IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.23 (2) 2010 Spectrophotometric Determination of Amiodarone Hydrochloride in Pharmaceutical Preparations D. M. Salh Department of Chemistry, College of Science, University of Sulaimaniyah, Abstract Amiodarone hydrochloride (AH) has been determined spectrophotometrically Using methyl orange (MO). In our previous researches MO was used for determination of Mexiletine Hydrochloride [1]. The method based on complexation between MO and AH. After shaking and diluting the complex solution with D.W, the pH was adjusted with NaOH and HCl to pH 3. The colored complex formed between AH and the reagent were transferred into separating funnels and extracted using 5.5ml CH2Cl2 and were shaken for (5 minutes). The extracted organic layer was used for preparation of the calibration curves for spectrophotometric measurements of AH at 434nm. The blanks were carried out in exactly the same way throughout the whole procedure. Molar absorptivity (ε L.mol-1.cm-1), detection limit, limit of linearity (µg.ml-1) and r2 were, 5.74 *105, 0.04 and 0.982 for (AH-MO) respectively. The method was used with reasonable accuracy and precision for the determination of (AH) in synthetic samples of tablets, capsules and ampoules. Keywords: spectrophotometric determination of amiodarone hydrochloride, methyl orange. Introduction Amiodarone 2-butyl-3-benzofuranyl-4-[2-(diethyl amino) ethoxy] -3-5diiodophenyl ketone or (Cordarone), have the chemical structure as shown in (Fig.1): It was introduced as an antiaginal agent. It has very pronounced class III action and is especially effective in maintaining sinus rhythm in patients who have been treated by direct current shock for a trial fibrillation. Like class III antiarrhythmic drug, amiodarone lengthens the effective refractory period by prolonging the action potential 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 chromatography method in this review was used for amiodarone metabolites in biological fluids [3] using Liquid-Solid Extraction and HPLC techniques, appeared in (1988). Later, many chromatographic 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 steps of analysis, or other improvements, with the limit of linearity ranged between [0.05 mg ml -1 – 20 mg ml -1 ]. During the literature review found that there was a method for determination of amiodarone using Amiodarone-selective membrane electrode [10], they applied a liquid membrane electrode based on an amiodarone-dipicrylamine ion-pair complex Extensive search in the literature has showed many spectrophotometric methods for the determination of MH [11-14]. Among them, how used derivative UV spectrophotometry, and visible spectrophotometry, with different type of optimizations to obey beers law. Preliminary practical tests on many reagents revealed that methyl orange which the chemical structure as shown in the (Fig.2), was suitable reagents which have the following chemical structure [15] to form colored complexes with the drug AH and were exploited for its quantitative determination in capsules, ampoules, serum, and tablets. IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.23 (2) 2010 Experimental Apparatus All measurements were taken with TU-1800S UV-Vis spectrophotometer, with (1cm) path length quartz cells. The pH of the solutions was adjusted by using Hanna pH-meter with combined glass electrode (910600) Orion Comb pH, Water bath Thermostat Shaker (GFL 1083) was used during the extraction steps and Micro pipettes (variable and fixed). Chemicals, reagents, and drugs Both Analar and general purpose reagents were used from [Fluka, Rohm and Haas, GCC (Gainland Chemical Company), and Merck] without further purification. Ordinary distilled water prepared in all glass still and stored in polyethylene container was used. Amiodarone hydrochloride ampoule (150mg/3ml), [sanofi ~ synthelabo], was taken as a stock solution; other concentrations were prepared by usual dilution. Methyl orange 0.005% aqueous MO. Phthalate buffer (pH=3) was prepared by mixing 50ml of 0.1M (potassium hydrogen phthalate), with 22.3 ml of 0.1M HCl [16, 17], and pH was adjusted with a pH meter. The recommended procedures A volume of 0.2ml 0.1% MO reagent was added to a certain amount of AH standard or samples containing between (0.04-0.22 μgml -1) AH. The mixture was shaking for (30 sec.) and diluted to 23ml in volumetric flasks using D.W. The pH was adjusted by adding 0.5ml phthalate buffer (pH 3) to the MO mixture and finally completed to 25ml. The resulting complex formed between AH and the reagent were transferred into separating funnels (100ml capacity) and extracted with 5.5ml CH2Cl2 in two portions to wash out the volumetric flasks for quantitative transfer of the solution and was shaken for 5 minutes. After separation, the organic layer was used for preparation of the calibration curves using spectrophotometric measurements of AH at 434nm. The blanks were carried out in exactly the same way throughout the whole procedure. Results and Discussion Preliminary work The absorption spectrum of the complex (AH-MO) against blank is shown in Figure (3) showing λ- max 434nm. A clear spectrum of the AH-MO with no observed shoulder and blank spectrum are seen in Figure (3). The spectrum also shows some background of the reagent in the region of the complex which will have a negative effect on the sensitivity of the method. The complex; or an ion-pair formation between the AH and MO, has show an increasing intensity of the spectrum. This is certainly a negative analytical phenomenon; since no high sensitivity could be expected with this system. However, the reagent was expected to show promising results therefore, studies were continued for optimization of the conditions. Optimization 1. pH Optimization A volume of 0.2 of 0.1% MO was added to 0.5ml of 1mg.ml -1 AH, shaking for 30 seconds, then diluted to 25ml in volumetric flasks. The pH was then adjusted between 1.5 to 5, by using 0.1M NaOH or 0.1M HCl. The rest of the test was then followed according to the procedure. The results reveal that the optimum pH for (AH-MO) complex is 3, shown in (Fig.4). 2. Type of buffer used (pH-adjustment) For the pH adjustment of (AH-O) system different buffers were tried, such as; acetic acid- sodium acetate and citric acid-sodium hydroxide, but Phthalate buffer pH = 3 [28, 27] was found suitable to adjust pH of the complex (AH-MO). Different volumes of this buffer were added in two ways; either before completing the volumetric flask by D.W to the mark, or until a small volume about 2ml was remaining then the buffer was added and completed to the IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.23 (2) 2010 mark. The results indicated that optimum volume and suitable time of addition were equal to 0.5ml phthalate 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 Solvent for Extraction Many solvents were tested for extracting the complex formed between the MO reagents with AH and the best solvent among many organic solvents like [Carbon tetrachloride, chloroform, Dichlorobenzene, Acetophenone) was found is dichloromethane. 4. Optimum amounts of the Reagents Preliminary test shows that 0.1% MO was suitable. Experiments were then performed 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 (Fig.6) shows the ranges between 4 – 6 mls the complex. A volume of 5.5ml CH2Cl2 was found suitable and also sufficient to complete the analysis. 6. Stability of the complexes The stability of the complex formed between (AH—MO) was followed by measuring absorbance against time. As shown in (Fig.7). It was found that the complex (AH—MO) was stable for a period of 35 minutes, after separation and only 10 minutes were needed to reach the true absorbance, and through this time there was time for shaking and for complete complexation that shown in the (Fig.8). Absorbance has, then increased after that due to the vaporization of the solvent. Stoichiometry of the [AH-MO] complex The stoichiometry of the drug and MO complexing reagent was examined by the mole ratio method at wave length of 434nm: The result of mole ratio method was obtained in two ways first by adding constant amount of MH to a series of different amounts of MO 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 that the ratio of (AH-to-MO) was about (2 to 1). Structure suggestion It was not possible to put forward a reasonable structure for the stoichiometry of AH: MO of 2:1. According 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 thus:- 2 AH + MO ---------►MO (AH) 2 Calibration curves: The calibration curves obtained according to the recommended procedure were drawn for both (AH + MO) complex. The linear range was 0.04 – 0.24 µg.ml -1 of AH with (r 2 = 0.982) for (AH + MO) complex as shown in (Fig. 10). Determination of Amiodarone hydrochloride in synthetic sample solutions: The recovery test was performed for different concentration of AH with MO reagent, results shown in (table 2). Determination of mexiletine hydrochloride in tablets: Amiodarone tablets were powdered, mixed thoroughly and weighed accurately to an equivalent to 200mg of AH. The mixture was stirred well with dichloromethane and filtered through a piece of Whatmann No. 42 filter paper. The residue was washed with dichloromethane for complete recovery of the drug. The filtrate was diluted to known volume of standard flask. In preparation of tablets, AH has been removed from the additives to make a solution. Therefore AH could be determined by calibration curve. Three different volumes (0.5, 1 and IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.23 (2) 2010 1.5) mls of the sample of AH were determined by MO according to the recommended procedure. The results are shown in tables 3. To test for the existence of a systematic error in the results shown in tables 3, the actual difference between (X) and (μ) was compared by t-test with the term [t.S / √N] at 95% confidence limit DOF = 2. From the results of t-test MO reagent the following conclusion were made: The direction of the errors (-) suggest the existence of a systematic error. This may be due to the extraction steps. Nearly non dependence of these errors on sample size between (0.5 to 1.5)mls suggests the existence of both constant and proportional systematic 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 present of a very small systematic error. which is mainly due to the extraction steps. If the recovery tests were considered only as it is the case with research workers, the value of (R %) in tables are quite reasonable. Precision and Accuracy: The precision of AH determination by MO complexing reagent was performed on three synthetic 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. The precision was also found on 10 times repeating of the whole operation on the same sample. This will show the precision of the operation. The relative standard deviation for the (AH- MO) complex ranged between 0.39 – 0.92% showing reasonable precision even at lower concentrations of AH. The accuracy shown in the previous sections also revealed reasonable accuracy giving sufficient validity for the application of both reagents to be used for the determination of AH in the tablets. Sensitivity of the methods: The results of the proposed method were statistically compared with those obtained by the spectrophotometric 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 (Values of molar absorptivity (ε), slopes of the calibration curves (m), limits of linearity and detection limit (D.L= 3 S.D)). The results indicate reasonable sensitivity of the methods with no significant deference between the methods compared. References 1. Dler,M. Salh; (2007) “Spectrophotometric Determination of Mexiletine Hydrochloride in Pharmaceutical Preparations, Urine and Serum” M.Sc. thesis, Sulaimaniyah University . 2. Block,J.H. and Beale, J.M . (2004). “Organic Medical and Pharmaceutical Chemistry”11th Edition p.641, by Lippincott Williams & Wilkins, ISBN: 0-7817- 3481-9. 3. Petrarulo , ,M.; Pellegrino, S. ; Mentasti,E. July (1988), J.Chromato., 25: ( 7),593 4. Ching-Nan Ou, Cheryl L Rognerud, 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. Maes ,A. ; Baert, K.; Croubels,S. ; De Clercq,D.; van Loon, G. Deprez ,P. and De Backer;P. 19 M ay (2006), J Chromato B; 836 Issue 1-2, , p.47-56. 8. Shuijun Lia, Gangyi Liua, Jingying Jiaa, Yun Liua, Cheng Panb, Chen Yua, Yongbao Caia and Jianying Renb; March (2007), J Chromato B; 847 Issue 2, 1, p.174-181. 9. Pérez-Ruiz T, Martínez-Lozano C, García-Martínez MD.; (2008),Anal Chim Acta. 8:623 (1):89-95. 10. Raluca-Ioana Stefan, Hassan Y. Aboul-Enein and George-Emil Baiulescu; December (1996), Article Outline, 37, Issue 3, p.141-144 IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.23 (2) 2010 11. Anna, M. Di Pietra, Vanni Cavrini, Rita Gatti, and Maria, 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 RAHMAN,† Nadeem Ahmad KHAN, and Syed Najmul Hejaz AZMI; Aug(2004), ANALYTICAL SCIENCES , 20: 1231-1235. 14. Pérez-Ruiz T, Martínez-Lozano C, Martín 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); Dept. of Chem., Univ. of Exeter printed in Germany, 08-016617-2. 16. Perrin D.D. and Dempsey B.; (1974), “Buffers for pH and Metal Ion Control” First published, p.123-156, By Chapman and Hall Ltd, ISBN: 0-412-21890-9. 17. Kolthoff, I.M.; Sandell, E.B.; Meehan, E.J.,and Stanley Bruckenstein; (1969), “Quantitative Chemical Analysis” Fourth Edition, p.1161 – 1165, Macmillan Company, Collier-Macmillan Limited. Table (1): Result of adding different volume phthalate buffer before and after adding D.W to the mixture of AH and MO. Adding the buffer before completing the volumetric flask by D.W (after mixing). Adding the buffer after mixing and after adding D.W until about 2ml were remaining to complete the volume. 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 synthetic samples using MO [AH-MO] complex. 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. SCI. VOL.23 (2) 2010 Table (3): Results of different volumes of AH sample (tablets) determined by MO reagent Conc. Of AH ppm found in Tablets by MO Using 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): Sensitivity of the proposed method Type of calibration ε (L.mol-1.cm-1) Limits of linearity (μg/ml) Recovery % R.S.D D.L.= 3S.D (μg/ml) r2 calibration curve for (AH+MO)compl ex 5.74*105 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 *103 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 absorptivity ε (L.mol-1.cm-1), b. Limits of linearity, c. (Recovery % and R.S.D) of different volumes of AH sample (tablets) using proposed method, d D.L with correlation coefficient O CH2CH2CH2 CH3 C O I I OCH2CH2N(CH2CH3)2 Amiodarone (Cordarone) Fig.(1) Amiodarone Chemical Structure N NNaSO3 N CH3 CH3 Methyl Orange pK1 = 3.76 pH transition interval = 3.1 (Red) – 4.4 (Yellow) Fig.(2) Methyl Orange Chemical Structure IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. SCI. 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 Wave length 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 00 450 500 550 600 650 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 optimization for (MH+MO) complex Optimum 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. of 0.005% M .O A bs . Fig. (5) Optimization of volume of 0.1% MO Optimum Vol. is Less than 5ml . 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0 1 2 3 4 5 6 7 8 9 10 11 Volume (ml) A bs . Fig. (6): Optimization of CH2Cl2 volume to be added for extraction the (AH-MO) complex Optimum pH = 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. SCI. 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. Stabili ty of c omp le x Fig.(7) Stability 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) Suitable 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 Mole Rati o [AH / MO] A bs . [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) Determination of the stoichiometry of the (AH to MO) by mole ratio y = 0.8764x + 0.0006 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 ppm 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 ــم اذ .MOعمال المثـی ي بحوثِنـا السـابقِة ت ان ـف ین . MHلتقدیرھایدروكلورید المكسیلیتین MOعمال كاشف المثیل البرتقالَي تاس ون ـب د مـل الطریقة مسـتندة علـى تكـوین معـق MO ع ــاء المقطــر . AHـم ــِد بالم ول المعقّ ــرج وتَخفیــف مَحـل ــى pHالـــ ، ثبّـتD.Wبعــد ال . 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 Molarان اـل absorptivity (ε L.mol -1.cm-1) ،حد الكشف وdetection limit ، المـدى الخطـيlimit of linearity (µg.ml-1) �ة . عل�ى الت�والي) 0.982(و ) 0.04(، ) 510* 5.74(عبارة عن ) r2(و �ِة والض�بط المعق�ولین لتق�دیر االطریق س�تعملْت بالدق )AH ( ِفي العیناِت الصناعیِة ِمْن األقراِص والكبسوالِت واألنبوبات IHJPAS