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Vol. 3 (1) Jan – Mar 2012 www.ijrpbsonline.com 90

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701

___________________________________________Research Paper

Development and Validation of a Dissolution Test with

Spectrophotometric Analysis for Gemifloxacin in Tablet

Dosage Form

Khan Hajera*

Y.B. Chavan College of Pharmacy, Dr. Rafiq Zakaria Campus, Rauza Bagh, Aurangabad, Maharashtra, India. _____________________________________________________________________________________ ABSTRACT The aim of this work was to develop and validate a dissolution test for Gemifloxacin mesylate tablets using spectrophotometric method. The dissolution established conditions were: 900 mL of 0.01M HCl pH 2.0 as dissolution medium, using a paddle apparatus at a stirring rate of 50 rpm. The drug release was evaluated by UV spectrophotometric method at 271 nm for Gemifloxacin mesylate. The method was validated to meet requirements for a global regulatory filing which includes linearity, precision, accuracy robustness and ruggedness. In addition, filter suitability and drug stability in medium were demonstrated. The comparison of the obtained dissolution profiles of tablets, obtained from three different batches (A, B and C) of 320 mg Gemifloxacin mesylate was performed and the results showed no significant difference among the products. Key Words: In vitro release, Stability, Gemifloxacin mesylate, Spectrophotometry, Derivative method, Validation. INTRODUCTION Gemifloxacin (GEM) (Fig. 1A) chemically R,S-7-(3 amino methyl 4- syn methoxyimino-1pyrrolidinyl)-1cyclopropyl-6-flouro1,4,dihydro 4- oxo-1,8 napthyridine-3-carboxylic acid methane- sulphonate1-

3 is a new flouroquinolone antibacterial compound with enhanced affinity for bacterial topoisomerase-IV and is being used for the treatment of respiratory and urinary tract infections, light brown powder, freely soluble in water and slightly soluble in Methanol. Literature survey revealed that few analytical method have been reported for the estimation of gemifloxacin, rapid and sensitive LC method for analysis of gemifloxacin in human plasma4, spectrophotometric determination of gemifloxacin mesylate in pharmaceutical formulation trough ion-pair complexation5and validated stability indicating assay of gemifloxacin and lomefloxacin in tablet formulation by capillary electrophoresis6. Drug absorption from a dosage form after oral administration depends on the release of the drug from the pharmaceutical formulation, the dissolution and/or its solubilization under physiologicalconditions, and the permeability across the gastrointestinal tract. Because of the critical

nature of the first two of these steps, in vitro dissolution may be relevant to the prediction of in vivo performance7. The dissolution test is a very important tool in drug development and quality control. At present time there are no official monograph for Gemifloxacin raw material and tablets and no dissolution test has been described in literature for these drugs. No single method is available for Gemifloxacin mesylate. The present paper describes the development and validation of dissolution test for quality control of Gemifloxacin in immediate release tablets. The best dissolution conditions were used to evaluate development and validation of a dissolution method with dissolutions profile of three different batches of tablets.

N N

F

OO

OH

N NO C H 3

N H 2

Fig. 1A: Structure of Gemifloxacin



MATERIAL AND METHODS Materials Standard gift sample of Gemifloxacin mesylate was provided by Hetero pharma Ltd., Himachal Pradesh. Tablets (Gemone, Gemez) Gemifloxacin mesylate 320 mg were purchased from local market for analysis. All reagents and solvents used were analytical grade. 0.01 M HCl of pH2.0, pH 4.5 sodium acetate, pH 6.8 sodium phosphate and 2.1 simulated gastric fluid buffer solutions were prepared according to USP Pharmacopoeia8. Instrument9

Make: Electrolab Model: TDT-06L Specification: USP Standards tablet dissolution test apparatus multi-bath (n=6) Dissolution test was performed in accordance to USP Pharmacopoeia [8] general method. The medium were vacuum degassed under in house vacuum and were maintained at 37.0 ± 0.5°C by using a thermostatic bath. A double-beam UV-Visible double beam spectrophotometer, make: SHIMADZU (model UV‐1800) with a pair of 1 cm matched quartz cells with spectral band width of 1 nm, was used for all absorbance measurements. Elico pH analyzer (Model: Elico 11610) was used to determine the pH of all solutions. Solubility/stability determination and dissolution test optimization Gemifloxacin solubility was determined in 900 mL of Purified Water, 0.01M HCl, simulated gastric fluid (SGF) pH 2.1, sodium acetate buffer pH 4.5 and sodium phosphate buffer pH 6.8, using an amount of the drug equivalent a three times of dose in the pharmaceutical formulation13. Drug release tests were carried out according to conventional dissolution procedures recommended for single entity products,using paddle(USP Apparatus II) at 25 and 50 rpm. Sampling aliquots of 10.0 mL were withdrawn at0, 5, 10, 20, 30, and 45 minutes, and replaced with an equal volume of the fresh medium to maintain a constant total volume. At the end of each test time, samples aliquots were filtered and diluted with dissolution medium, when necessary, and quantified. The assay of the Gemifloxacin product was performed using previously validated spectrophotometric method [, and the content results were used to calculatethe percentage release on each time of dissolution profile. The cumulative percentage of drug released was plotted against time, in order to obtain the release profile and to calculate the in vitro dissolution data (n=12). The filtration procedure of Gemifloxacin and samples (tablets

dissolved in dissolution medium, n=3) were evaluated using 0.1 μm, 0.2 μm, 0.45 μm cellulose acetate membrane filter (Phenomenex), and quantitative filter. The absorbance of filtered and unfiltered (centrifuged) solutions in dissolution medium was measured using concentration 35.6μg/mL. To assess the stability of Gemifloxacin in dissolution medium, samples were diluted in 0.01 M HCl, and tested after 24 h at room temperature and also kept at 37 ± 0.5 ºC for 2 h after dissolution. The stability of these solutions was studied by comparing values obtained with freshly prepared solutions and the respective result are shown in table 1. METHODS 10

Dissolution Study of Gemifloxacin from Tablets Using Derivative method The release kinetic of Gemifloxacin from Tablets was studied by conducting dissolution tests. Dissolution tests performed using USP type II dissolution apparatus and 900 ml of 0.01M HCL at 37± 0.5 0 C at 50 rpm. 10 ml sample were withdrawn at the intervals of 0,5,10, 20, 30, and 45 min. sampling was carried out and every time replaced with fresh 10 ml with 0.01M HCL. The absorbance of solution was recorded at 271nm using 0.01M HCL as blank (fig 1). The dissolution studies were performed in triplicate (n=3).and result was calculated as % drug release of GEM (fig 2) and table 2. Dissolution Study of Gemifloxacin from Tablets Using Area under curve method Following the above procedure the area of solutions was recorded at 265-276nm using 0.01N HCL as blank .the dissolution studies were performed in triplicate (fig 3) table 3. METHOD VALIDATION The UV spectrophotometric method used to analyze the Gemifloxacin samples in 0.01 M HCl dissolution medium was validated for specificity, linearity, precision, ruggedness and robustness according to USP Pharmacopoeia11 and ICH guidelines12. All absorbance were measured at 271 nm for Gemifloxacin. Linearity The linearity of Gemifloxacin response is evaluated from the range of 10–60 μg/mL and showed a good correlation coefficient. To assess linearity, the standard curves of Gemifloxacin was constructed by plotting concentration (μg/mL) versus absorbance is shown in (fig 4). Linear regression is also calculated and the obtained equation is y = 0.059x - 0.019 where



x is the concentration in μg/mL, y is amplitude for UV spectrophotometry (fig 4). Linearity or calibration curve of GEM. Precision The precision of the method is evaluated by measuring the repeatability in two different UV Vis spectrophotometers have shown %RSD value of GEM. These results demonstrated the good precision of the proposed methods for dissolution test (table 3). Accuracy The accuracy is evaluated by applying proposed method to the analysis of mixture of the tablet and with known amount of the Gemifloxacin working standard, corresponding to the concentrations of 80, 100 and 120%, which were subjected to dissolution test conditions described above. The accuracy was assessed from three replicate determinations of samples containing 35.6 μg/mL of GEM .recoveries obtained with a mean value of 99.97-100.37% for GEM .demonstrated that the method is accurate for intended use. The percent recoveries obtained (Table 4) are considered acceptable Ruggedness Ruggedness of the method is determined by carrying out the analysis by two different analysts and the respective dissolution values are calculated (table 5). Robustness The evaluation of robustness should be considered during the development phase and depends upon the type of procedure under study. It should show the reliability of an analysis with respect to deliberate variations in the method parameters. The solution containing 35.6 mcg/ml of GEM are analyzed under different condition as above and results are represented in (table 6).

Fig. 1: UV Spectrum of Gemifloxacin in 0.01 M HCl

Fig. 2: Derivative Method Graph

Fig. 3: AUC Method Graph

Fig. 4: Calibration curve of Gemifloxacin



Table 1: Stability of Gemifloxacin tablet in different medium at two time point S. No. Dissolution

Medium Stability after 2 h

at 37 ºC (%) Stability after 24 h at room temp (%)

Gemez Gemone Gemez Gemone 1 5 Purified water 99.5 99.2 99.1 99.7 2 01 M HCl 99.02 99.6 99.04 99.4 3 2.1 SGF 98.4 98.4 98.5 98.8 4 pH 4.5 Acetate buffer 98.2 98.1 98.1 99.7 5 pH 6.8 Phosphate buffer 102.02 101.8 102.04 102.3

Table 2A: Calculation by Derivative Method S. No. Sampling Time(Min) Absorbance of

Gemifloxacin at 271nm Percentage Released of

Gemifloxacin (%) Gemaze Gemone Gemaze Gemone 1 5 0.7668 0.6578 14.24 13.09 2 10 1.7209 1.6543 44.73 39.90 3 20 2.8128 2.7645 80.01 79.07 4 30 3.3812 3.3789 99.99 99.98 5 45 3.1923 3.2021 92.96 93.97

Table 2B: Calculation by AUC method S. No. Sampling Time(Min) Area of Gemifloxacin

(265-276) nm Percentage Released of

Gemifloxacin (%) Gemaze Gemone Gemaze Gemone 1 5 3.2133 1.8389 16 9 2 10 7.0171 8.4342 34 41 3 20 15.091 16.012 73 77 4 30 20.532 20.448 100 99 5 45 19.654 19.884 96 98

Table 3: Repeatability and intermediate precision of the dissolution method % Gemifloxacin release

Samples INTER-DAY PRESION BETWEEN EQUIPMET Day 1 Day 2 UV A UV B

Gemez Gemone Gemez Gemone Gemez Gemone Gemez Gemone 1 99.03 99.76 100.07 99.94 100.05 99.84 99.90 99.82 2 99.90 99.45 99.87 99.83 99.98 99.88 99.95 99.99 3 98.98 99.69 99.99 99.57 99.73 100.32 100.45 99.85 4 99.97 100.08 98.96 100.23 99.86 100.08 99.76 100.09 5 100.04 98.92 100.01 100.06 99.99 98.99 99.01 100.06

Mean 99.58 99.58 99.78 99.92 99.92 99.82 99.81 99.96 SD 0.47508 0.38652 0.41511 0.22205 0.11409 0.449552 0.464701 0.109072 SE 0.21246 0.17286 0.18564 0.09930 0.0510 0.2010 0.2078 0.0487

%RSD 0.4770 0.3881 0.4160 0.2226 0.1141 0.4503 0.4655 0.1091 * n=5 SD= standard Deviation %RSD= Relative Standard Deviation SE=Standard Error



Table 4: Results from accuracy as recovery studies

Level of %

Recovery

Amt. Present

(mcg/tab)

Amt. of standard added (mcg/tab)

Total Amt. recovered (mcg)

% Recovery

Gemone Gemez Gemone Gemez Gemone Gemez 80 35.6 28.48 28.48 64.31 64.06 100.37 99.98

100 35.6 35.6 35.6 71.28 71.17 100.12 99.97 120 35.6 42.42 42.42 77.78 77.81 99.95 99.99

Gemone Gemez Mean 100.14 99.98 SD 0.211 0.01 SE 0.1212 0.005

%RSD 0.210 0.010 * n=3 SD= standard Deviation %RSD= Relative Standard Deviation SE=Standard Error

Table 5: Ruggedness results S. No. Analyst 1 Analyst 2

Gemez Gemone Gemez Gemone 1 99.03 99.76 100.05 99.84 2 99.67 98.67 98.62 100.07 3 99.45 100.09 99.54 99.89 4 100.02 99.78 98.34 99.51 5 98.78 99.57 99.87 98.99

Mean 99.39 99.57 99.28 99.66 SD 0.495126 0.538637 0.762843 0.425676 SE 0.2214 0.2408 0.3411 0.1903

% RSD 0.4981 0.5409 0.7683 0.4271 * n=5 SD= standard Deviation %RSD= Relative Standard Deviation SE=Standard Error

Table 6: Robustness result S. No. Filters % Dissolved % Difference

Gemez Gemon Gemez Gemon 1 0.1μm 99.2 99.3 2.4 2.5 2 0.2 μm 99.6 98.9 2.8 2.7 3 0.45μm 100.1 100.2 2.6 2.9

4 Whatmann filter paper 94.8 95.7 6.1 6.4

CONCLUSION A simple dissolution test developed and validated for Gemifloxacin tablets are considered satisfactory. The conditions that allowed the dissolution determination are 900mL of 0.01 M HCl at 37.0 ± 0.5 ºC, paddle apparatus, 50 rpm stirring speed and filtration with 0.45 μ cellulose acetate membrane filters. In these conditions, the Gemifloxacin stability is good. The percent drug delivery is higher than 90% in 30 minutes in evaluated products. Therefore, the proposed method is successfully applied and suggested for the quality control studies of

Gemifloxacin in pharmaceutical dosage forms contributing to assure the therapeutic efficacy of the drug. ACKNOWLEDGEMENTS The authors are very much thankful to the Chairman, Mrs.Fatma Rafiq Zakaria, Maulana Azad Educational Trust,for providing necessary facilities for the project work. The authors are also thankful to Hetero Drugs Ltd. H.P for providing gift samples of Gemifloxacin mesylate.



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5. Krishna MV and Dannana GS. E-J Chem. 2007;5(3):515.

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