Method development and validation of Tinidazole and Ciprofloxacin HCl in bulk and tablet dosage form by Rp-HPLC

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1 Available online at ISSN: INTERNATIONAL JOURNAL OF NOVEL TRENDS IN PHARMACEUTICAL SCIENCES Method development and validation of Tinidazole and Ciprofloxacin HCl in bulk and tablet dosage form by Rp-HPLC RESEARCH ARTICLE Murugan S*, Sunil kumar V, Vineela Ruth Madhuri P, Niranjan Babu M and Kathiravan M. K *Department of Pharmaceutical, Seven Hills College of Pharmacy, Tirupathi , Andhra Pradesh, India. Article Info Article history Received 07 Oct 2014 Revised 10 Oct 2014 Accepted 12Oct 2014 Available online 30Oct 2014 Keywords Ciprofloxacin Hydrochloride, Tinidazole, Method Development, RP-HPLC. Abstract A facile and rapid isocratic reverse phase high performance liquid chromatography assay method has been developed for simultaneous estimation of Ciprofloxacin Hydrochloride and Tinidazole in tablet formulation. The column was equilibrated for at least 30 min and separation was achieved by using inerstil- BDS C 18 ( nm, 5µ). The column was maintained at ambient temperature (27 C). The mobile phase employed was Methanol: Orthophosphoric acid (0.28ml) in 1000 ml of water (55:45 v/v). The eluent was monitored using PDA detector at 245 nm. A volume of 20 µl of standard and sample solutions was injected in to the HPLC. The flow rate was 1.0ml /min. The retention times were min and for Tinidazole and ciprofloxacin HCl respectively. The developed method was validated as per ICH guidelines. The developed method was found to be accurate, precise and reproducible. INTRODUCTION Ciprofloxacin (CPX), 1-cyclopropyl-6-fluoro-1,4- dihydro-4-oxo-7-(1-piperazinyl)-3- quinoline carboxilic acid [1], is a broad spectrum fluoroquinolone antibacterial agent used in the treatment of various bacterial infections caused by gram-positive and gram-negative microorganisms [2]. Tinidazole (TNZ) [1-(2-ethylsulfonylethyl)-2- methyl-5-nitroimidazole] is a 5- nitroimidazole derivative, an anti-parasitic drug used against protozoan infections. It is also used in the treatment of a variety of amoebic and parasitic infections [3]. Both drugs are official in Indian pharmacopoeia, British Pharmacopoeia and United States Pharmacopoeia. The combination of Ciprofloxacin Hydrochloride and Tinidazole is widely used in treatment of microbial infections. Literature survey revealed a few UV-Visible Spectrophotometric [4-7] and HPLC [8-11] methods for the estimation of Ciprofloxacin Hydrochloride and Tinidazole alone or in combination with other drugs. Hence the study aimed at developing the a suitable cost effective method and validation of Tinidazole and Ciprofloxacin in tablet formulation using RP-HPLC. MATERIALS AND METHODS Materials All reagents used were of analytical-reagent grade. Sonicator (Fast clean) for degassing of HPLC grade Methanol and Orthophosphoric acid (S.D. Finechem Limited, Mumbai, India) and pure Tinidazole and Ciprofloxacin drug. Instrumentation and chromatographic condition A prominence isocratic HPLC system (Waters high performance liquid chromatography with autosampler and PDA detector) column Inertsil -BDS C 18 (250 x 4.6 mm, 5 µ). A 20µL Rheodyne injection syringe was used for sample injection. HPLC grade, methanol:orthophosphoric acid (0.28 ml) in 1000ml of water (55:45 v/v) were used for the preparing the mobile phase. A freshly prepared, methanol:orthophosphoric acid (0.28 ml) in 1000ml of water (55:45 v/v) was used as the mobile phase. The solvent was filtered through a 0.45µ membrane filter and sonicated before use. The flow rate of the mobile phase was maintained at 1.0 ml/min., column temperature was maintained at room temperature and the detection of the drug was carried out at 245nm. Preparation of stock solution Reference solution: The solution was To whom correspondence should be addressed: S.Murugan msm_apcp07@yahoo.com VOLUME 4 NUMBER 5 OCT

2 prepared by dissolving 20.0 mg of accurately weighed Tinidazole and 25.0 mg Ciprofloxacin HCl in mobile phase, in two ml volumetric flasks separately and sonicate for 20 min. From the above solutions take 10.0 ml from each solution into a 50.0 ml volumetric flask and then makeup with mobile phase and sonicate for 10 min. Preparation of working standard solution The stock solutions equivalent to 20 ppm to 80 ppm with respect to both drugs were prepared in combination of Tinidazole and Ciprofloxacin HCl above, sonicated and filtered through 0.45µ membrane. Preparation of sample drug solution for pharmaceutical formulation Twenty tablets were weighed accurately and a quantity of tablet powder equivalent to 20 mg Tinidazole and Ciprofloxacin HCl was weighed and dissolved in the 70 ml mobile phase with the aid of ultrasonication for 20 min. The content was diluted to 100 ml with mobile phase to furnish a stock test solution. The stock solution was filtered through a 0.45 µm nylon syringe filter and 10.0 ml of the filtrate was diluted into a 50.0 ml volumetric flask to give a test solution containing 40 µg/ml Tinidazole and 50 µg/ml Ciprofloxacin HCl. Procedure for calibration curve The contents of the mobile phase were filtered before use through 0.45micron membrane and pumped from the respective solvent reservoirs to the column at a specified flow rate. Prior to injection of the drug solutions, the column was equilibrated for at least 30min with the mobile phase flowing through the system. The chromatographic separation was achieved using a mobile phase consisting of Methanol : Orthophosphoric acid (0.28 ml) in 1000ml of water (55:45 v/v) the eluent was monitored using PDA detector at a wavelength of 245nm.The column was maintained at ambient temperature (27 0 C) and an injection volume of 20µl of each of standard and sample solutions were injected into the HPLC system to get the chromatograms. The retention time, peak areas of drug was recorded graph was plotted by taking concentration of the drug on x- axis and peak area on y-axis. RESULTS AND DISCUSSION Optimization of chromatographic conditions Analytical method used for assay of Tinidazole and Ciprofloxacin HCl used in Alcip-TZ Tablet by using High performance liquid chromatography technique was validated. Validation was carried out on HPLC WATERS Model NO.2690/5 series Compact System Consisting of Inertsil-C18 BDS column. The validation of the method was assayed by establishing validation criteria such as System suitability, Specificity, Linearity, Accuracy, Precision, Ruggedness, Robustness, LOD and LOQ. VALIDATION SYSTEM SUITABILITY A Standard solution was prepared by using Tinidazole and Ciprofloxacin HCl working standards as per test method and was injected five times into the HPLC system. The system suitability parameters were evaluated from standard chromatograms by calculating the % RSD from five replicate injections for Tinidazole and Ciprofloxacin HCl, retention times and peak areas SPECIFICITY Tinidazole and Ciprofloxacin Solutions of standard and sample were prepared as per the test method are injected into chromatographic system. LINEARITY A Series of solutions are prepared using Tinidazole and Ciprofloxacin HCl working standards at concentration levels from 20 ppm to 80 ppm of target concentration. Measure the peak area response of solution at Level 1 and Level 6 six times and Level 2 to Level 5 two times. ACCURACY (RECOVERY) A study of Accuracy was conducted. Drug Assay was performed in triplicate as per test method with equivalent amount of Tinidazole and Ciprofloxacin into each volumetric flask for each spike level to get the concentration of Tinidazole and Ciprofloxacin equivalent to 50%, 100%, and 150% of the labeled amount as per the test method. The average % recovery of Tinidazole and Ciprofloxacin were calculated. PRECISION 1. Repeatability a. System precision VOLUME 4 NUMBER 5 OCT

3 Standard solution prepared as per test method and injected five times. b. Method precision Prepared six sample preparations individually using single as per test method and injected each solution. RUGGEDNESS OF TEST METHOD a) System to system variability System to system variability study was conducted on different HPLC systems, under similar conditions at different times. Six samples were prepared and each was analyzed as per test method. Comparison of both the results obtained on two different HPLC systems, shows that the assay test method are rugged for system to system variability. b) Column to column variability Column to column variability study was conducted by using different columns. Six samples were prepared and each was analysed as per test method. The results obtained by comparing with both two types were within limit chromatographed at 25ºC temperature. Tinidazole and Ciprofloxacin were resolved from all other peaks and the retention times were compared. The tailing for Tinidazole and Ciprofloxacin HCl is found to be within the limits. LIMIT OF DETECTION AND QUANTITATION (LOD and LOQ) From the linearity data calculate the limit of detection and quantitation, using the following formula. LOD = σ = standard deviation of the response S = slope of the calibration curve of the analyte. LOQ = σ = standard deviation of the response Tinidazole From the linearity plot the LOD and LOQ are calculated LOD = ROBUSTNESS a) Effect of variation of flow rate A study was conducted to determine the effect of variation in flow rate. Standard solution prepared as per the test method was injected into the HPLC system using flow rates, 1.0ml/min and 1.2ml/min. The system suitability parameters were evaluated and found to be within the limits for 1.0ml/min and 1.2ml/min flow. Tinidazole and Ciprofloxacin and was resolved from all other peaks and the retention times were comparable with those obtained for mobile phase having flow rates 1.0ml/min. Ciprofloxacin HCl LOQ = LOD = b) Effect of variation of temperature A study was conducted to determine the effect of variation in temperature. Standard solution prepared as per the test method was injected into the HPLC system at 20ºC temperature. The system suitability parameters were evaluated and found to be within the limits for a temperature change of 20ºc. Similarly sample solution was LOQ = S = slope of the calibration curve of the analyte. VOLUME 4 NUMBER 5 OCT

4 Fig 1. Chromatogram of sample (Optimized) AU Tinadazole Ciprofloxacin Hcl Minutes Fig 2. Chromatogram of standard (Specificity) AU T inadaz ole Ciproflox ac in Hc l Minutes Inference: Got a peak for standard at an Rt of 2.9min for Tinidazole and 3.5min for Ciprofloxacin HCl Fig 3. Chromatogram of sample (Specificity) AU Tinadazole Ciprofloxacin Hcl Minutes Inference: Got a peak for sample at an Rt of 2.9min for Tinidazole and 3.5min for Ciprofloxacin HCl VOLUME 4 NUMBER 5 OCT

5 Fig 4(a). Linearity Plot (Concentration Vs Response) of Tinidazole Fig 4(b). Linearity Plot (Concentration Vs Response) of Ciprofloxacin HCl Table 1(a). Data of System Suitability for Tinidazole Injection RT Peak Area USP Plate count USP Tailing Mean SD % RSD VOLUME 4 NUMBER 5 OCT

6 Table 1(b). Data of System Suitability for Ciprofloxacin Injection RT Peak Area USP Plate count USP Tailing Mean SD % RSD Table 2(a). Data of Linearity (Tinidazole) Concentration (ppm) Average Area 0 0 Slope y-intercept Correlation Coefficient Table 2(b). Data of Linearity (Ciprofloxacin HCl) Concentration (ppm) Average Area 0 0 Slope y-intercept Correlation Coefficient Table 3(a). Data of Accuracy for Tinidazole Concentration % of spiked level Amount added (ppm) Amount found(ppm) % Recovery of % Recovery 50% Injection Mean % Injection % Injection %RSD % Injection Mean 100 % Injection % Injection %RSD % Injection Mean % Injection % Injection %RSD 0.31 VOLUME 4 NUMBER 5 OCT

7 Table 3(b). Data of Accuracy for Ciprofloxacin HCl Concentration % of spiked level Amount added (ppm) Amount found (ppm) % Recovery of % Recovery 50% Injection Mean % Injection % Injection %RSD % Injection Mean % Injection % Injection %RSD % Injection Mean % Injection % Injection %RSD 0.09 Table 4(a). Data of Repeatability (System precision) for Tinidazole Injection Peak Areas of Tinidazole %Assay Mean SD % RSD Table 4(b). Data of Repeatability (System precision) for Ciprofloxacin HCl Injection Peak Areas of Ciprofloxacin HCl %Assay Mean SD % RSD Table 5(a). Data of Repeatability (Method precision) for Tinidazole Injection Peak Areas of Tinidazole %Assay Mean SD % RSD VOLUME 4 NUMBER 5 OCT

8 Table 5(b). Data of Repeatability (Method precision) for Ciprofloxacin HCl Injection Peak Areas of Ciprofloxacin HCl %Assay Mean SD % RSD Intermediate precision (analyst to analyst variability)a study was conducted by two analysts as per test method Table 6(a). Data of Repeatability (Method precision) for Tinidazole Injection Peak Areas of Tinidazole %Assay Mean SD % RSD Table 6(b). Data of Repeatability (Method precision) for Ciprofloxacin HCl Injection Peak Areas of Ciprofloxacin HCl %Assay Mean SD % RSD Table 7(a). Data of Intermediate precision (Analyst 2) for Tinidazole Injection Peak Areas of Tinidazole %Assay Mean SD % RSD VOLUME 4 NUMBER 5 OCT

9 Table 7(b). Data of Intermediate precision (Analyst 2) for Ciprofloxacin HCl Injection Peak Areas of Ciprofloxacin HCl %Assay Mean SD % RSD Table 8(a). Data of system to system variability (Tinidazole) System-2 S.No Peak area Assay % of Tinidazole Mean %RSD Table 8(b). Data of system to system variability (Ciprofloxacin HCl) System-2 S.No Peak area Assay % of Ciprofloxacin HCl Mean %RSD Table 9(a). Data for Effect of variation in flow rate (Tinidazole) Flow 0.8 ml Flow 1.0 ml Flow 1.2 ml Avg Avg Avg SD SD SD %RSD %RSD %RSD VOLUME 4 NUMBER 5 OCT

10 Table 9(b). Data for Effect of variation in flow rate (Ciprofloxacin HCl) Flow 0.8 ml Flow 1.0 ml Flow 1.2 ml Avg Avg Avg SD SD SD %RSD %RSD %RSD CONCLUSION The developed method showed good recovery % and was found to be linear and precise over the range. Both system and method precision was found to be accurate and well within range. Detection limit was found to be 0.56 for Tinidazole and 0.57 Ciprofloxacin HCl. The analytical method was found linearity over the range of ppm of the target concentration for both the drugs. The analytical passed both REFERENCES 1. Maryadele J O Neil. The Merck Index: An Encyclopaedia of chemicals, drugs and biologicals, 14 th ed. Whitehouse Station, NJ. Merck and Co. Inc. 2006, Sweetman SC. The Martindale: The Complete Drug Reference. 35th ed. London, UK, Pharmaceutical Press. 2007, Alvarez-Lueje A, Lopez C, Nunez- Vergara LJ, Squella JA. Voltammetric behavior and analytical application oflomefloxacin, an antibecterial fluroquinalone. J AOAC Inst, 2001, 84, Diao YH. Determination of ciprofloxacin hydrochloride with UV method and quality investigation of its tablets. Chinese Journal of Hospital Pharmacy, 1994, 14, Karunasree A, Thejomoorthy K, Jaffer M, Reddy YP and Ramalingam P. In vitro protein binding study of ciprofloxacin by new UV - spectrophotometric method. Int J Pharm Tech Res, 2010, 2(2), Ibrahim A, Darwish MA, Sultan HA, Al-Arfaj. Kinetic spectrophotometric method for determination of ciprofloxacin and lomefloxacin in their pharmaceutical dosage forms. Int J Res Pharm Sci., 2010, 1(1), robustness and ruggedness tests. On both cases, relative standard deviation was well satisy. ACKNOWLEDGEMENT The authors would like to thank the Principal, Seven Hills College of Pharmacy, Venkataramapuram, Tirupathi, India for his continuous support and encouragement throughout this work. 7. Patel SA, Patel MM., Simultaneous spectrophotometric estimation of ciprofloxacin and ornidazole in tablets. Indian J Pharm Sci, 2006, 68(5), Patel SA. Development and Validation of RP-HPLC Method for Simultaneous Determination of Ciprofloxacin and Ornidazole in Tablets. Int J Current Pharmaceutical Res, 2011, 3(4), Singha R, Maithani1a M, Saraf SK, Saraf S and Gupta RC. Simultaneous Estimation of Ciprofloxacin Hydrochloride, Ofloxacin, Tinidazole and Ornidazole by Reverse Phase High Performance Liquid Chromatography. Eurasian J. Anal. Chem., 2009, 4(2), Dharuman J, Vasudevan M, Somasekaran KN, Dhandapania B, Ghodea PD and Thiagarajana M. RP-HPLC method development and validation for the simultaneous estimation of ofloxacin and tinidazole in tablets. Int J Pharm Tech Res., 1(2), Meshram DB, Bagade SB, Tajne MR. Simple HPLC method for simultaneous estimation of fluconazole and tinidazole in combined dose tablet. J Chromatogr Sci., 2000, 47(10), VOLUME 4 NUMBER 5 OCT

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