Determination Of Tilmicosin Research Articles

Determination of Tilmicosin in Bovine, Swine, Chicken, and Turkey Tissues by Liquid Chromatography With Tandem Mass Spectrometry, Single-Laboratory Validation.

An LC-MS/MS method was developed for determination and confirmation of tilmicosin in bovine, swine, chicken, and turkey tissues (liver, kidney, muscle, and skin/fat) and bovine milk. The method was subjected to single-laboratory validation to establish method performance parameters. Animal tissues and bovine milk were fortified at four concentrations ranging from 0.5 times the lowest maximum residue limit (MRL) or tolerance to 2 times the highest MRL or tolerance considering the Codex and EU MRLs and the US tolerances in the various tissues and milk studied. Incurred tissues were analyzed to verify the precision of the method. The data demonstrated linearity of matrix-matched calibration curves using a weighted (1/×) regression. Recoveries varied from 83.3 to 107.1%. Repeatability precision (RSDr) ranged from 0.465 to 13.4% and intermediate precision (RSDi) ranged from 2.24 to 14.7% in fortified tissue. Repeatability of the method was verified in incurred tissues, ranging from 3.41 to 16.0%. The limits of detection and quantitation of the method are presented and vary by matrix. One confirmatory transition ion was examined across all matrixes and met US and EU criteria for mass spectrometry confirmation. The method was shown to be robust when small changes in method parameters were made, and stability of the analyte in fortified tissues, extracts, standard solutions, and matrix-matched standards was estimated. The data satisfy the requirements of the AOAC Stakeholder Panel for Veterinary Drug Residue Methods for single-laboratory validation studies and the U.S. Food and Drug Administration Center for Veterinary Medicine Guidance for Industry #208 (VICH GL49). The LC-MS/MS method was demonstrated to be suitable for determination and confirmation of tilmicosin residues in bovine, swine, chicken, and turkey tissues and bovine milk based on Codex and EU MRLs and US tolerances.

Determination of Tilmicosin by Fluorescence-Based Immunochromatography

ABSTRACTTilmicosin has been widely used in the dairy industry, but this compound is hazardous to human health. Therefore, it is urgent to develop a rapid, convenient, sensitive, cost-effective, and user-friendly method to determine tilmicosin. A fluorescent microsphere-based immunochromatographic assay was developed for this analyte. A 200-nm red fluorescent microspheres were selected to be the label, and the mass of the labeled antibody was 4.48 µg/mL. 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide was used to catalyze carboxyl groups on the surface of the microspheres. The optimum reaction time between the antibodies and fluorescent microspheres was 30 min, and under these conditions, high coupling ratios and stable conjugations were obtained. The linear dynamic range of the developed protocol was from 0.02 to 2.10 µg/L. The half maximal inhibitory concentration was 0.19 µg/L. The recoveries were from 87 to 98% with a coefficient of variation less than 7.14%. In comparison with a colloidal gold immunochromatographic assay, the developed method offered a higher coupling rate, lower amounts of antibodies, and higher sensitivity. This approach is simple, sensitive, and suitable for on-site applications on farms for rapid screening.

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.

Determination of Tilmicosin in Bovine and Porcine Sera by Liquid Chromatography

A liquid chromatographic (LC) assay is described for determining tilmicosin in bovine and porcine blood sera. Tilmicosin is isolated from the serum matrix and purified by solid-phase extraction with C18 sorbent. Sample is analyzed by LC using a gradient system with a phenyl reversed-phase column that separates tilmicosin from the matrix in 30 min. Tilmicosin is measured by UV absorbance at 280 nm. Validation of assay included evaluation of accuracy, precision, linearity, specificity, sensitivity, range, and sample stability. The method has a limit of quantitation of 0.1 ppm and a validated range of 0.1 to 10.0 ppm. Recoveries were 91-95% for bovine serum and 85-93% porcine serum. The limit of detection was 0.05 microgram/mL. Limits of detection and quantitation were based on 3 and 6 times the baseline noise of control serum samples, respectively. Relative standard deviations of precision samples (n = 6) were 2% or less for both sera. The method has better specificity and analysis time than previous microbiological methods for tilmicosin in sera.

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%.

Determination of Tilmicosin in Bovine Milk by Liquid Chromatography with Ultraviolet Detection

A method is described for determining tilmicosin in bovine milk by liquid chromatography with ultraviolet (UV) detection. Samples are defatted by centrifugation. The lower skim layer is cleaned on a C18 solid-phase extraction cartridge. The extract is concentrated and analyzed with a reversed-phase phenyl column with UV detection at 280 nm. The method was validated with control milk fortified at 50, 100, and 200 ng/mL. Average recoveries (and intralaboratory coefficients of variation) were 97% (9%), 98% (5%), and 101% (3%), respectively. The limit of quantitation is approximately 20 ng/mL, and the limit of detection is approximately 13 ng/mL. The method was tested on milk from a cow dosed with tilmicosin.

Microbiological Plate Assay for Determination of Tilmicosin in Bovine Serum

A microbiological agar plate assay is described for determination of tilmicosin in bovine blood serum. The serum or serum dilution is added directly to wells cut in the agar plates. Tilmicosin activity is determined by measuring the zone of bacterial growth inhibition in agar medium inoculated with Micrococcus luteus, ATCC 9341. The assay was validated by evaluating the following parameters: accuracy, precision, linearity, parallelism, ruggedness, storage stability, and relative activity of isomers. Accuracy was evaluated with freshly collected bovine serum and with commercially available sera. Recoveries ranged from 93.4 to 97.5% across a fortification range of 0.08 to 1.28 micrograms/mL. Precision was estimated over a 6-day period with serum obtained from a tilmicosin-treated animal. Relative standard deviations were 0.63 to 3.13% within day and 5.23% across 6 days. Standard curves were linear with little variation in slope. No parallelism was observed between tilmicosin in serum and tilmicosin in buffered saline. The limit of detection was estimated to be 0.05 micrograms/mL, and the validated limit of quantitation was 0.08 micrograms/mL. Ruggedness was evaluated with different lots of antibiotic medium, different lots of sera, and different analysts. These variables did not affect method performance. Analyses of tilmicosin in frozen sera demonstrated that tilmicosin is stable for up to 16 days when stored at -20 degrees C. A comparison of the relative microbiological activities of the purified cis and trans isomers of tilmicosin to that of the reference standard indicated no differences in microbiological activities, and showed a parallel response among the 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of tilmicosin in ovine milk using high-performance liquid chromatography.

Tilmicosin is a novel macrolide antibiotic with a wide range of therapeutic uses against gram positive (+ve) and gram negative (-ve) bacteria and mycoplasmae causing pneumonia and mastitis and can be used to treat these diseases in sheep. After its use there may be residues present in ovine milk that interfere with cheese making and processing of other milk products. It is important to monitor for the presence of tilmicosin in ovine milk and a method has been optimized and validated for its determination. Tilmicosin is extracted from milk into methanol. The methanol extract is acidified and non-polar co-extractives removed using hexane followed by carbon tetrachloride. The pH is adjusted to 9.0 and the tilmicosin partitioned into chloroform. The chloroform extract is evaporated to dryness and the residue resuspended in high-performance liquid chromatography (HPLC) mobile phase. Tilmicosin is determined using reversed-phase HPLC and ultraviolet (UV) detection at 280 nm. Recovery of tilmicosin from ovine milk fortified over the range 50 to 250 micrograms l-1 is in the range 84.3-104.8%, with a relative standard deviation ranging from 6.6 to 12.9%. The proposed procedure allows the determination of residues of tilmicosin in ovine milk at levels less that 50 micrograms l-1 and satisfies the quality criteria specified in European Commission Decision 93/526/EEC with the exception of reproducibility data from interlaboratory trials.