INTRODUCTION:

Stability indicating methods have playing a vital role in the analytical method validation of pharmaceutical formulations. Gemifloxacin mesylate is a synthetic broad-spectrum antibacterial agent having affinity towards bacterial topoisomerase IV¹. Gemifloxacin is a compound related to the fluoroquinolone class of antibiotics, is available as the mesylate salt in the sesquihydrate form. Chemically, gemifloxacin is (R,S)-7[(4Z)-3-(aminomethyl)-4-(methoxyimino)-1-pyrrolidinyl]-1-cyclopropyl-6-fluoro-1,4-dihydro-4oxo-1,8-naphthyridine-3-carboxylic acid² and the chemical structure was shown in Figure 1. Gemifloxacin mesylate is useful in treatment of chronic bronchitis and mild to moderate pneumonia³. The literature survey revealed that analytical methods reported for the estimation of gemifloxacin mesylate by spectrophotometric^4-9, spectrofluorimetric¹⁰, HPLC^11-15, HPTLC¹⁶ and LC-MS^17-18. The objective of this study was to develop a rapid and sensitive RP-HPLC method for the estimation of gemifloxacin mesylate in bulk drug and in its tablet formulation.

MATERIALS AND METHODS:

Reagents and chemicals:

Gemifloxacin mesylate was provided as gift sample from Sumages Pharma Pvt. Ltd., Bhimavaram, India. Gemifloxacin mesylate tablets were purchased from local market. HPLC grade methanol and ammonium acetate were purchased from Merck Specialities Pvt. Ltd., Mumbai. HPLC grade water obtained from Milli Q water purification system was used throughout the study and all the other reagents used were of analytical reagent grade. All dilutions were performed in standard volumetric flasks.

Figure 1: Structure of gemifloxacin mesylate

Chromatographic conditions:

The HPLC system consisted of Waters equipped with a 2695 binary pump and a 2487 dual absorbance detector having Waters Empower2 software and Rheodyne injector with 20 µl fixed loop. The analytical column was a Symmetry C₁₈, with 150 x 4.6 mm, 5 μm particle size. The isocratic mobile phase consisted of acetate buffer (pH 3.5 adjusted with Glacial acetic acid) and methanol (38:62, v/v) was run at a flow rate of 1ml/min. Detection was accomplished with an UV detector at 273 nm.

Preparation of standard solution:

Accurately weigh and transfer 10 mg of gemifloxacin mesylate working standard into a 10 ml volumetric flask, add about 7 ml of diluent, sonicate to dissolve it completely and make volume upto the mark with diluent. Further pipette 0.3 ml of the above stock solution into a 10 ml volumetric flask and dilute upto the mark with diluent. Mix well and filter through 0.45 µm filter.

Preparation of sample solution:

Twenty tablets were finely powdered and quantity equivalent to one tablet was accurately weighed. Weigh and transfer the sample equivalent to 10 mg of gemifloxacin mesylate into a 10 ml volumetric flask, add about 7 ml of diluent, sonicate to dissolve it completely and make volume upto the mark with diluent. Further pipette 0.3 ml of the above stock solution into a 10 ml volumetric flask and dilute upto the mark with diluent. Mix well and filter through 0.45 µm filter. Inject 20 µl of the above solutions into the HPLC system. All experiments were conducted in triplicate.

Calibration curve for gemifloxacin mesylate:

Appropriate aliquots of standard stock solution were taken in different 10 ml volumetric flasks and diluted up to the mark with diluent to obtain final concentrations of 10, 20, 30, 40 and 50 µg/ml of gemifloxacin mesylate respectively. The solutions were injected using a 20 µl fixed loop system and chromatograms were recorded. Calibration curve was drawn by plotting average peak area versus concentrations as shown in Figure 2. The linearity table of gemifloxacin is shown in Table 1.

Table 1. Linearity of gemifloxacin mesylate

Concentration(µg/ml)	Area
10	1052098
20	2192790
30	3285161
40	4310106
50	5573770

Degradation studies:

Standard gemifloxacin mesylate at a concentration of 20 mg was used in all the degradation studies. The standard gemifloxacin were subjected to stress conditions in 0.1 M HCl and 0.1 M NaOH, at the temperature 60°C at different time intervals, after completion of the degradation processes, the solutions were neutralized and diluted with mobile phase. For the peroxide degradation studies, standard gemifloxacin of 20 mg was dissolved in 3% hydrogen peroxide and subjected to stress condition at the temperature 60°C at different time intervals. Thermal degradation was performed by exposing solid standard drug to dry heat at 105°C for 48 hrs. These degraded solutions were withdrawn periodically and subjected to analysis after suitable dilution with mobile phase to get 40 μg/ml. 20 μl of this degraded solution were injected using the same chromatographic conditions.

Method development and validation:

A variety of mobile phases were investigated in the development of an HPLC method suitable for analysis of gemifloxacin mesylate in the bulk drug and in the formulation. The suitability of the mobile phase was decided on the basis of the sensitivity of the assay, suitability for stability studies, time required for the analysis, ease of preparation and use of readily available cost-effective solvents. The analytical method validation was carried out as per ICH method validation guidelines. The validation parameters addressed were linearity, limit of detection, limit of quantitation, accuracy, precision (intra-day and inter-day), selectivity, specificity, robustness and stability of gemifloxacin in mobile phase. Consequently, the following were performed.

Linearity:

The linearity range was found 10-50 µg/ml. The response for the drug was linear and the regression equation was found to be y=-65413+111606.6x and correlation coefficient was found to be 0.999.

Limit of detection (LOD) and limit of quantitation (LOQ):

The limit of detection and limit of quantitation for gemifloxacin mesylate were found to be 0.012 and 0.042 µg/ml, respectively.

Accuracy:

The percentage recovery ranges from 100-102 %. The results show that there is no interference from excipients for the proposed method, thus making the method simple, less time consuming and suitable for routine quantitative estimation of gemifloxacin mesylate in tablet formulation. The recovery study data is given in Table 2.

Precision:

Intra-day and Inter-day precision of the gemifloxacin mesylate samples were analyzed five times in the same day and for three consecutive days by different analysts. The precision data was found to be within the limits of acceptance criteria. For the analytical method and system precision the relative standard deviation was found to be 0.25 % and 0.27 % respectively. The data for intra- and inter- day precision is given in Table 3.

Solution stability:

The stability of solution under study was established by keeping the solution at room temperature for 24 hrs. The result showed no significant change in concentration and thus confirms the stability of the drug in the solvent used for the analysis.

Robustness:

Robustness was determined by varying the mobile phase flow rate to ±0.2 ml/min (i.e. 0.8, 1 and 1.2 ml/min), the concentration of acetate buffer in the mobile phase to ±2 parts (acetate buffer: methanol as 36:64, 38:62 and 40:60) and pH of the mobile phase to ±1 (pH 2.5, 3.5 and 4.5). The deliberate changes in the flow rate, mobile phase concentration and the change in pH did not affect the recovery of the drug which indicated the robustness of the method.

Analysis of the marketed formulations:

The proposed method was applied to the determination in gemifloxacin mesylate in pharmaceutical formulatons of two different manufacturers. In both cases assay obtained is more than 99 % and no interference of impurity peak observed in gemifloxacin peak. The results of analysis are given in Table 4.

RESULTS AND DISCUSSION:

Development and optimization of the stability-indicating HPLC method:

An isocratic method validation was found necessary to optimize the separation of major degradation products formed under various stress conditions. The best resolution was achieved with a run of acetate buffer (pH 3.5) and methanol in the ratio of 38:62, v/v at a flow rate of 1 ml/min, the retention time was observed at 2.044 min. The method worked well with the mixture of degradation solutions and was even applicable to tablet formulation. The typical chromatogram of gemifloxacin mesylate was shown in Figure 3.

Figure 2: Calibration curve of gemifloxacin mesylate

Figure 3: Typical chromatogram of gemifloxacin mesylate

Figure 4: Acid degradation chromatogram of gemifloxacin mesylate

Figure 5: Alkali degradation chromatogram of gemifloxacin mesylate

Figure 6: Oxidative degradation chromatogram of gemifloxacin mesylate

Figure 7: Thermal degradation chromatogram of gemifloxacin mesylate

Degradation behavior:

HPLC studies on the combination under different stress conditions indicated the following degradation behavior. The results of analysis are given in Table 5. The typical chromatograms of degradation behavior of gemifloxacin mesylate are shown in Figure 4-7.

Acidic degradation:

The standard drug gemifloxacin at 60°C in 0.1 M HCl was found to be degrade 7.01 % at 1 hour, the degradation product was appeared at retention 2.037 min was identified.

Alkaline degradation:

The gemifloxacin drug was found to be degrade in alkaline hydrolysis of 0.1 M NaOH at 60°C and degraded to an extent of 9 % at 1 hour, the degradation product was appeared at retention 2.035 min was identified.

Oxidative degradation:

The drug gemifloxacin was found to be labile to hydrogen peroxide at 60°C and 18 % of the drug was decomposed at 1 hour, the degradation product was appeared at retention 2.019 min was identified.

Thermal degradation:

In the thermal degradation, more degradation was seen on subjecting the drug to dry heat 105°C for 48 hrs and 13 % of the drug was decomposed, the degradation product was appeared at retention 2.038 min was identified.

System suitability:

A system suitability test was performed to evaluate the chromatographic parameters (number of theoretical plates, tailing of the peak) before the validation runs. The results of system suitability parameters were given in Table 6.

CONCLUSION:

In the present work, a new rapid and simple reverse phase high performance liquid chromatographic method has been developed, optimized and validated for the estimation of gemifloxacin mesylate in pharmaceutical formulations using UV detector. The short peak retention time of 2.044 min cuts down on overall time of sample analysis and the method was more cost effective as it utilizes very less quantity of mobile phase. Linearity was found in a range of 10-50 µg/ml with LOD and LOQ of 0.012 and 0.042 µg/ml, respectively. The results of t-test applied to accuracy and precision was achieved. From the extraction efficiency data, the recovery of the active component was found to be quantitative. Selectivity of the method was demonstrated by the absence of any interfering peaks from other coexisting excipient substances at the retention time of the drug. Hence this method can be easily and conveniently used for the routine analysis of the drug in pharmaceutical formulations.

Table 2. Recovery data of gemifloxacin mesylate

Concentration (at specification level)	Peak area	Amount added (mg)	Amount found (mg)	% Recovery	Mean recovery
50 %	1679354	5.0	5.09	101.9
100 %	3321373	10.0	10.0	100.7	101.1 %
150 %	4783711	14.4	14.5	100.7

Table 3. Intra- and inter-day precision of gemifloxacin mesylate

Concentration (µg/ml)	Intra-day precision		Inter-day precision
Concentration (µg/ml)	SD	% RSD	SD	% RSD
30 µg/ml	8217.2	0.25	9096.8	0.27

Table 4. Assay result of commercial formulations of gemifloxacin mesylate

Formulation	Labelled strength (mg)	Amount found (mg)	% Assay
Formulation-1	10	9.97	99.7
Formulation-2	10	10	100

Table 5. Degradation studies of gemifloxacin mesylate

Stress conditions	Degradation time	Area of peak	% Degradation	% of active drug present after degradation
Standard Drug	-	3186025	-	-
Acidic	1 hour	2963003	7.01 %	92.99 %
Alkaline	1 hour	2899283	9 %	91 %
Oxidative	1 hour	2612541	18 %	82 %
Thermal	48 hours	2771842	13 %	87 %

Table 6. Summary of validation parameters

System suitability	Results
Theoretical plates (N)	2321
Linearity range (μg/mL)	10-50
Retention time (min)	2.044
Tailing factor	1.6
Correlation coefficient	0.999
LOD (μg/mL)	0.012
LOQ (μg/mL)	0.042

ACKNOWLEDGEMENTS:

The authors are grateful to Sumages Pharma Pvt. Ltd., Bhimavaram for providing authentic sample of gemifloxacin mesylate.

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Received on 03.09.2011 Modified on 11.02.2012

Asian J. Research Chem. 5(2): February 2012; Page 200-204