IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (3) 2011 Spectrophotometric Determination of Ketotifen Fumarate in Pure and Pharmaceutical Preperations by Bromophenol Blue Reagent O. S. Al-Khazrajy Department of Chemistry,College of Education Ibn- Alhaitham, University of Baghdad Received in : 18 May 2011 Accepted in : 20 September 2011 Abstract A spectrophotometric reliable, rapid and sensitive method has been developed and validated for the determination Ketotifen fumarate . A method was described for the determination of Ketotifen Fumarate in pure form or pharmaceutical formulations, a colored ion-pair complex formation reaction among ketotifen fumarate and acid-dye bromophenol blue at pH 3.0 was used for the colorimetric determination of the drug. The complex formed was extracted into chloroform and the maximum absorbance of the solution was measured at 413 nm against blank. The calibration curve calculated obey Beer's law over the concentration range of 0.4-16 μg/ml and the regression equation was A=0.069x+0.036 (R 2=0.998). The recovery of the drug from a commercial tablet was 100.66-104.26 % of the label claim with a relative standard deviation of 0.867-1.472 %. The Sandell sensitivity values, limits of detection (LOD) and limit of quantification (LOQ) values have also been reported. No interference was observed from common excipients p resent in pharmaceutical formulations. Keywords: Ketotifen fumarate, bromophenol blue, ion-pair complex, spectrophotometry Introduction Ketotifen fumarate [KTF; 10H-benzo(4,5)cyclohepta(1,2-b)thiophen- 10-one, 4,9- dihydro-4-(1-methyl-4- piperidinylidene)-(E)-2-butenedioate (1:1)] fig.1[1] is a nonspecific, oral mast cell stabilizer introduced in 1972.Its Histamine H1 Antagonists that selectively bind to but do not activate histamine H1 receptors, thereby blocking the actions of endogenous histamine. Included here are the classical antihistaminics that antagonize or prevent the action of histamine mainly in immediate hypersensitivity . They act in the bronchi, capillaries, and some other smooth muscles, and are used to prevent or allay motion sickness, seasonal rhinitis, and allergic dermatitis and to induce somnolence. [2-4] . Several methods have been reported for the determination of KTF in bulk and pharmaceutical formulations or biological samples, these methods include high performance liquid chromatography HPLC [5-7] , GC [8-11] , chemiluminescence [12] , atomic absorption spectrometry [13] , differential pulse polarographic method [14] , polymer membrane[15], capillary electrophoresis [16], flow IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (3) 2011 injection using PVC membrane selective electrodes [17] , carbon paste electrode [18 ] and square wave adsorptive stripping voltammetry [19]. A revision of the literature revealed that methods that have been developed for the analysis of KTF fumarate both in pharmaceutical or biological samples [5-19] are often based on instrumental methods and there are only few reports exist in literature specifically developed for the determination of in pharmaceutical preparation which are based on spectrophotometric methods [20-25]. On other hand ion pair extractive spectrophotometric methods have also been reported for the estimation of the drugs in pharmaceutical preparations which are based on colored complex of the drugs with reagents like bromophenol blue, bromocresol green , bromothymol blue and eriochrome black T [26-30]. So far, there has been no ion-pair extractive spectrophotometry method reported for an estimation of KTF fumarate with BPB reagent. The aim of present study was to develop and validate a simple method for the determination of KTF fumarate using spectrophotometric method which can be used as an alternative to the official method or other recommended procedures in quality control labs. Materials and Methods Apparatus A double beam UV-Visible recording spectrophotometer (Cintra 5) with matched 1cm quartz cuvettes was used for absorbance measurements. PH-meter DW-9421 from Philips instrument, a Sartorius BL 210S balance, and a Pentium 4 computer (acer) was used for data processing. Materials and Reagents All chemicals used were of analytical reagent grade except , Ketotifen fumarate which was provide as standard powder from the state company for drug industries and medical appliances Sammara – Iraq (SDI) . Standard KTF solution A stock standard solution containing1000 μg ml -1 of KTF was prepared in water by dissolving 1.3747 g of KTF in in 50 ml of water and diluting to 1000 ml by using volumetric flask . Working solution of 10 μg ml -1 was freshly prepared by subsequent dilutions . Phthalate buffer, pH 3.0, was prepared by dissolving 2.04 g of potassium hydrogen phthalate in 100 ml of water and the pH was adjusted by using 0.1 M hydrochloric acid and NaOH [21]. A 0.04% w/v solution of Bromophenol Blue BPB was prepared by dissolving 0.04 g in 100 ml of water. Spectroscopic grade chloroform was for extractio Pharmaceutical preparations from local markets. Analytical Procedure Absorption S pectra Fig. (2) shows the absorption spectra of the KTF-BPB ion-pair complex and of the reagent blank in chloroform. The absorption maximum of the ion-pair in chloroform is at 413 nm where the absorbance of the reagent blank is insignificant. Therefore, a wavelength of 413 nm was used for the examination of the conditions for the determination of KTF. IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (3) 2011 Calibration curve Aliquots of the standard solution containing 2 to 75 μg KTF were transferred into a 125 ml separating funnel and to each one 0.5 ml of phthalate buffer (pH 3.0) . and1.0 ml of 0.04% w/v BPB reagent solution were added. The separating funnel was shaking gently with 5.0 ml of chloroform for 4 min. The two layers were allowed to separate, the absorbance was measured at 413 nm against a reagent blank which was prepared similarly. Calibration curve was p lotted using absorbance-values versus concentration Fig.3. Assay procedure for tablets Twenty tablets were weighed and pulverized to a fine powder. An aliquot equivalent to about 1 mg of KTF was transferred into a 100-ml volumetric flask. A suspension of the drug with 5 mL ethanol and 50 ml water was shaken for 10 min and mixed well filtered using Whatman No.41 filter paper to a second100-ml volumetric flask. Final solution was diluted to 100 ml with water. Results and Discussions The proposed procedure is based on the reaction between KTF and BPB resulting in the formation of an ion-pair complex which could be extracted into chloroform and measured spectrophotometrically . The experimental conditions were optimized and the methods validated. The formation of the complex is shown in the reaction scheme1. given below. Effect of pH In order to confirm the optimum pH range 0.5 ml of phthalate buffer solution on the development and stability of the colored was used and the pH of the reaction mixture was adjusted exactly to values range between 2-5 with few drops of 0.1 N NaOH or 0.1 N HCl a plot of absorbance versus pH showed maximum color intensity and highly absorbance obtained at pH 3.0 Fig 4. On the other hand the absorbance decreased at pH above and below 3.0 . hence this pH was used in all the subsequent experimental work . Effect of BPB Concentration The formation colored complex was found to be affected by the concentration of BPB. To examine these, different concentrations of 1 ml BPB solution were added to a solution containing 1 ml of 20 μg ml -1 of KTF. A gradual increase in the absorbance was observed up to0.04%, beyond which a plateau was obtained.. Hence, 1 ml of 0.04% BPB solution was maintained Fig.5. Extraction S olvent and Shaking Time Several organic solvents (chloroform, toluene, carbon tetra chloride ,1,2dichloroethane and dichloromethane) were examined for their ability to extract drug - BPB ion-pair complex. Among those organic solvents, chloroform was found to be the most suitable for quantitative extraction. An organic phase was required for times of 1 to 6 min produced constant absorbance, hence a shaking time of 4 min was chosen for use. The drug-dye complex in the aqueous phase was extracted with 5 ml of chloroform. The absorbance was measured each time under the optimum conditions and only one extraction was found to be adequate to achieve a quantitative recovery of the complex. Reaction time and addition sequence The effect of the reaction time was studied by preparation of KTF-PB colored complex and measured under the optimum conditions from 1 to 10 minutes and there were no significant changes in absorbance under the optimal conditions for the sequence of addition and the maximum absorption was for the sequence (Drug + Buffer +Reagent) . IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (3) 2011 Composition of ion-pair complexes Anionic dyes such as BPB form ion-pair complex with the positively charged nitrogen containing molecule such as KTF. Each drug– dye complex, with two oppositely charged ions, behaves as a single unit held together by ions, behaves as a single unit held together by an electrostatic interaction. The suggested mechanism of KTF - BPB ion - pair complex formation is displayed in Scheme 1. The composition of the ion pairs associates was established by Job’s method of continuous variation. In the present study, different amounts of KTF and BPB were added to each flask and extracted in the same manner as recommended procedure. The absorbance of formed KTF-BPB ion-pair complex was measured at 413 nm. The absorbance was p lotted against [KTF]/[KTF]+[BPB] for Job’s method .In Job’s p lot, the plot reached a maximum value at a mole fraction of 0.5, which indicated the formation of 1:1 (KTF-BPB) complex Fig.6 . The extraction equilibrium can be represented as follows: KTF + (aq) + Dye - (aq) ↔ KTF + Dye - (aq) ↔ KTF + Dye (org) where KTF + and Dye- represent the protonated KTF and the anion of the BPB respectively.The subscript (aq) and (org) refer to the aqueous and organic phases . The absorbance of each solution was plotted against the mole fraction of the drug ,VKTF/VKTF+VBPB (Fig. 6). Analytical data Under the optimized experimental condition, calibration curve was constructed by plotting the absorbance at λmax against the concentration of KTF. Beer's law range, molar absorptivity, Sandell's sensitivity, regression equation, and correlation coefficient were determined for proposed method and are given in Table 1. A linear relationship was found between the absorbance at λmax and the concentration of the drug in the range of 0.4-15 μg ml-1 for KTF in the final measured with molar absorption coefficients of 2.94×10 4 l/mol.cm. Regression analysis of the Beer's law plots at λmax revealed a good correlation (R2 = 0.998). The graph showed negligible intercept and were described by the regression equation, y =0.069 C + 0.036 .The high molar absorp tivity of the resulting colored complex indicates and the high sensitivity of the method(Table 1). Sensitivity and Validation of the method The limit of quantification that can be determined was found to be 0.461 μg ml-1. The limit of detection that can be reliably detected of 3 replicates was found to be 0. 162 μg ml-1 Samples of pure KTF was prepared and tested in 3 replicates using the proposed procedure. The complete set of validation assays was performed. The results are given in Table 1. Application to dosage forms The proposed method was successfully applied to the determination of KTF in commercial tablets. The applicability of the proposed method for assay of KTF in formulations was examined by analyzing various formulations and the results are tabulated in Table 2. three replicates determinations were made. Satisfactory results were obtained and were in a good agreement with the label claims for different batches. The results were reproducible with low RSD values less than (1.472 %). The accuracy of the method is indicated by the good recovery (100.66-104.26%). Effect of additives and excipients The results of analysis of the commercial formulation and the recovery study of drug suggested that commonly used additives and excipients (lactose, magnesium stearate, starch, sucrose, fructose and cellulose) do not interfere with the assay procedure. The proposed IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (3) 2011 method is sufficiently sensitive to permit determination of low concentration of KTF (0.162 μg/ml) did not interfere in the assay. Conclusions Unlike GC and HPLC techniques, spectrophotometry is simple and inexpensive. The importance of the technique also lies in the chemical reactions upon which the procedures are based rather than upon the sophistication of the instrument. This aspect of spectrophotometric analysis is of major interest in analytical pharmacy since it offers distinct possibility in the assay of a particular component in complex dosage formulations. The proposed methods require only dyes as reagents which are cheaper and readily available and the procedures do not involve any critical reaction conditions or tedious sample preparation. Moreover, method is simple, fast, accurate, adequately sensitive and free from interference by common additives and excipients. The present methods are superior with respect to both sensitivity and selectivity . The calculated ε values of the proposed method is 2.94 × 104 L/mol/cm . The present method, one of the characteristic features of green analytical chemistry. The wide applicability of the new procedures for routine quality control is well established by the assay of KTF in pure form and in pharmaceutical preparations. In comparison with HPLC method the recoveries were 51.7–95.5% , precisions for the drugs in plasma were not greater than 9.5% [5] , that means the proposed method has a good recovery and precision . + KTF BPB + Scheme1. KTF – BPB proposed complex IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (3) 2011 References 1. Budavari S. (1996) The Merck Index. 12th ed. Whitehouse Station, NJ: Merck and Co. Inc, p. 836. 2. Grahnén, A.; Lönnebo, A.; Beck, O.; Eckernäs, S.A.; Dahlström, B. and Lindström, B. (1992), Pharmacokinetics of ketotifen after oral administration to healthy male subjects, Biopharm Drug Dispos 13 (4): 255–262. 3. Galindez, O.A. and Kaufman, H.E. (1996) The management of allergic conjunctivitis. Ophthalmology, 103:1335-1336. 4. Ciprandi, G.; Buscaglia, S. and Cerqueti, P.M . (1992), Drug treatment of allergic conjunctivitis: a review of the evidence. Drugs. 43:154-176. 5. Koichiro Fujimaki; Xiao-Pen Lee; Takeshi Kumazawa; Junichi Sato and Keizo Sato; (2006), Determination of some anti allergic drugs in human plasma by direct- injection high-performance liquid chromatography-tandem mass spectrometry. Forensic Toxicology 24(1):8-16 6. Mustafa, M. A. Elsayed , (2006), Development and Validation of a Rapid HPLC Method for the Determination of Ketotifen in Pharmaceuticals, Drug Dev Ind Pharm 32(4):457-461. 7. Nnane, I. P. ; Damani, L. A. and Hutt, A. J. , (1998), development and validation of stability indicating high performance liquid chromatography assay for ketotifen in aqueous and silicon oil formulation , Chromatographia 48(11):797-802. 8. Seno, H.; Kumazawa, T. and Suzuki, O.(1991) Rapid isolation with Sep-Pak C18 cartridges and capillary gas chromatography of ketotifen and mequitazine in body fluids Jpn. J. Forensic Toxicol. 9:163-167. 9. Leis, H.J. and Malle, E. (1991) Deuterium-labelling and quantitative measurement of ketotifen in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry, Biol Mass Spectrom 20:467 10. Tzvetanov, S.; Vatsova, M. ; Drenska,A.; Gorantcheva, J. and Tyutyulkova, N., (1999), Gas chromatographic-mass spectrometric method for quantitative determination of ketotifen in human plasma after enzyme hydrolysis of conjugated ketotifen, Journal of Chromatography B: Biomedical Sciences and Applications, 732(1)251-256. 11. Julien-Larose, C.; Guerret, M.; Lavene, D. and Kiechel, J. R.(1983) Quantification of ketotifen and its metabolites in human plasma by gas chromatography mass spectrometry, Biological Mass Spectrometry, 10(3): 136–142. 12. NIE Fei and LÜ Jiu-Ru, (2006), Determination of Ketotifen with Potassium Ferricyanide-Calcein Chemiluminescence System , Spectrochim Acta A Mol Biomol Spectrosc. 5(5922):168-172 13. El-Kousy, N. and Bebawy, L. I. (1999) Determination of some antihistaminic drugs by atomic absorption spectrometry and colorimetric methods, J Pharm Biomed Anal. ,20:4,671-679 14. Mohamed, E. Mohamed and Hassan, Y. Aboul-Enein (1986) Spectrophotometric and Differential Pulse Polarographic Methods of Analysis for Ketotifen Hydrogen Fumarate, Drug Dev Ind Pharm, 12(5):733-746. IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (3) 2011 15. Sayed Mehdi Ghoreishi; Mohsen Behpour; Hamid Ahmadi Zahrani and Mahshid Golestaneh, (2010) , Preparation and Optimization of a Ketotifen Sensor and its Pharmaceutical Applications, Anal. Bioanal. Electrochem., 2(3):112- 124 16. Zhou M.; Li YJ.; Ma YJ.; Wang WF.; Mi Jand Chen H. (2010) Determination of ketotifen fumarate by capillary electrophoresis with tris(2,2'-bipyridyl) ruthenium(II) electrochemiluminescence detection. Luminescence,[Epub ahead of print] 17. Khater M.M.; Issa Y.M. and Mohammed S.H. (2009) Flow injection determination of ketotifen fumarate using PVC membrane selective electrodes. Bioelectrochemistry,77(1):53-9. 18. Tabrizivand, G.; Sabzi, R.E. and Farhadi, K. (2007),Preperation and characterization of a new carbon paste electrode based on ketotifen- hexacyanoferrate, j.Solid State Electrochem 11:103-108 19. Daneshgar, P.; Norouzi, P. and Ganjali, M.R. (2009) ,Application of a continuous square-wave potential program for sub nano molar determination of ketotifen. Chem Pharm Bull (Tokyo). 57(2):117-121. 20. Singhvi, I. and Sachdeva, D. (2009) Spectrophotometric Estimation of Ketotifen Fumarate from Tablet Formulations ,Indian J Pharm Sci. , 71(1): 66–68. 21. Massoud Amanlou; Mehdi Hoseinzadeh Nazlou; Homa Azizian; Effat Souri and Hassan Farsam , (2007),Determination of Ketotifen Fumarate in Raw Material and Pharmaceutical Products Using Ion-pair Formation , Analytical Letters, 40(17): 3267 - 3279 22. Hamed, A. Abu-shdy ; Sonia, T hassib; Safinaz, A. Abass and Abdulazim M.M. (2005) Spectrophotometric Determination of Diphenylhydramine Hydrochloride and Ketoifen Hydrogen Fumarate , Bull. Fac. Pharm,43 (1):41-51. 23. Sastry, C.S.P.; Naidu, P.Y. and Murty, SSN. (1997) Visible spectrophotometric methods for the determination of ketotifen fumarate. Eastern Pharmacist. 40,133–5. 24. Szczepaniak ,W.; Cychowska, T. and Przadka, T. (1992) Spectrophotometric determination of ketotifen in pharmaceutical preparations after isolation on ion- exchanger, Acta Pol Pharm,49(4):3-5. 25. Chilukuri, S. P. Sastry and Petla, Y. Naidu, (1997), Spectrophotometric estimation of ketotifen fumarate in pharmaceutical formulations, Microchimica Acta , 127(3- 4):219-223 26. Alaa S. Amin and Yousry , M. Issa,(2005) Spectrophotometric determination of 6- aminopenicillanic acid using bromophenol blue and bromothymol blue, Microchimica Acta, 117(3-4:187-194. 27. ncilay, S sl and Ayla, Tamer , (2003) ,Application of Bromophenol Blue and Bromocresol Purp le for the Extractive-Spectrophotometric Determination of Ofloxacin, Analytical Letters, 36(6):1163 – 1181 28. Kanakapura , Basavaiah and Vaidyanathan, Shakunthala Charan, (2004)Ion-pair Complexometric Determination of Cyproheptadine Hydrochloride Using Bromophenol Blue ScienceAsia 30:163-170 29. Amit, Kumar; Sanjo, Nanda and Rajive, Chomwal, (2010),Spectrophotometeric Methods for Determination of Doxycycline in Tablet Formulation, International Journal of PharmTech Research, 2:599-602. IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (3) 2011 30. Rahman, N.; Khan, N.A. and Azmi, SNH.(2004) Extractive spectrophotometric methods for the determination of nifedipine in pharmaceutical formulation using bromocresol green , bromophenol blue , bromothymol blue and eriochrome black T , farmaco 59 :47-54 . Table (1): Sensitivity and regression parameters. Parameter KTF – BPB complex λmax (nm) 413nm Color yellow Linearity range (µg.mL -1 ) 0.4-15 Molar absorpitivites (l.mol-1.cm-1) 2.94x104 Regression equation A = 0.069 [ complex.µg.mL-1] + 0.036 Calibration Sensitivity 0.036 Sandell's Sensitivity (µg.cm -2 ) 0.01447 Correlation of Linearity (R2) 0.998 Correlation coefficient (R) 0.9989 Detection limit LOD (µg.mL -1 ) 0.162 Limit of quantifi cation (LOQ), μg mL-1 0.461 Table (2): Application of proposed method. Sample Labeled amount (mg) Conc. taken (µg.mL-1) Conc.* Found (µg.mL-1) Recovery % R.S.D* a % Accuracy b Ketotifen fumarate 1mg/ tablet india 1 5 5.213 104.26 1.242 4.26 10 10.253 102.53 1.138 2.53 Ketotifen fumarate 1mg/ tablet UAE 1 5 5.124 102.48 1.472 2.48 10 10.066 100.66 0.867 0.66 aRelative standard deviation (%). b (found – taken / taken) ×100. n= 3 IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (3) 2011 Fig.(1): Chemical structure of ketotifen fumarate (KTF) MWt. 425.5 gmol -1 Conc.ppm Fig. (2): The spectrum of 10µg/mL of ketotifen fumarate Fig.(3):Calibration curve of KTF against reagent blank and the reagent blank ketotifen fumarate KTF against chloroform. Fig.(4): The effect of pH on absorption of KTF Fig.(5): The effect of reagent 10µg/mL concentration(BPB) on absorption of KTF 10µg/mL IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (3) 2011 Fig.(6): The effect of shaking time of complex Fig.(7): Job’s method of continuous variation plot on absorption of KTF 10µg/mL for ion-pair complexe للعلوم الصرفة والتطبیقیة 2011) 3( 24المجلد مجلة ابن الھیثم التقدیر الطیفي لعقار الكیتوتیفین فیوماریت بشكله النقي وفي المستحضرات نیة باستعمال البروموفینول األزرق كاشفاالصیدال عمر صبیح الخزرجي قسم الكیمیاء ،كلیة التربیة ابن الهیثم ،جامعة بغداد 2011آیار 18:في استلم البحث 2011أیلول 20: قبل البحث في الخالصة ة قـــطری عملتاســت تـــم وصـــف الطریقـــة . اریـــت مطـــورة لتقــدیر عقـــار كیتـــوتیفین فیوم و حساســـة وســـریعة ة طیفیـــ ـدیر العقــار بصــورته النقیــة وفـي بعــض المستحضــرات الصــیدالنیة مــن خـالل تكــوین معقــد لواسـتعمالها بــین اصــفر اللــونتقـ المعقــد اللــوني المتكــون اســتخلص باســتعمال . 3=الكیتـوتیفین فیوماریــت وكاشــف بروموفینــول االزرق عنــد اس هیـدروجیني منحنـى المعـایرة اسـتجاب لقـانون بیـر بمسـتوى تركیـز .نـانومتر 413اعلى امتصاصیة ضـوئیة لـه عنـد تالكلوروفورم و قیس R)ومعامـل التـرابط A=0.069x+0.036وكانت معادلة الخط المسـتقیم ، ملیلیتر / میكروغرام 15-0,4من 2=0.998) ـبة االســتر . یم قـــ تكــذلك حســب. 104,26 – 100,66للمستحضــرات الدوائیــة علــى شــكل حبــوب بحــدود جاعیة كانــت نسـ ـاري ـاندل وكــذلك االنحـراف المعیـ ـدیر ،وحــد الكشـف ،حساسـیة سـ لـم یالحــظ اي تــداخل للمضـافات الموجــودة فــي . و حــد التقـ .ى النتائج المستحصلة لالمستحضرات الصیدالنیة ع . لمزدوجات األیونیة معقدات ا، البروموفینول األزرق ، عقار الكیتوتیفین فیوماریت : الكلمات المفتاحیة