Determination of tilmicosin residues in chicken, cattle, swine, and sheep tissues by liquid chromatography.

A method was developed and validated for determination and quantitation of tilmicosin residues in swine, cattle, and sheep edible tissues, as well as chicken fat, skin, and muscle over a concentration range of 0.025 microg/g-20 microg/g. For chicken kidney and liver, the method was validated over a range of 0.060 microg/g-20 microg/g. The tissue sample was extracted with methanol and a C18 cartridge was used for solid-phase extraction cleanup. A reversed-phase gradient liquid chromatographic method with detection at 280 nm was used to separate the tilmicosin from matrix components in 30 min run time. The limit of quantitation (LOQ) of the method was 0.025 microg/g for all tested tissues except chicken kidney and liver, for which the LOQ was 0.06 microg/g. Average recoveries for tissue samples ranged from 73 to 98%. Relative standard deviation values ranged from 0.6 to 14.7%.

Determination of tilmicosin residues in cow and sheep milk by liquid chromatography.

This method was developed and validated to detect and quantitate tilmicosin residues in cow milk over a range of 0.010-10 microg/mL, and in sheep milk over a range of 0.025-0.5 microg/mL. The procedure involves extracting the milk sample with acetonitrile and using a C18 cartridge to perform a solid-phase extraction cleanup of the extract. A reversed-phase gradient liquid chromatography method with detection at 280 nm is used to separate the tilmicosin from matrix components in a 30 min run time. The limit of quantitation of the method is 0.010 microg/mL for cow milk, and 0.025 microg/mL for sheep milk. Average percentage recoveries for milk samples ranged from 82 to 94%. Percentage relative standard deviation values ranged from 3.1 to 17.2%.

Determination of tilmicosin in swine feeds by liquid chromatography.

This method determines tilmicosin in feeds over a concentration range of 100 to 600 mg/kg. Tilmicosin is extracted from swine feeds by adding 200 mL of a swine feed extractant (20 + 80, acetonitrile-Millipore water, pH 2.5, with 25 mM dibutylammonium phosphate) to 20 g feed and placing on a shaker table for 1 h. This extractant is filtered and analyzed by liquid chromatography (LC). A gradient LC method is used to separate tilmicosin from the feed matrix in 30 min of run time. The recovery of tilmicosin from fortified feeds ranged from 96.7 to 112%, with the coefficients of variation (CVs) ranging from 1.4 to 3.9%. The determination of tilmicosin in medicated feeds resulted in an average recovery of 92.7% of labeled claim for pelleted feeds at 200 mg/kg and 99.1% of labeled claim for mash feeds at 400 mg/kg. Determination of tilmicosin in medicated feeds resulted in CVs ranging from 2.6 to 3.8%. The method has shown no interference with 18 other drugs.

Liquid chromatographic determination of tilmicosin in swine feed at 200-400 mg/kg level: interlaboratory study.

An analytical method for the determination of tilmicosin at 200-400 mg/kg, the intended use concentration range, was evaluated in an interlaboratory study involving 5 laboratories, including the sponsor. The interlaboratory study evaluated the intra- and interlaboratory precision and accuracy of a tilmicosin feed method. The method procedure involved extracting tilmicosin from feed by adding 200 mL extractant to 20 g feed and shaking for 1 h. The extract is filtered and analyzed by gradient liquid chromatography which separates tilmicosin from feed matrix in 30 min. Each laboratory assayed 5 replicates of fortified feed at concentrations of 0, 100, 200, 400, and 600 mg/kg. The mean recovery among fortified samples ranged from 81.4 to 98.8%, with a percent coefficient of variation (%CV) ranging from 0.3 to 4.0%. For all blank control feed samples no significant interferences were observed. In addition, each laboratory assayed 5 replicates of medicated feed samples prepared at 2 levels (200 and 400 mg/kg) with either a horizontal or vertical mixer. Along with the medicated feed samples were included 5 replicates of a blank control feed. The identities of the medicated and blank control feed samples were blinded to the analysts. The results for the medicated feed samples ranged from 95.8 to 106% of label claim, with a %CV ranging from 2.1 to 6.7%.

Effect of bentonite incorporated in a feed ration with tilmicosin in the prevention of induced Mycoplasma gallisepticum airsacculitis in broiler chickens.

A factorial arrangement of tilmicosin and bentonite was evaluated for efficacy in broiler chickens infected with Mycoplasma gallisepticum and correlated to tilmicosin recovery in a feed assay method. Tilmicosin at 300-500 g/ton prevented development of airsacculitis. The addition of 2% bentonite to the ration caused tilmicosin at 300 g/ton to be ineffective in controlling air-sac lesions, whereas 400 and 500 g/ton were moderately effective. Six percent bentonite rendered tilmicosin completely ineffective at all dose levels. There was a direct correlation between the percentage of bentonite in the feed rations, the percentage of the tilmicosin recovered in the assay procedure, and the increased incidence of air-sac lesions.