Indirect Spectrophotometric Determination of Oxymetazoline Hydrochloride

A simple and sensitive spectrophotometric method for the quantitative determination of trace amounts of oxymetazoline hydrochloride (OMZ) was developed .The method based on the reaction of (OMZ) with Fe(III) in acidic medium.The librated Fe(II) coupled with potassium ferricyanide, the reaction needs heating for 25 minutes at 50 o C, to form Prussian blue which is more stable by adding 1ml of 1% ethylendiaminetetraacetic acid. The maximum absorption of the product was measured at 766 nm .Beers law is obeyed in the concentration range 5-125 μg/10ml with a molar absorptivity of 5.07x10 l. mol.cm,with a relative error of +1.74 to +2.34 % and a relative standard deviation (RSD%) of the of ±1.272 to ±1.982 %.The method was applied successfully for the assay of OMZ in pharmaceutical preparation.

OMZ is available as a topical decongestant in nasal spray, it is also used to treat epistaxis and eye redness (Wikipedia, 2008 ).
Different methods have been used for the spectrophotometric determination of oxymetazoline hydrochloride.
The construction and analytical application of plastic membrane electrode (ion selective electrode) for the determination of OMZ was applied (Issa and Zayed, 2004), also a flow injection analysis of OMZ with inhibited chemiluminscent detection (Garcia et al., 2004;Wang et al., 2009)was also reported.The chromatographic method, high performance liquid chromatography technique has been used in the determination of OMZ (Sudsakorn et al., 2006;Hong, 2009;Hoffmaann,1989;Hayes,1995;Stanisz and Nowinki, 2000).
The British pharmacopoeia reported a potentiometric method using perchloric acid solution for the determination of OMZ (British Pharmacopeia, 2007) The objective of the investigation reported in this paper is to evaluate a sensitive and accurate method for the assay of oxymetazoline in a pure form or in dosage forms.The method was based on the oxidation of OMZ using Fe (III) in acidic medium and the librated Fe (II) which subsequently complexes with potassium ferricyanide to form the Prussian blue complex.

EXPERIMENTAL Instruments
Spectrophotometric measurements are performed using Shimadzu UV-160 UV-Visible recording spectrophotometer using 1.0cm quartz cells.The pH measurements are performed by using pH meter type HANNA 211 pH-ion meter.Waterbath (Memmert, Germany) has also been used.

Reagents
All chemicals used in this investigation are of analytical grade.Oxymetazoline hydrochloride standard material was provided from state company for Drug Industries and Medical Appliance (N.D.I), Ninavah-Iraq.

Working OMZ solution (50 µg.ml -1 )
This solution is prepared by dissolving 0.005 g of OMZ in distilled water and the volume is completed to the mark with distilled water in 100ml volumetric flask.

Ferric chloride solution, 0.1%
This solution is prepared by dissolving 0.1 g of ferric chloride (Fluka) in distilled water and completed with distilled water to the mark in a 100ml volumetric flask.

Potassium ferricyanide ,0.1%
This solution is prepared by dissolving 0.1 g of potassium ferricyanide in distilled water and completed with distilled water to the mark in a 100ml volumetric flask .

Recommended procedure and calibration curve
To a series of 10ml calibrated flasks ,0.1 to 2.5 ml of 50 µg/ml OMZ solution were added followed by the addition of 3 ml of potassium ferricyaninde (0.1%), 3ml of Fe (III) (0.1%) and 0.5ml (1M) acetic acid were added.Then the solution stood for 25 minutes in water bath adjusted at 50 o C,then 1ml of EDTA(1%) was added and diluted to the mark with distilled water.The absorbance was measured at 766 nm against the reagent blank.
The calibration graph is linear over the concentration range of 5-125 µg /10 ml while higher concentrations show a negative deviation from Beer's law (Fig. 1), the apparent molar absorptivity referred to OMZ has been found to be 5.07x10 4 l.mol -1 .cm - Fig. 1: Calibration graph for OMZ determination using the proposed method.

Determination of OMZ in nasal drop
The contents of 3 containers of Oxymet drops were mixed well, a 5 ml which is equivalent to 0.0025 g OMZ was diluted to 50 ml with distilled water in a volumetric flask.A suitable aliquot of drug solution was analyzed as described in the recommended procedure.

RESULTS AND DISCUSSION
The effect of various variables on the color development of 12.5-100µg OMZ/10ml reacted with ferric chloride (0.1%) and potassium ferricyanide reagent (0.1%) in acidic medium was tested to establish the optimum conditions for the determination of OMZ by Prussian blue complex formation reaction.

Effect of the amount of ferric chloride
Increasing volumes of ferric chloride solution (0.1%) were added to the standard amounts of OMZ, the results indicated that 3 ml of ferric chloride solution gave the highest intensity of colored product at 720 nm, and this volume was considered as an optimum (Table 1).

Effect of the amount of potassium ferricyanide
Various volumes of potassium ferricyanide solution (R) ( 0.1%) were added.The results indicated that using 3 ml of this solution gave maximum absorbance of colored product at 720 nm, and this volume was recommended in the subsequent experiments (Table 2).

Choice of acid and its amount
The effect of different acids(1M) on the absorbance of the colored product was studied.The results indicated that 0.5ml of 1M acetic acid solution (pH=2.70 in final dilution) was the optimum and recommended in the subsequent experiments according to the highest intensity of the colored product ( Table 3).

Table 3: Selection of acid and its optimum volume
Effect of buffer solution on absorbance and stability.
According to the optimum pH found in Table 3,different types of buffers of pH 2.70 have been tested.The results shown in Table 4 indicated that all these types of the buffer solutions used decrease the absorbance or forming a turbid solution, so that the use of a buffer solution is not recommended.

Effect of temperature
The rate of reaction is observed to be slow when carried out at room temperature.However, enhanced reaction rate is observed when the reaction mixture is heated.The effect of temperature on the absorbance of the final reaction mixture is shown in (Table 5).The results indicated that absorbance of the product increased with increasing temperature and heating to 50 o C was considered as an optimum temperature.

Effect of heating time
The time needed to complete the reaction had been studied at 50 o C. The results in Table 6 showed that a maximum intensity occurred at 25 minutes heating before the dilution of the solution with distilled water to the mark.

Effect of surfactant
The effect of different types of surfactants (positive, negative and neutral) on the color intensity was studied.The results showed that using Triton X-100 decreases the absorbance of the colored product whereas CPC gives a turbid solution and using SDS increases the absorbance but the solution becomes turbid after heating to 50 0 C. Therefore, it has been recommended to eliminate the use of surfactants in the subsequent experiments.

The stability of product
The stability of the formed colored product was investigated under the optimum conditions for the determination of OMZ and compared with the stability of the formed colored complex after adding 1ml of NaF (1%) and EDTA(1%) (Table 7).The results indicated that adding EDTA or NaF solutions which act as masking agent for the unreacted amount of ferric ions causes an increase in the absorbance and also the resulting product becomes more stable, so that adding EDTA solution is recommend for the subsequent experiments with an optimum volume of 1ml (Table 8) and (Fig. 2)

Accuracy and precision
To check the accuracy and precision of the calibration curve, OMZ was determined at two different concentrations.The results shown in Table 9 indicated that the results are satisfactory.The nature of the reaction product Job ' s and mole-ratio methods (Delevie, 1997) have been used in determination of reaction ratio of OMZ with Fe(III).The obtained results (Fig. 4) and (Fig. 5) showed that a 1:3 OMZ to Fe (III) ratio is obtained.The ferrous ion produced from the above reaction was reacted with potassium ferricyanide to form the well-known product (KFe [Fe(CN) 6 ], Prussian blue) as in the following equation.

Effect of dilution with organic solvents
Different organic solvents are examined in the dilution of the solution to evaluate their effects on the absorbance of the resulting colored complex and the data are shown in Table ( 10) Water is chosen in the subsequent experiments due to its availability, non-toxicity as well as from the sensitivity point of view.

Effect of interferences
In order to test the efficiency and selectivity of the proposed method, the effect of some foreign substances (e.g.gum acacia, glucose, lactose and starch) that usually present in dosage forms were studied by adding different amounts of foreign substances to 50 µg OMZ/10ml.The results indicated that gum acacia and lactose monohydrate interference at high concentration in the determination of OMZ.

Application of the method
The proposed method was successfully applied in the determination of oxymetazoline hydrochloride in pharmaceutical preparation (nasal drops), the results which are shown in Table 12 indicate that good recoveries are obtained.

Evaluation of the proposed method:
According to the difficulties of using the standard method for the determination of OMZ which includes potentiometric titration, so standard addition method was applied in order to prove that the proposed method is free from interference in the determination of OMZ in pharmaceutical preparations.(Table 13 and Fig. 6).The result in Table 13 and Fig. 6 indicated that the proposed method can be used to the determination OMZ in pharmaceutical preparation with a satisfactory result.

Comparison of the methods
Table (14) shows the comparison between some of analytical variables for the present method with that of other literature spectrophotometric methods.The results in Table 14 indicated that the suggested method was sensitive and accurate.

CONCLUSION
The proposed method for the determination of OMZ in pharmaceutical formulation is simple, sensitive, accurate and precise.The method is based on oxidation -reduction between OMZ and Fe (III), then the subsequent reaction of librated Fe (II) with potassium ferricyanide to form Prussian blue complex which is water soluble, stable and shows a maximum absorption at 766 nm.The proposed method has been applied successfully to the determination of the intended compound in its pharmaceutical formulation (nasal drop)

Fig. 2 :
Fig. 2: The effect of EDTA and NaF on absorbanceAbsorption spectraThe absorption spectra of the formed product showed a maximum absorption at 766 nm, against the corresponding reagent blank (Fig3).

Fig. 3 :
Fig. 3: Absorption spectra of 50µg OMZ treated according to the recommended procedure and measured against (A) blank,(B) distilled water and (C) blank measured against distilled water.

Table 12 : Analytical application.
Average of six determinations Note : Added 0.004g of NaH 2 PO 4 and 0.004g of Na 2 HPO 4 to a standard solution. *