Spectrophotometric Determination of Salbutamol Sulphate and Isoxsuprine Hydrochloride in Pharmaceutical Formulations

: A simple, sensitive and accurate spectrophotometric method has been developed for the determination of salbutamol sulphate (SAB) and isoxsuprine hydrochloride (ISX) in pure and pharmaceutical dosage. The method involved oxidation of (SAB) and (ISX) with a known excess of N-bromosuccinamid in acidic medium, and subsequent occupation of unreacted oxidant in decolorization of Evans blue dye (EB). This, in the presence of SAB or ISX was rectilinear over the ranges 1.0-12.0, 1.0-11.0 µg/mL, with molar absorptivity 4.21×10 4 and 2.58×10 4 l .mol -1 .cm -1 respectively. The developed method had been successfully applied for the determination of the studied drugs in their pharmaceutical dosage resulting in a good agreement with certified value and standard addition procedure.


Isoxsuprine
(ISX); p-hydroxy-N-(1-methyl2-phenoxyethyl) norephedrine hydrochloride, Fig. 2, is a vasodilator that produces the effects of β-adrenoceptor stimulation and αadrenoceptor antagonism; the former effect is the more predominant. It is used in the treatment of cerebral and peripheral vascular diseases [3][4] . It has a direct relaxant effect on the smooth muscular tissue of the blood vessels and uteruses also; it is used to arrest premature labor 5 .

Experimental: Instrument
All the absorption spectral measurements were employing UV-1650 PC UV-Visible spectrophotometer equipped with 1.0 cm matched quartz cells.

Reagents and Chemicals
Salbutamol sulphate and isoxsuprine hydrochloride are from the State Company for Drug Industries and Medical Appliances. Solutions of SAB and ISX were prepared in a concentration of 50μg/mL by dissolving 0.01 g of each drug in 200 ml distilled water in volumetric flasks. Evans blue dye (100μg/mL) was prepared by dissolving 0.025 g in distilled water in a 250 mL volumetric flask. N-Bromosuccinimide (NBS) was prepared of 200 μg/mL by dissolving 0.02 g in 100 mL distilled water. Hydrochloric acid was prepared in a concentration of 2 M by diluting 50 mL of conc. HCl (10 M) with distilled water in a 250 ml volumetric flask.

General Procedure
Into two series of volumetric flasks (10 mL) aliquots of solutions containing 1-9 and 1-10 μg/mL of SAB and ISX respectively, were added separately, followed by addition of 1 mL of 2M HCl and 2 mL of 200 μg/mL NBS to each flask. The solutions were gently shaken and left for 10 min at room temperature for oxidation. 2.5 mL of 100μg/mL EB were added to the solutions. The flasks were diluted to the mark with distilled water, mixed well and measure the absorbance at 600 nm after 5 min at room temperature versus reagent blank

Procedure for the Pharmaceutical Preparations Analysis of Tablet
Ten tablets were weighed and pulverized into a fine powder from each of butadin (each tablet containing 2 mg SAB) and dulivan (each tablet containing 10 mg ISX). An accurate weight, equivalent to 10 mg of pure drug, was dissolved in 100 mL distilled water, and then filtered after spraying in the ultrasonic device for 5 minutes to obtain a solution with a concentration of 100 μg/mL. A suitable volume was diluted with distilled water and followed the recommended procedure. Syrup 25 mL of butadin syrup of 2mg/ 5mL was transferred into a100 mL volumetric flask and diluted with distilled water to obtain 100 μg/mL of SAB. Aliquots of this solution were treated as described under the recommended proceedings.

Results and Discussion:
This suggested method is the indirect determination of phenolic drugs SAB and ISX involves the oxidation of these drugs by NBS in acidic medium, scheme 1 and the residual oxidant bleaches the blue color of EB dye 19 , scheme 2. When the known volume of NBS is added to an increasing amount of drug, there was a decrease in the concentration of oxidant and increasing the absorbance of EV dye, Scheme1 and Fig. 3.

Optimization of Experimental Variables Optimum Amount of Evans Blue Dye
The preliminary experiments were performed to optimize the useful and optimum concentration of EB dye that can be determined spectrophotometrically. The results indicated that 250 μg/mL from EB dye was found to be a useful agent for reaction, Fig. 4.   Figure 5 shows that 1.5 mL of 200 μg/mL NBS solution was enough to obtain maximum bleaching of the color of EB dye therefore it was recommended in the subsequent experiments.

The Effect of Acid
Experimental results show that the oxidation of EB dye and the studied drugs by NBS take place in an acidic medium. Therefore, the effect of different amounts from various acids (2 M) have been tested, using 8 μg/mL of each drug, to obtain high sensitivity. It was found that HCl is the best acid for the system (Table 1). In addition, 1 mL of 2M HCl was selected as optimum amount for two drugs as shown Fig. 6.

The Effect of Time on Oxidation
The effect of oxidation time of SAB and ISX drugs was studied by adding 1.5 mL of 200 μg/mL NBS to 8 μg/mL for each drug in the presence of 1mL of 2N HCl. The solutions were shaken and left at room temperature for different periods. Then 2.5 mL of 100 μg/mL EB were added to each drug and the solutions were agitated and diluted to 10 ml in volumetric flasks. The absorbance of the residual EB was measured after 5 min standing time at 600 nm against blank solution. The results obtained in Table 2 indicated that 10 min is sufficient for the oxidation of drugs and the absorbance remain constant for one hours.

Calibration Curves
Under the described experimental conditions, standard calibration curves for, salbutamol and Isoxsuprine drugs with EB dye were constructed by plotting absorbance against concentration (Fig.7). Beer's law limits, molar absorptivity values and Sandell Sensitivity 20 were evaluated and given in Table.3, as well as the limit of detection (LOD) and limit of quantitation (LOQ) 21 were calculated according to the following equations: LOD = 3σClow/X ̅ , LOQ = 10σClow/X ̅ Where (σ) is the standard deviation of absorbance of low concentration and X ̅ is the average absorbance of low concentration.

Accuracy and Precision
The accuracy and precision was evaluated by calculating the recovery ratios and the relative standard deviation of three different concentrations of each drug compound. The results in Table 4, indicate the method is precise accurate.

Analytical Applications
The suggested method was tested for the determination salbutamol and isoxuprine in some of their pharmaceutical dosage. The concentration of drugs was calculated by direct measurement on appropriate standard calibration curve ( Table 5). The similar results were obtained by applying the standard addition technique Fig. 8, indicating that suggested method is free from interferences.

Conclusion:
The suggested method described the successful development of simple, accurate and sensitive spectrophotometric method for the determination of salbutamol and isoxuprine using N-bromosuccinamid as oxidant agent of the two drugs. The unreacted N-bromosuccinamid bleached the Evans blue dye. The method has been applied successfully to determine salbutamol and isoxuprine in various pharmaceutical formulations.