Spectrophotometric determination of Sulphamethoxazole Via Charge Transfer Complex Formation Reaction

ةصلاخلا ف مم فسممفي ستف ممت ف ف فمملو ةفقف الستفيفبمميتسفسمملفلس فسطفممل فسممف فطفسمميفيطفةممط فصممت ف فم يتوستسفسمس فقفمبفمقوف فملو ةفقف الستف عف تفىاعفسيفيطستفبقتعتف.سف لابفطستف تت يضستلق ةي أفعق فميعقفنف وتسف ف يتوستسفسا يتلقفمبفقوف ف ت ي او ف مل ستفن ماستفا فسمايت قستفس سمشستفب ف مقستف طستفب عفصفطتقتفىطقأفكاتقففا ست 539 ف فىماةقستفة يم ستفبم عفبم فبميسف.يتفق فم فب بمممممس ف ممممم ط ففيممممف فن فممممقفنأ 2 60 ف فسمممممفيلا قفسفمممممطفطتقف فيتااقرصت يم يوففمممممق 3.675 × ف 3 10 ف ف ممممقوستفبممممسست فةممممشوستفبممممسفبمممم ففممممقوف.صممممل. قريتس 0.371 ف ف 1.236 ف ت يم يوففممممق فىمممماعفيتااقرص فسفعف يتمللاتفس مل ف بمعقفنفموف. ست متست 100.95 ف مل ستف ملففيستفةت يمس لات ف% ≤ %1.06 ف فصمتف. ف م ف فملو ةفقف الستفيفبميتف م فقفم فسميفيطستف مي طف. معف تستفسمفوف فوفق فبميعقستفسعف طفسلت يب .نفبف ف ت ت فسقب فبت يش فصت يقأف وشفىاعفسف لابفطستف تت يضستلق ف ف

The methods that are based on charge-transfer complexation are usually sensitive, rapid and simple to perform.Charge transfer reactions have been widely used for the determination of electron donating compounds through interaction with π-acceptors.This work describes a simple, rapid and sensitive spectrophotometric method for the determination of SMZ, in its pure form and pharmaceutical formulations containing trimethoprim and other excepients, by exploiting its electron donating property.The method is based on the charge transfer complexation reaction of SMZ with ochloranil.

Apparatus
Shimadzu UV-1650 PC UV-Visible spectrophotometer equipped with a 1.0-cm path length silica cell, Philips PW (9421) pH-meter with a combined glass electrode was used for pH measurements, All calculations in the computing process were done in Microsoft Excel for Windows.Weighing was carried out on a balance type of Mettler H 54 AR.Chemicals Sulphamethoxazol and its pharmaceutical formulations (tablet and syrup) were kindly provided by state company for Drug Industries and Medical Appliance-(SDI) Sammara-Iraq.o-Chloranil was obtained from MOLEKULA and other chemicals were obtained from Fluka and BDH companies.All solvents were analytical reagent grade and water was distilled.
Working standard solution of sulphamethoxazol: 250 μgml -1 sulphamethoxazole solution was prepared by dissolving of 25 mg of its pure form in 5 ml ethanol and diluted to 100 ml with distilled water in a volumetric flask.Further dilution, to obtain 100 μgml -1 , has been prepared.
Reagent solution: 5×10 -3 M o-chloranil solution was prepared by dissolving 0.123 g in absolute ethanol and diluted to 100 ml in a calibrated flask with the same solvent.
Basic solutions: 0.01 M sodium carbonate and sodium hydroxide were prepared in distilled water.

Recommended procedure
Aliquots of the working solution of sulphamethoxazol (2-60 μgml -1 ) were transferred into a series of 5 ml calibrated flasks.Then, 1ml of 5×10 -3 o-chloranil and 0.75 ml of 0.01 M Na 2 CO 3 were added and the solutions were diluted to the mark with ethanol.The absorbance was measured at 539 nm at room temperature against reagent blank.

Tablet
Ten tablets (each tablet containing 400 mg sulphamethoxazole) were accurately weighed and pulverized.A portion of the fine and homogenized powder equivalent to 400 mg sulphamethoxazol was accurately weighed and dissolved in about 10 ml of water-ethanol (50:50 v/v) mixture with mixing and heating for 5 min, then filtered with Whatmann filter paper no.1.The filtrate was diluted to the 100 with distilled water in a volumetric flask obtaining 4000 μgml -1 .a suitable volume was diluted, and the above procedure was followed.

Syrup
The appropriate volume of the syrup containing SMZ equivalent to 10 mg was transferred into a 100 ml measuring flask, diluted, filtered and made up to the mark with distilled water.An aliquot of the solution was analyzed, as described in recommended procedure.

Results and discussion
The proposed method involves the and reaction of SMZ with ochloranil reagent in the presence of Na 2 CO 3 to form a violet colored charge transfer complex having maximum absorption at 539 nm.This wavelength was used for all subsequent measurements.The absorption spectra of the reaction product are shown in Figure 1.The corresponding reagent blank have low absorbance at this wavelength.

Optimization of experimental conditions
The optimum conditions for the color development of the o-CA-SMZ complex were established by varying the parameters one at a time, keeping the others fixed and observing the effect produced on the absorbance of colored species.The following experiments were conducted for this purpose and conditions so obtained were incorporated in general procedure.

Effect of solvents
Different solvents such as methanol, ethanol, acetonitrile, acetone and water as medium for the reaction, between SMZ (20 μgml -1 ) and o-CA (1ml of 5×10 -3 M), in the presence of NaOH (1ml of 0.01M) in final volume of 5 ml, have been tried in order to achieve maximum sensitivity and complex stability.The absorbance of solutions were measured against corresponding blank after 5 min at room temperature.As shown in table1.It was found that on using water as solvent for sulphamethoxazole and ethanol as solvent for o-CA and dilution with ethanol were gave maximum color intensity and recommended in this method.However; dilution with water gave turbid solutions.

Effect of pH
The effect of pH on the absorption of the complex was studied using different pH values.It was observed that the complex was formed with low sensitivity at pH 3.34, but this sensitivity was increased by addition of NaOH and reached its maximum absorption at pH 5.1 (Fig. 2).Therefore different buffers of pH 5.1 were prepared to examine the sensitivity.A negative effect was observed on the color intensity.To obtain high sensitivity for the complex, different bases such as sodium hydroxide, potassium hydroxide, sodium carbonate and sodium bicarbonate with fixed volume and a concentration of 0.01M were examined by addition to a fixed amount of SMZ.It was found that sodium carbonate gave maximum color intensity (Figure 3), and the optimum amounts of this base were found to be 0.75 ml which was used in the subsequent experiments.

Effect of o-CA concentration
The effect of changing the o-CA concentration on the absorbance of solution containing a fixed amount of SMZ was studied.It was observed that the absorbance increases with increasing o-CA concentration and reached maximum on using 1.0 ml of 5×10 -3 M o-CA (Figure 4).Therefore, this volume of this concentration was used in the subsequent work.

Effect of surfactant
Effect of various surfactants including sodium dodecyl sulphate (SDS), cetylperydinum chloride (CPC), cetyltrimethylammonium bromide (CTAB), Tween-80 and Triton x-100 were tested.It was found that these surfactants decreased the absorbance of solutions.The reaction time was determined by following the color development at room temperature and in thermostatically controlled water-bath at different temperatures up to 60 ○ C. The absorbance was measured at 5 and 10 minutes intervals against reagent blank treated similarly.It was observed that the complex was formed after addition of o-CA immediately at room temperature and no effect of high temperatures on the maximum absorbance and stability of the complex (>5hr), therefore room temperature (25 ○ C) was selected.

Order of addition
The order of addition of reactants on the color development was examined.Maximum sensitivity was achieved when SMZ and o-CA were added before adding the sodium carbonate as shown in Fig. 5. Hence, the method was performed in the order: SMZ + o-CA + Na 2 CO 3 .

Quantification
In order to investigate the range in which the colored complex adhere to Beer's law, the absorbance of the complex was measured at 539 nm after developing the color by following the general procedure calibration graph for a series of solutions containing increasing amounts of SMZ.The Beer's law limits and molar absorptivity values were evaluated and given in Table 2, which are indicated that the method is sensitive.The linearity was represented by the regression equation and the corresponding correlation coefficient for the studied determined drugs by the proposed method represents excellent linearity.The relative standard deviation (RSD) and accuracy (average recovery %) for the analysis of five replicates of each three different concentrations for SMZ indicated that the method is precise and accurate.Limit of detection (LOD) are in the accepted range below the lower limit of Beer's law range.

Interference
The extent of interference by some excipients which often accompany pharmaceutical preparations were studied by measuring the absorbance of solutions containing fixed amount of SMZ and various amounts of diverse species, including trimethoprim which is present the pharmaceutical formulations of SMZ, in a final volume of 5 ml.It was found that the studied excipients up to 22.5 fold excess did not interfere seriously (Table 3).However; an error of 5.0 % in the absorbance readings was considered tolerable.

Stoichiometry, stability constant and mechanism
The molar ratio of the n-π charge transfer complex formed between the SMZ and o-CA reagent was investigated by applying the continuous variation (Job's) and mole ratio methods [16].The results indicated that Parameter SMZ Beer's law limits (μg ml -1 ) 2-60 Molar absorptivity (l.mol -1 .cm -1 ) 3.675×10 complex was formed in the ratio of 1:1 (Figures 6).This finding supports that the n-π CT complex is formed through amino group.The stability constant (K st ) of the complex was determined according to the previous ratio and found 1.897×10 3 l.mol -1 .However; the probable reaction mechanism based on the reported method [17] is given in scheme 1.

Analytical applications
The proposed method was successfully applied to determine SMZ in pharmaceutical tablets and syrup preparations.The validity of the method was confirmed by applying the standard addition procedure, (Fig. 7) and the results suggested that there is no interference from any excipients, which are present in commercial dosage forms, Table 4.

Conclusion
The proposed method is sensitive (trace amounts can be determined), accurate (average recovery range 100.95 %), precise (RSD ≤ 1.06) and simple since it does not need neither temperature control nor solvent extraction step.Analysis of authentic samples containing SMZ showed no interference from common additives and auxiliary substances in general.Hence, this method could be considered for the determination of SMZ both in pure form and in pharmaceutical preparations.

Figure 2 :
Figure 2: Effect of pH on the absorption of 20 μgml -1 SMZ complex with o-CA Effect of bases

Figure 3 :
Figure 3: Effect of Different bases on the intensity of 20 μgml -1 SMZ complex with o-CA

Table 2 :
Summary of optical characteristics and statistical data for the proposed method* Average of five determinations.** Y = a X + b, where X is the concentration of drug in μg ml -1 .

Fig. 7 :
Scheme 1: Probable mechanism for the reaction of o-CA with SMZ

Table 1 : Effect of solvents on the absorbance of o-CA-SMZ complex
* Sample against blank ** Blank against solvent at λmax of complex

Table 4 : Assay of SMZ in pharmaceutical preparations using the proposed method and comparison with the standard addition method
a Average of three determinations.