Conseguences of soil crude oil pollution on some wood properties of olive trees Chemistry |102 https://doi.org/10.30526/30.3.1606 7302(عام 0العدد ) 03مجلة إبن الهيثم للعلوم الصرفة والتطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (3) 2017 Spectrophotometric Determination of Sulfamethoxazole Based on Charge-Transfer Complexation with Sodium Nitroprusside Tariq Y. Mahmoud Sarmad B. Dikran Alaa K. Mohammed Dept. of Chemistry/College of Education for Pure Science (Ibn Al- Haitham)/ University of Baghdad Received in:2/February/2017,Accepted in:9/April/2017 Abstract A simple, accurate and precise spectrophotometric method has been developed for the analysis of sulfamethoxazole (SMZ) in pure form and pharmaceutical preparation. The method involves a direct charge transfer complexation of sulfamethoxazole (SMZ) with sodium nitroprusside (SNP) in alkaline medium and the presence of hydroxyl amine hydrochloride. Variables affecting the formation of the formed orange colored complex were optimized following two approaches univariate and central composite experimental design (CCD) multivariate. Under optimum recommended conditions, the formed complex exhibits λmax at 512 nm and the method conforms Beer's law for SMZ concentration in the range of 5.0-150.0 (µg.mL -1 ) with molar absorptivity 1.139×10 3 L.mol -1 .cm -1 , and r = 0.9997. Analysis of SMZ pharmaceutical dosages shows a good agreement with the real amounts. Keywords: spectrophotometric determination; sulfamethoxazole; sodium nitroprusside; charge-transfer complexation. Chemistry |103 https://doi.org/10.30526/30.3.1606 7302(عام 0العدد ) 03مجلة إبن الهيثم للعلوم الصرفة والتطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (3) 2017 Introduction Sulfamethoxazole, 4-amino-N-(5-methylisoxazol-3-yl)-benzenesulfonamide (M. wt. 253.279 gm. mol -1 ) [1]. Sulfamethoxazole an antibacterial drug which approved for treatment of the oversensitive forms of Hemophilic influenza, Staphylococcus aurous, Streptococcus, Escherichia coli, and for oral anaerobes. It also represents therapeutic approach to the treatment of the urinary tract infections as an alternative to amoxicillin-based antibiotics. The literature contains various methods for the determination of sulfamethoxazole in its pharmaceutical formulations. This involves spectrophotometric [2-7], HPLC [8-11], HPTLC [12], Capillary zone electrophoresis [13], Micellar electrokinetic chromatography [14], derivative ratio spectrometry [15], flow injection sensor [16], sulfamethoxazole-imprinted polymer [17], spectrofluorometry [18], fluorescence spectrophotometric [19] and NMR [20] methods. The structural formula of SMZ is shown in Scheme (1). In this work a simple and sensitive visible spectrophotometric method was developed for quantitative determination of sulfamethoxazole in its pure form and in pharmaceutical formulation. The suggested method is based on the formation of charge transfer molecular complex of drug with sodium nitroprusside in alkaline medium in the presence of hydroxylamine hydrochloride [21], [22]. Experimental Apparatus A CECLL UV / VIS double beam spectrophotometer (model CE 7200. UK (7000 series) with 1 cm quartz cells, a Sartorius-BL 210 scientific balance(Germany), a heater with magnetic starrier (IKA- Combimag Rct.), a lab tech water bath (Korea) were used for this study. The experimental design and the coefficients of the response surface equation were determined by statistical 12 (Stat. Soft. Inc., release 2013) software. Reagents All reagents and chemicals used of analytical grade. Sulfamethoxazole powders were provided from the State Company for Drug Industries and Medical Appliances Samara- Iraq (SDI) in pure form (99.99%). Reagents solutions  Sodium Carbonate monohydrate (6% w/v): 6g of Na2CO3.H2O was dissolved in 100 mL of distilled water.  Sodium Nitroprusside 0.2% (w/v): 0.2g of SNP was dissolved and diluted to the mark with distilled water in a 100 mL volumetric flask.  Hydroxylamine hydrochloride 0.4% (w/v): 0.4g of NH2OH.HCl was dissolved in 100 mL of distilled water. Preparation of standard stock solution (1000 µg.mL -1 ) An accurately weight 0.1 g of SMZ was dissolved in 5 mL of 0.1M Na2CO3.H2O and diluted in 100mL-volumetric flask with distilled. Other dilute solutions were freshly prepared by subsequent dilution with distilled water as required. Chemistry |104 https://doi.org/10.30526/30.3.1606 7302(عام 0العدد ) 03مجلة إبن الهيثم للعلوم الصرفة والتطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (3) 2017 Preparation of sample stock solution Different commercial SMZ pharmaceutical dosages (i.e. tablets and syrup) were analyzed to assay their contents of the drug. 10 tablets were powdered and an amount of the powder equivalent to 0.1 gm sulfamethoxazole dissolved in 5 mL of 0.1 M Na2CO3.H2O solution in 100 mL volumetric flask. The mixture was shaken, and left for 5 min. before dilution with distilled water to the mark. Any undissolved materials were filtered-out via Whatman No.41 filter paper. Five milliliters of the syrup sample solution mixed with 5mL of 0.1M Na2CO3.H2O solution in 100mL-volumetric flask. The flask was shaken and left to stand for five minutes before diluting it's contained with distilled water to the mark. Any undissolved materials were filtered-out via Whatman No.41 filter paper. All the samples were further diluted to achieve the concentration of SMX in the working range. General procedures Under conditions obtained by univariate optimization To a series of 10mL calibrated flasks containing (50-1500) μg of SMZ, one milliliter of 0.2 % (w/v) SNP solution and one milliliter of 0.4% (w/v) hydroxylamine hydrochloride were added respectively. The solutions were shaken thoroughly; then 0.2mL of 6% (w/v) Na2CO3.H2O solution was added to each. After letting the mixtures to stand for seven minutes at 25 °C in the dark, the volumes were brought to the mark with distilled water, mixed well and values of absorbance at 512.0 nm were measured versus the blank. Under conditions obtained by multivariate CCD conditions To a series of 10mL-calibrated flasks containing (50-1500) μg of SMZ, 2.3mL of 0.2% (w/v) SNP solution and 1.8mL of 0.4% (w/v) hydroxylamine hydrochloride were added respectively. The solutions were shaken thoroughly; then 0.6mL of 6% (w/v) Na2CO3 solution was added to each. After letting the mixtures to stand for seven minutes at 25 °C in the dark, the volumes were brought to the mark with distilled water, mixed well and values of absorbance at 512.0 nm were measured versus the blank. Results and Discussion Absorption Spectrum and reaction scheme When SMZ is reacted with SNP according to the recommended experimental conditions Scheme (2), the absorption spectrum of the formed colored complex compound showed a maximum absorption at 512.0 nm against reagent blank solution. While, the blank has no significant absorbance in this region, as it is shown in Figure (1). Optimization of reaction conditions Two different approaches i.e. one-factor-a time univariate, and CCD multivariate, were followed to establish optimum reaction conditions. Univariate method The effect of the amounts of SNP, Hydroxylamine hydrochloride, Na2CO3, and reaction time on the formation of SMZ-SNP complex were studied. The results show that 1.0 mL of 0.2% (w/v) sodium nitroprusside was needed to give best results (Figure 2a). The effect of volume of 0.4% (w/v) of NH2OH.HCl solution was examined, 1.0 mL of this solution gave the maximum intensity Figure (2b). The results on Figure 2c shows that 0.2 mL of 6% Na2CO3.H2O was sufficient for attaining the maximum and constant absorption intensities. Under these conditions the optimum time Chemistry |105 https://doi.org/10.30526/30.3.1606 7302(عام 0العدد ) 03مجلة إبن الهيثم للعلوم الصرفة والتطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (3) 2017 and temperature for the complex-formation reaction was found to be seven minutes at 25°C, Tables (1) and (2). Design of experiment method A design of experiments (DOE) was used to determine the relationship between three experimental factors (namely the amounts of reagent, NH2OH.HCl, and Na2CO3.H2O) affecting the studied reaction yield. in central composite design, around the central point (which is assumed to be 0) for each of the studied factors, the design is assumed to be symmetric. The studied variables with levels are given in Table (3). According to CCD, a cube could represent a three factors system (k=3), and its axes correspond to the three factors. The design consists of a number of 20 experiments (2 k factorial points = 8, 2 × k axial points = 6, and six center points). Table (4) shows the values of the studied experimental variables and their corresponding measured absorbance at 512 nm. Tables (5) illustrates the analysis of variance of the experimental results in Table (4). Table (5) show that at 95% confidence level (p < 0.05), the overall effect of the linear terms in the model is significant while the other terms i.e. the interaction terms are not. Moreover, Table (5) shows the standard error of coefficients of the second order polynomial model and t and p values. Moreover, according to the obtained second order polynomial model, three dimensional plots of the response surfaces for any two pairs of the studied variables against the response were constructed to illustrate the relationship between them and the response at the critical level (optimum value) of other factors, Figure (3). Calibration curves and analytical data To evaluate the proficiency of the recommended method for determination of sulfamethoxazole, calibration curves were constructed by plotting the absorbance values for a series of solutions containing varying concentrations of SMZ under the optimal conditions measured against the corresponding reagent blank at 512 mn. The graphs obtained under conditions established via univariate optimization and multivariate CCD method, Figures (3) and (4) respectively, were linear in the range of SMZ concentration (5.0 -150.0) μg.mL -1 evaluated by linear regression. Some of optical and statistical parameters of the SMZ-SPN complex and the constructed calibration plots are summarized in Table (6). In both procedures, the values of correlation coefficient (r) for the regression equation were high indicating the linearity of the plotted curves. Detection and quantification limits were calculated according to ICH guide. The high values of the molar absorptivities and the low values of detection limits indicate that both procedures are sensitive. Accuracy and precision The accuracy of the method was established by analyzing five replicate of the pure drug at three concentration levels and the precision was examined by determining the coefficient of variation (C.V. %) on the same solutions of drug sample. Results in Table (7) show low values of C.V % (in the range of 0.137%-0.435%) and for the relative error percent (which did not exceed ±0.8%) for both procedures, indicate high accuracy and precision of the proposed method. Chemistry |106 https://doi.org/10.30526/30.3.1606 7302(عام 0العدد ) 03مجلة إبن الهيثم للعلوم الصرفة والتطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (3) 2017 Interference study To assess the analytical potential of the proposed method, the effect of some common excipients; sucrose, vanillin, glucose, lactose, and starch which often accompany drug, were examined by carrying out the determination of 50.0 μg.mL -1 of SMZ in the existences of mentioned compounds. The results presented in Table (8) indicate that no significant interferences were found from any of the studied excipients in determination of SMZ except vanillin that completely interferes. Application of the method For examining the suitability of the recommended method for SMZ determination in real samples, it was applied to determine the drug in some pharmaceutical preparations. the collected results of the analyses shown in Table 9, indicate that the recovery percent values are ranged between (96.80-100.26) and those for C.V.% does not exceed 1.136 which illustrates that the proposed method is satisfactory. References 1. "British Pharmacopoeia". III, (2008), Her Majesty's Stationary Office British Pharmacopoeia Commission, London, 2058-2059. 2. Hassouna, MEM. (1997), "Simultaneous spectrophotometric determination of sulfamethoxazole and trimethoprim in drugs". Anal lett. 30: 2341-2352. 3. Nagaraja, P.; Sunitha, KR.; Vasantha, RA. and Yathirajan HS. (2002), "Iminodibenzyl as a novel coupling agent for the spectrophotometric determination of sulfonamide derivatives". Eur J Pharm Biopharm.; 53:187-192. 4. Nagaraja, P.; Yathirajan, HS.; Raju, CR.; Vasantha, RA.; Nagendra, P. and Kumar MS.H. (2003), "3-Aminophenol as a novel coupling agent for the spectrophotometric determination of sulfonamide derivative". Farmaco Sci.; 58:1295-1300. 5. Issa, YM. and Amin, AS. (1994), "Spectrophotometric micro determination of sulfamethoxazole and trimethoprim using alizarin and quinalizarin". Anal Lett.; 27:1147- 1158. 6. Granero, G.; Garnero, C. and Longhi, M. (2002), "Second derivative spectrophotometric determination of trimethoprim and sulfamethoxazole in the presence of hydroxypropyl-β- cyclodextrin (HP-β-CD)". J Pharm Biomed Anal.; 29:51-59. 7. Nalewajko, E.,; Gdlvez, AM.; Benito, CG. and Calatayud, JM. (2003), "FIA and batch simultaneous determination of sulfamethoxazole and trimethoprim in pharmaceutical formulations by derivative spectrophotometry". J Flow Inj Anal.; 20:75-80. 8. Bedor ,DC.; Goncalves, TM.; Ferreira, ML., de Sousa CE., Menezes AL., Oliveira EJ. and de Santana DP. (2008), "Simultaneous determination of sulfamethoxazole and trimethoprim in biological fluids for high-throughput analysis: comparison of HPLC with ultraviolet and tandem mass spectrometric detection". J Chromatogr B Analyt Technol Biomed Life Sci.; 863:46-54. 9. Nelis, HJ., Léger, F., Sorgeloos, P. and De leenheer AP. (1991), "Liquid chromatographic determination of efficacy of incorporation of trimethoprim and sulfamethoxazole in brine shrimp (Artemia spp.) used for prophylactic chemotherapy of fish". Antimicrob Agents Chemother.; 35:2486-2489. Chemistry |107 https://doi.org/10.30526/30.3.1606 7302(عام 0العدد ) 03مجلة إبن الهيثم للعلوم الصرفة والتطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (3) 2017 10. Amini, H. and Ahmadiani, A. (2007), "Rapid and simultaneous determination of sulfamethoxazole and trimethoprim in human plasma by high-performance liquid chromatography". J Pharm Biomed Anal. 43:1146-1150. 11. Hua, T. (2003), "HPLC determination of dissolution of compound sulfamethoxazole and trimethoprim". Chinese Journal of Pharmaceutical Analysis.. 12. Datta, K. and Das, SK., (1988), "Thin layer chromatographic method for rapid quantification and identification of trimethoprim and sulfamethoxazole in pharmaceutical dosage forms". J Liq Chromatogr Relat Technol.; 11:3079-8309. 13. Teshima, D., Otsubo, K., Makino, K., Itoh, Y. and Oishi, R. (2004), "Simultaneous determination of sulfamethoxazole and trimethoprim in human plasma by capillary zone electrophoresis". Biomed Chromatogr. 18:51-54. 14. Juan José, B. N., Gregorio, C. P. and Francisco, G. B. (2005), "Micellar electrokinetic chromatography method for the determination of sulfamethoxazole, trimethoprim and their main metabolites in human serum". J Sep Sci.; 28:543-548. 15. Sun, Z., Li R, Li Y, Wang, K., Zhang ,Q. and Zhou, J. (2001), "Determination of sulfamethoxazole in compound sulfamethoxazole tablet by first derivative ratio spectrometry". Guang Pu Xue Yu Guang Pu Fen Xi. 21:713-715. 16. de Cordova, M. F., Barrales, P. O., Torne, G. R. and Molina D. A. (2003), "A flow injection sensor for simultaneous determination of sulfamethoxazole and trimethoprim by using Sephadex SP C-25 for continuous on-line separation and solid phase UV transduction". J Pharm Biomed Anal.; 31:669-677. 17. Zheng, N.; Yuan-Zong, Li. and Mei-Juan Wen. (2004), "Sulfamethoxazole-imprinted polymer for selective determination of sulfamethoxazole in tablets". J Chromatogr.; 1033:179-182. 18. Lichtenwalner, DM.; Suh, B.; Lorber, B. and AM. Sugar. (1979), "Rapid assay for determination of trimethoprim and sulfamethoxazole levels in serum by spectrofluorometry". Antimicrob Agents Chemother.; 16:579-583. 19. Feng, Li M. and Chemical, Y. (2006), "Fluorescence spectrophotometric determination of sulfamethoxazole in sinomin". Chemical world-shanghai.; 47:215-216. 20. Rodriguez, M. R., Pizzorno, M. T. and S. M. Albonico, (2006), "NMR determination of trimethoprim and sulfamethoxazole in tablets and powders". J Pharm Sci.; 66:121-123. 21. MR. Prasad, M. Sugmaran and CS. Vidyanadhan. (1977), "Profiles of Drug Substances, Excipients and Related Methodology" Analyst, Biochem. 80, 483,. 22. Viplava Prasad, U.; Divakar, TE; Ramireddy, RV and. CSP Sastry. (1988), "Visible Spectrophotometric Analysis of Almotriptan Malate In Bulk And Formulations" J. food Sc. Technol., 25(1), 42-43. Chemistry |108 https://doi.org/10.30526/30.3.1606 7302(عام 0العدد ) 03مجلة إبن الهيثم للعلوم الصرفة والتطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (3) 2017 Table (1) : Effect of reaction time on the color development in the estimation of (50.0 μg.mL -1 ) SMZ. Time (min.) Absorbance 3 0.185 5 0.211 6 0.225 7 0.243 8 0.233 10 0.218 15 0.201 Table (2) : Effect of temperature on the color development in the determination of (50.0 μg.mL -1 ) SMZ. Temperature °C Absorbance 20 0.213 25 0.243 30 0.207 35 0.165 40 0.168 45 0.161 50 0.146 Table (3) : The levels of the experimental variables. Factors Levels -1 0 +1 Vol. of 0.2% SNP solution(mL) 0.5 2.0 3.5 Vol 0.4% NH2OH.HCl solution (mL) 0.5 1.5 2.5 Vol. of 6% Na2CO3 solution (mL) 0.1 0.6 1.0 Chemistry |109 https://doi.org/10.30526/30.3.1606 7302(عام 0العدد ) 03مجلة إبن الهيثم للعلوم الصرفة والتطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (3) 2017 Table (4) : The experimental conditions and their response based on CCD using of (50 μg.mL -1 ) SMZ solution. No Vol. of 0.2% SNP sol.(mL) Vol. of 0.4% NH2OH sol. (mL) Vol. of 6% Na2CO3 sol. (mL) Abs 1 2.0 1.5 0.6 0.132 2 3.5 2.5 1.1 0.092 3 0.5 2.5 0.1 0.000 4 0.5 1.5 0.6 0.034 5 2.0 1.5 0.6 0.135 6 0.5 0.5 1.1 0.108 7 2.0 2.5 0.6 0.200 8 2.0 1.5 0.6 0.139 9 2.0 1.5 0.6 0.135 10 2.0 1.5 0.6 0.133 11 2.0 0.5 0.6 0.076 12 2.0 1.5 1.1 0.110 13 3.5 0.5 1.1 0.000 14 3.5 0.5 0.1 0.181 15 2.0 1.5 0.1 0.000 16 0.5 0.5 0.1 0.199 17 0.5 2.5 1.1 0.020 18 3.5 2.5 0.1 0.018 19 3.5 1.5 0.6 0.122 20 2.0 1.5 0.6 0.133 Table (5) : The coefficients of the nonlinear polynomial model and p values. Variable Regression coefficient Standard error of coefficient t-value P Constant 0.23160 0.09502 2.43732 0.03501 SNP vol. 0.03187 0.07159 0.44512 0.66572 (SNP vol.)2 -0.01295 0.01622 -0.79823 0.44328 NH2OH vol. -0.20689 0.11761 -1.75919 0.10905 (NH2OH vol.)2 0.03086 0.03650 0.84555 0.41757 Na2CO3 vol. 0.11141 0.19885 0.56025 0.58764 (Na2CO3 vol.)2 -0.20855 0.14600 -1.42835 0.18367 SNP vol.× NH2OH vol. 0.01800 0.01427 1.26164 0.23571 SNP vol.× Na2CO3 vol. -0.00600 0.02853 -0.21027 0.83768 NH2OH vol.× Na2CO3 vol. 0.09150 0.04280 2.13779 0.05825 Chemistry |110 https://doi.org/10.30526/30.3.1606 7302(عام 0العدد ) 03مجلة إبن الهيثم للعلوم الصرفة والتطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (3) 2017 Table (6) : Some optical characteristics and statistical data for the determination of SMZ via the recommended procedures. Parameter Univariate CCD λmax (nm) 512.0 Color Orange Linearity range (µg.mL -1 ) 5.0-150.0 Regression equation y = 0.0045 [SMZ ug.mL -1 ] + 0.0052 y = 0.0049 [SMZ ug . mL -1 ] + 0.0001 Calibration sensitivity (mL.µg -1 ) 0.0045 0.0049 Correlation coefficient (r) 0.9997 0.9993 Correlation of linearity (R 2 ) 0.9995 0.9988 Molar absorptivity (L.mol -1 .cm -1 ) ε =1.1399×10 3 ε = 1.2412 x 10 3 Sandell's sensitivity (µg.cm -2 ) 0.222 0.204 Detection limit (µg.mL -1 ) 0.76 0.69 Quantification limit (µg.mL -1 ) 2.533 2.326 Table (7) : Accuracy and precision for the estimation of SMZ by the recommended method. Method Conc. of SMZ (µg.mL -1 ) *Relative Error % C.V. % Taken Found* For univariate 30 29.79 -0.700 0.183 70 70.51 0.729 0.391 120 119.36 -0.533 0.435 For CCD 30 30.105 0.350 0.137 70 70.321 0.459 0.265 120 120.112 0.093 0.282 *Average of five determinations. Table (8): Percent recovery for (50.0 µg.mL -1 ) of sulfamethoxazole in the presence of 200 µg.mL -1 of excipients. Sulfamethoxazole Conc. Taken (50.0 µg.mL -1 ) Taken SMZ concentration (µg.mL -1 ) Excipients Recovery* % Conc. Found* (µg.mL -1 ) 100.22 50.11 50 Sucrose Dark color with precipitate Vanillin 100.50 50.25 Glucose 98.82 49.41 Lactose 99.66 49.83 Starch *Average of three determinations. Chemistry |111 https://doi.org/10.30526/30.3.1606 7302(عام 0العدد ) 03مجلة إبن الهيثم للعلوم الصرفة والتطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (3) 2017 Table (9) : Application of the recommended method for the determination of SMZ in pharmaceutical preparations. Sample Conc. taken (µg.mL -1 ) *Conc. found (µg.mL -1 ) Recovery % C.V % TRIMOL/tablet Julphar, U.A.E 50.00 49.95 99.90 1.101 Trimoks syrup ATABAY, Turkey 50.00 50.13 100.26 1.097 Methoprim tablet (SDI-Iraq) 50.00 48.4 96.80 1.136 *Average of three determinations. Figure (1): Absorption spectra of (A) reaction product of (50.0 μg.mL -1 ) SMZ against reagent blank, (B) reagent blank against distilled water, under the optimum conditions. Figure (2): Effect of (a) volume 0.2% (w/v) SNP solution, (b) volume of 0.4% (w/v) NH2OH.HCl solution, (c) volume of 6% Na2CO3.H2O on the formation of SMZ-SNP complex. 0 0.05 0.1 0.15 0.2 0.25 0 0.5 1 1.5 2 2.5 3 A b so r b a n c e Chemistry |112 https://doi.org/10.30526/30.3.1606 7302(عام 0العدد ) 03مجلة إبن الهيثم للعلوم الصرفة والتطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (3) 2017 Figure (3): The response surface for the absorbance of sulfanilamide-SNP complex as a function of any pair of variables and keeping third variable constant at its optimum value. Figure (4): Calibration plot for SMZ under optimal conditions established by univariate optimization. Figure (5): Calibration plot for SMZ under optimal conditions established by multivariate CCD optimization. y = 0.0045x + 0.0052 R² = 0.9995 0 0.2 0.4 0.6 0 30 60 90 120 150 A b so r b a n c e Concentration of SMZ (µg.mL-1) y = 0.0049x + 0.0001 R² = 0.9988 0 0.2 0.4 0.6 0.8 0 25 50 75 100 125 150 A b so r b a n c e Concentration SMZ (µg.mL-1) Chemistry |113 https://doi.org/10.30526/30.3.1606 7302(عام 0العدد ) 03مجلة إبن الهيثم للعلوم الصرفة والتطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (3) 2017 Scheme (1): The structural formula of sulfamethoxazole. Scheme (2) : The suggested reaction mechanism for charge-transfer reaction between SMZ and sodium nitroprusside.