A fast magnetic bead-based colorimetric immunoassay for the detection of tetrodotoxins in shellfish

Tetrodotoxin (TTX) is a potent neurotoxin responsible for many food poisoning incidents and some fatalities. Although mainly associated with the consumption of pufferfish, in recent years, TTX has been found in shellfish, particularly in Europe. In this work, a magnetic bead (MB)-based colorimetric immunoassay was applied to the detection of TTX in Pacific oysters (Crassostrea gigas), razor clams (Solen marginatus) and mussels (Mytilus galloprovincialis). Effective LODs (eLODs) for TTX of 1 μg/kg in oysters and razor clams and 3.3 μg/kg in mussels, significantly below the EFSA guidance threshold (44 μg/kg), were obtained. The strategy was applied to the analysis of naturally-contaminated Pacific oysters (Crassostrea gigas) and mussels (Mytilus edulis) from the Netherlands, and TTX was detected in all samples. The approach, which takes less than 1.5 h, proved to be useful as a rapid and simple method to detect TTX, support shellfish safety and protect consumers.info:eu-repo/semantics/acceptedVersio

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

Ultra-fast searching assists in evaluating sub-ppm mass accuracy enhancement in U-HPLC/Orbitrap MS data

A strategy, detailed methodology description and software are given with which the mass accuracy of U-HPLC-Orbitrap data (resolving power 50,000 FWHM) can be enhanced by an order of magnitude to sub-ppm levels. After mass accuracy enhancement all 211 reference masses have mass errors within 0.5 ppm; only 14 of these are outside the 0.2 ppm error margin. Further demonstration of mass accuracy enhancement is shown on a pre-concentrated urine sample in which evidence for 89 (342 ions) potential hydroxylated and glucuronated DHEA-metabolites is found. Although most DHEA metabolites have low-intensity mass signals, only 11 out of 342 are outside the ±1 ppm error envelop; 272 mass signals have errors below 0.5 ppm (142 below 0.2 ppm). The methodology consists of: (a) a multiple internal lock correction (here ten masses; no identity of internal lock masses is required) to avoid suppression problems of a single internal lock mass as well as to increase lock precision, (b) a multiple external mass correction (here 211 masses) to correct for calibration errors, (c) intensity dependant mass correction, (d) file averaging. The strategy is supported by ultra-fast file searching of baseline corrected, noise-reduced metAlign output. The output and efficiency of ultra-fast searching is essential in obtaining the required information to visualize the distribution of mass errors and isotope ratio deviations as a function of mass and intensity

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

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

Evaluation of the shucking of certain species of scallops contaminated with domoic acid with a view to the production of edible parts meeting the safety requirements foreseen in the Union legislation

Acknowledgements: The Panel wishes to thank the following hearing experts for the support provided to this scientific output: Marina Nicolas and Micheal O'Mahony. The Panel wishes to acknowledge all European competent institutions, Member State bodies and other organisations that provided data for this scientific output.Peer reviewedPublisher PD

Evaluation of the shucking of certain species of scallops contaminated with lipophilic toxins with a view to the production of edible parts meeting the safety requirements foreseen in the Union legislation

Acknowledgements: The Panel wishes to thank the following hearing experts for the supportprovided to this scientic output: Laurent Guillier, Mari na Nicolas and Micheal O’Mahony. ThePanel wishes to acknowledge all European competent institutions, Member State bodies and otherorganisations that provided data for this scientic outputPeer reviewedPublisher PD

Immunomagnetic microbeads for screening with flow cytometry and identification with nano-liquid chromatography mass spectrometry of ochratoxins in wheat and cereal

Multi-analyte binding assays for rapid screening of food contaminants require mass spectrometric identification of compound(s) in suspect samples. An optimal combination is obtained when the same bioreagents are used in both methods; moreover, miniaturisation is important because of the high costs of bioreagents. A concept is demonstrated using superparamagnetic microbeads coated with monoclonal antibodies (Mabs) in a novel direct inhibition flow cytometric immunoassay (FCIA) plus immunoaffinity isolation prior to identification by nano-liquid chromatography–quadrupole time-of-flight-mass spectrometry (nano-LC-Q-ToF-MS). As a model system, the mycotoxin ochratoxin A (OTA) and cross-reacting mycotoxin analogues were analysed in wheat and cereal samples, after a simple extraction, using the FCIA with anti-OTA Mabs. The limit of detection for OTA was 0.15 ng/g, which is far below the lowest maximum level of 3 ng/g established by the European Union. In the immunomagnetic isolation method, a 350-times-higher amount of beads was used to trap ochratoxins from sample extracts. Following a wash step, bound ochratoxins were dissociated from the Mabs using a small volume of acidified acetonitrile/water (2/8 v/v) prior to separation plus identification with nano-LC-Q-ToF-MS. In screened suspect naturally contaminated samples, OTA and its non-chlorinated analogue ochratoxin B were successfully identified by full scan accurate mass spectrometry as a proof of concept for identification of unknown but cross-reacting emerging mycotoxins. Due to the miniaturisation and bioaffinity isolation, this concept might be applicable for the use of other and more expensive bioreagents such as transport proteins and receptors for screening and identification of known and unknown (or masked) emerging food contaminants

Marine Toxins: Chemistry, Toxicity, Occurrence and Detection, with Special Reference to the Dutch Situation

Various species of algae can produce marine toxins under certain circumstances. These toxins can then accumulate in shellfish such as mussels, oysters and scallops. When these contaminated shellfish species are consumed severe intoxication can occur. The different types of syndromes that can occur after consumption of contaminated shellfish, the corresponding toxins and relevant legislation are discussed in this review. Amnesic Shellfish Poisoning (ASP), Paralytic Shellfish Poisoning (PSP), Diarrheic Shellfish Poisoning (DSP) and Azaspiracid Shellfish Poisoning (AZP) occur worldwide, Neurologic Shellfish Poisoning (NSP) is mainly limited to the USA and New Zealand while the toxins causing DSP and AZP occur most frequently in Europe. The latter two toxin groups are fat-soluble and can therefore also be classified as lipophilic marine toxins. A detailed overview of the official analytical methods used in the EU (mouse or rat bioassay) and the recently developed alternative methods for the lipophilic marine toxins is given. These alternative methods are based on functional assays, biochemical assays and chemical methods. From the literature it is clear that chemical methods offer the best potential to replace the animal tests that are still legislated worldwide. Finally, an overview is given of the situation of marine toxins in The Netherlands. The rat bioassay has been used for monitoring DSP and AZP toxins in The Netherlands since the 1970s. Nowadays, a combination of a chemical method and the rat bioassay is often used. In The Netherlands toxic events are mainly caused by DSP toxins, which have been found in Dutch shellfish for the first time in 1961, and have reoccurred at irregular intervals and in varying concentrations. From this review it is clear that considerable effort is being undertaken by various research groups to phase out the animal tests that are still used for the official routine monitoring programs

First Report on the Occurrence of Tetrodotoxins in Bivalve Mollusks in The Netherlands

Tetrodotoxin (TTX) is traditionally associated with seafood from tropical regions, but recently TTX was detected in bivalve mollusks in more temperate European waters. In The Netherlands it was therefore decided to monitor TTX in shellfish harvested from Dutch production areas. All shellfish production areas were monitored in 2015, 2016 and 2017. Samples were analyzed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In total 1063 samples were investigated, and the highest concentrations were observed in 2016, i.e., 253 µg TTX/kg in oysters and 101 µg TTX/kg in mussels. No TTX analogues, with the exception of 4-epi-TTX in one single sample, were found and contaminated samples also showed positive results in the neuro-2a bioassay. The occurrence of TTX seems to be consistent over the last three years with the highest concentrations observed annually in late June. The causative organism and the reasons why specific Dutch production areas are affected while others are not, are still unclear. Initially in The Netherlands an action limit of 20 µg TTX/kg was used to ensure the safety of consumers (2016), but recently The European Food Safety Authority (EFSA) established an acute reference dose, and based on a high portion size of consuming 400 g mussels, this dose was translated into a safe concentration of 44 µg TTX per kg for shellfish. This concentration is now used as an action limit and TTX is formally included in the Dutch shellfish monitoring program

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.</p