Development and applicability of a multi-residue method for dyes, including new residue markers, to detect drug misuse in aquaculture.

A qualitative and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the sensitive and exhaustive analysis of residues from triarylmethane dyes, triarylmethane-derivative dyes, phenothiazines, phenoxazines and xanthenes in aquaculture samples. For a wider and more robust detection of dye misuse on farms, other residue markers were also included the leuco forms of brilliant green, crystal violet and malachite green; one direct metabolite of Victoria pure blue BO and methylene blue and three bile acids, which are endogenous markers of the effects of dye contamination in fish. We optimised the extraction method by comparing several extraction solvents and sample solvents reported in the literature to have the best extraction efficiency. The residues were determined using a positive electrospray ionisation source. We assessed the parameters of this LC-MS/MS method by evaluating the matrix effects, identification and quantitative parameters according to the criteria stipulated in the European Commission Decision No. 2002/657/EC. A study on the applicability of the method was conducted on various aquaculture species and on a positive catfish.

Validation approach for a fast and simple targeted screening method for 75 antibiotics in meat and aquaculture products using LC-MS/MS.

An approach is described to validate a fast and simple targeted screening method for antibiotic analysis in meat and aquaculture products by LC-MS/MS. The strategy of validation was applied for a panel of 75 antibiotics belonging to different families, i.e., penicillins, cephalosporins, sulfonamides, macrolides, quinolones and phenicols. The samples were extracted once with acetonitrile, concentrated by evaporation and injected into the LC-MS/MS system. The approach chosen for the validation was based on the Community Reference Laboratory (CRL) guidelines for the validation of screening qualitative methods. The aim of the validation was to prove sufficient sensitivity of the method to detect all the targeted antibiotics at the level of interest, generally the maximum residue limit (MRL). A robustness study was also performed to test the influence of different factors. The validation showed that the method is valid to detect and identify 73 antibiotics of the 75 antibiotics studied in meat and aquaculture products at the validation levels.

The Monitoring of Triphenylmethane Dyes in Aquaculture Products Through the European Union Network of Official Control Laboratories.

Aquaculture has been the fastest growing animal production industry for the past four decades, and almost half of the fish eaten in the world are now farmed fish. To prevent diseases in this more intensive aquaculture farming, use of therapeutic chemicals has become a basic choice. The monitoring of malachite green, a triphenylmethane dye and one of the oldest and widely used chemicals in fish production, has gained more interest since the mid 1990s when this substance was finally proven to be toxic enough to be prohibited in seafood products destined for human consumption. The enforcement of the European Union (EU) regulation of this banned substance along with some other triphenylmethane dye congeners and their metabolites in its domestic production and in seafood imports was undertaken through the National Residue Monitoring Plans implemented in nearly all of the 28 EU member states. The reliability of the overall European monitoring of this dye contamination in aquaculture products was assessed by using the results of proficiency testing (PT) studies provided by the EU Reference Laboratory (EU-RL) in charge of the network of the EU National Reference Laboratories (NRLs). The proficiency of each NRL providing analytical support services for regulating dye residues was carefully checked during three PT rounds. In the process, the analytical methods developed and validated for this purpose have gradually been improved and extended over the last two decades.

Development and validation of a confirmatory method for the determination of 12 non steroidal anti-inflammatory drugs in milk using liquid chromatography-tandem mass spectrometry.

A rapid and reliable LC-MS/MS method for the simultaneous confirmation of twelve non steroidal anti-inflammatory drugs (NSAIDs) in bovine milk was developed and fully validated in accordance with the European Commission Decision 2002/657/EC. The validation scheme was built in accordance with the MRLs or target analytical levels (EU-CRL recommended concentrations and detection capabilities) of the analytes, except for diclofenac for which the lower level of validation achieved was 0.5 µg kg(-1) whereas its MRL is 0.1 µg kg(-1). The NSAIDs investigated were as follows: phenylbutazone (PBZ), oxyphenylbutazone (OPB), naproxen (NP), mefenamic acid (MF), vedaprofen (VDP), flunixin (FLU), 5-hydroxyflunixin (FLU-OH), tolfenamic acid (TLF), meloxicam (MLX), diclofenac (DC), carprofen (CPF) and ketoprofen (KTP). Several extraction procedures had been investigated during the development phase. Finally, the best results were obtained with a procedure using only methanol as the extraction solvent, with an evaporation step included and no further purification. Chromatographic separation was achieved on a C18 analytical column and the run was split in 2 segments. Matrix effects were also investigated. Data acquisition implemented for the confirmatory purpose was performed by monitoring 2 MRM transitions per analyte under the negative electrospray mode. Mean relative recoveries ranged from 94.7% to 110.0%, with their coefficients of variation lying between 2.9% and 14.7%. Analytical limits expressed in terms of decision limits (CCα) were evaluated between 0.69 µg kg(-1) (FLU) and 27.54 µg kg(-1) (VDP) for non-MRL compounds, and at 0.10 (DC), 15.37 (MLX), 45.08 (FLU-OH), and 62.96 µg kg(-1) (TLF) for MRL compounds. The validation results proved that the method is suitable for the screening and confirmatory steps as implemented for the French monitoring plan for NSAID residue control in bovine milk.

Validation of a multi-residue liquid chromatography-tandem mass spectrometry confirmatory method for 10 anticoccidials in eggs according to Commission Decision 2002/657/EC.

A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the simultaneous detection and confirmation of halofuginone, robenidine, diclazuril, nicarbazin, monensin, narasin, lasalocid, salinomycin, maduramicin and semduramicin in whole egg has been developed and validated. The anticoccidial residues were extracted by acetonitrile, evaporated and dissolved in a sodium acetate/acetonitrile mixture. Then, the samples were injected on a C8 column in a gradient mode. Diclazuril-bis, DNC-d8 and nigericin were used as internal standards. The results of the full validation in accordance with the guidelines of the Commission Decision no 2002/657/EC are presented. This rapid and sensitive method was found suitable to confirm the anticoccidials at 1 and at 75 microg kg(-1) for the MRL compound lasalocid.