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Acta Pharm. 57 (2007) 221230 10.2478/v10007-007-0018-4 Short communication KANAKAPURA BASAVAIAH* HULLIKAL CHANDRASHEKAR PRAMEELA BANKAVADI CHIKKASWAMY SOMASHEKAR Department of Chemistry University of Mysore Manasagangotri Mysore-570 006, India A spectrophotometric method is described for assay of pefloxacin mesylate (PFM) in bulk drug and in tablets. The method is based on back extraction of the bromophenol blue dye at pH 5.2 from the dye-drug ion pair followed by measurement of the dye absorbance at 590 nm. The working conditions of the method were investigated and optimized. Beer's law plot showed a good correlation in the concentration range of 0.151.25 mg mL1. Sensitivity indices such as molar absorptivity, limits of detection and quantification are reported. Intra-day and inter-day precision, and accuracy of the methods were established according to the ICH guidelines, and the er values were in the range of 1.7 to 1.8% with RSD values ranging from 1.0 to 1.1%. The method was successfully applied to the assay of PFM in tablet preparations with recoveries varying from 97.5 to 101.9%, with standard deviation in the range of 0.6 to 1.9. The results were statistically compared with those of the reference method by applying Student's t-test and F-test. Accuracy evaluated by means of the spike recovery method, range from 97.0 to 106.0%, with precision better than 3%. Keywords: pefloxacin mesylate, assay, spectrophotometry, bromophenol blue, pharmaceuticals Accepted January 11, 2007 Pefloxacin mesylate dihydrate (PFM) is a fluorinated quinolone structurally related to nalidixic acid. Chemically, PFM, is 1-ethyl-6-fluoro-1,4-dihydro-7-(4-methyl-1-peperazinyl)-4-oxo-3-quinolinecarboxylic acid, methane sulfonate dihydrate (Fig. 1). It is an antibacterial drug, which is highly effective against both Gram-negative and Gram-positive pathogens that are resistant to other antibacterials (1). The therapeutic importance of PFM has necessitated the development of analytical methods for its determination in dosage forms in compliance with good manufacturing standards. The drug is official in British and European Pharmacopoeias which describe a potentiometric non-aqueous titration procedure for its assay (2, 3). The drug has been determined in pharmaceutical formulations by a variety of analytical techniques such as UV-spectrophotometry (4, 5), * Correspondence, e-mail: firstname.lastname@example.org O HOOC 2 CH3SO3H N C2H5 N N CH3 Br H F HO Br Br OH Br SO3 PFM-BPB complex (pH 3.0) O HOOC F 2 CH3SO3H N C2H5 N N CH3 + Br HO Br Br OH Br O SO2 pH 5.2 Fig. 1. Tentative reaction scheme. spectrofluorimetry (6, 7), capillary electrophoresis (8), polarography (9) and high performance liquid chromatography (1014). A few visible spectrophotometric methods based on acid-base (15), redox (7, 1618), oxidative coupling (19), complex formation (2024) reactions have been earlier reported for the determination of PFM in dosage forms. The reported methods have their respective advantages and disadvantages as indicated in Table 1. Therefore, it was considered desirable to develop an additional assay method suitable for rapid and reliable quality control of PFM formulations. In this study, the visible spectrophotometric approach was followed to develop a sensitive and selective procedure for the determination of PFM in commercial dosage forms. EXPERIMENTAL Apparatus A Systronics model 106 digital spectrophotometer (Systronics India Ltd., India) with 1-cm matched quartz cells was used for all absorbance measurements. Table I. Comparision of performance characteristics of the proposed method with the reported methods Beer's law e range (L mol1 cm1) 1) (mg mL 1040 540 1045 5.91 ´ 103 2.8 ´ 103 Reagent Vanadium(V) NaOH-phenol red Folin-Ciocalteau reagent lmax (nm) 766 560 670 Remarks Heating, 30 min, narrow linear range, less sensitive Critical dependence on NaOH concentration Less selective, less sensitive and narrow linear and dynamic range Less sensitive, shorter wavelength Narrow linear range, shorter wavelength Ref. 7 15 16 Iron(III) chloride Iron(III) chloride a) Cerium(IV)-MBTH 10100 814 414 1060 4.713.7 1.33 ´ 104 3.00 ´ 104 4.8 ´ 103 b) Ammonium molybdate 365 Iron(III) nitrate/NaNO3 a) Chloranilic acid b) Tetracyanoethylene c) DDQ Ammonium reineckate 404 520 290 460 527 Critical acid concentration, narrow linear range Critical acid concentration, shorter wavelength Shorter wavelength, narrow linear range, less sensitive Non-aqueous medium, usually less sensitive 236 Cumbersome procedure precipitation, filtration, dissolution in acetone Methyl orange Bromocresol green Bromophenol blue 440 216 0.151.25 2.40 ´ 105 Highly sensitive 23 24 this paper MBTH 3-methylbenzothiazoline-2-one hydrazone DDQ 2,3-dichloro-5,6-dicyano-p-benzoquinone Reagents and materials All chemicals used were of analytical reagent grade and all solutions were freshly prepared in doubly distilled water. Spectrophotometric grade chloroform (Ranbaxy Fine Chem, Ltd., India) was used for extraction. Phthalate buffers (pH 3.0 and 5.2) were prepared by adjusting the pH of 0.1 mol L1 potassium hydrogen phthalate (S. d. Fine Chem., India) with 0.1 mol L1 hydrochloric acid and 0.1 mol L1 sodium hydroxide, respectively. A 0.1% solution of Bromophenol blue (BPB) was prepared by dissolving the dye (Rankem, India, 95% dye content) in water and filtering it to remove the insoluble residue. Pharmaceutical grade PFM was kindly gifted by Cipla India Ltd. (India). A stock standard solution containing 1 mg mL1 of PFM was prepared in water. Working standard solution equivalent to 5 mg mL1 PFM was obtained by appropriate dilution of stock solution. The following dosage forms containing PFM were purchased from local commercial sources: A Peflex tablets (Wockhardt, India), 200 and 400 mg of PFM, B Pefbid tablets (Megacarem, India), 400 mg of PFM, C Qucin tablets (Aristo Pharma Pvt. Ltd. India), 400 mg of PFM, and D Peflobid injections equivalent to 2 mg mL1 PFM (Cadila, India). Analytical procedures Calibration graph. Twenty mL of PFM solution (5 mg mL1) was pipetted into a 125-mL separating funnel, 4 mL of BPB (0.1%) and 20 mL phthalate buffer (pH 3.0) were added and the total volume of aqueous phase was made up to 60 mL with water. Chloroform (20 mL) was added and the contents were shaken for 1 min; the separated chloroform layer was dried over anhydrous sodium sulphate and collected in a 25-mL calibrated flask. Different aliquots of the chloroform extract (drug-dye ion-pair equivalent to 5 mg mL1 PFM) were accurately measured and transferred into a series of 125-mL separating funnels and then, 5 mL of phthalate buffer (pH 5.2) added to each separating funnel. The contents were shaken for 1 min and the absorbance of the separated aqueous layer was measured at 590 nm against a reagent blank. Detection and quantification limits. The limit of detection (LOD) and limit of quantification (LOQ) were calculated according to the current ICH guidelines as the ratio of 3.3 and 10 standard deviations of the blank (n = 7), respectively, and the slope of the calibration line (25). Procedure for dosage forms. Twenty PFM tablets were chosen randomly from a total number of 50. They were weighed accurately and ground into a fine powder. An amount of powder equivalent to approximately 20 mg of PFM was accurately weighed into a 100-mL calibrated flask, 50 mL of water was added and the content was shaken for 1520 min. Then, the volume was finally diluted to the mark with water, mixed well and filtered using a Whatman No. 42 filter paper. The first 10-mL portion of the filtrate was discarded, and the tablet extract (200 µg mL1 in PFM) was diluted with water to get 5 mg mL1 PFM solution for assay. The contents of 20 ampoules containing injectables were pooled and an aliquot equivalent to 20 mg of PFM was accurately measured and diluted to 100 mL in a calibrated flask and mixed. After necessary dilution, the solution was assayed as stated before. Selectivity studies. As synthetic mixture consisting of PFM, talc, starch, lactose, gum acacia, sodium alginate, magnesium stearate, calcium gluconate, and calcium dihydrogenorthophosphate in the ratio 1:1.5:2:1.5:0.5:2.5:3.0:1:0.5 was prepared by thorough mixing of the constituents. An amount of the mixture equivalent to 20 mg of PFM was accurately weighed into a 100-mL calibrated flask and the drug was extracted with water and the steps described under the assay of dosage forms were followed to determine the percent recovery of PFM. The results are presented in Table V. RESULTS AND DISCUSSION The described method is based on the formation of a chloroform soluble ion-pair between PFM and BPB followed by measurement of the back-extracted dye. Optimization of experimental conditions While studying the effect of pH, the drug-dye ion-pair formation was found to be critically dependent on the pH of the aqueous phase and at pH values higher than 4.8 no ion-pair was formed, i.e., the ion-pair was found to break into its constituent ions. This observation was exploited to develop a highly sensitive method for PFM. In this method, the ion-pair formed at pH 3.0 and extracted into chloroform was back-extracted with a buffer of pH 5.2 and the absorbance of the aqueous buffer phase was measured at 590 nm (Fig. 2). Several organic solvents such as benzene, toluene, cyclohexane, carbon tetrachloride, methylene chloride and i-amylalcohol in addition to chloroform were examined for their ability to extract the drug-dye ion-pair. The latter was found to be the most suitable solvent in terms of extraction efficiency. Optimum conditions for quantitative extraction of the dye from the drug-dye ion-pair were also investigated. The absorbance was maximal and constant over the pH range 4.85.6; hence, pH 5.2 was selected. The extraction was also affected by the nature of the buffer employed. Of the several (pH 5.2) buffers tested, such as phthalate-NaOH, 1 0.9 0.8 0.7 a Absorbance 0.6 0.5 0.4 0.3 0.2 0.1 0 400 420 440 460 480 500 520 540 560 c 580 600 620 640 660 680 700 b Wavelengh (nm) Fig. 2. Absorption spectra of back extracted dye: a) 1.13 mg mL1 PFM, b) 0.63 mg mL1 of PFM, c) blank. acetate-acetic acid, succinic acid-borax, the first buffer was found to provide efficient extraction. A single extraction with 5 mL of buffer was found to be adequate for the concentration range investigated. Shaking times ranging from 0.5 to 3 min produced no change in absorbance; so a one min shaking time was selected. The absorbance of the aqueous phase was found to be constant over several weeks. Method validation Under the experimental conditions described, Beer's law was obeyed over 0.151.25 mg mL1 PFM. The calculated molar absorptivity was 2.40 × 105 L mol1 cm1. The LOD and LOQ values were calculated to be 0.03 and 0.10 mg mL1, respectively. The equation relating the absorbance to concentration was: A = 0.025 + 0.826 g (R = 0.9986, n = 7) where A is the absorbance, g is concentration of PFM in mg mL1 and R is the coefficient of correlation. In order to determine the intra-day accuracy and precision of the methods, solutions containing three different concentrations (within the working limits) of the drug were prepared and analyzed in seven replicates. The results obtained from this investigation are summarized in Table II. The relative standard deviation (£ 1.1%) indicates the high intra-day precision of the methods. The accuracy defined in terms of percent deviation of the calculated concentrations (£ 2%) can be considered satisfactory. The inter-day precision was evaluated by performing replicate analyses on pure drug solution at three concentration levels over a period of five days by preparing all solutions afresh each day. The inter-day RSD values ranged from 1.53.5%, reflecting the usefulness of the method in routine use (Table II). Table II. Accuracy and precision of the proposed method PFM taken (mg mL1) 0.200 0.600 1.000 PFM recoverd (%)a 98.0 98.3 101.8 Intraday RSD (%) 1.0 1.1 1.0 CL (mg mL1) 0.196 ± 0.002 0.590 ± 0.006 1.018 ± 0.009 Interday precision RSD (%) 1.5 2.8 3.5 a Average value of seven determinations. CL Confidence limits at the 95% confidence level for six degrees of freedom. The results of the selectivity studies as shown in Table V confirm that the proposed method is accurate and precise even in the presence of various excipients (recovery 97.6 ± 0.8%). Application to dosage forms In order to demonstrate the usefulness of the proposed method, PFM was determined in authentic samples (pharmaceuticals). The results agreed with the nominal contents (recovery 97.5101.9%) (Table III). The results were also compared statistically by Student's t-test for accuracy and the variance ratio F-test for precision with the official potentiometric method (3) at the 95% confidence level with four degrees of freedom. The results showed that the proposed method and the official method are comparable in terms of accuracy and precision. The reliability and accuracy of the methods were further confirmed by performing recovery studies. To a fixed and known quantity of the pre-analyzed tablet powder or injectable product, pure PFM (standard) was added at three different concentration levels, and the total was found by the proposed methods. The experiment was repeated three times at each level. The percent recoveries of the pure drug added (97.0106.0%), (Table IV), additionaly reveal the fair selectivity of the method. The proposed method is simple, rapid, accurate and precise. From Table I, it is clear that the proposed spectrophotometric method is highly selective compared to most existing methods (7, 1525). The most striking feature of the method is its extraordinary Table III. Determination of pefloxacin mesylate in formulations Recovery ± SD, n = 5 (%) Formulation Nominal amount (mg per tablet or mg per mL) 200 Official method (3)a 98.7 ± 0.9 Proposed spectrophotometric method 97.5 ± 1.2 t = 1.78 F = 2.03 99.0 ± 1.9 t = 0.21 F = 2.41 99.2 ± 0.6 t = 1.52 F = 2.12 101.9±1.32 t = 2.77 F = 5.76 99.2 ± 1.3 t = 1.76 F = 4.75 99.2 ± 1.3 98.7 ± 0.4 100.2 ± 0.6 98.3 ± 0.6 Tabulated t-value at 95% confidence level is 2.77; tabulated F-value at 95% confidence level is 6.39. a Non-aqueous potentiometric titration. Table IV. Recovery studies by the standard addition procedure PFM in formulation (mg) 0.98 0.98 0.98 A (400 mg) 1.0 1.0 1.0 D 0.99 0.99 0.99 Formulation A (200 mg) PFM added (mg) 1.0 2.0 3.0 1.0 2.0 3.0 1.0 2.0 3.0 PFM recovered (%)a 103.3 ± 1.1 97.0 ± 2.1 103.3 ± 1.3 102.0 ± 1.5 106.0 ± 1.1 97.3 ± 2.6 101.0 ± 2.7 97.0 ± 3.4 103.3 ± 4.1 Mean ± SD, n = 3. Table V. Selectivity data Composition of synthetic mixture (mg) PFM 100 Talc 150 Starch Lactose 200 150 Gum acacia 50 Sodium alginate 250 PFM Magnesium Calcium Calcium recovered (%)a stearate gluconate DHP 300 100 50 97.6 ± 0.8 Mean ± SD, n = 5. sensitivity, which is comparable to that of the spectrofluorometric method. Further, owing to the high sensitivity and selectivity of the method, it might be applied for measuring PFM levels in biological fluids. Other advantages of the method include a wide linear dynamic range, unassailable stability of the colour measured (colour stable for several weeks) compared to many procedures reported earlier. This is demonstrated by the high precision of the results. Further, once the drug-dye ion-pair is prepared, further extractions seldom use chloroform, unlike reported method (25). CONCLUSIONS Peflaxacin mesylate has been assayed in dosage forms using visible spectrophotometry. The method can be used to monitor the content uniformity of tablets and injections, and purity of pefloxacin raw material. Acknowledgements. The quality control manager, Cipla India ltd., Mumbai, India is thanked for providing the pure drug as a gift. Two of the authors (HCP, BCS) thank the authorities of the University of Mysore, Mysore, for facilities. 228
Acta Pharmaceutica – de Gruyter
Published: Jun 1, 2007
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