Microbiological Plate Assay for Determination of Tilmicosin in Bovine Serum

Abstract

Abstract 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 μg/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 μg/mL, and the validated limit of quantitation was 0.08 μg/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°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. The validation data demonstrate that this method is a rugged, reliable, and simple assay for tilmicosin in serum.