Validation of a liquid chromatography-electrospray ionization tandem mass spectrometric method to determine six polyether ionophores in raw, UHT, pasteurized and powdered milk.

This study aimed to validate a method developed for the determination of six antibiotics from the polyether ionophore class (lasalocid, maduramicin, monensin, narasin, salinomycin and semduramicin) at residue levels in raw, UHT, pasteurized and powdered milk using QuEChERS extraction and high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). The validation was conducted under an in-house laboratory protocol that is primarily based on 2002/657/EC Decision, but takes in account the variability of matrix sources. Overall recoveries between 93% and 113% with relative standard deviations up to 16% were obtained under intermediate precision conditions. CCα calculated values did not exceed 20% the Maximum Residue Limit for monensin and 25% the Maximum Levels for all other substances. The method showed to be simple, fast and suitable for verifying the compliance of raw and processed milk samples regarding the limits recommended by Codex Alimentarius and those adopted in European Community for polyether ionophores.

Innovative mixture of salts in the quick, easy, cheap, effective, rugged, and safe method for the extraction of residual macrolides in milk followed by analysis with liquid chromatography and tandem mass spectrometry.

A simple extraction technique has been developed for seven macrolide antibiotics in milk. The procedure involves a modified quick, easy, cheap, effective, rugged, and safe method based on acetonitrile extraction, followed by the addition of a mixture of salts (sodium sulfate, sodium chloride, and potassium carbonate) not yet reported in literature. The method was validated for tylosin and was selective, free of matrix effect, and linear in the range of 0.78-18.75 ng/mL in the final extract, corresponding to 0.125-3 times the maximum residue limit. The limit of detection, limit of quantification, decision limit, and detection capability were, respectively, 0.84, 2.79, 58.4, and 71.7 µg/kg. The overall average recovery at 25, 50, and 75 µg/kg ranged from 89-97%. Repeatability and intermediate precision expressed by relative standard deviations were below 10.5 and 12%, respectively. The extension of the validation for spiramycin, throleandomycin, oleandomycin, roxithromycin, erythromycin, and clarithromycin is under consideration since the procedure proved to be able to efficiently extract all studied macrolides, with recoveries from 74-104% at 50 µg/kg for tylosin, erythromycin, spiramycin, and oleandomycin and 20 µg/kg for throleandomycin, roxithromycin, and clarithromycin.

Simultaneous determination of polyether ionophores, macrolides and lincosamides in hen eggs by liquid chromatography-electrospray ionization tandem mass spectrometry using a simple solvent extraction.

A liquid chromatography-electrospray ionization tandem mass spectrometric (LC-ESI-MS/MS) method was developed and validated for the determination of residues of 6 polyether ionophores (lasalocid, maduramicin, monensin, narasin, salinomycin, semduramicin), 3 macrolides (erythromycin, tylosin, clarithromycin) and 1 lincosamide (lincomycin) in eggs. Nigericin was used as qualitative internal standard. Samples were deproteinizated/extracted with acetonitrile without pH adjustments. Aliquots of the extracts were evaporated and reconstituted for injection in the instrument operated in positive multiple reaction monitoring (MRM) mode. The stability of the antibiotics and the intensity of the formed ions were considered in order to select a suitable solvent for the reconstitution of the obtained dry extracts. No clean-up steps were required and matrix effects were controlled by sample dilution, selection of appropriate chromatographic conditions and reduced injection volume. Good within-laboratory reproducibility was obtained, with relative standard deviations (RSD(R)) from 4.0 (semduramicin at 5 µgkg(-1)) to 18.6 (erythromycin at 25 µgkg(-1)) for the ionophores and macrolides. Lincomycin showed the least precise results, with a maximum RSD(R) of 20.2% at 75 µgkg(-1)). Satisfactory decision limits (CCα) and detection capabilities (CCß) were also attained. Method limits of detection (LODs) from 0.04 (salinomycin) to 1.6 µgkg(-1) (lincomycin) were achieved. Method limits of quantification (LOQs) were from 0.14 to 5.3 µgkg(-1) for the same drugs, respectively. All the LOQs, except that obtained for maduramicin were remarkably below the lowest validation level. The proposed method is suitable for routine application in commercial egg samples.

Pilot survey of hen eggs consumed in the metropolitan area of Rio de Janeiro, Brazil, for polyether ionophores, macrolides and lincosamides residues.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which has recently been developed and validated, was used for the identification and quantification of polyether ionophore, macrolide and lincosamide residues in commercial eggs sold in the metropolitan area of Rio de Janeiro, Brazil. The method was applied to 100 samples and the results showed a high incidence of polyether ionophore residues (25%). Salinomycin was detected in 21% of samples, but only two non-compliant results (5.3 and 53 µg kg(-1)) were found if maximum limits (tolerances) established by European Union were adopted in Brazil and if a method decision limit (CCα) of 3.4 µg kg(-1) was considered. In 8% of analyzed samples, more than one studied coccidiostat was found. The lincosamide, lincomycin, and the macrolide, tylosin, were detected at trace levels in 4 and 1% of the samples, respectively. Lasalocid, clarithromycin and erythromycin were not found.

Pilot survey of commercial pasteurized milk consumed in the metropolitan area of Rio de Janeiro, Brazil, for tetracyclines residues, including the 4-epimers of oxytetracycline, tetracycline and chlortetracycline.

This pilot survey aimed to assess the occurrence of tetracyclines and the 4-epimers of oxytetracycline, tetracycline and chlortetracycline in commercial pasteurized milks sold in the metropolitan area of Rio de Janeiro, Brazil, between October 2009 and March 2010. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, developed and validated in our laboratory, was used. All 100 analyzed samples were compliant, but 14 contained oxytetracycline in concentrations ranging from the method limit of detection (3.7 µg l(-1)) to the method limit of quantification (12.2 µg l(-1)). One sample contained oxytetracycline and tetracycline simultaneously (at a concentration slightly higher than method limit of quantification, 7.0 µg l(-1)). The presence of 4-epioxytetracycline and 4-epitetracycline in contaminated samples with the parent drugs could not be confirmed as traces were detected only in the quantification MRM transition. No other tetracyclines were detected.

A liquid chromatography-tandem mass spectrometry confirmatory assay for the simultaneous determination of several tetracyclines in milk considering keto-enol tautomerism and epimerization phenomena.

A liquid chromatography-electrospray ionization tandem mass spectrometric (LC-ESI-MS/MS) method for the analysis of several tetracyclines residues in bovine milk has been developed. Milk deproteinization/extraction of samples was performed with acidified acetonitrile. After diluting and purification by solid-phase extraction (SPE), the extracts were injected into the instrument operated in Multiple Reaction Monitoring (MRM) acquisition mode. The reversible epimerization at C-4 of oxytetracycline, tetracycline and chlortetracycline and the keto-enol tautomerism of chlortetracycline between C-11a and C-12 were considered for reliable quantification. Degradation was also taken in account and minimized for the same purpose. A central composite (response surface) design with desirability function was employed for the optimization of extraction and clean-up steps. The optimization improved the extraction efficiency of the more polar analytes reaching 93.9% for 4-epioxytetracycline and 95.8% for oxytetracycline at 100 microg L(-1). The validation was performed following the criteria established by Commission Decision 2002/657/EC.