Shellfish Matrices Research Articles

Determination of neurotoxic shellfish poisoning toxins in shellfish by liquid chromatography-tandem mass spectrometry coupled with dispersive solid phase extraction

An innovative method based on dispersive solid phase extraction (d-SPE) in conjunction with LC-MS/MS had been developed for the simultaneous quantitative determination of three brevetoxins (BTXs), which can result in neurotoxic shellfish poisoning (NSP), in shellfish. The toxins were extracted with a 50 % acetonitrile (v/v) and cleaned by alumina-neutral sorbent. After chromatographic separation on a C18 column, the analytes were qualitatively and quantitatively detected using multiple reaction monitoring (MRM) in positive ionization mode. The created approach was validated by SANTE 11312/2021. The LOQs were 5 μg/kg for each toxin, below the advised regulatory limit of 800 μg BTX-2/kg. The mean recoveries of brevetoxins were in the range of 75.9 %–114.1 %, and the ranges of their intra- and inter-day precisions were 0.9–9.7 % and 0.6–7.2 %, respectively. The matrix effects for three BTXs in four shellfish matrices were in the range of 85.6 %–114.8 %. The method demonstrated great consistency and high sensitivity, and it can meet the requirements of daily monitoring.

Preparation of 18O-labelled azaspiracids for accurate quantitation using liquid chromatography–mass spectrometry

Azaspiracids (AZAs) are a group of polyether marine algal toxins known to accumulate in shellfish, posing a risk to human health and the seafood industry. Analysis of AZAs is typically performed using LC–MS, which can suffer from matrix effects that significantly impact the accuracy of measurement results. While the use of isotopic internal standards is an effective approach to correct for these effects, isotopically labelled standards for AZAs are not currently available. In this study, 18O-labelled AZA1, AZA2, and AZA3 were prepared by reaction with H218O under acidic conditions, and the reaction kinetics and sites of incorporation were studied using LC–HRMS/MS aided by mathematical analysis of their isotope patterns. Analysis of the isotopic incorporation in AZA1 and AZA3 indicated the presence of four exchangeable oxygen atoms. Excessive isomerization occurred during preparation of 18O-labelled AZA2, suggesting a role for the 8-methyl group in the thermodynamic stability of AZAs. Neutralized mixtures of 18O-labelled AZA1 and AZA3 were found to maintain their isotopic and isomeric integrities when stored at −20 °C and were used to develop an isotope-dilution LC–MS method which was applied to reference materials of shellfish matrices containing AZAs, demonstrating high accuracy and excellent reproducibility. Preparation of isotopically labelled compounds using the isotopic exchange method, combined with the kinetic analysis, offers a feasible way to obtain isotopically labelled internal standards for a wide variety of biomolecules to support reliable quantitation.Graphical

Exploring the Astrovirome of Shellfish Matrices Using Nanopore Sequencing.

Astroviruses are important human enteric pathogens transmissible with contaminated food and water. Astroviruses have also been identified in mammals, birds, lower vertebrates and invertebrates. The genetic diversity of human and animal astroviruses poses a challenge for diagnostics and taxonomy. As a proof of concept, we used a panastrovirus consensus primer set, able to amplify in a nested RT-PCR protocol a 400-nt-long fragment of the RNA-dependent RNA polymerase of most members of the Astroviridae family, in conjunction with a nanopore sequencing platform, to generate information on the astrovirome in filter-feeding mollusks. Amplicons generated from bivalve samples were used to generate libraries for deep sequencing. In three samples, only one unique RdRp sequence type was obtained. However, in seven samples and in three barcodes with eleven pooled samples, we identified a variety of known and unknown RdRp sequence types, in most cases distantly related to astrovirus sequences available in the databases. In total, 37 different sequence contigs were generated. Avian-origin astrovirus sequences were predominant, likely due to contamination of shellfish harvesting waters by marine birds. Astroviruses of the aquatic eco-system were also identified, whereas human astroviruses were not detected.

Preparation and characterization of an immunoaffinity column for the selective extraction of azaspiracids

The presence of azaspiracids (AZAs) in shellfish may cause food poisoning in humans. AZAs can accumulate in shellfish filtering seawater that contains marine dinoflagellates such as Azadinium and Amphidoma spp. More than 60 AZA analogues have been identified, of which AZA1, AZA2 and AZA3 are regulated in Europe. Shellfish matrices may complicate quantitation by ELISA and LC–MS methods. Polyclonal antibodies have been developed that bind specifically to the C-26–C-40 domain of the AZA structure and could potentially be used for selectively extracting compounds containing this substructure. This includes almost all known analogues of AZAs, including AZA1, AZA2 and AZA3. Here we report preparation of immunoaffinity chromatography (IAC) columns for clean-up and concentration of AZAs. The IAC columns were prepared by coupling polyclonal anti-AZA IgG to CNBr-activated sepharose. The columns were evaluated using shellfish extracts, and the resulting fractions were analyzed by ELISA and LC–MS. The columns selectively bound over 300 ng AZAs per mL of gel without significant leakage, and did not retain the okadaic acid, cyclic imine, pectenotoxin and yessotoxin analogues that were present in the applied samples. Furthermore, 90–92% of the AZAs were recovered by elution with 90% MeOH, and the columns could be re-used without significant loss of performance.

Bioaccessibility of paralytic shellfish toxins in different cooked shellfish using the simulated digestive model

Current regulatory limit values for paralytic shellfish toxins (PSTs) in shellfish products are not considering the bioaccessibility of PSTs in seafood matrix during the gastrointestinal digestion for human beings. In this study, the bioaccessibility of PSTs in the shellfish cooked by different ways was assessed using a static in vitro human digestion model. Results showed that the dissolution of PSTs from shellfish tissues was not significantly affected by digestion time, ratios of solid weight to liquid volume (S/L) and cooking methods, but obviously facilitated by digestive enzymes. Different cooking ways reduced the contents of PSTs in shellfish by 45% to 88%, but did not significantly change the high bioaccessibility of PSTs that ranged from 80% to 95% in four different shellfish matrices. Transformation or degradation of PSTs occurred during the simulated digestion process. This work will help us to objectively assess the potential risks of PSTs to human health.

A Longitudinal Study of Bacteriophages as Indicators of Norovirus Contamination of Mussels (Mytilus edulis) and Their Overlying Waters

Sewage pollution leads to the contamination of bivalve shellfish by pathogenic microorganisms. Bacterial indicators support the management of risks associated with the consumption of shellfish; however, they often fail to indicate adequately the potential hazard to human health posed by certain human enteric viruses. Bacteriophages have been proposed as alternative indicators that may more effectively predict the presence of enteric viral pathogens. This study explored the relationships between bacterial indicators (Escherichia coli (E. coli), faecal coliforms (FC) and intestinal enterococci (IE)), phages (somatic (SOMPH), F-specific RNA (F + PH) and human-specific Bacteroides GB-124 phages (GB124PH)) and Norovirus (NoV) (GI/GII) in mussels (Mytilus edulis) and their overlying waters. The bioaccumulation of these indicators and Norovirus in shellfish matrices (e.g., flesh, digestive gland) was investigated bimonthly over a 12-month period in an English estuary. The findings revealed a marked seasonality in the distribution of all organisms, with the highest levels occurring during the autumn/winter months. The levels of all phages in shellfish and their overlying waters correlated better with the levels of Norovirus than with those of bacterial indicators. Somatic coliphages were the indicator that exhibited the strongest correlations with NoV (rho = 0.929). This study suggests that relatively low-cost culture-based phage enumeration appears to offer a more accurate indication of the likely presence of Norovirus in mussels than traditional bacterial indicators.

Method performance verification for the combined detection and quantitation of the marine neurotoxins cyclic imines and brevetoxin shellfish metabolites in mussels (Mytilus edulis) and oysters (Crassostrea gigas) by UHPLC-MS/MS

A single laboratory method performance verification is reported for a rapid sensitive UHPLC-MS/MS method for the quantification of eight cyclic imine and two brevetoxin analogues in two bivalve shellfish matrices: mussel (Mytilus edulis) and Pacific oyster (Crassostrea gigas). Targeted cyclic imine analogues were from the spirolide, gymnodimine and pinnatoxin groups, namely 20-Me-SPX-C, 13-desMe-SPX-C, 13,19-didesMe-SPX-C, GYM-A, 12-Me-GYM, PnTx-E, PnTx-F and PnTx-G. Brevetoxin analogues consisted of the shellfish metabolites BTX-B5 and S-desoxy-BTX-B2. A rapid dispersive extraction was used as well as a fast six-minute UHPLC-MS/MS analysis. Mobile phase prepared using ammonium fluoride and methanol was optimised for both chromatographic separation and MS/MS response to suit all analytes. Method performance verification checks for both matrices were carried out. Matrix influence was acceptable for the majority of analogues with the MS response for all analogues being linear across an appropriate range of concentrations. In terms of limits of detection and quantitation the method was shown to be highly sensitive when compared with other methods. Acceptable recoveries were found with most analogues, with laboratory precision in terms of intra- and inter-batch precision deemed appropriate. The method was applied to environmental shellfish samples with results showing low concentrations of cyclic imines to be present. The method is fast and highly sensitive for the detection and quantification of all targeted analogues, in both mussel and oyster matrices. Consequently, the method has been shown to provide a useful tool for simultaneous monitoring for the presence or future emergence of these two toxin groups in shellfish.

Performance Characteristics of Refined LC-FLD and HILIC-MS/MS Methods for the Determination of Paralytic Shellfish Toxins in Shrimp, Whelk, and Crab.

Paralytic Shellfish Poison (PSP) toxins have been reported in non-bivalve shellfish species, including crustaceans and gastropods. Routine surveillance of these species is currently conducted in parts of England. To date, detection methods have not been validated for these matrices. Validation is required to ensure the test is fit for purpose, to give greater confidence in any results generated and ultimately facilitates accreditation. The aim was to test and validate two independent PSP toxin detection methods previously validated for bivalve shellfish matrices, for applicability to commercial non-bivalve species of interest. Matrices were shrimp (Crangon crangon), common whelk (Buccinum undatum), and edible crab (Cancer pagurus). The two methods assessed were the pre-column oxidation high-performance liquid chromatography-fluorescence detection AOAC 2005.06 Official Method of analysis and an internationally validated hydrophilic interaction chromatography-tandem mass spectrometry method. Brown and white crab meat were assessed separately. A refined extraction protocol was implemented with an increased solvent to sample ratio. The same extraction protocol was utilized for both methods, allowing both methods to be run simultaneously. Method sensitivity, recovery, repeatability, and method uncertainty were characterized in all matrix/toxin combinations. Overall, both methods performed similarly to that previously reported in bivalve mollusks. Acceptability of the majority of toxin/matrix combinations was evidenced through comparison of method performance characteristics against specific performance criteria, including Horwitz ratio values. Both PSP toxin detection methods were found to provide acceptable performance for the monitoring of shrimp, whelk, and crab species. Two PSP toxin detection methods have been single-laboratory validated successfully for three non-bivalve shellfish species.

Evaluation of different strategies to minimize the matrix effects on LC-MS/MS analysis of multiple lipophilic shellfish toxins in both acidic and alkaline chromatographic conditions

Lipophilic shellfish toxins (LSTs) accumulated by shellfish pose a potential threat to consumer health. A mandatory routine monitoring of LSTs has been adopted for seafood products by liquid chromatography–mass spectrometry (LC-MS) in many countries. In this study, two methods developed on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) under acidic and alkaline chromatographic conditions were assessed for the determination of multiple LSTs. Different strategies including matrix solid-phase dispersion (MSPD), solid phase extraction (SPE) and sample dilution were applied and evaluated the matrix effects of mussel, scallop, clam, and oyster samples on the signal response of mass spectrometry. Results showed that the alkaline method achieved a lower limit of detection (LOD) and more robust compared to the acidic method. The obvious signal suppression of OA and DTX1 (55%–76%) and signal enhancement of PTX2 (27%–34%) occurred in the crude extracts of shellfish under acidic chromatography. In the alkaline method, no remarkable matrix effects of crude extracts were found except for the scallop matrix on the signal intensity of DTX1, AZA3 and GYM-A (121%–130%). Clean-up methods MSPD, SPE and sample dilution obviously reduced the inhibition of shellfish matrices on the signal response of OA and DTX1, however, which were still subject to signal inhibition under acidic condition. Sample dilution was more effective than SPE and MSPD in minimizing the matrix interference in both acidic and alkaline methods. Furthermore, sample dilution in combination with the alkaline chromatography was the most effective method. Bivalve mollusks harvested from Beibu Bay, South China Sea, were generally contaminated by GYM-A and SPX1 at low concentrations.

Improved culture enrichment broth for isolation of Arcobacter-like species from the marine environment

Arcobacter-like species are found associated with many matrices, including shellfish in marine environments. The culture media and conditions play a major role in the recovery of new Arcobacter-like species. This study was aimed to develop a culture media for isolation and enhanced growth of Arcobacter-like spp. from marine and shellfish matrices. For this purpose, 14 different Arcobacter-like spp. mostly isolated from shellfish, were grown in 24 different formulations of enrichment broths. The enrichment broths consisted of five main groups based on the organic contents (fresh oyster homogenate, lyophilized oyster either alone or in combination with other standard media), combined with artificial seawater (ASW) or 2.5% NaCl. Optical density (OD420nm) measurements after every 24 h were compared with the growth in control media (Arcobacter broth) in parallel. The mean and standard deviation were calculated for each species in each broth and statistical differences (p < 0.05) among broths were calculated by ANOVA. The results indicated that shellfish-associated Arcobacter-like species growth was significantly higher in Arcobacter broth + 50% ASW and the same media supplemented with lyophilized oysters. This is the first study to have used fresh or lyophilized oyster flesh in the enrichment broth for isolation of shellfish-associated Arcobacter-like spp.

Development and Application of Immunoaffinity Column Purification and Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry for Determination of Domoic Acid in Shellfish.

Domoic acid (DA) is a neurotoxin associated with amnesic shellfish poisoning (ASP). Though LC coupled to tandem mass spectrometry (LC-MS/MS) has become the preferred method for DA determination, traditional sample pretreatment is still labor-intensive. In this study, a simple, efficient and selective method for LC-MS/MS analysis of DA in shellfish was established by optimizing clean-up procedures on a self-assembly immunoaffinity column (IAC). Shellfish was extracted with 75% methanol twice and diluted with phosphate buffered saline (PBS, 1:2). The mixture was purified on IAC as follows: preconditioned with PBS, loaded with sample, washed by 50% MeOH, and eluted with MeOH containing 2% ammonium hydroxide. Concentrated analyte was monitored by multiple reaction monitoring (MRM) using electrospray (ESI) positive ion mode throughout the LC gradient elution. Based on the post-extraction addition method, matrix effects for various shellfish matrices were found to be less than 8%. The developed method was fully validated by choosing mussel as the representative matrix. The method had a limit of detection (LOD) of 0.02 µg·g−1, showed excellent linear correlation in the range of 0.05–40 µg·g−1, and obtained ideal recoveries (91–94%), intra-day RSDs (6–8%) and inter-day RSDs (3–6%). The method was successfully applied to DA determination in 59 shellfish samples, with a detection rate of 10% and contaminated content of 0.1–14.9 µg·g−1.

A rapid competitive ELISA assay of Okadaic acid level based on epoxy-functionalized magnetic beads

ABSTRACTUsing newly designed epoxy group activated carbon shell magnetic beads as a goat against mouse antibody (G-Mab) carrier, a competitive immunoassay was conducted on the surface of G-Mab-coated magnetic beads (G-Mab-MBs). Mouse monoclonal antibody against Okadaic acid (MAb), Okadaic acid-horseradish peroxidase conjugate (OA-HRP) and OA were simultaneously added into a reaction system. The enzyme-linked immunosorbent assay (ELISA) was performed in a 96-well microplate incorporated with magnetic bars. Detection of the OA level by using the current approach provided an excellent linear relationship in the range of 0.35–25.0 µg L−1 with a half-maximal inhibitory concentration IC50 of 3.27 µg L−1 and LOD values of 0.35 µg L−1. The shellfish matrix and other algal toxins presented in the sample extraction did not produce obvious interference. As a comparison, tests were conducted via commercial ELISA and liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the corresponding analytical results demonstrated the reliability of the current protocol.

A Sensitive LC-MS/MS Method for Palytoxin Using Lithium Cationization.

Palytoxin (PlTX) and analogues are produced by certain dinoflagellates, sea anemones, corals and cyanobacteria. PlTX can accumulate in the food chain and when consumed it may cause intoxication with symptoms like myalgia, weakness, fever, nausea, and vomiting. The analysis of PlTXs is challenging, and because of the large molecular structure, it is difficult to develop a sensitive and selective liquid chromatography-mass spectrometry (LC-MS/MS) method. In this work, an LC-MS/MS method was developed to analyse PlTXs with use of lithium iodine and formic acid as additives in the mobile phase. For method development, initially, LC-hrMS was used to accurately determine the elemental composition of the precursor and product ions. The main adduct formed was [M + H + 2Li]3+. Fragments were identified with LC-hrMS and these were incorporated in the LC-MS/MS method. A method of 10 min was developed and a solid phase extraction clean-up procedure was optimised for shellfish matrix. The determined limits of detection were respectively 8 and 22 µg PlTX kg−1 for mussel and oyster matrix. Oysters gave a low recovery of approximately 50% for PlTX during extraction. The method was successfully in-house validated, repeatability had a relative standard deviation less than 20% (n = 5) at 30 µg PlTX kg−1 in mussel, cockle, and ensis, and at 60 µg PlTX kg−1 in oyster.

Using the radioligand-receptor binding assay for paralytic shellfish toxins: A case study on shellfish from Morocco

Paralytic shellfish poisoning (PSP) events occur regularly along the Mediterranean and Atlantic coast of Morocco, and have been responsible for several severe cases of human intoxication. Along the southern Atlantic coast of Morocco, aquaculture and intensive artisanal fishing practices have recently been particularly heavily impacted, and toxic species have been observed in increasing intensity and frequency. In the 1990's a regulatory monitoring program was established for the coastal waters off Morocco by the National Institute of Fisheries Research (INRH), to reduce the risk of intoxication with biotoxins. The regulatory monitoring is conducted weekly and includes toxic phytoplankton enumeration and identification, as well as saxitoxin (STX) analysis in seafood using the mouse bioassay (MBA). Animal testing remains the most widely used screening method for PSP toxin detection, yet its use is being reconsidered for animal-related ethical issues, as well as for practical considerations. To be able to better evaluate alternatives to animal testing, the performance of a nuclear-based radioligand-receptor binding assay (RBA) for paralytic shellfish toxins was assessed and compared with the MBA using four commercially important shellfish matrices, including cockles Cerastoderma edule, razor shells Solen marginatus, oysters Crassostrea gigas, and mussels Perna perna.Over 50 samples were collected and analysed as part of the regulatory monitoring framework including a suite of monthly samples from 2017 and all samples identified as toxic by MBA since 2011. Testing of reference material and evaluation of assay-critical parameters (e.g. slope of calibration curve, internal quality control QC and IC50) confirmed the robustness of the RBA methodology. With this RBA method, STX-like activity detected in shellfish samples ranged from 33 to 8500 μg STX equivalents per kg. RBA data were significantly correlated (P < 0.0001, Pearson r = 0.96) with the MBA-derived dataset. Importantly, the RBA method allowed for the detection and quantification of PSP toxins at levels not detectable by using the mouse bioassay. The limits of quantification of the RBA was calculated and found to be 10-fold lower than that of the MBA, respectively 35.24 ± 5.99 and 325 μg STX equivalents per kg of tissue. In addition, the RBA was easier to use and produced reliable results more rapidly than the MBA and without use of live animals.Considering the increasing risks associated with harmful algal blooms, globally and in Morocco, together with the increased development of aquaculture production and seafood consumption and the difficulties of live animal testing, these findings indicate that the RBA method is a reliable and effective alternative to the MBA method.

Rapid analysis of paralytic shellfish toxins and tetrodotoxins by liquid chromatography-tandem mass spectrometry using a porous graphitic carbon column

Although paralytic shellfish toxins (PSTs) have traditionally been analyzed by liquid chromatography with either pre- or post-column derivatization, and these methods have been validated successfully through inter-laboratory studies, mass spectrometry methods have also been described in literature for use in monitoring programs. However, methods using liquid chromatography coupled with mass spectrometry (LC-MS) need to be improved in terms of sensitivity, analyte recovery and retention time stability because of undesirable matrix effects. Furthermore, tetrodotoxin (TTX) has been found in northern European bivalves, so it is important to analyze TTX compounds alongside PSTs because characteristics of their toxicity are similar. This paper describes, for the first time, a chemical method that allows determination of PSTs, both hydrophilic and hydrophobic, alongside TTX and its analogue 4,9-anhydro tetrodotoxin (4,9-anhTTX) with LC-MS/MS using a Hypercarb® column. The method was validated for 13 hydrophilic PSTs and TTXs and was able to discriminate six hydrophobic PSTs in 20 min. The method was developed for four shellfish matrices: mussel (Mytillus galloprovincialis), clam (Ruditapes decussatus), scallop (Pecten maximus) and oyster (Ostrae edulis). Clean-up procedure used in this work allowed us to obtain good results for validation parameters for both PSTs and TTXs. No standards were available so strains of Gymnodinium catenatum (G. catenatum) were used instead.

Capillary electrophoresis-tandem mass spectrometry for multiclass analysis of polar marine toxins.

Polar marine toxins are more challenging to analyze by mass spectrometry-based methods than lipophilic marine toxins, which are now routinely measured in shellfish by multiclass reversed-phase liquid chromatography-tandem mass spectrometry (MS/MS) methods. Capillary electrophoresis (CE)-MS/MS is a technique that is well suited for the analysis of polar marine toxins, and has the potential of providing very high resolution separation. Here, we present a CE-MS/MS method developed, with use of a custom-built interface, for the sensitive multiclass analysis of paralytic shellfish toxins, tetrodotoxins, and domoic acid in seafood. A novel, highly acidic background electrolyte (5 M formic acid) was designed to maximize protonation of analytes and to allow a high degree of sample stacking to improve the limits of detection. The method was applied to a wide range of regulated and less common toxin analogues, and exhibited a high degree of selectivity between toxin isomers and matrix interference. The limits of detection in mussel tissue were 0.0052 mg/kg for tetrodotoxins, 0.160 mg/kg for domoic acid, and between 0.0018 and 0.120 mg/kg for paralytic shellfish toxins, all of which showed good linearity. Minimal ionization suppression was observed when the response from neat and mussel-matrix-matched standards was corrected with multiple internal standards. Analysis of shellfish matrix reference materials and spiked samples demonstrated good accuracy and precision. Finally, the method was transferred to a commercial CE-MS/MS system to demonstrate its widespread applicability for use in both R & D and routine regulatory settings. The approach of using a highly acidic background electrolyte is of broad interest, and can be considered generally applicable to simultaneous analysis of other classes of small, polar molecules with differing pKa values. Graphical abstract ᅟ.

Differential Mobility Spectrometry for Improved Selectivity in Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry Analysis of Paralytic Shellfish Toxins

Paralytic shellfish toxins (PSTs) are neurotoxins produced by dinoflagellates and cyanobacteria that cause paralytic shellfish poisoning in humans. PST quantitation by LC-MS is challenging because of their high polarity, lability as gas-phase ions, and large number of potentially interfering analogues. Differential mobility spectrometry (DMS) has the potential to improve the performance of LC-MS methods for PSTs in terms of selectivity and limits of detection. This work describes a comprehensive investigation of the separation of 16 regulated PSTs by DMS and the development of highly selective LC-DMS-MS methods for PST quantitation. The effects of all DMS parameters on the separation of PSTs from one another were first investigated in detail. The labile nature of 11α-gonyautoxin epimers gave unique insight into fragmentation of labile analytes before, during, and after the DMS analyzer. Two sets of DMS parameters were identified that either optimized the resolution of PSTs from one another or transmitted them at a limited number of compensation voltage (CV) values corresponding to structural subclasses. These were used to develop multidimensional LC-DMS-MS/MS methods using existing HILIC-MS/MS parameters. In both cases, improved selectivity was observed when using DMS, and the quantitative capabilities of a rapid UPLC-DMS-MS/MS method were evaluated. Limits of detection of the developed method were similar to those without DMS, and differences were highly analyte-dependant. Analysis of shellfish matrix reference materials showed good agreement with established methods. The developed methods will be useful in cases where specific matrix interferences are encountered in the LC-MS/MS analysis of PSTs in complex biological samples. Graphical Abstract ᅟ.

Quantification of PSP toxins in toxic shellfish matrices using post-column oxidation liquid chromatography and pre-column oxidation liquid chromatography methods suggests post-column oxidation liquid chromatography as a good monitoring method of choice

Different shellfish samples were analyzed by Pre- and Post-Column Oxidation Liquid Chromatography to compare the toxins profiles and get information about the degree of accomplishment of both methods. Comparison of the results obtained, the linear correlation coefficient (r2 = 0.94) and the paired t test (two tails, α = 0.05), indicated that there were not significant differences between both sets of data. Nevertheless, important differences related to toxins profiles were found: it was remarkable the difference in results for both Gonyautoxins 1 and 4 and Decarbamoylgonyautoxins 2 and 3, depending on the method of choice, due to an overestimation in the Pre-Column method. It was necessary to modify the elution conditions in the Post-Column method to avoid the interference of matrix peaks at retention times closer to the retention times of the calibrants, mostly when working with oyster and scallop matrices, although it is a good method to use routinely.

A mussel tissue certified reference material for multiple phycotoxins. Part 4: certification.

A freeze-dried mussel tissue (Mytilus edulis) reference material (CRM-FDMT1) was produced containing multiple groups of shellfish toxins. Homogeneity and stability testing showed the material to be fit for purpose. The next phase of work was to assign certified values and uncertainties to 10 analytes from six different toxin groups. Efforts involved optimizing extraction procedures for the various toxin groups and performing measurements using liquid chromatography-based analytical methods. A key aspect of the work was compensating for matrix effects associated with liquid chromatography-mass spectrometry through standard addition, dilution, or matrix-matched calibration. Certified mass fraction values are reported as mg/kg of CRM-FDMT1 powder as bottled for azaspiracid-1, -2, and -3 (4.10 ± 0.40; 1.13± 0.10; 0.96 ± 0.10, respectively), okadaic acid, dinophysistoxin-1 and -2 (1.59 ± 0.18; 0.68 ± 0.07; 3.57± 0.33, respectively), yessotoxin (2.49 ± 0.28), pectenotoxin-2 (0.66 ± 0.06), 13-desmethylspirolide-C (2.70 ± 0.26), and domoic acid (126 ± 10). Combined uncertainties for the certified values include contributions from homogeneity, stability, and characterization experiments. The commutability of CRM-FDMT1 was assessed by examining the extractability and matrix effects for the freeze-dried material in comparison with its equivalent wet tissue homogenate. CRM-FDMT1 is the first shellfish matrix CRM with certified values for yessotoxins, pectenotoxins or spirolides, and is the first CRM certified for multiple toxin groups. CRM-FDMT1 is a valuable tool for quality assurance of phycotoxin monitoring programs and for analytical method development and validation. Graphical Abstract CRM-FDMT1 is a multi-toxin mussel tissue certified reference material (CRM) to aid in development and validation of analytical methods for measuring the levels of algal toxins in seafood.

Laser ablation electrospray ionization high-resolution mass spectrometry for regulatory screening of domoic acid in shellfish.

RationaleDomoic acid (DA) is a potent neurotoxin that accumulates in shellfish. Routine testing involves homogenization, extraction and chromatographic analysis, with a run time of up to 30 min. Improving throughput using ambient ionization for direct analysis of DA in tissue would result in significant time savings for regulatory testing labs.MethodsWe assess the suitability of laser ablation electrospray ionization high‐resolution mass spectrometry (LAESI‐HRMS) for high‐throughput screening or quantitation of DA in a variety of shellfish matrices. The method was first optimized for use with HRMS detection. Challenges such as tissue sub‐sampling, isobaric interferences and method calibration were considered and practical solutions developed. Samples included 189 real shellfish samples previously analyzed by regulatory labs as well as mussel matrix certified reference materials.ResultsDomoic acid was selectively analyzed directly from shellfish tissue homogenates with a run time of 12 s. The limits of detection were between 0.24 and 1.6 mg DA kg−1 tissue, similar to those of LC/UV methods. The precision was between 27 and 44% relative standard deviation (RSD), making the technique more suited to screening than direct quantitation. LAESI‐MS showed good agreement with LC/UV and LC/MS and was capable of identifying samples above and below 5 mg DA kg−1 wet shellfish tissue, one quarter of the regulatory limit.ConclusionsThese findings demonstrate the suitability of LAESI‐MS for routine, high‐throughput screening of DA. This approach could result in significant time savings for regulatory labs carrying out shellfish safety testing on thousands of samples annually. © 2016 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.