Determination of avermectins: a QuEChERS approach to the analysis of food samples.

We present a simple method for extracting avermectines from meat, based on a QuEChERS approach followed by liquid chromatography (LC) coupled to triple quadrupole (QqQ) tandem mass spectrometry (MS/MS). The compounds considered are ivermectin, abamectin, emamectin, eprinomectin, doramectin and moxidectin. The new method has been fully validated according to the requirements of European Decision 657/2002/CE (EU, 2002). The method is suitable for the analysis of avermectins at concentration as low as 2.5 µg kg(-1), and allows high sample throughput. In addition, the detection of avermectins by high resolution mass spectrometry using a quadrupole-Orbritrap (Q-Orbitrap) hybrid instrument has been explored, and the target Selected Ion Monitoring data dependent MS/MS (t-SIM-dd MS/MS) mode has been found to provide excellent performance for residue determination of target analytes.

High-throughput method for the determination of nitroimidazoles in muscle samples by liquid chromatography coupled to mass spectrometry.

We present a high-throughput confirmatory method for the analysis of 11 nitroimidazoles in muscle samples (metronidazole, MNZ; dimetridazole, DMZ; ronidazole, RNZ; tinidazole, TNZ; carnidazole, CRZ; secnidazole semihydrate, SCZ; ornidazole, ORZ; metronidazole-hydroxy, MNZ-OH; ipronidazole, IPZ; ipronidazole-hydroxy, IPZ-OH; and dimetridazole-hydroxy, HMMNI). Extraction with acetonitrile is efficient and simple, with the majority of recoveries ranging between 65 and 101%, and without the need for clean-up of the extracts. The chromatographic analysis is performed using UPLC-QqQ-MS in the MRM mode, with an electrospray source operated in positive mode. Total chromatographic analysis time is 12 min. This method has been fully validated in muscle according to Decision 657/2002/CE guidelines, and the CCα values obtained were in the range of 0.04-0.4 µg·kg(-1). The method is currently used for the analysis of muscle samples and has been tested in other matrices of animal origin, such as kidney, retina and egg, with excellent results.

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.

Correlation of precursor and product ions in single-stage high resolution mass spectrometry. A tool for detecting diagnostic ions and improving the precursor elemental composition elucidation.

Monitoring of common diagnostic fragments is essential for recognizing molecules which are members of a particular compound class. Up to now, unit resolving tandem quadrupole mass spectrometers, operating in the precursor ion scan mode, have been typically used to perform such analysis. By means of high-resolution mass spectrometry (HRMS) a much more sensitive and selective detection can be achieved. However, using a single-stage HRMS instrument, there is no unequivocal link to the corresponding precursor ion, since such instrumentation does not permit a previous precursor selection. Thus, to address this limitation, an in silico approach to locate precursor ions, based on diagnostic fragments, was developed. Implemented as an Excel macro, the algorithm rapidly assembles and surveys exact mass data to provide a list of feasible precursor candidates according to the correlation of the chromatographic peak shape profile and other additional filtering criteria (e.g. neutral losses and isotopes). The macro was tested with two families of veterinary drugs, sulfonamides and penicillins, which are known to yield diagnostic product ions when fragmented. Data sets obtained from different food matrices (fish and liver), both at high and low concentration of the target compounds, were investigated in order to evaluate the capabilities and limitations of the reported approach. Finally, other possible applications of this technique, such as the elucidation of elemental compositions based on product ions and corresponding neutral losses, were also presented and discussed.

Use of gel permeation chromatography for clean-up in the analysis of coccidiostats in eggs by liquid chromatography-tandem mass spectrometry.

An analytical method for determination and confirmation of nine coccidiostatics in eggs is reported. Ethyl acetate is used as extraction solvent, with satisfactory results, and simple automated clean-up is based on gel-permeation chromatography (GPC) . The target compounds are then analysed by liquid chromatography-electrospray ionization-tandem mass spectrometry. The method was validated in-house in accordance with Commission Decision 2002/657/EC. Trueness and precision were determined at four concentrations, and the mean errors obtained were <10 %, with relative standard deviations ranging from 3 to 18 %. For three non-authorized coccidiostatics (clopidol, ethopabate, and ronizadole), decision limit and detection capability were in the ranges 0.12-0.16 and 0.18-0.23 µg kg(-1), respectively. The results obtained prove the suitability of this new analytical method for routine monitoring of these substances in eggs.

Development and validation of an LC-UV method for the determination of sulfonamides in animal feeds.

A simple LC-UV method was developed for the determination of residues of eight sulfonamides (sulfachloropyridazine, sulfadiazine, sulfadimidine, sulfadoxine, sulfamethoxypyridazine, sulfaquinoxaline, sulfamethoxazole, and sulfadimethoxine) in six types of animal feed. C18, Oasis HLB, Plexa and Plexa PCX stationary phases were assessed for the clean-up step and the latter was chosen as it showed greater efficiency in the clean-up of interferences. Feed samples spiked with sulfonamides at 2 mg/kg were used to assess the trueness (recovery %) and precision of the method. Mean recovery values ranged from 47% to 66%, intra-day precision (RSD %) from 4% to 15% and inter-day precision (RSD %) from 7% to 18% in pig feed. Recoveries and intra-day precisions were also evaluated in rabbit, hen, cow, chicken and piglet feed matrices. Calibration curves with standards prepared in mobile phase and matrix-matched calibration curves were compared and the matrix effects were ascertained. The limits of detection and quantification in the feeds ranged from 74 to 265 µg/kg and from 265 to 868 µg/kg, respectively.

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.

High-throughput multiclass method for antibiotic residue analysis by liquid chromatography-tandem mass spectrometry.

A simple and rapid method has been developed for the residue analysis of 39 antibiotics (tetracyclines, quinolones, penicillins, sulfonamides and macrolides) in foodstuffs of animal origin. The method combines an effective extraction technique, which uses water-methanol as extracting solvent, with ultra-high-pressure liquid chromatography-tandem mass spectrometry, allowing both confirmation and quantification in a single chromatographic run. The multiresidue method has been validated in chicken muscle matrix according to European Union Decision 2002/657/EC. It has been implemented as a routine method in a Public Health Laboratory, instead of the five plates test and LC methods previously used.

Uncertainty estimation in organic elemental analysis using information from proficiency tests.

We evaluate the uncertainty in organic elemental analysis of C, H, N, and S. We use data from six proficiency tests (PTs), in which some 35 Spanish laboratories participated. The uncertainty of the technique is estimated from the relative within-laboratory and between-laboratory variances for pure substances and samples with complex matrices (soil, powdered milk, oil, ash, and petroleum coke). We also calculate the relative standard uncertainties for individual laboratories when analysing pure substances using historical data from the participation of each laboratory in different editions of PTs. The uncertainty values obtained for the individual laboratories are comparable with the uncertainty of the technique and correlate with the combined z-scores. The evolution over time of those laboratories participating in common editions of PTs is also evaluated.

Analysis of thyreostatic drugs in thyroid samples by liquid chromatography tandem mass spectrometry: comparison of two sample treatment strategies.

A method based on ultra-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry (UPLC-MS/MS) for the determination of six thyreostatic drugs in thyroid tissue has been optimised and validated in accordance with Decision 2002/657/EC. Sample extraction was evaluated in methanol and in ethyl acetate, the latter which gave better results. Two clean-up strategies were compared: one based on silica cartridges (SPE), and the other, on gel permeation chromatography (GPC). Recoveries ranged from 40% to 79% for the SPE approach and from 80% to 109% for GPC. Quantification was performed with blank tissue samples spiked with the analytes in the range 50-500microgkg(-1). 5,6-Dimethyl-2-thiouracil and 2-mercaptobenzimidazole-d(4) were used as internal standards. Decision limit (CCalpha) and detection capability (CCbeta) ranged from 1 to 15microgkg(-1) and from 6 to 25microgkg(-1), respectively. The accuracy of the method was calculated as percent error, which was less than 10%. The relative standard deviation in reproducibility conditions ranged between 2% and 14%.

Analysis of thyreostatic drugs in thyroid samples by ultra-performance liquid chromatography tandem mass spectrometry detection.

A method based on ultra-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry for the determination of six thyreostatic drugs in thyroid tissue has been optimised and validated in accordance with the Decision 2002/657/EC. Samples are extracted with methanol and the extracts cleaned-up on silica cartridges. The recoveries range from 40% for 6-phenyl-2-thiouracil to 79% for 2-thiouracil. Quantification is carried out with blank tissue samples spiked with the analytes in the range 25-500 microg kg(-1). 5,6-Dimethyl-2-thiouracil is used as internal standard. CCalpha and CCbeta are in the ranges 4.3-16.1 microg kg(-1) and 8.7-20.7 microg kg(-1), respectively. Accuracy, expressed as percentage of error, is lower than 6% and relative standard deviation in reproducibility conditions falls between 5.6 and 10.3%. Nowadays, the proposed method is routinely implemented in the laboratory of the Agència de Salut Pública de Barcelona and allows processing of up to 20 samples per day.

Restricted access materials for sample clean-up in the analysis of trace levels of tetracyclines by liquid chromatography. Application to food and environmental analysis.

Restricted access materials (RAM) based on alkyl diol silica (ADS) porous particles were evaluated for the sample clean-up in tetracyclines analysis in milk and water samples by liquid chromatography. RAM with C(4), C(8) and C(18) bonded to the inner pore surfaces were tested, with C(8) providing the best performance. A switching column LC system which combines an ADS C(8) RAM column and an analytical C(18) column connected to a fluorimetric detector was used for tetracyclines analysis. The RAM clean-up removed large peaks that otherwise appeared in the initial time window of the chromatograms, attributed to proteins in milk samples and humic substances in water samples. Thus, quantification of analytes in real samples, especially of the most polar compounds such as oxytetracycline and tetracycline, was clearly improved.