IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (2) 2010 Spectrophotometric Determination of Amiodarone Hydrochloride in Pharmaceutical Preparations D. M. Salh Departme nt of Chemistry, College of Science, Unive rsity of Sulaimaniyah, Abstract Amiodarone hy drochloride (AH) has been determined sp ectrop hotometrically Using methy l orange (M O). In our p revious researches M O was used for determination of M exiletine Hy drochloride [1]. The method based on complexation between M O and AH. Aft er shaking and diluting the comp lex solution with D.W, the pH was adjust ed with NaOH and HCl to p H 3. The colored comp lex formed between AH and the reagent were transferred into sep arating funnels and extracted usin g 5.5ml CH2Cl2 and were shak en for (5 minutes). The extracted or ganic lay er was used for p reparation of the calibr ation curves for sp ectrop hotometric measurements of AH at 434nm. The blanks were carried out in exactly the same way throughout the whole p rocedure. M olar absorp tivity (ε L.mol-1.cm-1), detection limit, limit of linearity (µg.ml -1 ) and r 2 were, 5.74 *10 5 , 0.04 and 0.982 for (AH-M O) resp ectively. The method was used with reasonable accuracy and p recision for the determination of (AH) in sy nthetic samp les of t ablets, cap sules and ampoules. Keywords: sp ectrop hotometric determination of amiodarone hy drochloride, methy l orange. Introduction Amiodarone 2-buty l-3-benzofuranyl-4-[2-(diethy l amino) ethoxy ] -3-5diiodop henyl ketone or (Cordarone), have the ch emical structure as shown in (Fig.1): It was introduced as an antiaginal agent. It has very p ronounced class III action and is esp ecially effective in maintaining sinus rhy thm in p atients who have been treated by direct current shock for a trial fibr illation. Like class III antiarrhythmic drug, amiodarone lengthens the effective r efractory p eriod by p rolonging the action p otential duration in all myocardial tissues. Amiodarone is eliminated very slowly from the body , with a half-life of about 25 to 30 days after oral doses [2]. The oldest chromato graphy method in this review was used for amiodarone metabolites in biolo gical fluids [3] usin g Liquid- Solid Extraction and HPLC techniques, appeared in (1988). Later, many chromatogr aphic methods for the determinations of this drug and its metabolites have been reported [4-9] attempting different modifications in the method to increase sensitivity, reducing st eps of analysis, or other imp rovements, with the limit of lin earity ranged between [0.05 mg ml -1 – 20 mg ml -1 ]. During the literature review found that there was a method for determination of amiodaron e usin g Amiodaron e-selective membrane electrode [10], they applied a liqu id membran e electrode based on an amiodarone-dipicry lamin e ion-p air comp lex Extensive search in the literature has showed many sp ectrop hotometric methods for the determination of M H [11-14]. Among them, how used deriv ative UV sp ectrop hotometry , and visible sp ectrop hotometry , with different type of optimizations to obey beers law. Prelimin ary p ractical tests on many reagents revealed that methy l oran ge which the chemical st ructure as shown in the (Fi g.2), was suitable r eagents which have the following chemical st ructure [15] to form colored comp lexes with the drug AH and were exp loited for its quantitative determination in cap sules, amp oules, serum, and tablets. IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (2) 2010 Experime ntal Apparatus All measurements were taken with TU-1800S UV-Vis sp ectrop hotometer, with (1cm) p ath length quartz cells. T he pH of t he solutions was adjust ed by using Hanna pH-meter with combined glass electrode (910600) Orion Comb p H, Water bath Thermost at Shaker (GFL 1083) was used during the extraction steps and M icro p ipettes (variable and fixed). Chemicals, reagents, and drugs Both Analar and general p urp ose reagents were used from [Fluka, Rohm and Haas, GCC (Gainland Ch emical Company), and M erck] without further p urification. Ordinary distilled water p rep ared in all glass st ill and st ored in p olyethy lene container was used. Amiodaron e hy drochloride amp oule (150mg/3ml), [sanofi ~ sy nthelabo], was taken as a stock solution; other concentrations were p rep ared by usual dilution. M ethy l orange 0.005% aqueous M O. Pht halate buffer (p H=3) was p rep ared by mixing 50 ml of 0.1M (p otassium hy drogen p hthalate), with 22.3 ml of 0.1M HCl [16, 17], and pH was adjusted with a p H meter. The recommended procedures A volume of 0.2 ml 0.1% M O reagent was added to a certain amount of AH st andard or samples containing between (0.04-0.22 μgml -1 ) AH. The mixture was shaking for (30 se c.) and diluted to 23ml in volumetric flasks usin g D.W. The p H was adjust ed by adding 0.5ml p hthalate buffer (p H 3) to the M O mixture and finally comp leted to 25ml. The resulting comp lex formed between AH and the reagent were transferred into separating funn els (100m l cap acity ) and extracted with 5.5ml CH2Cl2 in two p ortions to wash out the volumetric flasks for quantitative transfer of the solution and was shaken for 5 minutes. Aft er sep aration, the organic lay er was used for p reparation of the calibration curves usin g sp ectrop hotometric measurements of AH at 434nm. The blanks were carried out in exactly the same way throughout the whole p rocedure. Results and Discussion Preliminary work The absorption sp ectrum of the comp lex (AH-M O) against blank is shown in Fi gure (3) showing λ- max 434nm. A clear sp ectrum of the AH-MO with no observed shoulder and blank sp ectrum are seen in Figure (3). The sp ectrum also shows some background of the reagent in the region of the complex which will have a n egative eff ect on the sensitivity of the method. The comp lex; or an ion-pair formation between the AH and M O, has show an increasin g intensity of the sp ectrum. This is certainly a negative analytical p henomenon; since no high sensitivity could be exp ected with this sy stem. However, the reagent was exp ected to show p romising results therefore, studies were continued for optimization of the conditions. Optimization 1. pH Optimi zation A volume of 0.2 of 0.1% M O was added to 0.5ml of 1mg. ml -1 AH, shaking for 30 seconds, then diluted to 25ml in vo lumetric flasks. The p H was then adjust ed between 1.5 to 5, by using 0.1M NaOH or 0.1M HCl. The rest of the test was then followed accordin g to the p rocedure. The results reveal that the optimum p H for (AH-MO) comp lex is 3, shown in (Fig.4). 2. Type of buffe r use d (pH-adjustment) For the pH adjust ment of (AH-O) sy stem different buffers were tried, such as; acetic acid- sodium acetate and citric acid-sodium hy droxide, but Pht halate buffer p H = 3 [28, 27] was found suitable to adjust pH of the comp lex (AH-M O). Different volumes of this buffer were added in two way s; either before completing the volu metric flask by D.W to the mark, or until a small vo lume about 2ml was remainin g then the buffer was added and comp leted to t he IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (2) 2010 mark. The results indicated that op timum volume and suitable time of addition were equal to 0.5ml p hthalate buffer added buffer before completing the volumetric flask by D.W (after mixing), and the result is shown in table (1). 3. Choosing a Suitable S olvent for Extraction M any solvents were tested for extracting the comp lex formed b etween the M O reagents with AH and the best solvent among many organic solvents like [C arbon tetrachloride, chloroform, Dich lorobenzene, Acetop henone) was found is dichloromethane. 4. Optimum amounts of the Reagents Prelimin ary test shows that 0.1% MO was suitable. Exp eriments were then p erformed with different volumes of the different concentrations to a constant volume 0.5ml 1mgml -1 AH. The results shown in (Fig.5) represent the optimum amount of MO. 5. Volume optimization of dichloromethane CH2Cl2 Different volumes of the solvent dichloromethane between 4 to 10 mls was used for extraction of the (AH-MO) complex as shown on (Fi g.6) shows t he ran ges between 4 – 6 mls the comp lex. A volume of 5.5ml CH2Cl2 was found suitable and also sufficient to comp lete the analy sis. 6. S tability of the complexes The stability of the comp lex formed between (AH—MO) was followed by measuring absorbance against time. As shown in (Fi g.7). It was found that the comp lex (AH—M O) was st able for a p eriod of 35 minutes, after sep aration and only 10 minutes were needed to reach the true absorbance, and through this time there was t ime for shaking and for comp lete comp lexation that shown in the (Fig.8). Absorbance has, then increased after that due to the vaporization of the solvent. S toichiometry of the [AH-MO] complex The stoichiometry of the drug and M O complexing reagent was examined by the mole ratio method at wave len gth of 434nm: The result of mole ratio method was obt ained in two way s first by adding constant amount of M H to a series of different amounts of M O solutions, and second by adding constant amount of MO to a series of different amounts of AH solutions. Fig.(9a and 9b) show the results of mole ratio method. It was found t hat the ratio of (AH-t o-MO) was about (2 to 1). S tructure suggestion It was not p ossible to p ut forward a reasonable st ructure for the stoichiometry of AH: M O of 2:1. Accordin g to their chemical structure, it was thought that a 1:1 or 1:2 ratios would be reasonable. This will make a salt-like formation between the AH and MO t hus:- 2 AH + MO ---------►M O (AH) 2 Calibration curves: The calibration curves obtained according to the recommended p rocedure were drawn for both (AH + M O) complex. The linear range was 0.04 – 0.24 µ g. ml -1 of AH with (r 2 = 0.982) for (AH + MO) comp lex as shown in (Fig. 10). De termination of Amiodarone hydrochloride in synthe tic sample solutions: The recovery test was p erformed for differ ent concentration of AH with M O reagent, results shown in (table 2). De termination of mexiletine hydrochloride in tablets: Amiodarone tablets were p owdered, mixed thorough ly and weighed accurately to an equivalent to 200mg of AH. The mixture was st irred well with dichloromethane and filtered through a p iece of Whatmann No. 42 filter p ap er. The residue was washed with dichloromethane for comp lete recovery of the drug. The filtrate was diluted to known volume of st andard flask. In p rep aration of tablets, AH has been removed from the additives to make a solution. Therefore AH could be determin ed by calibration curve. Three different volumes (0.5, 1 and IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (2) 2010 1.5) mls of the sample of AH were determined by MO accordin g to the recommended p rocedure. The results are shown in tables 3. To test for the existence of a sy st ematic error in the results shown in tables 3, the actual difference between (X) and ( μ) was comp ared by t-test with the term [t.S / √N] at 95% confidence limit DOF = 2. From the results of t-test MO reagent t he following conclusion were made: The direction of the errors (-) su ggest the existence of a sy st ematic error. This may be due to the extraction st eps. Nearly non dependence of these errors on sample size b etween (0.5 to 1.5)mls suggests t he existence of both const ant and p rop ortional sy stematic errors. The difference between (X - μ) and [t.s / √N] was not significant at 95% C. L. in all cases (0.5 to 1.5)mls of AH sample, indicating the non existence, or p resent of a very small systematic error. which is mainly due to t he extraction st ep s. If the recovery tests were considered only as it is t he case with research workers, t he value of (R %) in tables ar e quite reasonable. Precision and Accuracy: The p recision of AH determination by M O comp lexing reagent was p erformed on three sy nthetic samples containing AH in the range of the calibration curve, and their absorbance were measured 10 times for the same unknown, showing the precision of measurements. T he p recision was also found on 10 times repeating of the whole op eration on the same sample. This will show the p recision of the op eration. The relative st andard deviation for the (AH- MO) comp lex r anged b etween 0.39 – 0.92% showing r easonable p recision even at lower concentrations of AH. The accuracy shown in the p revious sections also rev ealed reasonable accuracy giving sufficient validity for the application of both reagents to be used for the determination of AH in the tablets. Se nsi tivity of the methods: The results of t he prop osed method were statist ically comp ared with those obtained by the sp ectrop hotometric method for determination of AH using two methods [13] and are summarized in (Table 4), the table show the results concerning sensitivity of the methods (Valu es of molar absorp tivity (ε), slop es of the calibration curves (m), limits of linearity and detection limit (D.L= 3 S.D)). T he results indicate reasonable sensitivity of the methods with no significant deference between the methods comp ared. Re ferences 1. Dler,M . Salh; (2007) “Spectrop hotometric Determination of M exiletine Hy drochloride in Pharmaceutical Prep arations, Urine and Serum” M .Sc. thesis, Su laimaniyah University . 2. Block,J.H. and Beale, J.M . (2004). “Organic M edical and Pharmaceutical Chemistry ”11th Edition p .641, by Lipp incott Williams & Wilkins, ISBN: 0-7817- 3481-9. 3. Petrarulo , ,M .; Pellegrino, S. ; M entast i,E. July (1988), J.Chromato., 25: ( 7),593 4. Ching-Nan Ou, Chery l L Ro gn erud, Ly T. Duong, and Vicki L Frawley ; (1990) CLIN. CHEM . 36:(3), 532-534 5. Al-Dhawailie,A. A. Oct. (1995), Analytical letters, 28, Issue 13 ,2391-2400 6. Andrew, S.; Jun, Dion, R. Brocks; (2001), J Pharm Pharmceut. Sci , 4 (3) : 263-268. 7. M aes ,A. ; Baert, K.; Croubels,S. ; De Clercq,D.; van Loon, G. Dep rez ,P. and De Backer;P. 19 M ay (2006), J Chromato B; 836 Issue 1-2, , p .47-56. 8. Shuijun Lia, Gan gy i Liua, Jin gy ing Ji aa, Yun Liu a, Chen g Panb, Ch en Yua, Yon gb ao Caia and Ji anying Renb; M arch (2007), J Chromato B; 847 Issue 2, 1, p.174-181. 9. Pérez-Ruiz T, M artínez-Lozano C, García-M artínez M D.; (2008),Anal Chim Acta. 8:623 (1):89-95. 10. Raluca-Io ana Stefan, Hassan Y. Aboul-Enein and George-Emil Baiulescu ; December (1996), Art icle Out line, 37, Issue 3, p.141-144 IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (2) 2010 11. Anna, M . Di Pietra, Vanni Cavrini, Rita Gatti, and M aria, A. Raggi; (1988), Pharma. Research, 5: ( 11), 709 12. Rao ,T.S., Sastry ,C .S.P. (2002) Sep .-Oct, Indian J. Pharm. Scie.: 64(5): 482-5 13. Nafisur RAHM AN,† Nadeem Ahmad KHAN, and Sy ed Najmu l Hejaz AZM I; Aug(2004), ANALYTICAL SCIENCES , 20: 1231-1235. 14. Pérez-Ruiz T, M artínez-Lozano C, M artín J,and Ruiz E; .Sep.( 2006), J Pharm Biomed Anal. 11 ;42(1):143-7. 15. Edmund Bishop ; (1972), “Indicators” First , pp . (79 – 81, and 358); Dep t. of Chem., Univ. of Exeter printed in Germany, 08-016617-2. 16. Perrin D.D. and De mpsey B.; (1974), “Buffers for p H and M etal Ion Control” First p ublished, p .123-156, By Chap man and Hall Ltd, ISBN: 0-412-21890-9. 17. Kolthoff, I.M .; Sandell, E.B.; M eehan, E.J.,and Stanley Bruckenst ein; (1969), “Quantitative Chemical Analysis” Fourt h Edition, p .1161 – 1165, M acmillan Company, Collier-M acmillan Limited. Table (1): Result of adding different volume phthalate buffe r before and after a dding D.W to the mixture of AH and MO. Adding the buffe r be fore comple ting the volume tri c flask by D.W (after mixi ng). Adding the bu ffer after mixing an d afte r adding D.W until abou t 2ml were remaining to com plete the volu me. 0.25 ml Abs.= 0.58 0.25 ml Abs.= 0.44 0.5 ml Abs.= 0.807 0.5 ml Abs.= 0.51 0.75 ml Abs.= 0.736 0.75 ml Abs.= 0.52 1 ml Abs.= 0.723 1.ml Abs.= 0.49 1.25 ml Abs.= 0.68 1.25 ml Abs.= 0.48 1.5 ml Abs.= 0.523 1.5 ml Abs.= 0.12 Table( 2): The accuracy of AH determination in syntheti c samples using MO [AH-MO] com plex. AH Present ppm AH Found ppm R% E% 0.2 0.191 95.5 -4.5 0.2 0.191 95.5 -4.5 0.2 0.189 94.5 -5.5 IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (2) 2010 Table (3): Results of different volumes of AH sample (tablets) determined by MO reagent Conc. Of AH ppm found in Table ts by MO Usi ng the Eqn. Y = 0.8764X + 0.0006 Vol.(ml) Of the sample taken AH ppm R% E% 0.5 0.0758 94.75 -5.25 1 0.1613 100.81 0.81 1.5 0.23 95.83 -4.17 Table (4): Se nsitivity of the propose d method Typ e of calibration ε (L.mol -1 .cm -1 ) Limits of lin earity (μg/ml) Recovery % R.S.D D.L.= 3S. D (μg/ml) r2 calibration curve for (AH+MO)compl ex 5.74*10 5 0.04 94.75 0.39 - 0.92 0.00253 0.982 100.81 95.83 Reference method (Two Methods A&B) A= 1.42 *10 3 B= 7.50 *10 3 A= 10 B= 2 A 100.11 A= 0.11 - 0.91 B= 0.16 - 1.41 A= 1.091 B= 0.161 0.9999 100.06 100.07 B 100.10 100.17 100.06 a. Molar absorpt ivit y ε (L.mo l-1.cm-1), b. Limit s of linearit y, c. (Recovery % and R.S.D) of different volumes of AH samp le (tablets) using propo sed method, d D.L with correlat ion coefficient O CH2CH2CH2 CH3 C O I I OCH2CH2N(CH2CH3)2 Amio darone (Co rdarone) Fig.(1) Amiodarone Chemi cal S tructure N NNaSO3 N CH 3 CH 3 M ethy l Orange p K1 = 3.76 p H transition interval = 3.1 (Red) – 4.4 (Yellow) Fig.(2) Methyl Orange Che mical Structure IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (2) 2010 43 4 nm 0 0. 2 0. 4 0. 6 0. 8 1 1. 2 1. 4 1. 6 1. 8 2 350 400 450 500 550 600 650 Wav e len gth λ (nm) A b s . 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 3 50 4 00 45 0 500 550 600 650 Wa ve len gth λ (nm ) A b s . Fig. (3) The spectrum of the (AH+MO) complex, and blank spectrum Fig. (4) The pH optimizati on for (MH+MO) complex Optim um amount of M .O. 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2. 4 Vol. o f 0.005% M .O A bs . Fig. (5) O ptimization of volume of 0.1% MO Optimum Vol. i s Les s t han 5 ml . 0 0.0 5 0 .1 0.1 5 0 .2 0.2 5 0 .3 0.3 5 0 .4 0.4 5 0 .5 0 1 2 3 4 5 6 7 8 9 10 11 Vol um e (m l) A b s. Fig. (6): O ptimization of CH2Cl 2 volume to be added for extraction the (AH-MO) complex Op timum p H = 3 0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 6 pH A b s . IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (2) 2010 0.55 0.555 0.56 0.565 0.57 0.575 0.58 0.585 0.59 0.595 0.6 0 5 10 15 20 25 30 35 4 0 45 50 55 6 0 65 70 75 80 85 90 9 5 10 0 10 5 11 0 Time (min.) A b s. Stab ili ty o f c omp le x Fig.(7) S tability of the (AH –MO) complex after separation time needed for shaking. 0 0.1 0.2 0.3 0.4 0.5 0.6 0 1 2 3 4 5 6 7 8 9 Time (min.) Ab s. Fig.(8) S uitable time for shaking the (AH –MO) complex after separation 0.1 0 .12 0 .14 0 .16 0 .18 0.2 0 .22 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2 .1 2 .2 2.3 2.4 2.5 M ole Rati o [AH / MO] A b s . [AH / MO] = 2.03 0 0 .02 0 .04 0 .06 0 .08 0.1 0 .12 0 .14 0 .16 0 .18 0.2 0 0.04 0 .08 0 .12 0.16 0.2 0 .24 0 .28 0.32 0 .36 0 .4 0 .44 0.48 Mole Ratio MO / AH A b s. [MO / AH] ≈ 0. 32 (a) (b) Fig.(9a and b) De terminati on of the stoichiometry of the (AH to MO) by mole ratio y = 0.876 4x + 0.00 06 R 2 = 0.982 0 0.05 0.1 0.15 0.2 0.25 0 0.02 0.04 0. 06 0. 08 0.1 0. 12 0.14 0.16 0. 18 0. 2 0.22 0.24 0. 26 p pm AH A b s . Fig.(10) Calibration curve for (AH-MO) complex IHJPAS 2010) 2( 32المجلد مجلة ابن الهیثم للعلوم الصرفة والتطبیقیة التقدیر الطیفي لھایدروكلورید االمیودارون في المستحضرات الصیدالنیة دلیر محمد صالح شریف جامعةالسلیمانیة ِم، قسم كیمیاِء، كلیَّة الِعلو الخالصة ودارون قـدر ــالَي تطیفیـاً باسـ AHھایدروكلوریـد االمـی ل البرتق Mعمال المثـی O. ــم اذ ي بحوثِنـا السـابقِة ت ان ـف Mعمال كاشف المثیل البرتقالَي تاس O لتقدیرھایدروكلورید المكسیلیتینM H . ین ون ـب د مـل الطریقة مسـتندة علـى تكـوین معـق M O ع ــر . AHـم ــاء المقط ــِد بالم ول المعقّ ــف مَحـل ــرج وتَخفی ــد ال pالـــ ، ثبّـتD.Wبع H ــى . HClو NaOHبالـــ ) 3(عل َل 5.5والكاشف یحول إلى قمع الفَْصل ویسـتخلص باسـتعمال AHالمرّكب المُلَوَّن التكوَن بین د المثـی ر مـن ثنـائي كلورـی ملیلـت CH2Cl2 ـ اتخاصـة الطبقة العضویة المس). دقائقِ 5(ثم الرج مدة ى المعـایرة للتقـدیر الطیفـي ـل AHسـتعملْت لتحضـیِر منحـن ـ . كافـة فـي الجوانـب نفسـھا الطریقِـة الكاملـةبنُفّذت بالضبط Blankان الـ ). nm 434(في الطول الموجي Mان اـل olar absorp tivity (ε L.mol -1 .cm -1 limit of linearityالمـدى الخطـي ، detection limitحد الكشف و، ( (µg.ml -1 ) r(و 2 * 5.74(عبارة عن ) 5 �ة . عل�ى الت�والي) 0.982(و ) 0.04(، ) 10 �ِة والض�بط المعق�ولین لتق�دیر االطریق س�تعملْت بالدق )AH ( ِالِت واألنبوبات في العیناِت الصناعیِة ِمْن األقراِص والكبسو IHJPAS