Spectrophotometric Determination of Oxymetazoline Hydrochloride Via Oxidative Coupling Reaction with 4-Aminoantipyrine in the Presence of Potassium Periodate

A simple spectrophotometric method is developed for the determination of oxymetazoline hydrochloride (OMCl) as pure and in its pharmaceutical preparations. The method is based on the oxidative coupling reaction of OMCl with 4-aminoantipyrine (4-A.A.P) in the presence of potassium periodate as oxidizing agent in an alkaline medium to produce a coloured water soluble product that is stable and has a maximum absorption at 480 nm. Beer's law is obeyed in a concentration range 20 -400μg OMCl/20 ml with a molar absorptivity of 5.34 x 10 1.mol.cm, a relative error of -0.50 to -1.47% and a relative standard deviation of ± 0.36 to ± 1.58%, depending on the concentration level. The optimum conditions for full colour development are described and the proposed method was applied successfully to the assay of OMCl in two pharmaceutical preparations.


INTRODUCTION
Oxymetazoline hydrochloride is 3-[(4,5 -dihydro-1H-imidazol-2-yl)methyl]-6-(1,1dimethyl ethyl )-2,4-dimethyl-phenol hydrochloride(OMCl) ,it is a white, crystalline powder, freely soluble in water and in alcohol, practically insoluble in ether (British Pharmacopia, 2000) ,its chemical formula and structure are as follows : OMCl is available as a topical decongestant in nasal sprays, it is also used to treat epistaxis and eye redness due to minor irritation, OMCl was developed by German patent Fruhstofer in 1961(Wikipedia, 2008. The large doses of OMCl may cause hypotension, presumably because of a central clonidine-like effect (Katzung, 2004). Different methods have been used for the determination of OMCl such as a titrimetric method using ammonium metavandate as a reagent in acidic medium (Dwivedi et al., 2006), ion selective membrane electrode (Issa and Zayed, 2004), also, a flow injection method employing chemiluminescence detection (Garcia-Campana et al., 2004),the chromatographic methods are often the most analytical methods used such as high performance liquid chromatography (HPLC) (Stanisz andNowinski, 2000 andSundsakom et al., 2006), also, reversed-phase-HPLC (RP-HPLC) (Sane et al., 1990) and RP-HPLC used ion pair (Hoffmann et al., 1989) have been used in the determination of OMCl in pharmaceutical preparations.
The literature review revealed that up today, nothing has been published concerning the use of 4-A.A.P as coupling reagent in the presence of potassium periodate in oxidative coupling reaction for determination of OMCl so that it is used in the present work to satisfactorily be applied for the determination of OMCl in dosage forms.

Instruments :
Spectrophotometric measurements are performed using Shimadzu UV-160 UV-Visible Recording Spectrophotometer and CEClL-CE 7200 UV-visible spectrophotometer using 1cm silica cells. The pH measurements are performed on pH meter type HANNA 211 pH-Ion meter.

Reagents :
All chemicals used in this investigation are analytical grade reagent. Working OMCl solution, (200 µg/ml). A 0.02 g of OMCl is dissolved in distilled water and the volume is completed to the mark with distilled water in a 100-ml volumetric flask, the solution is kept in a brown bottle, where it is stable for at least one week.

4-Aminoantipyrine reagent, (1.25x10 -3 M).
This solution is prepared by dissolving 0.025 g of pure 4-A.A.P reagent in distilled water and the volume made up to the mark in a 100-ml volumetric flask with the same solvent.
Sodium periodate solution, (0.015M). This solution is prepared by dissolving 0.3448 g of sodium periodate in distilled water and made up to 100 ml in a volumetric flask with the same solvent.

Sodium hydroxide solution, (4N).
This solution is prepared by appropriate dilution of the concentrated volumetric (Fluka) solution with distilled water to 250 ml in a volumetric flask and then transferred to a plastic bottle.
A 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 50 ml in a volumetric flask to prepare a solution of 200 µg/ml OMCl. Oxymet drops, (200 µg/ml). Provided from pharaonia pharmaceuticals .
A three containers of drug (each contains 15 ml of 0.025% OMCl) are mixed, then 40 ml of the above solution was diluted with distilled water to 50 ml in a volumetric flask to prepare a solution of 200 µg/ml OMCl.

RESULTS AND DISCUSSION
The effect of various variables on the colour development of 200µg of OMCl ,1ml of 4-A.A.P and 2ml of KIO 4 in alkaline medium(1ml 4N NaOH )was tested to establish the optimum conditions.

Choice of oxidizing agent with its concentration
Different types of oxidizing agents were used to select the best one which will give the highest colour intensity (Table 1). The results illustrated in Table 1 indicated that KIO 4 give the highest intensity of coloured product and a good colour contrast.
The effect of different volumes (0.5-6 ml) of KIO 4 solution (0.015M) on the colour intensity has been studied, it was observed that 4 ml of KIO 4 is the most suitable amount, since it gives the highest intensity of the formed product therefore it is chosen for further studies (Table 2).

Effect of 4-A.A.P concentration
Various volumes of 4-A.A.P (1.25x10 -3 M) were tested, the results indicated that using 2 ml of 4-A.A.P solution gives maximum absorbance of the coloured product at 480 nm and the volume was considered as an optimum value(Table3).

Choice of base and its amount
The preliminary experiments have shown that OMCl can give high intensity of coloured dye with (4-A.A.P) in the presence of potassium periodate in alkaline medium,so that different types of bases are examined (Table 4). The results shown in Table 4 indicated that NaOH gives the highest colour intensity of product and a good colour contrast. Also, the effect of different volumes (0.5-4 ml) of 4N NaOH solution on the colour intensity has been studied,a 1 ml of 4N NaOH with a final solution pH (13.15) gives the highest intensity of the formed product therefore it is used in subsequent experiments (Table 5).

Effect of surfactant
The effects of different surfactants on the colour intensity were studied in four orders by using 3 ml of various types of surfactant. The results showed that no effect of the surfactant had on the intensity (Table 6).

Order of addition
The effect of different orders of reagents addition were studied (Table 7).It was found that the order of reagents addition be followed as given under the general procedure give highest colour intensity, otherwise a loss in colour intensity takes place.

Effect of temperature
The effect different of temperatures on the colour intensity of resulting complex were investigated. The results indicated that the absorbance of complex increased with increasing temperature, the high value of absorbance was obtained at 70 o C then a decrease in colour intensity was observed as the temperature increase above70 o C (Table 8). The effect of the time needed to complete the oxidative coupling reaction had been studied at 70 o C and the results showed that a maximum intensity occurred at 25 minutes before dilution of the flask with distilled water (Table 9).

Development time and stability period
The stability time of the formed coloured complex is investigated under the optimum conditions for the determination of OMCl, the experimental results (Table 10) showed that the coloured complex formed is complete after 15 minutes from removing of the flasks from water bath and the absorbance remained constant at least for one hour.

Final absorption spectrum
When OMCl was treated according to recommended procedure, the absorption spectrum showed a maximum absorption at 480 nm versus the reagent blank (Fig. 1).

Fig1
: Absorption spectrum of (A) the coloured product (from 200µg OMCl) against blank, (B) complex against distilled water and (C) blank against distilled water.

Recommended procedure and calibration curve
To a series of 20-ml volumetric flasks, 0.1-3 ml of 200 µg.ml -1 OMCl solution are transferred then 2 ml of 4-A.A.P reagent (1.25x10 -3 M) and 4 ml of potassium periodate (0.015 M) are added. After that a 1 ml of 4N sodium hydroxide solution was added. The solutions were left for 25 minutes in water bath adjusted at 70 o C, then the volumes were completed to the mark with distilled water and left to stand for 15 minutes at room temperature, after that the absorbances are measured at 480 nm against the reagent blank.

Accuracy and precision
To determine the accuracy and precision of the method, OMCl was determined at three different concentrations. The results shown in table (11), indicate that a satisfactory accuracy and precision could be obtained with the proposed method. The nature of the reaction product Job's method of the continuous variations (Fig.3) indicates that the coloured product has a compostion of 1:2 OMCl to 4-A.A.P reagent at 480 nm. Absorbance 0-6 ml of 6.7x10 -4 [OMCl] 6-0 ml of 6.7x10 -4 [4-A.A.P]

Evaluation of the proposed method :
Because the standard method for the determination of OMCl included potentiometric titration, according to difficulties of availability of using it, so that standard addition method was used in order to prove that the proposed method can be applied to determination of OMCl in pharmaceutical preparations. (Table 12 and Fig. 4). The results in Table 12 and Fig.4 indicated that the proposed method can be used to determine OMCl in pharmaceutical preparation with satisfactory results. Table 13 shows the comparison between some of analytical variables obtained from the present method with that of a recent spectrophotometric method. The results indicate that the proposed method is sensitive and stable compared with the literature method .

CONCLUSION
A simple and sensitive spectrophotometric method for the determination of OMCl in aqueous solution based on the reaction of OMCl with 4-A.A.P in the presence of KIO 4 is developed. The proposed method has been successfully applied to assay OMCl in pharmaceutical preparations.