STABILITY-INDICATING METHODS FOR THE DETERMINATION OF GEMIFLOXACIN IN PRESENCE OF ITS ACID DEGRADATION PRODUCT(S)

3698 | P a g e M a y 2 5 , 2 0 1 5 STABILITY-INDICATING METHODS FOR THE DETERMINATION OF GEMIFLOXACIN IN PRESENCE OF ITS ACID DEGRADATION PRODUCT(S) Ezzat M.Abdel-Moety, Amr M. Badawey, Hebatallah M. Essam, Fatma M. AboulAlamain*. 1 Cairo University, Faculty of Pharmacy, Analytical Chemistry Department, Kasr el Aini St, Cairo, Egypt. eamoety@gmail.com 2 Future University, Faculty of Pharmaceutical Sciences and Pharmaceutical Industries, Pharmaceutical Chemistry Department, 1211, Cairo, Egypt. amrbadawey2014@hotmail.com 3 Cairo University, Faculty of Pharmacy, Analytical Chemistry Department, Kasr el Aini St, Cairo, Egypt. Heba_essam80@hotmail.com 4 The Holding Company for Biological Products & Vaccines VACSERA, Giza, Egypt. Corresponding author E-mail: falamine2004@yahoo.com


Instrumentation
Dual-beam UV-visible spectrophotometer, UV-1650 PC (Shimadzu) with matched 1 cm quartz cells, connected to an IBM compatible personal computer (PC), software is UV Probe version 2.21

2.3.1Standard solution of Gemifloxacin
A standard stock solution of GEM was prepared by transferring accurately 100 mg of pure drug into 100-mL volumetric flask, dissolving in 20 mL methanol and then the volume was completed to the mark with the same solvent to provide standard stock solution containing 1mg mL -1 .

Working solution of Gemifloxacin
GEM working solution was prepared by transferring 10 mL of the standard stock solution into100-mL volumetric flask and then the volume was completed to the mark with methanol to obtain standard working solution containing 0.1mg mL -1 of GEM, and then further dilution was applied to reach final concentration of 10μg mL -1 .

Stock solution of acid degraded GEM
Accurately weighed 100 mg of pure GEM was transferred to a 250-mL round bottomed flask and 25 ml of 3N HCL was added, and then refluxed with continuous magnetic stirring for 10 hrs. The solution was neutralized to pH 7 with 4N NaOH, concentrated nearly to dryness and cooled to room temperature. The residue was dissolved with least amount of methanol, and then filtered, the filtrate was collected into 100 ml measuring flask and the volume was made up with methanol. Complete acid degradation of GEM was confirmed by TLC -fractionation on silica gel F 254 plates using ethyl acetate : methanol : ammonia ( 8:4:3 by volumes as a developing solvent , visualization was carried under UV -lamp 254.0 nm [14] . Then the degradation products were elucidated by mass spectrometry.
Aliquot portion of this solution was diluted with distilled water to prepare working stock solution of 10μg mL -1 .

Construction of calibration curves
Accurately measured volumes of intact GEM working solution (0.1mg mL -1 ) were transferred into a series of 10-mL volumetric flasks and diluted to the mark with methanol to obtain concentrations from 2 to 12 μg mL -1 . These prepared concentrations were used to construct the calibration curve for each theory as follows: M a y 2 5 , 2 0 1 5

For D 1 spectrophotometric method.
The D 1 spectra of each solution was recorded using Δλ = 4 and scaling factor = 20. For determination of GEM in presence of its acid degradation products, calibration curve was obtained by plotting the peak amplitudes of D 1 at 254.6 nm (corresponding to zero-crossing of the degradation products) versus the corresponding drug concentrations, and regression equation was computed.
The absorption spectra of these solutions were divided by the absorption spectrum of 8 μg mL -1 of the acid degradation products (as divisors). The obtained ratio spectra were then differentiated with respect to wavelength usingΔλ= 4 and scaling factor = 1. The peak amplitudes at peak 273.0 nm and trough 284.0 nm were recorded for the determination of GEM in presence of its acid degradation products. The calibration curves representing the relationship between the measured amplitudes and the corresponding concentrations of the drug were constructed and the regression equations were computed.

For dual wave length method.
The Zero-order spectrum of each dilution was recorded against methanol as blank. The absorbance difference of GEM was measured between the selected wavelength pair (271.8 nm & 325.0 nm) for each dilution separately,and then the obtained differences were plotted against the corresponding concentrations of the drug and the regression equation was then computed.

For ratio difference spectrophotometric method:
The zero order spectra of the prepared concentrations of GEM were divided by the spectra of 8 μg mL -1 GEM acid degradation product, the difference in the peak amplitudes at the ratio spectra was measured at 355.0 and 270.0 nm (ΔP) . Calibration graph relating ΔP at the chosen wavelength couples to the corresponding concentrations of GEM was constructed, and the corresponding regression equation was computed.

For bivariate spectrophotometric method.
A concentration series of the acid degradation products was prepared as well. Accurately measured volumes from its working solution were transferred separately into a series of 10-mL volumetric flasks and diluted to the mark with methanol to obtain concentrations from 2 to12 μg mL -1 . The Zero-order spectrum of each dilution was recorded against methanol as blank. The absorbance of GEM and its acid degradation products were measured at 255.0 nm and 277.0 nm for each dilution separately, and then the absorbance at the selected wavelengths were plotted against the corresponding concentrations and the regression equations were then computed.

Laboratory prepared mixtures
Laboratory synthetic mixtures containing GEM and different percentages of its degradation product were prepared by transferring aliquot portions (1 -9 mL) of the GEM working solution in a concentration of (10 μg mL -1 ) into a series of 10-mL measuring flasks. Different portions of the acid degraded solution its concentration is 10 μg mL -1 were also added to prepare different mixtures containing 10 -90% of the degradation products then the volumes were made up with methanol.

Application to pharmaceutical preparation
Ten Factive ® tablets were powdered finely, a weight equivalent to ~ 100 mg GEM was transferred accurately into a 100-mL volumetric flask and sonicated for~20 min with about 50 mL methanol, followed by filtration through filter paper then the volume was completed with methanol to 100 mL. Further dilutions were applied with methanol in order to reach the linearity rang. The general procedures were followed and the concentration of GEM was calculated from its corresponding regression equations.

RESULTS AND DISCUSSION
The focus of the present work was to develop accurate, specific, and sensitive stability indicating methods for the determination of GEM in pure form and in pharmaceutical formulation in presence of its potential acid degradation products.

Separation and identification of degradation products
Accelerated stability studies were applied to GEM to explore its inherent stability characteristics and it was found that GEM is susceptible to hydrolysis under acidic conditions. The expected scheme of acid degradation of gemifloxacin may be illustrated as shown in Scheme (1).The structure of the acid-induced degradation products was confirmed using mass spectral analysis as shown in Figure (2).The zero-order absorption spectra of GEM and its acid degradation products show sever overlapping, Figure (3), which interferes with the direct determination of GEM. This problem suggested that these proposed methods are suitable method for the simultaneous determination of GEM in presence of its acid degradation products.

D 1 method:
The obtained spectra showed good resolutions allowing clear determination of GEM at 254.6 nm without any contribution of its degradation products that show zero crossing at the selected wavelength, Figure (4). Linear calibration curves were obtained for the suggested method in concentration range of (2-12 μg mL -1 ). The regression equation was calculated and found to be: 1 D: Peak amplitude of first derivative. C: Concentration (μg mL -1 ), r: Correlation Coefficient.

1 DD method:
1 DDvalues showed good linearity and reproducibility at a peak 273.0 nm and a trough 284.0 nm without interference from its acid degradation products Figure (5). Linearity of the peak amplitudes of the 1 DD curves at both wavelengths was obtained in the range of 2-12 μg mL

3.4.Dual wavelength method:
Dual wavelength method copes with the level of interference when the spectra as strongly overlapped as shown in Figure  (3), It uses the analytical signal data at two accurately selected wavelengths, thus appropriate selection, thus appropriate selection of wavelength pair is very important. To select the appropriate wavelength pair, the following principles were applied: at the selected wavelengths, the difference in analyte signals had to be linear while the difference in interferent signal is remaining zero with changing the concentration. In addition, the analytical signal obtained from a mixture of the analyte and the interferent should be equal to the sum of the individual signals of the two species. Also, the difference in absorbance due to the analyte signal at the two selected wavelengths should be as large as possible in order to reach good accuracy and sensitivity [26] .
To optimize the method, different pairs of wavelengths were selected and tried to show zero absorbance difference for the GEM acid degradation products with maximum difference in absorbance due to GEM, 271.8 nm and 325.0 nm were selected for the determination of GEM, where the acid degradation product shows the same absorbance. A linear Calibration curve was obtained in the range (2-12 μg mL -1 ) relating the difference between the absorbance at the two selected wavelengths 271.8 nm and 325.0 nm to the corresponding drug concentrations in presence of acid degradation product and the regression equation was calculated and found to be: Δ PGEM = 0.0581 C -0.0253 (r = 0.9998).
Where, Δ P: Absorbance difference at the two selected wavelengths (271.8 and 325.0 nm).

Ratio difference spectrophotometric method:
A smart ratio-difference method has been developed recently with the advantages of minimal data processing and wide range of application, in addition to the advantages of ratio spectra manipulation [27] , Figure (6). The linear regression data for the calibration curve showed a good linear relationship over a concentration range of 2 -12 μg mL -1 and the regression equation was computed and found to ΔP = 0.7113 C + 0.0927 (r = 0.9996). Where, ΔP: Absorbance difference at the two selected wavelengths. C: Concentration (µg mL -1 ), r : Correlation coefficient.

Bivariate Calibration Method
GEM was also determined and resolved from its acid degradation products by using bivariate calibration spectrophotometric method, which is based on a simple mathematical algorithm. The method has been successfully applied to resolve different binary mixtures [28,29] , in which the data is used derives from four linear regression calibration equations, two calibrations for each component at two wavelengths selected using the method of Kaiser [30] , whichwas used for the selection of optimum wavelength set which assured the best sensitivity for the quantitative determination of the studied drug . In order to apply this method, the signals GEM and its acid degradation products located at six wavelengths: 255.0, 270.0, 277.0, 310.0, 320.0, and 340.0 nm were selected. M a y 2 5 , 2 0 1 5 The determinants of these matrices were calculated as shown in Table (1). The wavelength set (255.0 and 277.0) nm was selected for which the highest matrix determinant value was obtained.

Method validation
Method validation is an integral part of the analytical procedure; therefore full validation study was done for each proposed method. Analytical figures of merits were applied in agreement with ICH guidelines [31] by measuring range, accuracy, precision, repeatability, interday precision, linearity, application of dosage form, application of standard addition technique and specificity. Results obtained are depicted in Tables (3-5).

4.CONCLUSION:
The proposed methods are accurate, precise and reproducible. They are stability-indicating methods. These methods complied with the validation guidelines of the International Conference on Harmonization and could be used for purity testing, stability studies, quality control, and routine analysis of GEM either in its bulk powder or in dosage form without noticeable interference from other common dosage form additives. The obtained results were statistically non-significant when they have been compared with those of the reference method.      (4) ** UV-Spectrophotometric method; a methanolic solution of Gemifloxacin was scanned between (200-400 nm), measured at 263.8 nm [4] .