IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 Spectrophotometric Determination of Isoptin (Verapamil Hydrochloride) in Pharmaceutical Preparations D. M. Salh Departme nt of Chemistry, College of Science, Sulaimaniyah Unive rsity Abstract Verap amil Hy drochloride (VH) has been determin ed sp ectrop hotometrically by using M ethy l Orange (MO). In our p revious researches MO was used for determination of M exiletine Hy drochloride [1]. The method was based on comp lexation between (M O and VH). Aft er shaking and d iluting the comp lex solution with D.W, the pH was adjust ed with NaOH and HCl to p H 4. The colored comp lex formed between VH and the reagents were transferred into separating funnels and extracted using 4.5 ml CH2Cl2 and were shaken for (4 minutes). The extracted organic layer was used for the prep aration of the calibration curves for sp ectrop hotometric measurements of VH at 437nm. The blanks were carried out in exactly the same way throughout the whole p rocedure. M olar absorptivity (ε L.mol-1.cm-1), detection limit, limit of linearity (µg.ml -1 ) and r 2 wer e, 1.75*10 5 , 0.009253, 0.08 and 0.993 for (VH-M O) resp ectively. The method was used with reasonable accuracy and p recision for the determination of (VH) in sy nthetic samp les of t ablets, cap sules and ampoules. Keywords: sp ectrop hotometric determination of Verapamil hy drochloride, methy l orange. Introduction Verap amil Hy drochloride 5-[N-(3, 4-dimethoxy -p henethy l)-N-methy l-amino]-2-(3,4- dimethoxy p henyl)-2-isop ropy lvaleronitrile hy drochloride, or (Isop tin) [2], have the chemical st ructure shown in Fig.1: Verap amil was introduced in 1962 as a coronary vasodilator & it is the p rototype of the Ca 2+ antagonists used in cardiovascu lar diseases. Verap amil major effect is on the slow Ca chann el. The inhibition of the action p otential inhibits one limb of the reentry circuit believ ed to underlie most p aroxy smal Sup eraventricular tachy cardia t hat uses the AV node as a reentry p oint. It is categorized as a class I V antiarrhythmic dru g .Hemodynamically ; verap amil causes a chan ge in the p reload. Aft er load contractility , heart rate, & coronary blood flow .The drug reduces sy stemic vascular resistance & mean blood p ressure, with minor effect on cardiac outp ut [2]. The oldest chromatography method in this review was Gas-Liquid Chromatography Using Nitrogen-Phosp horous Detection applied for determination of Verapamil in Hum an Plasma [3], which was appeared in (1984). Later, many chromatographic methods for the determinations of this drug and its metabolites have been reported [4-8] att empting different modifications in the method to increase sensitivity , reducing st eps of analysis, or other imp rovements, with the limit of linearity ranged between [2 n g L -1 – 1000 n g L -1 ]. Extensive search in the literature has showed many sp ectrop hotometric methods for the determination of VH [9-13]. Amon g them, how to use Sp ectrofluorometric and visible sp ectrop hotometry , with different types of optimizations to obey beers law, and limit of linear ity ranged between [0–340 μg ml -1 ]. IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 Prelimin ary p ractical tests on many reagents revealed that methy l orange whi ch has the chemical st ructure shown in Fig.2, was suitable reagent [14] to form colored comp lexes with the drug VH and were exp loited for their quantitative determination in cap sules, amp oules and tablets. Experime ntal Apparatus All measurements were taken with TU-1800S UV-Vis sp ectrop hotometer, with (1cm) p ath length quartz cells. T he pH of t he solutions was adjust ed by using Hanna pH-meter with combined glass electrode (910600) Orion Comb p H, Water bath Thermost at Shaker (GFL 1083) was used during the extraction steps and M icro p ipettes (variable and fixed). Chemicals, reagents, and drugs Both analar and general p urp ose reagents were used from [Fluka, Rohm and Haas, GCC (Gainland Chemical Company), and M erck] without further p urification. Ordinary distilled water p rep ared in all glass st ill and st ored in p oly ethy lene container was used. Verap amil hy drochloride ampoule [Knoll] which was (5 mg/2ml), was taken as a st ock solution, other concentrations were prepared by usual dilution. M ethy l orange 0.05% aqueous MO. Phthalate buffer (pH=4) was p rep ared by mixing 50ml of 0.1M (p otassium hy drogen p hthalate), with 49.9 ml of D.W and 0.1 ml of 0.1M HCl [15-16], and pH was adjust ed with a p H meter. The recommended procedures A volume of 500 µL 0.1% (MO) reagent was added to a certain amount of VH st andard or samples containing between (0.08-0.8 μgml -1 ) VH. The mixture was shaken for (2 min.) and diluted to about 24ml in volumetric flasks using D.W. The pH was adjust ed by addin g 1 ml p hthalate buffer (pH 4) to the MO mixture and finally comp leted to 25ml. The resulting complex formed b etween VH and the reagent were transferred into sep arating funnels (100ml cap acity ) and extracted with 4.5 ml CH2Cl2 in two p ortions to wash out the volumetric flasks for quantitative transfer of the solution and was shaken for 4 minutes. Aft er sep aration, the organic layer was used for the p reparation of the calibration curves us ing sp ectrop hotometric measurements of VH-M O comp lex at 437nm. The blanks wer e carried out in exactly the same way throughout the whole p rocedure. Results and Discussion Preliminary work The absorption sp ectra of the comp lex (VH-M O) against blank with λ-max at 437nm, and blank against D.W (accordin g to the recommended p rocedure) are shown in Fi g.3. The sp ectra also show some back ground of the r eagent in the r egion of the comp lex whi ch will have a negative effect on the sensitivity of the method. The comp lex; or an ion-pair formation between the VH and M O has shown an increasin g intensity of the sp ectrum. This is certainly a negative analytical p henomenon; since no high sensitivity could be exp ected with this sy stem. However, the reagent was exp ected to show p romising results therefore, st udies were continued for op timization of the conditions. Optimization 1. pH Optimi zation A volume of 500 µL of 0.1% M O was added to 1 ml of 0.1mg.ml -1 VH, shaking for 2 min., then diluted to about 24ml in volumetric flasks usin g D.W. The pH was then adjusted IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 between 2 to 6, by using 0.1M NaOH or 0.1M HCl. The rest of the test was then followed according to the p rocedure. The results reveal that the optimum p H for (VH-M O) comp lex is 4, as shown in Fig.4. 2. Type of buffe r use d (pH-adjustment) For p H adjust ment of (VH-M O) sy stem different buffers were tried, such as; acetic acid-sodium acetate and citric acid-sodium hy droxide, but Pht halate buffer of pH = 4, was found suitable to adjust pH of the comp lex (VH-M O). Different volumes of this buffer were added in two way s; either before completing the volu metric flask by D.W to the mark, or until a small volume about 2ml was remaining then the buffer was added and comp leted to the mark. The results indicated that optimum volume wer e equal to 1 ml phthalate buffer with no difference in order of addition before or after comp leting the volumetric flask by D.W (after mixing), and the result is shown in table (1). 3. Choosing a Suitable S olvent for Extraction: M any solvents were tested for extracting the comp lex formed between the M O reagents with VH [Carbon tetrachloride, Benzene, Ethy l acetate, Dioxane, Acetone, chloroform] and d ichloromethane was found t o be the best. 4. Amounts of the Reagent: Prelimin ary test shows that 500 µL of 0.1% M O was suitable. Exp eriments were then p erformed with different volumes of the d ifferent concentrations to a constant volume 1 ml 0.1 mgml -1 VH. The results shown in Fig.5 indicate that optimum volumes were 1.2 ml for 0.05% MO. 5. Effect of dichloromethane CH2Cl2 volume : Different volumes of dichloromethane between (4 to 10 ml) were used for extraction. The results are shown in Fig.6, the op timum volume was found between 4 – 5 mls of dichloromethane. A volume of 4.5 ml CH2Cl2 was found suitable and also sufficient for comp lete extraction. 6. S tability of the complexes: The stability of the comp lex formed between (VH—M O) was followed by measuring absorbance against time. As shown in (Fi g.7). It was found that the comp lex ( VH—M O) was stable for a p eriod of 35 minutes, after sep aration and only 5 minutes were needed to reach the true absorbance, and throu gh this time there was time for shaking and for comp leteting complexation that was shown in the (Fig.8). Absorbance has, then increased after that which may be due to the vap orization of the solvent. S toichiometry of the [VH-MO] complex: The stoichiometry of the drug and M O complexing r eagent was examined by the mole ratio method at wave len gth of 437nm: The result of mole r atio method was obt ain ed in two ways first by addin g constant amount of VH to a series of differ ent amounts of M O solutions, and second by addin g constant amount of MO to a series of different amounts of VH solutions. Fig.9a and 9b show the results of mole ratio method. It was found t hat the ratio of (VH-t o-MO) was about (2 to 1). S tructure suggestion: It was not possible to p ut forward a reasonable st ructure for the stoichiometry of VH: MO of 2:1. According to their chemical st ructure, it was thought that a 1:1 or 1:2 ratios would be reasonable. This will mak e a salt-like formation between the VH and M O t hus:- IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 2 VH + MO ---------►M O (VH) 2 Calibration curves: The calibration curves obtained according to the recommended procedure were drawn for VH-M O comp lex. The linear range was 0.08 – 0.8 µ g.ml -1 of VH with (r 2 = 0.993) for VH-M O comp lex shown in Fig.10. De termination of Verapamil hydrochl oride [Isoptin] in syntheti c sample solutions: The recovery test was p erformed for different concentrations of VH with M O reagent; the results obtained are shown in table 2. De termination of Verapamil hydrochl oride in tablets: Two t ablets were powdered and mixed thorough ly. An amount equivalent to 160 mg of verap amil hy drochloride was we ighed accurately and extracted with chlorofor m to eliminate any interference from excipients. It was filtered through Whatmann No. 42 filter p ap er and the residue was washed well with chloroform for comp lete recovery of the dru g. The chloroform was evap orated to dry ness and the drug was dissolved in doubly distilled water and diluted to 1000mL with dist illed water, It was further diluted (1 to 10) [12]. Three different volu mes (0.2, 0.5 and 1) mls of the samp le of VH were determined by MO accordin g to the recommend ed procedure. The results obtained are shown in tables 3. To test for the existence of a sy stematic error in the results shown in tables 3, the actual difference between (X) and (μ) was compared by t-test with the term [t.S / √N] at 95% confidence limit DOF = 2. From the results of t-test MO reagent t he following conclusions were made: The direction of the errors (+) suggest the existence of a sy st ematic error. This may be due to the extraction st ep s. Nearly non dep endence of these errors on samp le size between (0.2, 0.5 and 1) mls suggest s t he existence of both const ant and p rop ortional sy stematic errors. The difference between (X - μ) and [t.s / √N] was not significant at 95% C. L. in all cases (0.2 to 1) mls of VH sample, indicating the non existence, or p resence of a very small systematic error, which is mainly due to the extraction st ep s. If the recovery tests were considered only as it is t he case with research workers, t he value of (R %) in tables ar e quite reasonable. Precision and Accuracy: The p recision of VH determin ation by M O comp lexing reagent was p erformed on three sy nthetic samples containin g VH in the range of the calibration curve, and their absorbance were measured 10 times for the same unknown, showing the p recision of measurements. T he precision was also found on 10 t imes repeating of the whole operation on the same sample. This will show the p recision of the op eration. The relative st andard deviation for the (VH-M O) comp lex ranged between 0.75 – 1.75% showing r easonable p recision even at lower concentrations of VH. The accuracy shown in the p revious sections also revealed reasonable accuracy givin g sufficient validity for the application of both reagents to be used for the determination of VH in the tablets. Se nsi tivity of the methods: The results of t he p rop osed method were statistically comp ared with those obtained by the sp ectrop hotometric method for determination of VH usin g two methods [12] and are summarized in (Table 4), the table shows the results concernin g sensitivity of the methods (Valu es of molar absorp tivity (ε), slop es of the calibration curves (m), limits of linearity and detection limit (D.L= 3 S.D)). T he results indicate reasonable sensitivity of the methods with no significant deference between the methods comp ared. IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 References 1. Dler M . Salh; (2007) “Spectrop hotometric Determination of M exiletine Hy drochloride in Pharmaceutical Prep arations, Urine and Serum” M .Sc. thesis, Su laimaniyah University . 2. Block J.H., Beale J.M ; (2004). “Organic M edical and Pharmaceutical Chemist ry ”11th Edition p. 629-630, by Lipp incott Williams & Wilkins, ISBN: 0-7817-3481-9. 3. Darrell, R. A.; Todd E. L.and M itchell, J. R., (1984) International journal of exp erimental and clinical Pharmacology ; Vol.29( 5): p264-268. 4. Shukla, U.A.; Stetson, P.L. and Ensminger, W.D., (1985), J. of Chrom; 342 :. 406- 410. 5. Darrell, R. A., ;Todd, E. L.and M itchell J. R., (1988), Clin ical Chemist ry Vol.34: 2502-2503. 6. Ho-San g S. ; Yun- Suk ,Oh.; Hy un-Jin ,K. and Yoon-Koo, K., M arch 1996, J. of Chrom. B: Biom. Sciences and App l. Vol. 677, Issue 2, 3; P.369-373. 7. Yalçın ,Ö.; Niyazi ,Y.; Sibel ,A. Ö. and Biry olb, N., M ay 2000; J.Il Farmaco, Vol. 55: Issue 5, P.376-382. 8. Dorot a ,K.; Sept-Oct, (2005), J.of AOAC International, Vol. 88: 1525-1529. 9. Issa ,Y. M . ; Salem, A. A. and Bahbouh, M . S., Ap ril 1997, Analy tical Letters; Vol. 30, Issue 6 , p. 1153 - 1166. 10. Nafisur, R.and M d Nasrul, H..(2002), J Anal Bioanal Chem, Vo l. 374: p.484–489. 11. Nafisur, R. and Sy ed, N. H. A.. (July 2004), J.Il Farmaco, Vol. 59, Issue 7, P. 529-536 12. Nafisur, R., Nadeem, A. k. and Sy ed N. H. A.(2005), ScienceAsia 31: P. 341-348. 13. M ohamed W., Fathalla B., Nahed E., Amina A.; (2006), J.of AOAC INT ERNATIONAL, Vol. 89, Issue 6, P. 1565-1572. 14. Edmund Bishop ; (1972), “Indicators” First , pp . (79 – 81); Dep t. of Chem., Univ. of Exeter printed in Germany, 08:016617-2. 15. Perrin ,D.D. and Dempsey, B.(1974) “Buffers for p H and M etal Ion Control” First p ublished, p .123-156, By Chap man and Hall Ltd, ISBN: 0-412-21890-9. 16. Kolthoff, I.M .; Sandell, E.B.;M eehan, E.J. and Stanley, B. (1969) “Quantitative Chemical Ana lysis” Fourt h Edition, p .1161 – 1165, M acmillan Company, Collier- M acmillan Limited. Table (1): Result of adding diffe rent volume of phthalate buffe r before and after adding D.W to the mixture of VH and MO. Adding the bu ffer be fore dilution . Adding the bu ffer afte r dilution. 0.5 ml Abs.= 0.137 0.5 ml Abs.= 0.135 1 ml Abs.= 1.378 1.ml Abs.= 1.380 1.5 ml Abs.= 1.168 1.5 ml Abs.= 1.169 2 ml Abs.= 1.12 2 ml Abs.= 1.12 IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 Table (2): The accuracy of VH determination in syntheti c samples using MO [VH-MO] com plex. VH Prese nt ppm VH Found ppm R% E% 0.4 0.413 103.25 3.25 0.4 0.413 103.25 3.25 0.4 0.404 101 1.00 Table(3): Results of different volumes of VH sample (tablets) dete rmined by MO reage nt Conc. Of VH ppm found in Table ts by MO Usi ng the Eqn. Y = 0.3568X + 0.0077 Vol.(ml) Of the sample taken VH ppm R% E% 0.2 0.128 8 10 0.6487 0.648 7 0.5 0.332 4 10 3.9039 3.903 9 1 0.645 4 10 0.8530 0.853 0 Table (4): Se nsitivity of the propose d method Type of calibra tion ε (L.mol -1 .cm -1 ) Limits of line arity (μg/ml) Re covery % R.S .D % Detection Limit (μg/ml) r2 calibr ation curve for (VH+MO)com p lex 1.75 * 10 5 0.08 100.6487 0.75 – 1.75 D.L.= 3S. D 0.009253 0.993 103.9039 100.8530 Reference method (Two M ethods A&B) A = 1.18 * 10 3 B = 9.82 * 10 3 A = 12.50 B = 2.00 A 100.30 A = 0.28 – 0.23 B = 0.15 – 0.27 A = 0.77 B = 0. 4 A = 0.9999 B = 0.9998 100.21 100.30 B 99.96 99.93 99.96 a. Molar absorpti vity ε (L.mol-1.cm-1), b. Limits of linearity, c. (Recovery % an d R.S .D) of different volumes of AH sample (tablets) using propose d method, d D.L with correl ation coefficient Fig. (1): Verapamil Hydrochl oride Chemi cal S tructure IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 N NNaSO3 N CH 3 CH 3 M ethy l orange p K1 = 3.76 p H transition interval = 3.1 (Red) – 4.4 (Yellow) Fig. (2): Methyl Orange Che mical Structure 437 nm 0 0. 1 0. 2 0. 3 0. 4 0. 5 0. 6 0. 7 0. 8 0. 9 1 350 400 450 500 550 600 650 Wave len gth λ ( nm) A b s . 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 350 400 450 500 550 600 650 Wave length λ (nm) A b s . A B Fig. (3): (A) The spectrum of the (VH+MO) complex against blank, and (B) blank against D.W spectrum 0 0. 2 0. 4 0. 6 0. 8 1 1. 2 1. 4 0 1 2 3 4 5 6 7 pH A b s . Optimum pH=4 Fig. (4): The pH optimizati on for (VH+MO) complex 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 Vol. MO ml A b s. ----Optim um am ount ------ of MO Fig. (5): O ptimization of volume of 0.05% MO IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 3 4 5 6 7 8 9 10 11 Vol. ml A bs . Otim um Volume = 4 .5 ml Fig. (6): O ptimization of CH2Cl 2 volume to be added for extraction the (VH-MO) complex 0 .25 0 .27 0 .29 0 .31 0 .33 0 .35 0 .37 0 .39 0 1 0 20 3 0 40 50 6 0 70 8 0 Ti m e (mi nute s) A b s . Stab litity o f C om pl e x Fig.(7): S tability of the (VH –MO) complex after separation 0. 9 1 1. 1 1. 2 1. 3 0 1 2 3 4 5 Time (minutes) A b s . Time nee ded for Shaking Fig.(8): S uitable time for shaking the (VH –MO) complex after separation IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 0 0 .1 0 .2 0 .3 0 .4 0 .5 0 .6 1 1.1 1.2 1.3 1.4 1 .5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2 .5 [VH] / [MO] A b s . [VH] / [MO] = 1.9 ≈ 2 0 0 .05 0.1 0 .15 0.2 0 .25 0.3 0 0.1 0 .2 0 .3 0 .4 0.5 0.6 0.7 0.8 0 .9 1 1 .1 1.2 [MO] / [VH] A b s . [ MO ] / [VH] ≈ 0 .5 (a) (b) Fig.(9a and b):De terminati on of the stoichiometry of the (VH to MO) by mole ratio y = 0.3568x + 0. 0077 R 2 = 0. 993 0 0.05 0. 1 0.15 0. 2 0.25 0. 3 0.35 0 0. 1 0. 2 0. 3 0. 4 0.5 0. 6 0.7 0.8 0. 9 1 ppm VH A b s . Fig.(10): Calibration curve for (VH-MO) complex IHJPAS 2010) 3( 23 مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة المجلد في المستحضرات ) ھایدروكلورید الفیرابامیل(التقدیر الطیفي لالیزوبتین الصیدالنیة دلیر محمد صالح شریف كیمیاِء، كلیَّة العلوِم، جامعةالسلیمانیة القسم لخالصةا ان في بحوثِنا السابقِة اذ ،MOعمال المثیل البرتقالَي تطیفیاً باس VHتم تقدیر ھایدروكلورید الفیرابامیل ن الىالطریقة مستندة . MHلتقدیرھایدروكلورید المكسیلیتین MOعمل كاشف المثیل البرتقالَي تاس تكوین معقد ملون بی MO معVH . بعد الرج وتَخفیف مَحلول المعقِّد بالماء المقطرD.W ثبت الـ ،pH بالـ ) 4(علىNaO H وHCl . ن التكوَن بین ملیلتر من ثنائي كلورید المثیَل 4.5والكاشف یحول إلى قمع فَْصل ویستخلص باستعمال VHالمرّكب المُلَوَّ CH2Cl ل). دقائقِ 4(ثم الرج مدة 2 لـ الطبقة العضویة المستخ صة إستعملْت لتحضیِر منحنى المعایرة للتقدیر الطیفي VH 437(في الطول الموجي nm .( ان الـBlank ان الـ . كافة في الجوانب نفسھا نُفّذت بالضبط الطریقِة الكاملة Molar absorptivity (ε L.mol -1 .cm -1 limiالمدى الخطي ، detection limitحد الكشف ، ( t of linearity (µg.ml-1) و)r 2 10*1.75(عبارة عن ) 5 ْت . على التوالي) 0.993(و ) 0.008(، )0.009253 (،) الطریقة إستعمل ن الدوائیة) VH(بدقِة ونجاح في تقدیر .في العیناِت الصناعیةِ على األقراِص والكبسوالِت والحق IHJPAS IHJPAS