Chemistry - 226 مجلة إبن الهيثم للعلوم الصرفة و التطبيقية 2012 السنة 25 المجلد 3 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 3 Vol. 25 Year 2012 Development Of Two Different Spectrophotometric Methods For The Determination Of Atropine Drug In Pure Form And Pharmaceutical Preparations A.Kh.Mahmood Department of Chemistry, College of Education Ibn Al- Haitham, University of Baghdad E- mail: alslam_aaa@yahoo.com Abstract Two methods have been applied for the spectrophotometric determination of atropine, in bulk sample and in dosage form. The methods are accurate, simple, rapid, inexpensive and sensitive. The first method depending on the extraction of the formed ion-pair complex with bromphenol blue (BPB) as a chromogenic reagent in chloroform, use phthalate buffer of pH 3.0; which showed absorbance maxima at 413 nm against reagent blank. The calibration graph is linear in the ranges of 0.5-40 µg.mL-1 with detection limit of 0.363µg.mL-1. The second method depending on the measure of the absorbance maxima of the formed charge- transfer complex with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) at 457 nm against reagent blank; with linearity range 2.5-50.0 µg.mL-1, and detection limit of 2.143 µg.mL-1. The results show the absence of interferences from the excipients on the determination of the drug. The proposed methods have been successfully applied for the determination of atropine in pharmaceutical preparations. Keywords: Spectrophotometric, atropine, ion-pair, charge-transfer. Introduction Atropine(Scheme 1), was first isolated as an active principle from the roots of belladonna in 1831 by K. Mein, a German apothecary, [1], This compound, which have the chemical structure of tropane alkaloids[2], has two main types of actions, one on the central nervous system to cause respiratory stimulation, and the other, to suppress smooth muscles and secretary glands innervated by parasympathetic nerves[3]. It had been used as ingredients in many gastrointestinal drugs owing to their anticonvulsant and analgesic properties [1, 4]. Also it was used for bradicardia, following myocardial infection or over dosage of β-blockers and can be produced by typical application of anti cholinergic agent for treatment of irititis causing paralysis of ciliary muscle, leading to blurred vision [5]. For most of the alkaloids have special and distinct physiological properties and toxicity, the determination of atropine is of great importance not only in clinical application but also in pharmaceutical analysis. Scheme 1: The chemical structure of atropine http://www.sciencedirect.com.tiger.sempertool.dk/science/article/pii/S0021967305003043#bib9 Chemistry - 227 مجلة إبن الهيثم للعلوم الصرفة و التطبيقية 2012 السنة 25 المجلد 3 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 3 Vol. 25 Year 2012 Several methods have been reported for the determination of atropine in bulk and pharmaceutical dosage forms, these methods include high performance liquid chromatography [6-8] gas chromatography [9], potentiometry[10], Flow-injection post chemiluminescence [11] and thin-layer scanning method[12]. Some of these methods are time-consuming, tedious, and/or dedicated to sophisticated and expensive analytical instruments. Spectrophotometry[13-19]; are most convenient techniques because of their inherent simplicity, adequate sensitivity, low cost and wide availability in all quality control laboratories. The present work describes the utility of BPB and DDQ reagents for spectrophotometric determination of atropine in pure form as well as in dosage form. In addition, the optimization of chemical dependent variables of affecting absorbance has been studied. Apparatus: A Cintra 5 spectrophotometer with 1 cm quartz cells was used for absorbance measurements. PH-meter DW-9421 from Philips instrument, a Sartorius BL 210S balance, and a Pentium 4 computer (DELL) was used for data processing. Experimental Material and Reagents: All Chemicals used were of analytical reagent grad unless otherwise is mentioned, Atropine sulfate standard powder (purity 99.8%) were kindly provided by the State Company for Drug Industries and Medical Appliances, Samara-Iraq (SDI). Bromophenol blue (BPB) (Aldrich), 0.1% (w/v) solution was prepared by dissolving 0.1 g of the dye in 5 mL of methanol and then the solution was diluted to a final volume of 100 mL with distilled water. Working solutions were freshly prepared by subsequent dilutions. 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ)(BDH); 0.1 %( w/v) solution was prepared by dissolving 0.01 g of the DDQ in 5 mL of acetonitrile and then the solution was diluted to a final volume 10 mL with acetonitrile. Working solutions were freshly prepared by subsequent dilutions. This solution is prepared daily using red- glass volumetric flask because it is a light sensitive reagent. Hydrochloric Acid (Aldrich), ~0.1 M, a 0.85 mL of concentrated hydrochloric acid (37%, sp.gr1.18) was added to 50 mL distilled water and diluting to the mark in a 100 mL calibrated flask. Potassium Hydroxide (fluka), ~ 0.1 M, was prepared by dissolving 0.56 g of potassium hydroxide in 25 mL distilled water and diluted to 100 mL in volumetric flask with distilled water. Phthalate buffer 0.2M solution was prepared by dissolved 4.08 g of potassium hydrogen phthalate (MERCK) 25 mL distilled water and diluted to 100 mL in volumetric flask with distilled water, the pH was adjust to 5.5 by using few drops of 0.1M HCl and\or 0.1M KOH. Atropine standard solution (250µg.mL-1) For BPB method It was prepared by dissolving weighed amount of salt equivalent to 25 mg of atropine base in 20mL distilled water and diluting to 100mL in a volumetric flask with distilled water. Working solutions were freshly prepared by subsequent dilutions. For DDQ method An accurately weighed amount of atropine salt equivalent to 25 mg of the base was dissolved in 20 ml distilled water. The solution was quantitatively transferred into a separating funnel, made alkaline (pH=9) with ammonia solution [20, 21] and shaken with four 20 ml portions of chloroform. The extracts were pooled by filtration through a filterer paper Chemistry - 228 مجلة إبن الهيثم للعلوم الصرفة و التطبيقية 2012 السنة 25 المجلد 3 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 3 Vol. 25 Year 2012 containing anhydrous sodium sulphate into a 100 ml standard flask and made up to volume with chloroform. Working solutions were freshly prepared by subsequent dilutions. General recommended procedure For BPB method A suitable amount of atropine standard solution was transferred into a series of 50 mL separating funnels, to each funnel 0.5 mL of phthalate buffer of pH 3.0 and 0.3 mL of 0.05% BPB reagent solutions were added. The separating funnels were shaken vigorously with 5 mL chloroform for 4 mints. The two phases were then allowed for clear separation and the absorbance of the yellow colored organic phase was measured at 413nm against a reagent blank prepared similarly without addition of atropine. The calibration graph was constructed by plotting the measured absorbance of the organic phase against the drug concentration. For DDQ method A Suitable volume of the standard stock solution of the drug were pipette into 5-mL calibrated flasks, 0.2 ml of 0.1% DDQ solution was added to each, and then diluted to volume with acetonitrile. Absorbance measurements of resulting solutions were done at the wavelength of maximum absorption at 457 nm against reagent blank which prepared by the same manner, but without addition of atropine. Solution for the analysis of atropine in pharmaceutical preparations [22,23] I. In Ampoules For BPB method The contents of 15 ampoules were mixed well. A volume equivalent to 10 mg of atropine base was quantitatively transferred into 50 mL volumetric flask and diluted to the mark with distilled water. Working solutions were freshly prepared by subsequent dilutions and analyzed by the recommended procedures. For DDQ method The contents of 15 ampoules were mixed well. A volume equivalent to 10 mg of atropine base was quantitatively transferred into 20 mL volumetric flask and diluted to the mark with distilled water, quantitatively transferred it into a separating funnel, made alkaline(pH=9) with ammonia solution, extract the drug base as under (standard solution). Working solutions were freshly prepared by subsequent dilutions and analyzed by the recommended procedure. II. In Eye Drops For BPB method The volume of 5 drops was quantitatively transferred into 100 mL volumetric flask and diluted to the mark with distilled water. A volume equivalent to 25 mg of atropine base was transferred into 100 mL volumetric flask and diluted to the mark with distilled water. Working solutions were freshly prepared by subsequent dilutions and analyzed by the recommended procedure. For DDQ method The volume of 5 drops were quantitatively transferred into 100 mL volumetric flask and diluted to the mark with distilled water. A volume equivalent to 25 mg of atropine base was quantitatively transferred into a separating funnel, made alkaline (pH=9) with ammonia solution. Extract the drug base as under (standard solution).Working solutions were freshly prepared by subsequent dilution sand analyzed by the recommended procedure. Chemistry - 229 مجلة إبن الهيثم للعلوم الصرفة و التطبيقية 2012 السنة 25 المجلد 3 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 3 Vol. 25 Year 2012 Results and discussion Spectrophtometric procedures are popular for their sensitivity in the assay of drugs and hence, ion pair and charge transfer complexes formation has received considerable attentions for the quantitative determination of many pharmaceutical compounds [24-29]. Atropine reacts with BPB in acidic buffer to give yellow color chloroform soluble ion-pair complex, which exhibits absorption maxima at 413 nm against their reagent blank; (Figure1). Some amines salts don’t react with π-acceptors because they don’t possess a lone pair of electrons[29]. Similarly, atropine sulfate unable to react with DDQ; unless it is extracted with chloroform in basic medium[29], resulting formation of atropine base in the chloroform layer; which acts (as n-donors) react with DDQ (as π-acceptors) to give red brown color acetonitrile soluble charge transfer complex, which exhibits absorption maxima at 457 nm against their reagent blank (Figure2). Under the experimental conditions the reagent blank showed in both cases negligible absorbance thereby permit good analytical conditions for quantitative determination of atropine in pharmaceutical dosage forms. For BPB method Effect of pH In order to establish the optimum pH range, atropine was mixed with specified volumes of phthalate buffer. The pH was then adjusted to a value between (2.0 -4.5) with few drops of 0.1M KOH or 0.1M HCl. It was noticed that maximum color intensities and constant absorbance values were found at pH 3.0 (Figure 3). Low absorbencies were observed in solutions with higher or low pH than the optimum value. Hence, a pH of 3.0 was used in all the subsequent experimental work. Effect of reaction time The optimum reaction time was determined by following the color development at ambient temperature (25±2). It was found that the reaction was instantaneous. Hence the product attained maximum and constant absorbencies immediately after atropine have been mixed with BPB and the developed color, remained strictly unaltered for at least 24 hours. Effect of reagent volume The influences of reagent volume on the absorbance of complex are illustrated in (Figure 4). 0.3 mL of 0.05% solutions of BPB were found to be optimum to develop the maximum color intensities for atropine ion-pair complex, after which no more increase in absorbance values was obtained; therefore, the cited volume of BPB solution were used. Effect of shaking time The optimum shaking times for the complete extraction of the formed ion pair complex with chloroform were studied for the period of 1-5 minutes (Table 1). It was found that the optimum shaking times for complete extraction of atropine ion pair complex, at room temperature for minutes. Effect of the extraction solvent: Several organic solvents, such as, chloroform, toluene, carbon tetrachloride, benzene, 1, 2- dichloroethane and Dichloro methane were examined for their ability to extract the drug-BPB ion-pair complex. It was found to be chloroform the most suitable solvent in terms of extraction efficiency (Table 2). On the other hand, it was observed that only a single extraction with 5 mL portion of chloroform was adequate to achieve a quantitative recovery of the complex. Chemistry - 230 مجلة إبن الهيثم للعلوم الصرفة و التطبيقية 2012 السنة 25 المجلد 3 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 3 Vol. 25 Year 2012 For DDQ method Effect of pH The effect of pH on the development of the colored complex, between the cited drug and DDQ was investigated by adjusting the pH to a value between 6.0 and 10 with few drops of 0.2M NH4OH or 0.1M HCl. It was noticed that maximum color intensities and constant absorbance values were found at pH 9.0 (Figure 5). Low absorbencies were observed in solutions with higher or low pH than the optimum value. Hence, a pH of 9.0 was used in all the subsequent experimental work. Effect of reaction time: It was found the reaction was instantaneous. Hence the product attained maximum and constant absorbancies immediately after atropine have been mixed with DDQ and the developed color, remained strictly unaltered for at least 8 hours in drake place. Effect of reagent Volume The effect of the volume of DDQ on the color development was studied by adding different volumes(0.05- 0.30) mL of 0.1%DDQ solution to 20µg.ml of atropine .The results revealed the fact that 0.2 ml of 0.1%DDQ solution was required to achieve the maximum intensity of the color (Figure 6). Effect of solvent Several organic solvents such as acetonitrile , acetone, methanol, chloroform, 1,2-dichloro ethane, and dichloro methane was studied to choose the preferred diluting solvent for the quantitative measurements.(Table3). The results show that Acetonitrile was considered as an ideal diluting solvent as it gives good solvating capacity for atropine, and gives the highest yield of the radical anion. Stoichiometry of the complexes To establish molar ratio for both complexes, Job's method of continuous variation has been used (Figures 7 and 8). The results showed that 1:1 ratios for both complexes were formed; through the electrostatic attraction between the positive protonated atropine with the anion of BPB for ion pair complex formation[30], and the complex between the studied drugs, as n- donors, with DDQ, as π acceptors for charge transfer complex formation[20],(Scheme 1 and 2). Chemistry - 231 مجلة إبن الهيثم للعلوم الصرفة و التطبيقية 2012 السنة 25 المجلد 3 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 3 Vol. 25 Year 2012 Scheme 2: Proposed reaction pathway between ِ◌◌ِatropine –BPB ion pair complex, under recommended procedure. Scheme 3: Proposed reaction pathway between atropine –DDQ charge transfer complex. under optimum recommended procedure. Chemistry - 232 مجلة إبن الهيثم للعلوم الصرفة و التطبيقية 2012 السنة 25 المجلد 3 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 3 Vol. 25 Year 2012 Calibration graphs: Employing the experimental conditions, linear calibration graphs for both complexes; were obtained (Figures 9 and 10), which show that Beer's law was obey in the concentration range of 0.5-40 and 2.5-50 µg.mL-1 for atropine BPB ion pair and atropine DDQ charge transfer complexes respectively. Spectral characteristics of the proposed methods: According to the optimum experimental conditions of the proposed methods, the regression plots showed linear dependence of absorbance signals on the concentrations of the studied drug in the range given. The regression equations, correlation coefficients, molar absorptivities, detection limits and sandell sensitivities in addition to other parameters are given in Table 4. Accuracy and precision: The accuracies of the proposed methods were confirmed by analyzing five replicate analyses of three different amounts of the drug (within Beer's law) by calculating the relative error percentage (Table 5). The results indicated good accuracies for both of the methods. The precision was determined in each case by calculating the percentage relative standard deviation (RSD %) for five determinations at each of the studied concentration level and were found to be in the range of 1.352-1.826% and 1.778-2.103% for atropine BPB ion pair and atropine DDQ chrge transfer complexes respectively. The values of the mean error(xi-μ) were less than the values of indeterminate error (±ts/√n), indicating that no significant differences between the mean and the true values; at 95% confidence level. Interferences Study: The results showed that no interferences were found in the presence of 250 μg of the studied excipients (lactose, sucrose, starch, glucose, magnesium stearate, sodium citrate, and sodium chloride) in the determination of atropine for both methods, (Table 6). Analysis of dosage forms: It is evident from the aforementioned results that the proposed methods gave satisfactory results with the investigated drug. Thus, their pharmaceutical dosage forms were subjected to analysis of their contents of the active ingredient by the proposed methods (ion-pair and charge transfer complexes formation). The results given in (Table 7 and 8) were satisfactory. Reference 1.Donald, J. A.(2003) Burger's Medicinal Chemistry and Drug Discovery Sixth Edition, Wiley & Sons, Inc. Virginia,pp121.122. 2.Ashutosh, K. (2007) Pharmacognosy and pharmacobiotechnology, second edition, New Age International (P) Ltd., Publishers, New Delhi, p 396, 398,464,465. 3.Das, G. (1989) Therapeutic review. Cardiac effects of atropine in man: an update Int J. Clin Pharmacol Ther Toxicol, 27(10):473-7. 4.British Pharmacopeia (1998) CD-ROM Her Majesty,s Stationary office, London. 5.Alwan, Ala,dine, A. S. and Abou, Yousif, Z.(1990) Iraqi Drug Guide, 1st edition, NBSD , Iraq, 22:212. 6.Yoshiyuki, S.; Katsuhiro, Y.; Takaomi, T.; Masami, K. and Shuzo, T.(2011) Rapid determination of atropine and scopolamine content in scopolia extract powder by HPLC, J. of Nat Med 65:395–399. 7.Shaoyoug, Li. and Khalil, W. S. (1990) An HPLC Method for Determination of Atropine in Human Plasma, J. of Liquid Chromatography & Related Technologies, 13(7):1339-1350. http://www.google.iq/search?hl=ar&tbo=p&tbm=bks&q=inauthor:%22Ashutosh+Kar%22&source=gbs_metadata_r&cad=5 http://www.ncbi.nlm.nih.gov/pubmed?term=%22Das%20G%22%5BAuthor%5D http://www.ncbi.nlm.nih.gov/pubmed/2684867 http://www.ncbi.nlm.nih.gov/pubmed/2684867 Chemistry - 233 مجلة إبن الهيثم للعلوم الصرفة و التطبيقية 2012 السنة 25 المجلد 3 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 3 Vol. 25 Year 2012 8.Takanori, O.; Masafumi, N.; Ichiro, S.; Kazunori, K.; Kenji, H.; Yoshifumi ,T. and Kazuaki, K.(1991) Determination of atropine in biological specimens by high-performance liquid Chromatography, J. of Chromatography B: Biomedical Sciences and Applications,567(1):141-149. 9.Majlát, P. (1984) Gas chromatography determination of atropine, theophylline, phenobarbital and aminophenazone in tablets, J of Pharmazie 39(5):325-326. 10.Mostafa, G. A. E.; Abbas, M. N. (2008) PVC Membrane Sensor for Potentiometric Determination of Atropine in Some Pharmaceutical Formulations, J. of Instrumentation Science & Technology, 36( 2): 209 – 221. 11.Shuwen, S. and Jiuru, Lu.(2006) Flow-injection post chemiluminescence determination of atropine sulfate, J. of Analytica Chimica Acta, 580 (1): 9-13. 12.Gilpin, R. K. (1979) determination of atropine by thin-layer scanning method, J. of Anal. Chem, 51 (5): 257-187. 13.Polomik, M. ;Sober, M. ; Pleho, A. and Nikolin، B. (1993) Spectrophotometric Determination of atropine-sulfate in eyedrops using bromthymol blue, J. of Med Arh.,47(1-2):25-27. 14.El-Shahat, M.,F.; Abdel B. M. and Daifullah A., A. (1992) Spectrophotometric determination of ephedrine HCl, cinchonine HCl, chlorpheniramine maleate, atropine sulphate and diphenhydramine HCl by solvent extraction of reineckate complexes, J. of Chem Technol Biotechnol, 45(2):175-181. 15.Suraj P. Agarwal and M. Abdel-Hady Elsayed (1981)Utility of π -acceptors in charge- transfer complexation of alkaloids: chloranilic acid as a spectrophotometric titrant in non-aqueous media, J. of Analyst, 106: 1157 – 1162. 16.Tehseen, A.; Ahmad, H.; Irshad, K. and Rashid, A.(1994) Spectrophotometric Determination Of Atropine J. of Analytical Letters, 27 (10): 1833 – 1845. 17.Sarah, C.; Yves. P.; Patrick , M. and Bertrand, B.(2010) Determination of atropine and scopolamine contents in wild and ornamental varieties of Datura, J. of Ann Toxicol Anal., 22(4): 173-179. 18.Elsayed, M. A. and Agarwal S. P. (1982) Spectrophotometric determination of atropine, pilocarpine and strychnine with chloranilic acid, J. of Talanta, 29(6):535-537. 19.Wang, Y. and Zhang, Y. (2007) Determination of atropine sulfate in atropine sulfate gel solution by binary derivative spectrophotometry, Chinese Journal of Pharmaceutical Analysis 27(1):139-140. 20.Walash, M.; Sharaf-El Din, M.; Metwalli, M. E.-S. and Reda, S. M. (2004) Spectrophotometric Determination of Nizatidine and Ranitidine Through Charge Transfer Complex Formation, J. of Arch Pharm Res, 27(7): 720-726. 21.Basavalahy, K. and Charan, V. S. (2002) The Use of Chloranilic Acid for the Spectrophotometric Determination of Three Antihistamines, Turk J. Chem 26: 653 -661. 22.Darwish, Ia.; Husein, Sa. ; Mohmoud, Am. and Hassan, Ai.(2007) Sensative Spectrophotometric method for the determination of H2- receptor antagonists in Pharmaceutical Formulation, International J. of Biomedical Science, 3(2): 123-130. 23.Geffken, D. and Salem, H. (2006) Spectrofluorimetric Study of the Charge-transfer Complexation of Certain Fluoroquinolones with 2,3,5,6-tetrafluoro-p-bezoquinone ,American J. of Applied Sciences 3 (8): 1952-1960. 24.Siddappa, K.; Mallikarjun, M.; Reddy, T. and Tambe, M. (2008) Simple and Sensitive Extractive Spectrophotometeric Method for the Assay of Mebeverine Hydrochloride in Pure and Pharmaceutical Formulations Journal of the Chinese Chemical Society, 55:1062-1068. 25. Darwish, Ia; Husein, Sa. ; Mohmoud Am. And Hassan, Ai.(2008) A Sensative Spectrophotometric method for the determination of H2- receptor antagonists by means of N- brmosuccinimide and P- aminophenol, J. of Acta Pharm., 58:87-97. http://www.sciencedirect.com/science/journal/03784347 http://www.sciencedirect.com/science/journal/03784347 http://www.ncbi.nlm.nih.gov/pubmed?term=%22Majl%C3%A1t%20P%22%5BAuthor%5D http://www.informaworld.com/smpp/content~db=all~content=a790769853~frm=titlelink http://www.sciencedirect.com/science/journal/00032670 http://www.sciencedirect.com/science?_ob=PublicationURL&_tockey=%23TOC%235216%232006%23994199998%23635602%23FLA%23&_cdi=5216&_pubType=J&view=c&_auth=y&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=884f54355284e6eb894516992ebdd5f5 http://www.ncbi.nlm.nih.gov/pubmed?term=%22Polomik%20M%22%5BAuthor%5D http://www.ncbi.nlm.nih.gov/pubmed?term=%22Sober%20M%22%5BAuthor%5D http://www.ncbi.nlm.nih.gov/pubmed?term=%22Pleho%20A%22%5BAuthor%5D http://www.ncbi.nlm.nih.gov/pubmed?term=%22Nikolin%20B%22%5BAuthor%5D http://www.ncbi.nlm.nih.gov/pubmed/7934217 http://www.ncbi.nlm.nih.gov/pubmed/7934217 http://www.ncbi.nlm.nih.gov/pubmed?term=%22el-Shahat%20MF%22%5BAuthor%5D http://www.ncbi.nlm.nih.gov/pubmed?term=%22Abdel%20Badei%20MM%22%5BAuthor%5D http://www.ncbi.nlm.nih.gov/pubmed?term=%22Daifullah%20AA%22%5BAuthor%5D http://www.informaworld.com/smpp/title~db=all~content=t713597227~tab=issueslist~branches=27#v27 http://www.informaworld.com/smpp/title~db=all~content=t713597227~tab=issueslist~branches=27#v27 http://www.ncbi.nlm.nih.gov/pubmed?term=%22Elsayed%20MA%22%5BAuthor%5D http://www.ncbi.nlm.nih.gov/pubmed?term=%22Agarwal%20SP%22%5BAuthor%5D http://www.ncbi.nlm.nih.gov/pubmed/18963183 Chemistry - 234 مجلة إبن الهيثم للعلوم الصرفة و التطبيقية 2012 السنة 25 المجلد 3 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 3 Vol. 25 Year 2012 26.Julic, M.and Cardso, S. C. (2005) Spectrophotometric determination of oxiconazole in topical lotion using methylorange, J. of Pharmaceutical and Biomedical Analysis, 37 (4,1):639-642. 27.Zhao Yanqing, Li Hua McCain, Gui-Zhi Zhao, (2005) Spectrophotometric determination of erythromycin ethylsuccinate based on the charge transfer reaction between erythromycin ethylsuccinate and quinalizarin”, J.of China Modern Applied Pharmacy, 22 (3): 229-303. 28.Hesham, S. (2008) Analytical study for the charge-Transfer complexes of gabapentin African J.of Pharmacy and Pharmacology, 2(7):136-144. 29.Nafisur, R. and Syed, N.H. AZMI (2000) Spectrophotometric Determination of Amlodipine Besylate by Charge- Transfer Complex Formation with p-Chloranilic Acid, J.of Analytical Shines (The Japan Society for Analytical Chemistry), 16:1353-1356. 30.Kanakapura, B. and Vaidyanathan, Sh. C. (2004) Ion-Pair Complexometric Detrmination of Cyproheptadine Hydrochloride Using Bromophenol Blue J. of Science Asia, 30, 163-170. Table(1): Effect of shaking time on extraction of 20 µg.mL-1 Atropine; 0.3mL of 0.05%BPB, pH(3.0). Shaking time(minute) Absorbance 1 0.4121 2 0.4168 3 0.4202 4 0.4329 5 0.4325 6 0.4325 Table (2): Effect of type of extraction solvent on absorbance of 20 µg.mL-1 atropine; 0.3mL of 0.05%BPB, pH(3.0). Extraction solvent Absorbance Chloroform 0.4329 Toluene 0.0221 Carbontetrachloride 0.0297 Benzene 0.0096 1,2-Dichloro ethane 0.3054 Dichloro methane 0.2153 Table (3): Effect of type of organic solvent on absorbance of 20 µg.mL-1 atropine; 0.2mL of 0.1%DDQ, pH(9.0). Organic Solvent Absorbance Acetonitrile 0.3031 Acetone 0.2322 Methanol 0.0799 Chloroform 0.1413 1,2-dichloroethane 0.0976 Dichloro methane 0.2155 http://www.sciencedirect.com/science/journal/07317085 http://www.sciencedirect.com/science?_ob=PublicationURL&_tockey=%23TOC%235266%232005%23999629995%23589391%23FLA%23&_cdi=5266&_pubType=J&view=c&_auth=y&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=f78a82d0e44c01893af11409f564837c http://www.jstage.jst.go.jp/search/?typej=on&typep=on&typer=on&d3=au&dp3=Nafisur+RAHMAN&ca=999999&alang=all&rev=all&pl=20&search=%8C%9F%8D%F5%8E%C0%8Ds http://www.jstage.jst.go.jp/search/?typej=on&typep=on&typer=on&d3=au&dp3=Syed+Najmul+Hejaz+AZMI&ca=999999&alang=all&rev=all&pl=20&search=%8C%9F%8D%F5%8E%C0%8Ds Chemistry - 235 مجلة إبن الهيثم للعلوم الصرفة و التطبيقية 2012 السنة 25 المجلد 3 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 3 Vol. 25 Year 2012 Table(4): Spectral characteristics and statistical data of the regression equations for determination of atropine by ion–pair and charge transfer complexes formation. Parameter Ion–Pair Complex Formation Charge Transfer Complex Formation λma x (nm) 413 457 Color Yellow Red-brown Linearity range (µg.mL-1) 0.5 – 40 2.5 – 50 Molar absorpitivites(l.mol-1.cm-1) 5787.6 4051.32 Regression equation A = 0.020 x + 0.037 A= 0.014x + 0.012 Calibration Sensitivity 0.020 0.014 Sandell's Sensitivity(µg.cm-2) 50.000 71.439 Correlation of Linearity (R2) 0.9998 0.9982 Correlation coefficient (R) 0.9999 0.9991 Detection limit (µg.mL-1) 0.363 2.143 Table (5): Evaluation of accuracies and precisions of the two proposed procedure. Method Drug Concentration (µg.mL-1) Relative Error % R.S.D.* % xi-μ ±ts/√n Taken Found* Ion–Pair Complex 2.5 2.488 -0.480 1.352 -0.012 0.039 10 9.932 -0.680 1.414 -0.068 0.161 30 30.205 +0.683 1.826 +0.205 0.634 Charge Transfer Complex 5 4.962 -0.760 1.833 -0.038 0.105 20 19.871 -0.645 1.778 -0.129 0.406 40 40.283 +0.708 2.103 +0.283 0.974 *Average of five determinations t = 2.571 for n=5 at 95% confidence level. Table(6): Percent recovery for 20 µg.mL-1 of atropine in the presence of 250 µg.mL-1of Excipients by ion–pair and charge transfer complexes formation. Excipients Ion–Pair Complex Formation Method Charge Transfer Complex Formation Method Conc. Fund (µg.mL-1) Recovery% Conc. Fund (µg.mL-1) Recovery% lactose 19.779 98.895 19.828 99.140 Sucrose 20.231 101.155 20.197 100.985 Starch 19.888 99.444 20.302 101.510 Glucose 19.862 99.310 20.340 101.700 Magnesium Stearate 20.316 101.580 19.863 99.315 Sodium Citrate 19.803 99.015 20.265 101.325 Sodium Chloride 20.334 101.670 19.798 98.990 *Average of three determinations. Chemistry - 236 مجلة إبن الهيثم للعلوم الصرفة و التطبيقية 2012 السنة 25 المجلد 3 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 3 Vol. 25 Year 2012 Table (7): Spectrophotometric determination of atropine in pharmaceutical compounds by ion–pair complex formation. Ion–Pair Complex Drug Concentration (µg.mL-1) Recovery% R.S.D.* % Taken Found* ATROPINE Sulfate (1mL Ampoules) 1mg/1mL PAYAL - Uk 2.5 2.555 102.200 1.572 10 9.989 99.890 1.521 30 30.562 101.873 1.884 ATROPINE Sulfate (1mL Ampoules) 1mg/1mL BELCO - India 2.5 2.576 103.040 1.776 10 9.883 98.830 1.902 30 29.505 98.350 2.091 Atropina 0.5% 8mL Eye Drops Atropine sulfate 5mg/1mL Dalta Co.Syria 2.5 2.468 98.720 1.769 10 9.876 98.760 1.814 30 29.658 98.860 2.143 Apitropine1% 10mL Eye Drops Atropine sulfate 10mg/1mL API - Jordan 2.5 2.622 104.88 1.852 10 10.264 102.640 2.056 30 30.792 102.650 2.442 *Average of five determinations. Table (8): Spectrophotometric determination of atropine in pharmaceutical compounds by charge transfer complex formation. Charge Transfer Complex Drug Concentration (µg.mL-1) % Recovery R.S.D.* % Taken Found* ATROPINE Sulfate (1mL Ampoules) 1mg/1mL PAYAL - Uk 5 5.146 102.900 1.989 20 20.315 101.575 2.183 40 40.472 101.180 2.552 ATROPINE Sulfate (1mL Ampoules) 1mg/1mL BELCO - India 5 5.178 103.560 2.286 20 20.617 103.085 2.602 40 40.992 102.480 2.814 Atropina 0.5% 8mL Eye Drops Atropine sulfate 5mg/1mL Dalta Co.Syria 5 5.123 102.460 1.792 20 20.287 101.435 2.521 40 40.790 101.975 2.746 Apitropine1% 10mL Eye Drops Atropine sulfate 10mg/1mL API - Jordan 5 5.222 104.440 2.473 20 20.451 102.255 2.756 40 41.243 103.108 2.967 *Average of five determinations. Chemistry - 237 مجلة إبن الهيثم للعلوم الصرفة و التطبيقية 2012 السنة 25 المجلد 3 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 3 Vol. 25 Year 2012 Fig. (1): Absorption spectra of A: 20 µg.mL-1 atropine –BTB Ion –Pair Complex against reagent blank, B: reagent blank against chloroform, under optimum conditions. Fig. (2): Absorption spectra of A: 15 µg.mL-1 atropine–DDQ charge transfer complex, against reagent blank, B: reagent blank against acetonitrile, under optimum conditions. Chemistry - 238 مجلة إبن الهيثم للعلوم الصرفة و التطبيقية 2012 السنة 25 المجلد 3 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 3 Vol. 25 Year 2012 Fig. (3): Effect of pH on the absorbance of 20 µg.mL-1atropine; 0.05% BPB. Fig. (4): Effect of reagent volume (0.05% BPB) on the absorbance of 20 µg.mL-1 atropine; pH 3.0 Fig. (5): Effect of pH on the absorbance of 20 µg.mL-1 atropine; 0.1% DDQ. Fig. (6): Effect of reagent volume (0.1% DDQ) on the absorbance of 20 µg.mL-1 atropine; pH 9.0 Chemistry - 239 مجلة إبن الهيثم للعلوم الصرفة و التطبيقية 2012 السنة 25 المجلد 3 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 3 Vol. 25 Year 2012 Fig. (7): Continuous variation of atropine –BPB ion pair complex, (each 3.456x10-4M), pH 3.0. Fig. (8): Continuous variation of atropine –DDQ charge transfer complex, (each 3.456x10-4M), pH 9.0 Fig. (9): Calibration graph of atropine-BPB ion-pair complex, under optimum recommended Procedure. Chemistry - 240 مجلة إبن الهيثم للعلوم الصرفة و التطبيقية 2012 السنة 25 المجلد 3 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 3 Vol. 25 Year 2012 Fig. (10): Calibration graph of atropine –DDQ charge transfer complex, under optimum recommended Procedure. Chemistry - 241 مجلة إبن الهيثم للعلوم الصرفة و التطبيقية 2012 السنة 25 المجلد 3 العدد Ibn Al-Haitham Journal for Pure and Applied Science No. 3 Vol. 25 Year 2012 طريقتين طيفيتين مختلفتين لتقدير دواء األتروبين بصورته النقية وفي تطوير المستحضرات الصيدالنية علي خليل محمود ابن الهيثم ، جامعة بغداد -قسم الكيمياء ، كلية التربية 2012حزيران 17قبل البحث في : 2012نيسان 15استلم البحث في : الخالصة ان طيفيتان لتقدير دواء األتروبين في عينات نقية وبعض المستحضرات الصيدالنية باالعتماد على اقترحت طريقت تكوين معقدات األزدواج األيوني و أنتقال الشحنة. كانت الطريقتان اعاله دقيقة ، وبسيطة، وسريعة، وغير مكلفة وحساسة. معقد االزدواج االيوني المتكون بين العقار اعاله اعتمدت الطريقة االولى على استعمال الكلوروفورم في أستخالص ، أذ اظهر المعقد المتكون اقصى امتصاص له 3.0والكاشف بروموفينول االزرق من وسط مائي عند دالة حامضية مقدارها نامومتر، ضد محلول الخلب، وأظهر منحني المقايسة عالقة خطية لمدى من التراكيز تراوحت بين 413عند الطول الموجي مايكروغرام /مل. في حين اعتمدت الطريقة الثانية على قياس اقصى 0.363مايكروغرام/مل و بحد كشف 0.5-40 داي -6-5 –داي كلورو -3و2امتصاص لمعقد انتقال الشحنة المتكون بين العقار قيد الدراسة(كواهب لاللكترونات) و نانومتر ضد محلول الخلب وبخطية تراوحت 457جي طول الموبنزوكوينون (كمستقبل لاللكترونات) عند ال -بارا –سيانو مايكروغرام/مل. أظهرت الدراسة أيضآ أن الطريقتين المقترحتين 2.143مايكروغرام/مل، وبحد كشف 50 -2.5بين خالية من تأثير المتداخالت المتعارف على وجودها في المستحضرات الصيدالنية، فقد أمكن تطبيق الطريقتين بنجاح لتقدير تروبين في بعض تلك المستحضرات.األ طيفي، اتروبين، األزدواج األيوني ،انتقال الشحنة الكلمات المفتاحية: 14.El-Shahat, M.,F.; Abdel B. M. and Daifullah A., A. (1992) Spectrophotometric determination of ephedrine HCl, cinchonine HCl, chlorpheniramine maleate, atropine sulphate and diphenhydramine HCl by solvent extraction of reineckate complexes, J. of Chem Technol Biotechnol, 45(2):175-181.