LATVIJAS ĶĪMIJAS ŽURNĀLS 2005, Nr. 4, 3.—269. lpp. A.M. El-Didamony1, M.A.F. El-Mosallamy1, A. Amin2, M. Mesherf1 SPECTROPHOTOMETRIC DETERMINATION OF BENZYDAMINE AND CHLORPHENOXAMINE HYDROCHLORIDES IN BULK AND PHARMACEUTICAL PREPARATIONS USING SULFOPHTHALEIN DYES 1Chemistry Department, Faculty of Science, Zagazig University, Zagazig, Egypt 2Chemistry Department, Faculty of Science, Benha University, Benha, Egypt Benzydamine hydrochloride (BZH), I is hydrochloride of N,N-dime- thyl-3-[1-phenylmethyl-1H-indazol-3-yl]oxy-1-propanamine (1-benzyl-3-(3-dime- methylaminopropoxy-1H-indazole, also named 1-benzyl-1H-indazol-3-yl 3-di- methylaminopropyl ether). It is a white crystalline powder, readily soluble in water, freely soluble in ethanol (95%), and in chloroform, practically insoluble in ether. N HCl N CH3 O N I CH 3 Benzydamine hydrochloride is a non-steroidal anti-inflammatory agent with analgesic, anti-exudative and local anesthetic activity. It is used topically on the skin in concentration of 3—5% in case of painful musculoskeletal and soft tissue disorder, also used as mouth wash or spray in concentration of 0.15% for relief of inflammatory conditions of mouth and throat. Chlorphenoxamine hydrochloride (II) is hydrochloride of 2-[1-(4-chlorophe- nyl)-1-phenylethoxy]-N,N-dimethylethanamine (2-[(p-chloro--methyl--phe- nylbenzyl)oxy]-N,N-dimethylethylamine, also named -dimethylaminoethyl (p-chloro--methyl-benzhydryl) ether). Cl O CH3 N CH3 HCl CH3 II Chlorphenoxamine hydrochloride is a white crystalline powder, readily so- luble in water, alcohol, and its aqueous solutions are stable. Chlorphenoxamine 263 hydrochloride possesses antimuscarinic and antihistaminic properties. It has been used in cases of nausea, vomiting and vertigo, and was formerly used in the symptomatic treatment of parkinsonism. It has also been used in hypers- ensitivity reaction. Chlorphenoxamine hydrochloride is not official in either the USP XXIV or the BP 2002. A few methods for the analysis of compounds I and II have been reported, including spectroscopic methods based on formation of coloured complexes [1—3]; other spectrophotometric methods that do not involve formation of coloured complexes [4—6]; IR spectroscopy [7], atomic absorption spectro- scopy [8], cation exchange [9], high-performance liquid [10, 11] and thin-layer chromatography [12], potentiometry [13], polarography [14]. The aim of the present work is to develop three simple, rapid and sensitive extraction spectrophotometric methods for the assay of benzydamine hydro- chloride and chlorphenoxamine hydrochloride. The methods are based on formation of ion-pair complexes of compounds I and II with dyestuffs such as bromophenol blue, bromocresol green, and bromocresol purple and subsequent extraction into chloroform under reaction conditions used. EXPERIMENTAL Apparatus. All the absorption spectra were taken using Shimadzu (UV-Visible 1601) spectrophotometer (Japan) with scanning speed of 400 nm/min, and band-width of 2.0 nm equipped with 10 mm matched quartz cells. The pH values of buffer solutions were measured using Jenway pH-meter (combined electrode). Reagents. All the chemicals used were of analytical or pharmaceutical grade, and water was twice-distilled. Pure benzydamine hydrochloride (BZH) (Tantum and Tantum rose) and chlorphenoxamine hydrochloride (CPH) (Allergics and Emeral), were obtained from Egyptian International Pharmaceutical Industrial Company (EPICO). Stock solutions of pure benzydamine hydrochloride and chlorphenoxamine hydrochloride (100 µgml–1) were prepared by dissolving 10 mg of pure drugs in water and filling up to the mark in a 100 ml volumetric flask with water. Working solutions of lower concentrations were prepared by serial dilution. Solutions of 5.010–4 moll–1 of bromocresol green (BCG), bromocresol purple (BCP) and bromophenol blue (BPB) (Aldrich) were prepared by dissolving an accurate weight of the dyes in few drops of acetone and then filling up to the mark with water in 100 ml volumetric flasks separately. A series of buffer solutions of CH3COONa–HCl (pH 1.99—5.2) and CH3COONa– CH3COOH (pH 3.42—5.89), and NaH2PO4–Na2HPO4 (pH 6.0—8.0) were prepared by the following standard methods. General procedures Benzydamine hydrochloride (I) Aliquot of compound I solution containing up to 2.0—28 μgml–1 of the drug was transferred into a series of 50 ml separating funnels, 2.0 ml of bromophenol blue, bromocresol green and bromocresol purple solutions (5.010–4 moll–1) were added followed by acetate buffer solution of pH = 3.1 (2.0 ml) when using bromophenol blue, pH = 3.3 (3.0 ml) when using bromocresol green and pH = 3.2 (2.0 ml) when using bromocresol purple. The formed ion pairs were extracted with 5.0 ml of CHCl3 under shaking for 3.0 min. The two phases were allowed to separate and the chloroform layer was dried by running through anhydrous sodium sulphate. The absorbance of the extracted solutions was measured at the corresponding max against extracted blank solution prepared similarly but without addition of the drug under study. Chlorphenoxamine hydrochloride (II) Aliquot of compound (II) solution containing up to 2.0—26 µgml–1 of the drug was transferred into a series of 50 ml separating funnels, 2.0 ml of bromophenol blue, bromocresol green or bromocresol purple solutions (5.010–4 moll–1) were added followed by acetate buffer solution of pH = 3.1 (2.5 ml) when using bromophenol blue, pH = 3.8 (2.0 ml) when using bromocresol green and pH = 3.2 (2.0 ml) when using bromocresol purple. The formed ion pairs were extracted with 5.0 ml of CHCl3 under shaking for 3.0 min. The two phases were allowed to 264 separate and the chloroform layer was dried by running through anhydrous sodium sulphate. The absorbance of the extracted solutions was measured at the corresponding max against extracted blank solution prepared similarly but without addition of the drug under study. A calibration graph was constructed for each drug and the concentration of unknown samples was deduced by using of the calibration graph. Procedure for Dosage Forms Bulk samples were prepared by weighing and pulverizing 20 tablets or capsules, or sachets and finding the average weight of each one. An amount of the sample equivalent to about 10 mg of the drug was weighed and dissolved in 100 ml of water, shaken well and filtered through a sintered glass crucible (G4). A 1.0 ml aliquot of the test solution was diluted to 100 ml in a volumetric flask. The drug content in this solution was obtained by applying the general procedure to aliquot containing 4.0 µgml–1 of the drug as described above and then followed by the standard addition method to confirm the obtained results. RESULTS AND DISCUSSION The nitrogenous drugs are present in positively charged protonated forms and anionic dyes of sulfophthalein group are present mainly in anionic form at pH 3.0. So when treated with an acid dye such as bromophenol blue, bromocresol green or bromocresol purple in acetate buffer solutions at pH ranged from 3.1 to 3.8, a yellow ion-pair complex which is extracted with chloroform is formed. The absorption spectra of the ion-pair complexes formed between benzydamine hydrochloride, chlorphenoxamine hydrochloride and each of sulfophthalein dyes (bromophenol blue, bromocresol green and bro- mocresol purple), were measured at 320—600 nm against a blank solution and are shown in Fig. 1, 2. The developed methods were applied to pharmaceutical preparations and the obtained results were evaluated statistically. 0.700 0.600 2 3 0.400 1 Absorbance, a.u. Absorbance, 0.200 0.000 320.0 400.0 500.0 600.0 Wavelength, , nm Fig. 1. Absorption spectra of benzydamine hydrochloride solution (4.0 gml–1) with dyestuff: 1 — bromophenol blue, 2 — bromocresol green, 3 — bromocresol purple Optimization of the reaction conditions The optimization of the reaction conditions of the proposed methods was carefully studied to achieve the complete reaction, highest sensitivity and maximum absorbance. Reaction conditions of formation of the ion-pair com- plexes were found by preliminary experiments varying pH of buffer solutions, nature of organic solvent, concentration of the sulfophthalein due and shaking time for the extraction of ion-pair complexes. 265 0.700 3 0.600 1 2 0.400 Absorbance, a.u. Absorbance, 0.200 0.000 335.0 350.0 400.0 450.0 500.0 555.0 Wavelength, , nm Fig. 2. Absorption spectra of chlorphenoxamine hydrochloride solution (4.0 gml–1) with dyestuff: 1 — bromophenol blue, 2 — bromocresol green, 3 — bromocresol purple Effect of pH The effect of pH was studied by extracting the coloured complex in the presence of various buffer solutions such as KCl–HCl (pH = 1.0—2.2), CH3COONa–HCl (pH = 1.99—6.0), CH3COONa–CH3COOH (pH = 3.7—5.8) and potassium hydrogen phthalate – HCl (pH = 2.2—3.6). The maximum colour intensity was developed and constant absorbance values were found in CH3COONa–HCl buffer solutions (see Fig. 3). It is evident that the absorbance of complexes with benzydamine hydrochloride was found to be maximal at pH 3.1, 3.3 and 3.2 using bromophenol blue, bromocresol green and bromocresol purple, respectively. As follows from Fig. 4, the absorbance of complexes with chlorphenoxamine hydrochloride is maximal at pH 3.1, 3.8 and 3.2 with bromophenol blue, bromocresol green and bromocresol purple, respectively. The optimum amount of buffer solution added to the aqueous layer of total volume of 5.0 ml was found to be 2.0 ml in case of benzydamine hydrochloride with bromophenol blue and bromocresol purple, and 3.0 ml with bromocresol green; 2.5 ml in case of chlorphenoxamine hydrochloride with bromophenol blue and 2.0 ml in case of chlorphenoxamine hydrochloride with bromocresol green and bromocresol purple that gave marginally the best results. Effect of the extracting solvent nature A number of organic solvents such as chloroform, dichloromethane, carbon tetrachloride, benzene and toluene were examined for extraction of the ion-pair complexes in order to provide an applicable extraction procedure.