Introduction

The aim of present work is to develop reliable and accurate spectrophotometric methods for the quality control of the pharmaceutical formulations containing the cited drug.Phenazopyridine (2, 6-diamino-3-phenylazopyridine), is frequently used as an adjunct to sulfonamides, antibiotics and other urinary tract antiseptics  to treat bacterial mucosal infections of the lower urinary tract^1-4, because of its putative analgesic effect on the mucosa of the urinary tract, high selectivity and no anti-choline activity. The drug has been determined by a variety of analytical techniques such as GC-MS⁵, Spectrofluorimetric⁶, Electro analytical methods⁷, UV spectrophotometric⁸, Ratio spectra derivative spectrophotometry⁹ and High performance liquid chromatography¹⁰. By exploiting the various functional groups in the Phenazopyridine the authors had developed three simple and sensitive spectrophotometric methods for the determination of Phenazopyridine in pharmaceutical formulations and bulk.

Meterials and Methods

Apparatus

Elico UV – Visible Double beam spectrophotometer model SL-159.

Reagents

All the chemicals used were of analytical grade. All the solutions were freshly prepared in distilled water.

1)  0.1% Bromophenolblue (method A)

2 %Amidoblack (method B)

2 %Bromothymol blue (method C)

1N Hcl

Standard drug solution

Accurately weighed 100mg of Phenazopyridine was dissolved in 100mL-distilled water to give a concentration of 1mg/mL. The final concentration was brought to 100 µg/mL for both Methods A, B and C.

Analytical procedure

Into a series of 125 mL separating funnels, aliquots of drug sample (0.2-1.0mL) was added. 2mL of bromophenol blue (Method A), Amidoblack (Method B) and Bromothymol blue (Method C) and 0.1N HCl. The funnels were shaken vigorously with 5 mL chloroform for 2 min, and then allowed to stand for clear separation of the two phases. The separated organic phase was transferred to a 50 mL beaker, dried over anhydrous sodium chloride, and transferred to a 10 mL volumetric flask. Then the combined extract was made up to the mark with chloroform and mixed well. The absorbance of the organic phase was measured at 370nm (Method A), 620nm (Method B) and 400nm (Method C) respectively against  reagent blank .The standard calibration curve was prepared to calculate the amount of the drug.

Assay of pharmaceutical formulations

Powdered   drug equivalent to 100 mg was accurately weighed and dissolved in water and filtered. The filtrate was made up to 100 mL and appropriate aliquots of the drug solution were treated as described above and the results were tabulated in Table-2

Results and Discussion

The proposed methods are based on the formation of ion-pair complex between the positively charged drug and anionic dyes bromophenol blue (Method A), Amidoblack (Method B) and Bromothymol blue (Method C). Each drug-dye complex, with two oppositely charged ions, behaves as a single unit held together by an electrostatic force of attraction.

X⁺ _(aq) + d ^–_(aq) ↔ X ⁺ D ^–_(aq) ↔ X⁺D^–_(org)

Where x⁺ and d⁺ represent the protonated drug and anion form of the dye respectively and subscript (aq) and (org) refer the aqueous and organic phases respectively. The different optical parameters were calculated for all the methods and the results were summarized in table -1. The precision and accuracy were found by analyzing six replicate samples containing known amounts of the drug and the results are summarized in Table-1

Table 1: Optical and regression characteristics, precision and accuracy of the proposed methods.

* Y= a + bx, where Y is the absorbance and x is the concentration of Phenazopyridine in

μg/ mL

$ = for six replicates

The accuracy of these methods was ascertained by comparing the results obtained with the proposed and reference methods in the case of formulation are presented in Table-2. As an additional check on the accuracy of these methods, recovery experiments were performed by adding known amounts of pure drug to pre-analyzed formulation and percent recovery values obtained are listed in Table-2. Recovery experiments indicated the absence of interferences from the commonly encountered pharmaceutical additives and excipients.

Table 2: Results of analysis of tablet formulations containing Phenazopyridine.

* Reference method was UV method developed in the laboratory.

** Recovery amount was the average of six determinants.

Conclusion

The proposed methods were found to be simple, economical and sensitive. The statistical parameters and recovery study data clearly indicate the reproducibility and accuracy of the methods. Analysis of the authentic samples containing Phenazopyridine showed no interference from the common excipients. Hence, these methods could be considered for the determination of Phenazopyridine in the quality control laboratories.

Acknowledgements

The authors are grateful to the centre for biotechnology, Acharya Nagarjuna University, Guntur and Management of Sri Venkatewara Institute of Medical Sciences, Tirupathi for their continuous support and encouragement and for providing the necessary facilities.

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