Multiclass method for antimicrobial analysis in animal feeds by liquid chromatography-tandem mass spectrometry.

A rapid multiclass method that covers 50 antimicrobials from 13 different families in animal feeds was developed. Samples were extracted using a mixture of methanol, acetonitrile and a McIlvaine buffer combined with sonication. Feed extracts were simply diluted prior to injection, since the clean-up strategies that were tested, based on either solid-phase extraction or dispersive solid-phase extraction, were ineffective at minimizing matrix-related signal suppression/enhancement. Analysis was carried out by liquid chromatography coupled to tandem mass spectrometry using an electrospray ionization source operating in positive and negative modes. For the quantification, matrix-fortified standard calibration curves were used to compensate for matrix effects and losses in sample preparation. The method was validated in-house in pig, poultry and cattle feed matrices and showed satisfactory performance characteristics. Thus, the proposed approach was suitable for application in a routine high-throughput laboratory for the official control of feeds.

Analysis of quinolone antibiotics in eggs: preparation and characterization of a raw material for method validation and quality control.

A quality control material for the analysis of quinolone residues in egg samples has been prepared. Homogenized fresh whole egg spiked with nine quinolones (marbofloxacin, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid and flumequine), at the concentration level of 500 µg kg(-1) was lyophilized and homogenized to obtain the reference material. The homogeneity of both the bulk and the packed material was verified. Two different strategies, classical and isochronous, were used for the stability study. Conclusions obtained with the classical and isochronous approaches were similar, but the variability of the isochronous results was lower and this led to lower material uncertainty. The reference material was found to be stable for at least 1 year when stored at either room temperature, 4 °C or -20 °C; -80 °C was taken as reference temperature in all cases. The determination of quinolones in eggs was carried out by a previously developed and validated method making use of a pressurized liquid extraction followed by liquid chromatography with fluorescence detection. The suitability of the material prepared was demonstrated by using a lyophilized egg material spiked with nine quinolones as sample in a proficiency test.

Validation of a method for the analysis of nine quinolones in eggs by pressurized liquid extraction and liquid chromatography with fluorescence detection.

A multiresidue method for the analysis, in egg matrices, of residues of nine quinolones used in veterinary medicine, has been developed and validated according to the provisions of Council Decision 2002/657. Compounds were extracted by a pressurized liquid extraction (PLE) technique using a 1:1 mixture of acetonitrile and a phosphoric acid buffer (pH 3.0) at 70°C. The obtained extract was clear enough, so that no further clean-up was necessary. Analytes were determined by liquid chromatography (LC) with fluorescence detection (FL). Two chromatographic columns were compared: a high-purity silica Inertsil C(8) column and a newly developed Kinetex C(18) core-shell technology column. Validation was carried out at four concentration levels, using both chromatographic columns. Precision in terms of reproducibility standard deviation was between 7% and 23%, and good recoveries were obtained. Decision limit (CCα) and detection capability (CCß) values obtained with the Inertsil and Kinetex columns were in the 0.2-19.8 µg kg(-1) and 0.4-33.5 µg kg(-1) concentration ranges, respectively. The proposed method allows residues of quinolones banned for use with laying hens to be detected and quantified in eggs. About twenty-four samples per day can be processed.

Development and validation of a multiclass method for the analysis of antibiotic residues in eggs by liquid chromatography-tandem mass spectrometry.

A multiclass method for the analysis of residues, in egg matrices, of 41 antimicrobial agents belonging to seven families (sulfonamides, diaminopyridine derivates, quinolones, tetracyclines, macrolides, penicillins and lincosamides) was developed and validated according to the requirements of European Commission Decision 2002/657. Compounds were extracted with a pressurized liquid extraction (PLE) technique using a 1:1 mixture of acetonitrile and a succinic acid buffer (pH 6.0) at 70 °C. As this resulted in clear extracts, no further clean-up was necessary. Analytes were determined by ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPL-MS/MS) in a chromatographic run of 13 min. Calibration was carried out with spiked blank samples subjected to the entire analytical procedure. Five compounds, two of them isotopically labelled, were used as internal standards. Most analytes were quantified with errors below 10%. Precision in terms of reproducibility standard deviation was between 10% and 20% in most cases. CCα values were in the range 0.5-3.8 µg kg⁻¹ for the non-authorized compounds. The proposed method would enable an experienced analyst to process about 25 samples per day.

Preparation of quality control materials for the determination of sulfonamides in animal feed.

A feasibility study of the preparation of quality control materials for the analysis of medicated feeds has been carried out. Two analytical methodologies for the analysis of sulfonamides in feeds were developed, validated and applied to homogeneity and stability studies. Pig feeds spiked with sulfadiazine and sulfadimidine were prepared. The drugs were spiked at 500 microg g(-1), representing what can be expected in a commercial medicated feed, and at 2 and 5 microg g(-1), which roughly correspond to drug-free feeds cross-contaminated during the fabrication process. The homogeneity of both the bulk and the bottled materials was verified. A stability study of the materials containing 2 and 5 microg g(-1) of sulfonamides was carried out over an 18-month period at room temperature, at 4 degrees C and -20 degrees C. The determination of sulfadiazine and sulfadimidine in samples coming from these homogeneity and stability studies of the quality control materials was carried out by high-performance liquid chromatography (HPLC) with either ultraviolet light or fluorimetric detection, depending on the concentration of the analytes in the samples.

Antibiotics in food: legislation and validation of analytical methodologies.

In this paper, European food safety legislation is presented, and special attention is devoted to monitoring residues of veterinary drugs in foodstuffs of animal origin. After a short review of the state of the art of analytical methodology for antibiotic residue analysis, the paper focuses on validation of analytical methods, with Decision 657/2002/EC as reference document. Finally, the main issues of the quality control of the analytical data, i.e. analysis of reference materials and participation in proficiency tests, are briefly addressed.

Determination of pK(a) values of some hydroxylated benzoic acids in methanol-water binary mixtures by LC methodology and potentiometry.

An accurate estimation of pK(a) values in methanol-water binary mixtures is very important for several separation techniques such as liquid chromatography and capillary electrophoresis that use these solvent mixtures. In this study, the pK(a) values of 11 polyphenolic acids have been determined in methanol-water binary mixtures (10%, 20% and 30% (v/v)) by potentiometry, liquid chromatography (LC) and LC-DAD methodology. The results show a similar trend for the pK(a) values of all the studied compounds, as they increase with increasing concentration of organic modifier, which allows a linear relationship between pK(a) values and mole fraction of methanol to be obtained. The pK(a) values obtained in aqueous medium have been compared with those given in the literature, and also with the values predicted by the SPARC on-line pK(a) calculator. The data obtained have been used to test the feasibility of an estimation of dissociation constants in a methanol-water medium from the relationship between pK(a) values and the organic cosolvent fraction in the mixtures.

Multivariate calibration applied to synchronous fluorescence spectrometry. Simultaneous determination of polycyclic aromatic hydrocarbons in water samples.

Synchronous fluorescence spectra of mixtures containing ten polycyclic aromatic hydrocarbons (anthracene, benz[a]anthracene, benzo[a]pyrene, chrysene, fluoranthene, fluorene, naphthalene, perylene, phenanthrene and pyrene) have been used for the determination of these compounds by Partial Least Squares Regression (PLSR), using both PLS-1 and PLS-2. Different procedures have been used for the pretreatment of the data in order to obtain better models, and the size of the calibration matrix has also been studied. The best models have been used for the determination of the above mentioned PAHs in spiked natural water samples at concentration levels between 4 and 20 ng ml(-1). Recoveries ranged from 80 to 120% in most cases, although fluorene gave significantly lower results.

Three-way multivariate calibration procedures applied to high-performance liquid chromatography coupled with fast-scanning fluorescence spectrometry detection. Determination of polycyclic aromatic hydrocarbons in water samples.

Three-way partial least-squares and n factor parallel factor analysis have been compared for the analysis of polycyclic aromatic hydrocarbons in water samples. Data were obtained with a chromatographic system set to record short-time chromatograms containing several unresolved peaks. The detection system consisted of a fast-scanning fluorescence spectra detector, which allows one to obtain three-dimensional data, where retention time, emission wavelengths, and fluorescence intensity are represented. The combined use of a multivariate calibration method and the three-dimensional data obtained from the HPLC-FSFS system allows resolution of closely eluting compounds, thus making a complete separation unnecessary. The procedure has been applied to tap water samples (spiked at 0.10 and 0.20 µg L(-)(1) levels) with good results, similar to those obtained with a HPLC system with a conventional fluorescence detector.

Spectrofluorimetric determination of zinc in foods with 8-(p-toluenesulphonamido)quinoline in micellar medium.

A new method for the spectrofluorimetric determination of zinc with 8-(p-toluene- sulphonamido)quinoline in a Brij-35 micellar medium has been developed. This method allows the determination of 8-550 ng/ml Zn(II) and is relatively free from interferences. It has been applied to food samples with satisfactory results.

Spectrofluorimetric method for the simultaneous determination of zinc and cadmium.

The simultaneous determination of zinc and cadmium using derivative synchronous and multiwavelength synchronous spectrofluorimetry is described. Effects of experimental variables on the fluorescence intensities and on the spectral resolution of the fluorescent chelates of these metals with 8-(p-toluenesulfonamido)-quinoline (TSQ) in a sodium lauryl sulfate micellar medium have been studied. Mixtures containing 10-200 µg L(-1) Zn and 15-250 µg L(-1) Cd have been analyzed with good results. The proposed method has been applied to a biological sample.