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Indian Journal of Chemical Technology Vol. 13, July 2006, pp. 360-366 Application of bromate-bromide mixture and methyl orange in the titrimetric, spectrophotometric and kinetic assay methods for cyproheptadine in pharmaceuticals K Basavaiah* Department of Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India Email: [email protected] Received 4 October 2005; revised received 12 April 2006; accepted 28 April 2006 Three new methods have been developed for the assay of cyproheptadine in bulk drug and in tablet formulation based on the bromination of cyproheptadine hydrochloride (CPH) by bromine generated in situ by the action of acid on bromatebromide mixture. In titrimetry, the drug is treated with a known excess of bromate-bromide mixture in hydrochloric acid medium followed by the determination of unreacted bromine iodometrically. Spectrophotometry involves the addition of a measured excess of bromate-bromide reagent in acid medium to CPH, and after the reaction is ensured to be complete, the residual bromine is determined by reacting with a fixed amount of methyl orange, and measuring the absorbance at 520 nm. In kinetic method, a mixture containing CPH and methyl orange dye in 1 M sulphuric acid is mixed with bromate-bromide reagent at 4-5°C and time required for bleaching of methyl orange colour is measured. In titrimetry and spectrophotometry, the amount of bromine reacted corresponds to the amount of CPH, and in the former method, quantitation is based on a 1:1 reaction stoichiometry (CPH:KBrO3) and the method is applicable over 2-15 mg range. The spectrophotometric method permits the determination of CPH in 0.5-4.0 μg mL-1 range with an apparent molar absorptivity of 5.25× 104 L mol-1 cm-1 and a Sandell sensitivity of 0.0062 μg cm-2. The limits of detection (LOD) and quantification (LOQ) are calculated to be 0.04 and 0.12 μg mL-1, respectively. Quantitation in kinetic method is based on the linear relationship that exists between the CPH concentration and the time for bromination as indicated by the bleaching of methyl orange acid color. The method is applicable over 10-50 μg mL-1 range with LOD and LOQ values being 1.42 and 4.74 μg mL-1 , respectively. The methods were statistically evaluated by calculating the relative error for accuracy and intra-day and inter-day coefficients of variation for precision, and were applied successfully to the determination of CPH in tablets and syrup with mean recoveries in the range of 97.58-100.9%. The accuracy and reliability of the methods were further ascertained by performing recovery tests via standard-addition technique. There was no interference from tablet excipients and additives. Keywords:Bromate-bromide mixture, Methyl orange, Cyproheptadine, Pharmaceuticals IPC Code: G01J3/00 Cyproheptadine hydrochloride (CPH) is 4-(5H-dibenzo [a, d] cyclohepten-5-ylidene)-1-methyl piperidine hydrochloride. CPH is a serotonin and a histamine antagonist with anticholinergic and sedative effects. In addition, it exhibits antimuscarine and calcium channel blocking activity. CPH is used to treat some hormonal disorders and may be used for treating side-effects of taking antidepressants. It has also been shown to stimulate appetite and weight gain in children and adults. CPH is some times used as an adjunct in therapy in children who are taking human growth harmone1. Various techniques have been developed for the assay of CPH in body fluids and pharmaceuticals. High performance liquid chromatography (HPLC) has been applied for the determination of the drug in human milk and other biological fluids2-4. The drug metabolites5 have been assayed by ultracentrifugation-mass spectrometry. The drug is official in the Indian Pharmacopeia6 which describes a UV- spectrophotometric method for its assay in tablets. Literature survey revealed the availability of few methods for the assay of CPH in pharmaceutical formulations. Liquid chromatography (LC)7, GLC8, and HPLC9 have been used to assay CPH. Ion-selective based potentiometry is another technique which has found application in the analysis of CPH-containing tablets. The drug has been assayed by potentiometry using CPH-tetraphenyl borate10, and CPH-dinonylnaphthalene sulphonic acid11 as electroactive compounds. Application of derivative UV-spectrophotometry for the assay of CPH in two- BASAVAIAH: ASSAY METHODS FOR CYPROHEPTADINE IN PHARMACEUTICALS component12 and multi- component13 mixtures is also reported. There is no report on the titrimetric assay of CPH, and all the visible spectrophotometric methods reported are based on the extraction of the ion-pair complex into an organic solvent before measuring the absorbance. Adamski14 was, perhaps, the first to report a colorimetric method for the assay of CPH in tablets. The method involved the extraction of the drug with chloroform, the chloroform extract with bromocresol green in phosphate buffer of pH 5.4, reextraction of the aqueous layer with chloroform, and finally with 0.1 N sodium hydroxide followed by absorbance measurement at 615 nm. In another extractive colorimetric method15, the drug was precipitated with reineckate, the ion-pair complex was filtered, dissolved in acetone and absorbance measured at 520 nm. Sane et al.16, have reported a similar ion-pair extraction photometric method using three dyes Solochrome Black-T, Solochrome Dark Blue and Fast Sulphon Black FF, the absorbance of the complex being measured at 520 nm. The drug has also been determined spectrophotometrically based on ion-pair complex formation with benzyl orange17 at pH 4.7-4.9 followed by extraction into dichloromethane and measurement at 404 nm. Basavaiah and Charan18 have also reported a spectrophotometric method based on complexation reaction using Bromophenol Blue. Thus, the reported chromatographic techniques, although sensitive, require expensive instrumental set-up. The visible spectrophotometric methods currently available involve multi-step extraction, rigid pH control and use of organic solvents. Additionally, their sensitivity is poor. This communication reports three methods based on bromination of CPH by in situ generated bromine, the reaction being followed by titrimetry, spectrophotometry or reaction rate measurement, and use of bromate-bromide mixture and methyl orange as reagents. The methods are simple, rapid and sensitive, and can be used conveniently in laboratories which lack modern expensive instruments such as those required for LC, HPLC or GLC. Experimental Procedure Apparatus A Systronics Model 106 digital spectrophotometer provided with matched 1 cm quartz cells was used for all absorbance measurements. 361 Reagents and materials All chemicals used were of analytical reagent grade and distilled water was used to prepare the solutions. Bromate-bromide mixture equivalent to 0.02 M KBrO3-0.2 M KBr was prepared by dissolving accurately weighed 3.34 g of KBrO3 (Sarabhai M. Chemicals, Baroda, India) and 23.8 g of KBr (S. d. Fine Chem. Ltd., Mumbai, India) in water and diluting to 1 litre in a calibrated flask. The solution was used in titrimetric work and for kinetic work the same was diluted accurately in 1:1 ratio with water. Sodium thiosulphate solution (0.15 M), hydrochloric acid (5 M), 1% starch indicator and potassium iodide (10%) were prepared in the usual manner. For spectrophotometric work, a stock standard solution equivalent to 1000 μg mL-1 KBrO3 containing a large excess of bromide was prepared by dissolving 100 mg of KBrO3 and 1 g of KBr in water and diluting to the mark with water in a 100 mL calibrated flask (KBrO3 to be weighed exactly). The stock solution was diluted stepwise with water to get a working concentration of 10 μg mL-1 KBrO3. A stock standard solution equivalent to 500 μg mL-1 methyl orange solution was prepared by dissolving accurately weighed 55.5 mg of the dye (S. d. Fine Chem. Ltd., Mumbai, India; dye content 90%) in water and diluting to the mark in a 100 mL calibrated flask, mixed, and filtered using glass-wool. The filtrate was diluted 10-fold to obtain a working concentration of 50 μg mL-1 and used in spectrophotometric work. For kinetic investigations, a 400 μg mL-1 methyl orange was similarly prepared in exactly 1 M H2SO4 and diluted 10-fold with same acid to obtain a working concentration of 40 μg mL-1 dye. Pharmaceutical grade CPH was received as gift from Cipla India Ltd., Mumbai, India, and was used as received. A stock standard solution equivalent to 2 mg mL-1 CPH was prepared by dissolving accurately weighed 500 mg of pure drug in water and diluting to volume in a 250 mL calibrated flask, and used in titrimetric studies. The stock solution (2000 μg mL-1 CPH) was diluted appropriately with water to get working concentrations of 20 and 200 μg mL-1 for spectrophotometric method and kinetic method, respectively. Assay methods Titrimetry A 10 mL aliquot of pure drug solution containing 2-15 mg of CPH was accurately measured and transferred into a 100 mL titration flask. The solution was acidified by adding 5 mL of 5 M HCl. Then, 362 INDIAN J. CHEM. TECHNOL., JULY 2006 10 mL of KBrO3–KBr mixture (0.02M in KbrO3)was added, the contents were mixed well and left to stand for 10 min with occasional swirling. Finally, 5 mL of 10% KI solution was added and the liberated iodine was estimated. A reagent blank experiment was performed without CPH. The amount of CPH in the measured aliquot was calculated based on the amount of bromate reacted. orange solution without CPH, and the time required to bleach the color was noted (Tb). The corrected time was computed using Tc=Ta-Tb. A calibration graph was prepared by plotting the corrected time, Tb versus concentration of CPH, or a regression equation was derived using Tb and concentration data. The concentration of the unknown was read from the calibration graph or computed from the regression equation. Spectrophotometry Different aliquots (0.25, 0.50---------2.0 mL) of 20 μg mL-1 standard CPH solution were accurately transferred into a series of 10 mL calibrated flasks by means of micro burette, and the total volume was adjusted to 2 mL by adding requisite volume of water. To each flask was added 2 mL of 5 M HCl followed by 1 mL of bromate-bromide mixture (10 μg mL-1 with respect to KBrO3). The contents were mixed well and let stand for 10 min with periodic shaking. Finally, 1 mL of 50 μg mL-1 methyl orange solution was added to each flask, the volume was diluted to the mark with water, mixed and absorbance of each solution was measured at 520 nm against a reagent blank after 5 min. A standard graph was prepared by plotting the measured absorbance as a function of CPH concentration. The concentration of the unknown was read from the calibration graph or computed from the regression equation derived using the Beer’s law data. Kinetic method Different aliquots ( 5, 10,-----------25 mL) of 200 μg mL-1 standard CPH solution were accurately measured and transferred into separate 50 mL calibrated flasks each containing 25 mL of methyl orange solution (40 μg mL-1 in 1 M H2SO4) and diluted to the mark with water. Into separate test tubes of similar dimensions, pipetted out 5 mL of the above drug-dye solution and 5 mL of bromate-bromide mixture (0.01 M with respect to KBrO3). Both the tubes were immersed in an ice bath until they reached 4-5°C. A stop-clock (accurate to 1 s) was started and the two solutions were mixed thoroughly noting the time of mixing (initial time Ti). The solution was stirred gently with the thermometer used for temperature measurements. The time at which methyl orange acid colour was discharged was noted (final time, Tf). The actual time required for bleaching of the dye color is given by Ta= Tf –Ti. A blank experiment was carried out simultaneously by mixing equal volumes of bromate-bromide mixture and methyl Method for tablets Twenty tablets containing CPH were accurately weighed and ground into a fine powder. A portion of the powder equivalent to 250 mg of CPH was accurately weighed into a 250 mL calibrated flask, 150 mL of water was added to the flask and shaken for 15-20 min, the volume was diluted to the mark with water, mixed well and the mixture was filtered using a Whatman No 42 filter paper. The first 10 mL portion of the filtrate was discarded, and a convenient aliquot of the subsequent portion was assayed by the titrimetric procedure. The filtrate equivalent to 1000 μg mL-1 CPH was diluted with water so as to get 20 and 200 μg mL-1 w. r. t. CPH for assay by spectrophotometric method and kinetic method, respectively. Method for syrup The contents of five bottles of ciplactin syrup containing CPH were pooled and mixed. A 250 mL portion of the syrup equivalent to 100 mg of CPH was measured accurately and transferred into a separator. The contents were rendered alkaline to litmus paper with 6 M ammonia and then one mL added in excess. The contents were then exctracted with four 25 mL portions of chloroform, the extracts were pooled and evaporated to dryness on a water bath. The residue was dissolved in 0.1 M HCl and diluted to 100 mL with water in a calibrated flask. A suitable aliquot was then assayed by titrimetry. The syrup solution (1000 μg mL-1 CPH) was diluted appropriately as described under tablets and analysed by spectrophotometric and kinetic methods. Results and Discussion The bromate-bromide mixture and methyl orange combination has successfully been employed for the assay of several bioactive substances19,20. From preliminary experiments, CPH was found to undergo bromination by bromine, generated in situ by the action of acid on bromate-bromide mixture. Based on BASAVAIAH: ASSAY METHODS FOR CYPROHEPTADINE IN PHARMACEUTICALS 363 this reaction, three new methods were developed for CPH. In titrimetry, the reaction was followed by back titration of the unreacted bromine iodometrically, and in spectrophotometry, it was followed by measuring the increase in absorbance; the change being caused by bleaching action of bromine. The same bleaching action was used to follow the bromination reaction kinetically. Method development Optimisation of reaction conditions Titrimetry The quantitative nature of reaction between CPH and in situ generated bromine was tested by treating 2-15 mg of drug with a known excess of bromatebromide mixture in acid medium, and backtitrating the residual bromine iodometrically after ensuring the completion of reaction. The reaction stoichiometry was calculated to be 1:1(CPH:KBrO3) in conformity with the probable reaction scheme given in Fig. 1. Hydrochloric acid medium was found to be better suited and the reaction stoichiometry was unaffected when 3-7 mL of 5 M HCl was used in a total volume of 25 mL. Hence, 5 mL of 5 M HCl was used in the assay procedure. At the laboratory temperature (30±2°C), the time required for complete bromination was found to be 5 min and contact time up to 15 min did not affect the reaction stoichiometry and the results. For the range studied (2-15 mg), 10 mL of 0.02 M-KBrO3-0.2 M KBr solution was found adequate for a quantitative reaction. Spectrophotometry In the proposed spectrophotometric method for CPH, varying amounts of drug were reacted with a fixed and excess amount of bromate-bromide in HCl medium, and after a predetermined time, the unreacted bromine was determined by treating with a fixed amount of methyl orange and measuring the absorbance at 520 nm. A linear relation was found between absorbance and concentration of CPH which formed the basis for quantification of the drug. A preliminary study showed that 1 mL of 50 μg mL-1 methyl orange acid form in a total volume of 10 mL produced a convenient maximum absorbance at 520 nm. This color was completely and irreversibly bleached by 1 mL of 10 μg mL-1 of KBrO3 in the presence of a large excess of bromide in HCl medium. Hence, different amounts of CPH were reacted with 1 mL of 10 μg mL-1 of KBrO3 (plus excess of KBr) and after the bromination was judged complete, the surplus Fig. 1⎯ Possible reaction scheme bromine (in situ generated) was determined by treating with 1 mL of 50 μg mL-1 methyl orange, and measuring the absorbance at 520 nm. This step enabled to determine the concentration range of CPH which could be quantitated. CPH, when added in increasing amounts to a fixed amount of bromate-bromide mixture in acid medium, consumes bromine proportionately, and there will be a concomitant fall in the bromine concentration. When a fixed amount of methyl orange was added to decreasing concentrations of bromine, a proportional increase in the dye concentration occurred as indicated by the proportional increase in the absorbance at 520 nm as a function of CPH concentration. Hydrochloric acid medium was ideally suited for both bromination and bleaching steps. Two mL of 5 M HCl in a total volume of ~ 5 mL was found adequate for the bromination step which was complete in 5 min, and the same quantity of acid was maintained in the final volume for bleaching of the dye color. A contact time of 5 min was necessary for the bleaching of methyl orange acid color. The measured color was stable for several days in the presence of the reaction product. Kinetic method The bleaching of methyl orange acid color by in situ generated bromine has previously been applied for the kinetic determination of various drugs21. Applying the same concept of reaction rates, micro amounts of CPH were determined. In the proposed method for CPH, different amounts of drug were mixed with a fixed amount of methyl orange and made 0.5 M with respect to sulphuric acid. Equal volumes of drug-dye solution and 0.01 M KBrO3 -0.1 M KBr mixture, each maintained at 4-5°C were mixed and the time required for the red color of methyl orange to be discharged was measured and corrected for blank experiment. When increasing amounts of CPH were reacted with a fixed amount of bromate- INDIAN J. CHEM. TECHNOL., JULY 2006 364 bromide, the concentration of the latter decreased concomitantly after the bromination reaction. This resulted in an increase in time required for bleaching the color of a fixed amount of methyl orange. This is observed as proportional increase in corrected time, Tc with increasing concentration of CPH. A linear relationship was found between the corrected time, Tc and concentration of CPH which served as the basis for the quantitation of the drug. Method validation Quantitative data as a function of CPH concentration, and the linearity was found in the range of 10-50 μg mL-1 and can be described by the equation: Y= -0.36+ 0.258 X (n = 5, r = 0.9992) where Y is the corrected time, Tc, in seconds and X concentration in μg mL-1. The LOD and LOQ were calculated to be 1.42 and 4.74 μg mL-1, respectively. Precision and accuracy Titrimetry was found applicable over 2-15 mg range. Outside these limits deviant results were obtained. The relationship between the titration end point and the drug amount was evaluated by calculating the correlation coefficient, r, via linear least squares treatment, and the r value was found to be -0.9917 suggesting that the reaction between CPH and KBrO3 proceeds stoichiometrically in the ratio 1:1 in the investigated range. In spectrophotometry, the Beer’s law was obeyed over the concentration range of 0.5-4.0 μg mL-1. The apparent molar absorptivity and Sandell sensitivity were 5.25× 104 L mol-1 cm-1 and 0.0062 μg cm-2, respectively. The linear plot gave the regression equation A= 0.005 + 0.1573 C (r ═ 0.9886, n═ 8) Intra-day precision was assessed from the results of seven replicate analyses on pure drug solution. The mean values and the relative standard deviation (RSD) values for replicate determinations at three different levels (concentration/amount) were calculated. To evaluate the inter-day precision, analysis was performed over a period of five days preparing all solutions afresh each day. The accuracy of the methods was established by calculating the percentage deviation observed in the analysis of pure drug solution and expressed as the percent relative error (RE). Table 1 summarizes the intra-day precision and accuracy data for the determination of CPH in pure drug solution by the proposed methods, which were within 2% (except kinetic method which was less precise). The inter-day precision was less than 3%. Application to assay in dosage forms where A is the absorbance and C, concentration in μg mL-1. The limit of detection, LOD (K=3) and the limit of quantification, LOQ (K=10) were established according to ICH guidelines 22 and were calculated to be 0.04 and 0.12 μg mL-1, respectively. The calibration graph for kinetic method was obtained by plotting the corrected time (Tc) in seconds To demonstrate the applicability of the proposed methods to the assay of CPH in tablets, commercially available proprietary drugs containing CPH were subjected to analysis. The results of the assay compiled in Table 2 reveal good agreement with the declared content and percent found. The results were also compared with those obtained by the official Table 1⎯ Evaluation of intra-day precision and accuracy Method* CPH taken CPH found Range RE % SD SDM RSD % ROE** % Titrimetry 5.0 9.0 13.0 1.0 2.0 4.0 10.0 30.0 50.0 5.04 9.13 13.29 0.99 2.03 4.05 10.17 30.55 50.24 0.15 0.28 0.33 0.04 0.05 0.12 0.28 0.61 1.81 0.80 1.44 2.23 1.00 1.50 1.25 1.70 1.83 0.48 0.11 0.03 0.15 0.015 0.013 0.034 0.24 0.96 1.43 0.04 0.01 0.06 0.0056 0.0049 0.0128 0.090 0.36 0.54 2.18 0.32 1.12 1.51 0.65 0.83 2.33 3.13 2.85 ±2.17 ±0.31 ±1.11 ±1.50 ±0.64 ±0.82 ±2.24 ±3.01 ±2.75 Spectrophotometry Kinetic method *In titrimetry, CPH taken/found, range, SD, SDM are in mg and in the other two methods the said quantities are in μg mL-1 . RE. relative error; SD. Standard deviation; SDM. Standard deviation of the mean; RSD. Relative standard deviation and ROE. Range of error. * * At 95% confidence level for six degrees of freedom. BASAVAIAH: ASSAY METHODS FOR CYPROHEPTADINE IN PHARMACEUTICALS 365 Table 2⎯ Result of assay of CPH tablets by the proposed methods Brand name of Formulation* Label claim, Practin tabletsa 4mg/tablet Ciplactin syrupb 2mg/5mL Found** (% of label claim ±SD) Titrimetry Spectrophotometry Kinetic method Reference method 100.9±0.84 t=1.91 F=1.16 97.58±1.22 t=2.96 F=2.21 99.69±1.85 t=3.24 F=5.62 98.12±1.28 t=1.89 F=2.43 100.9±1.66 t=1.26 F=4.53 98.46±1.78 t=0.73 F=4.7 101.6±0.78 99.05±0.82 ** Mean value of five determinations * Marketed by : a. Merind b. Cipla India Ltd. Table 3⎯ Results of recovery experiment Formulation studied Practin tablets (4 mg) Ciplactin Syrup (2 mg/5mL) Titrimetry Spectrophotometric method Kinetic method CPH Pure Total Pure CPH CPH in Pure Total Pure CPH CPH in Pure Total Pure CPH informulation, CPH found, recovered* formulation, CPH found recovered* formulation, CPH found recovered* mg added, mg % µg added, µg % µg mL-1 added, µg % mg µg µg mL-1 mL-1 5.05 5.05 5.05 4.88 4.88 4.88 2.0 5.0 10.0 2.0 5.0 10.0 7.12 10.19 15.08 6.77 9.57 14.64 103.6 102.8 100.3 94.66 93.73 97.58 9.97 9.97 9.97 9.81 9.81 9.81 5 10 20 5 10 20 15.30 20.80 30.67 14.65 19.25 29.76 106.7 108.3 103.5 96.76 94.38 99.75 19.62 19.62 19.62 19.69 19.69 19.69 10 20 30 10 20 30 30.07 40.88 51.90 29.44 39.15 47.99 104.5 106.3 107.6 95.73 97.28 94.33 *Mean value of three determinations Indian Pharmacopoeial method6 by applying the Student’s t-test and F-test at the 95% confidence level. The calculated Student’s t-test and F-values did not exceed the tabulated values for four degrees of freedom suggesting that there was no significant difference between the proposed methods and the reference method in respect of accuracy and precision. The accuracy and validity of the methods were further ascertained by performing recovery experiments via standard–addition technique. When the tablet powder or syrup (pre-analysed) spiked with known amounts of pure CPH was analysed, the recoveries of pure drug added to the tablet were quantitative (Table 3), revealing that common additives and excipients such as talc, starch, gumacacia, lactose, sodium alginate, magnesium stearate, calcium gluconate, calcium dihydrogenorthophosphate etc, did not interfere in the determination. Conclusions The assay results demonstrate that it is possible to use bromate-bromide mixture and methyl orange as quantitative reagents for the determination of cyproheptadine in authentic samples. The methods allow determination of as small an amount as 5 μg and as large an amount as 15 mg of CPH with a fair degree of accuracy and precision. The methods can be rapidly carried out, are simple to perform, and do not involve any specific/special sample treatment or do not require any expensive experimental set-up. The titrimetric method is the first ever assay procedure reported for cyproheptadine and is applicable over a wide dynamic range. The spectrophotometric method is easier to perform than all the currently available methods which involve liquid-liquid extraction step and use of organic solvents. The method is the most sensitive ever developed for cyproheptadine and its sensitivity comparable with that of many HPLC procedures reported earlier. Even the kinetic method is reasonably sensitive and does not require any sophisticated equipment to measure the bleaching time but can be followed visually. All the three methods use eco-friendly chemicals, are fairly accurate and hence can be used for routine quality control. Acknowledgements The author gratefully acknowledges the receipt of pure cyproheptadine from Cipla India Ltd., Mumbai, India. 366 INDIAN J. CHEM. 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