Development of a Spectrophotometric Technique for The Estimation of Sulfamethoxazole (SMX) in its Pure also in Pharmaceutical Formulation, using organic reagent (A1)

   A sensitive, simple, as well as accurate spectrophotometric technique to the estimation of Sulfamethoxazole (SMX) drug in pure also pharmaceutical formulations was developed. It was created by the reaction of (SMX) and organic reagent (A1) after oxidation by iron chloride and potassium periodate in acidic medium. The absorption of the product of SMX and A1 was measured at 423 nm. Linearity ranged between (0.5-22.5 μg. mL-1), molar absorptivity was (6.839 *10+3L / mol.cm), limits of detection and quantification were (0.204, 0.612 μg/ml ), respectively, the method was applied successfully to the estimation of SMX in pure and pharmaceutical formulations.


Apparatus
PG Instrumental Ltd UV-Visible Spectrophotometer, UK T90 using 10-3 m quartz cell for all spectrophotometric quantities, 210 S kern sartorius balance using all weight measurements were used.

Chemicals
Sulfamethoxazole was provided by the state Company for Drugs Industry and Medical Appliances Samarra-Iraq.The solutions were prepared by using distilled water; as 1000 µg.ml -1 of A1 was ready via dissolving 0.05 gm in 50ml of hot distill water with a stirrer in volumetric bottle 50mL.A 500 µg.mL -1 SMX of solution ready by dissolving the exact weights of in 10mL absolute ethanol then added distilled water to the mark in volumetric bottle 100 mL, kept in dark place then used, for at least 10 days , as standard solution.More dilute working solutions of the SMX solution prepared by serial dilutions by distilled water Solutions of iron chloride and potassium periodate (with 0.01M for each) were prepared and used,0.1M of each of hydrochloric acid , sulfuric acid nitric acid and acetic acid prepared then used .

Solutions of Interference 1000 μg/mL
These solutions ready via dissolving 0.1gm of (glucose, fructose, lactose, sucrose and vanillin) in right solvent (water or ethanol) completed volume to 100ml by D.W.

General procedure for estimation of Sulfamethoxazole SMZ with A1 reagent
The primary test for the present method involved oxidation of 0.3ml A1 organic reagent 250μg/mL by 0.1ml FeCl3 0.1ml KIO4 of 1x10 -2 M and then add 0.5ml of SMX drug 250 μg/mL in acid medium the contents were diluted to the mark with D.W in 10 ml volumetric bottle.Absorbance and ʎmax of brown color solution at 423nm against blank organized in same manner without SMX Figure2 shows that the maximum absorption was obtained at a wavelength 423 nm.

6. Sample preparation
A standardized powder prepared from 10 exactly weighed metheprim tablets (0.4934gm).A suitable quantity of the powder dissolved in absolute ethanol and added D.W to the mark in volumetric bottle 100mL.Dissolution of sample by an ultrasonic bath.the mixture filtered and made up to the mark with D.W in 100mL volumetric bottle to obtain solution (250 µg/mL) 2.7.Condition's reaction optimization Numerous conditions studied are affecting remove of the absorbance.The effect of the kind of acids and reagent of oxidation, the quantity of (HCl, , potassium periodate, FeCl3 and A1 reagent) was studied.Result that use 1.00 ml of 0.1 M HCl i.e pH=4,0.500ml of 0.01M of KIO4, and 0.6 ml of FeCl3 and 0.6 of 250µg.ml-ofA1 reagent give best results Figures.3-5 respectively.The order of adding reactants must be followed as mentioned in the mentioned procedure solution was left 5 minutes before adding distilled water.Figure.6.

The statistical data &calibration curve
The Calibration curve was fabricated Using-conditions Figure .7 product follows Beer's law in A variety of concentrations 0.5-22.5 μg/ml SMX.Table 1.

Precision and accuracy
The method's efficiency was statistically calculated by evaluating accuracy as relative error percentage (Erel % and precision as RSD percent of the suggested methods.Table 2 shows that the results found for 6 repeats at 3 concentrations of SMX sample which show that the suggested methods ensure a good accuracy and precision.Tabte 1 illustrate the statistical information to the calibration curve such wave length , linear rang ,Molar absorptivity …etc.

Stoichiometry of reaction
Figure 8 Proposed mechanism of reaction.

Study of Interference
For test selectivity towards excipients added to the pharmaceutical formulation, such as (glucose, fructose, lactose, sucrose and vanillin), which do not interfere the estimation of SMX and do not affect the reaction (10μg.ml - ) of SMX.Thus, interference was analyzed.The results in Table 3.

Figure 2 .
Figure 2. SB: Spectrum of sample solution against blank, and SW: Spectrum of solution against water.

Figure. 6
Figure.6Effect of time on absorbance at 423nm.

Figure. 7
Figure.7 Calibration curve for the estimation of SMX.

Table . 2
Valuation of accuracy and precision. *n=6

Table . 3
Rec. for (10μg.ml - ) of SMX in the presence of diverse concentration of excipients SMX

Table . 4
Estimation SMX in formulation pharmaceutical Results confirmed that a proposed technique is simple with sensitivity to the estimation of SMX.Colored product showed an absorption maximum at 423 nm.It was found that the proposed method was highly efficient and recoverable with a high linear range.It did not use solvent extraction or organic solvents.It could be applied successfully to the estimation of SMX in formulations of pharmaceutics.