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.

MRM3-based UHPLC-MS/MS method for quantitation of total florfenicol residue content in milk and withdrawal study profile of milk from treated cows.

Florfenicol is a broad spectrum antibacterial, licensed globally for treatment of animal and aquaculture diseases. In the EU, Canada and US it is not permitted for use in animals producing milk or eggs. There are no published methods for analysis of total florfenicol content in milk/milk products as these lack a hydrolysis step, failing to meet the marker residue definition. A method for determining total florfenicol content in milk that meets this definition is reported for the first time. Use of a UHPLC-MS/MS multiple reaction monitoring-cubed method improved the selective detection and quantitation of lower levels of florfenicol amine in milk compared to MRM only. Single laboratory validation data and withdrawal profile in bovine milk are presented. A withdrawal period of over 50 days is indicated in case of off-label use. Requirement for hydrolysis is demonstrated.

Screening method for the detection of residues of amphenicol antibiotics in bovine milk by optical biosensor.

An immunobiosensor assay was developed for multi-residue screening in bovine milk of the parent amphenicols, thiamphenicol and florfenicol, along with the metabolite florfenicol amine. A polyclonal antibody raised in a rabbit after immunisation with a florfenicol amine-protein conjugate was employed in the assay. Milk samples were subjected to acetonitrile extraction, reconstituted in buffer and diluted prior to biosensor analysis. Validation data obtained from the analysis of fortified samples has shown that the method has a detection capability of less than 0.25 µg kg-1 for florfenicol and less than 0.5 µg kg-1 for florfenicol amine and thiamphenicol. The cross-reactivity profile and validation data for the detection of these amphenicols is presented together with results obtained following the analysis of florfenicol incurred samples using the developed screening method along with a comparison of results obtained from the analysis of the same incurred samples using an MRM3 UPLC-MS/MS confirmatory method. Results are also presented obtained from the analysis of samples from both treated and non-treated animals which were co-housed and which show the potential for cross-contamination.

Detection of Tetrodotoxin Shellfish Poisoning (TSP) Toxins and Causative Factors in Bivalve Molluscs from the UK.

Tetrodotoxins (TTXs) are traditionally associated with the occurrence of tropical Pufferfish Poisoning. In recent years, however, TTXs have been identified in European bivalve mollusc shellfish, resulting in the need to assess prevalence and risk to shellfish consumers. Following the previous identification of TTXs in shellfish from southern England, this study was designed to assess the wider prevalence of TTXs in shellfish from around the coast of the UK. Samples were collected between 2014 and 2016 and subjected to analysis using HILIC-MS/MS. Results showed the continued presence of toxins in shellfish harvested along the coast of southern England, with the maximum concentration of total TTXs reaching 253 µg/kg. TTX accumulation was detected in Pacific oysters (Crassostreagigas), native oysters (Ostreaedulis) common mussels (Mytilusedulis) and hard clams (Mercenariamercenaria), but not found in cockles (Cerastodermaedule), razors (Ensis species) or scallops (Pectenmaximus). Whilst the highest concentrations were quantified in samples harvested during the warmer summer months, TTXs were still evident during the winter. An assessment of the potential causative factors did not reveal any links with the phytoplankton species Prorocentrumcordatum, instead highlighting a greater level of risk in areas of shallow, estuarine waters with temperatures above 15 °C.

Screening method for the detection of residues of amphenicol antibiotics in bovine, ovine and porcine kidney by optical biosensor.

An immunobiosensor assay was developed for the multi-residue screening of the parent amphenicols, thiamphenicol and florfenicol, along with the metabolite florfenicol amine, in bovine, ovine and porcine kidney. A polyclonal antiserum raised in a rabbit after inoculation with a florfenicol amine-protein conjugate was employed in the assay. Sample homogenates were extracted directly into acetonitrile, reconstituted in buffer and diluted prior to biosensor analysis. Validation data obtained from the analysis of fortified samples has shown that the method has a detection capability (CCß) of less than 25µgkg-1 (1/2 MRL) for thiamphenicol in the kidney of all three species, less than 150µgkg-1(1/2 MRL) for florfenicol and florfenicol amine and less than 250µgkg-1 (1/2 MRL) for florfenicol and florfenicol amine in bovine/ovine and porcine kidney respectively. Intra-assay variation (n=10) was calculated at 4.5% and 2.6% at concentrations of 10µgkg-1 and 150µgkg-1respectively for bovine kidney while inter-assay variation (n=3) was determined to be 5.0% and 16.5% respectively at the same concentrations. The cross-reactivity profile and validation data for the detection of these amphenicols is presented together with the results obtained following the analysis of florfenicol incurred samples using the developed method.

Evidence of non-extractable florfenicol residues: development and validation of a confirmatory method for total florfenicol content in kidney by UPLC-MS/MS.

The parent compound florfenicol (FF) is a broad-spectrum antibacterial compound licensed in the UK for use in cattle, pigs and the aquaculture industry. The analysis of porcine tissues in this study demonstrates that significant amounts of solvent non-extractable FF-related residues are present in incurred tissues (kidney and muscle) from treated animals. The results indicate that methods based on solvent extraction alone may carry a significant risk of reporting false-negative results. The use of a strong acid hydrolysis step prior to solvent extraction of tissue samples is necessary for an accurate estimate of the total tissue FF content. A robust and sensitive method for the determination of total FF residue content in kidney samples by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has been developed and validated. This method covers the synthetic amphenicol drug FF and its metabolites, measured as the marker residue florfenicol amine (FFA) as per Commission Regulation (EU) No. 37/2010. Non-extractable and intermediate metabolites are converted to the hydrolysis product FFA, and then partitioned into ethyl acetate. Extracts are solvent exchanged prior to a dispersive solid-phase extraction step, then analysed using an alkaline reverse-phase gradient separation by UPLC-MS/MS. The method was validated around the maximum residue levels (MRLs) set out in Regulation (EU) No. 37/2010 for bovine kidney in accordance with Commission Decision No. 2002/657/EC. The following method performance characteristics were assessed during a single laboratory validation study: selectivity, specificity, sensitivity, linearity, matrix effects, accuracy and precision (decision limit (CCα) and detection capability (CCß) were determined).

The occurrence of semicarbazide in the meat and shell of Bangladeshi fresh-water shrimp.

There is evidence that semicarbazide (SEM), a marker for the banned nitrofuran nitrofurazone, can arise from other, unrelated sources. Recently, Belgium rejected 54 consignments of Bangladeshi freshwater shrimp (Macrobrachium rosenbergii), following a laboratory decision to test meat and exoskeleton combined. To study the possible natural occurrence of SEM in wild shrimp, samples were collected and analysed from 29 sites across Bangladesh. SEM (<1.0 µg/kg) was detected in ∼65% of meat samples. However, SEM concentrations were approximately 100 times higher in the exoskeleton, and were unrelated to sampling location, strongly suggesting natural occurrence. In meat, most SEM was surface-associated. When the shrimp was shelled, some of the epidermal layer (which synthesises new exoskeleton) remained with the shell and some remained with the meat--leading to differing levels of natural SEM on the shrimp surface. This has implications for the use of SEM and the analytical strategy used to control nitrofuran use.