Optimisation and validation of a quantitative and confirmatory method for residues of macrolide antibiotics and lincomycin in kidney by liquid chromatography coupled to mass spectrometry.

A solid phase extraction followed by a liquid chromatography (LC)-tandem mass spectrometry (MS/MS) detection method for the confirmatory analysis of lincomycin (LIN), clindamycin (CLI), tilmicosin (TIM), erythromycin (ERI) and tylosin (TYL) residues in kidney were optimised and validated for monitoring and controlling the use of these antibiotics in food producing-animals. The method optimisation was carried out by testing changes in the extraction buffer pH and in the ammonium/acetonitrile concentrations on SPE eluent solutions. The optimised extraction procedure involved the extraction of the analytes with a pH 8 phosphate buffer, clean-up on a reversed-phase mixed-cation exchange cartridge, followed by the elution of the analytes in a 98:2 acetonitrile/ammonia solution, concentration in air flow and re-dissolved with an 1:1 methanol/water solution. The analytes were detected in an LC-MS/MS system in electrospray positive ionisation mode. The validation was performed according to the European Commission Decision 2002/657/EC. Linearity was established for all analytes using the method of least weighted squares and CCα values ranged from 5.3% to 21.1% higher than the minimum residue limit (MRL) values. The addition levels varied from 0.5 to 1.50 MRL for all analytes, with recoveries exceeding 92.5%. The relative standard deviations (RSD%) in terms of repeatability (n = 54) and reproducibility (n = 108) for all analytes were less than 21.6% and 21.4%, respectively. The uncertainties were calculated by simplified methods using the calibration curve uncertainty and the intermediate precision to obtain the combined measurement uncertainty. The results of the validation process demonstrated that this method is suitable for the quantification and confirmation of antibiotic residues for the Brazilian Residue and Contaminant Control Plan (PNCR).

Occurrence of antimicrobial residues in Brazilian food animals in 2008 and 2009.

Brazil is one of the most important countries as a producer and exporter of cattle and poultry. In 2009 cattle accounted for 30% of the export market and 41.4% for poultry meat. The Brazilian National Residues and Contaminants Control Plan (PNCRC) follows the guidelines set by the Codex Alimentarius Commission and checks compliance maximum residue limits (MRLs) to ensure the quality of these commodities. Kidney samples (n = 2978) were analysed between January 2008 and December 2009. Fifteen antibiotics of the macrolide and aminoglycoside groups (clindamycin, eritromycin, lincomycin, tylmicosin, tylosin, amikacin, apramycin, dihydrostreptomycin, gentamycin, higromycin, kanamycin, neomycin, spectinomycin, streptomycin, tobramycin) were determined by a microbiological screening method (FAST) and confirmed/quantified using liquid chromatography (LC-MS/MS and UPLC-MS/MS). In 2008, 1459 samples were analysed by a screening test and liquid chromatography with only one sample (0.07%) exceeded Brazilian legislation limits (>MRL). In 2009, 1519 samples were analysed and none exceeding Brazilian legislation limits (>MRL). The slaughterhouses of 16 states were monitored during the year of 2008, and 18 states were monitored in 2009, being the major producing states most sampled by the PNCRC.

Optimisation and validation of a quantitative and confirmatory LC-MS method for multi-residue analyses of ß-lactam and tetracycline antibiotics in bovine muscle.

A multi-residue method for the determination of the ß-lactam antibiotics ampicillin, cefazolin, cloxacillin, dicloxacillin, nafcillin, oxacillin, penicillin G, penicillin V and the tetracyclines chlotetracycline, tetracycline and oxytetracycline was optimised and validated in bovine muscle. The method is based on the extraction of the residues from muscle using water/acetonitrile (2/8, v/v) with subsequent use of dispersive solid-phase C18 and hexane for purification. Extracts were analysed using ultra-performance liquid chromatography (UPLC-MS/MS) coupled with the mass spectrometer in positive electrospray ionisation mode (ESI+) for all analytes. The method was validated according to the requirements of European Commission Decision 2002/657/EC. The validation results were obtained within the MRL range of 0-1.5 of the MRL, with recoveries varying from 90% to 110% and CV < 20% (n = 54), except for cloxacillin, dicloxacillin and nafcillin. However, matrix interference was observed. The decision limit (CCα) ranged from 10% to 15% of the MRL. The uncertainty measurement was estimated based on both bottom-up and top-down strategies and the uncertainty values were found to be lower than 20% of the MRL. The method has a simple extraction procedure whereby analytes are separated with reasonable resolutions in a single 11-min chromatographic run. According to the validation results, this method is suitable for monitoring ß-lactams and tetracyclines according to National Program for Residue and Contaminant Control - Brazil (NPRC-Brazil) in bovine muscle.

Validation of a quantitative and confirmatory method for residue analysis of aminoglycoside antibiotics in poultry, bovine, equine and swine kidney through liquid chromatography-tandem mass spectrometry.

The use of aminoglycoside antibiotics in food animals is approved in Brazil. Accordingly, Brazilian food safety legislation sets maximum levels for these drugs in tissues from these animals in an effort to guarantee that food safety is not compromised. Aiming to monitor the levels of these drugs in tissues from food animals, the validation of a quantitative, confirmatory method for the detection of residues of 10 aminoglycosides antibiotics in poultry, swine, equine and bovine kidney, with extraction using a solid phase and detection and quantification by LC-MS/MS was performed. The procedure is an adaptation of the US Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) qualitative method, with the inclusion of additional clean-up and quantification at lower levels, which proved more efficient. Extraction was performed using a phosphate buffer containing trifluoroacetic acid followed by neutralization, purification on a cationic exchange SPE cartridge, with elution with methanol/acetic acid, evaporation, and dilution in ion-pair solvent. The method was validated according to the criteria and requirements of the European Commission Decision 2002/657/EC, showing selectivity with no matrix interference. Linearity was established for all analytes using the method of weighted minimum squares. CCα and CCß varied between 1036 and 12,293 µg kg(-1), and between 1073 and 14,588 µg kg(-1), respectively. The limits of quantification varied between 27 and 688 µg kg(-1). The values of recovery for all analytes in poultry kidney, fortified in the range of 500-1500 µg kg(-1), were higher than 90%, and the relative standard deviations were lower than 15%, except spectinomycin (21.8%). Uncertainty was estimated using a simplified methodology of 'bottom-up' and 'top-down' strategies. The results showed that this method is effective for the quantification and confirmation of aminoglycoside residues and could be used by the Brazilian programme of residue control.