|Year : 2013 | Volume
| Issue : 4 | Page : 298-308
A novel validated stability indicating high performance liquid chromatographic method for estimation of degradation behavior of ciprofloxacin and tinidazole in solid oral dosage
Bhupendrasinh K Vaghela, Surendra Singh Rao
Department of Chemistry, JJT University, Jhunjhunu, Rajasthan, India
|Date of Submission||26-Oct-2012|
|Date of Decision||13-Dec-2012|
|Date of Acceptance||25-Apr-2013|
|Date of Web Publication||19-Oct-2013|
Bhupendrasinh K Vaghela
Department of Chemistry, JJT University, Jhunjhunu, Rajasthan
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: The objective of current investigation was to study the degradation behavior of Ciprofloxacin and Tinidazole. The study was performed as per International Conference on Harmonization recommended stress condition. A novel stability-indicating reverse phase HPLC method was developed for the determination of Ciprofloxacin and Tinidazole purity in the presence of its impurities and forced degradation products. This method is also capable to separate placebo peaks as well in pharmaceutical dosage forms. The solid oral dosage form was subjected to the stress conditions such as oxidative, acid, base hydrolysis, heat and photolytic degradation. Materials and Methods: The method was developed using Waters symmetry shield, Reverse Phase (RP) C18, 250mm x 4.6mm, 5΅ as a stationary phase. The mobile phase containing a gradient mixture of solvent A and B. 10mM phosphate buffer, adjusted pH 3.0 with phosphoric acid was used as a buffer. Buffer pH 3.0 was used as solvent A and buffer pH 3.0: Acetonitrile in the ratio of 20: 80 v/v were used as solvent B. The eluted compounds were monitored 278 nm (Ciprofloxacin), 317 nm (Tinidazole). The run time was 50 minute. Results: In the precision study the % RSD for the result of Ciprofloxacin, Tinidazole and its impurities was below 10%. The method was linear with the correlation coefficient greater than 0.997. The percentage recoveries were calculated and observed from 93.0% to 106.7%.The peak purity of Ciprofloxacin, Tinidazole peak had not shown any flag, thus proved the stability-indicating power of the method. Conclusion: The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness.
Keywords: Ciprofloxacin, degradation, related substances, tinidazole, validated
|How to cite this article:|
Vaghela BK, Rao SS. A novel validated stability indicating high performance liquid chromatographic method for estimation of degradation behavior of ciprofloxacin and tinidazole in solid oral dosage. J Pharm Bioall Sci 2013;5:298-308
|How to cite this URL:|
Vaghela BK, Rao SS. A novel validated stability indicating high performance liquid chromatographic method for estimation of degradation behavior of ciprofloxacin and tinidazole in solid oral dosage. J Pharm Bioall Sci [serial online] 2013 [cited 2019 Nov 12];5:298-308. Available from: http://www.jpbsonline.org/text.asp?2013/5/4/298/120082
Ciprofloxacin and tinidazole in tablet formulation (antibacterial) is the combination of a ciprofloxacin a fluoroquinolone, which synthetic broad spectrum antimicrobial agents and Tinidazole is a synthetic antiprotozoal and antibacterial agent. 
Ciprofloxacin is described chemically as: 1-cyclopropyl-6-fluoro-1, 4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylic acid. Its empirical formula is C 17 H 18 FN 3 O 3 and molecular weight is 331.4.
Tinidazole is 1-[2-(ethylsulfonyl) ethyl]-2-methyl-5- nitroimidazole, a second-generation 2-methyl-5-nitroimidazole. Its molecular formula is C 8 H 13 N 3 O 4 S and molecular weight is 247.27.
Each tablet contains ciprofloxacin (500 mg) and tinidazole (600 mg). Ciprofloxacin, tinidazole, and impurities chemical structure is shown in [Figure 1].
Methods are available for estimation of simultaneous estimation of ciprofloxacin hydrochloride and tinidazole in bulk and pharmaceutical dosage. ,,,, A number of high performance liquid chromatographic (HPLC) methods for determination of ciprofloxacin and its metabolites in human plasma are also available. ,,,,,,, A HPLC method available for simultaneous determination of norfloxacin and tinidazole.  Similarly determinations of tinidazole by HPLC methods are also available. ,, Pharmacopeial methods are also available for estimation of both drugs separately. ,,, No analytical method is available, which deals degradation study for combination solid dosage form. Attempts were made to develop a Liquid Chromatographic method for the estimation of degradants and known impurities in ciprofloxacin hydrochloride and tinidazole tablet in the presence of placebo. This paper deals with the validation of the developed method for the accurate quantification of degradants in dosage form.
|Figure 1: Structure of ciprofloxacin and its related compound (I-II). Structure of tinidazole and its related compound (III-V)|
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This paper also deals with the forced degradation of ciprofloxacin and tinidazole solid dosage formulation under stress condition such as acid hydrolysis, base hydrolysis, photolytic, oxidation, and heat.
A reproducible stability indicating HPLC method was developed for the quantitative determination of ciprofloxacin and tinidazole impurities in solid dosage formulation.
| Experimental|| |
Chemicals and reagents
Tablets, ciprofloxacin, and tinidazole working standard and impurities were supplied by Dr. Reddy's laboratories limited, Hyderabad, India. Deionized water was prepared using a Milli-Q plus water purification system from Millipore (Bedford, MA, USA). The HPLC grade acetonitrile, analytical grade KH 2 PO 4 , and ortho-phosphoric acid were purchased from Merck, Mumbai, India.
The Waters HPLC photodiode array (PDA) 2996 system (Waters Corporation, Milford, MA, USA) used consists of a quaternary solvent manager, a sample manager, and a PDA detector. The output signal was monitored and processed using Empower 2 software. The specificity study was conducted by using heating oven (MACK Pharmatech, Hyderabad, India), and water baths equipped with Milli Volt controller (Julabo, Seelbach, Germany) were used for hydrolysis studies.
Photo stability studies were carried out in a photo stability chamber Sanyo, Leicestershire, UK. The pH of the solutions was measured by a pH meter (Mettler-Toledo, Switzerland) all samples were centrifuged by Thermo scientific multifuge machine.
The method was developed using waters symmetry shield, RP18, 250 mm × 4.6 mm, 5 μ, column as stationary phase. 0.01 M phosphate buffer, adjusted pH 3.0 with phosphoric acid was used as a buffer. Buffer pH 3.0 was used as solvent A and buffer pH 3.0 acetonitrile in 20:80 v/v ratios were used as solvent B. The gradient program (T (min)/%B) was set as 0/5, 35/30, 40/5 and 50/5 respectively. The mobile phase pumped at 1.5 mL/min. The eluted compounds were monitored at 278 nm (ciprofloxacin), 317 nm (tinidazole). The column temperature was maintained at 30°C. The sample injection volume was 10 μL.
Preparation of stock solutions
A mixture of buffer pH 3.0 (0.01 M phosphate buffer, adjusted pH 3.0 with phosphoric acid) and acetonitrile in 50:50 v/v was used for diluent to prepare solutions. A stock solution of ciprofloxacin (0.24 mg/mL) and tinidazole (0.4 mg/mL) was prepared by dissolving appropriate amount of drug in diluent. Working solutions of 2.4 and 4.0 μg/mL were prepared from the above stock solution for the related substance determination. A stock solution of impurity mixture of imp-A and imp-B for tinidazole at 0.1 mg/mL and Ethylenediamine (EDA) impurity for ciprofloxacin at 0.1 mg/mL was also prepared in the diluent.
System suitability solution prepared by mixing (1 mg/mL) ciprofloxacin with 10 μg/mL impurities and 1.2 mg/mL tinidazole with 12 μg/mL impurities from above impurity stock.
Preparation of sample solution
Crush not less than 20 tablets to fine powder in a mortar with pestle. Transfer an accurately weighed amount of tablet powder equivalent to about 25 mg of ciprofloxacin to 100 mL volumetric flask. Add about 50 mL of diluent and sonic ate for 15 min, dilute to volume with diluent and mix well.
Filter about 2 mL through 0.45 μm durapore hydrophilic membrane filters or 0.45 μm Pal Pharmalab nylon 66 membrane filter.
Force degradation studies
Forced degradation studies were performed at 250 μg/mL for ciprofloxacin and 410 μg/mL for tinidazole on tablet formulation to provide an indication of the stability indicating property and specificity of proposed method. Peak purity test was carried out for the ciprofloxacin and tinidazole peak by using PDA detector on stress. All solutions used in forced degradation studies were prepared by dissolving the drug product in small volume of stressing agents. After degradation, these solutions were diluted with diluents to yield stated concentration of respective actives. Conditions employed for performing the stress studies were as follows. ,,
Transfer an accurately weighed amount of tablet powder equivalent to about 25 mg of ciprofloxacin to 100 mL volumetric flask, dissolved in 50 mL of diluents then add 5 mL of 0.5 N HCl and mixed. The flask was placed at 50°C in a water bath for 5 h, After 5 h, the flask was removed and placed on bench-top to attain the laboratory temperature, add 5 mL 0.5 N NaOH to neutralized and finally made up to the volume with diluents and mixed well.
Transfer an accurately weighed amount of tablet powder equivalent to about 25 mg of ciprofloxacin to 100 mL volumetric flask, dissolved in 50 mL of diluents then add 5 mL of 0.5 N NaOH and mixed. The flask was placed at 50°C in a water bath for 5 h, After 5 h, the flask was removed and placed on bench-top to attain the laboratory temperature, add 5 mL 0.5 N HCl to neutralized and finally made up to the volume with diluent and mixed well.
Transfer an accurately weighed amount of tablet powder equivalent to about 25 mg of ciprofloxacin to 100 mL volumetric flask, dissolved in 10 mL of water. The flask was placed at 50°C in a water bath for 5 h, After 5 h, the flask was removed and placed on bench-top to attain the laboratory temperature, and finally made up to the volume with diluents and mixed well.
Transfer an accurately weighed amount of tablet powder equivalent to about 25 mg of ciprofloxacin to 100 mL volumetric flask, dissolved in 10 mL of 3% H 2 O 2 . The flask was placed at 25°C in a water bath for 24 h, After 24 h, the flask was removed and finally made up to the volume with diluents and mixed well.
Thermal degradation study
Transfer an accurately weighed amount of tablet powder equivalent to about 25 mg of ciprofloxacin to 100 mL volumetric flask and placed in hot air oven at 80°C for 5 h. After 5 h, the flask was removed and placed on bench-top to attain the laboratory temperature; dissolved in 50 mL diluent. The mixture was then sonicated for about 30 min, finally made up to the volume with diluents and mixed well.
Photolytic degradation study
Transfer an accurately weighed amount of tablet powder equivalent to about 25 mg of ciprofloxacin to 100 mL volumetric flask and placed in photo stability camber and exposed to white florescent lamp with an overall illumination of 1.2 million lux hours and near ultraviolet (UV) radiation with an overall illumination of 200 watt/m 2 /h at 25°C. Following removal of the flask from photo stability chamber and the drugs were finally dissolved in 50 mL diluent. The mixture was then sonicated for about 30 min, finally made up to the volume with diluents and mixed well.
Placebo (without actives, contains only excipint), placebo for ciprofloxacin (containing tinidazole with excipient), and placebo for tinidazole (containing ciprofloxacin with excipient) were prepared in similar way to identify degradation pathways.
The proposed method was validated as per ICH guidelines. 
System suitability was determined before sample analysis from system suitability solution; resolution should be minimum 2.0 between ciprofloxacin Impurity Chemical Reference Substance (CRS) (EDA compound) and ciprofloxacin peak. USP tailing factor less than 2.0 for ciprofloxacin, tinidazole peak from resolution solution. Theoretical plate count for ciprofloxacin, tinidazole peak from resolution solution should be not less than 4000.
Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities and excipients. Placebo interference was evaluated by analyzing the placebo prepared as per test method. No peak due to placebo detected at the retention time of active peaks and their impurities. Peak purity test was carried out for the ciprofloxacin, tinidazole peaks by using PDA detector in stress samples.
The precision of the related substances method was checked by injecting six individual preparations of ciprofloxacin (0.25 mg/mL) and tinidazole (0.4 mg/mL) and its impurities in tablets. Spiked with 1% of imp-EDA for ciprofloxacin and imp-A, imp-B for tinidazole with respect to respective analyte concentration. %Relative Standard Deviation of area for each impurity was calculated.
The intermediate precision of the method was also evaluated using different analyst and different instrument in the same laboratory
Limits of detection and quantification
The LOD and LOQ for impurities and analyte (with respect to unknown impurities) were determined at a signal-to-noise ratio of 3:1 and 10:1, respectively, by injecting a series of dilute solutions with known concentrations. Precision and accuracy studies were also carried out at the LOQ level by injecting six individual preparations of ciprofloxacin and it's known impurities (imp-EDA) and tinidazole and it's known impurities (imp-A, imp-B) and calculating the % RSD of the area.
Linearity test solutions for the related substance method were prepared by diluting stock solutions (1000 ppm) to the required concentrations. The solutions were prepared at five concentration levels from LOQ to 200% of the specification level 0.2%. For tinidazole and its impurities Loq to 4.0 ppm solution has been prepared and for ciprofloxacin and its impurity Loq to 2.5 ppm solution prepared. Correlation coefficient, value for the slope, Y-intercept and % bias of the calibration curve was calculated.
The accuracy study of all impurities was carried out in triplicate at LOQ, 50%, 100%, 125% and 150% of the target concentration level 0.2%. The percentages of recoveries for impurities were calculated.
To determine the robustness of the developed method, experimental conditions were deliberately altered and the resolutions between all peaks were recorded; and system suitability parameters were recorded. The variable evaluated in the study was pH of the mobile phase (±0.2), column temperature (±5°C), flow rate (±0.2).
The solution stability of ciprofloxacin, tinidazole, and its impurities in the related substance method was carried out by leaving spiked sample solutions in tightly capped volumetric flasks at room temperature for 24 h. Content of ciprofloxacin and it's known impurities (imp-EDA) and tinidazole and its known impurities (imp-A, imp-B) determined for 24 h interval up to the study period.
| Results and Discussion|| |
Method development and optimization
The main objective of the chromatographic method was to separate all degradants from both active peaks. The maximum absorption wavelength of the reference drug solution, related substances and force degradation product is 278 nm (ciprofloxacin), 317 nm (tinidazole). Initially a mobile phase composed of 0.01 M KH2PO4 (pH 3.5) and acetonitrile (90:10) (v/v) with a flow rate of 1.0 mL/min over inertsil octadecyl silane (ODS)-3V C18, 150 mm × 4.6 mm, 5 μm column was employed for separation. The EDA peak was not separate from ciprofloxacin peak. The pH of the buffer of mobile phase decreased to 3.0 and gradient elution selected. Peak of EDA and tinidazole resolution decreased. To separates the EDA and tinidazole with sufficient resolution Waters symmetry shield, RP8, 250 mm × 4.6 mm, 5 μ, column used as stationary phase. Resolution between EDA peak and ciprofloxacin peak was greater than 2.5 and EDA peak separates from tinidazole with more than 2.0 resolutions. Finally, the mobile phase containing a mixture of buffer (0.01 M potassium di-hydrogen ortho-phosphate pH adjusted to 3.0 with ortho-phosphoric acid) used as mobile phase A and buffer pH 3.0 acetonitrile in 20:80 v/v ratios were used as solvent B at a flow rate of 1.5 mL/min was found to be an appropriate mobile phase allowing adequate separation of all three impurities, degradation products as well as ciprofloxacin and tinidazole.
Validation of the method
System suitability parameters were measured so as to verify the system, method and column performance. Results of other system suitability parameters such as relative retention time of each impurity, tailing factor and similarity factor (between two preparations) are presented in [Table 1].
As seen from this data, the acceptable system suitability parameters would be: relative retention time (RRT) of each impurity should comparable, tailing factor for ciprofloxacin, tinidazole in standard solution is not more than 2.0, and resolution between all peaks should be more than 2.0 Presented in [Table 1]. Standard chromatogram of ciprofloxacin and tinidazole are presented in [Figure 2]c and j. Spiked chromatogram of impurity/degradation products with ciprofloxacin, tinidazole is presented in [Figure 2]d and k.
|Figure 2: Typical chromatograms ciprofloxacin at 278 nm (placebo, resolution solution, standard solution, test spiked with impurities and degradation test) at optimized chromatographic conditions (a-h) typical chromatograms tinidazole at 317 nm (placebo, standard solution, test spiked with impurities and degradation test) at optimized chromatographic conditions (i-o)|
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All forced degradation samples were analyzed at an initial concentration of ciprofloxacin, tinidazole with HPLC conditions mentioned in Section chromatographic conditions using PDA detector to ensure the homogeneity and purity of ciprofloxacin, tinidazole. Significant degradation of ciprofloxacin, tinidazole was observed in Heat (80°C for 5 h), photolytic UV light (200 Wh/m 2 ), sun light (1.2 million lux hours), oxidative (3% H 2 O 2 at room temperature for 24 h), acid (0.5 N HCl at 50°C for 5 h), and base (0.5 N NaOH at 50°C for 5 h) conditions leading to the formation of impurities (% degradation should be < 0.1% >20%). The % degradation values presented in [Table 2]a and b and degradation chromatograms presented in [Figure 2] (Ee-Hh and Lll-Oo).
Placebo interference was not observed at retention time of ciprofloxacin and tinidazole and its impurities. Placebo chromatograms presented in [Figure 2]a and i.
The % RSD for the area of ciprofloxacin and it's known impurities (imp-EDA) and tinidazole and it's known impurities (imp-A, imp-B) in related substances method precision was found less than 10% (should be less than 15.0%) conforming good precision of the method. The % RSD values were presented in [Table 3].
LOD and LOQ
The determination of limit of detection and limit of detection (LOD) of all impurities namely ciprofloxacin and it's known impurities (imp-EDA) and tinidazole and its known impurities (imp-A, imp-B) are reported in [Table 3]. The precision at the LOQ concentrations for ciprofloxacin and its known impurities (imp-EDA) and tinidazole and its known impurities (imp-A, imp-B) were found below 10% (should be less than 15.0%). Limit of detection, LOQ of all impurities values presented in [Table 3].
The result shows that an excellent correlation existed between the peak area and concentration of the analyte. Linear calibration plot for the related substance method was obtained over the calibration ranges tested, i.e., LOQ to 200% for impurity (ciprofloxacin and its known impurities (imp-EDA) and tinidazole and its known impurities (imp-A, imp-B)). The correlation coefficient obtained was greater than 0.997 [Table 3]. The above result show that an excellent correlation existed between the peak area and the concentration. The % bias also calculated for all related compounds and main analytes and found less than 5% [Table 3].
The percentage recovery of ciprofloxacin and its known impurity and tinidazole and it's known impurities in tablet varied from 90% to 110% at LOQ, 50%, 100%, 125% and 150% levels of target 0.2% level of respective target concentrations. The LC chromatogram of spiked sample at 0.2% level of all three impurities in the sample solution is shown in [Figure 2]. % Recovery values for impurities are presented in [Table 4] (% recovery should be in between 90% and 110%).
In all deliberate varied chromatographic conditions (flow rate, column temperature and pH of mobile phase buffer); the resolution between critical pairs was greater than 2.0, illustrating the robustness of the method.
Stability in solution
No significant changes were observed in the content of impurities namely ciprofloxacin and its known impurities (imp-EDA) and tinidazole and its known impurities (imp-A, imp-B) during solution stability experiments when performed using the related substances method. The solution stability experiment data confirms that the sample solutions used during the related substances determination were stable for 24 h.
| Conclusions|| |
The gradient RP-HPLC method developed for ciprofloxacin and tinidazole and related substances in solid pharmaceutical dosage forms is found precise, accurate, linear, robust, rugged, and specific. Satisfactory results were obtained from validation of the method. Hence, the method is stability-indicating and can be used for routine analysis of production samples and to check the stability of samples.
| Acknowledgments|| |
The authors wish to thank the management of Dr. Reddy's Laboratories Ltd. for supporting this work. Cooperation from Nitish Sharma of Analytical Research and Development of Dr. Reddy's Laboratories Ltd. is appreciated.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]