Interlaboratory Evaluation of Multiple LC-MS/MS Methods and a Commercial ELISA Method for Determination of Tetrodotoxin in Oysters and Mussels.

BACKGROUND: Given the recent detection of tetrodotoxin (TTX) in bivalve molluscs but the absence of a full collaborative validation study for TTX determination in a large number of shellfish samples, interlaboratory assessment of method performance was required to better understand current capabilities for accurate and reproducible TTX quantitation using chemical and immunoassay methods. OBJECTIVE: The aim was to conduct an interlaboratory study with multiple laboratories, using results to assess method performance and acceptability of different TTX testing methods. METHODS: Homogenous and stable mussel and oyster materials were assessed by participants using a range of published and in-house detection methods to determine mean TTX concentrations. Data were used to calculate recoveries, repeatability, and reproducibility, together with participant acceptability z-scores. RESULTS: Method performance characteristics were good, showing excellent sensitivity, recovery, and repeatability. Acceptable reproducibility was evidenced by HorRat values for all LC-MS/MS and ELISA methods being less than the 2.0 limit of acceptability. Method differences between the LC-MS/MS participants did not result in statistically different results. Method performance characteristics compared well with previously published single-laboratory validated methods and no statistical difference was found in results returned by ELISA in comparison with LC-MS/MS. CONCLUSION: The results from this study demonstrate that current LC-MS/MS methods and ELISA are on the whole capable of sensitive, accurate, and reproducible TTX quantitation in shellfish. Further work is recommended to expand the number of laboratories testing ELISA and to standardize an LC-MS/MS protocol to further improve interlaboratory precision. HIGHLIGHTS: Multiple mass spectrometric methods and a commercial ELISA have been successfully assessed through an interlaboratory study, demonstrating excellent performance.

Development and Validation of a Hybrid Screening and Quantitative Method for the Analysis of Eight Classes of Therapeutants in Aquaculture Products by Liquid Chromatography-Tandem Mass Spectrometry.

A method using reverse-phase ultra-high-performance liquid chromatography coupled with tandem mass spectrometry is described for eight classes of therapeutants that are used in marine aquaculture products. Validation studies to evaluate recovery, precision, method detection limits, and measurement uncertainty were performed at three levels, using three representative matrices [salmon (fatty fish), tilapia (lean fish), and shrimp (crustaceans)] to assess the method performance for use as a screening or determinative (quantitative and confirmatory) method. A total of 16 sulfonamides (plus 2 potentiators), 2 tetracyclines, 11 (fluoro)quinolones, 7 nitroimidazoles, 3 amphenicols, 5 steroids, and 3 stilbenes met the quantitative criteria for method validation. An additional 5 triphenylmethane dyes, 2 sulfonamides, 2 tetracyclines, and 1 amphenicol met the required performance for use as a screening method. Limits of detection (LODs) for the compounds that met the quantitative criteria ranged from 0.1 to 5 µg/kg, while LODs for compounds from the screening group ranged from 0.1 to 30 µg/kg. This method provides a comprehensive approach to the determination of different classes of compounds in aquaculture products.

Hydrophilic interaction liquid chromatography-tandem mass spectrometry for quantitation of paralytic shellfish toxins: validation and application to reference materials.

Paralytic shellfish toxins (PSTs) are potent neurotoxins produced by marine dinoflagellates that are responsible for paralytic shellfish poisoning (PSP) in humans. This work highlights our ongoing efforts to develop quantitative methods for PSTs using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). Compared with the commonly used method of liquid chromatography with post-column oxidation and fluorescence detection (LC-ox-FLD), HILIC-MS/MS has the potential of being more robust, sensitive and straightforward to operate, and provides unequivocal confirmation of toxin identity. The main driving force for the present work was the need for a complementary method to LC-ox-FLD to assign values to shellfish tissue matrix reference materials for PSTs. Method parameters that were optimized included LC mobile and stationary phases, electrospray ionization (ESI) conditions, and MS/MS detection parameters. The developed method has been used in the detection and identification of a wide range of PSTs including less common analogues and metabolites in a range of shellfish and algal samples. We have assessed the matrix effects of shellfish samples and have evaluated dilution, standard addition and matrix matched calibration as means of mitigating them. Validation on one LC-MS/MS system for nine common PST analogues (GTX1-4, dcGTX2&3, STX, NEO, and dcSTX) was completed using standard addition. The method was then transferred to a more sensitive LC-MS/MS system, expanded to include five more PSTs (C1&2, dcNEO and GTX5&6) and validated using matrix matched calibration. Limits of detection of the validated method ranged between 6 and 280 nmol/kg tissue using standard addition in extracts of blue mussels, with recoveries between 92 and 108%. Finally, this method was used in combination with the AOAC Official Method based on LC-ox-FLD to measure PSTs in a new mussel tissue matrix reference material.

The Development and Single-Laboratory Validation of a Method for the Determination of Steroid Residues in Fish and Fish Products.

Due to potential use in aquacultured fish products, the Canadian Food Inspection Agency has identified residue testing for steroids as a priority. These compounds are used in aquaculture primarily to direct sexual differentiation with both androgens and estrogens applied depending on the desired outcome. Published research is lacking with respect to steroid residue testing in fish; however, recent studies in other matrixes provided transferable cleanup techniques. A simple, rapid, and sensitive method was developed and validated for use in monitoring aquacultured fish products for the presence of methyltestosterone, nandrolone, epi-nandrolone, boldenone, and epi-boldenone residues. The developed method consists of solvent extraction followed by cleanup using hexane and dual cartridge SPE with analysis by ultra-HPLC-MS/MS. The method is capable of detecting and confirming steroid residue levels ranging from 0.05 to 25 ng/g in salmon and tilapia, depending on the analyte. Recoveries ranged from 88 to 130% for the analytes. Instrument repeatability was less than 13% for all compounds, while intermediate precision ranged from 5 to 25% RSD. HorRat values were within acceptable ranges.

Acute toxicities of saxitoxin, neosaxitoxin, decarbamoyl saxitoxin and gonyautoxins 1&4 and 2&3 to mice by various routes of administration.

Saxitoxin and its derivatives, the paralytic shellfish toxins (PSTs), are known to be toxic to humans, and maximum permitted levels in seafood have been established by regulatory authorities in many countries. Until recently, the mouse bioassay was the reference method for determining the levels of these toxins in seafood, but this has now been superseded by chemical methods of analysis. The latter methods are able to determine the levels of many PSTs in shellfish, but for risk assessment an estimate of the relative toxicities of the individual components of the PST mixture is required. The relative toxicities are expressed as "Toxicity Equivalence Factors" (TEFs). At present, TEFs are based on relative specific activities in the mouse bioassay, rather than on acute toxicity determinations, as measured by median lethal doses (LD50s). In the present study, the median lethal doses of saxitoxin, neosaxitoxin, decarbamoyl saxitoxin and equilibrium mixtures of gonyautoxins 1&4 and gonyautoxins 2&3 have been determined by intraperitoneal injection, gavage and feeding. The results indicate that specific activities in the MBA do not consistently correlate with acute toxicities by any of the routes of administration, and TEFs, particularly for neosaxitoxin, require revision.

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.

Comparison of AOAC 2005.06 LC official method with other methodologies for the quantitation of paralytic shellfish poisoning toxins in UK shellfish species.

A refined version of the pre-column oxidation liquid chromatography with fluorescence detection (ox-LC-FLD) official method AOAC 2005.06 was developed in the UK and validated for the determination of paralytic shellfish poisoning toxins in UK shellfish. Analysis was undertaken here for the comparison of PSP toxicities determined using the LC method for a range of UK bivalve shellfish species against the official European reference method, the PSP mouse bioassay (MBA, AOAC 959.08). Comparative results indicated a good correlation in results for some species (mussels, cockles and clams) but a poor correlation for two species of oysters (Pacific oysters and native oysters), where the LC results in terms of total saxitoxin equivalents were found to be on average more than double the values determined by MBA. With the potential for either LC over-estimation or MBA under-estimation, additional oyster and mussel samples were analysed using MBA and ox-LC-FLD together with further analytical and functional methodologies: a post-column oxidation LC method (LC-ox-FLD), an electrophysiological assay and hydrophilic interaction liquid chromatography with tandem mass spectrometric detection. Results highlighted a good correlation among non-bioassay results, indicating a likely cause of difference was the under-estimation in the MBA, rather than an over-estimation in the LC results.

Liquid chromatographic post-column oxidation method for analysis of paralytic shellfish toxins in mussels, clams, scallops, and oysters: single-laboratory validation.

A single-laboratory validation study was conducted for the LC post-column oxidation analysis of paralytic shellfish toxins (PST): saxitoxin (STX); neosaxitoxin (NEO); gonyautoxins (GTX) 1-5; decarbamoyl gonyautoxins (dcGTX) 2 and 3; decarbamoyl saxitoxin (dcSTX); and N-sulfocarbamoyl-gonyautoxin-2 and 3 (C1 and C2) in mussels (Mytilus edulis), soft shell clams (Mya arenaria), scallops (Placopectin magellanicus), and oysters (Crassostrea virginicus). The instrumental technique was developed for the analysis of PST in shellfish as an alternative to the precolumn oxidation method, AOAC Official Method 2005.06, and a replacement for the current AOAC biological method 959.08. The method used reversed-phase LC with post-column oxidation and fluorescence detection. Test materials for method recovery were prepared by fortification of blank material with a cocktail of PST. Materials used to determine method repeatability and intermediate precision were prepared by blending blank material with naturally incurred material. The target total toxicity levels evaluated in the study were 0.40, 0.80, and 1.60 mg STX x diHCl equivalents per kilogram [(eq/kg) 1%, 1, and 2 times the regulatory limit]. Linearity, recovery, and within-laboratory precision parameters of the method were evaluated. Correlation coefficients of the calibration curves for all toxins studied were > 0.99. Total toxin recovery ranged from 94 to 106% at the three levels of interest. Repeatability and intermediate precision RSD ranged from 2 to 7% and 2 to 8%, respectively. The method LOD and LOQ (assuming the presence of all toxins) were determined to be equivalent to 0.18 and 0.39 mg STX x diHCl eq/kg. The method is intended for a regulatory framework and will be submitted for an AOAC collaborative study.