Novel stability indicating RP-HPLC method for the simultaneous estimation of tobramycin and loteprednol in pharmaceutical dosage forms

A simple, accurate, rapid and precise isocratic stability indicating reversed-phase high-performance liquid chromatographic method has been developed and validated for simultaneous determination of Tobramycin and Loteprednol in combined tablet dosage form. The chromatographic separation was carried out on Zodiac C18 (150 x 4.6mm, 5μ) with a mixture of Phosphate buffer: acetonitrile (60:40%v/v) as a mobile phase at a flow rate of 1.0 mL/min. UV detection was performed at 243nm. The retention times were 2.442 and 3.269 min for Tobramycin and Loteprednol respectively. Calibration plots were linear (r2=0.999) over the concentration range of 3.75-22.5 μg/mL for Tobramycin 6.25-37.5μg/mL for Loteprednol. The method was validated for accuracy, precision, specificity, linearity, robustness, LOD and LOQ. The proposed method was successfully used for quantitative analysis of tablets. No interference from any component of pharmaceutical dosage form was observed. Validation studies revealed that developed method is specific, rapid, reliable, and reproducible. The high recovery and low relative standard deviation confirm the suitability of the method for routine determination of Tobramycin and Loteprednol in bulk and tablet dosage form.


Chemicals and reagents
The reference samples of TOBR and LOTE (API) were obtained from Pulse Pharmaceuticals, Hyderabad. The branded formulations LACNE gel was procured from the local market. Gel claimed to contain 0.3% TOBR and 0.5% LOTE have been utilized in the present work. All chemicals and reagents used were HPLC grade and purchased from Merck chemicals, India.

Chromatographic conditions
Separation was performed on an isocratic waters HPLC 2695 system instrument equipped with a with binary pump and variable wavelength PDA detector with auto injector. Data was analysed by using Empower2 software. Degassing of the mobile phase was done by using bath sonicator. A Shimadzu balance was used for weighing the materials. The separation was achieved on a Zodiac C18 (150 x 4.6 mm, 5) analytical column. The mobile phase consisted of phosphate buffer: acetonitrile (60:40%v/v). The flow rate was 1.0 mL/min and UV detection was performed at 243 nm. The mobile phase was shaken on an ultrasonic bath for 30 min. The resulting transparent mobile phase was filtered through a 0.45 µ membrane filter (Millipore, Ireland). The injection volume was 10 µL and all the experiments were performed at ambient temperature.

Preparation of Standard stock solutions
Accurately weighed 3.75mg of TOBR and 6.25mg of LOTE standard drugs was transferred 25ml clean and dry volumetric flask containing 3/4 th volume of diluent and sonicated for 10 minutes. Flask was made up with diluent and labeled as Standard stock solution. (150µg/ml of TOBR and 250µg/ml of LOTE).

Preparation of Sample stock solutions
20 tablets were weighed and the average weight of each tablet was calculated, then the weight equivalent to 1 tablet was transferred into a 100 ml volumetric flask, 50ml of diluents was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC filters (150µg/ml of TOBR and 250µg/ml of LOTE).

Method validation
The developed method was validated according to ICH guidelines. The system suitability was evaluated by six replicate analysis of TOBR and LOTE mixture at concentrations of 1000 µg/mL and 100µg/mL. The acceptance criteria are number of theoretical plates (N) at least 2000 per each peak and tailing factor is not more than 2.0.

Linearity
Standard calibration curves were plotted against the concentration ranging from 3.75-22.5 µg/mL for TOBR and 6.25-37.5 µg/mL for LOTE. Different linearity levels were prepared and injected into the HPLC system keeping the injection volume constant.

Precision
Precision of assay was determined by System and Method Precision. Every sample was injected six times. The repeatability of sample application and measurements for peak area were expressed in terms of %RSD.

Specificity
All chromatograms were examined to determine whether compound of interest co-eluted with each other or with any additional excipient peaks. Marketed formulation was analysed to determine the specificity of the optimized method in presence of common excipients.

Limit of detection and limit of quantification
Limit of detection (LOD) and limit of quantification (LOQ) were estimated from signal-to-noise ratio. LOD and LOQ were calculated using 3.3 σ/s and 10 σ/s formulae, respectively. Where, σ is the standard deviation of the peak areas and S is the slope of the corresponding calibration curve.

Robustness
To evaluate robustness of HPLC method a few parameters were deliberately varied. The parameters included are variation of flow rate and Detection Wavelength.

Force Degradation studies
Oxidation To 1 ml of stock solution, 1 ml of 20% hydrogen peroxide (H2O2) was added separately. The solutions were kept for 30 min at 60 0 c. For HPLC study, the resultant solution was diluted to obtain 100µg/ml and 10µg/ml solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Acid Degradation Studies
To 1 ml of stock solution of TOBR and LOTE, 1 ml of 2N Hydrochloric acid was added and refluxed for 30mins at 60 0 c .The resultant solution was diluted to obtain 100µg/ml and 10µg/ml solution and 10 µl solutions were injected into the system and the chromatograms were recorded to assess the stability of sample.

Alkali Degradation Studies
To 1 ml of stock solution of TOBR and LOTE, 1 ml of 2N sodium hydroxide was added and refluxed for 30mins at 600c. The resultant solution was diluted to obtain 100µg/ml and 10µg/ml solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Dry Heat Degradation Studies
The standard drug solution w a s placed in oven at 105 0 c for 6 h to study dry heat degradation. For HPLC study, the resultant solution was diluted to 100µg/ml and 10µg/ml solution and10µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Photo Stability studies
The photochemical stability of the drug was also studied by exposing the 300µg/ml, 10µg/ml and 25µg/ml solution to UV Light by keeping the beaker in UV Chamber for 7days or 200 Watt hours/m 2 in photo stability chamber . For HPLC study, the resultant solution was diluted to obtain 100µg/ml and 10µg/ml solutions and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Neutral Degradation Studies
Stress testing under neutral conditions was studied by refluxing the drug in water for 6h r s at a temperature of 60º. For HPLC study, the resultant solution was diluted to 100µg/ml and 10µg/ml solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Results and discussion
During the optimization of HPLC method, two columns symmetry C-18 and C-8 analytical column (4.6×250 mm; 5 µm) and (4.6×150 mm; 5 µm), organic solvent (acetonitrile), one buffer (phosphate) were tested. Initially Water: Acetonitrile and Phosphate buffer were tried in different ratios. Finally mobile phase consisting of mixture of acetonitrile: Phosphate buffer in ratio 40:60% v/v was selected as mobile phase to achieve clear separation and sensitivity. Flow rates between 0.8 to 1.2 mL/min were studied. A flow rate of 1.0 mL/min gave an optimum signal to noise ratio with reasonable separation time using a C18 Zodiac column (4.6×150 mm; 5 µm), the retention times for TOBR and LOTE were observed to be 2.442 and 3.269 min respectively. Total run time was less than 7 min. The chromatogram at 243 nm showed a complete resolution for all peaks (Fig. 3).

Figure 3 Typical chromatogram of standard for TOBRA and LOTE
Validity of the analytical procedure as well as the resolution between different peaks of interest is ensured by the system suitability tests. All critical parameters tested meet the acceptance criteria on all days. As shown in chromatogram, two analytes are eluted by forming symmetrical peaks.
Linearity was obtained for TOBR and LOTE in the range of 3.75-22.5 µg/mL and 6.25-37.5 µg/mL. The correlation coefficient (r 2 ) was found to be greater than 0.999 in all instances. The results of calibration studies are summarized in Table 1. The proposed method afforded high recoveries for TOBR and LOTE in dosage form. Results obtained from recovery studies presented in Table 2 indicate that this assay procedure can be used for routine quality control analysis of binary mixture in sample. Precision of the analytical method was found to be reliable based on %RSD (<2%) corresponding to peak areas and retention times. As can be seen in Table 3 the %RSD values were less than 2 for System and Method precision. Hence, the method was found to be precise for these two drugs. The chromatograms were checked for appearance of any extra peaks under optimized conditions, showing no interference from common excipients and impurities. Also the peak areas were compared with standard and percentage purity calculated was found to be within limits. LOD and LOQ were found to be 0.07µg/mL and 0.21µg/mL for TOBR, 0.15µg/mL and 0.45µg/mL for LOTE. In all deliberately varied conditions, the %RSD for replicate injections of TOBR and LOTE were found to be within the acceptable limit. The tailing factors for two peaks were found to be less than 1.5 and the results are shown in Table 4. The validated method was used in analysis of marketed tablet dosage form. The results for the drugs assay showed good agreement with label claims and the results are shown in Table 5. Degradation studies results were shown in Table 6 and 7.

Conclusion
The developed stability indicating RP-HPLC method is simple, specific, accurate and precise for the simultaneous determination of TOBR and LOTE in combined tablet dosage form. The developed method provides good resolution between TOBRA and LOTE. It was successfully validated in terms of system suitability, linearity, precision, accuracy, specificity, LOD, LOQ and robustness in accordance with ICH guidelines. Thus the described method is suitable for routine analysis and quality control of pharmaceutical preparations containing these drugs either as such or in combination.