Spectrophotometric Determination of Sulfamethoxazole Based on Charge-Transfer Complexation with Sodium Nitroprusside

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.

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 Na 2 CO 3 .H 2 O 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 NH 2 OH.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 Na 2 CO 3 .H 2 O and diluted in 100mL-volumetric flask with distilled.Other dilute solutions were freshly prepared by subsequent dilution with distilled water as required.

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) Na 2 CO 3 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.

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, Na 2 CO 3 , 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 NH 2 OH.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% Na 2 CO 3 .H 2 O was sufficient for attaining the maximum and constant absorption intensities.Under these conditions the optimum time 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, NH 2 OH.HCl, and Na 2 CO 3 .H 2 O) 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.