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

Available online at www.ijntps.org ISSN: 2277 2782 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-517561, 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 (250 4.6 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 2.955 min and 3.539 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 Email: msm_apcp07@yahoo.com VOLUME 4 NUMBER 5 OCT 2014 130

prepared by dissolving 20.0 mg of accurately weighed Tinidazole and 25.0 mg Ciprofloxacin HCl in mobile phase, in two 100.0 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 2014 131

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 2014 132

Fig 1. Chromatogram of sample (Optimized) AU 0.18 0.16 0.14 0.12 0.10 0.08 0.06 Tinadazole - 2.960 Ciprofloxacin Hcl - 3.548 0.04 0.02 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 Minutes Fig 2. Chromatogram of standard (Specificity) AU 0.40 0.30 0.20 T inadaz ole - 2.954 Ciproflox ac in Hc l - 3.526 0.10 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 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 0.40 0.30 0.20 Tinadazole - 2.955 Ciprofloxacin Hcl - 3.525 0.10 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 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 2014 133

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 1 2.955 2748977 9478.317159 1.021108 2 2.953 2748357 9452.196217 1.080574 3 2.954 2748360 9569.928335 1.090824 4 2.955 2748206 9619.633847 1.089932 5 2.957 2748407 9749.907462 1.108610 Mean 2.954 2748461 9573.997 1.07821 SD 0.00148 297.998 ------- ------- % RSD 0.050 0.0108 ------- ------- VOLUME 4 NUMBER 5 OCT 2014 134

Table 1(b). Data of System Suitability for Ciprofloxacin Injection RT Peak Area USP Plate count USP Tailing 1 3.532 729374 10953.609752 1.604407 2 3.527 729587 10951.014286 1.604878 3 3.526 729020 10003.278630 1.590957 4 3.525 729174 10986.906427 1.584354 5 3.536 729744 10946.878423 1.566451 Mean 3.529 729379.8 10768.34 1.590209 SD 0.004658 294.7104 ------- ------- % RSD 0.131 0.040 ------- ------- Table 2(a). Data of Linearity (Tinidazole) Concentration (ppm) Average Area 0 0 Slope 5140 20 102965 y-intercept 114.7 30 154371 Correlation Coefficient 1 40 205856 50 257167 60 308577 70 359903 80 411306 Table 2(b). Data of Linearity (Ciprofloxacin HCl) Concentration (ppm) Average Area 0 0 Slope 18600 20 372546 y-intercept 276.2 30 558296 Correlation Coefficient 1 40 744400 50 930308 60 1116282 70 1302046 80 1488277 Table 3(a). Data of Accuracy for Tinidazole Concentration % of spiked level Amount added (ppm) Amount found(ppm) % Recovery of % Recovery 50% Injection1 20 20.15 100.75 Mean 99.69333 50% Injection2 20 19.86 99.31 50% Injection3 20 19.80 99.02 %RSD 0.92 100 % Injection1 40 39.88 99.70 Mean 100 % Injection2 40 40.12 100.30 99.83333 100% Injection 3 40 39.80 99.50 %RSD 0.41 150% Injection 1 60 60.12 100.21 Mean 99.97333 150% Injection 2 60 59.76 99.61 150% Injection 3 60 60.06 100.10 %RSD 0.31 VOLUME 4 NUMBER 5 OCT 2014 135

Table 3(b). Data of Accuracy for Ciprofloxacin HCl Concentration % of spiked level Amount added (ppm) Amount found (ppm) % Recovery of % Recovery 50% Injection 1 20 20.04 100.22 Mean 100.06 50% Injection 2 20 19.97 99.85 50% Injection 3 20 20.02 100.11 %RSD 0.18 100 % Injection 1 40 40.01 100.02 Mean 100.04 100 % Injection 2 40 40.05 100.14 100% Injection 3 40 39.98 99.96 %RSD 0.091 150% Injection 1 60 60.08 100.14 Mean 100.02 150% Injection 2 60 59.97 99.96 150% Injection 3 60 59.98 99.98 %RSD 0.09 Table 4(a). Data of Repeatability (System precision) for Tinidazole Injection Peak Areas of Tinidazole %Assay 1 205625 99.95 2 206225 100.24 3 205840 100.06 4 204283 99.30 5 205735 100.00 Mean 205541.6 99.91 SD 739.0046 0.35819 % RSD 0.35 0.35 Table 4(b). Data of Repeatability (System precision) for Ciprofloxacin HCl Injection Peak Areas of Ciprofloxacin HCl %Assay 1 734360 98.66 2 739098 99.30 3 755696 101.53 4 748289 100.53 5 744147 99.98 Mean 744318 100.00 SD 8241.164 1.107678 % RSD 1.1 1.10 Table 5(a). Data of Repeatability (Method precision) for Tinidazole Injection Peak Areas of Tinidazole %Assay 1 202110 98.6 2 203700 99.02 3 201851 98.12 4 202255 98.31 5 203283 98.81 6 202349 98.36 Mean 202687.6 98.48 SD 771.5483 0.352647 % RSD 0.38 0.35 VOLUME 4 NUMBER 5 OCT 2014 136

Table 5(b). Data of Repeatability (Method precision) for Ciprofloxacin HCl Injection Peak Areas of Ciprofloxacin HCl %Assay 1 733495 98.55 2 735992 98.88 3 739828 99.40 4 739098 99.30 5 748289 100.53 6 731322 98.28 Mean 738004 99.278 SD 5988.879 0.827236 % RSD 0.81 0.83 2. 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 1 202110 98.6 2 203700 99.02 3 201851 98.12 4 202255 98.31 5 203283 98.81 6 202349 98.36 Mean 202687.6 98.48 SD 771.5483 0.352647 % RSD 0.38 0.35 Table 6(b). Data of Repeatability (Method precision) for Ciprofloxacin HCl Injection Peak Areas of Ciprofloxacin HCl %Assay 1 733495 98.55 2 735992 98.88 3 739828 99.40 4 739098 99.30 5 748289 100.53 6 731322 98.28 Mean 738004 99.278 SD 5988.879 0.827236 % RSD 0.81 0.83 Table 7(a). Data of Intermediate precision (Analyst 2) for Tinidazole Injection Peak Areas of Tinidazole %Assay 1 205267 99.78 2 205625 99.95 3 205840 100.00 4 202735 98.55 5 208991 101.50 6 208543 101.37 Mean 206333.5 100.19 SD 2572.599 1.100898 % RSD 1.24 1.09 VOLUME 4 NUMBER 5 OCT 2014 137

Table 7(b). Data of Intermediate precision (Analyst 2) for Ciprofloxacin HCl Injection Peak Areas of Ciprofloxacin HCl %Assay 1 736792 99.99 2 734360 99.66 3 755696 101.53 4 744147 99.98 5 744127 99.97 6 752525 101.10 Mean 744607.8 100.37 SD 8392.59 0.753536 % RSD 1.1 0.75 Table 8(a). Data of system to system variability (Tinidazole) System-2 S.No Peak area Assay % of Tinidazole 1 203625 99.98 2 202225 99.30 3 202840 98.60 4 204283 99.30 5 202735 98.55 6 203110 98.73 Mean 203136.3 99.07667 %RSD 0.35 0.56 Table 8(b). Data of system to system variability (Ciprofloxacin HCl) System-2 S.No Peak area Assay % of Ciprofloxacin HCl 1 734360 98.65 2 734098 98.63 3 735696 98.86 4 733289 98.52 5 734147 98.63 6 733495 98.55 Mean 734180.8 98.64 %RSD 0.11 0.12 Table 9(a). Data for Effect of variation in flow rate (Tinidazole) Flow 0.8 ml Flow 1.0 ml Flow 1.2 ml 273707 1.362089 206349 1.280574 166195 1.285372 273211 1.352617 205267 1.279932 165885 1.299385 273948 1.376926 205625 1.261721 166303 1.308063 273465 1.345752 205840 1.276089 167243 1.274662 273862 1.374925 205735 1.250640 165762 1.267630 Avg 273638.6 1.362462 Avg 205763.2 1.269791 Avg 166277.6 1.287022 SD 301.369 0.013609 SD 392.1635 0.01314 SD 582.9758 0.016786 %RSD 0.11 0.99 %RSD 0.19 1.03 %RSD 0.35 1.3 VOLUME 4 NUMBER 5 OCT 2014 138

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 1120286 1.322089 734322 1.604878 602077 1.285372 1119282 1.331920 735792 1.584354 601854 1.319385 1121337 1.296438 734360 1.543805 602403 1.292055 1120456 1.315454 735696 1.568590 603421 1.304561 1120765 1.326551 733147 1.559986 602465 1.294621 Avg 1120425 1.31849 Avg 734663.4 1.572323 Avg 602444 1.299199 SD 754.0018 0.013728 SD 1100.917 0.023367 SD 599.8833 0.013223 %RSD 0.06 1.04 %RSD 0.14 1.48 %RSD 0.09 1.01 CONCLUSION The developed method showed good recovery 99.02-100.75 % 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 20-80 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, 386. 2. Sweetman SC. The Martindale: The Complete Drug Reference. 35th ed. London, UK, Pharmaceutical Press. 2007, 219. 3. Alvarez-Lueje A, Lopez C, Nunez- Vergara LJ, Squella JA. Voltammetric behavior and analytical application oflomefloxacin, an antibecterial fluroquinalone. J AOAC Inst, 2001, 84, 649-658. 4. Diao YH. Determination of ciprofloxacin hydrochloride with UV method and quality investigation of its tablets. Chinese Journal of Hospital Pharmacy, 1994, 14, 212-214. 5. 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), 1150-1154. 6. 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), 43-50. 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), 665-667. 8. 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), 72-75. 9. 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), 161-167. 10. 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), 121-124. 11. 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), 885-888. VOLUME 4 NUMBER 5 OCT 2014 139