IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (3) 2011 Spectrophotometric Determination of Ketotifen Fumarate in Pure and Pharmaceutical Preperations by Bromophenol Blue Reagent O. S. Al-Khazrajy Departme nt of Chemistry,College of Education Ibn- Alhaitham, Unive rsity of Baghdad Re ceived in : 18 May 2011 Accepted in : 20 Septembe r 2011 Abstract A sp ectrop hotometric 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 p ure form or p harmaceutical formulations, a colored ion-pair comp lex formation reaction among ketot ifen fumarate and acid-dye bromophenol blue at p H 3.0 was used for the colorimetric determination of the drug. The comp lex 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 st andard 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 excip ients p resent in p harmaceutical formulations. Keywords: Ketotifen fumarate, bromophenol blue, ion-pair comp lex, sp ectrop hotometry Introduction Ketotifen fumarate [KT F; 10H-benzo(4,5)cyclohepta(1,2-b)thiop hen- 10-one, 4,9- dihydro-4-(1-methy l-4- p iperidinylidene)-(E)-2-butenedioate (1:1)] fig.1[1] is a nonsp ecific, oral mast cell st abilizer introduced in 1972.Its Histamine H1 Ant agonists t hat 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 p revent the action of histamine mainly in immediate hy p ersensitivity . They act in the bronchi, cap illaries, and some other smooth muscles, and are used to p revent or allay motion sickness, seasonal rhinitis, and allergic dermatitis and to induce somnolence. [2-4] . Several methods have been reported for the determination of KT F in bulk and p harmaceutical formulations or biological samples, these methods include high p erformance liquid chromatography HPLC [5-7] , GC [8-11] , chemiluminescence [12] , atomic absorp tion sp ectrometry [13] , differential p ulse p olarographic method [14] , p olymer membrane[15], capillary electrop horesis [16], flow IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (3) 2011 injection using PVC membrane selective electrodes [17] , carbon p aste electrode [18 ] and square wave adsorp tive st ripp ing voltammetry [19]. A revision of the literature revealed that methods that have been developed for the analysis of KT F fumarate both in p harmaceutical or biological samples [5-19] are often based on instrumental methods and there are only few reports exist in literature sp ecifically developed for the determination of in p harmaceutical p reparation which are based on sp ectrop hotometric methods [20-25]. On other hand ion p air extractive sp ectrop hotometric methods have also been reported for the estimation of the drugs in p harmaceutical p reparations which are based on colored comp lex of the drugs with reagents like bromophenol blue, bromocresol green , bromothy mol blue and eriochrome black T [26-30]. So far, there has been no ion-pair extractive sp ectrop hotometry method reported for an estimation of KT F fumarate with BPB reagent. The aim of p resent study was to develop and validate a simple method for the determination of KT F fumarate using sp ectrop hotometric method which can be used as an alternative to the official method or other recommended p rocedures in quality control labs. Materials and Methods Apparatus A double beam UV-Visible recording sp ectrop hotometer (Cintra 5) with matched 1cm quartz cuvettes was used for absorbance measurements. PH-meter DW-9421 from Philip s instrument, a Sartorius BL 210S balance, and a Pentium 4 comp uter (acer) was used for data p rocessing. Materials and Reagents All chemicals used were of analy tical reagent grade excep t , Ketotifen fumarate which was p rovide as standard p owder from the state comp any for drug industries and medical appliances Sammara – Iraq ( SDI) . S tandard KTF sol uti on A st ock st andard solution containing1000 μg ml -1 of KT F was p repared in water by dissolving 1.3747 g of KT F in in 50 ml of water and diluting to 1000 ml by using volumetric flask . Working solution of 10 μg ml -1 was freshly p repared by subsequent dilutions . Pht halate buffer, pH 3.0, was p repared by dissolving 2.04 g of p otassium hydrogen p hthalate in 100 ml of water and the p H was adjust ed by using 0.1 M hy drochloric acid and NaOH [21]. A 0.04% w/v solution of Bromophenol Blue BPB was p repared by dissolving 0.04 g in 100 ml of water. Sp ectroscop ic grade chloroform was for extractio Pharmaceutical p reparations from local markets. Anal ytical Procedure Absorption S pectra Fig. (2) shows the absorp tion sp ectra of the KT F-BPB ion-pair comp lex and of the reagent blank in chloroform. The absorp tion 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 t he determination of KTF. IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (3) 2011 Cal ibration curve Aliquots of the st andard solution containing 2 to 75 μg KT F were transferred into a 125 ml sep arating funnel and to each one 0.5 ml of p hthalate buffer (p H 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 sep arate, the absorbance was measured at 413 nm against a reagent blank which was p repared similarly. Calibration curve was p lott ed using absorbance-values versus concentration Fig.3. Assay procedure for tablets Twenty tablets were weighed and pulverized to a fine p owder. An aliquot equivalent to about 1 mg of KT F was t ransferred into a 100-ml volumetric flask. A susp ension 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 p aper to a second100-ml volumetric flask. Final solution was diluted to 100 ml with water. Results and Discussions The prop osed p rocedure is based on the reaction between KTF and BPB resulting in the formation of an ion-pair comp lex which could be extracted into chloroform and measured sp ectrop hotometrically . The exp erimental conditions were op timized and the methods validated. The formation of the comp lex is shown in the reaction scheme1. given below. Effect of pH In order to confirm the op timum p H range 0.5 ml of p hthalate buffer solution on the development and st ability of the colored was used and the p H of the reaction mixture was adjust ed exactly to values range between 2-5 with few drop s of 0.1 N NaOH or 0.1 N HCl a p lot of absorbance versus p H showed maximum color intensity and highly absorbance obtained at p H 3.0 Fig 4. On t he other hand the absorbance decreased at p H above and below 3.0 . hence this p H was used in all the subsequent exp erimental 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 KT F. A gradual increase in the absorbance was observed up to0.04%, beyond which a p lateau was obtained.. Hence, 1 ml of 0.04% BPB solution was maintained Fig.5. Extraction S olvent and S haking Ti me Several organic solvents (chloroform, toluene, carbon tetra chloride ,1,2dichloroethane and dichloromethane) were examined for their ability to extract drug - BPB ion-pair comp lex. Among those organic solvents, chloroform was found to be the most suitable for quantitative extraction. An organic p hase was required for times of 1 to 6 min p roduced constant absorbance, hence a shaking time of 4 min was chosen for use. The drug-dy e comp lex in the aqueous p hase was extracted with 5 ml of chloroform. The absorbance was measured each time under the op timum conditions and only one extraction was found to be adequate to achieve a quantitative recovery of the complex. Reaction time and addition seque nce The effect of the reaction time was st udied by p reparation of KT F-PB colored comp lex and measured under the op timum conditions from 1 to 10 minutes and there were no significant changes in absorbance under the op timal conditions for the sequence of addition and the maximum absorp tion was for the sequence (Drug + Buffer +Reagent) . IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (3) 2011 Composition of i on-pair complexes Anionic dy es such as BPB form ion-pair comp lex with the positively charged nitrogen containing molecule such as KT F. Each drug– dy e comp lex, with two op p ositely charged ions, behaves as a single unit held together by ions, behaves as a single unit held together by an electrost atic interaction. The suggested mechanism of KT F - BPB ion - p air comp lex formation is disp lay ed in Scheme 1. The composition of the ion p airs associates was established by Job’s method of continuous variation. In the p resent study , different amounts of KT F and BPB were added to each flask and extracted in the same manner as recommended p rocedure. The absorbance of formed KT F-BPB ion-pair comp lex was measured at 413 nm. The absorbance was p lott ed against [KT F]/[KT F]+[BPB] for Job’s method .In Job’s p lot, the p lot reached a maximum value at a mole fraction of 0.5, which indicated the formation of 1:1 (KT F-BPB) comp lex Fig.6 . The extraction equilibrium can be rep resented as follows: KT F + (aq) + Dy e - (aq) ↔ KTF + Dy e - (aq) ↔ KTF + Dy e (org) where KTF + and Dy e- represent t he p rotonated KT F and the anion of the BPB resp ectively.The subscrip t (aq) and (org) refer to the aqueous and organic p hases . The absorbance of each solution was p lott ed against the mole fraction of the drug ,VKTF/VKTF+VBPB (Fig. 6). Anal ytical data Under the op timized exp erimental condition, calibration curve was constructed by p lott ing the absorbance at λmax against the concentration of KT F. Beer's law range, molar absorp tivity , Sandell's sensitivity , regression equation, and correlation coefficient were determined for p rop osed 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 KT F in the final measured with molar absorp tion coefficients of 2.94×10 4 l/mol.cm. Regression analysis of the Beer's law p lots at λmax revealed a good correlation (R 2 = 0.998). The graph showed negligible intercep t and were described by the regression equation, y =0.069 C + 0.036 .The high molar absorp tivity of the resulting colored comp lex indicates and the high sensitivity of the method(T able 1). Se nsi tivity 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 Samp les of p ure KT F was p repared and tested in 3 replicates using the p rop osed p rocedure. The comp lete set of validation assay s was p erformed. The results are given in Table 1. Application to dosage forms The p rop osed method was successfully app lied to the determination of KT F in commercial tablets. The app licability of the p rop osed method for assay of KT F 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 st udy of drug suggested that commonly used additives and excip ients (lactose, magnesium st earate, st arch, sucrose, fructose and cellulose) do not interfere with the assay p rocedure. The p rop osed IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (3) 2011 method is sufficiently sensitive to p ermit determination of low concentration of KT F (0.162 μg/ml) did not interfere in the assay . Conclusions Unlike GC and HPLC techniques, sp ectrop hotometry is simple and inexp ensive. The imp ortance of the technique also lies in the chemical reactions up on which the p rocedures are based rather than up on the sop histication of the inst rument. This asp ect of sp ectrop hotometric analysis is of major interest in analytical p harmacy since it offers distinct p ossibility in the assay of a p articular comp onent in comp lex dosage formulations. The p rop osed methods require only dy es as reagents which are cheaper and readily available and the p rocedures do not involve any critical reaction conditions or tedious sample p reparation. M oreover, method is simple, fast, accurate, adequately sensitive and free from interference by common additives and excip ients. The p resent methods are sup erior with resp ect to both sensitivity and selectivity . The calculated ε values of the p rop osed method is 2.94 × 10 4 L/mol/cm . The p resent method, one of the characterist ic features of green analytical chemist ry . The wide app licability of the new p rocedures for routine quality control is well established by the assay of KT F in p ure form and in p harmaceutical preparations. In comp arison with HPLC method the recoveries were 51.7–95.5% , precisions for the drugs in p lasma were not greater than 9.5% [5] , that means t he prop osed method has a good recovery and precision . + KT F BPB + S cheme1. KTF – BPB propose d complex IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (3) 2011 Re ferences 1. Budavari S. (1996) The M erck Index. 12th ed. Whitehouse Station, NJ: M erck and Co. Inc, p. 836. 2. Grahnén, A.; Lönnebo, A.; Beck, O.; Eckernäs, S.A.; Dahlström, B. and Lindst röm, B. (1992), Pharmacokinetics of ketot ifen after oral administ ration to healthy male subjects, Biop harm Drug Disp os 13 (4): 255–262. 3. Galindez, O.A. and Kaufman, H.E. (1996) The management of allergic conjunctivitis. 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Seno, H.; Kumazawa, T. and Suzuki, O.(1991) Rapid isolation with Sep-Pak C18 cartridges and cap illary gas chromatography of ketot ifen and mequitazine in body fluids Jp n. J. Forensic Toxicol. 9:163-167. 9. Leis, H.J. and M alle, E. (1991) Deuterium-labelling and quantitative measurement of ketot ifen in human p lasma by gas chromatography /negative ion chemical ionization mass sp ectrometry , Biol M ass Spectrom 20:467 10. Tzvetanov, S.; Vatsova, M . ; Drenska,A.; Gorantcheva, J. and Ty uty ulkova, N., (1999), Gas chromatographic-mass sp ectrometric method for quantitative determination of ketot ifen in human p lasma after enzy me hy drolysis of conjugated ketot ifen, Journal of Chromatography B: Biomedical Sciences and Ap p lications, 732(1)251-256. 11. Julien-Larose, C.; Guerret, M .; Lavene, D. and Kiechel, J. R.(1983) Quantification of ketot ifen and its metabolites in human p lasma by gas chromatography mass sp ectrometry , Biological M ass Spectrometry , 10(3): 136–142. 12. NIE Fei and LÜ Jiu-Ru, (2006), Determination of Ketotifen with Pot assium Ferricy anide-Calcein Chemiluminescence Sy st em , Sp ectrochim Acta A M ol Biomol Sp ectrosc. 5(5922):168-172 13. El-Kousy , N. and Bebawy , L. I. (1999) Determination of some antihistaminic drugs by atomic absorp tion sp ectrometry and colorimetric methods, J Pharm Biomed Anal. ,20:4,671-679 14. M ohamed, E. M ohamed and Hassan, Y. Aboul-Enein (1986) Sp ectrop hotometric and Differential Pulse Polarographic M ethods of Analysis for Ketotifen Hy drogen Fumarate, Drug Dev Ind Pharm, 12(5):733-746. IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (3) 2011 15. Sayed M ehdi Ghoreishi; M ohsen Behpour; Hamid Ahmadi Zahrani and M ahshid Golestaneh, (2010) , Prep aration and Op timization of a Ketotifen Sensor and its Pharmaceutical Ap p lications, Anal. Bioanal. Electrochem., 2(3):112- 124 16. Zhou M .; Li YJ.; M a YJ.; Wang WF.; M i Jand Chen H. (2010) Determination of ketot ifen fumarate by cap illary electrop horesis with tris(2,2'-bipy ridyl) ruthenium(II) electrochemiluminescence detection. Luminescence,[Ep ub ahead of p rint] 17. Khater M .M .; Issa Y.M . and M ohammed S.H. (2009) Flow injection determination of ketot ifen fumarate using PVC membrane selective electrodes. Bioelectrochemistry ,77(1):53-9. 18. Tabrizivand, G.; Sabzi, R.E. and Farhadi, K. (2007),Prep eration and characterization of a new carbon p aste electrode based on ketot ifen- hexacy anoferrate, j.Solid State Electrochem 11:103-108 19. Daneshgar, P.; Norouz i, P. and Ganjali, M .R. (2009) ,Ap p lication of a continuous square-wave p otential p rogram for sub nano molar determination of ketot ifen. Chem Pharm Bull (Toky o). 57(2):117-121. 20. Singhvi, I. and Sachdeva, D. (2009) Sp ectrop hotometric Estimation of Ketotifen Fumarate from Tablet Formulations ,Indian J Pharm Sci. , 71(1): 66–68. 21. M assoud Amanlou; M ehdi Hoseinzadeh Nazlou; Homa Az izian; Effat Souri and Hassan Farsam , (2007),Determination of Ketotifen Fumarate in Raw M aterial and Pharmaceutical Products Using Ion-p air Formation , Analytical Letters, 40(17): 3267 - 3279 22. Hamed, A. Abu-shdy ; Sonia, T hassib; Safinaz, A. Abass and Abdulazim M .M . (2005) Sp ectrop hotometric Determination of Dip henylhydramine Hy drochloride and Ketoifen Hy drogen Fumarate , Bull. Fac. Pharm,43 (1):41-51. 23. Sastry , C.S.P.; Naidu, P.Y. and M urty , SSN. (1997) Visible sp ectrop hotometric methods for t he determination of ketotifen fumarate. East ern Pharmacist. 40,133–5. 24. Szczep aniak ,W.; Cy chowska, T. and Prz adka, T. (1992) Sp ectrop hotometric determination of ketot ifen in p harmaceutical p reparations after isolation on ion- exchanger, Acta Pol Pharm,49(4):3-5. 25. Chilukuri, S. P. Sastry and Petla, Y. Naidu, (1997), Sp ectrop hotometric estimation of ketot ifen fumarate in p harmaceutical formulations, M icrochimica Acta , 127(3- 4):219-223 26. Alaa S. Amin and Yousry , M . Issa,(2005) Sp ectrop hotometric determination of 6- aminopenicillanic acid using bromophenol blue and bromothy mol blue, M icrochimica Acta, 117(3-4:187-194. 27. ncilay , S sl and Ay la, Tamer , (2003) ,Ap p lication of Bromophenol Blue and Bromocresol Purp le for the Extractive-Sp ectrop hotometric Determination of Ofloxacin, Analytical Letters, 36(6):1163 – 1181 28. Kanakap ura , Basavaiah and Vaidy anathan, Shakunthala Charan, (2004)Ion-p air Complexometric Determination of Cy p roheptadine Hy drochloride Using Bromophenol Blue ScienceAsia 30:163-170 29. Amit, Kumar; Sanjo, Nanda and Rajive, Chomwal, (2010),Sp ectrop hotometeric M ethods for Determination of Doxy cycline in Tablet Formulation, International Journal of PharmTech Research, 2:599-602. IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (3) 2011 30. Rahman, N.; Khan, N.A. and Az mi, SNH.(2004) Extractive sp ectrop hotometric methods for the determination of nifedipine in p harmaceutical formulation using bromocresol green , bromophenol blue , bromothy mol blue and eriochrome black T , farmaco 59 :47-54 . Table (1): S ensitivity and regressi on parameters. Parameter KTF – BP B complex λmax (nm) 413nm Color yellow Linearity range (µg.mL -1 ) 0.4-15 Molar absorpitivites (l.mol -1 .cm -1 ) 2.94x10 4 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 (R 2 ) 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 propose d method. Sample Lab eled amount (mg) Conc. taken (µg.mL-1) Conc.* Foun d (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 a Relative standard deviation (%). b (found – taken / taken) ×100. n= 3 IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (3) 2011 Fig.(1): Chemical structure of ke totifen fumarate (KTF) MWt. 425.5 gmol -1 Conc.ppm Fig. (2): The spectrum of 10µg/mL of ke totifen fumarate Fig.(3):Calibration curve of KTF against reagent blank and the reagent blank ke totifen 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/m L IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL. 24 (3) 2011 Fig.(6): The effect of shaking time of complex Fig.(7): Job’s method of continuous variati on plot on absorption of KTF 10µg/m L for ion-pair com plexe 2011) 3( 24المجلد مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة 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 تـــم وصـــف الطریقـــة . اریـــت مطـــورة لتقــدیر عقـــار كیتـــوتیفین فیوم و حساســـة وســـریعة ة طیفیـــة قـــطری عملتاســت بــین اصــفر اللــونتقــدیر العقــار بصــورته النقیــة وفـي بعــض المستحضــرات الصــیدالنیة مــن خـالل تكــوین معقــد لواسـتعمالها 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) یم قـــ تكــذلك حســب. 104,26 – 100,66للمستحضــرات الدوائیــة علــى شــكل حبــوب بحــدود جاعیة كانــت نســبة االســتر . لـم یالحــظ اي تــداخل للمضـافات الموجــودة فــي . و حــد التقــدیر ،وحــد الكشـف ،حساسـیة ســاندل وكــذلك االنحـراف المعیــاري .ى النتائج المستحصلة لالمستحضرات الصیدالنیة ع . لمزدوجات األیونیة معقدات ا، البروموفینول األزرق ، عقار الكیتوتیفین فیوماریت : الكلمات المفتاحیة