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. Dik ran, A. K. Mohammed ,A. K.M. Al-Jumaily Departme nt of Chemistry, College of Education Ibn Al Haitham, Unive rsity of Baghdad Abstract The ion-pair formation method has been app lied for the sp ectrop hotometric determination of Cimetidine and Erythromy cin ethy lsuccinate, in bulk samples and in dosage form. The methods are accurate, simple, rapid, inexp ensive and sensitive dep ending on the extraction of the formed ion-pair with brompthy mol blue (BTB) as a chromogenic reagent in chloroform, use p hthalate buffer of p H 5.5 and 4.0 for Cimetidine and Erythromycin ethy lsuccinate resp ectively. The formed comp lexes show absorbance maxima at 427.5 nm and 414.5 nm for Cimetidine and Erythromy cin ethy lsuccinate resp ectively against reagent blank. The calibration graphs are linear in the ran ges of 0.5-15 µ g.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 Erythromy cin ethy lsuccinate. The results show the absence of interferences from the excipients on t he determination of these drugs. T he prop osed methods have been successfully applied for the determination of Cimetidine and Erythromy cin ethy lsuccinate (with two of its derivatives) in pharmaceutical p reparations. Key words, Simplex, Sp ectrop hotometric, Cimetidine, Erythromy cin ethy lsuccinate, Ion-pair. Introduction Cimetidine was the first histamine H2-recep tor antagonist app roved by the Food and Drug Administ ration, USA, for the treatment of duodenal ulcers, Zollinger-Ellison sy ndrome, and othergastric hy p ersecretory st ates [1] . It ' s also indicated for the relief heartburn in p eptic, duodenal ulcers and p revents rebleeding in p atients which reduce the secretion of gastric acid [2] , cimetidine, due to its effects on the immune sy st em and as an H2-recep tor antagonist, can inhibit growt h of carcinogen-induced colonic tumors in rats, as well as the in vitro human colon cancer cell lines[1]. The Chemical st ructure 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 ch emical structure of Cimetidine Erythromy cin is the most emp loy ed macrolide antibiotic for treating a myriad of infections caused by gr am-p ositive bacteria such as anthra x, tonsillitis, otits media and syp hilis [2], it is oft en prescribed as an alternative for p atient allergic to p enicillin [2-3]. It has been also emp loyed a as p art of therap eutic cocktails together with amino glycoside antibiotics that covers gr am-negative microor ganisms [3]. The Chemical st ructure of Erythromy cin is given in (Scheme 2), which shows t he lactone rin g on it. Scheme 2: The ch emical Structure of Erythromy cin Ethy lsuccinate Sever al methods have b een r eported for the determination of C imetidine and Erythromy cin in bulk and p harmaceutical dosa ge forms, t hese methods include titrimetry [4], high p erformance liquid chromato graphy [5], high p erformance thin lay er chro matogr aphy [6], liquid chromato graphy [7], cap illary electrop horesis[8,9] and chemiluminesence[10].So me of these methods are time-consuming, tedious, and/or dedicated to sop histicated and exp ensive analytical instruments. Sp ectrop hotometry [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 exp erimental chemistry , the op timization of technical sy st em is the p rocess of adjust ing the control variables to find the levels that achieve the best op timization. Usually, many conflicting resp onse must be op timized simultaneously . In lack of syst ematic approaches the optimization is done by trial and error, or by changing one control variable at a time while hold ing the rest constant, such methods require a lot of e xp eriments t o be carried out. IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 IHJPAS Simp lex op timization of exp erimental p arameters was first introduced by Sp endley[14], and then modified by Nelder[15] and Aber g[16]. A si mplex is a geometric f igure in whi ch there are n +1 vertices, where (n) represents t he number of variables [17], the method found a lot of applications in field of analy tical chemist ry [18-20], because it offers the cap ability of optimizing several factors simultaneously dep ending on a st atistical design search to find out the maxima or minima of resp onse, by rejecting the point p roducing the worst resp onse and a replacement of it by the new p oint which is obtained st atist ically. The present work describes the utility of bromothy mol blue (BTB) r eagent for sp ectrop hotometric determination of Cimetidine and Erythromy cin in p ure form as well as in these dosage form. In addition , the optimization of chemical dependent variables of affecting absorbance h ave b een st udied by using modified simplex method (M SM ) via comp uter p rogram. Apparatus A Cintra 5 sp ectrop hotometer with 1 cm quartz cells was used for absorbance measurements. PH-meter DW-9421 from Philip s instrument, a Sartorius BL 210S balance, and a Pentium 4 comp uter (DELL) was used for data p rocessing. Experime ntal Material and Reagents: All Chemicals used were of analy tical reagent grad un less otherwise is mentioned, Cimetidine and Erythromycin ethy lsuccinate st andard p owders (p urity 99.8%) were kindly p rovided by the State Company for Drug Indust ries and M edical App liances, Samara-Iraq (SDI). Bromothy mol blue(BTB) (Aldrich), 0.1% (w/v) solution is p repared by dissolving 0.1 g of the dye in 5 mL of methanol and then the solution was diluted to a fin al vo lume of 100 mL with distilled water. Working solutions were freshly p repared by subsequent dilutions. Hy drochloric Acid (Aldrich), ~ 0.1 M , a 0.85 mL of concentrated hy drochloric acid (sp .gr1.18.37%) was added to 50 mL distilled water and diluted to the mark in a 100 mL calibr ated flask . Pot assium Hy droxide (fluka), ~ 0.1 M , p repared by dissolving 0.56 g of p otassium hy droxide in 25 mL d istilled water and d iluted to 100 mL in volumetric flask with distilled water. Pht halate buffer 0.2M solution was p rep ared by dissolved 4.08 g of p otassium hy drogen p hthalate (M ERCK) 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 drop s of 0.1M HCl and\or 0.1M KOH [21] . S tandard drugs solutions: Cimetidine st ock solution (250 µg. mL -1 ), was p repared by dissolving 25 mg of Cimetidine in 5mL methanol and diluting to 100mL in a vo lumetric flask with dist illed water . Workin g solutions were freshly p rep ared by subsequent dilutions. Erythromy cin st ock solution (250 µ g.mL -1 ), was p repared by dissolving an accurate weigh ed 25 mg of Erythromycin ethy lsuccinate 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 Ci metidine 1 mL aliquots of Cimetidine standared solution containing (2.5-75) μg were transferred into a series of 50 mL separating funn els, t o each funnel 0.5 mL of p hthalate buffer of p H 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 p hases were then allowed for clear sep aration and the absorbance of the yellow color ed organic p hase was measured at 427.5 n m against a reagent blank p repared similarly without addition of Cimetidine. The calibration graph was constructed by p lotting the measured absorbance of the or ganic p hase against the drug concentration. Assay procedure for pure Erythromycin ethylsuccinate 1 mL aliquots containin g (2.5-250) μg Erythromycin ethy lsuccinate were t ransferred into a series of 50 mL separating funnels and to each one, 0.5 mL of p hthalate buffer of p H 4.0 and 1 mL of 0.02% BTB reagent were added. The sep arating funn els were shak en vigorously with 5 mL chloroform for 3 mints. T he two p hases were then allowed for clear sep aration and the absorbance of the y ellow colored or ganic p hase, measured at 414.5 nm against a r eagent blank, was p lotted against the concentration of the drug. Anal ysis of Cime tidine and Erythromycin e thylsuccinate in pharmaceutical preparations i. In table ts and capsules: The content of 10 tablets or capsules were mixed we ll and a certain amount of fine p owder was accurately weighted to give an equivalent to 200 mg for tablets and 250 mg for cap sules 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 b efore use. Working solutions were freshly p repared by subsequent dilutions with distilled water and analyzed by the recommended procedure. ii. In Ampoul es: The volume of 10 ampoules were quantitatively transferred into 250 m L volu metric f lask and diluted to the mark with distilled water. An accurately measured vo lume (2.5mL) was transferred into 100 mL volumetric flask and diluted to the mark with distilled water. Workin g solutions were freshly p repared by subsequent dilutions with distilled water and analyzed by the recommended procedure. Results and Discussion Extractive sp ectrop htometric p rocedures are p op ular for their sensitivity in the assay of drugs and h ence, ion pair extractive sp ectrop hotometry has received considerable att ention for the quantitative determination of many p harmaceutical co mpounds [22- 24] . IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 Cimetidine and erythromy cin ethy lsuccinate reacts with BTB in acidic buffer to give y ellow color chloroform solub le ion-pair complex, which exhibits absorp tion maxima at 427.5 and 414.5 nm r esp ectively against their reagent blanks (Fi gur e1). Under the exp erimental conditions the reagent blank showed in both cases ne gligible absorbance thereby p ermit good analytical conditions for quantitative determination of Cimetidine and Erythromycin ethy lsuccinate in p harmaceutical dosage forms. Optimization of experimental variable s: i. Uni variable method: The exp erimental variables affecting the development and st abilities of both ion-p air comp lexes were achieved throu gh a number of preliminary exp eriments. Such factors includ e p H, reaction time, reagent concentration, order of mixing, shaking time and the type of organic solvent used for extraction. For this reason, a variable was modif ied while maintainin g the other variables at their constant values, t hen by maintainin g that variable at its optimized value, another was modif ied; all variables were op timized via this method. Effect of pH In order to establish the op timum pH range, Cimetidine and Erythromycin ethy lsuccinate were mixed sep arately with sp ecified volumes of BTB. The p H was then adjust ed to a value between (4.5 -7.5) and (3-6.0) with few drop s of 0.1M NaOH or 0.1M HCl for Cimetidine and Erythromycin ethy lsuccinate resp ectively. It was noticed that maximu m color intensities and const ant absorbance values wer e found at p H 5.5 and 4.0 for C imetidine and Ery thromycin ethy lsuccinate resp ectively (Figure 2). Low absorbencies were observed in solutions with higher or low p H than the optimum values for each dru g. Hen ce, a p H of 5.5 and 4.0 was used in all the subsequent e xp erimental work. Effect of reaction time The optimum reaction times for both dru gs were determin ed by followin g the color development at ambient temp erature (25±2). It was found that both reactions were instantaneous. Hence the products attained maximu m and constant absorbancies immediately after the Cimetidine and Erythromycin ethy lsuccinate 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 comp lexes 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 ethy lsuccinate ion- p air comp lexes r esp ectively, after which no more in crease in absorban ce v alues was obt ained; therefore, the cited concentrations of BT B solution were used. Effect of t he 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 p lacin g the cited dru g, the buffer and fin ally 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 comp lexes with chloroform were st udied for the period of 1-8 minutes (Table 1). It was found that the minimu m shaking times for complete extraction of Cimetidine and Erythromycin ethy lsuccinate complexes, at room temp erature, were 6 and 3 minutes resp ectively. Effect of the extraction sol vent Sever al or ganic solvents, namely toluene, carbon tetrachloride, methy lene chloride, 1, 2- dichloroethane, benzene in addition to chloroform, were examined for their ability to extract the drug-dy e ion-p airs. 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 sin gle extraction with 5 mL p ortion of chloroform was adequate to achiev e a quantitative recov ery of both comp lexes. ii. Si mplex me thod Simp lex method is used to confirm the optimum conditions, which were obtained by the univariate p rocedure. Three major p arameters (p H, reagent concentration, and shaking time) were optimized by the simp lex p rocedure, while t he other minor p arameters were obtained by the univariate method. To set simplex p rogram for the three studied variables (Table 3), four arbitrary exp erimental conditions should be chosen. The v alues of these p arameters wer e selected within sp ecified boundaries for each at which they affected the measured absorption signal of the colored products. The absorbencies of these four exp eriments were measured and the results were feed to the simplex p rogram. Points (1 to 4) in Tables 4 and 5 rep resent the first four exp eriment cycle with t heir measured absorbencies. The Simplex p rogram starts to reflect the worst p oint through the centroid of other p oints to obtain a new p oint 5. An exp eriment was then p erformed utilizing the variable setting as a ref lected p oint; because this value was b ett er than that at p oint 3, the latter was rejected and replaced by p oint 5. A measured absorp tion signal was feeding again to the p rogram and the p rocess is repeated successively until optimum conditions were obtained similarly to those obtained by the univariate method. S toichiometry of the complexes To establish molar ratio between Cimetidine and Erythromycin ethy lsuccinate with BTB, Job's method of continuous variation has b een used (Fi gure 4). The r esults showed that 1:1 comp lexes were formed with BTB through the electrostatic attraction between the p ositive p rotonated Cimetidine and Erythromy cin ethy lsuccinate with the anion of BTB [25, 26] . The formation of the ion-pair comp lex can be represented by taking Cimetidine as an examp le (Scheme 3): IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 Scheme 3: Prop osed reaction p athway between Cimetidine and BTB. Calibration graphs Emp loying the exp erimental conditions, linear calibration graph for Cimetidine and Erythromy cin 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 µ g. mL -1 for Cimetidine and Erythromy cin ethy lsuccinate resp ectively. S pectral characteristics of the propose d methods Accordin g to the op timum exp erimental conditions of the p rop osed methods, the regr ession p lots showed linear dependence of absorban ce signals on the con centrations of t he st udied drugs in the range given (Table 6). The regression equations, correlation coeff icients, molar absorp tivities, detection limits and sandell sensitivities in addition to other p arameters are given in Table 5. Accura cy and precisi on The accuracies of the p rop osed methods were confirmed by analyzing three r eplicate analyses of t hree different amounts of each drug (within Beer's law) by calculating the relative error percentage (Table 7). T he results indicated good accuracies of the method for both cited drugs. The p recision was determined in each case by calcu lating the p ercentage relative st andard deviation (RSD %) for three determin ations at each of the st udied concentration level and were found to be in the range of 1.158-2.003% and 0.173-2.276% for C imetidine and Ery thromy cin ethy lsuccinate resp ectively. The p rop osed method was comp ared st atistically 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 Inte rferences S tudy The results showed that no interferences were found in the p resence of 1000 μg of the st udied excipients (lactose, sucrose, st arch, glucose, magnes ium st earate and sodium citrate) in the determination of Cimetidine and Erythromy cin ethy lsuccinate (Table 9). Anal ysis of dosage forms It is evident from the aforementioned results that the prop osed method gave satisfactory results with the investigated dru gs. T hus, t heir p harmaceutical dosage forms were subjected to analysis of their contents of the active ingredient by the p rop osed method (ion-p air formation). The results given in table 8 were satisfactory . The recommended method was st atist ically comp ared with official, st andard 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). Re ferences 1. Slobodan, R. (1999) Drug Interactions of H2-Recep tor Antagonists Involvin g Cytochrome( P450 -CYPs) Enzymes from the Laboratory t o the Clinic, Journal of the Croatian M edical, 40( 3, 1):4 2. Alwan, Ala , dine, A. S. and Abou, Yousif, Z.(1990) Iraqi Drug Guide, 1 st edition, NBSD , Iraq, 40-41,100-102, 115. 3. Kanfer, I.; SKiner, M . F. and Walker, R. B. (1998) Analy sis of macrolide antibiotics , Journal of Chromatogr aphy A, 812 (1/2): 255-286. 4. Girish-Kumar K. and Karp aselvi, L. (1994) Determination of cimetidine in p ure form and in dosage forms using N,N-dibromo,dimethy lhy dantoin Journal of Analyst,119(6):1375-1376. 5. Griessmann , K.; Kaunzinger, A.; Schubert-Z silavecz M .and Abdel-Tawab,M . (2007) A rapid HPLC-UV method for the quantification of ery thromy cin in dermatologica l p rep arations, Journal of Pharmazie,Die, 62 (9) :668-671. 6. Kelani, Km.; Az iz, Am.; Hegazy , M a. and Farrah, La. (2002) Determination of cimetidin e, famotidine, and ranitidin e hy drochloride in the p resence of their sulfoxide derivatives in p ure and dosa geforms by high-p erformance thin-layer chromato graphy and scanning densitometry , Journal of AOAC int, Sep -Oct,85(5):1015-1020. 7. Deubel, A.; Frand ino, AS.; Sor gel, F. and Holzgggrabe, U. (2006) Determination o f erythromy cin and related substances in commercial samples using liquid chromatogr aphy /ion trap mass sp ectrometry , Journal of Chromatogr A.; Dec; 1136(1):39-47. 8. Thanh, Ha. PT.; Van Schpdael, A.; Rots E. and Hwgmartens,J (2004) Invest igating th e p otential of erythromycin and derivatives as chiral selector in capillary electrop horesis Journal of Phrm. Biomed anal. M ar 10 34(5): 861-870. 9. Luo, J-W.; Chen, H-W. and He, Q-H. (2001) Originals - Electrop horesis - Determination of Cimetidine in Human Plasma by Use of Coup led-Flow Injection, Solid-Phase Extraction, and Capillary Zone Electrop horesis, Journal of Chromatographia., 53:295-300. 10. Jennifer, S. ; Ridlen ; David, R. Skotty ; Peter T. Kissinger and Timothy A. Nieman (1997) Determination of ery thromy cin in urine and p lasma using microbore liquid chromatogr aphy with tris(2,2′-bipy ridyl)ruthenium(II) electrogenerated IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 chemilumin escence detection, Journal of Chromatography B, Volume 694, Issue 2, 4 July , Pages 393-400. 11. Dabrowska, D.; Regosz , A.; Piekos, R.; M ierzwa, M .;and Paruch, B.(1990) A st udy of ion-pair formation between erythromy cin and bromothy mol blue, methy lthy mol blue, and thy mol blue and their use for assay ing erythromy cin in dosage forms, Journal o f microch emical, 41, 210-218. 12. Darwish, Ia; Husein, Sa. ; M ohmoud Am. And Hassan, Ai.(2008) Sp ectrop hotometric determination of H (2)-recep tor antagonists via their oxidation with cerium (I V) , Journal of Sp ectrochim Acta a M ol Biomol Sp ectrosc; Jan; 69(1): 33-40. 13. Virginata, de L. M . Finete; M arcia, Aissawa and Ricardo, Q. Aucelio (2008) Fluorimetric method for the determination of erythromycin using a p hotochemical derivatization approach, Journal of Beazilian Chem. Soc. volume 19(7) : 1418-1422. 14. sp endley,W.; Hext, G.R.; and Himusworth, F.R.T . (1962) application of Simp lex designs in op timisation and evolutionary Sequential op eration, Journal of Technometrics. 4: 441-462. 15. Nelder, J. A.; and M ead, R. A. (1965) Asimp lex M ethod for Function M inimization, 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.; M organ, S. L.; and Demin g, S. N. (1991) Sequential Simp lex Op timization, 1 st , CRC Press,Inc., Boca Raton, Florida, 44. 18. M omenbeik, F.; M omeniz, Z. and Kharasani, H., J.,(2005) Separation and determination of Vitamins E and A in multivitamin sy rup using micellar liquid chromatogr aphy and simp lex op timization, Journal of Pharmaceutical and Biomedical Analysis, , 37( 2): 383-387. 19. M urillo Pulgarn, J.A.; Alanon M olina, A.; Alanon Pardo. M .T.(2002) The use of modified si mplex method to optimize the room temp erature p hosp horescence variables in the determination of an antihyp ertensive drug, Journal of Talanta, 57, 795-805. 20. Tinoi, J.; Rakariy atham, N. and Demin g, R.L. (2005) Simplex op timization of carotenoid p roduction by Rhodotorula glutinis using hy drolyzed mung bean wast e flour as subst rate, Journal of Process Biochemistry 40, Issue 7: 2551-2557. 21. Basavaiah, K.; and Shakunthala,C., V.(2004) Ion-p air Complexometric Determination of Cyp roheptadine Hy drochloride Using bromophenol blue, Journal of ScienceAsia, 30 :163-170. 22. Julic, M .; Cardso, S. C. (2005) Sp ectrop hotometric determination of oxiconazole in top ical lotion using methy lorange, Journal of Pharmaceutical and Biomedica l Analysis, 37, Issue 4, 1 : 639-642. 23. Siddap p a, K.; M allikarjun, M .; Reddy T. and Tambe M . (2008) Simp le and Sensitiv e Extractive Sp ectrop hotometeric M ethod for the Assay of M ebeverine Hy drochloride in Pure and Pharmaceutical Formulations Journal of the Chinese Chemical Society , 55: 1062-1068. 24. Basavaiah, K.; Prameela, H. C. ; Somashek ar, B. C. (2007) Sp ectrop hotometric determination of p efloxacin mesy late in p harmaceuticals, 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 Ay ad M agda, M . (2002)Colorimetric methods for the determination of some tricy clic antidepressant drugs in their p ure and dosa ge forms), Journal of M icrochim. Acta, 140:175-182. 26. .Shah, J. ; R asul, M . and M anzoor S. (2008) Extractive Sp ectrop hotometric M ethods for Determination of Clarithromy cin in Pharmaceutical Formulations Using Bromothy mol Blue and Cresol Red, Journa l of the Chinese Chemical Society 55(5): 1107-1112. 27. Darwish, Ia; Husein, Sa. ; M ohmoud Am. And Hassan, Ai.(2008) A Sensativ e Sp ectrop hotometric method for the determination of H2- receptor antagonists by means of N- brmosuccin imide and P- aminophenol, J. of Acta Pharm. 58: 87-97. 28. Darwish, Ia; Husein, Sa. ; M ohmoud Am. And Hassan, Ai.(2007) Sensativ e Sp ectrop hotometric method for the determination of H2- receptor antagonists in Pharmaceutical Formulation, International J. of Biomed ical Science, 3(2): 123-130. 29. Helali , N. and M onser, L.(2006)Simultaneous Determination of Cimetidine and Related Compounds in Pharmaceuticals by HPLC on a PorousGraphitic Carbon Column, J.of chromamatogr aph:a 63(9-10): 425-430. 30. Kelly , MT .; M cGuirk, D. and Bloomfield, FJ.(1995) Determination of cimetidine in human p lasma by high-p erformance liqu id chromato graphy following liquid-liquid extraction J Chromato gr B Biomed App l. Jun 9;668(1):117-23. 31. Kelani, K. M . ; AZ IZ, A. M . ; Hegazy , M . A. and Laila, Abde l F. (2002), Different sp ectrop hotometric methods for the determin ation of cimetidine, ranitidine hy drochloride, and famotidine. J. of Sp ectroscopy letters 35, n o 4: 543-563. 32. Ratt ay a, R.; Wachiranee, V. ; Worap ot S. and Leena, S. 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Khashaba (2002) Sp ectrofluorimetric analysis of certain macrolid e antibiotics in bulk and p harmaceutical formulations, Journal of Pharmaceutical and Biomedical Ana lysis, 27, Issue 6: 923-932. 37. Zhao Yanqin g, Li Hua M cCain, Gui-Zhi Zhao(2005) Sp ectrop hotometric determination of erythromycin ethy lsuccinate based on the char ge transfer reactio n between erythromy cin ethy lsuccinate and quinalizarin, J. of China M odern App lied 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) Sp ectrop hotometric Determination of Erythromy cin Ethy lsuccinate with Charge Transfer Reaction between Erythromy cin Ethy lsuccinate and Alizarin, J. of Spectroscopy Laboratory , 21(06): 1115-1116. 39. British Pharmacop eia (1998), CD-ROM Her M ajesty , s Stationary office, London. 40. British Pharmacop eia (2002), CD-ROM , BP, Co.-UK. Table(1): Effect of shaking time on e xtraction of 5 µg.mL -1 Ci mitidine; (0.038%BTB, pH5.5) and 10µg.m L -1 Erythromycin ethylsuccinate (0.02%BTB, pH 4) M ixing Time/mint Absorbance Cimetidine Absorbance Erythromy cin 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 e thylsuccinate Organic p hase Absorbance of Drug-BTB ion pair comp lex Cimetidine Erythromy cin ethy lsuccinate 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 conditi ons for the studied variable s Variable Range for Cimetidine Range for Ery thromycin p H 5.0-8.5 3-6 Reagent Conc.(%) 0.01-.038 0.01-0.025 Shakin g 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 (Si mplex) for determination of Cimitidine Exp . No. p H %Reagent Conc. M ixing 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 (Si mplex) for determinati on of Erythromycin ethylsuccinate Exp . No. p H %Reagent Conc. M ixing 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): S pectral characteristi cs and statistical data of the regression equati ons for determination of Ci metidine and Erythromycin e thylsuccinate using i on–pair formation Parameter Cimetidine Erythromy cin ethy lsuccinate λm ax (nm) 427.5 414.5 Color Yellow Yellow Linear ity range (µg.mL -1 ) 0.5 – 15.0 0.5 – 50.0 M olar absorp itivites (l.mol -1 .cm -1 ) 13172 18103 Regr ession 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 Lin earity (R 2 ) 0.9970 0.9985 Correlation coeff icient (R) 0.9984 0.9992 Detection limit (µg. mL -1 ) 0.222 0.286 Table (7): Evaluati on of accuracies and precisi ons of the two propose d 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 Erythromy cin 5 4.921 -1.580 2.276 20 19.858 -0.710 1.695 40 40.272 +0.680 0.173 Average of three determin ations* IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 Table (8): S pectrophotome tric determination of Cimetidine and Erythromycin Ethyl succinatein pharmaceutical compounds using Ion –Pair Formation Samp le Amount (mg) Concentration (µg.mL - 1 ) Relative Error % R.S.D.* % Labeled Found taken Found* Tagadin e (Cimetidine) 200mg/ tablet SDI/Iraq 200 204.840 5 5.121 +2.400 0.937 200.760 10 10.038 +0.380 0.508 Cimedne R (Cimetidine) 200mg/ tablet DAD Jordan 200 180.320 5 4.508 -9.840 0.615 179.820 10 8.991 -10.090 0.316 Hist ale (Cimetidine Hy drochloride) 200mg/Amp oule IBH/Sy ria 200 199.000 5 4.975 -0.500 0.582 198.840 10 9.942 -0.580 1.579 Erythrosam (Erythromy cin 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 (Erythromy cin 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 (Az ithromy cin dehydrate) 250mg/ tablet R.P/Egy p t 250 249.600 10 9.984 -0.160 1.202 246.588 20 19.727 -1.365 0.487 Erythromy cin Stearate (p ure p owder) SDI/Iraq 250 249.200 10 9.968 -0.320 1.204 246.375 20 19.710 -1.450 0.639 *Average of three determin ations IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Table (9): Percent Recovary for 10 µg.m L -1 of Ci metidine and 20 µg.mL -1 of Erythromycin Ethyl succinate in the presence of 1000 µg.mL -1 of Excipie nts Excipients Cimetidine Con c. Taken10 µg.mL -1 Erythromy cin Ethy lsuccinate 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 st arch 9.932 99.320 19.857 99.285 Glucose 9.929 99.290 19.857 99.285 M agnesium Stear ate 9.935 99.350 19.839 99.195 Sodium C itrate 9.933 99.330 19.843 99.215 Average of three determin ations* Table(10): Anal ytical Parameters for the anal ysis of Cime tidine by the propse d and othe rs methods Ref. No. methods Linear range µg.mL -1 ε L.mo l . - cm - 1 Correlation Coefficient ( R) Recoveri es range% RS D% 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.T L.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): Anal ytical Parameters for the anal ysis of Erythromycin Ethyl succinate by the propse d and others methods Ref. No. methods Linear r ange µg.mL -1 εL.mo l . - cm -1 Correlation Coefficient ( R) Recovery % RSD% - Prop osed method 0.5-50 18103 0.9992 98.4- 100.6 0.17-2.27 32 Sp ectrop hotometric 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 Sp ectrop hotometric Ion-Pair 2-61 - - 98.4- 103.6 1.4-4.4 33 high-p ressure 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 Sp ectroflurimetric 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- Value s for analysis of Ci metidine in Pharmaceytical Copmpounds Cimetidine Prop osed M ethod, (S.D.I) T-Values a F-values b Ot her M ethods (N=5) S.D Ref. No. Ion-Pair 0.671 157.692 Official 0.640 27,39 N=3 0.567 357.692 Ot her 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 valu es for F-t est at 99.5% (199.25), 99.9%(999.25) and 95% (19.274) confidence limit resp ectively, were N=(4,2). Table(13): t- and F- Value s for analysis of Erythromycin Ethyl succinate in Pharmaceytical Copmpounds Erythromy cin Ethy lsuccinate Prop osed M ethod, (S.D.I) T-Values a F-values b Ot her M ethods 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)resp ectively b Theoretical valu es 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 resp ectively. IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 Fig.(1): Absor ption spectra of (A) 4 µg.m L -1 Ci metidine-BTB ion-pair complex, (B) 10 µg.mL -1 Erythromycin e thylsuccinate-BTB ion-pair complex. Fig.(2): Effect of pH on the Absor bance of: (A) 5 µg.m L -1 Ci metidine; 0.04% BTB. (B) 10 µg.mL -1 Erythromycin e thylsuccinate ; 0.04 % BTB. Fig.(3): Effect of Reagent Concentration on the Absorbance of: (A) 5 µg.m L -1 Ci mitidine pH 5.5, (B) 10 µg.mL -1 Erythromycin e thylsuccinate pH 4. IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI VOL. 23 (3) 2010 Fig. (4): Continuous Variati on 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 e thylsuccinate; underder optimum e xperimental 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 ظم امتصاص لها عند االطوال المتكونة للسمیتیدین واالرثرومایسین ایثایل سوكسینیت اعلقد اظهرت المعقدات . على التوالي 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