Spectrophotometric Determination of Salbutamol Sulphate by Coupling with Diazotized 5-Amino-2-chlorobenzotrifluoride – Application to Pharmaceutical Preparations

A simple, rapid and sensitive spectrophotometic method for the estimation of trace amounts of salbutamol sulphate (SBS) in pharmaceutical preparations has been proposed .The method is based on the coupling reaction of the intended compound with diazotized 5-amino-2chlorobenzotrifluoride in alkaline medium and in the presence of Triton X-100 to form a yelloworange dye that shows maximum absorption at 467 nm. Beer’s law is obeyed over the range 5 – 300 μg / 10 ml (i.e.,0.5-30 ppm) with a molar absorptivity of 4.20 × 10 l.mol.cm and Sandell’s sensitivity index of 0.0139 μg.cm, a relative error of +1.08 to +3.46 % and a relative standard deviation of ±0.76 to ±3.50 %, depending on the concentration. The method has been applied to estimate salbutamol sulphate in syrup, tablet and ventolin.


Salbutamol sulphate
The assay of Salbutamol sulphate officially listed in British Pharmacopoeia describes a potentiometric titration procedure (British Pharmacopoeia, 2007).

THE EXPEREMENTAL Apparatus:
The spectrophotometric measurements were carried out on Jasco V-630 using 1cm glass cells.

Reagents
All chemicals used are of the highest purity available. Salbutamol sulphate solution, 100 µg. ml -1 .
A 0.0100g amount of salbutamol sulphate was dissolved in distilled water, then the volume was completed to 100 ml in a volumetric flask with distilled water. This solution was prepared daily by dissolving 0.0978 g of 5-amino-2-chlorobenzotrifluoride in 10 ml of ethanol then a 3.0 ml of concentrated HCl was added, followed by dilution to 80 ml with distilled water, then the solution was transferred into a 100-ml volumetric flask and cooled to (0 -5) ºC in an ice-bath, a 0.0345 g of sodium nitrite was added then stirred vigorously, after 5 minutes the solution was made up to 100 ml with cooled distilled water and stored in a dark bottle. This solution was prepared by the dilution of the concentrated volumetric solution (1Ampoule, Fluka) to 500 ml with distilled water and then transferred to a plastic bottle. Triton X-100, (1%).
These solutions were prepared by dissolving 0.1 g of each of them in 100 ml of distilled water. Butadine syrup solution, 100 µg .ml -1 .
This solution was prepared by diluting 25 ml of butadine syrup (2 mg salbutamol sulphate per 5 ml) to 100 ml with distilled water in a volumetric flask. Butadin a tablets solution, 100 µg .ml -1 .
Finely 5 powdered tablets of butadine drug (each tablet contains 2 mg salbutamol sulphate) were dissolved in 80 ml of distilled water, and the solution was shaked and warmed. The solution was filtered into a 100-ml volumetric flask, the residue was washed with distilled water and diluted to volume with distilled water to obtain 100 mg/l salbutamol sulphate. Solution, 100 µg .ml -1 .Salb. Vent.

RESULTS AND DISCUSSION Principles of the method
The method included the following steps: -Preparation of diazotized 5-amino-2-chlorobenzotrifluoride.

Optimum reaction conditions
The effect of different factors on the formation of the colored dye is investigated and the reaction conditions have been optimized.
For the subsequent experiments, 100 µg of salbutamol sulphate is taken in 10 ml as a final volume and absorbance measurements are achieved directly after dilution with distilled water at 451.5 nm.

Selection of diazotized reagent
Some diazotized reagents (1 ml of 5x10 -3 M) have been selected for optimum conditions. The results in Table ( 1) show that 5-amino-2-chlorobenzotrifluoride gives the highest intensity with high value of color contrast, therefore, it has been selected for the subsequent experiments.

Effect of pH
Salbutamol sulphate undergoes complete diazo-coupling reaction in alkaline medium (Othman and Zakaria , 2004), so that several bases have been tested Table (3) for optimum conditions (1 ml of 2 M of each base was added). The experimental data in Table (3) showed that the reaction needs a strong alkaline medium and NaOH gives a highest sensitivity with best color contrast, therefore it has been fixed for the subsequent experiments.

The optimum amount of sodium hydroxide
The results in Table (4) indicate that 1ml of NaOH (2 M) gives the highest intensity of the colored dye and the value of determination coefficient (0.999), therefore this volume has been recommended for the subsequent experiments.

Effect of surfactant
The effect of several types of surfactants with different orders of addition on color intensity and color contrast of the dye has been investigated. (Table 5 and Fig. 1).

Table 5: The effect of surfactant on dye absorbance
Note: Absorbance = 0.5259 without surfactant and λ max. = 451.5 nm I.
S + R + C + B III.
S + R + B + C

Fig. 1: The effect of Triton X-100 on absorbance (order II ) A-Sample with Triton X-10 B-Sample without Triton X-100
The results in Table (5) and (Fig. 1) indicate that the addition of Triton X-100 in order (II) increases the intensity of the formed dye and color contrast from 141.5 nm To 158 nm, therefore it has been recommended in subsequent experiments.

The optimum amount of Triton X-100
From the results in Table (6), it was found that 1 ml of (1%) TritonX-100 solution was adequate for the maximum absorbance, therefore it has been used in the subsequent experiments.

Final Absorption Spectra
Absorption spectra of the colored dye formed from treatment salbutamol sulphate with diazotized 5-amino-2-chlorobenzotriflouride reagent in basic medium, in the presence of TritonX-100, according to the above recommended procedure, showed that the maximum absorption is obtained at 467 nm (Fig. 2).

Procedure and calibration graph
To a series of 10-ml volumetric flasks, increasing volumes of aqueous solution containing 5-300 µg salbutamol sulphate are transferred, 4 ml diazotized 5-amino-2-chlorobenzotrifluoride (5x10 -3 M) followed by the addition at 1 ml of (1%) Triton X-100 and 1 ml NaOH (2 M) then the volumes were completed to the mark with distilled water. The absorbance for each flask was measured directly after dilution at 467 nm against blank. The calibration graph is linear over the range 0.5 -30 µg.ml -1 and higher concentrations show negative deviation from Beer's law (Fig. 3). The apparent molar absorptivity referred to salbutamol sulphate, has been found to be 4.2 ×10 4 l. ol -1 .cm -1 .

Interference
The extent of interferences by some excipients which often accompany pharmaceutical preparations as studied by measuring the absorbance of solutions containing 100 µg ml -1 of salbutamol sulphate and various amounts (50,100 and1000) of exaplents in a final volume of 10 ml. It was found that the studied excipients do not interfere in the determination of salbutamol sulphate in its dosage forms. Typical results are given in Table (8).

Application of the method
The proposed method was applied to determine salbutamol sulphate in its pharmaceutical preparations (Butadin syrup, tablet and Salbu. Vent.). The results shown in Table (  The validity of the method was confirmed by applying the standard addition procedure (Al-Abachi, and Al-Ghabsha, 1986) ( Fig. 6). The recoveries calculated by using the equations of the linearity in (Fig. 6) and the results obtained are in agreement with the certified value Table (10).  Table (11) shows the comparison between some of analytical variables obtained from the present method with other spectrophotometric methods.

CONCLUSION
The advantage of the proposed method compared to the reference methods was a higher sensitivity than the refered methods which were linear from 0.5-30 µg.ml -1 .Moreover, the proposed method could be applied successfully to the determination of the salbutamol sulphate in a pure form as well as in its dosage forms.