Determination of robenidine in shrimp and chicken samples using the indirect competitive enzyme-linked immunosorbent assay and immunochromatographic strip assay.

In this study, the monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and an immunochromatographic strip assay were developed for the rapid screening of robenidine hydrochloride (ROBH) in samples. The 50% inhibitory concentration (IC50) of ROBH was 0.927 ng mL-1, and the standard curve showed a linear correlation coefficient of 0.99932. There was no cross-reaction between ROBH and other commonly used anticoccidial drugs, which indicated that the monoclonal antibody had high specificity. The recoveries of ic-ELISA were in the range of 87.8% to 102.0%. The immunochromatographic strip assay displayed cut-off values of 10, 5 and 10 ng g-1 for shrimp, chicken breast and chicken liver samples, respectively. In addition, the results can be obtained within 10 min by naked eye observations. And in sample analysis, the results of ic-ELISA and the immunochromatographic strip assay were in accordance with those of LC-MS/MS. Thus, the ic-ELISA and immunochromatographic strip assay are effective methods for the detection of ROBH in shrimp, chicken breast and chicken liver samples.

Analysis of veterinary drug and pesticide residues in animal feed by high-resolution mass spectrometry: comparison between time-of-flight and Orbitrap.

The use of medium-high-resolution mass spectrometers (M-HRMS) provides many advantages in multi-residue analysis. A comparison between two mass spectrometers, medium-resolution (MRMS) time-of-flight (TOF) and high-resolution (HRMS) Orbitrap, has been carried out for the analysis of toxic compounds in animal feed. More than 300 compounds belonging to several classes of veterinary drugs (VDs) and pesticides have been determined in different animal feed samples using a generic extraction method. The use of a clean-up procedure has been evaluated in both instruments, and several validation parameters have been established, such as the matrix effect, linearity, recovery and sensitivity. Finally, both instruments have been used during the analysis of 18 different feed samples (including chicken, hen, rabbit and horse). Some VDs (sulfadiazine, trimethoprim, robenidine and monensin sodium) and one pesticide (chlorpyrifos) have been identified. In general, better results were obtained using the Orbitrap, such as sensitivity (1-12.5 µg kg(-1)) and recovery values (60-125%). Moreover, this analyser had several software tools, which reduced the time for data processing and were easy to use, performing quick screening for more than 450 compounds in less than 5 min. However, some disadvantages such as the high cost and a decrease in the number of detected compounds at low concentrations must be taken into account.

Determination of robenidine residues in chicken muscle by high performance liquid chromatography with ultraviolet detection.

A simple, robust and reliable method for the determination of residual robenidine in chicken muscle using high performance liquid chromatography with ultraviolet (UV) detection was developed and validated according to the Codex Alimentarius Commission guidelines. Chicken muscle was extracted by acetonitrile/formic acid (98:2, v/v) and defatted with hexane. Analytes were isocratically separated on a Luna C18 column (4.6 × 150 mm, 5 µm) using 70 % methanol in water containing 0.1 % trifluoroacetic acid at a flow rate of 1.0 mL/min at 30 °C. UV detection was performed at 312 nm. The method was validated by assessing performance parameters including selectivity, linearity, limit of quantification (LOQ), precision, accuracy, recovery, stability and robustness. A calibration curve that was constructed over 0.05-0.5 µg/g showed correlation coefficients of more than 0.999. The intra- and inter-day precisions (as coefficient of variation) were 1.45-3.32 and 2.63-4.99 %, respectively. The intra- and inter-day accuracies were 99.4-105.3 and 98.3-101.6 %, respectively. The recoveries were in the range of 76.6-81.8 % and the LOQ was 0.05 µg/g. The developed method showed suitable performance for the determination of robenidine residues in chicken muscle.

Development and validation of a multiresidue liquid chromatography/tandem mass spectrometry method for 11 coccidiostats in feed.

A confirmatory method for the determination of 11 regulated coccidiostats including the ionophore antibiotics lasalocid, maduramicin, monensin, narasin, salinomycin, and semduramicin and the chemical coccidiostats decoquinate, diclazuril, halofuginone, nicarbazin, and robenidine in animal feed was developed and validated. The procedure was intended for the identification and quantification of the coccidiostats at concentrations relating both to the unintentional carryover as stated in Regulation 574/2011 and to the authorized levels in target feed. The analytes were determined by LC/MS/MS in the positive or negative electrospray ionization mode. The method performance characteristics were estimated in the relevant application field from 0.003 to 200 mg/kg. Validation criteria of linearity, specificity, trueness, precision, LOD, and LOQ along with measurement uncertainty were estimated for all analytes. Absolute and relative matrix effects were also studied. The results proved that the method performance was satisfactory, and it was successfully applied to carryover control by analyzing 165 feed samples collected within regulatory monitoring plans. Finally, since the carryover phenomenon in feed may result in the presence of residues in food products of animal origin, a survey has been carried out on the occurrence of coccidiostats in 167 eggs and animal muscles.

Biotic transformation of anticoccidials in soil using a lab-scale bio-reactor as a precursor-tool.

Two anticoccidial agents, salinomycin and robenidine, heavily used in the worldwide veterinary meat production, were investigated for their potential biotic degradation by cultured soil bacteria. The degradation-study was performed in lab-scale bio-reactors under aerobic and anaerobic conditions incubated for 200 h with a mixed culture of soil bacteria. Samples were analyzed by LC-MS/MS and potential transformation products were tentatively identified. Salinomycin was degraded under aerobic conditions and traces could be found after 200 h, however, seems more persistent under anaerobic conditions. Four transformation products of salinomycin were discovered. Robenidine was degraded under aerobic and anaerobic conditions, however, traces of robenidine were observed after 200 h. Five biotic transformation products of robenidine were discovered.

[Determination of robenidine residue in chicken tissues and eggs by high performance liquid chromatography].

A method for the determination of robenidine residue in chicken tissues and eggs by high performance liquid chromatography (HPLC) was established. The samples were extracted with acetonitrile and purified by an HLB solid-phase extraction (SPE) cartridge. The extract was analyzed by HPLC with acetonitrile-0.05 mol/L NH4H2PO4 buffer (6:4, v/v, pH 6.5) as the mobile phase. The flow rate of the mobile phase was 1.0 mL/min and detection wavelength was 317 nm. There was a good linear correlation between the peak areas and the concentrations of robenidine in the range of 10-1000 microg/L. The limit of detection (S/N = 3) was 10 microg/L, and the limit of quantification (S/N = 10) was 15 microg/kg. The recoveries of robenidine were 73.1%-88.7% at the spiked levels of 15, 50 and 100 microg/kg. The results demonstrate that the method is easy, fast, sensitive, and suitable for the confirmation and quantification of robenidine residue in chicken tissues and eggs. The clean-up effect, sensitivity, accuracy and precision can all meet the analysis requirement.

Polarographic determination of robenidine residues in chicken tissues, eggs, litter, soil, and plant tissue.

Polarographic residue methods have been developed for determining robenidine (Robenz), 1,3-bis[p-chlorobenzylidene)amino]-guanidine monohydrochloride, in chicken tissues, eggs, litter, soil, and plants. The compound is extracted from chicken fat, skin, muscle, liver, and eggs with ethyl acetate; from blood with acetone; from plant tissue, litter, and kidney with acidic acetone; and from soil with basic methanol. After extraction by high-speed blending or overnight shaking, the extract is cleaned up by evaporation, solvent partition and/or elution from CG-50 ion exchange resin. Robenidine is quantitated by differential cathode ray polarography, using acidic aqueous methanol or acetic acid (1+1) supporting electrolyte. Recoveries ranged from 64 to 125% with an average overall recovery of 90%. The validated sensitivity is 0.1 ppm for chicken tissues, soil, and plants, 0.01 ppm for eggs, and 1 ppm for litter.

Automated high-pressure liquid chromatographic assay of robenidine hydrochloride.

High-pressure liquid chromatography has been applied to the separation and analysis of robenidine hydrochloride in the presence of its chemical precursors. It provides a rapid and specific method for the analysis of robenidine in technical material as well as feed premixes and is capable of distinguishing the intact drug from its degradation products. The chromatographic system employs a mixture of methanol, acetic acid, and methylene chloride as the mobile phase and a controlled pore glass as the stationary phase. Sample manipulation has been automated in 2 ways. In the first, manually prepared samples are automatically injected onto the column for separation and quantitation. In the second, a totally automated sample handling system, weighed samples are extracted, diluted, filtered, and then injected onto the column, all in a sequential manner. These procedures are capable of giving quantitative results at a rate of 10 samples/hr with a relative standard deviation of 1.13%.