Development and validation of a liquid chromatography-UV detection method for the determination of sulfonamides in fish muscle and shrimp according to European Union Decision 2002/657/EC.

Sulfonamides are one class of antimicrobial agents used in aquaculture production. Sulfonamides are often overused because they are inexpensive and readily available. Their presence at a concentration above the legal limits is a potential hazard to human health. Brazilian authorities have included in the National Regulatory Monitoring Program the control of the three most widely used sulfonamides in aquaculture production, i.e., sulfathiazole, sulfamethazine, and sulfadimethoxine. An LC method with UV detection for the determination of residual sulfonamides in fish muscle, using sulfapyridine as an internal standard has been developed and validated. The validation was performed according to the Brazilian Regulation 24/2009 (equivalent to European Union Decision 2002/657/EC). The method meets the Brazilian regulatory requirement that establishes criteria and procedures for determination of parameters such as decision limit (CCalpha), detection capability (CCbeta), precision, and recovery. For fish muscle, CCalpha was determined at 3.63, 2.91, and 7.46 microg/kg for sulfathiazole, sulfamethazine, and sulfadimethoxine, respectively. CCbeta was 9.39, 14.54, and 9.39 microg/kg for sulfathiazole, sulfamethazine, and sulfadimethoxine, respectively. For shrimp, CCalpha was 11.5, 8.67, and 4.46 microg/kg for sulfathiazole, sulfamethazine, and sulfadimethoxine, respectively. CCbeta was 18, 11.93, and 5.24 microg/kg for sulfathiazole, sulfamethazine, and sulfadimethoxine, respectively. A complete statistical analysis was performed on the results obtained. The results indicate that the method is robust when subjected to day-to-day analytical variations.

Liquid chromatography/tandem mass spectrometry method to determine boldenone in bovine liver tissues.

Boldenone, an androgenic steroid, is forbidden for use in meat production in most countries worldwide. Residues of this drug in food present a potential risk to consumers. A sensitive LC/MS/MS method for analysis of 17ß-boldenone using boldenone-d3 as an internal standard was developed. An enzymatic hydrolysis and extraction using ethyl acetate, methanol, and hexane were performed in the sample preparation. Parameters such as decision limit (CCα), detection capability (CCß), precision, recovery, and ruggedness were evaluated according to the Brazilian Regulation 24/2009 (equivalent to European Union Decision 2002/657/EC) and International Organization for Standardization/International Electrotechnical Commission 17025:2005. CCα and CCß were determined to be 0.17 and 0.29 µg/kg, respectively. Average recoveries from bovine liver samples fortified with 1, 1.5, and 2 µg/kg were around 100%. A complete statistical analysis was performed on the results obtained, including an estimation of the method uncertainty. The method is considered robust after being subjected to day-to-day analytical variations and has been used as a standard method in Brazil to report boldenone levels in bovine liver.

Determination and confirmation of metronidazole, dimetridazole, ronidazole and their metabolites in bovine muscle by LC-MS/MS.

Nitroimidazoles are a class of veterinary drugs used for the treatment and prevention of certain bacterial and protozoal diseases in poultry, swine dysentery and genital trichomoniasis in cattle. Since the safety assessment of nitroimidazoles showed them to be genotoxic, carcinogenic and mutagenic, a number of nitroimidazoles have been banned for therapeutic purposes in different countries. Moreover, nitroimidazoles have also been extensively used as growth promoters in food-producing animals. Due to their efficacious improvement in meat production and feed conversion, deliberate use still exists. Therefore, the illegal use of nitroimidazoles in animal husbandry must be monitored. A sensitive method based on LC-MS/MS for the simultaneous determination and confirmation of five banned nitroimidazole drugs including metronidazole, ronidazole, dimetridazole, metronidazole-OH (metabolite of metronidazole), and 2-hydroxymethyl-1-methyl-5-nitroimidazole (metabolite of ronidazole and dimetridazole) in bovine muscle, using ronidazole-d3 as an internal standard, was developed and validated. After extraction with ethyl acetate and evaporation, the nitroimidazoles were reconstituted in petroleum ether and purified, and LC-MS/MS analysis was performed. The method was validated according to Brazilian Regulation 24/2009 (equivalent to European Union Decision 2002/657/EC). Parameters such as decision limit (CCα), detection capability (CCß), precision, accuracy, uncertaincy and ruggedness were determined. Average accuracy of the five nitroimidazoles from bovine muscle fortified at 5 levels (0.5, 1.0, 1.5, 2.0 and 2.5 µg kg(-1)) ranged from 96% to 103%. The calculated CCα ranged from 0.0 to 0.17 µg kg(-1); CCß ranged from 0.08 to 0.41 µg kg(-1). A complete statistical analysis was performed and the results indicate that the method is robust when subjected to day-to-day analytical variations.

Monitoring of florfenicol residues in fish muscle by HPLC-UV with confirmation of suspect results by LC-MS/MS.

Florfenicol, a derivative of thiamphenicol in which the hydroxyl group at C-3 has been replaced with fluorine, is listed by the World Health Organization as an antibacterial agent for human medicine that is critically important in risk management strategies for non-human use. AOAC International has also identified it as an important molecule for the development of effective methods for the seafood sector. Following inspection missions from the European Union and United States of America, it was introduced in the Brazilian Residues Control Program to fulfill export and internal national requirements with a Maximum Residue Limit of 800 µg/kg. A high performance liquid chromatography method with ultraviolet detection at a wavelength of 230 nm (λ = 230 nm) for the detection of florfenicol in fish muscle was developed and validated according to the Brazilian Regulation 24/2009 (equivalent to European Union Decision 2002/657/EC). Fish samples were extracted with ethyl acetate and hexane followed by C18 solid phase clean-up and chromatographic separation on a reversed-phase C18 LC column with acetonitrile:water as mobile phase. The method results were also was compared with those obtained using liquid chromatography-tandem quadrupole mass spectrometry. The method meets the Brazilian regulatory requirements with a decision limit (CCα) of 840 µg/kg and detection capability (CCß) of 879 µg/kg. This method is easy to use and has been implemented into Brazil's residue control program, with liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmation of any suspect samples using the same method.