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www.scielo.br/eq Volume 35, número 3, 2010
DEVELOPMENT AND VALIDATION OF SPECTROSCOPIC METHODS FOR SIMULTANEOUS ESTIMATION AND DISSOLUTION OF OFLOXACIN AND ORNIDAZOLE IN TABLET DOSAGE FORMS
R.C. Mashru, S.V. Saikumar*
Centre of Relevance and Excellence in Novel Drug Delivery System, Pharmacy Department, The Maharaja Sayajirao University of Baroda, G.H. Patel Building, Donor’s Plaza, Fatehgunj, 390002, Vadodara, India * firstname.lastname@example.org
Abstract: The aim of this work was to develop and validate simple, accurate and precise spectroscopic methods (multicomponent, dual wavelength and simultaneous equations) for the simultaneous estimation and dissolution testing of ofloxacin and ornidazole tablet dosage forms. The medium of dissolution used was 900 ml of 0.01N HCl, using a paddle apparatus at a stirring rate of 50 rpm. The drug release was evaluated by developed and validated spectroscopic methods. Ofloxacin and ornidazole showed 293.4 and 319.6nm as λ max in 0.01N HCl. The methods were validated to meet requirements for a global regulatory filing. The validation included linearity, precision and accuracy. In addition, recovery studies and dissolution studies of three different tablets were compared and the results obtained show no significant difference among products. Keywords: Dissolution • Oﬂoxacin • Ornidazole • Spectroscopy • Simultaneous estimation Introduction Chemically oﬂoxacin ( OFL, Fig. 1) is (±) 9-fluoro-2,3,dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-d,e]-1,4-benzoxazine-6-carboxylicacid, belongs to new generation of synthetic ﬂuorinated quinolone, structurally related to nalidixic acid [1-3]. This agent is a new broad spectrum antibacterial drug active against most Gram-negative, Gram-positive bacteria, and some anaerobes . Its bacterial action is based on its anti-DNA gyrase activity . This broad spectrum of antibacterial activity and widespread distribution to most tissues and body ﬂuids at relatively high concentrations after oral administration have made this drug useful for the
Ecl. Quím., São Paulo, 35 - 3: 123 - 132, 2010
treatment of systemic infections including urinary tract, respiratory, and gastro-intestinal infections [6-8]. Ornidazole (ORN ﬁg. 2)1-(3-chloro−2hydroxy)-propyl-2-methyl-5-nitroimidazole, is a nitroimidazole derivative with antiprotozoal and antibacterial properties. It is used for the treatment and prophylaxis of infections, which is induced by anaerobic and microaerophilic bacteria and protozoa . Combinations of oﬂoxacin and ornidazole are available in the market, which are highly active against many bacterial infections of enteritis and anaerobic bacteria . Both Oﬂoxacin and Ornidazole are almost completely absorbed from the small intestine when administered orally both having almost 100% bioavailability. Subsequent
and 0. When dissolution test is not deﬁned in the monograph of the dosage form.8%) were obtained from GLPL (Vadodara. Chemical structure of ornidazole. Method 1: Multi component analysis Two sampling wavelengths 293. 35 . The present paper describes the development and validation of analytical methods for the estimation and dissolution test for OFL and ORN tablet dosage form. solved in 0. Development of UV spectroscopic methods for the simultaneous estimation Three simple. and also the analytical methods are available for stability  studies of these drugs. The dissolution test is a very important tool in drug development and quality control.5°C by using a thermostatic bath.6nm for Ornidazole were selected for the estimation in the multicomponent mode in the instrument. Simultaneous equations have been developed for the simultaneous determination and dissolution studies of Oﬂoxacin and Ornidazole in tablet dosage forms.7 & 99. with a ﬁxed slit width (1 nm) using 1. The sample solutions were scanned in the selected wavelength region for respective methods.4nm for Oﬂoxacin and 319. Hydrochloric acid is typical medium used to dissolution test .01 M HCl solution was prepared according to USP Pharmacopoeia . There are many reported HPLC [16-18]. Beer’s law was obeyed in the concentration range of 2-12 μg ml for Oﬂoxacin and 5-30 μg ml for Ornidazole.Artigo Article Artigo Article plasma concentrations of Oﬂoxacin are obtained in 1-2 hours after oral administration. OFL is ofﬁcial in BP . or if the monograph is not available.. The ﬁltered solution was suitably diluted with distilled water to obtain mixed standards in the linearity range for each drug. Method 2: Dual wavelength method For estimation of one component.8(λ2) were selected. in accordance to USP Pharmacopoeia  general method. Because of the critical nature of the ﬁrst two of these steps. 2010 Ecl.01N HCl. The absorbance spectra of mixed standards and sample solutions were measured at selected wavelength. A double-beam UV-Vis spectrophotometer (Shimadzu. 303. Peak plasma concentrations of Ornidazole are obtained within 2 hours of administration. Quím.01N HCl.4 nm and Ornidazole shows at 319. and the permeability across the gastrointestinal tract. The purpose of in vitro dissolution studies in QC is batch to batch consistency and detection of manufacturing deviation while in R&D the focus is to provide some predictive estimate of the drug release in respect to the in vivo performance of a drug product. São Paulo.01N HCl and ﬁltered through Whatmann’s ﬁlter paper. However. India). Japan) model UV – 1700 PC. All reagents and solvents used were analytical grade. for prediction of the in vivo performance of drug product a dissolution test should be sensitive and reliable . Quím. Experimental Materials Oﬂoxacin and ornidazole chemical reference substances (CRS) (assigned purity 99. For Oﬂoxacin. for Ornidazole 284. The difference in the absorbances at the selected wavelengths. hence stock solution of OFL (100μg ml) and ORN (250 μg ml) were prepared in 0. Typical acceptance criteria for the amount of drug dissolved are in the range of 70 – 80 % dissolved .3: 123 . there are no ofﬁcial monographs for OFL and ORN dosage forms and no dissolution tests have been described in literature. Stock solutions OFL and ORN are present in 2:5 ratio in commercial dosage forms. Dissolution is an ofﬁcial test used by pharmacopoeias for drug evaluation release of solid and semisolid dosage forms. UV spectrophotometry  and spectroﬂuorimetry methods for the estimation of these drugs from pharmaceutical preparations and biological ﬂuids. accurate spectrophotometric methods Multi component.132. where the absorbances of other component were same.01N HCl. Oﬂoxacin shows absorption maximum at 293. Also HPLC [21-22]. The best dissolution conditions were used to evaluate the dissolutions testing of three different brands of tablets.01N HCl by dissolving 10mg and 25mg of drug in solvent. HPTLC  methods are available for the estimation of this combination. The mediums were vacuum degassed under house vacuum and were maintained at 37. were plotted against the respective concentration to obtain the calibration curves. Dual wavelength. Therefore the difference in the absorbances in the mixed spectra at the corresponding wavelength will be directly proportional to the concentration of that component. Drug absorption from a dosage form after oral administration depends on the release of the drug from the pharmaceutical formulation. The content was sonicated for 10min.132.2nm (λ1) and 334. two wave lengths were selected.0 cm quartz cells was used for all absorbance measurements. India). ORN is ofﬁcial in none of the Pharmacopoeias. 35 . Chemical structure of oﬂoxacin Figure 2. in vitro dissolution may be relevant to the prediction of in vivo performance [10-12]. Tablets were purchased at the local market and were claimed to contain 200 mg OFL and 500mg ORL each.0 ± 0.3: 123 .6nm in 0. ﬁnally the volume made up to the mark with 0. The assay procedure mentioned in these pharmacopoeias is non aqueous titration. and it is routinely used in Quality Control (QC) and Research & Development (R&D). At present. For QC. USP  and EP . model VDA 6DR multi-bath (n=6).01M HCl medium was selected. the selection of a dissolution medium may be based on the solubility data and dosage range of the drug product . Appropriate dilutions were made to the stock solution with distilled water to get the working standard solutions in the same linearity ranges for all three methods. The accomplishment of dissolution proﬁles is recommended as support in the development and optimization of drug formulation as well as in the establishment of in vitro/in vivo correlation. an over-discriminatory test might be suitable to detect even small production deviations.6(λ2) nm. Parameters to set up the dissolution test should be researched and deﬁned for drugs that do not possess ofﬁcial monographs . Distilled water obtained from a Lab Sil Water Distillation Unit (Bangalore.4nm (λ1) and 300. and volume made up to 100ml with 0. 0. Instrumentation Dissolution test was performed in a Veego dissolution test system. and the results were obtained are reported in the table.. 2010 125 . The powder sample equivalent to 10mg of Oﬂoxacin and 25mg of Ornidazole was weighed and transferred to 100ml volumetric ﬂask and dis- Figure 1. the dissolution and/or its solubilisation under physiological conditions. São Paulo. The concentration in sample solutions Sample solutions Average weight of twenty tablets was determined and these tablets were crushed to ﬁne powder. 124 Ecl.
27 0. samples aliquots were ﬁltered. μg/ml Intercept (a) Slope (b) Correlation coefﬁcient (r) 0.6nm 5 . 30.0283 0.592 0. 2010 Ecl. pure drugs and their mixture standards were scanned in UV-Visible Spectrophotometer in 200400nm wavelength region. 2010 127 .1464 -0.01M HCl were validated for linearity. shown as Figure 4. The cumulative percentage of drug dissolved was plotted against time.082 0.22 0.9999 Multi component method Dual wavelength method Mixture 296nm 2 . Overlay spectrum of mixture containing OFL and ORN (2+5 to12+30 ug/ml) 126 Ecl.4 nm and 319. Sampling aliquots of 5.3: 123 . The percent recovery of Oﬂoxacin and Ornidazole in the sample mixture were determined and reported in the table. Method 3: Simultaneous equations method The absorbances of the both the drugs at both wavelengths (respective absorption maximums 293. and the dissolution study in medium 0.15 0.6nm 5 .0018 0.065 0. Accuracy of the method was found on the basis of bias measurement and the precision of the method was determined by measuring the repeatability (intra-day precision) and the intermediate precision (inter-day precision). 45 and 60 minutes.54 0. precision.30μg 0. when necessary.The results were shown in tables 1&2. Results and discussion As described in the experimental section.12μg 0. Table 1. Overlay spectrum of oﬂoxacin (2-12 ug/ml) and ornidazole (5-30 ug/ml) Method validation The developed methods were validated for linearity. The contents results were used to calculate the percentage release on each time of dissolution proﬁle. São Paulo.0462 0.6nm) were measured. Quím. Validation parameters includes linearity. Method validation and recovery studies The developed UV spectrophotometric methods in 0. 15. Quím. μg/ml LOD. to 12ug/ml of OFL+ 30ug/ml of ORN) is shown as Figure 3.48 0.9999 ORN 319. 35 .27 0.0 ml were withdrawn at 0. Dissolution test conditions Dissolution testing was carried out according to conventional dissolution procedures recommended for immediate release products.0111 0. accuracy. Figure 4. and the absorptivity and molar absorptivity values were determined for OFL and ORN.15 0.30μg 0.0018 0.12μg 5 . and quantiﬁed.0293 -0.12μg 0.003 0. After the end of each test time. accuracy and precision.3: 123 . diluted in distilled water.082 0. 100% and 120% levels on a pre analysed tablet solution. The assay of the three tested products was performed using previously developed and validated spectrophotometric methods.48 0. using paddle (USP Apparatus 2) at 50 rpm. both expressed as RSD (%).Artigo Article Artigo Article of each component was obtained from the calibration curves of the respective drugs. limits of detection and quantiﬁcations Regression and analytical parameters Parameter λ max / nm Linearity.9999 OFL 293. y* * y = a+bx.4nm 2 .16 0.4nm 2 .9996 ORN 319. and replaced with an equal volume of the fresh medium to maintain a constant total volume.132.9992 Regression equation.9968 -1 Simultaneous equation method OFL 293.048 0. Recovery studies were carried out at 80%.. where y is the absorbance and x the concentration in μg ml Figure 3.0041 0.01M HCl was validated for precision according to USP Pharmacopoeia  and ICH guidelines. 35 . The overlay spectra of pure drug substances OFL (2-12 ug/ml) and ORL (5-30 ug/ml).. the overlay spectra of mixture (2ug/ml of OFL+ 5ug/ml of ORN.30μg 0. São Paulo. μg/ml LOQ.132.1481 0.
2010 Ecl.95 SIMULTANEOUS EQUATION METHOD OFL 6 ORN 15 a 2 4 6 8 10 12 Mean S. São Paulo.6nm 314.6 nm were 5351. while respective values for ORN at 293.98 0.D R.97±1.03±0.75 0.D M.62 1. 2010 129 .5 1.64 101.25 0.63 0.25 1.15 0.99 0.37 and 11331.62 98.6nm 399.26 0.02±0.132. Table 4. d relative standard deviation.87±1.21±1.21±0.50 99.50 313.55 METHOD 3 98.54 n = 6.14 1.25 245.A * Conc (μg/ml) Accuracy e Absorptivity for OFL 293.04±0.17 101.6 nm respectively.02 15.4nm 244.50 313.62 1.15±0.67 396.69 101. %.21±1.94 6.15 1. A1 = 5351.30C2 (1) (2) Method 1 .37 319.86 101.4 and 319.17 1.00 396.D R.32 DUAL WAVE LENGTH METHOD OFL 6 ORN 15 6.00 873.32 11331.2 0.24±0.96C1 + 31589.4nm 874.81 Estimation by spectroscopic methods For the multicomponent and dual wavelength methods. Recovery studies carried out on the pre analyzed tablets and the results were shown in table 5.85±1.50 873.54 101.20 242.48 0.32 0. Results for OFL and ORN in three different tablet dosage forms Found a (% of nominal amount ± SD) METHOD 1 98. Quím.36 1. c mean ± standard error.33 242.05 0.16±1.39 1. 35 .12 0.36 6.D M.45 0.12 15.00 397.11 31589.S.71±1. São Paulo.83 243.35 0.20±0.18 Precision d Accuracy e Found c Precision d MULTI COMPONENT METHOD OFL 6 ORN 15 6.36 15.68 98.14±0.68±1.6 nm were 31589.132.25 0.76±0.00 312.03 15.61±1.07±0.Artigo Article Artigo Article Table 2.00 876.69 1.54 METHOD 2 99.00 395.00 396.18 cm−1 mol−1 lit−1. and were used for the measurement of samples.90 314.54 0.Simultaneous Equation method a Mean value of six determinations where A1 and A2 are the values of absorbance of sample at 293.Multicomponent method Method 2 -Dual wave length method Method 3. 128 Ecl.75±1.A * 6.71 97.32±1.4 and 219. is molar absorptivity in cm-1 moles-1 lit-1 determined from mean value of absorptivity Three different tablet dosage forms were analysed by the developed methods and the results were shown in table 4.41 1. Absorptivity values of OFL and ORN for simultaneous equations method Inter-day b Conc (μg/ ml) 5 10 15 20 25 30 Mean S.33 312.64 0.37C2 and A2 = 8706.00 314.31 0.32±1.00 244.57 0. Molecular weight of OFL and ORN is 361.18C1 + 11331.86 102.3: 123 .41 8706.45 102..10 99.96 319.45 101..30 cm−1 mol−1 lit−1.30 Absorptivity for ORN 293.625 respectively. and C1 and C2 are concentrations of OFL and ORN in moles lit−1 respectively. Validation results includes accuracy and precision Taken (ug/ml) Intra-day a Found c Table 3.43 5351.31±0. Quím.96 and 8706.44 100.07 15. e bias %: (found − taken/taken)×100 *M. calibration curves were prepared at respective wavelengths selected.27±2.A.35±1.3: 123 .00 873. b n = 6. Dosage form Labelled amount (mg) ORNI O Tablet OSNO O Tablet OFLOX OZ Tablet OFL 200 ORN 500 OFL 200 ORN 500 OFL 200 ORN 500 Determination by simultaneous equations method The absorptivity values and molar absorptivity values for OFL and ORN are determined (shown in Table 3) and molar absorptivity values for OFL at 293.22±1.50 243.28±0.08±0.74±1.55 6. 35 .4 and 319.20±0.21 1.08 0.4 and 319.67 874.16 15.S.73 102.33 874.80 394.
85 22. São Paulo.23±0.35±1.5 12. samples were collected at 0. Quím.D* (μg/ml) 5 12.54 102.52 101.85 100.5 4 10 8.65±1.64 102.52 27.5 5 6 4 5 6 4 5 6 12.56 99.57±0.18 11.5 4 10 8. The cumulative percentage of drug dissolved was plotted against time.15 98.34 27.5 pure OFL added (μg/ ml) 4 5 6 4 5 6 4 5 6 pure ORN added (μg/ ml) 10 12.5 15 10 12.54±0.5 15 total OFL found (μg/ ml) 9.5 15 10.5 12.65±0.49±0.31±1.54±1.5 12.5 15 10 12.69 96.58 22.17 101.85 99.5 12.79 99.24 11.68 27. 35 .5 12.67 101.24 10.51 97. Quím.65 101.69 Figure 5.65±1.31 101.68±1.D* 100. Dissolution proﬁle comparison for OFL in three tablets by three methods SIMULTANEOUS EQUATION METHOD Dosage form pure OFL pure ORN ORN in pure ORN total OFL total ORN OFL in dosage pure OFL recovered % ± recovered % ± dosage form added (μg/ml) added (μg/ml) found (μg/ml) found (μg/ml) form (μg/ml) S.86 24.69 22.41 5 5 5 OSNO O tab 5 5 5 OFLOX OZ Tab * N=3 5 5 12.5 15 10 12.D* 5 12.14 11.78 102.24 102.61 24.79±0.12 pure OFL recovered % ± S. São Paulo.5 5 6 4 5 6 4 5 6 12.81 102.24±1.132.07 10.58 97.45 99.21 9..31 total ORN found (μg/ ml) 22.45 99.68±0.04 11.97 27.57±0.5 12.31 103.5 12.40 98. DUAL WAVELENGTH METHOD pure OFL pure ORN ORN in pure OFL pure ORN total OFL total ORN OFL in dosage recovered % ± recovered % ± Dosage form dosage form form (μg/ml) added (μg/ml) added (μg/ml) found (μg/ml) found (μg/ml) (μg/ml) S.65 100.35 22. 2010 131 .75±0.26 24.39 102.59 102.D* S.36±2.D* S.65±1.12 99.98 102.1 9. 2010 Ecl.58±1.85±1.59±1.5 12.56±1.5 15 10 12.20±0.08 22.60 97.76±1.08 10.3: 123 .26 11.39 23.5 12.01 24.78±1.5 12.21 10.5 12.23 103.14 101.54 ORNI O tab 5 5 5 OSNO O tab 5 5 5 OFLOX OZ Tab 5 5 12.3: 123 .5 12.35 Dissolution testing Dissolution was carried in 0.39 27.85±0.D* 101. Recovery results for OFL and ORN by using three different tablet dosage forms MULTICOMPONENT METHOD Dosage form OFL in dosage form (μg/ml) 5 ORNI O tab 5 5 5 OSNO O tab 5 5 5 OFLOX OZ Tab 5 5 ORN in dosage form (μg/ml) 12.96±1.45 98.15 11.36 25.89 10.39 103.36±1.81±0. 30.50 99.94 22.31 22.56±0.5 15 9.41 100.5 12.96 10.56±1.132.56±0.5 12.35±2.21 102. Dissolution proﬁle comparison for ORN in three tablets by three methods 130 Ecl.77 97.Artigo Article Artigo Article Table 5.05 8.66±1.15 9.25 99.97 102.36 102. shown in ﬁgures 5 & 6. 35 .37±1.35±1.57 98.5 12.56±1. 45 and 60 min intervals and the amount of drug dissolved was calculated by the developed three methods.67 101.5 12.15 ORNI O tablet Figure 6.65 24.26±1.96 pure ORN recovered % ± S.5 12.24 8.87 99.01N HCl for three different tablets.56±0.59 103.97 10.15±0.5 15 10 12.5 12.38±0.47 102.71 28.5 12.55±1.5 12.65±1.75 9.37 99.97±0.35 99.65±2.56±1.32 99.97 27.63±0.39 25.25 100..47 98.45±0.12 9.21 27.35±1.5 15 10 12.79 27.95 11.25±0.39 102.36 98. 15.25±0.58 11.38±0.68 25.5 12.08 101.85±1.53±1.5 12.67 24.
paddle apparatus. S.  British pharmacopoeia. Lett.. 42 (2005) 723. M. (2007) 1355. Krishnaiah..01M HCl. Ravi. J. S. 93 (2004) 28. D. Indian Drugs. Gandhimathi. Y. Chem. S. Sci. 35 . Desai. G. P. P. 68 (2006) 838-840. M. C. Bakshi. Kapadia. Drugs. 36 (2003) 1163–1181. Anal. Mudryc. Pharm.  P. B. (1998).0 ± 0. A. M. Rockville. Ahuja.  J. 1997. 32 (2004) 619–621.  V. 2010 . 50 rpm stirring speed. W.5 ºC. L. B. 22 (2005) 578–580. D. M. Kucers. A. H. Singh. Toxicol. Adcock. 9 (2006) 169–189. 76 (2008) 541–554.. Strasbourg. Chin.  Proceedings of the World Health Organisation Meeting on Use of Quinolones in Food  Animals and Potential Impact on Human Health. Liziane. M. Norwich. (2005) 2131.  Drug information USA. N. Biomed. M. S.Ayla. A. Finch. 1997. 67 (2005) 479. Venkatachalam. A. S.  Y.. Chem. The Use of Antibiotics. Chin. S. Xiang. Switzerland. Indian Drugs. M. The methods were validated and showed to be linear.. Anal. Pharm. M. Palermob. FDA Guidance for Industry. Sci. United State Pharmacopoeial Convention. S. Appl. Argekar. 55th Edn. Sílvia. Indira.  H.3: 123 . Shi. M. 33 (1996) 261. Pharm.. B. L. Nilima. Sci.Artigo Article Conclusions The analytical methods developed and validated for the simultaneous estimation and for dissolution testing of oﬂoxacin and ornidazole tablet dosage forms were considered satisfactory. U. J. Crowe. 5th edn. Li. M. Licensing division HMSO. Kunjur. G. B. J. Anal.. at 37. 54 (2005) 3–12. Patel. In Vitro. Quím. EDQM.. The conditions that allowed the dissolution determination were 900 ml of 0.. J. Pharm.  P. Journal of Drug Targeting. Marcela. Gao. Behl. F.Bethesda. M. Council of Europe. Acknowledgments We acknowledge our sincere thanks to GLPL. Patel.  European Pharmacopoeia.  R.05). 11 (2003)109. Indian J.  A. 35 (1998) 715. Bhaskar. D. Liu. São Paulo. Pharm. Singh. Raj. Simone. Liu. T.  N.  N. References  S. A. Indian J.  A. S. U. J. precise and accurate. Analytica Chimica Acta. Tong. Geneva. G.  A.  M. Shinde. Dhake.Incilay. The analysis of variance of the recovery of dosage forms and dissolution values showed that the methods developed were similar (p<0. 88 (1988) 415–420. J.Graysan.. Indian Drugs. 26 (2001) 891. Francis. Emami.  C. Carballo. J. 49 (1995) 144–151. L.132. Vadodara. D. J. M.  United State Pharmacopoeia. K. S. Hoy. Lopez. Directors of the American Society of Hospital Pharmacists. T. Singh. P. V. Subaghia. P. M. B. Indian Drugs.  P. (2003) 357. M. S. S. 17 (2003) 35–40.  M. Lutiane. 132 Ecl. S. Huang. N. S.  M. Z. N.. J.  M. Tendolkar. S. Kamble. Sci. The % drug delivery was higher than 80% in 30 minutes for all evaluated products..
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