Spectrophotometric Determination of Oxymetazoline Hydrochloride in Pure and Pharmaceutical Preparations Using Diazo-coupling Reaction

A ccurate and simple spectrophotometric method  has been suggested for the determination of oxymetazoline hydrochloride (OMCl) in aqueous solution is developed. The present process included coupling of OMCl with diazotized sulphanilic acid reagent in the presence of sodium hydroxide. The orange colored azo dye formed very stable and soluble in water gives maximum absorption at 496 nm. The lineratey is obeyed over the range 20 – 400 g / 25 ml (0.8 –16μg.ml) the molar absorptivity is equate to 2.30  10 l.molcm. The proposed method has been used to the determination of OMCl in two formulations with satisfactories results.

The large doses of OMCl may cause hypotension, presumably because of a central clonidine-like effect [5].
Our aim is to evaluate a simple spectrophotometric method for the determination of OMCl as pure and in pharmaceutical drop formulations included coupling with diazotised sulphanilic acid in an alkaline medium of NaOH . The product of orange azo dye formed proves to be intense, water-soluble and stable.

Materials and Methods Apparatus
Shimadzu UV-Vis.Recording spectrophotometric had been used in measurement of absorbance using 1-cm silica cells.

Reagents
chemical and solvents are chosed with high purity.
Working OMCl solution, 100 g / ml. A 0.01g of OMCl is dissolved in distilled water and then volume is completed to 100 ml in a calibrated flask. Diazotised sulphanilic acid reagent solution, 30mM. A 0.5190 g of sulphanilic acid is dissolved 75 ml of distilled water and the mixture is heated until the clear solution is obtained, then 1 ml of hydrochloric acid (conc.) is added, the mixture is left in ice bath at 0 -5C and 0.207g sodium nitrite is added and stirred vigorously and then the volume completed to 100 ml in a volumetric flask using cooled distilled water, and is stored in refrigerator. This solution is prepared freshly each day [17]. Alkaline solution of NaOH(1N),is prepared byThis solution is prepared by appropriate dilution of the concentrated (Fluka) solution with distilled in a plastic container. Nazodrin drops, (100µg /ml). Three containers of drug (each contains 10 ml of 0.05% OMCl) are mixed, then 20 ml of the above solution was diluted with distilled water to 100 ml in a volumetric flask to prepare a solution of 100 µg /ml OMCl. Oxymet drops, (100µg/ml). Provided from the Pharaonia pharmaceuticals. Three containers of drug (each contains 15 ml of 0.025% OMCl) are mixed, then 40 ml of the above prepared solution was diluted to 100 ml by adding distilled water in a volumetric flask .

Procedure and calibration graph.
To a series of 25 ml volumetric flasks add 20 -500 g (0.8 -20 ppm) of OMCl, 0.5 ml of diazotised sulphanilic acid (30mM) and 1ml of 1N NaOH are then added and the volumes are diluted to the mark using distilled water as a slovent. After 10 minutes the A are read virsus a reagent blank at 496 nm using 1-cm cell. The linearity is over the range 20 to 400 g/25ml (0.8-16 ppm) (Fig.1). The molar absorptivity is found to be 2.3010 4 l.mol -1 .cm -1 When OMCl in aqueous solution is treated with diazotized sulphanilic acid reagent solution, an absorption peak is obtained showing an intense orange dye with maximum absorption at 496 nm. The reagent blank shows no absorption at this wavelength (Fig.2).

Fig.2:Absorption spectrum of OMCl with diazotized sulphanilic acid at 496 nm (A) the azo dye against blank, (B) blank against distilled water
Study of the optimum reaction conditions.All parameters effecting and related to orange azo dye have been optimized .

Effect of base.
The preliminary experiments have shown that the azo dye develops only completely in using base solution . Different amounts of bases (strong and weak) have been used (Table1). The results in Table 1 indicated that 1ml of 1N sodium hydroxide is the more suitable amount which gives a high values of colour contrast.

Effect of diazotized sulphanilic acid reagent amount.
Various volumes of the diazotized sulphanilic acid (30mM) are tested, the results indicate that using 0.5 ml of diazotized sulphanilic acid reagent solution gives maximum A of the complex at 496 nm and the volume is considered as an optimum value (Table 2).

Effect of surfactant
Three orders by using 1 ml of various types of surfactants have been studied. The effects of different surfactants on the colour intensity are showed that no useful effect and a loss in colour intensity are observed. Therefore, it has been recommended to eliminate the use of surfactants in the subsequent experiments (Table3).

Stability of formed azo dye
The color development showed that the colour started to form within about five minutes. The formation of azo dye being complete after 15 minutes and the absorbance of the coloured species remained constant for at least 25 minutes, this stability period is sufficient for several measurements (Table4).

Accuracy and precision
In order to check the accuracy and precision of the proposed method three various amountes of OMCl where taken and determined. The results illustrated in tables show that suggested method gave satisfactory.

Nature of the dye.
Job's method indicated that the azo dye has a composition of 1:1 OMCl to diazotized sulphanilic acid [R] (Fig.3). Hence the dye might be the following structure. The excipients which frequently accompany pharmaceutical formulation are studied by adding three various amounts (100, 500 and 1000g) to 100g OMCl (Table 6). Table 6. Effect of foreign compounds for assay of OMCl Table 6 with its results indicated that there is no interference of any excipients added to the determination of OMCl using the suggested procedure.

Applications part
The formulations of OMCl as nasal drops have been selected in our applications part. Good recoveries are obtained (Table 7).
Table7. Result of applications part.

Evaluation of the proposed method
In order to prove that the suggested method can be applied to the determination of OMCl in formulations without interferences, a standard addition method is applied.
The results in Fig.6 and Table 8 shows that there is no significant different between the amounts taken and experimental results.  The various analytical parameters for our method and the same parameters for other literature methods (14,16) have been calculated and illustrated in Table 9. Table 9. Comparison of the methods Lit. method (14) Lit. method (

Conclusion
The proposed method is simple, sensitive and there is no previous separation or temperature controlled. The method has been successfully applied to the determination of OMCl in various pharmaceutical preparations.