Validated UV-Spectrophotometric Methods for Determination of Gemifloxacin Mesylate in Pharmaceutical Tablet Dosage Forms

Two simple, economic and accurate UV spectrophotometric methods have been developed for determination of gemifloxacin mesylate in pharmaceutical tablet formulation. The first UV-spectrophotometric method depends upon the measurement of absorption at the wavelength 263.8 nm. In second area under curve method the wavelength range for detection was selected from 268.5-258.5 nm. Beer’s law was obeyed in the range of 2 to 12 μgmL for both the methods. The proposed methods was validated statistically and applied successfully to determination of gemifloxacin mesylate in pharmaceutical formulation. Keyword: Gemifloxacin mesylate, UV-Spectrophotometry, AUC,


Introduction
Gemifloxacin (R, S)-7(3-aminomethyl-4-syn-methoxyimino-1-pyrrolidinyl)-1-cyclopropyl-6fluro-1, 4 dihydro-4-oxo-1, 2 naphthyridine-3-carboxylic acid is a new fluroquinolone antibacterial compound with enhanced affinity for bacterial topoisomerase IV and has been developed for the treatment of respiratory and urinary tract infection.The compound has a broad spectrum of activity against gram-positive and gram-negative bacteria.Literature survey revealed that few analytical method have been reported for the estimation of gemifloxacin mesylate including utility of σ and π -acceptors for the spectrophotometer determination of gemifloxacin mesylate in pharmaceutical formulation 1 , rapid determination in human plasma by HPLC-MS-MS 2,3 , rapid and sensitive LC method for analysis of gemifloxacin in human plasma 4 , spectrophotometric determination of gemifloxacin mesylate in pharmaceutical formulation trough ion-pair complexation 5 and validated stability indicating assay of gemifloxacin and lomefloxacin in tablet formulation by capillary electrophoresis 6 .Gemifloxacin mesylate is not official in any pharmacopoeia.These developed proposed methods are simple, rapid and sensitive.

Experimental
Pharmaceutical grade gemifloxacin mesylate was supplied by Matrix.Pharma Ltd., Sinner, Maharashtra, India and hydrochlorothiazide was supplied by Ajanta.Pharma, Paithan, Maharashtra, India.The methanol was purchased from Qualligens Fine Chemicals, Mumbai, India.Commercially available tablets Gem one (equivalent to 320 mg of gemifloxacin mesylate) of Dr. Reddy was purchased from market for analysis.

Instrumentation
Shimadzu UV-2450 double beam spectrophotometer with 1 cm path length supported by Shimadzu UV-Probe software, version 2.21 was used for all spectrophotometric estimations.Shimadzu balance (AUW-120D) was used for all weighing.

Preparation of standard solution of gemifloxacin mesylate
Standard stock solution of gemifloxacin mesylate was prepared by dissolving 25 mg of gemifloxacin mesylate in 25 mL of methanol to get concentration of 1.0 mg mL -1 in volumetric flask.Ten mL of stock solution was further diluted to 100 mL with methanol to get a working standard solution of concentration 100 µg mL -1 .This solution used as standard working solution.

Tablet sample preparation
Ten tablets were weighed accurately and powdered.Powder equivalent to 10 mg of gemifloxacin mesylate was weighed and transferred to 100 mL volumetric flask.Then it was dissolved in 25 mL of methanol by shaking the flask for 15 minutes and volume was made up to the mark with distilled water.The solution was filtered through Whatmann filter paper no.41.A 1 mL aliquot of sample stock solution was transferred to 10 mL standard volumetric flask and volume was made up to mark with distilled water.Procedure was repeated five times for analysis of sample solution.

Measurement of UV spectra
From the working stock solutions appropriate dilutions of gemifloxacin mesylate were made with distilled water.Solutions were scanned in the wavelength range of 400 -200 nm and recorded the UV-Spectrum of gemifloxacin mesylate using distilled water in the reference cell.The recorded UV-Spectrum of gemifloxacin mesylate, showed λ max at 263.8 nm (Figure 1).

Linearity
The wavelengths selected should be such that at one the wavelength having same λ max at different concentration of one of the drug, hence from the spectra of gemifloxacin mesylate 263.8 nm were selected as wavelength of detection for proposed method shown in Figure 1.The concentration range over which the drugs obeyed Beer's law was 2 to 12 µg mL -1 for gemifloxacin mesylate.The equation for calibration curve of gemifloxacin mesylate is y= 0.0604x+0.0256and correlation coefficients is 0.9991 (Table 1).0.12 0.12 Limit of quantitation (LOQ), µg mL -1 0.37 0.37

Precision and accuracy
The proposed method was used for the determination of drug in tablets and results indicating satisfactory recoveries and high precision (Table 2).Precision of the method was evaluated with five replicates of standard solution of 10 µg mL -1 of gemifloxacin mesylate.The relative standard deviation value for gemifloxacin mesylate was found to be 0.63 and 1.83 for intra-day and inter-day precision.Inter-day precision and accuracy of the method was tested for 3 days at the same concentration levels.Percentage recoveries for gemifloxacin mesylate are shown in Table 3.

Measurement of UV spectra
UV spectra for the solutions of gemifloxacin mesylate were recorded in a 10 mm cell over the range of 200-400 nm using distilled water in the reference cell.From the working stock solutions appropriate dilutions of gemifloxacin mesylate were made with distilled water.For the estimation of gemifloxacin mesylate by AUC method the wavelength ranges were selected from 268.8-258.8nm (263.8±5nm) for gemifloxacin mesylate (Figure 2).

Linearity
The AUC of each drug was measured in the range of 258.8-268.8nm.The concentration range over which the drugs obeyed Beer's law was chosen 2 to 12 µ g mL -1 for gemifloxacin mesylate.The equation for calibration curve of gemifloxacin mesylate was y= 0.0275x+0.00134and correlation coefficients is 0.9983.The LOD and LOQ values 0.123 µ g mL -1 and 0.374 µ g/mL for montelukast sodium and levocetrizine dihydrochloride showed good precision of gemifloxacin mesylate for the proposed method (Table 1).

Precision and accuracy
The proposed method was used for the determination of drug in tablets and results indicating satisfactory recoveries and high precision (Table 2).Precision of the method was evaluated with five replicates of standard solutions of 10µ g mL -of gemifloxacin mesylate.The percent relative standard deviation (%RSD) values for gemifloxacin mesylate was found to be 0.552 and 0.828 for intra-day and inter-day precision.Interday precision and accuracy of the method was tested for 3 days at the same concentration levels.Percentage recoveries for gemifloxacin mesylate are shown in Table 3.

Conclusion
UV spectrophotometric and AUC methods were developed and validated for the determination of gemifloxacin mesylate in pharmaceutical dosage forms.The selected methods were found to be sensitive, reproducible and accurate for the analysis of gemifloxacin mesylate in tablets.In general, both methods were found to be suitable for routine quality control of the drugs in a tablet dosage form.

Table 2 .
Result of analysis of commercially available tablet of gemifloxacin mesylate byUV-spectrometric and AUC method.

Table 3 .
Accuracy and precision of gemifloxacin mesylate by UV-spectrophotometric and AUC-method.