International Journal of Medicine and Nanotechnology
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1 International Journal of Medicine and Nanotechnology Access online at Original Article Method Development and Validation for the Simultaneous Estimation of Doxycycline and Ornidazole in Bulk and Pharmaceutical Dosage Form by Using RP-HPLC Method YELLANKI RUPA*,M.AKIFUL HAQUE,D SIREESHA,VASUDHA BAKSHI, S.HARSHINI Department of pharmaceutical analysis and quality assurance School of pharmacy,anurag group of institutions,venkatapur,rangareddy district,telangana,india. Article Received on: Accepted on: ABSTRACT A selective, specific and sensitive stability-indicating high-performance liquid chromatographic method was developed and validated for the determination of Doxycycline and Ornidazole. Reversed-phase chromatography was performed on Shimadzu(Japan) liquid chromatograph, equipped with LC-20 AD pump,lc-20a UV/VIS detector using Inertsil ODS C18 column (250 mm 4.6 mm, 5 µm). Mobile phase and flow rate selection was based on peak parameters (height, capacity, theoretical plates, tailing or symmetry factor), run and resolution. The system with mobile phase containing a mixture of 20Mm KH 2 PO 4 (ph adjusted to 5 with 0.1% OrthoPhosphoric acid): Acetonitrile (40:60% v/v) with 1 ml/min flow rate was quite robust. The optimum wavelength for detection was 324 nm at which better detector response for the drug was obtained. Chromatogram showed peak of Doxycycline at retention was 2.71 min and Ornidazole at 3.70 min. The LOD & LOQ of Doxycycline and Ornidazole was found to be 0.91 µg / ml & 2.86 µg / ml. The percentage relative standard deviation in precision and accuracy studies was found to be less than 2%. Keywords: Doxycycline and Ornidazole, RP-HPLC, Robustness & Precision. Corresponding Author Quick Response code YELLANKI RUPA * School of pharmacy, Anurag group of institutions Venkatapur,Rangareddy district, Telangana,India. Phone: yellanki.rupa@gmail.com INTRODUCTION Analytical method1 is a detailed description of different steps necessary to perform analytical tests which may include preparation of samples, reagents, use of IIII International Journal of Medicine and Nanotechnology, Mednano Publications Page 179
2 apparatus, generation of calibration curve and use of formulae for calculations. A method is to be developed with the goal to rapidly test preclinical samples, formulation prototypes and commercial samples. Once the HPLC method development was over, the method was validated 2,3 as per ICH guidelines4,5 in terms of parameters like system suitability, linearity, accuracy, precision, ruggedness and robustness. The validity of a specific method should be demonstrated in laboratory experiments using samples or standards that are similar to unknown samples analyzed routinely. Drug Profile 6-9 Doxycycline, like minocycline, is lipophilic and can pass through the lipid bilayer of bacteria. Doxycycline reversibly binds to the 30 S ribosomal subunits and possibly the 50S ribosomal subunit(s), blocking the binding of aminoacyl trna to the mrna and inhibiting bacterial protein synthesis. Doxycycline prevents the normal function of the apicoplast of Plasmodium falciparum, a malaria causing organism. Doxycycline, a long-acting tetracycline derived from oxytetracycline, is used to inhibit bacterial protein synthesis and treat non-gonococcal urethritis and cervicitis, exacerbations of bronchitis in patients with COPD, and adult periodontitis. Fig 1: Chemical Structure of Doxycycline. Ornidazole is a 5-nitroimidazole derive ative. Chemic ally, ornidazole is α - (chloro-methyl)- 2 - methy l 5 nitroimidazole 1 ethanol.it is a broad-spectrum antiprotozoal, antimicrobial and antifungal1 used for treatment in severe hepatic and intestinal amoebiasis, giardiasis, trichomoniasis of urogenital tract and bacterial vaginosis. It is not official in I.P., U.S.P. and B.P. till date. A survey of literature reveals that ornidazole is estimated by voltametry2 and H.P.L.C (in biological fluids3, plasma, red blood cell and human sreum. Fig 2: Chemical Structure of Ornidazole In the literature survey very few analytical methods have been reported for the determination of Doxycycline and Ornidazole which include simultaneous techniques and individual methods. So, at present the authors have developed a stability indicating RP-HPLC method which is more precise and reliable for the determination of Doxycycline and Ornidazole in presence of its degradation products. MATERIALS AND METHODS Chemicals and reagents Drug Sample: Were procured from 1. Doxycycline - Chandra Labs Pvt. Ltd., Hyderabad. 2.Ornidazole - Chandra Labs Pvt. Ltd., Hyderabad. Chemicals and Solvents Used: 1. Water HPLC grade 2. Methanol HPLC grade 3. Acetonitrile - HPLC grade 4. Triethyl Amine -HPLC grade 5. Potassium Dihydrogen Phosphate 6. DiPotassium Hydrogen Phosphate All the above chemicals and solvents were supplied by S.D. Fine ChemicalsLtd., India; Qualigens Fine Chemicals Ltd., Mumbai, India and Ranbaxy Chemicals Ltd., New Delhi, India. 2. Instruments Used: 1. Shimadzu ATY224 Digital Electronic Balance 2. Wensar Pvt. Limited, India, ph meter 3. Vacuum Pump Filter (PCI) 4. Life Care Sonicator. 5. Elico SL 210 Double beam UV/Vis Spectrophotometer 6. Hitachi Elite Lachrome HPLC model IIII International Journal of Medicine and Nanotechnology, Mednano Publications Page 180
3 Containing Pump: L2130 Detector: UV, L2400 Software: D2000 Elite Preparation of standard solution Weigh 25 mg Doxycycline and transfer it into 25 ml volumetric flask add sufficient amount of mobile phase and sonicated to dissolve make up the volume with mobile phase. Pipette out 1.0ml of resulting solution and transfer it into 10 ml volumetric flask make up the volume with mobile phase. Weigh 25 mg Ornidazole and transfer it into 25 ml volumetric flask add sufficient amount of mobile phase and sonicated to dissolve make up the volume with mobile phase. Pipette out 1.0ml of resulting solution and transfer it into 10 ml volumetric flask make up the volume with mobile phase. Preparation of sample solutions: Transfer 1.0 ml of Doxycycline and 5 ml of Ornidazole from their stock solutions (1000µg/ml) into 10 ml volumetric flask make up the volume with mobile phase. So the standard solution concentration is 100 µg/ml of Doxycycline and 500 µg/ml of Ornidazole. coefficient for Doxycycline and Ornidazole were found to be & respectively Table 1: Calibration Readings for Doxycycline Concentration Each value is mean of 5 replicates RESULTS AND DISCUSSION Method Validation The method was validated for system suitability, linearity, limit of quantitation (LOQ), limit of detection (LOD), precision, accuracy, selectivity and robustness, assay. Linearity To evaluate the linearity, serial dilution of analyte were prepared from the stock solution was diluted with mobile phase to get a series of concentration ranging from 15, 20, 25, 30 and 35µg/ml. The prepared solutions were filtered through whatmann filter paper (No.41). From these solutions, 20µl injections of each concentration were injected into the HPLC system and chromatographed under the optimized conditions. Calibration curve was constructed by plotting the mean peak (Y-axis) against the concentration (X-axis). The correlation IIII International Journal of Medicine and Nanotechnology, Mednano Publications Page 181
4 Fig 4: Calibration curve of Ornidazole Precision: Repeatability The Repeatability of each method was ascertained separately from the peak s obtained by actual determination of five replicates of a fixed concentration of Doxycycline and Ornidazole. The percent relative standard deviations were calculated for Doxycycline and Ornidazole. Table 3: Repeatability Readings for Doxycycline and Ornidazole concentration of Rt of DOX of Rt of of DOX+ORN in ppm DOX ORN ORN %RSD Table 5: Intraday Readings for Ornidazole Time 10am pm pm Avg RSD %RSD Each value is mean of 5 replicates Table 6: Interday Readings for Doxycycline Time AVG RSD Day %RSD Intraday: In the intra-day variation studies, six injections of standard solution (50µg/ml) were injected at different days. % RSD was calculated presented in Table below Table 4: Intraday Readings for Doxycycline Day Day Avg RSD %RSD Table 5: Interday Readings for Ornidazole Time 10am pm pm Avg RSD Time Day Day Day Avg IIII International Journal of Medicine and Nanotechnology, Mednano Publications Page 182
5 RSD %RSD Each value is mean of 5 replicates Accuracy The accuracy of the assay method was evaluated in triplicate at three concentration levels (80, 100 and 120%), and the percentage recoveries were calculated. Standard addition and recovery experiments were conducted to determine the accuracy of the method for the quantification of for Doxycycline and Ornidazole. in the drug product. The study was carried out in triplicate at 40, 50 and 60 µg/ml. The percentage recovery in each case was calculated. Robustness The robustness of the assay method was established by introducing small changes in the HPLC conditions which included wavelength (322nm and 326nm) & flow rate (0.8 and 1.2 ml/min). Robustness of the method was studied using six replicates at a concentration level of 100 µg/ml of Avidox-OZ. Table 7: Observation for 0.8ml/min S.NO for Rt for for Rt for AVG SD %RSD Table 8: Observation for 1.2 ml/min S.NO for Rt for for Rt for AVG SD Table 9: Observation at 322 nm S.NO for Rt for for Rt for AVG SD %RSD Table 10: Observation at 326 nm: S.NO for Rt for for Rt for AVG SD %RSD Ruggedness Ruggedness is a measure of the reproducibility of a test result under normal, expected operating condition from instrument to instrument and from analyst to analyst. The ruggedness of the method was determined by carrying out the experiment by different operators. Table 11: Readings by Analyst 1: S.No Injection Doxycycline Ornidazole Area Retention Area Retention AVG SD %RSD %RSD IIII International Journal of Medicine and Nanotechnology, Mednano Publications Page 183
6 Table 12: Readings by Analyst 2: Name of tablets Labeled amount of (±SD) amount Assay (n = Drug Doxycycline (mg) found by the6) & Ornidazole proposed method (n=6) Mean (± SD) AVIDOX-OZ 100mg+500mg (±0.13) (±0.39) (±0.09) (±0.42) Assay was performed as described in previous chapter. Results obtained are tabulated below: The assay of Avidox-OZ tablets containing Doxycyclinewas found to be 99.74% Ornidazole was found to be %. Pharmaceutical Dosage Form of Doxycycline and Ornidazole ppm: Fig 6: Chromatogram of pharmaceutical dosage form replicate-1 S.No Injection Doxycycline Ornidazole Area Retention Area Retention AVG Fig 7: Chromatogram of pharmaceutical dosage form replicate-2 SD %RSD CONCLUSION Fig 5: Chromatogram of optimized method Table 13: Readings of Assay S.NO.DRUG RT PEAK AREA TAILING FACTOR 1 Doxycycline Ornidazole THEORETICAL PLATES Assay of Atenolol and Losartan in Dosage Form: Based on the results, obtained from the analysis of samples using described method, it can be concluded that the absence of co-eluting peak along with the main peak of Doxycycline and Ornidazole indicated that the developed method is specific for the estimation of Doxycycline and Ornidazole. For routine analytical purpose, it is always necessary to establish methods capable of analyzing huge number of samples in a short period with due accuracy and precision.the proposed RP-HPLC method is more sensitive, accurate and precise. REFERENCES 1. H.H.Williard, L.L.Merit, F.A.Dean and F.A.Settle, Instrumental methods of analysis, 7th edition, C.B.S.Publishers, New Delhi,( 2002). 2. FDA, "Analytical Procedures and Methods Validation: Chemistry, Manufacturing and Controls IIII International Journal of Medicine and Nanotechnology, Mednano Publications Page 184
7 Documentation; Availability," Federal Register (Notices) 65(169), (2000). 3. P. A. Winslow and R. F. Meyer, Defining a master plan for the validation of analytical methods, J. Validation Technology, pp (1997). 4. ICH Validation of analytical procedures: Text and methodology Q2 (R1), International Conference on Harmonization, ICH Stability Testing of New Drug Substances and Products Q1A (R2), International Conference on Harmonization, /usp28nf23s0_m28220.htm The complete Drug reference; Martindale, Pharmaceutical press 32 edition;12th pg. 10. B.Dhandapani, N.Thirumoorthy, Shaik Harun Rasheed,Method Development and Validation for the Simultaneous Estimation of Ofloxacin and Ornidazole in Dosage form,international Journal of Pharma Sciences and Research (IJPSR),Vol.1(1), 2010, Snezana S. Mitci, Gordana Z. Milte1, Danijela A. Kostic*,Daniela Č. Naskovic,A rapid and reliable determination of doxycycline hyclate by HPLC with UV detection in pharmaceutical samples,j. Serb. Chem. Soc. 73 (6) (2008) UDC : :543 76,JSCS Ibrahim A Alsara,Esmail M Niazy HPLC Determination of Doxycycline in Dosage Forms and Human Plasma, Saudi journal of Pharmaceutical sciences., 2005, Vol. 13, No. 13. Satish A. Patel, Natvarlal J. Patel, Development and validation of HPTLC method for simultaneous estimation of levofloxacin and ornidazole in tablet dosage form,.journal of Pharm Education and Research, Vol. 2, Issue No. 2, December Nanda RK, Simultaneous Spectrophotometric Estimation of Cefixime and Ornidazole in Tablet Dosage form. International Journal of PharmTech Research CODEN( USA): IJPRIF ISSN : Vol.1, No.3, pp IIII International Journal of Medicine and Nanotechnology, Mednano Publications Page 185
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