Flubendazole residues in eggs were experimentally induced by providing groups of 8 laying hens feed with approximately 3, 10 and 30 mg kg-1 flubendazole for 21 days. Eggs were sampled during this period and one week after the administration. Samples of both whole egg and egg white/yolk were analysed separately. Flubendazole analysis was performed by reversed phase HPLC and UV detection at 250 nm (eggs) or 320 nm (feed). The limit of detection (LOD) for flubendazole in feed was 0.3 mg kg-1 and in whole egg 0.012 mg kg-1. Both the hydrolysed and reduced metabolites of flubendazole were also determined quantitatively. The eggs of control hens housed in the same room during the study period did not contain any detectable flubendazole or metabolite residue. The eggs from the lowest dosed group (3 mg kg-1 feed) did contain residues, but most of them were only slightly higher than the LOD. Residues in eggs collected from the laying hens that obtained feed with 10 and 30 mg kg-1 flubendazole reached a plateau level after some 10 days and there was a good dose response relation between levels in feed and those in eggs. The residues of parent compound and metabolites almost exclusively occurred in yolk, the metabolites accounting for some 60-65% of the total residue. The residues of the parent compound and its metabolites declined below 100 micrograms kg-1 5 days after the administration of dosed feed had ended.
A reversed-phase liquid chromatography method for determination of maduramicin in feedingstuffs and premixtures was developed, validated, and interlaboratory studied. The extraction solvent was methanol. Maduramicin was detected at 520 nm after postcolumn derivatization with vanillin.
A reversed-phase liquid chromatography (LC) method for narasin in feedingstuffs and premixtures was developed, validated, and interlaboratory studied. The extraction solvent was methanol–K2HPO4 solution (9 + 1, v/v). Narasin was detected at 600 nm after post-column derivatization with dimethylamino-benzaldehyde. Recovery was >90%. The repeatability (RSDr) in feed (20–140 mg/kg) ranged between 1.2 and 10.5%; the within-laboratory reproducibility (RSDR) ranged between 2.2 and 4.9%. The limit of determination was <20 mg/kg. Other feed additives did not interfere in the assay. The method showed ruggedness against changes in the composition of extraction solvent, eluent, and conditions for post-column reactions. In an interlaboratory study, 5 broiler feeds (4 positive, 1 blank) and 1 premixture were analyzed by 13 laboratories. The RSDr of the feedingstuffs (20–120 mg/kg) varied between 2.17 and 7.57%. The HORRAT ranged between 0.77 and 0.88, with recoveries between 82 and 104%. One laboratory detected small signals in the blank sample, calculated as 0.6 and 2.8 mg/kg. For the premixture, acceptable results for reproducibility could only be obtained after modification of the method: the RSDr was 4.42% and the HORRAT was 1.56 (12 laboratories).
A reversed-phase liquid chromatography method for nicarbazin in broiler feeds and premixtures was developed, validated, and interlaboratory studied. The extraction solvent was an acetonitrile–methanol (1 + 1) mixture. For feedingstuffs, water was also added. The 4,4′-dinitrocarbanilide moiety of nicarbazin was detected at a wavelength of 350 nm. Recovery was ≥ 87%. At 20 mg/kg, the repeatability was 0.7% and the within-laboratory reproducibility was 2.7%. The limit of determination was <20 mg/kg. Other feed additives did not interfere in the assay that proved to be applicable to broiler feeds from different European Union countries. In an interlaboratory study, 4 positive broiler feeds, 1 blank pig feed, and 1 broiler premixture were analyzed by 19 laboratories using the method developed in this study. The relative standard deviation for repeatability (RSDr) of the feedingstuffs (20–240 mg/kg) varied between 2.6 and 10.2%. The HORRAT ranged between 0.70 and 1.22. Recoveries were 91–108%. Three laboratories detected small signals in the blind blank samples, ranging from 0.4 to 2 mg/kg. For the premixture, acceptable results for reproducibility could only be obtained after the sample weight and volume of extraction had been doubled. To avoid excessive dilution of the extracts, the range of the calibration curve had also been doubled. With this modified method, the RSDr was 5.7% and the HORRAT was 1.95 (10 laboratories).
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