-AZAs and carboxy-AZAs, using state-of-the-art techniques including ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS). Blue mussels (Mytilus edulis) from a toxic event of the northwest coast of Ireland in 2005 were extracted and analysed using LC/MS. The mass spectra obtained from different instruments enabled identification of previously unknown analogs of azaspiracid with additional hydroxyl and carboxyl substituents. Mass fragmentation patterns of the dihydroxy-AZAs indicated the positions of these substituents to be at the C3 and C23 position. The previously theoretically postulated AZA12 was also observed in this study. Product ion spectra showed the presence of a unique fragment ion at m/z 408 for all C23-hydroxylated analogs. This fragmentation competes with the fragmentation leading to m/z 362, a fragment ion that has shown to be present in all AZAs. The novel analogs have not been seen in plankton or water samples and are believed to be metabolites of AZAs formed in mussels. All the new AZA analogs were present at low concentrations in the shellfish and it is probably safe to assume that they do not pose a risk for the shellfish consumer.     

Strategies for the elimination of matrix effects in the liquid chromatography tandem mass spectrometry analysis of the lipophilic toxins okadaic acid and azaspiracid-1 in molluscan shellfish

Considerable efforts are being made worldwide to replace in vivo assays with instrumental methods of analysis for the monitoring of marine biotoxins in shellfish. Analysis of these compounds by the preferred technique of liquid chromatography tandem mass spectrometry (LC-MS/MS) is challenged by matrix effects associated with the shellfish tissues. In methods validation, assessment of matrix interferences is imperative to ensure the validity and accuracy of results being produced. Matrix interferences for the analysis of okadaic acid (OA) and azaspiracid 1 (AZA1) were assessed using acidic methods on electrospray triple stage quadrupole (TSQ) and hybrid quadrupole time of flight (QToF) instruments by the use of matrix matched standards for different tissue types. Using an acidic method no matrix interference and suppression was observed on the TSQ for OA and AZA1 respectively, whilst the opposite was observed on the QToF; matrix enhancement for OA and no matrix interference for AZA1. The suppression of AZAs on the TSQ was found to be due to interfering compounds being carried over from previous injections. The degree of suppression is very much dependant on the tissue type ranging from 15 to 70%. Several strategies were evaluated to eliminate these interferences, including the partitioning of the extract with hexane, optimisation of the chromatographic method and the use of on-line SPE. Hexane clean up did not have any impact on matrix effects. The use of an alkaline method and a modified acidic method eliminated matrix suppression for AZA1 on the TSQ instrument while an on-line SPE method proved to be effective for matrix enhancement of OA on the QToF.     

Antibodies with broad specificity to azaspiracids by use of synthetic haptens

The development of general, sensitive, portable, and quantitative assays for the azaspiracid (AZA) class of marine toxins is urgently needed. Use of a synthetic hapten containing rings F-I of AZA to generate antibodies that cross-react with the AZAs via their common C28-C40 domain and use of these antibodies in ELISA and immunoaffinity columns are reported. This approach has many advantages over using intact azaspiracids (AZAs) derived from environmental samples or total synthesis as haptens for antibody development. A derivative of the levorotatory C28-C40 azaspiracid domain (1) was synthesized efficiently using a one-pot Staudinger reduction/intramolecular aza-Wittig reaction-imine capture sequence to form the H-I ring spiroaminal and a double intramolecluar hetero-Michael addition to assemble the F-G ring ketal. Conjugation of the hapten 1 to cBSA and immunization in sheep generated antibodies that recognized and bound to ovalbumin-conjugated 1 in the absence of AZA1. This binding was inhibited by 1 in a concentration-dependent manner. A mixture of AZA1, AZA2, AZA3, and AZA6 caused a degree of inhibition of antibody binding consistent with its total AZA content, rather than just its content of AZA1. This result suggests that the antibodies also have a similar affinity for AZA2, AZA3, and AZA6 as they do for AZA1 and that such antibodies are suitable for analysis of AZAs in shellfish samples.     

LC-MS-MS aboard ship: tandem mass spectrometry in the search for phycotoxins and novel toxigenic plankton from the North Sea

Phycotoxins produced by various species of toxigenic microalgae occurring in the plankton are a global threat to the security of seafood resources and the health of humans and coastal marine ecosystems. This has necessitated the development and application of advanced methods in liquid chromatography coupled to mass spectrometry (LC-MS) for monitoring of these compounds, particularly in plankton and shellfish. Most such chemical analyses are conducted in land-based laboratories on stored samples, and thus much information on the near real-time biogeographical distribution and dynamics of phycotoxins in the plankton is unavailable. To resolve this problem, we conducted ship-board analysis of a broad spectrum of phycotoxins collected directly from the water column on an oceanographic cruise along the North Sea coast of Scotland, Norway, and Denmark. We equipped the ship with a triple-quadrupole linear ion-trap hybrid LC-MS-MS system for detection and quantitative analysis of toxins, such as domoic acid, gymnodimine, spirolides, dinophysistoxins, okadaic acid, pectenotoxins, yessotoxins, and azaspiracids (AZAs). We focused particular attention on the detection of AZAs, a group of potent nitrogenous polyether toxins, because the culprit species associated with the occurrence of these toxins in shellfish has been controversial. Marine toxins were analyzed directly from size-fractionated plankton net tows (20 μm mesh size) and Niskin bottle samples from discrete depths, after rapid methanolic extraction but without any further clean-up. Almost all expected phycotoxins were detected in North Sea plankton samples, with domoic acid and 20-methylspirolide G being most abundant. Although AZA was the least abundant of these toxins, the high sensitivity of the LC-MS-MS enabled detailed quantification, indicating that the highest amounts of AZA-1 were present in the southern Skagerrak in the 3–20 μm size-fraction. The direct on-board toxin measurements enabled isolation of plankton from stations with high AZA-1 levels and from the most suspicious size-fraction, i.e. most likely to contain the AZA-producer. A large number (>100) of crude cultures were established by serial dilution and later screened for the presence of AZAs after several weeks growth. From one crude culture containing AZA, a small dinoflagellate was subsequently isolated and brought into pure culture. We have thus proved that even sophisticated mass spectrometers can be operated in ship laboratories without any limitation caused by vibrations of the ship’s engine or by wave movement during heavy seas at wind forces up to nine Beaufort. On-board LC–MS–MS is a valuable method for near real-time analysis of phycotoxins in plankton for studies on bloom dynamics and the fate of toxins in the food web, and for characterization and isolation of putatively toxigenic organisms.     

A mussel tissue certified reference material for multiple phycotoxins. Part 1: design and preparation

The development of multi-analyte methods for lipophilic shellfish toxins based on liquid chromatography–mass spectrometry permits rapid screening and analysis of samples for a wide variety of toxins in a single run. Validated methods and appropriate certified reference materials (CRMs) are required to ensure accuracy of results. CRMs are essential for accurate instrument calibration, for assessing the complete analytical method from sample extraction to data analysis and for verifying trueness. However, CRMs have hitherto only been available for single toxin groups. Production of a CRM containing six major toxin groups was achieved through an international collaboration. Preparation of this material, CRM-FDMT1, drew on information from earlier studies as well as improved methods for isolation of toxins, handling bulk tissues and production of reference materials. Previous investigations of stabilisation techniques indicated freeze-drying to be a suitable procedure for preparation of shellfish toxin reference materials and applicable to a wide range of toxins. CRM-FDMT1 was initially prepared as a bulk wet tissue homogenate containing domoic acid, okadaic acid, dinophysistoxins, azaspiracids, pectenotoxin-2, yessotoxin and 13-desmethylspirolide C. The homogenate was then freeze-dried, milled and bottled in aliquots suitable for distribution and analysis. The moisture content and particle size distribution were measured, and determined to be appropriate. A preliminary toxin analysis of the final material showed a comprehensive toxin profile.     

Determination of azaspiracids in shellfish using liquid chromatography/tandem electrospray mass spectrometry.

Azaspiracid (AZA1), a recently discovered marine toxin, is responsible for the new human toxic syndrome, azaspiracid poisoning (AZP), which is caused by the consumption of contaminated shellfish. A new, sensitive liquid chromatography/mass spectrometry (LC/MS) method has been developed for the determination of AZA1 and its analogues, 8-methylazaspiracid (AZA2) and 22-demethylazaspiracid (AZA3). Separation of these toxins was achieved using reversed-phase LC and coupled, via an electrospray ionisation (ESI) source, to an ion-trap mass spectrometer. Spectra showed the protonated molecules, [M + H]+, and their major product ions, due to the sequential loss of two water molecules, [M + H - H2O]+, [M + H - 2H2O]+, in addition to fragment ions that are characteristic of these cyclic polyethers. A highly specific and sensitive LC/MS(3) analytical method was developed and, using shellfish extracts containing AZA1, the detection limit (S/N = 3) was 4 pg on-column, corresponding to 0.8 ng/mL. Using the protocol presented here, this is equivalent to 0.37 ng/g shellfish tissue and good linear calibrations were obtained for AZA1 in shellfish extracts (average r2 = 0.9988). Good reproducibility was achieved with % RSD values (N = 5) ranging from 1.5% (0.75 microg/mL) to 4.2% (0.05 microg/mL). An efficient procedure for the extraction of toxins from shellfish aided the development of a rapid protocol for the determination of the three predominant azaspiracids.     

The development of a rapid method for the isolation of four azaspiracids for use as reference materials for quantitative LC–MS–MS methods

The azaspiracids are a family of lipophilic polyether marine biotoxins that have caused a number of human intoxication incidents in Europe since 1995 after consumption of contaminated shellfish (Mytilus edulis). Levels of azaspiracids in shellfish for human consumption are monitored in accordance with EU guidelines: only shellfish with less than 160 μg kg⁻¹ are deemed safe. The limited availability of commercially available standards for azaspiracids is a serious problem, because validated LC–MS methods are required for routine analysis of these toxins in shellfish tissues. The procedure described herein has been used for the separation and the isolation of four azaspiracid (AZA) toxins from shellfish, for use as LC–MS–MS reference materials. Five separation steps have been used to isolate azaspiracids 1, 2, 3, and 6. The purity of the toxins obtained has been confirmed by multiple mass spectrometric methods using authentic azaspiracid standards. The same techniques have been used for quantification of the toxins extracted. The isolation procedure involves several chromatographic purification techniques: solid-phase extraction (diol sorbent, 90% mass reduction, and 95 ± 1% toxin recovery); Sephadex size-exclusion chromatography (87% mass reduction and up to 95 ± 2% toxin recovery), Toyopearl HW size-exclusion chromatography (90% mass reduction and up to 92.5 ± 2.5% toxin recovery), and semi-preparative LC (78 ± 3% toxin recovery). The procedure effectively separates the toxins from the sample matrix and furnishes azaspiracid toxins (AZA1, AZA2, AZA3 and AZA6) of sufficient purity with an average yield of 65% (n = 5). Triple-quadrupole mass spectrometry was used for qualitative and quantitative monitoring of the isolation efficiency after each stage of the process. High-resolution mass spectrometric evaluation of the toxic isolated material in both positive and negative modes suggests high purity.     

液相色谱-串联质谱检测贝类组织中5种脂溶性贝毒素

建立了液相色谱-串联质谱分析贝类组织中米氏裸甲藻(GYM)贝毒素、螺环内酯毒素(SPX1)、大田软骨酸(OA)贝毒素、蛤毒素(PTX2)、原多甲藻酸(AZA1)贝毒素的方法.用甲醇-水(4: 1, V/V)溶液对贝类组织中GYM, SPX1, OA, PTX2和AZA1进行提取,MAX阴离子交换柱净化后,采用液相色谱分离,除OA以负离子选择反应监测外,GYM, SPX1, PTX2和AZA1以电喷雾离子源正离子选择反应监测模式进行质谱分析.5种脂溶性贝毒素GYM, SPX1, OA, PTX2和AZA1在各自相应浓度范围内线性良好,相关系数＞0.99.扇贝闭壳肌空白样品添加5种贝毒素的提取率均为78.6%～94.4%(n=6); 精密度(RSD)为6.8%～14.9%.贝类组织中5种贝毒素GYM, SPX1, OA, PTX2和AZA1的检出限分别为0.10, 0.21, 2.00, 0.32和0.04 μg/kg.     

Development of reference materials for paralytic shellfish poisoning toxins

A project was undertaken to develop mussel reference materials that were certified for their mass fractions of saxitoxin and decarbamoyl-saxitoxin. Fifteen laboratories from various European countries participated. Three of these had major responsibility for substantial parts of the work and overall coordination of the project. The project involved 4 main activities: (1) procurement and characterization of calibrants; (2) improvement of analytical methodology; (3) preparation of reference materials, including homogeneity and stability studies; (4) 2 interlaboratory studies and a certification exercise. The joint activities resulted in 3 homogeneous and stable reference materials: 2 lyophilized mussel materials with and without naturally incurred paralytic shellfish poisoning (PSP) toxins, and a saxitoxin enrichment solution. The reference materials were certified with respect to their saxitoxin and decarbamoyl-saxitoxin content. The lyophilized mussel material with PSP toxins (CRM 542) contained <0.07 mg saxitoxin x 2HCl/kg and 1.59 +/- 0.20 mg decarbamoyl-saxitoxin x 2HCl/kg. The lyophilized mussel material without PSP toxins (CRM 543) contained <0.07 mg saxitoxin x 2HCl/kg and <0.04 mg decarbamoyl-saxitoxin x 2HCl/kg. The certified value of the saxitoxin mass fraction in the saxitoxin enrichment solution (CRM 663) was 9.8 +/- 1.2 microg/g.     

In-house validation of a liquid chromatography tandem mass spectrometry method for the analysis of lipophilic marine toxins in shellfish using matrix-matched calibration

A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitative analysis of lipophilic marine toxins in shellfish extracts (mussel, oyster, cockle and clam) was validated in-house using European Union (EU) Commission Decision 2002/657/EC as a guideline. The validation included the toxins okadaic acid (OA), yessotoxin (YTX), azaspiracid-1 (AZA1), pectenotoxin-2 (PTX2) and 13-desmethyl spirolide-C (SPX1). Validation was performed at 0.5, 1 and 1.5 times the current EU permitted levels, which are 160 μg kg^-1 for OA, AZA1 and PTX2 and 1,000 μg kg^-1 for YTX. For SPX1, 400 μg kg^-1 was chosen as the target level as no legislation has been established yet for this compound. The method was validated for determination in crude methanolic shellfish extracts and for extracts purified by solid-phase extraction (SPE). Extracts were also subjected to hydrolysis conditions to determine the performance of the method for OA and dinophysistoxin esters. The toxins were quantified against a set of matrix-matched standards instead of standard solutions in methanol. To save valuable standard, methanolic extract instead of the homogenate was spiked with the toxin standard. This was justified by the fact that the extraction efficiency is high for all relevant toxins (above 90%). The method performed very well with respect to accuracy, intraday precision (repeatability), interday precision (within-laboratory reproducibility), linearity, decision limit, specificity and ruggedness. At the permitted level the accuracy ranged from 102 to 111%, the repeatability from 2.6 to 6.7% and the reproducibility from 4.7 to 14.2% in crude methanolic extracts. The crude extracts performed less satisfactorily with respect to the linearity (less than 0.990) and the change in LC-MS/MS sensitivity during the series (more than 25%). SPE purification resulted in greatly improved linearity and signal stability during the series. Recently the European Food Safety Authority (EFSA) has suggested that to not exceed the acute reference dose the levels should be below 45 μg kg^-1 OA equivalents and 30 μg kg^-1 AZA1 equivalents. A single-day validation was successfully conducted at these levels. If the regulatory levels are lowered towards the EFSA suggested values, the official methods prescribed in legislation (mouse and rat bioassay) will no longer be sensitive enough. The validated LC-MS/MS method presented has the potential to replace these animal tests.     

同位素稀释-液相色谱-串联质谱法在小麦粉细交链孢菌酮酸和腾毒素标准物质制备和定值中的应用

建立了小麦粉中细交链孢菌酮酸（TeA）和腾毒素（TEN）标准物质的研制和定值方法,为开展粮食中交链孢霉毒素基体标准物质的研制提供重要方法学借鉴。该标准物质样品为天然污染交链孢霉毒素的小麦籽粒,定值目标物为TeA和TEN,采用同位素稀释-液相色谱-串联质谱法（ID-LC-MS/MS）进行定值测量,多个实验室合作定值。所研制的标准物质具有常温避光保存、定值不确定度小等特点。该标准物质是目前国际上唯一一种天然污染TeA和TEN的小麦粉标准物质,可用于食品安全风险监测、产品质量检测等领域相关分析方法的评价和测量质量控制等。     

Multiresidue method for determination of algal toxins in shellfish: single-laboratory validation and interlaboratory study

A method that uses liquid chromatography with tandem mass spectrometry (LC/MS/MS) has been developed for the highly sensitive and specific determination of amnesic shellfish poisoning toxins, diarrhetic shellfish poisoning toxins, and other lipophilic algal toxins and metabolites in shellfish. The method was subjected to a full single-laboratory validation and a limited interlaboratory study. Tissue homogenates are blended with methanol-water (9 + 1), and the centrifuged extract is cleaned up with a hexane wash. LC/MS/MS (triple quadrupole) is used for quantitative analysis with reversed-phase gradient elution (acidic buffer), electrospray ionization (positive and negative ion switching), and multiple-reaction monitoring. Ester forms of dinophysis toxins are detected as the parent toxins after hydrolysis of the methanolic extract. The method is quantitative for 6 key toxins when reference standards are available: azaspiracid-1 (AZA1), domoic acid (DA), gymnodimine (GYM), okadaic acid (OA), pectenotoxin-2 (PTX2), and yessotoxin (YTX). Relative response factors are used to estimate the concentrations of other toxins: azaspiracid-2 and -3 (AZA2 and AZA3), dinophysis toxin-1 and -2 (DTX1 and DTX2), other pectenotoxins (PTX1, PTX6, and PTX11), pectenotoxin secoacid metabolites (PTX2-SA and PTX11-SA) and their 7-epimers, spirolides, and homoYTX and YTX metabolites (45-OHYTX and carboxyYTX). Validation data have been gathered for Greenshell mussel, Pacific oyster, cockle, and scallop roe via fortification and natural contamination. For the 6 key toxins at fortification levels of 0.05-0.20 mg/kg, recoveries were 71-99% and single laboratory reproducibilities, relative standard deviations (RSDs), were 10-24%. Limits of detection were <0.02 mg/kg. Extractability data were also obtained for several toxins by using successive extractions of naturally contaminated mussel samples. A preliminary interlaboratory study was conducted with a set of toxin standards and 4 mussel extracts. The data sets from 8 laboratories for the 6 key toxins plus DTX1 and DTX2 gave within-laboratories repeatability (RSD(R)) of 8-12%, except for PTX-2. Between-laboratories reproducibility (RSDR) values were compared with the Horwitz criterion and ranged from good to adequate for 7 key toxins (HorRat values of 0.8-2.0).     

Analysis of acidic drugs in the effluents of sewage treatment plants using liquid chromatography-electrospray ionization tandem mass spectrometry

Azaspiracid poisoning (AZP) is a new human toxic syndrome that is caused by the consumption of shellfish that have been feeding on harmful marine microalgae. A liquid chromatography–mass spectrometry (LC–MS) method has been developed for the determination of the three most prevalent toxins, azaspiracid (AZA1), 8-methylazaspiracid (AZA2) and 22-demethylazaspiracid (AZA3) as well as the isomeric hydroxylated analogues, AZA4 and AZA5. Separation of five azaspiracids was achieved on a C₁₈ column (Luna-2, 150×2 mm, 5 μm) with isocratic elution using acetonitrile–water containing trifluoroacetic acid and ammonium acetate as eluent modifiers. Using an electrospray ionisation (ESI) source with an ion-trap mass spectrometer, the spectra showed the protonated molecules, [M+H]⁺, with most major product ions due to the sequential loss of two water molecules. A characteristic fragmentation pathway that was observed in each azaspiracid was due to the cleavage of the A-ring at C₉–C₁₀ for each toxin. It was possible to select unique ion combinations to distinguish between the isomeric azaspiracids, AZA4 and AZA5. Highly sensitive LC–MS³ analytical methods were compared and the detection limits were 5–40 pg on-column. Linear calibrations were obtained for AZA1 in shellfish in the range 0.05–1.00 μg/ml (r²=0.9974) and good reproducibility was observed with a relative standard deviation (%RSD) of 1.8 for 0.9 μg AZA1/ml (n=5). The %RSD values for the minor toxins, AZA4 and AZA5, using LC–MS³ (A-ring fragmentation) were 12.3 and 8.1 (0.02 μg/ml; n=7), respectively. The selectivity of toxin determination was enhanced using LC–MS–MS with high energy WideBand activation.     

Liquid chromatography post-column oxidation (PCOX) method for the determination of paralytic shellfish toxins in mussels, clams, oysters, and scallops: collaborative study

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-sulfocarbamoyl-gonyautoxin-2 and -3. This instrumental technique has been developed as a replacement for the current AOAC biological method (AOAC Official Method 959.08) and an alternative to the pre-column oxidation LC method (AOAC Official Method 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 1/2 maximum level (ML), ML, or 2xML established by regulatory authorities (0.40, 0.80, and 1.60 mg STX diHCl 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 Method 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 MBAAOAC Official Method 959.08, and a detailed profile of the individual toxin present in the sample.     

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.     

Food contaminant analysis at ultra‐high mass resolution: application of hybrid linear ion trap – orbitrap mass spectrometry for the determination of the polyether toxins,azaspiracids, in shellfish

The biotoxins, azaspiracids (AZAs), from marine phytoplankton accumulate in shellfish and affect human health by causing severe gastrointestinal disturbance, diarrhea, nausea and vomiting. Specific and sensitive methods have been developed and validated for the determination of the most commonly occurring azaspiracid analogs. An LTQ Orbitrap mass spectrometer is a hybrid instrument that combines linear ion trap (LIT) mass spectrometry (MS) with high‐resolution Fourier transform (FT) MS and this was exploited to perform simultaneous ultra‐high‐resolution full‐scan MS analysis and collision‐induced dissociation (CID) tandem mass spectrometry (MS/MS). Using the highest mass resolution setting (100 000 FWHM) in full‐scan mode, the methodology was validated for the determination of six AZAs in mussel (Mytilus galloprovincialis) tissue extracts. Ultra‐high mass resolution, together with a narrow mass tolerance window of ±2 mDa, dramatically improved detection sensitivity. In addition to employing chromatographic resolution to distinguish between the isomeric azaspiracid analogs, AZA1/AZA6 and AZA4/AZA5, higher energy collisionally induced dissociation (HCD) fragmentation on selected precursor ions were performed in parallel with full‐scan FTMS. Using HCD MS/MS, most precursor and product ion masses were determined within 1 ppm of the theoretical m/z values throughout the mass spectral range and this enhanced the reliability of analyte identity. For the analysis of mussels (M. galloprovincialis), the method limit of quantitation (LOQ) was 0.010 µg/g using full‐scan FTMS and this was comparable with the LOQ (0.007 µg/g) using CID MS/MS. The repeatability data were; intra‐day RSD% (1.8–4.4%; n = 6) and inter‐day RSD% (4.7–8.6%; n = 3). Application of these methods to the analysis of mussels (M. edulis) that were naturally contaminated with azaspiracids, using high‐resolution full‐scan Orbitrap MS and low‐resolution CID MS/MS, produced equivalent quantitative data. Copyright © 2010 John Wiley & Sons, Ltd.     

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.