SPECTROPHTOTOMETRIC DETERMINATION OF PURE SULFAMETHOXAZOLE IN PHARMACEUTICAL PREPARATIONS BY OXIDATIVE COUPLING REACTION

A new, simple, rapid and sensitive spectrophotometric method for the determination of sulfamethoxazole in both pure form and pharmaceutical preparations has been reported.The adapted technique based on utilization 4-aminobenzene sulfonic acid as a new modern chromogenic through an oxidative coupling reaction with sulfamethoxazole and potassium iodate in basic media to form orange soluble dye product with absorption maxima at 490 nm. Subject to Beer's law in the range 2–32μg mL-1. The values of molarabsorption coefficient (ε) and correlation coefficient were found to be 9.118 × 103 and0.9999 respectively whereas the Sandels index was 0. 02778 µg.cm-2


INTRODUCTION
Sulfamethoxazoloe (SMZ) is an isoxazole (1,2-oxazole) compound having a methyl substituent at the 5-position and a 4-aminobenzenesulfonamido group at the 3-position. It has a role as an antibacterial agent. IUPAC Name is [4-

MATERIALS AND METHODS Instruments
Absorbance measurements were performed using T92 + Spectrophotometer wavelength at the range (190-900) nm. (Beijing, China).Measurement was performed by a spectrophotometer in an analytical chemistry laboratory. (Table 1)shows the chemicals used.

Pharmaceutical preparations of sulfamethoxazole
Pharmaceutical preparations from industrial sources were collected as follows: 1.Trimoks Pills of (SDI, Samarra-Iraq): 400 mg sulfamethoxazole and 80 mg trimethoprim for each Pills. 2. Sulprim syrup (Jerusalem Pharmacy products Co.Ltd-palestine):200 mg sulfamethoxazole and 40 mg trimethoprim for each 1 mL ofsyrup (100 mL). It was prepared by taking 1 mL of the Sulprim and diluting it with a small amount of distilled water and filtering and thecomplement filtrate with distilled water to the mark to obtain a concentration of 100 µg / mL.
The principle of the method is based on the coupling of the reagent (4-aminobenzene sulfonic acid) with (Sulfamethoxazole) in the presence of potassium iodate as oxidizing agent in basic medium.

Preliminary study
We put (1 mL, 2×10 -2 M) of reagent 4-aminobenzensulfonic acid and add an oxidizing agent potassium iodate(1 mL, 2×10 -2 M) to 2 mL Sulfamethoxazole (100µg/ mL) a orange product was formed. The intensity of the color increased in the presence of 1 mL (0.2 M) of NaOH solution. Absorbance measurements were performed after diluting the solution with

RESULTS AND DISCUSSION Study of the optimal reaction conditions
Experiments were performed using 2 mL of Sulfamethoxazole 100 µg/ mL in a final volume of 25 mL and the absorbance values of the solutions was measured at 490 nm wavelength versus a blank solution.

Effect of the best coupling reagent
Series of experiments were held (performed) by mixing 1mL of each of the reagent solutions shown below in (Table 2) with 1 mL of (2 × 10 -2 M) of potassium iodate as oxidizing agent solution with2 mL of (100µg/ mL) sulfamethoxazolesolution in the presence of 1 mL NaOH solution 0.2 M and the absorbance values were recorded at 490 nm wavelength. Oxidizing agent optimization: 1 mL, 2×10 -2 M of series of oxidizing agents were added to 1 mL, 4-amino benzene sulfonic reagent solution,2 mL of (100µg/ mL) sulfamethoxazole solution and 1 mL, 0.2 M NaOH solution in 25 mL volume flask followed by absorption measurements. For each sample,the blank solution was fixed at wavelength (800-400) nm. The best oxidizing agent was noted to be KIO 3 for giving the maximum absorption value of the colored product at the wavelength 490 nm. The results are shown in (Table 3).

Effect the base used for coupling
One mL of different types of bases (strong and weak) has been used; optimum results wererecorded in the case of NaOH as shown in the (Table 5).

Effect of the amount of base used
Various quantities of the used base (NaOH) have been added that have been selected to find the optimal amount that gives the highest absorption of the formed product. Maximum absorption was recorded in the case of (1 mL) with pH=11.86. This volume was adopted in the subsequent experiments and all the results are shown in the ( Table 6).

Sequence of addition
Sulfamethoxazole (D), reagent solution 4-aminobenzen sulfonic(R), potassium iodate solution (O), and sodium hydroxide (B) alkaline solution were added to each other in different sequence keeping the same volume and concentration thereof. Maximum absorption was recorded in the sequence mode of addition (IV) as shown in (Table 7).

Effect of temperature
The temperature of mixture and the colored product formed was varied at the range 10-70°C. Maximum and stable absorption of the formed colored product solution was observed at the temperature range 20-30°C. 25°C was chosen to be the optimal temperature for the reaction mixture. The details of the results are shown in the ( Table 8).

Stability of the reaction product:
The stability of the reaction product was studied throughout observing the values of absorption of the formed colored solution at duration of time 5-60min using 2 mL, 100µg/mL of the sulfamethoxazole solution and 2 mL of the drug. Results exhibited in (Table 9) shows that 60 min was very sufficient time period to hold the measurements.

Accuracy andprecision
The accuracy of the method represented by relative error (E%) and recovery(Rec%) was calculated to estimate sulfamethoxazole and the compatibility of the method represented by relative standard deviation (RSD%) (measuring three different concentrations) of Sulfamethoxazole 100 μg/ mL, as it appears from the results obtained. (Table10) it demonstrates that the technique has good accuracy andprecision.

Detection Limit
The detection limit was calculated for at the wavelength of 490 nm, by measuring the absorption of the lowest concentration (2 μg/ mL) taken from the calibration curve depicted from (for) seven readings under the same conditions. Results exhibited in (Table 11).

The nature of the formed product
To identify the nature of the formed product and the ratio of the drug's attachment to the detector, both continuous changes method (the Jop method) and molar ratio method (Niknia, 2018) have been applied. Different volumes of drug solution ranging from 1-9 mL were placed into series of 25 mL volumetric flasks containing decreasing volumes of the reagent 9-1 mL. The rest of the additives were in the optimal sizes according to the adopted method of work in this study, followed by filling the bottles to the mark with distilled water. According to absorption measurements of the solutions at 490 nm against their blank solutions the following (Figure 4) was depicted for JOP method. 1:1 ratio found to be the optimal mixing ratio.
According to this method mixture comprising of Sulfamethoxazole with the 4-amino benzensulfonic reagent in the presence of the oxidizing agent potassium iodate, and 1 mL of a NaOH-base solution 0.2M was estimated. To ensure that the interaction ratio between Sulfamethoxazole and the reagent 4-aminobenzen sulfonic is 1:1, the molar ratio method was used and as follows: 2 mL of the drug solution were placed in a series of 25 mL volumetric bottles containing different volumes of reagent solution 0.2-2 mL with the remaining additives at the optimum sizes, and diluted with distilled water to the point of the mark. Absorption measurements of these solutions at the wavelength of 490 nm against the formed solution for each of them were held. Molar ratio was found to be consistent with the method of continuous changes. (Figure 5) shows that the ratio is 1:1. The reaction equation is as ( Figure 6).

Applications
This method can be applied to the following pharmaceutical preparations containing: Sulprim syrups (Jerusalem Pharmaceuticals Co.Ltd-palestine):200 mg sulfamethoxazole and 40 mg trimethoprim for each 5 mL of suspension 100 mL.

The direct method
Three different concentrations were taken from the solution of each product (Suspensions ) whose preparation is indicated in its preparation in the paragraph, namely 10,20, 30µg/mL. The solutions were treated following the same steps in preparing the titration curve and measuring the absorbance for them at the wavelength of 490 nm against the blank solution. An average of five readings was calculated for each, as well as the calculation of retrospective and RSD according to the results shown in (Table 12). The results exhibited in the above table show that the proposed method was successful in estimating the pharmaceutical preparations that contain them. The value of the recovery rate was 100.405% for syrups.

Standard additions method
The standard addition method was used to demonstrate the efficiency of the proposed method, as the method includes the addition of constant quantities (1.3, 2.0 mL) of prepared pharmaceutical solutions at a concentration of 100µg/mL in two series of volumetric bottles of 25mL in capacity and adding increasing volume 1, 1.5,2.5 and 3.5 mLof a standard solution of 100 µg/mLconcentration and leaving one of the bottles without addition. The absorbance of the solutions was measured against the blank solution at the 490 wavelength. The results are shown in (Table 13) and (Figure 7).  From the results shown in the (Table 13), it is shown that the standard addition method is well in agreement with the direct method, within the range of acceptance of the error, which indicates that it is the satisfactory method.

Statistical evaluation of the results of the proposed method
A comparison was made between the proposed analytical method and the standard method to find out the accuracy and validity of the analytical application of the proposed method by applying the following two tests. The obtained result was less than the tabular value for F, t. F=4.36, t=2.84 (which is required). While the tabular value for F, t was F=7.25, t=4.776 at confidence limit 95% and for four degrees of freedom. Consequently, these values demonstrate the success of the proposed method asshown in (Table 13).