eclética química 35-3.indd ECLÉTICA química www.scielo.br/eq Volume 35, número 3, 2010 109 Artigo/Article NEW REAGENTS FOR THE SPECTROPHOTOMETRIC DETERMINATION OF RANITIDINE HYDROCHLORIDE B. Narayana*, K.Veena, K. Ashwani and Divya. N. Shetty Department of P.G. Studies and Research in Chemistry, Mangalore University, Mangalagangotri-574 199, India E.mail: nbadiadka@yahoo.co.uk; Fax: 0091-824-2287367 Abstract: A new spectrophotometric method is proposed for the assay of ranitidine hydrochlo- ride (RNH) in bulk drug and in its dosage forms using ceric ammonium sulphate (CAS) and two dyes, malachite (MAG) green and crystal violet (CV) as reagents. The method involves the addition of a known excess of ceric ammonium sulphate to ranitidine hydrochloride in acid medium, followed by the determination of unreacted CAS by reacting with a fixed amount of malachite green or crystal violet and measuring the absorbance at 615 or 582 nm respectively against the reagent blank. The Beer’s law is obeyed in the concentration range of 0.4-8.0 μg/ ml of ranitidine hydrochloride (RNH) for RNH-MAG system and 0.2-1.6μg/ml of ranitidine hydrochloride for RNH-CV system. The molar Absorptivity, Sandell’s sensitivity for each sys- tem were calculated. The method has been successfully applied to the determination of raniti- dine hydrochloride in pure and dosage forms. Key words: Spectrophotometry; Ranitidine hydrochloride; Ceric ammonium sulphate; Malachite green; Crystal violet Introduction Ranitidine hydrochloride (RNH), chemi- cally, is N, N-dimethyl-5-[2-(1-methylamino-2- -nitrovinyl) - ethylthiomethyl] furfurylamine hydrochloride. The drug was introduced in the market in 1981. The drug is offi cial in Indian Phar- macopoeia [1]. It is a H2- receptor antagonist and is widely used in short term treatment of duode- nal and gastric ulceration, refl ux oesophagitis and dyspepsia [2,3]. When used at the usual recomme- ned dose, ranitidine has been found to be safe [4]. Earlier studies have indicated that ranitidine was oxidized by the liver micosomal oxidases and was converted to its N-oxidase, S-oxide and desmethyl metabolites [5,6,7]. Among these hepatic micro- somal ranitidine metabolites, ranitidine N-oxide produced by the hepatic microsomal-fl avin-con- taining monooxygenase (FMO) has been found to be the major metabolite excreted in human urine. Thus measuring the amount of ranitidine N-oxide present in urine after administration of ranitidine has been used as a non-invasive method of deter- mining the human liver FMO activity in vivo [8]. Several techniques such as proton magnetic reso- nance spectroscopy [9], near infrared refl ectance spectrometry [10], scintillation proximity assay [11], fl ow injection fl uorimetry [12], polarogra- phy [13,14], differential pulse polarography [15], capillary electrophoresis [16], high performance liquid chromatography [17,18] have been used for the determination of RNH in pharmaceuticals. These techniques require sophisticated instru- ments and expensive reagents, and involve several manipulation steps and derivatization reactions. These methods, however, are not adaptable for use in pharmokinetic studies because of their lack of selectivity. Among these instrumental analyti- Ecl. Quím., São Paulo, 35 - 3: 109 - 115, 2010 Ecl. Quím., São Paulo, 35 - 3: 109 - 115, 2010110 Ecl. Quím., São Paulo, 35 - 3: 109 - 115, 2010 111 Artigo Article Artigo Article cal techniques, spectrophotometric techniques oc- cupies a unique position, because of its simplicity, sensitivity, accuaracy and rapidity. Literature survey revealed that the only ti- trimetric method [19] reported for RNH requires 300 mg of drug for each titration. There are few methods for the spectrophotometric determination of ranitidine. These are based on the reaction of ranitidine with some organic acidic dyes follo- wed by extraction of the colored ion-pairs into organic solvents and absorbance measurements. Spectrophotometric determination of ranitidine in tablets has been also suggested through chromo- genic reactions with 3-methylbenzothiozline-2- -one hydrazone [20], 3,5-dichloro-pbenzoquinone chlorimine [20], Folin-Ciocalteu [21] reagents. These methods, however, are not adaptable for use in automated systems due to the long reaction time for color development (15- 30 min) , they require prior extraction of the colored reaction product and involve a high reaction temperature (-90°C). In this communication, we demonstrate the use of spectrophotometric techniques for the de- termination of RNH. The present work involves sensitive, selective and cost effective methods for the determination of ranitidine hydrochloride. The method utilizes ceric ammonium sulphate and two dyes malachite green and crystal violet. Spectro- photometric techniques are in good agreement with the reported methods. In addition, it is not susceptible to interference from common tablet excipients. The developed method has been suc- cessfully applied to the determination of ranitidi- ne hydrochloride in pure and dosage form. Experimental Aparatus A SHIMADZU UV-2550 UV-VIS Spectro- photometer with 1 cm matched quartz cells were used for the absorbance measurements. Reagents and Solutions All reagents used were of analytical reagent grade and distilled water was used for the prepara- tion of all solutions. A 1000 μg/ml standard drug solution of ranitidine hydrochloride was prepared in distilled water. The stock solution was diluted appropriately to get the working concentration. Ceric ammonium sulphate (0.01 M) was prepared in 1M sulphuric acid and standardized. This was diluted stepwise to obtain the working concentrations containing 400 μg/ml (RNH- -MAG system) and 900 μg/ml (RNH-CV system). Hydrochloric acid (1M), malachite green (0.05%), crystal violet (0.05%) were also used. Procedure Method A Different aliquots (0.4- 8.0μg/ml) of RNH were transferred in to a series of 10 ml calibrated fl asks by means of a micro burette. Then, 1 ml of 5M HCl was added followed by 1ml of CAS solution. The contents were shaken well and were set aside for 15 minutes with occasional shaking. Then, 1.0 ml of malachite green was added to each fl ask, and the volume was adjusted up to the mark with distilled water and mixed well. The absor- bance of each solution was measured at 615 nm against the corresponding reagent blank. The ab- sorbance corresponds to the bleached color, which in turn corresponds to the drug solution, was ob- tained by subtracting the absorbance of the blank by that of the test solution. Method B Different aliquots (0.2-1.6 μg/ml) of RNH were transferred in to a series of 10 ml calibrated fl asks by means of a micro burette. Then, 1 ml of 5M HCl was added followed by 1ml of CAS solution. The contents were shaken well and were set aside for 15 minutes with occasional shaking. Then, 0.5 ml of crystal violet was added to each fl ask, and the volume was adjusted up to the mark with distilled water and mixed well. The absor- bance of each solution was measured at 582 nm against the corresponding reagent blank. The ab- sorbance corresponds to the bleached color, which in turn corresponds to the drug solution, was ob- tained by subtracting the absorbance of the blank from that of the test solution. Analysis of dosage forms Weighed an amount of the sample equiva- lent to about 168mg ranitidine hydrochloride and was dissolved in a suffi cient amount of distilled water. The solution was shaken and fi ltered throu- gh Whatman No.1 fi lter paper and washed with water. The fi ltrate was diluted up to the mark with distilled water and made up to 100 ml. Suitable aliquots of the sample solution were analyzed by applying general procedure with no modifi cation and the results are shown in table 2 and 3. Table 2. Results of assay of formulations by the proposed method using MAG as reagent Sample RNH certifi ed Found± SDa Recovery (%) at-test Zinetac 168.0 168.154±0.495 100.090 0.703 Rantac 168.0 167. 996±0.118 99.900 0.075 Zenloc 168.0 168.120±0.463 100.070 0.579 aMean ±Standard deviation (n=5) [mg/tablet], bTabulated t-value at 95% confi dence level is 2.78 Rantac- J.B.Chemicals Pharmaceuticals Limited, Gujarat Zinetac- GlaxoSmithKline Pharmaceuticals Limited, Nashik Zenloc- Relief Biotech (P) Ltd, Haridwar Table 3. Results of assay of formulations by the proposed method using CV as reagent Sample RNH certifi ed Found± SDa Recovery (%) at-test Zinetac 168.0 168.224±0.340 100.130 1.470 Rantac 168.0 167. 976±0.08 99.980 0.671 Zenloc 168.0 168.096±0.110 100.057 1.812 aMean ±Standard deviation (n=5) [mg/tablet], bTabulated t-value at 95% confi dence level is 2.78 Rantac- J.B.Chemicals Pharmaceuticals Limited, Gujarat Zinetac- GlaxoSmithKline Pharmaceuticals Limited, Nashik Zenloc- Relief Biotech (P) Ltd, Haridwar Result and discussion In this work, a method based on spectro- photometry was developed and validated for rani- tidine hydrochloride in pure and dosage form. In recent years, the development of spectrophotome- tric methods for determinations of drugs has in- creased considerable, due to their importance, low cost, and simplicity. Before applying an analytical method in the quality control, it is necessary to validate it. The validation testifi es that the pro- cedure is suitable for the intended purpose. The International Conference on the Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use (ICH 2005) and USP 30 (USP 30, 2007) guidelines describe the analytical parameters that should be evaluated in Ecl. Quím., São Paulo, 35 - 3: 109 - 115, 2010112 Ecl. Quím., São Paulo, 35 - 3: 109 - 115, 2010 113 Artigo Article Artigo Article a method validation. The type of method and its respective use determine which parameters should be evaluated. It is the responsibility of the analyst to select the parameters considered relevant for each method [28]. The experimental conditions were chosen after testing the different parameters that infl uence the analysis. The methods involve the addition of a known excess CAS to ranitidine hydrochloride in acid medium, followed by deter- mination of residual CAS by reacting with a fi xed amount of either malachite green measuring the absorbance at 615 nm (RNH-MAG system), or crystal violet measuring the absorbance at 582 nm, (RNH-CV system). In the present method all pa- rameters infl uencing the color development were investigated and are incorporated in the recomme- ned procedure. When added in increasing concen- trations to a fi xed concentration of CAS, ranitidi- ne consumes the latter proportionally and there is concomitant drop in the remaining concentration of CAS. When a fi xed dye concentration is added to decreasing concentrations of CAS, a concomi- tant increase in the dye concentration results. The reaction mechanism are shown in scheme 1. Scheme.1 Analytical data To assess the linearity, a standard curve for ranitidine hydrochloride was constructed by plot- ting concentrations versus absorbance and sho- wed good linearity in the range 0.4-8.0μg/ml of ranitidine hydrochloride for RNH-MAG system and 0.2-1.6 μg/ml of ranitidine hydrochloride for RNH-CV system. The correlation coeffi cients for each system were 0.9945 and 0.9903 indicating good linearity. The accuracy expresses the agre- ement between the accepted value and the value found. The precision and accuracy of the method was studied by analyzing the coupling solution containing known amounts of the cited reagents within Beer’s law limit. Molar absorptivity, Sandell’s sensitivity, slope, intercept for RNH- -MAG system is found to be 1.10×104 L mol-1 cm- 1, 0.028 μg cm-2, 0.0096, 0.0089 while that for RNH-CV system is found to be 4.09×104L mol-1 cm-1 0.007 μg cm-2, 0.0424, 0.0372 respectively. Results of evalution of accuracy and precision for each system are shown in table 4 and 5. The spe- cifi city test demonstrated that there was no inter- ference in the determination of the drug. Table 4. Evaluation of accuracy and precision Ranitidine hydrochloride (Using MAG as Reagent) Amount taken (μg mL-1) Amount founda (μg mL-1) Recovery (%) SD RSD (%) 1.00 0.99 99.00 0.02 2.02 2.00 1.96 98.00 0.04 2.04 3.00 2.97 99.00 0.02 0.67 4.00 3.94 101.60 0.05 1.27 5.00 5.08 98.50 0.03 0.59 6.00 6.02 100.30 0.02 0.33 a- average of fi ve determinations, SD- standard deviation, RE-relative error Table 5. Evaluation of accuracy and precision Ranitidine hydrochloride (Using CV as Reagent) Amount taken (μg mL-1) Amount found (μg mL-1) Recovery (%) SD RSD (%) 0.200 0.203 101.500 0.002 0.985 0.400 0.405 101.250 0.002 0.490 0.600 0.607 101.160 0.002 0.329 0.800 0.810 101.250 0.007 0.864 1.000 0.982 98.200 0.008 0.814 a- average of fi ve determinations, SD-standard deviation, RE-relative error Ecl. Quím., São Paulo, 35 - 3: 109 - 115, 2010114 Ecl. Quím., São Paulo, 35 - 3: 109 - 115, 2010 115 Artigo Article Artigo Article Applications A new method is described for the spec- trophotometric determination of ranitidine hydro- chloride. The proposed method is applied to the determination of ranitidine hydrochloride in pure and dosage forms. The comparisons of the repor- ted methods with earlier methods are shown in table 1. The percent recovery of added pure drug which lies between 98.0 and 101.60 reveals that the procedures are free from interference from usual tablet excipients like talc, starch, calcium gluconate, sucrose, etc. Table 1. Comparison of proposed method with earlier methods Reagent Remarks F-C reagent [21] Less sensitive Bromothymol blue [22] Involve extraction Rose Bengal [23] Involve extraction Hg(SCN)2-IRON(III) [24] Less sensitive KIO3-DCF [25] Requires strict pH control and less sensitive. KMnO4/NBS azine dyes [26] Involves extraction. Proposed methods Sensitive Selective, no interference from usual tablet excipients. Conclusions The method is sensitive, enabling the ac- curate and precise determination of the analytes over satisfactory concentration ranges without the need of special or laborious sample-pretreatment steps. The method, which is advantageously time- and cost-effi cient, was successfully applied to the quantifi cation of the analytes in commercial sam- ples, with results being in good statistical agree- ment with the reported methods; therefore, it is considered useful for routine quality monitoring of pharmaceuticals. Acknowledgement One of the author KV thank UGC for JRF References [1] The Indian Pharmacopeia, The Controller of Publications, Ministry of Health and family Welfare, Govt. of India, New Delhi, 1996, pp.659. [2] R.N.Brogden, A.A. Carmine, R.C. Heel, T.M. Speight, G.S. Avery, Drugs 24 (1982) 267 [3] W. Catterall, K. Mackie, Local anaesthetics. In J. G. Hardman & L.E. 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