Anodic Adsorptive Stripping Voltammetric Determination of Rafoxanide on Glassy Carbon Electrode.

AIMS AND OBJECTIVE: The development of easy, accurate, reliable technique which is characterized by low cost, minimal sample pre-treatment, and short analysis time to monitor RFX residues in milk samples before distribution to consumers. BACKGROUND: Literature survey reveals several analytical methods, including high-performance liquid chromatography (HPLC), ultra-performance liquid chromatography (UPLC) and thin-layer chromatography (TLC)-densitometry. These methods are time consuming, require additional steps like preconcentration or multisolvent extraction, trained technicians, and expensive instruments. MATERIALS AND METHODS: The electrochemical analysis of RFX was effectively established by the adsorptive stripping method on GCE due to the effective interfacial accumulation of RFX on the electrode surface. The RFX adsorptive accumulation is followed by electrochemical measurement of the accumulated analyte. RESULTS: The electrochemical oxidation of RFX was studied at glassy carbon electrodes (GCE) in Britton-Robinson buffer (BR) solutions over the pH range from 2.0-12.0 using cyclic and differential pulse voltammetry (DPV). The oxidation of the drug was accomplished in a single irreversible, adsorption-controlled step within the pH range 4.0-9.0. Therefore, the application of GCE for a sensitive and selective quantification of RFX by adsorptive stripping voltammetry was reported. This format was satisfactorily applied for the determination of RFX in bovine milk. Limit of detection (LOD) of 1.25 µg kg-1 of milk and mean recoveries of 97.8 to 107.5% were achieved. CONCLUSION: The proposed method might be competitive with the HPLC techniques. The detection limit found for RFX on GCE for milk samples, after medium exchange, was well below the MRLs, the maximum concentration of a veterinary drug residue legally permissible in food, are proposed by the European Medicines Agency.

Comparative study on the selectivity of various spectrophotometric techniques for the determination of binary mixture of fenbendazole and rafoxanide.

Five different spectrophotometric methods were applied for simultaneous determination of fenbendazole and rafoxanide in their binary mixture; namely first derivative, derivative ratio, ratio difference, dual wavelength and H-point standard addition spectrophotometric methods. Different factors affecting each of the applied spectrophotometric methods were studied and the selectivity of the applied methods was compared. The applied methods were validated as per the ICH guidelines and good accuracy; specificity and precision were proven within the concentration range of 5-50 µg/mL for both drugs. Statistical analysis using one-way ANOVA proved no significant differences among the proposed methods for the determination of the two drugs. The proposed methods successfully determined both drugs in laboratory prepared and commercially available binary mixtures, and were found applicable for the routine analysis in quality control laboratories.

Investigation of the persistence of rafoxanide residues in bovine milk and fate during processing.

Rafoxanide is an effective treatment for the control of fluke infections in animals, but it is currently not permitted for treating animals whose milk is intended for human consumption. In this study, the persistence of rafoxanide residues in milk, and their migration to dairy products, was investigated following the treatment of six lactating dairy cows with Curafluke 10% oral drench. The highest concentration of rafoxanide residues detected in the individual cows milk ranged from 249 to 627 µg kg(-1) and occurred at 2-3 days post-treatment. At 2 and 23 days post-treatment (representing high and low residue concentrations) the milk was pooled into two independent aliquots, each containing the full day's milk produced by three cows. Milk products were made from pasteurised and unpasteurised milk. Pasteurisation appeared to have little impact on the stability of the residues. Rafoxanide concentrated sixfold in the cheese (week 0) compared to the starting milk (2070 vs. 349 µg kg(-1)) but was four times lower in whey (75 µg kg(-1)). Rafoxanide residues were up to 14 times higher in butter (week 0) than in the starting milk (5468 vs. 376 µg kg(-1)). Residues were found to further concentrate in butter and cheese at longer storage and ripening times, respectively. Skim-milk powder was manufactured from skim milk, and residues were 10-fold higher than in the starting skim milk (5468 vs. 376 µg kg(-1)) despite the 185°C temperature required for the process. Rafoxanide residues were stable in this skim-milk powder when stored at ambient temperature for at least 1 year. Results showed that detectable rafoxanide residues were excreted in milk for 47 days, and concentrated in the fat-based products. The analytical ranges of the UHPLC-MS/MS method used were 1.0-200 µg kg(-1) (milk and whey) and 10-2000 µg kg(-1) (other dairy products).

Determination of closantel and rafoxanide in animal tissues by online anionic mixed-mode solid-phase extraction followed by isotope dilution liquid chromatography tandem mass spectrometry.

A rapid and high-throughput isotope dilution LC-MS/MS method with online sample pre-concentration and clean-up using anionic mixed-mode SPE was described for the determination of closantel and rafoxanide in edible bovine and ovine tissues. Tissue samples were extracted with acetonitrile and acetone mixture (60:40, v/v). Sample pre-concentration, clean-up and analysis were completed simultaneously with the online MAX SPE LC-MS/MS system. Closantel-(13) C(6) and rafoxanide-(13) C(6) were used as the internal standards to improve the precision of the method. The method was validated with edible ovine and bovine tissues (muscle, kidney and liver) fortified at three different levels. The accuracy and RSD were 86-106% and ≤14%, respectively. This high-throughput method was suitable for routine quantitative analysis of closantel and rafoxanide in food safety surveillance samples.

Quantitative analysis of closantel and rafoxanide in bovine and ovine muscles by high-performance liquid chromatography with fluorescence detection.

An HPLC method with a fluorescence detector (HPLC-FLD) was described for the quantitative determination of closantel and rafoxanide in bovine and ovine muscles. A structural analog closely related to rafoxanide, viz., N-[4-(4-chlorophenoxy)phenyl]-2-hydroxy-3,5-diiodobenzamide, was synthesized as an internal standard. Bovine and ovine muscles were extracted with acetonitrile-acetone (60 + 40, v/v) followed by cleanup on mixed mode anionic exchange SPE cartridges. After evaporation and reconstitution with the mobile phase, the sample was analyzed by HPLC-FLD using internal standard calibration. The method was validated by using fortified bovine and ovine muscles at 15, 30, and 60 microg/kg. The accuracy and RSD were 70-110% and < or =10%, respectively.

Screening of closantel and rafoxanide in animal muscles by HPLC with fluorescence detection and confirmation using MS.

A sensitive and selective LC-FLD method was described for determination of closantel and rafoxanide in bovine and ovine muscles. Bovine and ovine muscles were extracted with ACN and acetone mixture (80:20, v/v). After cleanup with Oasis MAX SPE cartridges, the sample was analyzed by LC-FLD using the control point approach. No false-negative result was observed at or below maximum residue limits and the false-positive rate was below 5%. Suspected positive sample was confirmed by LC-MS/MS. This method was suitable for screening of large batch of samples and hence considerably reduced the time and cost required for quantitation and confirmatory analyses.