IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Univariate and Simplex Optimization for the Spectrophotometric Determination of Cimetidine and Erythromycin ethylsuccinate Drugs Using Bromothymol Blue Via Ion-Pair Formation S. B. Dikran, A. K. Mohammed ,A. K.M. Al-Jumaily Department of Chemistry, College of Education Ibn Al Haitham, University of Baghdad Abstract The ion-pair formation method has been applied for the spectrophotometric determination of Cimetidine and Erythromycin ethylsuccinate, in bulk samples and in dosage form. The methods are accurate, simple, rapid, inexpensive and sensitive depending on the extraction of the formed ion-pair with brompthymol blue (BTB) as a chromogenic reagent in chloroform, use phthalate buffer of pH 5.5 and 4.0 for Cimetidine and Erythromycin ethy lsuccinate respectively. The formed complexes show absorbance maxima at 427.5 nm and 414.5 nm for Cimetidine and Erythromycin ethylsuccinate respectively against reagent blank. The calibration graphs are linear in the ranges of 0.5-15 µg.mL-1 with detection limit of 0.222 µg.mL-1 for Cimetidine and 0.5-50 µg.mL-1 with detection limit of 0.286 µg.mL-1 for Erythromycin ethylsuccinate. The results show the absence of interferences from the excipients on the determination of these drugs. The proposed methods have been successfully applied for the determination of Cimetidine and Erythromycin ethylsuccinate (with two of its derivatives) in pharmaceutical preparations. Keywords, Simplex, Spectrophotometric, Cimetidine, Erythromycin ethy lsuccinate, Ion-pair. Introduction Cimetidine was the first histamine H2-receptor antagonist approved by the Food and Drug Administration, USA, for the treatment of duodenal ulcers, Zollinger-Ellison syndrome, and othergastric hypersecretory states [1]. It's also indicated for the relief heartburn in peptic, duodenal ulcers and prevents rebleeding in patients which reduce the secretion of gastric acid[2], cimetidine, due to its effects on the immune system and as an H2-receptor antagonist, can inhibit growth of carcinogen-induced colonic tumors in rats, as well as the in vitro human colon cancer cell lines[1]. The Chemical structure of Cimetidine are given in (Scheme 1), which shows the imidazole ring on it. IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 IHJPAS N H NH3C S N H N H H3C N CN Scheme 1: The chemical structure of Cimetidine Erythromycin is the most employed macrolide antibiotic for treating a myriad of infections caused by gram-positive bacteria such as anthrax, tonsillitis, otits media and syphilis [2], it is often prescribed as an alternative for patient allergic to penicillin [2-3]. It has been also employed a as part of therapeutic cocktails together with amino glycoside antibiotics that covers gram-negative microorganisms [3]. The Chemical structure of Erythromycin is given in (Scheme 2), which shows the lactone ring on it. Scheme 2: The chemical Structure of Erythromycin Ethylsuccinate Several methods have been reported for the determination of Cimetidine and Erythromycin in bulk and pharmaceutical dosage forms, these methods include titrimetry[4], high performance liquid chromatography[5], high performance thin layer chromatography[6], liquid chromatography[7], capillary electrophoresis[8,9] and chemiluminesence[10].Some of these methods are time-consuming, tedious, and/or dedicated to sophisticated and expensive analytical instruments. Spectrophotometry[11,12]and fluorimetry[13] are most convenient techniques because of their inherent simplicity, adequate sensitivity, low cost and wide availability in all quality control laboratories. In experimental chemistry, the optimization of technical system is the process of adjusting the control variables to find the levels that achieve the best optimization. Usually, many conflicting response must be optimized simultaneously. In lack of systematic approaches the optimization is done by trial and error, or by changing one control variable at a time while holding the rest constant, such methods require a lot of experiments to be carried out. IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 IHJPAS Simplex optimization of experimental parameters was first introduced by Spendley[14], and then modified by Nelder[15] and Aberg[16]. A simplex is a geometric figure in which there are n +1 vertices, where (n) represents the number of variables [17], the method found a lot of applications in field of analytical chemistry [18-20], because it offers the capability of optimizing several factors simultaneously depending on a statistical design search to find out the maxima or minima of response, by rejecting the point p roducing the worst response and a replacement of it by the new point which is obtained statistically. The present work describes the utility of bromothymol blue (BTB) reagent for spectrophotometric determination of Cimetidine and Erythromycin in pure form as well as in these dosage form. In addition , the optimization of chemical dependent variables of affecting absorbance have been studied by using modified simplex method (MSM) via computer program. 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, Cimetidine and Erythromycin ethylsuccinate standard powders (purity 99.8%) were kindly provided by the State Company for Drug Industries and Medical Appliances, Samara-Iraq (SDI). Bromothymol blue(BTB) (Aldrich), 0.1% (w/v) solution is 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. Hydrochloric Acid (Aldrich), ~ 0.1 M , a 0.85 mL of concentrated hydrochloric acid (sp .gr1.18.37%) was added to 50 mL distilled water and diluted to the mark in a 100 mL calibrated flask . Potassium Hydroxide (fluka), ~ 0.1 M, 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[21]. Standard drugs solutions: Cimetidine stock solution (250 µg.mL-1 ), was prepared by dissolving 25 mg of Cimetidine in 5mL methanol and diluting to 100mL in a volumetric flask with distilled water . Working solutions were freshly prepared by subsequent dilutions. Erythromycin stock solution (250 µg.mL-1 ), was prepared by dissolving an accurate weighed 25 mg of Erythromycin ethylsuccinate in 5 mL methanol and diluting to 100mL in a IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 volumetric flask with distilled water. Working solutions were freshly prepared by subsequent dilutions. General Procedure Assay procedure for pure Cimetidine 1 mL aliquots of Cimetidine standared solution containing (2.5-75) μg were transferred into a series of 50 mL separating funnels, to each funnel 0.5 mL of phthalate buffer of pH 5.5 and 1 mL of 0.038% BTB reagent solutions were added. The separating funnels were shaken vigorously with 5 mL chloroform for 6 mints. The two phases were then allowed for clear separation and the absorbance of the yellow colored organic phase was measured at 427.5 nm against a reagent blank prepared similarly without addition of Cimetidine. The calibration graph was constructed by plotting the measured absorbance of the organic phase against the drug concentration. Assay procedure for pure Erythromycin ethylsuccinate 1 mL aliquots containing (2.5-250) μg Erythromycin ethylsuccinate were transferred into a series of 50 mL separating funnels and to each one, 0.5 mL of phthalate buffer of pH 4.0 and 1 mL of 0.02% BTB reagent were added. The separating funnels were shaken vigorously with 5 mL chloroform for 3 mints. The two phases were then allowed for clear separation and the absorbance of the yellow colored organic phase, measured at 414.5 nm against a reagent blank, was p lotted against the concentration of the drug. Analysis of Cimetidine and Erythromycin ethylsuccinate in pharmaceutical preparations i. In tablets and capsules: The content of 10 tablets or capsules were mixed well and a certain amount of fine powder was accurately weighted to give an equivalent to 200 mg for tablets and 250 mg for capsules was dissolve in 5 mL of methanol and diluted to 100mL in a volumetric flask with distilled water. The solution was filtered by using Whatman filter paper No.41 to avoid any suspended or un dissolved material before use. Working solutions were freshly prepared by subsequent dilutions with distilled water and analyzed by the recommended procedure. ii. In Ampoules: The volume of 10 ampoules were quantitatively transferred into 250 mL volumetric flask and diluted to the mark with distilled water. An accurately measured volume (2.5mL) was transferred into 100 mL volumetric flask and diluted to the mark with distilled water. Working solutions were freshly prepared by subsequent dilutions with distilled water and analyzed by the recommended procedure. Results and Discussion Extractive spectrophtometric procedures are popular for their sensitivity in the assay of drugs and hence, ion pair extractive spectrophotometry has received considerable attention for the quantitative determination of many pharmaceutical compounds [22-24]. IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Cimetidine and erythromycin ethy lsuccinate reacts with BTB in acidic buffer to give yellow color chloroform soluble ion-pair complex, which exhibits absorption maxima at 427.5 and 414.5 nm respectively against their reagent blanks (Figure1). Under the experimental conditions the reagent blank showed in both cases negligible absorbance thereby permit good analytical conditions for quantitative determination of Cimetidine and Erythromycin ethy lsuccinate in pharmaceutical dosage forms. Optimization of experimental variables: i. Univariable method: The experimental variables affecting the development and stabilities of both ion-pair complexes were achieved through a number of preliminary experiments. Such factors include pH, reaction time, reagent concentration, order of mixing, shaking time and the type of organic solvent used for extraction. For this reason, a variable was modified while maintaining the other variables at their constant values, then by maintaining that variable at its optimized value, another was modified; all variables were optimized via this method. Effect of pH In order to establish the optimum pH range, Cimetidine and Erythromycin ethy lsuccinate were mixed separately with specified volumes of BTB. The pH was then adjusted to a value between (4.5 -7.5) and (3-6.0) with few drops of 0.1M NaOH or 0.1M HCl for Cimetidine and Erythromycin ethy lsuccinate respectively. It was noticed that maximum color intensities and constant absorbance values were found at pH 5.5 and 4.0 for Cimetidine and Erythromycin ethy lsuccinate respectively (Figure 2). Low absorbencies were observed in solutions with higher or low pH than the optimum values for each drug. Hence, a pH of 5.5 and 4.0 was used in all the subsequent experimental work. Effect of reaction time The optimum reaction times for both drugs were determined by following the color development at ambient temperature (25±2). It was found that both reactions were instantaneous. Hence the products attained maximum and constant absorbancies immediately after the Cimetidine and Erythromycin ethylsuccinate have been mixed with BTB and the developed color, in each case, remained strictly unaltered for at least 24 hours. Effect of reagent concentration The influences of reagent concentration on the absorbancies of both complexes are illustrated in (Figure 3). 0.038% and 0.020% solutions of BTB were found to be optimum to develop the maximum color intensities for Cimetidine and Erythromycin ethylsuccinate ion- pair complexes respectively, after which no more increase in absorbance values was obtained; therefore, the cited concentrations of BTB solution were used. Effect of the order of mixing: The effect of order of addition of the reactant was also studied. It was found that best results were obtained in both cases by placing the cited drug, the buffer and finally the reagent instead of any other orders of addition. IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Effect of shaking time The optimum shaking times for the complete extraction of the formed ion pair complexes with chloroform were studied for the period of 1-8 minutes (Table 1). It was found that the minimum shaking times for complete extraction of Cimetidine and Erythromycin ethy lsuccinate complexes, at room temperature, were 6 and 3 minutes respectively. Effect of the extraction solvent Several organic solvents, namely toluene, carbon tetrachloride, methylene chloride, 1, 2- dichloroethane, benzene in addition to chloroform, were examined for their ability to extract the drug-dye ion-pairs. The latter was found to be 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 both complexes. ii. Simplex method Simplex method is used to confirm the optimum conditions, which were obtained by the univariate procedure. Three major parameters (pH, reagent concentration, and shaking time) were optimized by the simplex procedure, while the other minor parameters were obtained by the univariate method. To set simplex program for the three studied variables (Table 3), four arbitrary experimental conditions should be chosen. The values of these parameters were selected within specified boundaries for each at which they affected the measured absorption signal of the colored products. The absorbencies of these four experiments were measured and the results were feed to the simplex program. Points (1 to 4) in Tables 4 and 5 represent the first four experiment cycle with their measured absorbencies. The Simplex program starts to reflect the worst point through the centroid of other points to obtain a new point 5. An experiment was then performed utilizing the variable setting as a reflected point; because this value was better than that at point 3, the latter was rejected and replaced by point 5. A measured absorption signal was feeding again to the program and the process is repeated successively until optimum conditions were obtained similarly to those obtained by the univariate method. Stoichiometry of the complexes To establish molar ratio between Cimetidine and Erythromycin ethylsuccinate with BTB, Job's method of continuous variation has been used (Figure 4). The results showed that 1:1 complexes were formed with BTB through the electrostatic attraction between the positive protonated Cimetidine and Erythromycin ethylsuccinate with the anion of BTB [25, 26] . The formation of the ion-pair complex can be represented by taking Cimetidine as an example (Scheme 3): IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Scheme 3: Proposed reaction pathway between Cimetidine and BTB. Calibration graphs Employing the experimental conditions, linear calibration graph for Cimetidine and Erythromycin ethy lsuccinate was obtained (Figure 5 A and B), which showed that Beer's law was obeyed in the concentration range of 0.5-15 and 0.5-50 µg.mL-1 for Cimetidine and Erythromycin ethylsuccinate 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 drugs in the range given (Table 6). The regression equations, correlation coefficients, molar absorptivities, detection limits and sandell sensitivities in addition to other parameters are given in Table 5. Accuracy and precision The accuracies of the proposed methods were confirmed by analyzing three replicate analyses of three different amounts of each drug (within Beer's law) by calculating the relative error percentage (Table 7). The results indicated good accuracies of the method for both cited drugs. The precision was determined in each case by calculating the percentage relative standard deviation (RSD %) for three determinations at each of the studied concentration level and were found to be in the range of 1.158-2.003% and 0.173-2.276% for Cimetidine and Erythromycin ethy lsuccinate respectively. The proposed method was compared statistically with other methods found in the literature and the results are shown in tables 10 and 11. IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Interferences Study The results showed that no interferences were found in the presence of 1000 μg of the studied excipients (lactose, sucrose, starch, glucose, magnesium stearate and sodium citrate) in the determination of Cimetidine and Erythromycin ethy lsuccinate (Table 9). Analysis of dosage forms It is evident from the aforementioned results that the proposed method gave satisfactory results with the investigated drugs. Thus, their pharmaceutical dosage forms were subjected to analysis of their contents of the active ingredient by the proposed method (ion-pair formation). The results given in table 8 were satisfactory. The recommended method was statistically compared with official, standard and other methods, no significant differences were found between the calculated and theoretical values of t- test at 95%and F- test at 99.5%, 99.5% and 95% (Tables 12 and 13). References 1. Slobodan, R. (1999) Drug Interactions of H2-Receptor Antagonists Involving Cytochrome( P450 -CYPs) Enzymes from the Laboratory to the Clinic, Journal of the Croatian Medical, 40( 3, 1):4 2. Alwan, Ala,dine, A. S. and Abou, Yousif, Z.(1990) Iraqi Drug Guide, 1st edition, NBSD , Iraq, 40-41,100-102, 115. 3. Kanfer, I.; SKiner, M. F. and Walker, R. B. (1998) Analysis of macrolide antibiotics, Journal of Chromatography A, 812 (1/2): 255-286. 4. Girish-Kumar K. and Karpaselvi, L. (1994) Determination of cimetidine in pure form and in dosage forms using N,N-dibromo,dimethylhydantoin Journal of Analyst,119(6):1375-1376. 5. Griessmann , K.; Kaunzinger, A.; Schubert-Zsilavecz M.and Abdel-Tawab,M. (2007) A rapid HPLC-UV method for the quantification of erythromycin in dermatological preparations, Journal of Pharmazie,Die, 62 (9) :668-671. 6. Kelani, Km.; Aziz, Am.; Hegazy , Ma. and Farrah, La. (2002) Determination of cimetidine, famotidine, and ranitidine hydrochloride in the presence of their sulfoxide derivatives in pure and dosageforms by high-performance thin-layer chromatography and scanning densitometry , Journal of AOAC int, Sep-Oct,85(5):1015-1020. 7. Deubel, A.; Frandino, AS.; Sorgel, F. and Holzgggrabe, U. (2006) Determination of erythromycin and related substances in commercial samples using liquid chromatography/ion trap mass spectrometry, Journal of Chromatogr A.; Dec; 1136(1):39-47. 8. Thanh, Ha. PT.; Van Schpdael, A.; Rots E. and Hwgmartens,J (2004) Investigating the potential of erythromycin and derivatives as chiral selector in capillary electrophoresis Journal of Phrm. Biomed anal. Mar 10 34(5): 861-870. 9. Luo, J-W.; Chen, H-W. and He, Q-H. (2001) Originals - Electrophoresis - Determination of Cimetidine in Human Plasma by Use of Coupled-Flow Injection, Solid-Phase Extraction, and Capillary Zone Electrophoresis, Journal of Chromatographia., 53:295-300. 10. Jennifer, S.; Ridlen ; David, R. Skotty; Peter T. Kissinger and Timothy A. Nieman (1997) Determination of erythromycin in urine and plasma using microbore liquid chromatography with tris(2,2′-bipyridyl)ruthenium(II) electrogenerated IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 chemiluminescence detection, Journal of Chromatography B, Volume 694, Issue 2, 4 July, Pages 393-400. 11. Dabrowska, D.; Regosz, A.; Piekos, R.; Mierzwa, M.;and Paruch, B.(1990) A study of ion-pair formation between erythromycin and bromothymol blue, methylthymol blue, and thymol blue and their use for assaying erythromycin in dosage forms, Journal of microchemical, 41, 210-218. 12. Darwish, Ia; Husein, Sa. ; Mohmoud Am. And Hassan, Ai.(2008) Spectrophotometric determination of H (2)-receptor antagonists via their oxidation with cerium (IV), Journal of Spectrochim Acta a Mol Biomol Spectrosc; Jan; 69(1): 33-40. 13. Virginata, de L. M. Finete; Marcia, Aissawa and Ricardo, Q. Aucelio (2008) Fluorimetric method for the determination of erythromycin using a photochemical derivatization approach, Journal of Beazilian Chem. Soc. volume 19(7): 1418-1422. 14. spendley,W.; Hext, G.R.; and Himusworth, F.R.T. (1962) application of Simplex designs in optimisation and evolutionary Sequential operation, Journal of Technometrics. 4: 441-462. 15. Nelder, J. A.; and Mead, R. A. (1965) Asimplex Method for Function Minimization, Computer Journal, 7: 308-313. 16. Aberg, E. R.; and Gustavsson, A.G.T. (1982), Journal of Analytica Chemica Acta. 144: 39-53. 17. Walters, F. H.; Parker, L. R.; Morgan, S. L.; and Deming, S. N. (1991) Sequential Simplex Optimization, 1st , CRC Press,Inc., Boca Raton, Florida, 44. 18. Momenbeik, F.; Momeniz, Z. and Kharasani, H., J.,(2005) Separation and determination of Vitamins E and A in multivitamin syrup using micellar liquid chromatography and simplex optimization, Journal of Pharmaceutical and Biomedical Analysis, , 37( 2): 383-387. 19. Murillo Pulgarn, J.A.; Alanon Molina, A.; Alanon Pardo. M.T.(2002) The use of modified simplex method to optimize the room temperature phosphorescence variables in the determination of an antihypertensive drug, Journal of Talanta, 57, 795-805. 20. Tinoi, J.; Rakariyatham, N. and Deming, R.L. (2005) Simplex optimization of carotenoid production by Rhodotorula glutinis using hydrolyzed mung bean waste flour as substrate, Journal of Process Biochemistry 40, Issue 7: 2551-2557. 21. Basavaiah, K.; and Shakunthala,C., V.(2004) Ion-pair Complexometric Determination of Cyproheptadine Hydrochloride Using bromophenol blue, Journal of ScienceAsia, 30 :163-170. 22. Julic, M.; Cardso, S. C. (2005) Spectrophotometric determination of oxiconazole in topical lotion using methylorange, Journal of Pharmaceutical and Biomedical Analysis, 37, Issue 4, 1 : 639-642. 23. 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. 24. Basavaiah, K.; Prameela, H. C.; Somashekar, B. C. (2007) Spectrophotometric determination of pefloxacin mesylate in pharmaceuticals, Journal of Acta Pharm., 57:221–230. IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 25. Taha Elham, A., Soliman Susan, M. Abdellatef Hisham, E. and Ayad Magda, M. (2002)Colorimetric methods for the determination of some tricyclic antidepressant drugs in their pure and dosage forms), Journal of Microchim. Acta, 140:175-182. 26. .Shah, J. ; Rasul, M. and Manzoor S. (2008) Extractive Spectrophotometric Methods for Determination of Clarithromycin in Pharmaceutical Formulations Using Bromothymol Blue and Cresol Red, Journal of the Chinese Chemical Society 55(5): 1107-1112. 27. 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. 28. 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. 29. Helali, N. and Monser, L.(2006)Simultaneous Determination of Cimetidine and Related Compounds in Pharmaceuticals by HPLC on a PorousGraphitic Carbon Column, J.of chromamatograph:a 63(9-10): 425-430. 30. Kelly, MT.; McGuirk, D. and Bloomfield, FJ.(1995) Determination of cimetidine in human plasma by high-performance liquid chromatography following liquid-liquid extraction J Chromatogr B Biomed Appl. Jun 9;668(1):117-23. 31. Kelani, K. M. ; AZIZ, A. M . ; Hegazy , M. A. and Laila, Abdel F. (2002), Different spectrophotometric methods for the determination of cimetidine, ranitidine hydrochloride, and famotidine. J. of Spectroscopy letters 35, no4: 543-563. 32. Rattaya, R.; Wachiranee, V.; Worapot S. and Leena, S. (2007) Simple and Rapid Spectrophotometric Method for the Analysis of Erythromycin in Pharmaceutical Dosage Forms, Journal of Food and Drug Analysis, 15(1): 10-14. 33. Kiyoshi Tsuji and Michael P. Kane (2006) Improved high-pressure liquid chromatographic method for the analysis of erythromycin in solid dosage form, J. of Pharmaceutical sciences, 71 (10): 1160-1164. 34. Issa, Y. M. and Amin, A.S. (2001) EXtraction-Colirenetric Method for the Determination of Erythromycin and its easter Dosage formsUsing Chromtopic Acid Azo Dyes, J.of Analytical Letters, 34, Issue 7 : 1163 – 1173. 35. LIJun, LIU. Mei, DU and Xiang-Yan, LI. Quan-Mina(2009) Charge Transfer Reaction between Erythromycin Ethylsuccinate and Salicyl Fluorone, J. of Chinese Journal of Spectroscopy Laboratory, Issue 3: 519-523. 36. Pakinaz, Y. Khashaba (2002) Spectrofluorimetric analysis of certain macrolide antibiotics in bulk and pharmaceutical formulations, Journal of Pharmaceutical and Biomedical Analysis, 27, Issue 6: 923-932. 37. Zhao Yanqing, Li Hua McCain, Gui-Zhi Zhao(2005) Spectrophotometric determination of erythromycin ethy lsuccinate based on the charge transfer reaction between erythromycin ethylsuccinate and quinalizarin, J. of China Modern Applied Pharmacy, Volume 22, (3), 229-303. IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 38. SUN, Shu-Guang (2004) Spectrophotometric Determination of Erythromycin Ethylsuccinate with Charge Transfer Reaction between Erythromycin Ethylsuccinate and Alizarin, J. of Spectroscopy Laboratory, 21(06): 1115-1116. 39. British Pharmacopeia (1998), CD-ROM Her Majesty ,s Stationary office, London. 40. British Pharmacopeia (2002), CD-ROM , BP, Co.-UK. Table(1): Effect of shaking time on extraction of 5 µg.mL-1 Cimitidine; (0.038%BTB, pH5.5) and 10µg.mL-1 Erythromycin ethylsuccinate (0.02%BTB, pH 4) Mixing Time/mint Absorbance Cimetidine Absorbance Erythromycin ethy lsuccinate 1 0.272 0.247 2 0.274 0.249 3 0.268 0.255 4 0.269 0.243 5 0.294 0.242 6 0.298 0.242 7 0.264 0.241 8 0.264 0.240 Table (2): Effect of type of organic phase on extraction of 5 µg.mL-1 Cimetidine and 10 µg.mL-1 Erythromycin ethylsuccinate Organic phase Absorbance of Drug-BTB ion pair complex Cimetidine Erythromycin ethylsuccinate Chloroform 0.298 0.255 Toluene 0.014 0.168 Carbontetrachloride 0.022 0.066 Benzene 0.002 0.185 1,2-Dichloro ethane 0.089 0.097 Dichloro methane 0.077 0.067 Table(3): Boundary conditions for the studied variables Variable Range for Cimetidine Range for Erythromycin pH 5.0-8.5 3-6 Reagent Conc.(%) 0.01-.038 0.01-0.025 Shaking time (min.) 1-8 1-5 IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Table (4): Multivariate experiments (Simplex) for determination of Cimitidine Exp. No. pH %Reagent Conc. Mixing Time A 1 5.5 0.038 5 0.532 2 8.0 0.020 4 0.267 3 6.5 0.010 1 0.226 4 7.0 0.030 3 0.290 5 5.5 0.038 8 0.531 6 5.0 0.038 8 0.445 7 6.5 0.036 4 0.322 8 5.5 0.038 7 0.532 9 6.0 0.038 5 0.528 10 5.5 0.038 4 0.530 11 5.5 0.038 6 0.534 12 5.5 0.038 6 0.534 Table( 5): Multivariate experiment (Simplex) for determination of Erythromycin ethylsuccinate Exp. No. pH %Reagent Conc. Mixing Time A 1 4.0 0.010 1 0.164 2 5.5 0.020 2 0.229 3 6.0 0.015 4 0.023 4 3.0 0.025 3 0.226 5 6.0 0.025 5 0.239 6 6.0 0.020 5 0.200 7 4.5 0.025 4 0.246 8 5.0 0.025 5 0.235 9 4.0 0.025 5 0.254 10 3.0 0.025 5 0.210 11(8) 5.0 0.025 5 0.235 12 5.0 0.025 5 0.210 13(7) 4.5 0.025 4 0.246 14 3.5 0.025 5 0.240 15(7) 4.5 0.025 4 0.246 16(14) 3.5 0.025 5 0.240 IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Table (6): Spectral characteristics and statistical data of the regression equations for determination of Cimetidine and Erythromycin ethylsuccinate using ion–pair formation Parameter Cimetidine Erythromycin ethylsuccinate λmax (nm) 427.5 414.5 Color Yellow Yellow Linearity range (µg.mL -1 ) 0.5 – 15.0 0.5 – 50.0 Molar absorpitivites (l.mol -1 .cm -1 ) 13172 18103 Regression equation A = 0.052 [ Cim. µg.mL-1] + 0.013 A = 0.021[ Ery. µg.mL-1] + 0.018 Calibration Sensitivity 0.052 0.021 Sandell's Sensitivity (µg.cm -2 ) 19.157 47.620 Correlation of Linearity (R2) 0.9970 0.9985 Correlation coefficient (R) 0.9984 0.9992 Detection limit (µg.mL -1 ) 0.222 0.286 Table (7): Evaluation of accuracies and precisions of the two proposed procedure Drug Concentration ((µg.mL-1) Relative Error % R.S.D.* % Taken Found* Cimetidine 2 1.971 -0.145 1.538 4 3.972 -0.700 1.158 10 9.936 -0.640 2.003 Erythromycin 5 4.921 -1.580 2.276 20 19.858 -0.710 1.695 40 40.272 +0.680 0.173 Average of three determinations* IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Table (8): Spectrophotometric determination of Cimetidine and Erythromycin Ethylsuccinatein pharmaceutical compounds using Ion –Pair Formation Sample Amount (mg) Concentration (µg.mL- 1 ) Relative Error % R.S.D.* % Labeled Found taken Found* Tagadine (Cimetidine) 200mg/ tablet SDI/Iraq 200 204.840 5 5.121 +2.400 0.937 200.760 10 10.038 +0.380 0.508 CimedneR (Cimetidine) 200mg/ tablet DAD Jordan 200 180.320 5 4.508 -9.840 0.615 179.820 10 8.991 -10.090 0.316 Histale (Cimetidine Hydrochloride) 200mg/Ampoule IBH/Syria 200 199.000 5 4.975 -0.500 0.582 198.840 10 9.942 -0.580 1.579 Erythrosam (Erythromycin ethy lSuccinate) 250mg/ tablet SDI/Iraq 250 255.150 10 10.206 +2.060 0.970 256.35 20 20.508 +2.540 0.353 Erythronin (Erythromycin ethy lsuccinate) 250mg/ tablet NDI/Iraq 250 253.575 10 10.143 +1.430 0.473 254.363 20 20.349 +1.745 0.359 Zithrorive (Azithromycin dehydrate) 250mg/ tablet R.P/Egypt 250 249.600 10 9.984 -0.160 1.202 246.588 20 19.727 -1.365 0.487 Erythromycin Stearate (pure powder) SDI/Iraq 250 249.200 10 9.968 -0.320 1.204 246.375 20 19.710 -1.450 0.639 *Average of three determinations IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Table (9): Percent Recovary for 10 µg.mL -1 of Cimetidine and 20 µg.mL -1 of Erythromycin Ethylsuccinate in the presence of 1000 µg.mL-1of Excipients Excipients Cimetidine Conc. Taken10 µg.mL-1 Erythromycin Ethylsuccinate Conc. Taken20 µg.mL - Conc. Fundµg.mL -1 %Recovery Conc. Fundµg.mL -1 %Recovery lactose 9.928 99.280 19.829 99.145 Sucrose 9.934 99.340 19.842 99.210 starch 9.932 99.320 19.857 99.285 Glucose 9.929 99.290 19.857 99.285 Magnesium Stearate 9.935 99.350 19.839 99.195 Sodium Citrate 9.933 99.330 19.843 99.215 Average of three determinations* Table(10): Analytical Parameters for the analysis of Cimetidine by the propsed and others methods Ref. No. methods Linear range µg.mL -1 ε L.mo l . - cm - 1 Correlation Coefficient ( R) Recoveri es range% RSD% range - Proposed method 0.5-15.0 13172 0.9984 98.550-99.36 1.158-2.003 12 Spectrophotometric 1-20 0.9994 98.3-102.6 27 Spectrophotometric 8-30 6710 99.8-100.2 0.81-0.84 28 Spectrophotometric 2-16 13660 0.9989 99.8-100.7 0.74-0.92 6 H.P.TL.C. 5-50 - - 100.39 ± 1.33 29 H.P.L.C 0.25-83 0.998 99.2 - 100.8 30 H.P.L.C 50-3000 - - 71 -81 less than 6% 31 Spectrophotometric 1 st derivative 25-150 - - 100.27±0.679 - Spectrophotometric Complex formation 10-60 - - 99.84±0.858 - IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Table(11): Analytical Parameters for the analysis of Erythromycin Ethylsuccinate by the propsed and others methods Ref. No. methods Linear range µg.mL-1 εL.mol.- cm-1 Correlation Coefficient ( R) Recovery % RSD% - Proposed method 0.5-50 18103 0.9992 98.4- 100.6 0.17-2.27 32 Spectrophotometric Direct UV 1 st Derivative 3-15 37.43 0.9836- 0.9892 97.6 0.48 3-15 44.03 0.9917- 0.9967 106.5 0.65 11 Spectrophotometric Ion-Pair 2-61 - - 98.4- 103.6 1.4-4.4 33 high-pressure L.C. 60-120 - - 99.9 Lees than1% 34 Extraction 0.4-56 - - 1.,3 35 Charge transfer 1.724-129.3 8500 98.3 36 Spectroflurimetric 0.0426-1.2 - - 98.3- 100.8 0.014- 0.058 37 Charge transfer 5-60 11410 - 98 0.82 38 Charge transfer 0-80 9910 - 97 - IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Table(12): t- and F- Values for analysis of Cimetidine in Pharmaceytical Copmpounds Cimetidine Proposed Method, (S.D.I) T-Valuesa F-valuesb Other Methods (N=5) S.D Ref. No. Ion-Pair 0.671 157.692 Official 0.640 27,39 N=3 0.567 357.692 Other 0.930 28 S.D = 0.051 0.177 1.727 Stander 0.167 40 a Theoretical values for t-test at 95% confidence limit were N=6 (2.447). * b Theoretical values for F-test at 99.5% (199.25), 99.9%(999.25) and 95% (19.274) confidence limit respectively, were N=(4,2). Table(13): t- and F- Values for analysis of Erythromycin Ethylsuccinate in Pharmaceytical Copmpounds Erythromycin Ethylsuccinate Proposed Method, (S.D.I) T-Valuesa F-valuesb Other Methods Ref. No. N S.D Ion-Pair 0.388 200 (N=9) 1.400 11 N=3 1.735 13.151 (N=8) 0.359 35 S.D = 0.099 2.832 2 (N=6) 0. 140 38 * a Theoretical values for t at 95% confidence limit were N=10(2.228), 9(2.262) and 7(2.365)respectively b Theoretical values for F at 99.9%were N=(8,2)(999.31), 95% were N=(7,2)(19.353) and 95% were N = (5,2)(19.296) confidence limit respectively. IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Fig.(1): Absorption spectra of (A) 4 µg.mL-1 Cimetidine-BTB ion-pair complex, (B) 10 µg.mL-1 Erythromycin ethylsuccinate-BTB ion-pair complex. Fig.(2): Effect of pH on the Absorbance of: (A) 5 µg.mL-1 Cimetidine; 0.04% BTB. (B) 10 µg.mL-1 Erythromycin ethylsuccinate ; 0.04 % BTB. Fig.(3): Effect of Reagent Concentration on the Absorbance of: (A) 5 µg.mL -1 Cimitidine pH 5.5, (B) 10 µg.mL-1 Erythromycin ethylsuccinate pH 4. IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. SCI VOL. 23 (3) 2010 Fig. (4): Continuous Variation of (A) 9.907*10-5 M Cimetidine, 9.907*10-5 M BTB (B) 1.740*10-4 M Erythromycin ethylsuccinate, 1.740*10-4 M BTB. Fig.(6): Calibration graph of (A) Cimetidine, (B) Erythromycin ethylsuccinate; underder optimum experimental conditions. IHJPAS 2010) 3( 23 مجلة ابن الهیثم للعلوم الصرفة والتطبیقیة المجلد دوائيایجاد الظروف المثلى بدراسة المتغیرات االحادیة وبطریقة السمبلكس لتقدیر دین یالسمت واالرثرومایسین ایثایل سوكسینیت بأستخدام الكاشف بروموثایمول االزرق ج االیونياو االزدتكوین معقدات ب سرمد بهجت دیكران، عالء كریم محمد، علي خلیل محمود بغداد جامعة ،كلیة التربیة ابن الهیثم ،قسم الكیمیاء الخالصة طریقة طیفیة باالعتماد على تكوین معقدات أزدواج ایوني لتقدیر السمتیدین واالرثرومایسین ایثایل تلقد استخدم ة ،غیر مكلفة و سریعة، و بسیطة ، وكانت الطریقة دقیقة ،. صیدالنیةسوكسینیت في عینات نقیة ومستحضرات وحساس ستخالص معقدات االزدواج االیوني المتكونة بین العقارین قید الدراسة مع اتعتمد باالساس على استخدام الكلوروفورم في ت 4.0و 5.5الكاشف بروموثایمول االزرق من وسط مائي وبدوال حامضیة للسمیتیدین واالرثرومایسین ایثایل سوكسینی ل المتكونة للسمیتیدین واالرثرومایسین ایثایل سوكسینیت اعلقد اظهرت المعقدات . على التوالي ظم امتصاص لها عند االطوا واظهرت منحنیات المقایسة عالقات .على التوالي مقابل محالیل الخلب لها نانومتر 414.5و،نانومتر 427.5الموجیة 0.222وبحدود كشف مل / میكروغرام) 50 – 0.5( و ،مل/ مایكروغرام ) 15 – 0.5( خطیة في المدیات من أظهرت الدراسة أیضآ أن الطریقتین . مل للغقارین المذكورین على التوالي /مایكروغرام 0.286و ،مل/ مایكروغرام لهذین العقارین، وقد أمكن ةفي المستحضرات الصیدالنی غالباالمقترحتین خالیة من تأثیر المتداخالت المعروفة التي تتواجد في بعض ) ن من مشتقاته یع أثنم( تطبیق الطریقتین بنجاح لتقدیر السمتیدین واالرثروةمایسین ایثایل سوكسینیت .المستحضرات الصیدالنیة IHJPAS