Determination of fifteen coccidiostats in feed at carry-over levels using liquid chromatography-mass spectrometry.

A multi-residue method has been developed and validated for the simultaneous determination of authorized (decoquinate, diclazuril, halofuginone, lasalocid, maduramicin, monensin, narasin, nicarbazin, robenidine, salinomycin and semduramicin) and non-authorized (amprolium, clopidol, ethopabate and toltrazuril) coccidiostats in animal feed. Feed samples were extracted with basic followed by acidified solution in methanol and, after centrifugation, were injected directly into LC-MS/MS system. Detection was performed in selected reaction monitoring mode with both positive and negative electrospray ionization. The time efficient validation experiment has verified the robustness of a method in different types of feed and on two separate LC-MS/MS instruments. The comparison of different quantification methods demonstrated that, against expectations, the standard addition did not prove better in comparison with matrix-matched calibration curve. Although the sample preparation was very easy, the observed matrix effects were not significant for the most part but they could explain the problems with the quantification of some coccidiostats.

Deposition and depletion of decoquinate in eggs after administration of cross-contaminated feed.

Decoquinate, a chemical coccidiostat used as a feed additive, can occur in eggs due to cross-contamination of feedstuffs for laying hens. An experiment was designed to assess the transfer of decoquinate to hen eggs and its distribution between egg yolk and egg white. Hens were given the feed containing decoquinate at a cross-contamination level (0.34 mg kg(-1)) and collected eggs were analysed using an LC-MS/MS method. The plateau level was reached on the eighth day of the experiment and averaged 8.91 µg kg(-1), which is far below the maximum level established at 20 µg kg(-1) for whole eggs. Decoquinate was deposited mostly in egg yolks (26.2 µg kg(-1)) and did not deplete completely during 14 days of administration of decoquinate-free feed. The results confirmed that administration of cross-contaminated feed is associated with very low risk of non-compliant residue levels of decoquinate in eggs.

Distribution of semduramicin in hen eggs and tissues after administration of cross-contaminated feed.

Semduramicin is an ionophore coccidiostat used in the poultry industry as a feed additive. Cross-contamination of feeds for non-target animals with semduramicin is unavoidable. However, it is not known whether undesirable residues of semduramicin may occur in food after cross-contaminated feed is administered to animals. The aim of the work was to determine the levels of semduramicin in hen eggs (yolks and albumen) and tissues (liver, muscle, spleen, gizzard, ovarian yolks and ovaries) after administration of feed contaminated with 0.27 mg kg(-1) of this coccidiostat. The residues were determined using LC-MS/MS. The distribution pattern confirmed the high lipophilicity of semduramicin. Residues were found mainly in egg yolks (28.8 µg kg(-1)), ovarian yolks (19.5 µg kg(-1)) and liver (2.57 µg kg(-1)), while hens' muscle was free from semduramicin (LOD = 0.1 µg kg(-1)). Among edible tissues, the maximum level (2 µg kg(-1)) was exceeded only in the liver.

Semduramicin in eggs--the incompatibility of feed and food maximum levels.

The cross-contamination of non-target feeds with coccidiostats may result in the occurrence of their residues in food of animal origin. To assure food safety, maximum levels (ML) of coccidiostats have been set for both feed and food. However, scientific data are not available on the transfer of some coccidiostats from feed into food. This experiment was therefore designed to verify, whether the administration of compliant semduramicin-contaminated feed could cause the occurrence of volatile residues of coccidiostats in eggs. The laying hens received feed containing 0.27 ± 0.034 mg/kg of semduramicin (ML=0.25 mg/kg). Semduramicin residues were detected in whole eggs after two days of administration of semduramicin-containing diet. A plateau level was achieved (16.1 ± 5.19 µg/kg, mean ± SD) with the concentrations significantly exceeding the maximum level of semduramicin in eggs (2 µg/kg). The results of this experiment might be a signal for the revision of the ML value.

The determination of six ionophore coccidiostats in feed by liquid chromatography with postcolumn derivatisation and spectrofotometric/fluorescence detection.

The control of levels of anticoccidial feed additives in targeted feeds plays an important role in the assurance of efficiency of animal treatment, prevention of drug resistance, and food safety. The robust and labour-efficient method for the simultaneous determination of six ionophore coccidiostats (lasalocid, maduramicin, monensin, narasin, salinomycin, and semduramicin) in targeted feed has been developed. Properly grinded and homogenized feed sample was spiked with internal standard (monesin methyl ester) and extracted with methanol. The extract was analysed with reversed phase HPLC without any further purification. The separation of the analytes with conventional C18 and core-shell columns was compared. Lasalocid was analysed with fluorescence detection, whereas other ionophores were detected with UV-Vis detector after derivatisation with vanillin in the presence of sulfuric acid. Fortified samples and targeted feeds at authorized levels were used for method validation. Recovery was in the range of 85-110%, depending on the analyte. The within-laboratory reproducibility did not exceed the target value from Horwitz equation. The results of the proficiency tests (z-scores in the range of -1.0 to 1.9) confirmed the reliability of the developed protocol.

Rapid method for the determination of metamizole residues in bovine muscle by LC-MS/MS.

Metamizole is a pyrazolone non-steroidal anti-inflammatory drug allowed for use in food-producing animals. According to Council Directive 96/23, residues of this drug have to be monitored because of the potential risk to consumers' health. Metamizole is hydrolysed to its marker residue 4-methylaminoantypyrine.This compound is further metabolised to three main metabolites: 4-formylaminoantipyrine, 4-aminoantipyrine and 4-acetylaminoantipyrine. The MRL of 4-methylaminoantipyrine in animal tissues is 100 µg kg(-1). Considering the above points, a method for the detection of four metamizole metabolites in bovine muscles was developed. Analytes were extracted from muscle by a mixture of acetonitrile and sodium acetate buffer. After centrifugation, the supernatant was passed through alumina cartridges, diluted with mobile phase and analysed by using LC-MS/MS. Four metamizole metabolites were separated on a C8 column in 23 min with a gradient of methanol:acetonitrile:ammonium formate solution and analysed by using positive ionisation. Validation of the method indicated a within-laboratory reproducibility in the range of 7-30% and recovery in the range of 45-95%. The method fulfils the criteria for confirmatory methods and, thanks to its labour efficiency, may also be used for screening purposes.

Influence of matrix effect on the performance of the method for the official residue control of non-steroidal anti-inflammatory drugs in animal muscle.

RATIONALE: During the development and validation of mass spectrometry based method in residue control analysis, it is recommended to evaluate the level of the matrix effect. Often its level is relatively high, despite extensive sample purification. METHODS: The matrix effect in the method for the determination of non-steroidal anti-inflammatory drugs in animal muscle was tested using a post-extraction addition technique. The experiment was performed to assess the impact of chromatographic conditions and design of ion source on the results. Additionally, the impact of phospholipids was tested. RESULTS: The matrix effect signal varied from 36% (64% ion suppression) to 192% (92% ion enhancement), depending on the analyte and species. The internal standard corrected matrix effect was generally lower but was still high for some analytes. Both chromatographic conditions and ion source design have influence on the level of matrix effect; however, this effect was no longer observed after compensation with internal standards. CONCLUSIONS: Currently, no commonly accepted criteria exist for the interpretation of results of determination of matrix effects; such criteria have been proposed in this paper, based on guidelines for bioanalytical methods and results of the study.

Determination of non-steroidal anti-inflammatory drugs and their metabolites in milk by liquid chromatography-tandem mass spectrometry.

Non-steroidal anti-inflammatory drugs are widely used for treatment of animals. According to Council Directive 96/23/EC, residues of these drugs must be monitored because of the potential risk they pose to the consumers' health. For this reason an LC-MS-MS method was developed for detection of wide range of NSAIDs, including both "acidic" NSAIDs (carprofen, diclofenac, flunixin, meloxicam, phenylbutazone, oxyphenbutazone, tolfenamic acid, mefenamic acid, naproxen, ketoprofen, ibuprofen, firocoxib, rofecoxib, and celecoxib) and "basic" NSAIDs (four metamizole metabolites). Analytes were extracted from milk samples with acetonitrile in the presence of ammonium acetate. One portion of the extract was directly analyzed for the presence of metamizole metabolites; a second portion was cleaned with an amino cartridge. All NSAIDs were separated on a Phenomenex Luna C8(2) column and analyzed by LC-MS-MS in negative (acidic NSAIDs) and positive (metamizole metabolites) ion modes. The method was validated in accordance with the requirements of Commission Decision 2002/657/EC. Within-laboratory reproducibility was in the range 7-28%, and accuracy was in the range 71-116%. The method enabled detection of all the analytes with the expected sensitivity, below the recommended concentrations. The method fulfills the criteria for confirmatory methods and, because of its efficiency, may also be used for screening purposes. The procedure was also successfully verified in the proficiency test organized by EU-RL in 2010. As far as the authors are aware, this is one of the first methods capable of detecting diclofenac residues below the MRL in milk (0.1 µg kg(-1)). An additional advantage is the possibility of simultaneous determination of "acidic" NSAIDs and metamizole metabolites.

Residue control of coccidiostats in food of animal origin in Poland during 2007-2010.

Coccidiostats are widely used as feed additives to prevent coccidiosis. The off-label use of anticoccidials or feeding non-target animals with cross-contaminated feedingstuffs may result in the occurrence of coccidiostat residues in animal tissues and eggs. In EU countries, food of animal origin is subjected to official control of residues according to Council Directive 96/23/EC. In Poland, within the framework of the National Residue Control Plan, 3718 samples (3533 targeted and 185 suspect) of animal liver, eggs, drinking water and feed were tested for coccidiostats between 2007 and 2010. Violative residues of nicarbazin, lasalocid, maduramicin, salinomycin, semduramicin and robenidine were detected in 77 food samples (53 samples of chicken liver, 23 samples of eggs and 1 sample of turkey liver). A high percentage (31%) of non-compliant feed samples collected during follow-up investigations was observed, which confirms that feed cross-contamination may be the reason of the occurrence of coccidiostat residues in food.

Determination of non-steroidal anti-inflammatory drugs residues in animal muscles by liquid chromatography-tandem mass spectrometry.

A confirmatory method for the determination of residues of nine non-steroidal anti-inflammatory drugs and one metabolite in animal muscles has been developed. After enzymatic hydrolysis samples were extracted with acetonitrile and cleaned up using alumina and C(18) SPE cartridges. Liquid chromatography-tandem mass spectrometry was used for the separation and determination of analytes. The method was validated in bovine muscles, according to the Commission Decision 2002/657/EC criteria. Applicability of the method in the analysis of swine, horse and chicken muscles was checked by precision and recovery experiment. The influence of matrix effect on the quantification of non-steroidal anti-inflammatory drugs residues was investigated. The method was used for the confirmation of phenylbutazone and oxyphenbutazone in horse muscle sample.

Determination of benzimidazoles and levamisole residues in milk by liquid chromatography-mass spectrometry: screening method development and validation.

The screening method for the determination of residues of 19 benzimidazoles (parent drugs and their metabolites) and levamisole in bovine milk has been developed and validated. Milk samples were extracted with ethyl acetate, sample extracts were cleaned up by liquid-liquid partitioning with hexane and acidic ethanol. Liquid chromatography-single-quadrupole mass spectrometry was used for the separation and determination of analytes. The method was validated in bovine milk, according to the CD 2002/657/EC criteria. An alternative approach to the validation of the method was applied ("sum MRL" substances). The method was successfully verified in CRL proficiency test.

Multi-residue confirmatory method for the determination of twelve coccidiostats in chicken liver using liquid chromatography tandem mass spectrometry.

The confirmatory LC-MS/MS method for the determination of residues for twelve coccidiostats including ionophore antibiotics (lasalocid, maduramycin, monensin, narasin, salinomycin, semduramycin) and chemical coccidiostats (clazuril, decoquinate, diclazuril, halofuginone, nicarbazin and robenidine) in poultry liver has been developed. The sample preparation was based on extraction with acetonitrile, defatting with Alumina columns and clean-up on Oasis HLB spe. The separation of analytes was performed on PhenylHexyl column with a gradient of acetonitrile, methanol and the ammonium formate pH 4.0. For all analytes, at least 2 diagnostic fragmentation ions were monitored. The validation, performed according to the CD 2002/657/EC, proved the suitability of the method for the confirmatory analysis of the coccidiostats. For lasalocid, however, low reproducibility was observed and the proper quantification could not be performed with this method.

In-house reference materials: 5-hydroxyflunixin and meloxicam in cow milk-preparation and evaluation.

Reference materials are helpful to evaluate the performance of laboratories as well as being useful for the quality control of analytical procedures. Certified reference materials and other reference materials containing non-steroidal anti-inflammatory drugs in milk are however, not available. Therefore, production and evaluation of in-house reference materials with incurred residues of 5-hydroxyflunixin (5OHFLU) and meloxicam (MEL) in cow milk has been performed. The milk was collected 12h after dosing from cows which received meloxicam (0.5 mgkg(-1) b.w., i.v., single dose) or flunixin meglumine (2.2 mgkg(-1) b.w., i.v. during three days). The concentrations of analytes were checked in the milk samples. The milk was diluted with milk free from NSAIDs residues, homogenised, put into sterile 20 mL vials, frozen and lyophilised. The vials were weighed before and after lyophilisation, in order to calculate the amount of water necessary for reconstitution, and were stored at a temperature of -20+/-2 degrees C. For the homogeneity study, 10 random samples were analysed in duplicate and the results were interpreted using Cochran's test, Horwitz standard deviation and the test for a sufficient homogeneity. The assigned values, calculated from the results of the homogeneity test were 54.3 microgkg(-1) for 5OHFLU and 46.4 microgkg(-1) for MEL. The samples were tested for their stability every 14 days for 2 months and after 9 months. It has been confirmed that an appropriate homogeneity and stability of the produced in-house reference material has been obtained.