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Simultaneous estimation of multiple drug formulations have advantage that the methods are less time consuming and usage of solvent is minimized. Two simple, rapid, precise and accurate spectrophotometric and an isocratic RP - HPLC methods were developed and validated for the estimation of Gemifloxacin mesylate and Ambroxol Hcl in mixture and in combined tablet dosage form. The methods employed for the analysis of Gemifloxacin mesylate and Ambroxol Hcl were
The solubility of Gemifloxacin mesylate and Ambroxol was determined as per Indian Pharmacopoeia. The numerous polar and non - polar solvents were tried to dissolve the drugs. From the solubility profile Distilled water was chosen as a common solvent for the estimation of Gemifloxacin mesylate and Ambroxol. The solubility data is shown in Table 1 for Gemifloxacin mesylate and Ambroxol respectively.
From the working standard solutions 10 ïg /ml of Gemifloxacin mesylate and Ambroxol in distilled water prepared separately and the solutions were scanned in UV region in the wavelength range from 200 to 400 nm by using distilled water as blank it is shown in the fig and respectively
The drugs were dissolved in distilled water to produce 10 µg / ml. Scanned in the range of 400-200 nm and it shows constant lmax at 266.5 nm for Gemifloxacin mesylate and 244 nm for Ambroxol Hcl and overlain spectra was made. This is shown in Fig- and respectively. Stability of absorbance at their lmax was also checked.
The linearity of Gemifloxacin mesylate and Ambroxol was constructed in the range of 4-24 and 1-5 µg / ml and their calibration curves were shown in the Fig. to respectively. The optical characteristics such as Beer's law limit (4-24 and 1-5µg / ml), molar extinction co-efficient, sandell's sensitivity, correlation co-efficient, slope and intercept were calculated and are shown in Table-
The Limit of detection and the Limit of quantification were determined from the linearity studies which was done 6 times and calculated by using slope and standard deviation of response (Intercept).
The formulation G-CIN A TAB was selected for analysis. The amount present were determined by calculating the average of six replicate analysis and its percentage purity was found to be in the range of 98-102 % by the three methods. It is shown in Table- and respectively.
To evaluate the accuracy of the method, recovery studies were carried out, known amount of pure drug was added to the pre-analyzed solution containing formulation and the mixture was re-analyzed by the proposed methods, and their recoveries were calculated. The percentage recovery of Gemifloxacin mesylate and Ambroxol Hcl in the formulation G-CIN A TABwere found to be in the range of 98-102%. These values are shown in Table- and .
Precision of the method was studied by making repeated analysis of the same sample and it was carried out three times in a day and for three days. The % RSD and standard deviation for inter-day and intra day analysis was found to be less than 2 indicates the method is precise, which are shown in Table and .
An involvement was made in this project to device, a simple, accurate, less expensive and sensitive RP-HPLC method of estimation of Gemifloxacin mesylate and Ambroxol Hcl solid dosage form. Since the drug is polar reverse phase high performance liquid chromatography was selected.
SELECTION OF MOBILE PHASE
Acetonitrile was preferred because of its lower viscosity and high UV transparency. Methanol was selected due to its inexpensiveness. Acetonitrile: Methanol: Water in different ratios were tried and no elution of gemifloxacin was found. Hence choice of Tri fluro acetic acid was incorporated. An attempt was made in 0.1% Trifluro acetic acid instead of water in different ratio, this also gave good peaks. Hence Methanol: Acetonitrile : 0.1% Trifluro acetic acid: in the ratio 20:25:55 this gave sharper peaks and it was selected as mobile phase.
The detection wavelength was measured by scanning the solution of Gemifloxacin mesylate and Ambroxol Hcl in mobile phase. In UV-spectrophotometry, over lining spectra and select the wavelength of maximum absorption was selected as 248 nm.
The limit of detection and the limit of quantification were determined by using slope and standard deviation and it was calculated. The system suitability parameters such as theoretical plate, Tailing factor, Asymmetric factor and Resolution were calculated and shown in Table-, the parameters were found to be satisfactory as per guidelines.
With the optimised chromatographic conditions, stock solutions of Gemifloxacin mesylate and Ambroxol Hcl were prepared in HPLC water and the final dilution with mobile phase and prepared the mixture in the concentration range 4-24 µg / ml of Gemifloxacin mesylate and 1-6 µg / ml of Ambroxol Hcl. 20 µl of each solution was injected and records the chromatogram at 248 nm.
The chromatogram as Fig.. The calibration curve was plotted using concentration against peak area. The procedure was repeated for three times. The correlation coefficient was found to be above 0.99985 and 0.99956 Gemifloxacin mesylate and Ambroxol Hcl. The calibration graph of Gemifloxacin mesylate and Ambroxol Hcl are shown in Fig. and respectively. The optical characteristics of Gemifloxacin mesylate and Ambroxol Hcl shown in Table-
The tablet dosage form G-CIN A TAB was selected for the analysis. The ostensible concentration 8 µg / ml of Gemifloxacin mesylate and 2 µg / ml of Ambroxol Hcl in the mobile phase were prepared. 20 µl of each solution was injected and chromatograms were recorded. The percentage purity was found to be 99.85% for Gemifloxacin mesylate and 100.08 % of Ambroxol Hcl respectively.
The precision of the method was confirmed by repeatability of formulation for six times and the chromatograms are shown in Fig.25-30. The percentage RSD were found to be 0.21104 and 0.37361 for Gemifloxacin mesylate and Ambroxol Hcl respectively. The data is shown in Table-.
The accuracy of the method was performed by recovery studies to the pre analysed formulation, a known quantity of Gemifloxacin mesylate and Ambroxol Hcl raw material solutions were added at different levels, injected the solutions. The chromatograms were recorded as shown in the Fig.. The percentage recovery was found to in the range between 98.7-100.48% for Gemifloxacin mesylate and 99.7-100.2% for Ambroxol Hcl. The percentage RSD was found to be 0.89919 and 0.25116 for Gemifloxacin mesylate and Ambroxol Hcl respectively. The low percentage of RSD values for recovery indicated that the method was found to be accurate. The values given in the Table-. The high percentage recovery revealed that no interference was produced due to the excipients used in formulation. Therefore developed method was found to be accurate.
All the above parameters with the ease of operations ensure that the projected methods could be applied for the routine analysis of Gemifloxacin mesylate and Ambroxol Hcl pure form and in tablet dosage form.
SUMMARY AND CONCLUSION
Gemifloxacin mesylate and Ambroxol hydrochloride in combination used in treatment of lower respiratory tract infections in adult patients.
A rapid, precise and accurate analytical method for the simultaneous estimation of gemifloxacin mesylate and ambroxol hydrochloride in formulation was developed (G-CIN A TAB)
G-CIN A TAB has been selected for study
G-CIN A (Containing 320 mg of gemifloxacin mesylate and 75 mg of Ambroxol hydrochloride)
The methods proposed for this combination of drugs are
UV SPECTROSCOPIC METHOD:
UV spectrophotometric method for the estimation of gemifloxacin mesylate and amroxol hydrochloride in combined dosage form by
Simultaneous equation method
Absorbance correction method
First order derivative method
From the solubility data , Distilled water is used as common solvent. Gemifloxacin mesylate and Ambroxol Hcl were prepared seperatlely (10 mcg/ml) and scanned in UV region.From the overlain spectra, by the observation of spectral characteristics of gemifloxacin mesylate and ambroxol were selected for simultaneous estimation, absorbance correction method and Derivative spectroscopic method. The wavelengths selected for simultaneous estimation method were 266.5nm and 244 nm For the Absorbance correction method the wavelengths selected were 371 nm and 244 nm. In the same way the wavelengths selected for the first order derivative spectroscopic method are 276 nm and 220.5 nm.
SIMULTANEOUS EQUATION METHOD
The percentage label claim present in tablet formulation was found to be 100.37% and 99.65 % for Gemifloxacin and Ambroxol Hcl repectively. The percentage recovery was found to be in the range of 99.9 - 100.29% and 99.91- 100.3% for Gemifloxacin mesylate and Ambroxol Hcl.
ABSORBANCE CORRECTION METHOD
The percentage label claim present in tablet formulation was found to be 99.07% and 100.47% for Gemifloxacin and Ambroxol Hcl repectively. The percentage recovery was found to be in the range of 98.6 - 99.06% and 99.49- 101.57% for Gemifloxacin mesylate and Ambroxol Hcl.
DERIVATIVE SPECTROSCOPIC METHOD
The percentage label claim present in tablet formulation was found to be 100.03% and 100.02% for Gemifloxacin and Ambroxol Hcl repectively. The percentage recovery was found to be in the range of 99.93 -100.64 % and 99.74- 100.37% for Gemifloxacin mesylate and Ambroxol Hcl
RP - HPLC METHOD:
The drug samples containing gemifloxacin mesylate and ambroxol hydrochloride in combined dosage forms were analyzed by UV-spectrophotometric method using distilled water as a solvent and the contents of drug were determined in each formulation( G-CIN A TAB) were found to be satisfactory.
Simultaneously, RP-HPLC method has been developed for the estimation of both drugs in bulk and in formulation. The proposed method gives reliable assay results with short analysis time, using mobile phase Acetonitrile: Methanol: 0.1% Tri fluro acetic acid in the ratio of 20:25:55. The contents of drug present in the formulation were found to be satisfactory and system suitability parameters are in desired limit.
All the above methods do not suffer from any interference due to common excipients. It indicates that methods were accurate. Therefore the proposed methods could be successfully applied to estimate commercial pharmaceutical products containing Gemifloxacin mesylate and Ambroxol Hcl.
Thus the above studies findings would be helpful to the analytical chemists to apply the analytical methods for the routine analysis of the analyte in pharmaceutical dosage forms after their approval from FDA.