Keywords
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| Gatifloxacin , Loteprednol, Buffer pH 3.5 And Acetonitrile. |
INTRODUCTION
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| A study of the interaction of light (or other electromagnetic radiation) with matter is an important and versatile tool for the chemist. Indeed, much of our knowledge of chemical substances comes from their specific absorption or emission of light. In this experiment, we are interested in analytical procedures based on the amount of light absorbed (or transmitted) as it passes through a sample.1 Loteprednol and Gatifloxacin Ophthalmic Suspension is a combination eye drops, which contains antibiotic Gatifloxacin & an anti-inflammatory agent Loteprednol. ZYLOPRED name which contain these two drugs. Gatifloxacin is an antibiotic of the fourth-generation fluoroquinolone family that like other members of that family inhibits the bacterial enzymes DNA gyrase and topoisomerase IV. Bristol-Myers Squibb introduced Gatifloxacin in 1999 under the proprietary name Tequin® for the treatment of respiratory tract infections, having licensed the medication from Kyorin Pharmaceutical Company of Japan. Its Chemical name is (±) - 1- cyclopropyl-6-fluoro-1, 4- dihydro- 8- methoxy- 7- ( 3 –methyl - 1- piperazinyl )- 4- oxo - 3 quinolinecarboxylic acid sesquihydrate. Its Molecular formula and Molecular weight are C19H22FN3O4 and 402.42 respectevely. The structural formula is: |
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| Its water solubility is 60mg/ml at pH 4. Gatifloxacin is a synthetic broad-spectrum 8- methoxyfluoroquinolone antibacterial agent for oral or intravenous administration. is bactericidal and its mode of action depends on blocking of bacterial DNA replication by binding itself to an enzyme called DNA gyrase, which allows the untwisting required to replicate one DNA double helix into two. Notably the drug has 100 times higher affinity for bacterial DNA gyrase than for mammalian. Gatifloxacin is a broad-spectrum antibiotic that is active against Gram-positive and Gram-negative bacteria. Loteprednol is a white to off-white powder. Loteprednol as the ester Loteprednol etabonate. Its chemical name is chloromethyl 17-[(ethoxycarbonyl) oxy]-11- hydroxy-3-oxoandrosta-1,4-diene-17- carboxylate. Its Molecular formula and Molecular weight are C21H27ClO5 and 394.889 respectively. The structural formula is: |
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| Its solubility in water in 4mg/ml. Loteprednol etabonate is structurally similar to other glucocorticoids. However, the number 20 position ketone group is absent. It is highly lipid soluble which enhances its penetration into cells. Loteprednol etabonate is synthesized through structural modifications of prednisolone- related compounds so that it will undergo a predictable transformation to an inactive metabolite.2, 3, 4 there are spectroscopic and Chromatographic methods developed on single Loteprednol and Gatifloxacin 5-20. But up to now there is no HPLC methods develop for simultaneous estimation Gatifloxacin and Loteprednol Analysis of the drug is important for development of drugs in their formulation and their use in therapies, for which we require standard analytical procedures. The USP has published specific guidelines for method validation for compound evaluation. USP defines eight steps for validation: Accuracy, Precision, Specificity, Limit of detection, Limit of quantitation , Linearity and range, Robustness21-22 As quality control process is not static some form of validation/verification should continue till the validated procedure is in use. It should not be a concept that once the method is initially developed and validated it is forgotten. |
MATERIALS AND METHODS:
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| Chromatographic methods offer an advantage in terms of sensitivity and selectivity. These methods can be used for routine analysis of dosage forms where two or more drugs are present together. HPLC method was developed for simultaneous estimation of Gatifloxacin and Loteprednol. |
1. Reagents and Material
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| Loteprednol Gatifloxacin Acetonitrile for HPLC Water for HPLC Potassium Dihydrogen ortho phosphate AR grade Sodium hydroxide AR Grade |
2. Marketed formulation
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| The commercial formulation was purchased from Local pharmacy. Each ZYLOPRED Tablet contains 0.3% w/v Gatifloxacin and 0.5%w/v Loteprednol. |
3. Selection of Mobile Phase
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| After assessing the solubility of drugs in different solvents as well on the basis of literature survey, the standard solution of Gatifloxacin and Loteprednol were injected into the HPLC system by using different solvent systems. Different mobile phases were tried in order to find the best conditions for the separation of both the drugs. It was found that Buffer pH 3.5: ACN give satisfactory results as compared to other mobile Raval phases. Finally, the optimal composition of the mobile phase was determined to be Buffer pH 3.5: Acetonitrile (45:55) which show in table no.1 |
4. Selection of Detection Wavelength
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| The sensitivity of HPLC method that uses UV detection depends upon proper selection of detection wavelength. An ideal wavelength is the one that gives good response for the drugs that are to be detected. In the present study standard drug solutions of 15μg/ml Gatifloxacin and 25 μg/ml Gatifloxacin were, therefore, prepared in solvent mixtures of mixture of Buffer pH 3.5: Acetonitrile (45:55). This drug solution was than scanned in the UV region of 200-400 nm and the spectrum was recorded 275nm which is shown in figure no.1 |
5. Optimized Chromatographic Conditions
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| To optimize the chromatographic conditions, the effect of chromatographic variables such as mobile phase pH, flow rate, and solvent ratio were studied. The resulting chromatograms were recorded and the chromatographic parameters such as capacity factor, asymmetric factor and column efficiency were calculated. The conditions that gave the best resolution, symmetry and capacity factor were selected for estimation show in table no.1 and figure 2-7. |
| 1) Buffer preparation (0.1% triethylamine, pH3.5): |
| 2) Mobile phase: Buffer: acetonitrile 45:55 |
| 3) Flow rate: 1.0ml/mint |
| 4) Wavelength: 275nm |
| 5) Column: 250*4.6mm C18, 5micron (Hypersil BDS) |
| 6) Injection volume: 20 micro liter |
6. Preparation of standard solutions
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| Preparation of buffer pH 3.5 |
| are to be detected. In the present study standard drug solutions of 15μg/ml Gatifloxacin and 25 μg/ml Gatifloxacin were, therefore, prepared in solvent mixtures of mixture of Buffer pH 3.5: Acetonitrile (45:55). This drug solution was than scanned in the UV region of 200-400 nm and the spectrum was recorded 275nm which is shown in figure no.1 |
| 1ml of triethylamine was taken into a 1000ml beaker; add 800ml water and mix. Adjust pH3.5 with 1% orthophosphoric acid. Make up volume with water up to 1000ml. Preparation of mobile phase 450 ml of Buffer (pH 3.5) and 550 ml of Acetonitrile (HPLC grade) were mixed and filtered through 0.45μm filter, Sonicated for 10minutes to degas and used as mobile phase. Use mobile phase as a diluents. Preparation of STD Stock solution of Gatifloxacin: 30mg of Gatifloxacin was taken as working standard into a 100ml volumetric flask. Add 60ml mobile phase and dissolve, make up volume with mobile phase (300 μg/ml) Preparation of STD Stock solution of Lotepredlon: 50mg of Lotepredlon was taken as working standard into a 100ml volumetric flask. Add 60ml mobile phase and dissolve, make up volume with mobile phase (500 μg/ml) |
7. Calibration curve for the 15-45 μg/ml Gatifloxacin and 25-75 μg/ml Loteprednol
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| Appropriate volume of aliquots from standard Gatifloxacin and Loteprednol stock solutions were transferred to same volumetric flasks of 10 ml capacity. The volume was adjusted to the mark with mobile phase give a solution containing 15, 22.5, 30, 37.5 and 45 μg/ml Gatifloxacin and 25, 37.5, 50, 62.5, 75 μg/ml Loteprednol. Each of these mixed standard solutions was chromatographed for 10 minutes run time using mobile phase at 275nm at flow rate of 1 ml/min. The graphs were plotted for peak area vs. concentration for both the drugs. Data is recorded in table no. 2 and figure no. 8, 9 and 10. |
8. Analysis of marketed formulation:
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| Solution-1: Took sample equivalent to 30mg of Gatifloxacin (10ml sample) into a 100ml volumetric flask. Add 60ml of mobile phase and shake for 15 minutes to dissolve. Make up with mobile phase. Filter this solution with 0.45micron membrane filter (This solution contains 300 μg/ml of Gatifloxacin and 500 μg/ml of Lotepredlon) Solution-2: Take 1ml of solution-1 into a 10ml volumetric flask and make up with mobile phase. (This solution contains 30 μg/ml of Gatifloxacin and 50 μg/ml of Lotepredlon)Q |
| The prepared sample solution was chromatographed for 10 minutes run time using mobile phase at 275nm at flow rate of 1 ml/min. From the peak area obtained in the chromatogram, the amounts of both the drugs were calculated by fitting peak area responses into the equation of the straight line representingthe calibration curves for Gatifloxacin and Loteprednol. And result shown in table no 3 and figure no. 11. |
VALIDATION OF PROPOSED HPLC METHOD
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9. System suitability
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| System suitability testing is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such. System suitability test parameters to be established for a particular procedure depend on the type of procedure being validated. System suitability test was carried out to verify that the analytical system is working properly to give accurate and precise results. Standard solution (15μg/ml Gatifloxacin and 25μg/ml Loteprednol) was injected six times and the chromatograms were recorded in table no. 4 and figure no. 12. Acceptance Criteria: The % RSD for area response obtained from six replicate injections of Standard solution should be 2.0 %, Tailing factor should be 2.0, Theoretical plates should be 2000 and Resolution of drug peaks should be 2.0. |
10. Solvent suitability:
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| Recorded in table no.5 |
11. Linearity
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| The linearity of analytical method is its ability to elicit test results that are directly proportional to the concentration of analyte in sample within a given range. The range of analytical method is the interval between the upper and lower levels of analyte that have been demonstrated to be determined within a suitable level of precision, accuracy and linearity. The linearity peak area response was determined by analyzing solutions having concentrations in the range of 15-45 μg/ml and 25-75 μg/ml for Gatifloxacin and Loteprednol respectively from same solution. Peak area of each solution was measured using developed method. Calibration curve of peak area Vs concentration was plotted. The correlation coefficient and regression line equations for Gatifloxacin and Loteprednol were determined. Linearity is recorded in table no.6. |
12. Precision
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| I. Repeatability 6 replicates of standard mixture solution having and Gatifloxacin (15 μg/ml) and Loteprednol (25μg/ml) were prepared and chromatograms were recorded and RSD was calculated and shown in table no. 7. |
| II. Intraday precision Standard solutions containing 15, 30, 45 μg/ml Gatifloxacin and 25, 50, 75 μg/ml Loteprednol were analyzed 3 times on the same day. Chromatogram of each sample was recorded. SD and RSD were calculated and shown in table no. 8. |
| III. Interday precision Standard solutions containing 15, 30, 45 μg/ml Gatifloxacin and 25, 50, 75 μg/ml Loteprednol were analyzed on three different days. Chromatogram of each sample was recorded. SD and RSD were calculated and shown in table no. 9. |
13. Accuracy
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| Accuracy is the closeness of the test results obtained by the method to the true value. Recovery studies were carried out by addition of standard drug to the pre analysed sample at 3 different concentration levels (80, 100 and 120 %) taking into consideration percentage purity of added bulk drug samples. It was determined by calculating the recovery Gatifloxacin and Loteprednol by standard addition method. |
recovery:
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| An accurately weighed powder equivalent to about 30 mg of Gatifloxacin and 50 mg of Loteprednol was transferred to 100 ml volumetric flask; dissolved and the volume was made up to the mark using mobile phase. The solution was sonicated for 20 minutes. The solution was filtered through whatman Filter Paper No.42. First few ml of filtrate were discarded. 1 ml of the solution from above filtrate was diluted to 10 ml with mobile phase. The prepared sample solution was chromatographed for 10 minutes using mobile phase at flow rate of 1 ml/min. concentration of Gatifloxacin and Loteprednol is calculated which is known as pre-analyzed sample.In pre-analyzed sample 80, 100 and 120% of Gatifloxacin and Loteprednol was spiked. Chromatogram of each spiked solutions was taken and total amount of drug was calculated and from which % recovery was calculated. This is shown in table no. 10 and 11. |
14. Limit of Detection (LOD)
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| The LOD is estimated from the set of 6 calibration curves used to determine method linearity. The LOD may be calculated as; LOD = 3.3 x (SD / Slope) Where, SD = the standard deviation of Yintercept of 6 calibration curves. Slope = the mean slope of the 6 calibration curves. |
15. Limit of Quantification (LOQ)
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| The LOQ is estimated from the set of 6 calibration curves used to determine method linearity. The LOQ may be calculated as; LOQ = 10 x (SD / Slope) Where, SD = the standard deviation of Yintercept of 6 calibration curves. |
16. Robustness
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| The robustness of an analytical method was carried out to confirm that the method remained unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage. The standard solution was injected five times for each varied conditions of flow, column temperature, pH, and mobile phase ratio and chromatograms were recorded in table no. 13 & 14. |
Change in Conditions for Robustness
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| Change in flow rate: ± 0.2 ml/min Change in pH: ± 0.2 |
RESULT AND DISCUSSION:
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| High Performance Liquid Chromatographic (HPLC) method has been developed for simultaneous determination of Gatifloxacin and Loteprednol in bulk and dosage form. The linearity range was found to be in the range of 25-75 μg/ml for Loteprednol and 15-45 μg/ml for Gatifloxacin with using mobile phase Buffer pH 3.5: ACN (45:55). Correlation co-efficient for calibration curve Gatifloxacin and Loteprednol was found to be 0.9977 and 0.9983 respectively. The method is simple, accurate, reproducible and short and can be used for simultaneous analysis of Loteprednol and Gatifloxacin. |
Optimization of Mobile phase
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| Conclusion Different mobile phases were tried in order to find the best conditions for the separation of both the drugs. It was found that Buffer pH 3.5: Acetonitrile (45:55) gives satisfactory results as compared to other mobile phases. This is shown in table no.1 and figure 2-7. |
Conclusion:
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| The linearity range was found to be in the range of Gatifloxacin (15-45μg/ml). Loteprednol (25- 75μg/ml). Correlation co-efficient for calibration curve Gatifloxacin and Loteprednol was found to be 0.9977 and 0.9983 respectively. Data is recorded in table no. 2 and figure no. 8,9,10. |
System suitability:
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| Conclusion: The column efficiency was more than 2000 theoretical plates and the tailing factor was less than 2.0 for Gatifloxacin and Loteprednol drugs. Resolution is 4.169. The study concludes the suitability of the HPLC system being use. And data was recorded in table no.4 and figure no.12. |
Solvent suitability:
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| Conclusion: The % RSD of response for both drugs was found to be less than 2. So, it was concluded that proposed mobile phase Buffer pH 3.5: ACN (45:55) is suitable for estimation of Gatifloxacin and Loteprednol in combined dosage form and shown in table no 5 |
VALIDATION
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| Linearity: 6 times chromatogram were taken for all five concentration and data were recorded table no.6 |
Precision
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| I. Repeatability: Discussion: The % RSD for Repeatability of both the drugs was found to be less than 2. So, it was concluded that proposed method for estimation of Gatifloxacin and Loteprednol is précised in nature and shown in table no.7. |
LOD and LOQ:
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| Discussion: The proposed method can detect Gatifloxacin and Loteprednol at very low level .So, it was concluded that the proposed method is very sensitive in nature which shown in table no. 12. |
| Robustness: Discussion: The %RSD in both cases is less than 2.0. The study proves the reliability of test method for minor changes in chromatographic condition and which shown in table no. 13 and 14. |
ACKNOWLEDGEMENT:
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| The author wishes to thanks mates who helped me lot for my work. And how can I forget U.Srinivas, my guide who suggests me in all way. |
Figures at a glance
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