Spectrophotometric Determination of Phenylephrine Hydrochloride by Coupling with Diazotized 2-Aminobenzothiazole

A spectrophotometric method for determination of microgram amounts of phenylephrine-HCl(PE) has been proposed. The method is based on coupling of phenylephrine-HCl with diazotized 2-aminobenzothiazole in alkaline medium. The molar absorptivity of the formed dye is 6.620×10 l.mol. cm at λ max 510 nm and Beers law obeyed within in the range of 10-250 μg of phenylephrine-HCl /25 ml (0.4 – 10 ppm). The colour reaction was highly stable and did not show a significant change in absorbance(within acceptable analytical error) up to 48 h with a relative error +0.31 to +1.07% and a relative standard deviation of ±0.95 to ±3.09%, depending on concentration level. The method has been applied successfully to the determination of phenylephrine-HCl in pharmaceutical preparation (nose drop) .


INTRODUCTION
Phenylephrine hydrochloride[3-(hydroxylphenyl)-2-(methylamino)ethanol] ydrochloride is widely used as a decongestant drug (Al-Abachi and Al-Ward, 2002).The drug is available as an oral medicine or as a nasal spray.Phenylephrine is rarely used as a vasopressor due to its increase in the blood pressure for unstable patients with hypotension.(Louis, 1985) .
The flow injection methods have been used in the determination of phenylephrine with spectrophotometric detection (Yolanda et al., 2001;Knochen and Giglio, 2004;Beyene and Vanstaden, 2004).
Also, the high performance liquid chromatography technique has been applied to the determination of trace amounts of phenylephrine (Amer et al., 2008;Marin andBarbas, 2004, Senyuva andOzden, 2002).
The objective of investigation reported in this paper is to evaluate a sensitive and an accurate method for the assay of phenylephrine in an aqueous medium, either in pure form or in its pharmaceutical preparations.The method based on coupling of phenylephrine with diazotized 2-aminobenzothiazol in alkaline medium to produce an intense azo dye which was considered desirable to develop an additional assay method suitable for accurate and reliable quality control of phenylephrine formulations.

Apparatus
Shimadzu UV-Visible Recording Spectrophotometer UV-160 with 1.0 cm matched silica cells was used for all absorption measurements.

Reagent
All Chemicals used were of analytical-reagent grade .
A pure phenylephrine -HCl (PE) was obtained from the State company for Drug Industries and Medical Applicances(SDI), Sammara, Iraq.A solution of 100 μg ml −1 was prepared by dissolving 0.01g of phenylephrine-HCl in 100 ml distilled water.Sodium hydroxide (2N) and different interferences solution(1000 μg ml −1 ) were prepared by dissolving the proper amount in distilled water.
The diazotized 2-aminobenzothiazole(25 mM) solution was daily prepared by dissolving 0.1877 g of 2-aminobenzothiazole (Fluka) in 10 ml ethanol and 5 ml of concentrated sulphuric acid (Samir and Uma, 2005), followed by the addition of about 25 ml distilled PDF created with pdfFactory Pro trial version www.pdffactory.comwater.Finally the mixture was transferred to a 50 ml volumetric flask and is cooled at 0-5 O C in an ice-bath.A 0.0862 g sodium nitrite was added and the mixture was stirred vigorously.After 5 minutes, the solution was made up to 50 ml with cold distilled water.The solution was kept in a brown bottle in a refrigerator for 1 hour before using.

General procedure and calibration graph
The aqueous solution (0.1 -3ml) contain phenylephrine-HCl (100 μg .ml - ) was transferred to 25 ml calibrated flasks.A 2.5 ml of 2-aminobenzothiazole diazotized solution (25mM), and 4 ml of sodium hydroxide solution (2N) were added and the volume was made up to the mark with distilled water.The absorbance was measured at 510 nm against a blank solution which was prepared in a similar way but without the addition of phenylephrine-HCl .
The calibration graph as shown in Fig.

Procedure for the phenylephrine-HCl nose drop
The contents of three of phenylephrine -HCl nose drop containers (nasal drop, SDI, Sammara, Iraq) were mixed.An accurate volume containing 0.005 mg phenylephrine -HCl was transferred to a 50 ml volumetric flask, and the volume adjusted to the mark with distilled water and , then it was proceeded as described under procedure for calibration.

RESULTS AND DISCUSSION
The effect of various variables on the colour development was tested to establish the optimum conditions for determination of PE by coupling with diazotized 2aminobenzothiazole reagent.

PRINCIPLE OF THE METHOD
The method involves the coupling of phenylephrine drug with diazotized 2aminobenzothiazole in basic medium to form an intenselycoloured azo dye:-

Choice of diazotised agent
Several aromatic diazotised agents have been tested for optimum conditions.The results in Table 1 show that 2-aminobenzothiazole give the most sensitive reaction (ε = 8.810×10 3 l.mol - .cm - ) in alkaline medium.Therefore, it has been selected for subsequent experiments.

Effect of diazotized 2-aminobenzothiazole reagent amount
The effect of different amounts of diazotized 2-aminobenzothiazole reagent on the absorbance of solutions containing different amounts of phenylephrine-HCl (20-200       μg/25 ml) was studied.The obtained results indicated that the absorbance increases with increasing reagent concentration and reached a maximum on using a volume of 2.5 ml of 25 mM diazotized 2-aminobenzothiazole which also gives the highest value of coefficient of determination(r 2 ) (Table2).Therefore, the addition of 2.5 ml reagent was recommended for the subsequent experiments.

Effect of base
Preliminary experiments have shown that PE gives coloured dye with diazotized 2aminobenzothiazole only in basic medium.Different bases (strong and weak) have been used and the results (Table 3 ) indicate that the formation of the coloured dye needs a strong basic medium.Therefore 4 ml of 2N NaOH solution has been recommended for the subsequent experiments.
PDF created with pdfFactory Pro trial version www.pdffactory.com

Effect of surfactant
The effect of several types of surfactants on colour intensity of the dye has been investigated .The results indicate that addition of surfactants give no useful effect [increasing the intensity or improving the colour contrast (∆λ)], therefore it has not been used in the subsequent experiments.

Order of addition of reagents
The order of additions of reagents [phenylephrine-HCl (PE), NaOH(OH), 2aminobenzothiazole (R)] was examined.The results (shown in Table 4) indicated that order (I) of addition of reagents was the optimum order due to the high intensity of the formed azo dye.

Effect of time and amount of PE on absorbance
The effect of time on the development and stability period of the formed coloured dye was investigated under optimum experimental conditions described before.The formation of coloured dye being complete after mixing the component of reaction and the absorbance of the coloured species remained constant for, at least 48 hours (Table5).

Final absorption spectra
An absorption spectra of the formed coloured dye by coupling of PE with diazotized 2-aminobenzothiazole in basic medium, against its corresponding reagent blank show a maximum absorption at 510 nm in contrast to the reagent blank ( Fig 2).
PDF created with pdfFactory Pro trial version www.pdffactory.com

Interference
The criterion of interference was an error of not more than ± 5.0% in the absorbance.To test the efficiency and selectivity of the proposed analytical method, a systematic study of excipients (e.g., glucose, lactose, gum Arabic and starch) that usually present in dosage forms was performed.Experimental results showed that there was no interference from additives or excipients up to 1000 μg in the present method as shown in Table 6.

Accuracy and precision
To check the accuracy and precision of the calibration curve, PE was determined at three different concentrations.The results (illustrated in Table 7) indicate that the method is satisfactory.

Application of the Method
The proposed method was successfully applied to determine phenylephrine in its pharmaceutical prepration(nose drop)(Table 8).The performance of the proposed method was assessed by calculation of the t-test compared with the standard method(British Pharmacopeia,2000) (potentiometric titration with sodium hydroxide ) for 95% confidence level with four degrees of freedom.The results showed that the t-value was less than the critical value, indicated that there was no significant difference between the proposed and standard method for phenylephrine.Comparison of the methods Table(9) shows the comparison between some of analytical variables for the present method with that of another literature spectrophotometric method.
Table 9: comparison of the methods * Al-Abachi, M. Q. and Al-Ward, H. S. National J. Chem., 2002, 6 , 221.The results indicate that the proposed method is less sensitive than the literature method but the colour is much more stable.

CONCLUSION
The proposed method was a simple and has a good sensitivity.The proposed method has advantageous over some of the reported visible spectrophotometric methods with respect to, reproducibility, precision, accuracy and stability of the coloured species.The proposed method is suitable for the determination of phenylephrine in pure form and in nose drop-formulation without excipients interference .

Absorbance
PDF created with pdfFactory Pro trial version www.pdffactory.com Colour contrast = λ  maxS-λ  maxB where S=The dye, pdfFactory Pro trial version www.pdffactory.com

Fig. 2 :
Fig. 2: Absorption spectra of 250 µg PE treated according to the recommended procedure and measured against (A) blank (B) distilled water and (C) blank measured against distilled water.
pdfFactory Pro trial version www.pdffactory.com

Table 2 :
The effect of the amount of diazotized 2-aminobenzothiazole on dye absorbance

Table 3 :
The effect of base on dye absorbance   Δλ  = λ max S- λ max B , where S=The dye, B=Blank

Table 4 :
Effect of order of addition PDF created with pdfFactory Pro trial version www.pdffactory.com

Table 5 :
The effect of time on absorbance

Table 6 :
Effect of foreign compounds for assay of phenylephrine-HCl

Table 7 .
Accuracy and precision of the calibration curve

Table 8 :
Analytical applications of the proposed method and expremental t-value