eclética química 35-3.indd ECLÉTICA química www.scielo.br/eq Volume 35, número 3, 2010 93 Artigo/Article SPECTROPHOTOMETRIC DETERMINATION OF NEVIRAPINE USING TETRATHIOCYANATOCOBALT(II) ION AS A REAGENT T. V. Sreevidya & B. Narayana* Department of Post-Graduate Studies and Research in Chemistry, Mangalore University, Mangalagangotri-574 199, Karnataka, INDIA. * E-mail: nbadiadka@yahoo.co.uk Abstract: A simple and rapid spectrophotometric method for the determination of nevirapine is described. The method is based on the reaction of nevirapine with tetrathiocyanatocobalt(II) ion in buffer of pH 4 to form the corresponding complex. Beer’s law is obeyed in the range of 0.2 – 2.0 μg mL-1 for nevirapine. The optical parameters such as molar absorptivity, Sandell’s sensitivity, detection limit and quantitation limit were found to be 1.16× 104 Lmol-1cm-1, 2.09 X 10-3 μg cm-2, 0.073 μg mL-1 and 0.222 μg mL-1 respectively. The optimum reaction conditions and other analytical parameters were evaluated. The statistical evaluation of the method was examined by determining intra-day and inter-day precision. The proposed method has been successfully applied for the determination of nevirapine in pharmaceutical formulations. Keywords: Spectrophotometry, Nevirapine, Tetrathiocyanatocobalt(II) ion. Introduction Nevirapine (NVP) chemically 11-cyclopropyl-4-methyl-5,11-dihydro-6H- dipyrido[3,2-b:2′,3′-e][1,4]diazepin-6-one, is a non-nucleoside reverse transcriptase inhibitor (NNRTI) class of antiretrovirals used for the tre- atment of HIV-1 infections and AIDS [1-3]. Ne- virapine is structurally a member of the dipyrido- diazepinone chemical class of compounds. It is used in resource poor areas and it is available as a part in generic drug combinations. It is an in- ducer of cytochrome P450 isoenzymes CYP3A4 and CYP2B6 [4,5]. Nevirapine in triple combi- nation therapy has been shown to suppress viral load effectively when used as initial antiretrovi- ral therapy [6,7]. It is a potent and selective non- -competitive inhibitor of reverse transcriptase, an important therapeutic target for treatment of HIV-1. The introduction of highly effective com- bination regimens of antiretroviral drugs has led in recent years to substantial improvements in morbidity and mortality. Combination of antire- troviral therapy is the most effective approach to managing HIV infection [8-10]. Although early clinical trials with nevirapine found that its use as a monotherapy resulted in the rapid onset of resistance and hypersensitivity reactions [11,12], it is now evident that nevirapine is effective when used as part of highly active antiretroviral therapy [13] (HAART) and may offer an alternative to the inclusion of protease inhibitors (which have been associated with toxicity during long term use) in such treatment [14]. Furthermore, compared with the protease inhibitors, nevirapine offers more convenient administration regimens (once daily dosing is possible, and there are no food restric- Ecl. Quím., São Paulo, 35 - 3: 93 - 102, 2010 Ecl. Quím., São Paulo, 35 - 3: 93 - 102, 201094 Ecl. Quím., São Paulo, 35 - 3: 93 - 102, 2010 95 Artigo Article Artigo Article tions), which has meant that it is a particularly successful anti-HIV treatment for young children. Several analytical techniques have been reported for the determination of nevirapine [15- 25]. The method in the United States Pharmaco- poeia (USP)—monograph for determining nevira- pine and its related compounds, A and B—uses a reversed-phase separation with UV detection [26]. The method calls for a 4.6 × 150 mm column pa- cked with L60 (spherical, porous silica gel, 10 øm or less in diameter, the surface of which has been covalently modified with alkyl amide groups and endcapped). Due to the strong retention of impuri- ty C, the separation requires about 30 minutes. In the present work we discuss a rapid spectropho- tometric method for the routine determination of nevirapine using tetrathiocyanatocobalt(II) ion as a reagent in pure and dosage forms. Experimental Apparatus A Shimadzu UV-2550 UV-VIS Spectro- photometer with 1cm matched quartz cells was used for absorbance measurements. Reagents and Solutions All chemicals used were of analytical rea- gent grade. NVP drug was obtained as gift sample from SeQuent Scientifi c Ltd, Mangalore. Com- mercial tablets containing 200 mg were used for the study of dosage forms. A 1000 μg mL-1 standard drug solution was prepared by dissolving 0.1g of NVP in alcohol di- luting to the mark in a 100 mL standard fl ask. For the calibration samples, a working solution was prepared by appropriate dilution of the stock con- centration in ethanol. Buffer of pH 4 was prepared by transferring one buffer tablet of pH 4 in 100 mL. Tetrathiocyanatocobalt(II) ion (TTC) was prepared by mixing 4g cobalt(II) chloride with 20g KSCN and made up to 100 mL with distilled water in a standard fl ask. Procedure Determination of nevirapine using tetrathiocyanatocobalt(II) ion Different aliquots containing 0.2 - 2.0 μg mL-1 of NVP were transferred into a series of 10 mL standard fl asks using a micro burette. To this 4 mL of tetrathiocyanatocobalt(II) ion solution was added followed by 2 mL of pH 4 buffer solution. The contents were shaken well and set aside for 10 minutes and diluted up to the mark with distil- led water and mixed well. The absorbance of each solution was measured at 624.5 nm against the corresponding reagent blank. Assay of formulations To determine the content of nevirapine in conventional tablets (label claim: 200 mg/tablet), the tablets were powdered and powder equivalent to 100 mg of nevirapine was weighed. The drug from the powder was extracted with ethanol. To ensure complete extraction of the drug, it was so- nicated for 30 min and volume was made up to 100 mL. The analysis was repeated in triplicate. The possibility of excipients interference in the analysis was studied. Results and Discussion The method involves the reaction of NVP with TTC in pH 4 to form a complex [Scheme 1], which has an absorption maximum at 624.5 nm [Fig. 1]. Scheme 1. Reaction of NVP with TTC to form NVP-TTC complex Figure 1. Absorbance spectrum of NVP-TTC complex Ecl. Quím., São Paulo, 35 - 3: 93 - 102, 201096 Ecl. Quím., São Paulo, 35 - 3: 93 - 102, 2010 97 Artigo Article Artigo Article Analytical Data The adherence of Beer’s law was studied by measuring the absorbance values of the solutions va- rying analyte concentration [Fig. 2]. A linear relation was found between absorbance at λmax and concen- tration ranges given in table 1. Regression analysis of Beer’s law data using the method of least squares were made to evaluate the slope (a), intercept (b) and correlation coeffi cient (R), for each system of NVP and are also presented in table 1. Sensitivity parameters such as molar absorptivity, Sandell’s sen- sitivity, detection limit and quantifi cation limit are also compiled in table 1. The limit of detection and quantitation are calculated according to ICH guidelines. 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.2 0.4 0.6 0.8 1.0 A bs or ba nc e Concentration μg/mL Figure 2. Adherence of Beer’s law Table 1. Analytical parameters Parameters Nevirapine (NVP) λ max (nm) 624.5 Beer’s law limits (μg mL-1) 0.2 – 2.0 Molar Absorptivity (L mol-1 cm-1) 1.16× 104 Sandell’s sensitivity (μg cm -2) 2.09 × 10-3 Limit of detection** (μg mL-1) 0.073 Limit of quantifi cation** (μg mL-1) 0.222 Regression equation* y = ax + b Slope (a) 0.4508 Intercept (b) -0.0135 Correlation coeffi cient (R) 0.9993 * y is the absorbance and x is the concentration in μg mL-1 ** Calculated using ICH – Guidelines Stability of the Complex An aliquot containing 2 μg mL-1 (2 mL) of NVP was pipetted from the stock solution of NVP (10 μg mL-1) into a 10 mL calibrated fl ask. To this 2 mL buffer of pH 4 and 4 mL of TTC were added and mixed well for 10 minutes and made up to 10 mL. The absorbance was measured at 624.5 nm at frequent intervals of time [Fig. 3]. Constant absor- bances were obtained which indicated the stability of the complex and was found to be stable over two days. Ecl. Quím., São Paulo, 35 - 3: 93 - 102, 201098 Ecl. Quím., São Paulo, 35 - 3: 93 - 102, 2010 99 Artigo Article Artigo Article 0 1 2 3 4 5 6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Ab so rb an ce Volume of CTC added (mL) Figure 3. Optimum concentration of reagent TTC Optimum Volume of Reagent An aliquot containing 2 μg mL-1 (2 mL) of NVP was pipetted out from the stock solution of NVP (10 μg mL-1) into a 10 mL calibrated fl ask along with 2 mL of buffer solution of pH 4. Then it was mixed with TTC in the order of 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL and 6 mL. The com- plexes were made up to 10 mL and absorbance was measured at 624.5 nm. It was found that the volume between 4 and 6 mL is optimum volume of the reagent to get the maximum absorbance and 4 mL was chosen for the experiment [Fig. 4]. 0 10 20 30 40 50 0.0 0.5 1.0 1.5 A bs or ba nc e Time in hours Figure 4. Stability of NVP-TTC complex Stability Constant of the Complex (Turner- Anderson Method) Equimolar solutions of NVP and TTC are mixed in complimentary proportions such that the fi nal volume is 10 mL and the solutions are made up to the mark in 25 mL standard fl asks using bu- ffer of pH 4.0. The absorbances of these are mea- sured at 624.5 nm against the reagent blank. The experiment is repeated by changing the concen- tration of NVP and TTC. The graphs are plotted with absorbance versus mole ratio of NVP-TTC complex [Fig. 5]. The stability constant of the complex is calculated from the graph and is found to be 2.7 × 104. Ecl. Quím., São Paulo, 35 - 3: 93 - 102, 2010100 Ecl. Quím., São Paulo, 35 - 3: 93 - 102, 2010 101 Artigo Article Artigo Article Mole fraction of NVP-TTC Complex 0.0 0.2 0.4 0.6 0.8 1.0 Ab so rb an ce 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 Figure 5. Continuous variation graph for TTC-NVP Complex Method Validation The proposed method is applied to the as- say of NVP in three commercially available dieta- ry supplements. An aliquot containing 1.2 μg mL-1 drug solution is taken and assayed according to the proposed methods. The content of the tablet formulation is calculated by applying suitable di- lution factor. The proposed methods are checked by a thorough analysis of each spiked sample and the results are compiled in table 3 .The accuracy and reliability of the proposed method are fur- ther established by performing recovery studies. The relative error and relative standard deviation indicate the high accuracy and precision for the method and are compiled in table 2. For a better picture of reproducibility on a day- to-day basis, a series of experiments are performed in which standard drug solution at three levels is determi- ned each day for fi ve days with all solutions being prepared afresh each day. The day-to-day relative standard deviation values represent the best ap- praisal of the method in routine use. Table 2. Evaluation of accuracy and precision Amount taken (μg mL-1) Amount found* (μg mL-1) SD (μg mL-1) RE (%) RSD (%) 0.4 0.401 0.002 0.15 0.379 0.6 0.602 0.002 0.23 0.417 0.8 0.801 0.002 0.05 0.287 1.0 1.003 0.006 0.34 0.604 1.2 1.202 0.008 0.17 0.696 1.4 1.404 0.009 0.29 0.637 1.6 1.604 0.006 0.25 0.341 1.8 1.802 0.013 0.11 0.724 * Average of fi ve determinations SD- Standard deviation, RE- Relative error, RSD-Relative standard deviation Interference Study In pharmaceutical analysis, it is important to test the selectivity towards the excipients and fi llers added to the pharmaceutical preparations. Several species which can occur in the real samples together with drug were investigated. The level of interference was considered acceptable. Commonly encounte- red excipients such as talc, starch, glucose etc did not interfere in the determination. Applications The proposed methods were applied to the determination of NVP in three commercial dietary supplements (tablets). An aliquot containing 1.2 μg mL-1 drug solution was taken and assayed according to the proposed methods. The content of the tablet formulation was calculated by applying suitable di- lution factor. The accuracy of the proposed method was checked by a thorough analysis of each spiked sample and the results are compiled in table 3. The accuracy and reliability of the methods were further ascertained by recovery studies. Table 3. Results of assay of formulations by the proposed method Brand name of tablet Labeled amount (mg) Found* ± SD (mg) Nevimunea 200 200.25 ± 0.008 Nevipanb 200 200.59 ± 0.045 Neviretroc 200 198.25± 0.016 *Mean value of three determinations; aCipla Pharmaceuticals; bRanbaxy Pharmaceuticals; cAlkem Laboratories. ECLÉTICA química www.scielo.br/eq Volume 35, número 3, 2010 103 Artigo/Article Ecl. Quím., São Paulo, 35 - 3: 93 - 102, 2010102 Artigo Article Conclusions Simple spectrophotometric method for the determination of NVP have been developed and validated according to ICH guidelines. The me- thod is simple and easy to perform compared to other existing methods and do not entail any rigo- rous experimental variables which affect the relia- bility of the results. The ingredients usually pre- sent in the pharmaceutical formulations of these drugs seldom interfere in the proposed methods. The proposed method is simple, accurate and easy to perform and can be used for the routine deter- mination of NVP in bulk and in dosage forms. References [1] S. Staszewski, J. Morales-Ramirez, K. T. Tashima, A. Rachlis, D. Skiest, J. Stanford, R. Stryker, P. Johnson, D. F. Labriola, D. Farina, D. J. Manion, N. M. Ruiz, N. Engl. J. Med. 341 (1999) 1865. [2] J. S. G. Montaner, P. Reiss, D. Cooper, S. Vella, M. Harris, B. Conway, M. A. Wainberg, D. Smith, P. Robinson, D. Hall, M. Myers, J. M. A. Lange, JAMA, 279 (1998) 930. [3] D. Podzamczer, E. Ferrer, E. Consiglio, J. M. Gatell, P. Perez, J. L. Perez, E. Luna, A. Gonzalez, E. Pedrol, L. Lozano, I. Ocana, J. M. Llibre, A. Casiro, M. Aranda, P. Barrufet, J. Martinez-Lacasa, J. M. Miro, X. Badia, A. Casado, S. Lupo, P. Cahn, M. Manos, J. Estela, Antivir Ther. 7 (2002) 81. [4] L. I. Malaty, J. J. Kupper, Drug Sar. 20 (1999)147. [5] D. Back, S. Gibbons, S. Khoo, J. Acquir. Immune Defic. Syndr. 34 (2003) S8–14. [6] R. Panchagnula, S. Agrawal, Y. Ashokraj, M.V.S. Varma, K. Sateesh, V. Bhardwaj, S. Bedi, I. Gulati, J. Parmar, C. Kaul, B. Blomberg, B. Fourie, G. Roscigno, R. Wire, R. Laing , P. Evans, T. Moore, Methods Find. Exp. Clin. Pharmacol. 26 (2004) 703. [7] D. Rey, M. Partisani, H. -K. Georgette, V. Krantz, M. Priester, C. Christine, B. -H. Claudine, E. de Mautort, L. Decroix, J.-M. Lang, J. Acquir. Immune Defic. Syndr. 37 (2004) 1454. [8] C. C. J. Carpenter, M. A. Fischl, S. M. Hammer, M. S. Hirsch, D. M. Jacobsen, D. A. Katzenstein, J. S. G. Montaner, D. D. Richman, M. S. Saag, R. T. Schooley, M. A. Thompson, S. Vella, P. G. Yeni, P. A. Volberding, J. Am. Med. Assoc. 77 (1997) 1962. [9] R. M. Gulick, J. M. Mellors, D. Havlir, J. J. Eron, C. Gonzalez, D. McMahon, D. D. Richman, F. T. Valentine, L. Jonas, A. Meibohm, E. A. Emini, J. A. Chodekewitz, N. Engl. J. Med. 337 (1997) 734. [10] W. Cavert, D. W. Notermans, K. Staskus, W. W.Stephen, Z. Mary, G. Kristin, H. Keith, Z. –Q. Zhang, R. Mills, H. McDade, J. Goudsmit, S. A. Danner & T. H. Ashley, Science 276 (1997) 960. [11] A. Carr, D. A. Cooper, Adv. Exp. Med. Biol. 394 (1996) 299. [12] S. M. Hammer, J. Infect. Dis. 192 (2005) 1. [13] M. Hartmann, S. Witte, J. Brust, D. Schuster, F. Mosthaf, M. Procaccianti, J. A. Rump, H. Klinker, D. Petzoldt, Int. J. STD. AIDS 16 (2005) 404. [14] P. Barreiro, V. Soriano, F. Blanco, C. Casimiro, J. J. de la Cruz, J. Gonzalez-Lahoz, AIDS 14 (2000) 807. [15] N. L. Rezk, R. R. Tidwell, A. D. M. Kashuba, J. Chromatogr. B (2004) 805, 241. [16] C. F. Silverthorn, T. L. Parsons, Biomed. Chromatogr. 20 (2006) 23. [17] G. Ramachandran, A. K. Hemanthkumar, V. Kumaraswami, S. Swaminathan, J. Chromatogr. B 843 (2006) 339. [18] B. H. Chi, A. Lee, E. P. Acosta, L. E. Westerman, M. Sinkala, J. S. A. Stringer, HIV Clin. Trials 7 (2006) 263. [19] R. ter Heine, H. Rosing, E. C. M. van Gorp, J. W. Mulder, W. A. van der Steeg, J. H. Beijnen, A. D. R. Huitema, J. Chromatogr. B 867 (2008) 205. [20] R. Sekar, S. Azhaguvel, Chromatographia 67 (2008) 389. [21] S. Notari, C. Mancone, T. Alonzi, M. Tripodi, P. Narciso, P. Ascenzi, J. Chromatogr. B 863 (2008) 249. [22] G. Ramachandran, A. K. Hemanthkumar, V. Kumaraswami, S. Swaminathan, J. Chromatogr. B 843 (2006) 339. [23] G. R. Da Silva, G. P. Lages, G.A. Pianetti, E.D.A. Nunan, C. D. V. Soares, L. M. M. De Campos, Quimica Nova 29 (2006) 1159. [24] C. F. Silverthorn, T. L. Parsons, Biomed. Chromatogr. 20 (2006) 23. [25] P. Lemmer, S. Schneider, M. Schuman, C. Omes, V. Arendt, J.-C. Tayari, L. Fundira, R. Wennig, Therapeutic Drug Monitoring 27 (2005) 521. [26] U. S. Pharmacopeia 29 NF 24 (2006) 29 15-19-1520. BOUND STATE SOLUTIONS OF SCHRÖDINGER EQUATION FOR A MORE GENERAL EXPONENTIAL SCREENED COULOMB POTENTIAL VIA NIKIFOROV- UVAROV METHOD Benedict I. Ita, P. Ekuri Quantum Chemistry Group, Department of Pure and Applied Chemistry, University of Calabar, P. O. Box 3700, Calabar, CRS, Nigeria (Corresponding author: e-mail: iserom 2001@yahoo.com) Idongesit O. Isaac Department of Mathematics/Statistics and Computer Science, University of Calabar, Calabar, Cross River State, Nigeria. Abosede O. James Department of Pure and Industrial Chemistry, University of Port Harcourt, Nigeria Abstract: The arbitrary angular momentum solutions of the Schrödinger equation for a diatomic molecule with the general exponential screened coulomb potential of the form ( ) ( ) ( ){ }br2ebr11r/arV −++−= has been presented. The energy eigenvalues and the corresponding eigenfunctions are calculated analytically by the use of Nikiforov-Uvarov (NU) method which is related to the solutions in terms of Jacobi polynomials. The bounded state eigenvalues are calculated numerically for the 1s state of N2 CO and NO Keywords: Nikiforov-Uvarov method, Eigenvalues, Eigenfunctions, General Exponential Screened Cou- lomb Potential. Introduction The exact analytic solutions of the wave equations (non-relativistic and relativistic) are only possible for certain potentials of physical in- terest under consideration since they contain all the necessary information on the quantum system [1]. It is known that for certain potentials, the Schrödinger equation can be solved for the angu- lar momentum quantum numbers 0= [2]. How- ever, in some cases, like for the 0≠ states, some approximations are often used to obtain analytic solutions of the Schrödinger equation [3 – 5]. A more general exponential screened cou- lomb (MGESC) potential used in this paper is of the form [6]: ( ) ( ) ( ){ }br2expbr11 r a rV −++⎟ ⎠ ⎞⎜ ⎝ ⎛−= (1) where a is the strength coupling constant and b is the screened parameter. The potential in equation (1) is known to describe adequately the effective interaction in many-body environments of a variety of fi elds [6]. In this paper, we have de- cided to explore the possibility of also using it in obtaining bound state solutions of the Schrödinger equation for diatomics using Nikiforov-Uvarov (NU) method. Ecl. Quím., São Paulo, 35 - 3: 103 - 107, 2010