الصرفة والتطبیقیة 200) 4(2 2 المجلد مجلة ابن الھیثم للعلوم 9 تطویر طریقتین طیفیتین لتقدیر االمتربتلین في المستحضرات الصیدالنیة باالعتماد على دراسة المحددات أحادیا وبطریقة السمبلكس للوصول إلى الظروف الفضلى عالء كریم محمد ة التربیة ابن الهیثم،جامعة بغدادقسم الكیمیاء،كلی الخالصة متـربتلین بـشكله النقـي وفـي األقـراصان وبـسیطتان سـهلتان طیفیتـا طریقتت اقترح ى .ن لتقـدیر اال تعتمـد الطریقـة األولـى علـ بـ قــال شـحنة بــین االمتـربتلین واه ین اتكـوین معقـد انت تتراســیانواثل يمثـل لاللكترونــات مـع ستقبل بــا تفاعـل أقــصى اظهـر نــا.مـ تج ال وفـي الطریقـة الثانیــة . 9 نــانومیتر فـي مـذیب االسـیتونتریل عنـد دالــة حمـضیة مقـدارها 470امتـصاص عنـد طـول مـوجي مقـداره ي تقیـس طـول مـوج ضر عنـد زول األخـ راسـة مـع البرومـوكری ألیـوني المتكـون مـن تفاعـل الـدواء قیـد الد امتـصاصیة معقـد االزدواج ا 3. 5 دالة حمضیة مقدارها نانومیتر عند415مقداره فـي دراسـة المتغیـرات للوصـول إلـى الظـروف ا طریقتتطبق تغیر المحددات أحادیـا التقلیدیـة وطریقـة الـسمبلكس المحـورة حـصول علیهـ.الفـضلى للتفـاعلین باسـتخدام برنـامج الـسمبلكس الهندســي ثالثـي اإلبعـاد ا مطاوعـة لقــانون أظهـرت النتـائج التــي تـم ال مـل.م مــایكرو غـرا8 -100 و 6-70 بییـر فـي مـدى تركیـز یـتراوح مـن - 1 مقــدارها یم معامـل امتـصاص مـوالي 227 وبقـ و 5 147 كانـت قـیم حـدود الكـشف مـساویة إلـى . لطریقتـي تتراسـیانواثیلیین وبرومـوكریزول األخـضر علـى التـوالي1-سـم .1-مـول . لتـر5 لم.م مایكرو غرا0.034 و0.043 -1 سـم.م مـایكرو غـرا0.188 و 0.122 وقـیم حـساسیة سـاندل هـي -1 . للطـریقتین علـى التـوالي . ن بنجاح لتقدیر االمتربتلین في أقراص دوائیة من مناشى مختلفة وكانت دقة وتوافق النتائج مقبولةاطبقت الطریقت IBN AL- HAITHAM J. FO R PURE & APP L. SC I. VO L. 22 (4) 2009 Development of Two New Spectropho tometeric Methods for the Determination of Amitriptyline in Pharmaceutical Preparation Using Univariate and Simplex Optimization A. k . Mohamme d De partment of Chemistry, College of Education, Ibn Al-Haitham University of Baghdad Abstrac t Tw o simp le and sens itive sp ectrop hotometric methods are prop osed for the determination of amitripty line in its p ure form and in t ablets. T he first method is based on the formation of charge- transfer comp lex between amitrip ty line as n-donor and tetracyano-ethy lene (T CNE) as π- accep tor. T he p roduct exhibit absorbance maximum at 470 nm in acetonitrile solv ent (pH =9.0 ) . In the second method the absorbance of the ion- p air comp lex, which is form ed between the sought ed drug and bromocresol green (BCG), was measured at 415 nm at ( p H=3.5) . In addition t o classical univar iate optimization, modified simp lex method (M SM ) was app lied in the opt imization of the variable affectin g the color p roducin g r eaction by a geometric simp lex in three dimensions of sp ace. Beers , law was obey ed in the concent ration ranges 6.0-70 and 8.0-100 µg.m l -1 with molar absorbitivites of 2275 and 1475 l. mol -1 cm -1 for TCNE and BCG methods resp ectively . T he limits of detection of t he two methods are 0.043 and 0.034 µg.ml -1 and their Sandells sensit ivity values are 0.122 and 0.188 µ g.ml-1 resp ectively . Introductio n Amitripty line is chem ically 3-(10,11-dihy dro-5H-dibenz o[a,d]cy cloheptene-5-y lidene)- N,N-dimethy l-1-p rop anamine, a tricy clic antidepressant drug, widely used for treating clinical dep ression, neuropathic pain , nocturnal enuresis, and attention-deficit hyp eractivity disorder (ADHD) [1] , but it has also been used successfully for head ache, anxiety , smoking cessation, bulimia nervosa, p ersist ent hiccups and as an adjunct in schizophrenia [2]. The vital imp ort ance of this drug p romp ted the develop ment of various analytical methods for its determination , these methods include capillary electrop horesis [3,4] high p erformance liquid chromatography [5,6], gas chromato grap hy [7,8], p otentiometric [9,10] , chemom etric [11], and spectrop hotometry [12-18]. π-accep tors such as 2,3-dichloro-5,6-dicyano-p -benz o-quinone(DDQ), 7,7,8,8- tetracy anoquino-dimethane(T CNQ), tetracy ano-et hylene(TCNE), 2,4,7-trinitroflurene-9- one(TNF), 2,5-dihydroxy -3,6-dichloro-p -benz oquinone( p - chloranilic acid) are known to yield charge transfer comp lexes and r adical ions with a v ariety of electron donors such as cephalosp orins [19] , gabap entin [20] , lor atadine [21] and ranitidine [22] . On the other hand, ion- p air extraction sp ectrop hotometry has been received aconsid erable attention for quantitative est imation of p harmaceut ical comp ound . Bromop henol blue (BPB), methylene blue (M B) , bromocresol p urp le (BCP) and thy mol blue (T B) were widely used as ion- pairing reagents for the quant itative analy sis of many pharmaceutical co mp ounds [23-25] . IBN AL- HAITHAM J. FO R PURE & APP L. SC I. VO L. 22 (4) 2009 In exp eriment al chemist ry , the op timization of technical syst ems is the p rocess of adjust ing the control var iables t o find t he levels that achieve the best opt imization. Usually , many conflictin g r esp onse must be optimized simultaneously. In lack of syst ematic ap p roaches the opt imization is done by “ trial-and- error”, or by chan gin g on e cont rol var iable at a time while holdin g the rest const ant, such methods requires a lot of exp eriment s to be carried out. Simplex opt imization of exp eriment al p arameters was first introduced by Spendley [26] and then modif ied by Nelder [27] and A ber g [28] . T he method found a lot of ap p lications in the felid of analytical chemistry [29-32] , because it offers the cap ability of opt imizing several factors simultaneously depending on a statistical design search to find t he maxima or m inima of response, by rejectin g the point p roducing the worst response and a replacement of it by the new p oint which is obt ained statistically. The p resent work est ablished an imp roved sp ectrop hotometric method for the determination of amitrip tyline by exp loiting its basic nature and e lectron- donatin g p rop erty. The determination is based on charge- transfer reaction with TCNE and ion – association comp lexation with Bromocresol green (BCG) and the optimization of chemical sp ectrop hotometric variables of the p roposed methods namely p H, reagent amount and reaction time were studied by using bot h class ical univ ariate and modified simplex. The multivariate simplex op timization was carr ied out via comp uter p rogram [33] adapted to fit p ersonal micro- comput er. Experime ntal Apparatus: A Shim adz u ( model 1601 UV- visible sp ectrop hotometer from Shimadzu, Koy oto, Japan ) with 1cm glass cells was used for absorban ce measure ments.pH – meter model PW-9421 from Philip s was used for all pH measurements . M aterials and Reagents All chemical used were of analytical r eagent grad e unl ess ot herwise- amitrip ty line- hy drochloride st andard p owder materials (p urity 99.8%) were provided from the State Company for Drug Industries and M edical App liances S amar a-Iraq ( SDI). 1- TCNE 1.6x 10 -3 M solution, p repared by dissolving 20.5 mg of the reagent in 100 ml of acetonitrile by using volu metric flask. 2- BCG 1x10 -3 M solution p rep ared by dissolving 36.0 mg of the reagent in 50 ml dist illed water by using volum etric flask. 3- Pht halate buffer (p H=3.60).To 250 ml of 0.2M potassium hy drogen p hthalate 11.90 ml of 0.2M HCl was added and then the solution was diluted to a final volum e 1000m l with dist illed water [34] . Standard a mitriptyline solutions: 1- Solution for TCNE p rocedure (250 µg.ml 1- ): 50 m g of a mitriptyline base was dissolved in 50 ml of methanol, then the solut ion was made alkaline (p H=9.0) with a drop wise of 0.2N sodium hy droxide.The solution was quantitatively transferred into a sep arating funnel and shaken with four 10 ml p ort ions of chloroform.T he extracts were p ooled by filtration through a filter p ap er cont aining anhy drous sodium sulp hate into a 200 ml volumetric f lask and diluted to volum e with chlorofor m. This was diluted to get a working concentration of 100 µg/ml . IBN AL- HAITHAM J. FO R PURE & APP L. SC I. VO L. 22 (4) 2009 2- Solution for BCG procedure (250 µg. ml -1 ) : 50 mg of amitrip tyline base was diss olved in 200 ml of methanol by using vo lumetric flask. Procedures: Calibration graphs 1- TCNE me thod: Serial vo lumes of standard solution ran ging fro m 0.60 to 7.0 ml wer e transferred t o 10 ml volumetric flasks , then 1.50 ml of T CNE reagent was add ed, and allowed to stand for 30 min at 25 0 C and t hen diluted to volume wit h acetonitrile. The absorbance was measured at 470nm against reagent blank. 2- BCG me thod: Serial volumes of standard solution rangin g 0.32 to 4.0 ml were transferred individually into 25 ml sep arating funnel, then 1 ml of BCG solution and t hree ml p hthalate buffer (p H= 3.6 ) were added.T he for med comp lex was extracted for 2 min with two 5 m l p ortions of chloroform. The extracts were p ooled by filtration through a filter p ap er cont aining anhy drous sodium sulp hate int o a 10 ml volum etric flask and di luted to volume with chlorofor m, and then the absorbanc e was measured at 415 nm against reagent blank. Procedure for the dete rminati on of amitri ptyline in pharmaceutical preparati on: TCNE me thod: T en tablets were fine ly p owdered and m ixed .An accur ately weighed qu antity equivalent to the drug base conc entration ment ioned in the standard solution p reparation was dissolved by shaking with 50 ml d ist illed water . The solution was made alkaline (pH=9.0) with a drop wise addition of 0.2N sodium hy droxide. The resulted solution was quantitatively transferred into a sep arating funnel and shaken with four 10 ml p ortions of chloroform. T he extracts were p ooled by filtrat ion through a filter p ap er cont aining anhy drous sodium sulp hate into a 200 ml volumetric f lask and di luted to volum e with ch loroform. The p rocedure was cont inued as described under t he p rep aration of calibration grap hs [24]. BCG me thod : Ten tablets were finely p owdered and mixed .An accurately weighed quant ity equivalent to t he drug base concentration, ment ioned in the st andard solution p reparation, was dissolved in 200 ml of methanol by using volumetric flask .The p rocedure was continued as described under the prep aration of calibration gra ghs [24]. Results and Discussion Charge transfer complexation: The reaction of am itrip ty line with TCNE in acetonitrile solvent result s in t he formation an intense red brown color comp lex, which exh ibits an absorp tion maximum at 470 nm (Fig 1). This absorption band formed is the results of the formation of charge-transfer comp lex through the interaction of T CNE as a π - a ccept or and the st udied drug as n-donor fo llowed by the form ation of colored rad ical anion accord in g to t he following sch eme [22,24,35,36]: IBN AL- HAITHAM J. FO R PURE & APP L. SC I. VO L. 22 (4) 2009 Formation of a radical anion in such molecular interactions was confirmed by electron-sp in resonance measurement s [37]. O ptimization of experi mental variable s: i. Univariate me thod Effect of p H: The effect of p H on the develop ment of t he colored comp lex between amitripty line and TCNE is shown in (Fig 2). The p H being adjusted with few drop s of 0.1 M HCl and 0.1 M NaOH. M aximum and constant absorbance were obtained in the p H range 9.0-9.5.The absorbance de creased at p H value above 9.5 and below 9.0. H ence a p H of 9.0 was used in all the subsequent exp erimental work. Effect of reage nt: Various vo lumes of TCNE solution were add ed to 40µg.m l -1 of amitrip ty line solution. 1.5 ml of 1.6 x 10 -4 M of TCNE was found to be enou gh t o develop the co lor to its full intensity and was considered to be the op timum for the concentration ran ge of amitrip tyline 6.0-75 µg.ml -1 (Fig 3). Effect of reacti on time: The color intensit y reached a maximum after the amitrip ty line was reacted with T CNE for 30 minutes (Fig 4), therefore 30 m inutes develop ment time was selected as op timum in the gen eral p rocedure . The color obtained was st able for at least 2 hours . S toichiometry of the complex : The stoichiometry of the reaction between amitriptyline and TCNE was st udied by mole ratio method (Fig 5). The results obtained shows that 1:1 amitripty line to TCNE was formed at 470nm, therefore the formation of t he comp lex can be r ep resented as in followin g scheme [22,35] : IBN AL- HAITHAM J. FO R PURE & APP L. SC I. VO L. 22 (4) 2009 ii. Simplex optimization: To set the simplex op timization of the three st udied variables, four exp eriment al conditions should be chosen involve values for p H, reagent volume and st anding time. The values of the four exp eriment al condit ions were sel ected with sp ecific boundar ies for each at which it affects the absorpt ion s ignal of t he color ed p roduct (t able1). The absorbencies of these four initial exp eriments were measured and the results were feed to the comput er p rogram. The program then starts simp lex by searchin g the worse absorption signal and ref lects it in ahyp er-p lane of the remaining p oints t o p roduce a new set of exp erimental conditions, which were app lied to carry out the exp eriment and the measured absorption signal was feeded again to the p rogram. The p rocess is rep eated successively unt il opt imum conditions wer e obt ained (i.e. cond itions yieldin g h ighest absorpt ion signal). The p rocedure is cont inued for furt her few exp eriments to ensure that the opt imum conditions are reached (T able 2 and Figure 6). Values of the results obtained by app ly ing simp lex program are shown in Figure 7. Calibration graph: Employ ing the experimental conditions describ ed under p rocedure, a linear calibr ation graph for amitrip tyline is obtained (Fig 8), which shows that Beers law was obey ed in the concentration ran ge 6.0-70 µ g.m l -1 . Ion-pair com ple xation: The amitripty line solution reacted with BCG solution in aqu eous solution in acidic medium to form a yellow color ion p air complex, whi ch exhib its an absorp tion maximum band at 415nm against reagent blank (F ig 9). O ptimization of experi mental variable s: IBN AL- HAITHAM J. FO R PURE & APP L. SC I. VO L. 22 (4) 2009 i. Univariate me thod: Effect of p H: In order to est ablished the opt imum p H range , amitrip ty line solution was mixed with asp ecified volu me of BCG , and then the p H was adjusted t o a value between 2.0-6.0 with a few drops of 0.1N NaOH or 0.1N HCl .M aximum and constant absorbances were obtained in the pH range 3.5-4.0 (Fig 10) . The absorban ce was de creased at p H value above 4.0 and below 3.50. Hence a pH of 4.0 was used in all the subsequent exp erimental work. Effect of reage nt: The influence of excess reagent con centration on the absorbance of the comp lex is illustrat ed in (Fig 11). One ml of 1.6x10 -4 M solution of BCG was found enough to develop the color and reached its maximum intensity . Effect of s haki ng time: The op timum shaking time for the comp lete extraction of the ion pair comp lex with chloroform was st udied from 30 s econd to 3 minutes (Fig12). It was found t hat the minimum shaking time for comp lete extraction w as 2 minutes at room t emp erature (25± 1 C o ) . S toichiometry of the complex: The stoichiometry of t he reaction between amitrip ty line and BCG was st udied by mole ratio method (Fig13). The results obt ained shows that 1:1 amitrip ty line to BCG was formed at 415 nm, therefore the formation of the comp lex can be rep resent ed as in following scheme [24,25] : IBN AL- HAITHAM J. FO R PURE & APP L. SC I. VO L. 22 (4) 2009 ii. Simplex optimization: The same st ep s of simp lex p rogram, within t he given boundary condit ions (T able 3), were followed to op timize the exp eriment al conditions. Results are shown in Figure 14, 15 and Table 4. Calibration graph Employ ing the experimental conditions, a linear calibration gr ap h for amitrip ty line is obt ained (Fig 16), which shows that Beers law was obeyed in the concent ration ran ge of 8.0-100 µg.ml-1. S pectral characte ristic of the two propose d methods: Under the opt imum exp erimental cond itions of the two prop osed methods, the regression p lots show ed t hat there were linear d ep endence of absorbance sign als on the con centration of the drug in the ranges given in Table 5. The regression equations and correlation coefficient s, which were obt ained by the linear least –squares treatment of the result s in addition to the molar absorptivites, detection limits , and Sandell sensitivities are given in T able 5. The accuracies of the two p roposed methods were est ablished by p erforming seven rep licate analyses on st andard solutions containing three different amount s of drug and calculatin g the p ercentage error. The precisions were determined by calculatin g the relative st andard deviations (RSD) for seven determinations at each level Table 6. It is clear from the results that at all of the three studied levels , the values of the mean ( ) were less than the values of indet ermin ate error ( ), indicating that no si gnificant differ ences existed between the mean and t he true values. Table 7 shows that the two p rop osed methods have accept able linear ity ranges, acceptable p recisions and accura cies when they compared with ot her methods. Analyti cal application: The p rop osed methods were app lied to determine amitripty line in p harmaceutical p reparation tablets. T he results, p resented in T able 8, reveal t hat the recover ies were in the ran ge of 94.56 to 100.20 %, r eflecting hi gh accur acies and p recisions of the p rop osed methods as indicated by low RSD values. References 1- Biederman,J. and Spencer ,T . (1999). Biol Psychiatry , 46(9) :1234-42. 2- M ico,J.; Ardid,D. ; Berrocoso,E. and Eschalier, A. (2006) T rends Pharmacol Sci , 27(7 ): 348-54. 3- Kou,H .S. ; Chen,C.C. ; Huang,H.Y. ; Ko,W.K . ; Wu,H.I. and Wu S.M. (2004) Analyt ica Chimica Acta, 525(1): 23-30. 4- Wu,S.M . and Chen,S.H. (2000).Analy tica Chimica Acta, 443(1-2) :125-129. 5- Yoshida ,H. ; Hidak a ,K. ; Ishida ,J. ; Nohta ,H. and Yamaguch i , M . (2000).Analytica Chimica Acta, 413(1)18 M ay : 137-145(9). 6- Linden,R.; Ant unes, M .V .; Z iulkoski,A.L.; Win gert, M .; Tonello,P. ;Tzvetkov M . and A.A.Souto.(2008). J .Braz.Chem.Soc. 19(1): 34-41. 7- Ulrich,S. ; Isensee,T. and Pest er U. (1996). Journal of Chromatograp hy .B:Biomed ical App lication, 685(1): 81-89. 8- F eng, S.; Sun, J .; Liu, Y. ; Li,Y. and W an g, F. (1996).Yaowu Fenxi Zazhi 16(2):75-78. IBN AL- HAITHAM J. FO R PURE & APP L. SC I. VO L. 22 (4) 2009 9- El-Nashar,R.M .; Abdel Chani, N.T . and Bioumy ,A.A. (2004) M icrochemical Journal, 68(2):107-113. 10- Wan g,C.Y. ; Hu,X.Y. ; L en g,Z.Z and Jin ,G.D. (2003) .Electroanalysis, 15(8):709- 714. 11- Markopoulou,C.K. ; Malliou,E.T . and Koundourellis, J.E.(2005)J ournal of Pharmaceutical and Biomed ical Analy sis, 37(2) :383-387. 12- Karp inska,J. and Szostak, J. (2005).Spectrochim Act a A M ol Biol Spctrosc, 61(5): 975-81. 13- Mohamed ,G.G .; EL-Dein, F A.; M ohamed ,NA. (2007). Spectrochim Acta A M ol Biomol Spectrsc. 31:68(5): 1244-9. 14- Misiuk,W. and Tarasiewicz, M. (1998). Analytical letters, 31(7) : 1197-1207. 15- Aman,J. ; Kaz i,A.A. ; Hussain,M .I ; Firdous,S. and Khan ,I.U. (2000). Analytical letters, 33(12) : 2477-2490. 16- Misiuk,W. and Puz anowska-Tarasiewicz ,H. (2002). Analytical lett ers, 35(7):1163- 1170. 17- Karp inska,J. and Suszynska ,J. (2001). Journal of T race and M icroprobe Techniques, 9(3): 355-364. 18- Onah ,J.O. (2005). G lobal Journal of Pure and App lied Sciences, 11(2) : 237-240. 19- Gamal,A. ; Hussan,F . ; Ibrahim,A. and Abde l-Nasser A. (2003). Analytical sciences, 19 : 281-287. 20- Salem ,H. (2008) .African Journa l of Pharmacy and Pharmacolo gy , 2 (7):136-144. 21- Basavaiah ,K . and Ch aran , V.S (2002). Science Asia 28 : 359-364. 22- Walash,M .; Sharaf- EL-Din, M .; M etawalli ,E. S and Red ashabana ,M. (2004).A rch Pharm Res, 27(7): 720-727. 23- Betello ,J.C. and Perez, G. (1995). T alanta, 42(1): 105-108. 24- Elham, A.; Suzan,M . ; Hisham ,E. and M agda, M . (2002). Microchim .Acta, 140: 175-182. 25- Basavaiah ,K . and Ch aran, V.S. (2004). Scienc e As ia, 30 : 163-170. 26- Sp endely ,W. ; Hext ,G.R. and Himsworth F.R. (1962). T echnometrics, 4 : 441-461. 27- Nelder ,J.A. and M ead ,R.A (1965). Comp uter Journal, 7 : 308-313. 28- Alberg ,E.R. and Gustavsson A.G. (1982).Analytica Chimica Acta, 144: 39-53. 29- Sultan,S.M .; Suliman, F.E. ; Duffuaa S.O. and Abu-Abdoun ,I.I. (1992).Analyst, (7): 1179-1186. 30- Sultan ,S.M . and El-M ubarak ,A.H. (1996). Op timized by the modified simplex p rocedure, Talant a, 43 : 569-576. 31- Momenbeik,F. ; M omeniz ,Z. and Kharasani ,J.H. (2005). Journal of Pharmaceut ical and Biomedical Analy sis, 37(2) : 383-387. 32- Pulgar in,J.A.; Molina ,A. and Pardo ,M .I. (2002). Talanta, 57 : 795-805. 33- Wade,A.P. Universit y college Swansea, Wales, cited in S.Al-Najafi.(1984) .T hesis, university of Wales, university college of Swanse a 34- Vo gel,I. Atext books of macro and semimicro quant itative in or ganic ana lysis (1959).4 th .Edn.p age :645. 35- Abdellatef ,H.E. (1998). Journal of Pharmaceut ical and B iomed ical Analy sis, 17: 1267-1271. 36- Al-Ghannam ,S. and Belal ,F. (2002) J.AO AC Int , 85 : 1003. 37- Abdel Hamid, M.E.; Abdel Salam,M .;M ahrous, M .S. and A bdel-khalek, M.M . (1985). T alanta, 36 :1002-1004. IBN AL- HAITHAM J. FO R PURE & APP L. SC I. VO L. 22 (4) 2009 Fig.(1): Absor ption spectra of: (A) 40 µg.ml -1 amitriptyline, 2.4 x 10 -4 M TCNE at pH=9.0 agai nst reagent blank; (B) 2.4 x 10-4M TCNE in acetonitrile against distille d wate r. Fig.(2): Effe ct of pH on the absorbance of : 40 µg.ml -1 amitriptyline, 2.4 x 10-4 M TCNE at 470 nm Fig.(3): Effe ct of reage nt vol ume on the absorbance of 40 µg.ml -1 amitriptyline solution. IBN AL- HAITHAM J. FO R PURE & APP L. SC I. VO L. 22 (4) 2009 Fig.(4): Effect of standing ti me on the absorbance of 40 µg.ml -1 amitriptyline, 2.4 x 10 -4 M TCNE at pH=9.0. Fig.(5): Mole ratio plot of ami triptyl ine determined via charge-transfer me thod. Fig.(6): Experi mental si mplex study of variables resulting in pH=9, reagent vol ume=1.5ml, and standing time=30min. IBN AL- HAITHAM J. FO R PURE & APP L. SC I. VO L. 22 (4) 2009 Fig. (7): O ptimization of absorption signal via simplex changing of pH, reage nt volume, and standing time . Fig.(8): C alibrati on graph of amitriptyline with 2.4 x 10 -4 M TCNE at 460 nm. Fig.(9): Absor ption spectra of: (A) 45 µg.ml -1 amitriptyline, 1.0 x 10 -4 M BCG at pH=3.5 agai nst reagent blank; (B) 1.0 x 10 -4 M BCG in chloform against distilled wate r. IBN AL- HAITHAM J. FO R PURE & APP L. SC I. VO L. 22 (4) 2009 Fig.(10): Effe ct of pH on the absorbance of : 45 µg.ml -1 amitriptyline, 1.0 x 10 -4 M BC G at 415 nm Fig.(11): Effe ct of reagent vol ume on the absorbance of 45 µg.ml -1 amitriptyline solu tion. Fig.(12): Effe ct of sh aking ti me on the absorbance of 45 µg.ml -1 amitriptyline, 1.0 x 10 -4 M BCG. IBN AL- HAITHAM J. FO R PURE & APP L. SC I. VO L. 22 (4) 2009 Fig.(13): Mole ratio plot of amitriptyline determined via i on-pair method Fig.(14): Experi mental simplex study of variables resulting in pH=1.2, re agent vol ume=1.2ml, and shakin g time=90 sec. Fig.(15): O ptimization of absorption signal via simplex changing of pH, reage nt vol ume, and shaking ti me. IBN AL- HAITHAM J. FO R PURE & APP L. SC I. VO L. 22 (4) 2009 Fig.(16): Calibration graph of amitriptyline with 1.0 x 10 -4 M BC G at 415 nm. Table (1): Boundary conditi ons for the studied variable s Variable range p H 6-11 Reagent volume (ml) 0.3-3.0 Standin g time (min) 5-60 Table ( 2): Absorbance for e ach of the simplexes in the optimizati on of color producing reaction variables Table (3): Boundary conditi ons for the studied variable s Variable range p H 2-6 Reagent volume ( ml) 0.2-2.0 Standin g time (sec) 30-180 IBN AL- HAITHAM J. FO R PURE & APP L. SC I. VO L. 22 (4) 2009 Operation pH Reagent Vol ume(ml ) Time (min) Absorban ce Simpl ex 1 8.0 1.2 35 0.200 Simpl ex 2 10. 0 2.4 20 0.267 Simpl ex 3 8.5 2.1 15 0.239 Simpl ex 4 6.0 1.8 30 0.246 Simpl ex 5 9.0 3.0 5 0.212 Simpl ex 6 8.5 1.8 30 0.297 Simpl ex 7 8.0 1.8 35 0.287 Simpl ex 8 11. 0 2.1 30 0.279 Simpl ex 9 8.0 1.2 35 0.279 Simpl ex 10 10. 0 2.4 25 0.259 Simpl ex 11 8.5 1.5 35 0.310 Simpl ex 12 8.5 1.8 30 0.297 Simpl ex 13 6.0 1.2 35 0.246 Simpl ex 14 9.5 1.8 30 0.335 Simpl ex 15 10. 0 1.8 25 0.289 Simpl ex 16 9.5 1.8 30 0.335 Simpl ex 17 10. 0 1.8 30 0.311 Simpl ex 18 10. 5 2.1 25 0.259 Simpl ex 19 9.0 1.5 30 0.350 Simpl ex 20 9.5 1.8 30 0.335 Simpl ex 21 8.5 1.5 30 0.310 Simplex 22 9.5 1.8 30 0.335 Table (4): Absorbance for each of the simplexes in the optimizati on of color pro ducing re action variables Op eration p H Reagent Volu me(m l) Time (sec) Absorbance Simplex 1 3.0 1.6 90 0.203 Simplex 2 3.0 0.6 60 0.166 Simplex 3 3.5 1.4 120 0.240 Simplex 4 5.5 1.8 30 0.138 Simplex 5 2.0 0.8 120 0.180 Simplex 6 3.0 2.0 120 0.198 Simplex 7 4.5 2.0 120 0.198 Simplex 8 2.0 1.2 120 0.200 Simplex 9 3.0 0.8 120 0.207 Simp lex 10 4.5 1.4 120 0.221 Simp lex 11 4.0 1.0 120 0.250 Simp lex 12 5.0 1.6 120 0.212 Simp lex 13 4.5 0.6 120 0.193 Simp lex 14 3.5 1.2 90 0.250 Simp lex 15 3.0 0.8 120 0.207 Simp lex 16 4.0 1.2 120 0.250 Table (5): Analytical characteristics for the two methods. Parameter TCNE method BCG method linear dy namic ran ge (µg.ml -1 ) 6.0-70 8.0-100 Regr ession equation Abs= 0.008Conc.+0.027 Abs=0.005Conc.+0.007 Slop e (b) (l.m g -1 .cm -1 ) 0.008 0.005 Int ercep t (a) 0.027 0.007 Correlation coeff icient 0.9998 0.9999 M olar absorp tivity l.mol -1 .cm -1 2275 1475.50 Detection limit (µg.ml -1 )* 0.043 0.034 Sande ll sensit ivity (µg.ml-1) 0.122 0.188 *Calculated for single an alysis at 99.9 confidence limit. IBN AL- HAITHAM J. FO R PURE & APP L. SC I. VO L. 22 (4) 2009 Table(6): Evaluati on of accuracy and precision of the two methods. It em TCNE method BCG method Concentration of amitrip ty line t aken ( µg.ml- 1 ) 15.00 25.00 35.00 30.00 20.00 50.00 Concentration of amitrip ty line found ( µg.ml -1 ) * 15.03 24.85 34.75 30.13 20.24 50.21 Error % 0.20 0.60 0.71 0.43 0.24 0.42 Standard deviation 0.11 0.59 0.59 0.39 0.41 0.30 R.S.D % (n= 7) 0.76 2.40 1.71 1.29 2.06 0.61 0.03 0.15 0.25 0.13 0.24 0.21 0.10 0.52 0.52 0.34 0.37 0.27 * M ean value of s even det erminations (N) at each level. =mean value , µ= true value. t= 2.36 for n=7 at 95% confidence level. s =standard deviation. Table (7): Comparison of linearties of the two proposed me thods with those from other spectrophotome tric methods for dete rmination of amitriptyline in pharmace utical formulation. * Extra ctive pro cedure Reagent Beers law limit µg.m l-1 Referenc es Niobium (V) thiocy anate 1.0-12 16 - 1.0-30 12 Potassium thiocy anate * 3.0-60 14 Ammonium mo lybdate 1.0-140 15 Bromocresol p urple * 30-200 13 Chloranilic a cid 8.7-90 18 TCNE 6.0-75 This work BCG 8.0-100 This work IBN AL- HAITHAM J. FO R PURE & APP L. SC I. VO L. 22 (4) 2009 Table (8): Results of anal yses of pharmaceutical preparations containing amitriptyline by TCNE method and BCG me thod. § n = 4 * M arked by S.D.I, Iraq. ♦ M arked by MSD-USA. ◊ M arked by HM -Holden BV-Holland Dep resol * 25mg Tryp tizol ♦ 25mg Amitripty line ◊ 25 mg M ethod Taken (µ.ml - 1) Found (µ.ml - 1) § M ean Recovery% § R.S.D.% Taken (µ.ml - 1) Found (µ.ml - 1) § M ean Recovery % § R.S.D.% Taken (µ.ml - 1) Found (µ.ml - 1) § Mean Recovery % § R.S.D.% 15.00 14.95 99.68 0.98 15.00 14.84 98.94 1.98 25.00 24.84 99.38 1.18 25.00 24.84 99.38 1.18 30.00 29.42 98.09 0.90 35.00 34.84 94.56 0.81 Charge- transfer method 45.00 44.52 98.94 0.71 45.00 44.52 98.94 0.71 50.00 49.77 99.55 1.84 15.00 14.92 99.46 0.82 15.00 14.91 99.40 0.71 20.00 19.74 98.71 0.66 30.00 29.87 99.56 0.66 35.00 35.07 100.20 0.50 40.00 39.89 99.75 0.91 Ion-pair method 60.00 59.71 99.51 0.87 60.00 59.60 99.33 1.01 70.00 69.92 99.88 0.41 37-M .E.Abdel Hamid, M . Abdel Salam, M.S. M ahrous and M .M.Abdel-Khalek, Utility of 7,7,8,8-tetracy anoquinodimethane and p - chloranilic acid in the qualitative and qu antitative analysis