DETERMINATION OF PHENYLEPHRINE HYDROCHLORIDE IN PHARMACEUTICAL PREPARATIONS USING SPECTROPHOTOMETRIC METHOD

Objective: A simple and sensitive spectrophotometric method has been presented for the determination of phenylephrine hydrochloride by coupling reaction with diazotized sulfacetamide sodium. Methods: The method is based on the diazotization reaction of sulfacetamide sodium with sodium nitrite in the presence of hydrochloric acid to form diazonium salt, which is coupled with the drug in alkaline medium to form azo dye, showing absorption maxima at 425 nm. Results: Calibration plot was linear over the concentration range of 2–24 μg/mL and detection limit of 0.278 μg/mL with a correlation coefficient of 0.9929. All different chemical and physical experimental parameters affecting on the development and stability of the colored product were carefully studied. Conclusions: The proposed method was successfully applied to the determination of phenylephrine in nasal drops with good precision and sensitivity.

In this work, a spectrophotometric method for estimation of phenylephrine was described. The method was based on coupling reaction between diazotized sulfacetamide sodium with the medicine in alkaline medium to form a yellow water-soluble azo dye measured at 425 nm. This method has been successfully applied for the determination of phenylephrine in nasal drops.

Apparatus
An optima spectrophotometer ultraviolet-visible (Japan) double beam with 1 cm quartz cells was used in all absorbance measurements.

Preparation of solutions
PHP stock standard solution (1000 μg/mL) was prepared by dissolving 0.100 g of pure PHP in distilled water and made up to 100 mL volumetric flask with distilled water. Working standard solutions were prepared by suitable dilution of the stock standard solution with distilled water. Sulfacetamide sodium solution (0.1%) was prepared by dissolving 0.1 g of sulfacetamide sodium in distilled water and diluting to 100 mL volumetric flask with the same solvent.
NaOH solution (2 M) was prepared by dissolving 20 g of NaOH with distilled water in 250 mL volumetric flask.
General procedure for calibration About 2 mL of 0.1% sulfacetamide sodium was transferred into a series of 25 mL calibrated flask. To this solution, equimolar of sodium nitrite solution (3.9×10 −3 M) was added and the acidity was adjusted with 1 mL of 0.5 M HCl solution. The solution was shaken thoroughly. Then, an aliquot of a standard solution (500 μg/mL) containing 0.1-1.2 mL of PHP was Al-uzri transferred into this series of 25 mL calibrated flasks and 1 mL of 2 M NaOH solution was added, and the contents were diluted to the mark with distilled water and mixed well. After 15 min, the absorbance of the colored azo dye was measured at 425 nm against the corresponding reagent blank. For the optimization of conditions and in all subsequent experiments, 1 mL of 500 µg/mL of PHP in a final volume of 25 mL was used.

Procedure for PHP in nasal drops
The contents of three bottles of nasal drops were mixed. An aliquot corresponding to 50 mg of PHP was diluted to 50 mL with distilled water in a volumetric flask to obtain 500 μg/mL of PHP. Further, appropriate solutions of pharmaceutical preparations were made by simple dilution with distilled water.

Determination of absorption maximum
An aqueous solution of PHP is reacted with diazotized sulfacetamide sodium in alkaline medium giving yellow dye which became stable after 15 min and has a maximum absorption at 425 nm. Fig. 1 shows the spectra of the product formed.

Optimization of the experimental conditions
The effects of various parameters on the absorption intensity of the formed product were optimized.

Effect of the volume of HCl (0.5 M)
The effect of different volumes (0.3-3 mL) of HCl was examined on the maximum absorbance of the formed product. Fig. 2 shows that 1 mL of HCl (0.5 M) was enough to obtain a maximum absorbance.

Effect of the volume of sulfacetamide sodium (0.1%)
The effect of different volumes (0.5-4 mL) of sulfacetamide sodium was examined on the maximum absorbance of the formed azo dye.  shows that 2 mL of sulfacetamide sodium (0.1%) was enough to obtain a maximum absorbance.

Effect of the volume of NaOH (2 M)
The effect of different volumes (0.5-3 mL) of NaOH was examined on the maximum absorbance of the formed product. Fig. 4 shows that 1 mL of NaOH (2 M) was enough to obtain a maximum absorbance.

Effect of reaction time
The stability of the product was studied for 180 min following the mixing of the reagents. The colored product developed rapidly after mixing and attained maximum absorbance about 15 min at room temperature. The color was stable for a period of 180 min.

Structures of the products
The stoichiometry of the reaction between PHP and diazotized sulfacetamide sodium was investigated under the recommended optimum conditions using continuous variation method. The result obtained in Fig. 5 shows that a 1:2 azo dye was formed between PHP and diazotized sulfacetamide sodium.
A reaction subsequent based on the above result is shown in Scheme (1) [22].

Determination of stability constant and Gibbs free energy of the reaction
The

Scheme 1: Proposed mechanism of the reaction between phenylephrine hydrochloride and diazotized sulfacetamide sodium
ΔG was found to be −43.612 kJ/mole. The negative value of ΔG refers to the spontaneity of the reaction.

Interferences
The extent of interfering by some excipients which often accompanied pharmaceutical preparations was studied by measuring the absorbance of solutions containing 20 µg/mL of PHP and excess amounts (10-fold excess) of each excipient, none of these substances interfered seriously (Table 1).

Analytical characteristics of spectrophotometric method
Calibration graph (Fig. 6) was obtained after optimized all the reaction conditions mentioned previously and a series of standard solutions were analyzed in triplicates to test the linearity. The molar absorptivity (ε), the Sandell's sensitivity (S), the intercept (b), and the slope (a) were determined and are included in Table 2. The limit of detection was determined by taking the ratio of the standard deviation (SD) of the blank with respect to water and the slope of the calibration curve multiplied by the factor three [28].
The accuracy and precision of the proposed method were tested by analyzing five replicate of phenylephrine by proposed spectrophotometric method for two different concentrations of phenylephrine. The values of relative SD relative standard deviation% and relative error % are summarized in Table 3. These values indicated the high accuracy and precision of the proposed method.

Pharmaceutical application
The proposed method was successfully applied to determine phenylephrine in nasal drops. The obtained results were compared statistically by a Student's t-test for accuracy and a variance ratio F-test for precision with the standard method [29] at the 95% confidence level [30] as cited in Table 4. The results showed that the experimental t-test and F-test (t=1.962, F=3.079) were less than the theoretical value (F=19.00, t=2.776 (n 1 +n 2 -2=4)), indicating that there was no significant difference between the proposed method and standard method. Further, the proposed method is very economical when compared to chromatographic British pharmacopoeia methods [31].
In addition, a paired t-test [32] was conducted between the samples determined by proposed method with standard method. t-value (tab) for n-1 degree of freedom=4.303 calculated t-value=1.322 for n-1 at α 0.05 (95%), two tailed indicate that since 1.322<<4.303; therefore, it can be regarded that there is no difference between the results.

CONCLUSIONS
This research offers a simple spectrophotometric method for the determination of PHP hydrochloride in nasal drops. This method has the advantage of simplicity, speed, accuracy, and the use of inexpensive equipment. In addition, the present method, as compared with other expensive techniques such as HPLC-MS, electro-sensors, and capillary electrophoresis, is economical and cheap and has an excellent accuracy and precision.

ACKNOWLEDGMENT
The author is grateful to the Chemistry Department, College of Science, Baghdad University, for providing facilities.