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.     

A feasibility study into the production of a freeze-dried oyster reference material for paralytic shellfish poisoning toxins

Matrix reference materials are an essential component for the validation and quality control of analytical methodologies for the quantitation of marine biotoxins in shellfish. Given the potential advantages of reference materials in powder form, a study was conducted to assess the feasibility for the production of a freeze-dried oyster tissue reference material containing a range of important paralytic shellfish poisoning toxins. One bulk sample of a wet oyster tissue homogenate was generated following mass culturing of toxic Alexandrium and oyster feeding experiments. The bulk tissue was used to prepare untreated wet frozen aliquots with the remainder being freeze-dried and processed into appropriately-sized powder samples. A pre-column oxidation LC-FLD analysis was used to confirm the absence of any chromatographic artefacts resulting from the processing and to confirm acceptable homogeneity of the tissues. Excellent stability over both the short-term (1 month) and long-term (1 year) of the freeze-dried material was demonstrated as compared with the stability of the untreated wet tissue. A post-column oxidation LC-FLD method was used to confirm the absence of toxin epimerisation in freeze-dried tissues which were observed in the wet tissues. Overall the work showed the feasibility of an approach to produce a homogenous freeze-dried oyster matrix material with enhanced stability in comparison to the untreated wet tissue. The potential for use of the process for preparation of large scale production batches of a freeze-dried CRM for paralytic shellfish poisoning toxins has therefore been demonstrated.     

Derivatization of azaspiracid biotoxins for analysis by liquid chromatography with fluorescence detection

Azaspiracids (AZAs) are an important group of regulated lipophilic biotoxins that cause shellfish poisoning. Currently, the only widely available analytical method for quantitation of AZAs is liquid chromatography-mass spectrometry (LC-MS). Alternative methods for AZA analysis are needed for detailed characterization work required in the preparation of certified reference materials (CRMs) and by laboratories not equipped with LC-MS. Chemical derivatization of the amine and carboxyl groups on AZAs was investigated for the purpose of facilitating analysis by LC with fluorescence detection (FLD). Experiments towards chemical modification of AZA1 at the amine achieved only limited success. Derivatization of the carboxyl group, on the other hand, proved successful using the 9-anthryldiazomethane (ADAM) method previously applied to the okadaic acid (OA) group toxins. Extraction and clean-up methods were investigated for shellfish tissue samples and a post-reaction solid phase extraction procedure was developed for the AZA ADAM derivatives. Chromatographic separations were developed for the LC-FLD analysis of derivatized AZAs alone or in the presence of other derivatized toxins. This new analytical method for analysis of AZAs enabled verification of AZA1-3 concentrations in recently certified reference materials. The method demonstrated good linearity, repeatability and accuracy showing its potential as an alternative to LC-MS for measurement of AZAs.     

Feasibility of gamma irradiation as a stabilisation technique in the preparation of tissue reference materials for a range of shellfish toxins

The effect of γ-irradiation on concentrations of hydrophilic and lipophilic phycotoxins has been investigated by use of HPLC–UV and LC–MS. Pure toxins in organic solvents and toxins in mussel (Mytilus edulis) tissues were irradiated at three different doses. In solution all toxin concentrations were reduced to some extent. Most severe decreases were observed for domoic acid and yessotoxin, for which the smallest dose of irradiation led to almost complete destruction. For pectenotoxin-2 the decrease in concentration was less severe but still continuous with increasing dose. Azaspiracid-1 and okadaic acid were the least affected in solution. In shellfish tissue the decrease in toxin concentrations was much reduced compared with the effect in solution. After irradiation at the highest dose reductions in concentrations were between ca. 5 and 20% for the lipophilic toxins and there was no statistical difference between control and irradiated samples for azaspiracids in tissue. Irradiation of shellfish tissues contaminated with domoic acid led to a more continuous decrease in the amount of the toxin with increasing dose. The effect of irradiation on the viability of microbial activity in shellfish tissues was assessed by using total viable counting techniques. Microbial activity depended on the type of shellfish and on the pretreatment of the shellfish tissues (with or without heat treatment). As far as we are aware this is the first investigation of the effectiveness of irradiation as a technique for stabilising tissue reference materials for determination of phycotoxins. Our results suggest that this technique is not effective for materials containing domoic acid. It does, however, merit further investigation as a stabilisation procedure for preparation of shellfish tissue materials for some lipophilic toxins, in particular azaspiracids. Chemical structures of the toxins investigated in the study     

A mussel tissue certified reference material for multiple phycotoxins. Part 3: homogeneity and stability

A candidate certified reference material (CRM) for multiple shellfish toxins (domoic acid, okadaic acid and dinophysistoxins, pectenotoxins, yessotoxin, azaspiracids and spirolides) has been prepared as a freeze-dried powder from mussel tissues (Mytilus edulis). Along with the certified values, the most important characteristics for a reference material to be fit-for-purpose are homogeneity and stability. Acceptable between-bottle homogeneity was found for this CRM. Within-bottle homogeneity was assessed using domoic acid, and it was shown that repeated subsampling of the CRM can be performed precisely down to 0.35 g. Both short- and long-term stability studies carried out under isochronous conditions demonstrated excellent stability of the various toxins present in the material. While degradation of some analytes was observed at +60°C in short-term studies, it was determined that shipping at ambient temperature is adequate. No instability was detected in long-term stability studies, and it was shown that the material can be held at +18°C safely for up to 1 year. To guarantee stability of the CRM over its lifetime the stock will be maintained at −20°C. The results of the homogeneity and stability testing show that CRM–FDMT1 is appropriate for its intended use in quality assurance and quality control of shellfish toxin analysis methods.     

Potential use of gamma irradiation in the production of mussel and oyster reference materials for paralytic shellfish poisoning toxins

Bivalve shellfish samples containing paralytic shellfish poisoning toxins were subjected to gamma irradiation dosage trials in order to assess the potential suitability of the technique in the production of toxin reference materials. Two candidate reference materials of tissue homogenates, mussels (Mytilus sp.) and native oysters (Ostrea edulis), were prepared in-house. Both were subjected to gamma irradiation at four different dose levels, 3.0, 6.0, 13.0 and 18.1 kGy. Bacterial levels were shown to be eliminated in the mussels and significantly reduced in the oysters following irradiation at all four dose levels. Paralytic shellfish poisoning (PSP) toxin concentrations were not significantly reduced in any of the samples indicating the treatment had no adverse affect on the initial stability of any of the PSP toxins monitored. Chromatographic results showed near-identical profiles for treated and non-treated samples inferring that no fluorescent toxin degradation products or matrix interferences were produced during the irradiation process. Results therefore proved that gamma irradiation treatment reduced bacterial levels within paralytic shellfish poisoning reference materials without compromising analyte content, with the subsequent potential to enhance the stability of future candidate reference materials treated in this manner.     

Reference materials for domoic acid, a marine neurotoxin

Summary Domoic acid was recognized recently [1, 2] to be a marine neurotoxin associated with shellfish harvested or cultured for use as human food. Evidence about the occurrence of domoic acid and its importance to shellfish industries is reviewed. The preparation and certification of two reference materials for the determination of domoic acid, an instrument calibration solution (DACS-1, released by the Marine Analytical Chemistry Standards Program [MACSP] in May, 1989), and a tissue reference material (MUS-1, homogenized soft tissues of Mytilus edulis, released by the MACSP in August, 1989) are described. We believe these are the first certified standards or reference materials to be available for the determination of shellfish toxins, a problem of increasing importance to aquaculturists and the seafood industry, as well as to agencies concerned with the safety of food. The tissue homogenate preparation techniques we have developed and used may be of general interest for the preparation of other tissue reference materials for the determination of other organic compounds, since the sealed, fluid homogenate samples seem acceptably stable without being continuously frozen or refrigerated.NRCC No. 31925     

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.     

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.     

Assessment of MIST Alert,a commercial qualitative assay for detection of paralytic shellfish poisoning toxins in bivalve molluscs

A recently developed commercial rapid test kit (MIST Alert) was assessed for determination of the presence of paralytic shellfish poisoning (PSP) toxins in shellfish. Several commercially important shellfish species obtained from the UK shellfish toxin monitoring program, containing a range of total PSP toxicities as determined by the mouse bioassay (MBA), were tested. The kit detected toxin in all samples containing the European Community tolerance level of 80 microg saxitoxin (STX) equivalents/100 g shellfish flesh as determined by the MBA. With one exception, the kit detected toxin in all samples that contained >40 microg STX equivalents/100 g according to the MBA. Among samples in which the MBA did not detect toxin, the kit disagreed in 25% of the tests, although further analysis by liquid chromatography (LC) and MBA of some samples confirmed the presence of toxins. These results suggest that MIST Alert may be suitable as an initial screen for PSP toxins as part of routine monitoring programs, thereby greatly reducing the number of MBAs. Trials were also performed by nonscientific personnel to evaluate the ease of use and interpretation of results obtained by MIST Alert. The results indicated that the kits could be readily used and accurately interpreted by individuals with no technical or scientific background.     

Feasibility study for the production of certified calibrants for the determination of deoxynivalenol and other B-trichothecenes: intercomparison study

Thirteen European laboratories experienced in the analysis of mycotoxins participated in an intercomparison study within a European Commission-funded project. Goals of the study were to check the fitness for purpose of a small batch of gravimetrically prepared calibrants; to compare individually prepared calibrants with common calibrants; to check the feasibility of toxin mixtures as calibrant solutions; and to give recommendations on the production of future certified reference materials (CRMs) with regard to the nature of the calibrant and the means of certification. Each laboratory received ampules of each common calibrant containing single toxins [solution containing either deoxynivalenol (DON), 3-acetyl-DON (3-Ac-DON), nivalenol (NIV), or 15-acetyl-DON (15-Ac-DON)] and 3 ampules of toxin-mixture (solutions of DON + 3-Ac-DON + NIV in acetonitrile) of known concentrations (about 20 microg/mL). Ampules with single toxins (solution containing either DON, 3-Ac-DON, NIV, or 15-Ac-DON) and a toxin-mixture (solutions of DON + 3-Ac-DON + NIV in acetonitrile) of unknown concentrations were distributed to the participants for quantification. The participating laboratories used mainly high-performance liquid chromatography (HPLC)-diode array detection UV for DON, 3-Ac-DON, NIV, and 15-Ac-DON; gas chromatography-electron capture detection (GC-ECD) and GC-mass spectrometry methods were used sparingly. Linear calibration curves were achieved by >90% of the participants. Relative between-day variation (RSDr) of 26% of the laboratories was greater than the target value of 5% for HPLC, and RSDr of 32% of the laboratories was greater than the desired value of 10% for GC. Relative between-laboratory variation (RSDR) of the GC results obtained with single common calibrants was greater than the target value of 16% for all laboratories. RSDR of the HPLC results for the common unknown single toxin solutions was less than the target value of 8% except for 15-Ac-DON. Generally, better recoveries were observed from common calibrants (102% for mix calibrants and 98% for single calibrants) than from individually prepared calibrants (95%). This international comparison study clearly showed the high scattering of results in the analysis of type-B trichothecenes, particularly when GC was used. Obviously, this intercomparison study was not suited for the certification of B-trichothecenes. A certification of the proposed calibrant material was therefore recommended on the basis of its gravimetrical preparation.     

Evolving to the optoelectronic mouse for phycotoxin analysis in shellfish

Despite ethical and technical concerns, the in vivo method, or more commonly referred to mouse bioassay (MBA), is employed globally as a reference method for phycotoxin analysis in shellfish. This is particularly the case for paralytic shellfish poisoning (PSP) and emerging toxin monitoring. A high-performance liquid chromatography method (HPLC-FLD) has been developed for PSP toxin analysis, but due to difficulties and limitations in the method, this procedure has not been fully implemented as a replacement. Detection of the diarrhetic shellfish poisoning (DSP) toxins has moved towards LC-mass spectrometry (MS) analysis, whereas the analysis of the amnesic shellfish poisoning (ASP) toxin domoic acid is performed by HPLC. Although alternative methods of detection to the MBA have been described, each procedure is specific for a particular toxin and its analogues, with each group of toxins requiring separate analysis utilising different extraction procedures and analytical equipment. In addition, consideration towards the detection of unregulated and emerging toxins on the replacement of the MBA must be given. The ideal scenario for the monitoring of phycotoxins in shellfish and seafood would be to evolve to multiple toxin detection on a single bioanalytical sensing platform, i.e. ‘an artificial mouse’. Immunologically based techniques and in particular surface plasmon resonance technology have been shown as a highly promising bioanalytical tool offering rapid, real-time detection requiring minimal quantities of toxin standards. A Biacore Q and a prototype multiplex SPR biosensor have been evaluated for their ability to be fit for purpose for the simultaneous detection of key regulated phycotoxin groups and the emerging toxin palytoxin. Deemed more applicable due to the separate flow channels, the prototype performance for domoic acid, okadaic acid, saxitoxin, and palytoxin calibration curves in shellfish achieved detection limits (IC₂₀) of 4,000, 36, 144 and 46 μg/kg of mussel, respectively. A one-step extraction procedure demonstrated recoveries greater than 80 % for all toxins. For validation of the method at the 95 % confidence limit, the decision limits (CCα) determined from an extracted matrix curve were calculated to be 450, 36 and 24 μg/kg, and the detection capability (CCβ) as a screening method is ≤10 mg/kg, ≤160 μg/kg and ≤400 μg/kg for domoic acid, okadaic acid and saxitoxin, respectively.     

The BCR programme and measurements for food and agriculture

Summary The importance of accurate and comparable measurements to the removal of technical barriers to trade and the achievement of the single European Market is well established. The BCR Programme is recognized as having an important role in this area, a fact reflected in a substantial increase in the programme in recent years.There has been a consequential expansion of its activities for food and agricultur measurements which currently involves some 50 projects and will lead to the issue of a further 25 CRMs in the next 12 months.Topics covered include:Nutritional properties — fatty acids, sterols, total fat, protein, carbohydrates, dietary fibre, oil and fat soluble vitamins, and major elements.Undesirable substances — PAHs, PCBs, shellfish toxins, mycotoxins, heterocyclic amines, nitrate, pesticides and toxic element in food and animal feed.Technological properties — bread making quality of flour (farinograph, extensograph, etc.), determination of gluten in wheat, rapid NMR methods and reference materials for the oil content of rapeseed, reference colour tiles for calibration of colorimeters for tomato paste, and characterisation of natural products by isotopic determination (NMR and MS).For agriculture particular emphasis has been placed on methodology and RMs for the analysis of the very wide range of growth promoting compounds in farm animals (organs and fluids). Other projects concern amino-acids in feed and rapid method (XRF) for glucosinolates in rapeseed. Although most of the projects lead to CRMs, the common feature and indeed, the objective of the work, is the establishment of reliable reference values which form the basis for the accuracy and comparability of the results of these measurements.The work of the BCR Programme in this area is reviewed and the steps taken to ensure that the reference values are accurately certified with an acceptably small uncertainty are illustrated with examples taken from current projects.     

Freeze-drying for the stabilisation of shellfish toxins in mussel tissue (<Emphasis Type="Italic">Mytilus edulis</Emphasis>) reference materials

Two samples of mussels (Mytilus edulis) were collected from the southwest of Ireland. One sample contained domoic acid, the other sample contained okadaic acid, dinophysistoxin-2 and azaspiracid-1, -2 and -3. Wet and freeze-dried reference materials were prepared from each of the two samples to test for differences in homogeneity, stability and extractability of the analytes in either condition. Wet materials were homogenised, aliquoted and hermetically sealed under argon and subsequently frozen at −80 °C. Dry materials were similarly homogenised but frozen in flat cakes prior to freeze-drying. After grinding, sieving and further homogenisation, the resulting powder was aliquoted and hermetically sealed. Domoic acid materials were characterised using HPLC–UV, while LC–MS was used for the determination of lipophilic toxins. The extractabilities of all phycotoxins studied were comparable for wet and freeze-dried materials once a sonication step had been carried out for reconstitution of the freeze-dried materials prior to extraction. Homogeneity was assessed through replicate analysis of the phycotoxins (n = 10), and was found to be similar for wet and freeze-dried materials, for both hydrophilic and lipophilic toxins. Water contents were determined for both wet and freeze-dried materials, and particle size was determined for the freeze-dried materials. Stability was evaluated isochronously over eight months at four temperatures (−20, +4, +20 and +40 °C). The freeze-dried material containing domoic acid was stable over the whole duration at all temperatures, while in the wet material domoic acid degraded to some extent at all temperatures except −20 °C. In freeze-dried and wet materials containing lipophilic toxins, okadaic acid, dinophysistoxin-2, azaspiracid-1 and azaspiracid-2 were stable over the whole duration at all conditions, while concentrations of azaspiracid-3 changed significantly in both materials at some storage temperatures. Figure Aliquots of freeze-dried and wet mussel tissue reference materials containing the various shellfish toxins examined in the study     

Rapid detection of cyanobacterial toxins in precursor ion mode by liquid chromatography tandem mass spectrometry

We established an analytical method based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in the precursor ion mode for simultaneous qualitative monitoring of various groups of cyanobacterial toxins. The toxin groups investigated were paralytic shellfish poisoning (PSP) toxins, anatoxins (ANAs), cylindrospermopsins (CYNs), microcystins (MCs), and nodularins (NODs), including rare and uncharacterized derivatives found in plankton and water matrices. Alternative analytical methods based on tandem mass spectrometry commonly operate in multiple reaction monitoring (MRM) mode and depend on prior knowledge of putative toxigenicity of the cyanobacterium species and strain, and the expected toxin variants. In contrast, the precursor ion mode yields diagnostic mass fragments for the detection of characteristic compounds of the different toxin classes and thus allows monitoring of a large set of unspecified cyanotoxins of various groups, even when the species composition is undetermined or uncertain. This rapid method enables screening for a wide spectrum of toxic cyanobacterial metabolites and degradation products in a single chromatographic separation with detection limits at nanogram levels. The precursor ion technique is a valuable adjunct to existing mass spectrometric methods for cyanotoxins, although it is not a complete replacement for detailed quantitative analysis requiring comprehensive sample cleanup.     

Preparation of a reference mussel tissue material for polycyclic aromatic hydrocarbons and trace metals determination

Due to high cost of certified reference materials (CRMs), reference materials (RMs) are preferred to check the method performance in environmental analysis. In this work, a laboratory reference material (LRM) was prepared and characterised to carry out the quality control in monitoring analysis of eight polycyclic aromatic hydrocarbons (PAHs) and nine trace metals in mussel tissue. Mussels were collected in a naturally polluted area. Before the reference material was bottled, the mussel tissue was stabilised by freeze-drying, ground and sieved. For the material characterisation, several statistical tests were applied to check the homogeneity of the analytes in the tissue, and a stability test was performed to study the effect of the storage temperature in the analyte concentration. Other characteristics such as specific density, moisture and lipid contents as well as particle size distribution of the material were determined. Although the LRM had a homogeneous distribution for all PAHs and almost all metals, the stability study showed different results at both storage temperatures studied. For both PAHs and trace metals, the material was suitable to assure the quality control of the analysis.     

Certified reference materials for inorganic and organic contaminants in environmental matrices

Chemical measurements often constitute the basis for informed decision-making at different levels in society; sound decision-making is possible only if the quality of the data used is uncompromised. To guarantee the reliability and comparability of analytical data an intricate system of quality-assurance measures has to be put into effect in a laboratory. Reference materials and, in particular, certified reference materials (CRMs) are essential for achieving traceability and comparability of measurement results between laboratories and over time. As in any other domain of analytical chemistry, techniques used to monitor the levels and fate of contaminants in the environment must be calibrated using appropriate calibration materials, and the methods must be properly validated using fit-for-purpose matrix-matched CRMs, to ensure confidence in the data produced. A sufficiently large number of matrix CRMs are available for analysis of most elements, and the group of chemicals known as persistent organic pollutants, in environmental compartments and biota. The wide variety of analyte/level/matrix/matrix property combinations available from several suppliers enables analysts to select CRMs which sufficiently match the properties of the samples they analyse routinely. Materials value-assigned for the so-called emerging pollutants are scarce at the moment, though an objective of current development programmes of CRM suppliers is to overcome this problem.     

Liquid chromatography–tandem mass spectrometry method for the detection of marine lipophilic toxins under alkaline conditions

A new LC–MS/MS method for the separation and detection of the most prominent marine lipophilic toxin groups comprising okadaic acid, dinophysistoxins, yessotoxins, azaspiracids, pectenotoxins, spirolides and some okadaic acid fatty acid esters has been developed. With this method 28 different marine lipophilic biotoxins can be analysed in a single run. Separation was achieved with an acetonitrile/water gradient containing ammonium hydroxide (pH 11). All toxins were stable under these basic conditions. Compared to chromatography using an acidic gradient, the limit of detection (LODs) for okadaic acid, yessotoxin, gymnodimine and 13-desmethyl spirolide C were improved two- to three-fold, mainly due to better peak shapes. The azaspiracids and pectenotoxins-2 showed comparable LODs under acidic and basic conditions. A major advantage of the developed method is that toxins can be clustered in retention time windows separated for positively and negatively ionized molecular ions. Therefore, there is no need for rapid polarity switching or two separate runs for one sample. The new method showed good repeatability and reproducibility and is an important step in the development of alternatives to the animal tests currently in use for shellfish toxin analysis.     

Determination of domoic acid toxins in shellfish by biosense ASP ELISA--a direct competitive enzyme-linked immunosorbent assay: collaborative study

A collaborative study was conducted on the Biosense amnesic shellfish poisoning (ASP) enzyme-linked immunosorbent assay (ELISA) for the determination of domoic acid (DA) toxins in shellfish in order to obtain interlaboratory validation data for the method. In addition, a method comparison study was performed to evaluate the ASP ELISA as an alternative to the current liquid chromatography (LC) reference method for DA determination. The study material comprised 16 shellfish samples, including blue mussels, Pacific oysters, and king scallops, spiked with contaminated mussel homogenates to contain 0.1-20 mg DA/kg shellfish flesh. The shellfish samples were extracted with 50% aqueous methanol, and the supernatants were directly analyzed. Sixteen participating laboratories in 10 countries reported data from the ASP ELISA, and 4 of these laboratories also reported data from instrumental LC analysis. The participating laboratories achieved interlaboratory precision estimates for the 8 Youden paired shellfish samples in the range of 10-20% for RSD(r) (mean 14.8 +/- 4%), and 13-29% for RSDR (mean 22.7 +/- 6%). The precision estimates for the ELISA data did not show a strong dependence on the DA concentration in the study samples, and the overall precision achieved was within the acceptable range of the Horwitz guideline with HorRat values ranging from 1.1 to 2.4 (mean HorRat 1.7 +/- 0.5). The analysis of shellfish samples spiked with certified reference material (CRM)-ASP-MUS-b gave recoveries in the range of 88-122%, with an average recovery of 104 +/- 10%. The estimate on method accuracy was supported by a correlation slope of 1.015 (R2 = 0.992) for the determined versus the expected DA values. Furthermore, the correlation of the ASP ELISA results with those for the instrumental LC analyses of the same sample extracts gave a correlation slope of 1.29 (R2 = 0.984). This indicates some overestimation of DA levels in shellfish by the ELISA, but it is also a result of apparent low recoveries for the LC methods. This interlaboratory study demonstrates that the ASP ELISA is suitable for the routine determination and monitoring of DA toxins in shellfish, and that it offers a rapid and cost-effective methodology with high sample throughput.     

Determination of the reference value of nitrogen mass fraction in the reference material of Polish soil

Ensuring a traceability and meaningful of a measurements is one of the most important stages of each analysis, each measurement. It is especially important for measurement of the environmental samples, like soil, which is a very complex matrix. A certified reference materials (CRMs) should be routinely used for this purpose. The paper discusses the procedure for preparation of the soil samples for certification as CRM. As for agricultural reasons there is a growing demand for CRMs regarding a nitrogen mass fraction in the Polish soil, we prepared such a material and established the reference value with associated measurement uncertainty. Homogeneity and stability of the material were shown to be appropriate for the intended purpose. The presented approach can also be used in a process of manufacture of a laboratory reference material, which can be used for a routine quality control.