Stock solution EtOH A Stock solution was prepared by dissolving accurately weighed portions of about 666 mg of tobramycin in portions of the mobile phase and diluted to produce 100 ml solution. An appropriate portion of the stock solution was spiked into a blank placebo matrix to produce concentrations of 80, 100, and 120 of the target level. Mean recovery of spiked samples was 99.45 % for tobramycin [Table 2]. Table 2 Accuracy data (analyte recovery): Tobramycin Precision Instrumental precision was determined by six replicate determinations of standard solution and the relative standard deviation was 0.29 for tobramycin. Method precision or intra-assay precision was performed by preparing six different samples involving different weighing.
Each solution was injected in triplicate under same conditions and the mean value of peak area response for each solution was taken. Corrections in area were made for each weight that had been taken to prepare six sample solutions and relative standard deviation of the contents of tobramycin the six sample solutions was calculated. Relative standard deviation was 0.29 for tobramycin. Intermediate precision was performed by analyzing the samples by two different analysts employing different instruments. The standard solution and six different samples at 100% target level were prepared by each analyst. The relative standard deviation obtained from 12 assay results by two analysts was 0.42 for tobramycin. Robustness Robustness of the proposed method was performed by keeping chromatographic conditions constant with the following deliberate variations: (i) change in the column oven temperature (ii) change in flow rate from 1.
0 ml/min to 1.2 ml/min. The standard solution was injected six times in replicate for each minor change. System suitability parameters like peak asymmetry, theoretical plates, capacity factor, and relative standard deviation were recorded for each peak and found to be within acceptable limits. Six test samples at the target concentration level were prepared and analyzed for each change. The percentage difference in assay and relative standard deviations was calculated during each change and found to be �� 0.5% and less than 1.0, respectively. It was noted that slight addition of solvents in the mobile phase affects the method and does not produce results of similar system suitability, as in the proposed method it resulted in poor peak response or no elution of tobramycin peak.
System suitability System suitability tests were performed to chromatograms obtained from standard and test solutions to check parameters such as column efficiency, peak asymmetry, and capacity factor of tobramycin peaks. Results obtained from six replicate injections of standard solution as per the proposed method are summarized in AV-951 Table 3.