Spectrophotometric Determination of Bromhexine HCl in Pure and Pharmaceutical Forms

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Methods A, B, and C.
The methods A, B, and C are based on the interaction of the drug with bromothymol blue (BTB), bromophenol blue (BPB), and bromocresol green (BCG), respectively, to form chloroform-extractable ion pair complexes (Scheme 1) which absorb around 415 nm (Figure 1).The absorbance of this band increases with increasing the concentration of the drug and formed a basis for the quantification of the drug.The dyestuffs were used as 0.025% solutions in doubly distilled water.Sodium acetate hydrochloric acid buffers of pH 2.8, 2.5, and 3.5 were prepared by mixing 50 mL of 1.0 M sodium acetate solution with 49.50 mL, 50.50 mL, and 46.25 mL of 1.0 M HCl solution, respectively, and diluted to 250 mL with doubly distilled water.The pH of each solution was adjusted to an appropriate value with the aid of a pH meter.

Method D Interaction of Iodine with Drug.
The method depends upon the interaction of neutralized drug with iodine that generates iodide ion having an absorption band at 366 nm (Figure 2).The absorbance of this band increases Figure 1: Absorption spectra of bromhexine hydrochloride-dye complex extracted into 10 mL chloroform: (a) drug = 10 g mL −1 + 5 mL of 0.025% BTB + 5 mL of pH2.8 buffer, (b) drug = 9 g mL −1 + 5 mL of 0.025% BPB + 5 mL of pH2.5 buffer, and (c) drug = 9.5 g mL −1 + 5 mL of 0.025% BCG + 5 mL of pH3.5 buffer.with increasing the concentration of the drug and formed a basis for the quantification of the drug.Mixing the solution of iodine prepared in DCE with BRH resulted in a change of violet color of iodine into light brown to pale yellow color, and as a consequence, absorption spectra exhibited a new band of 366 nm.This is attributed due to I 3 − ion formed by the interaction of iodine with drug (Scheme 2).

Method E: Kinetic Method.
The method depends on the oxidation of the drug with alkaline KMnO 4 (1 × 10 −2 M) to produce manganate ion which absorbs at 610 nm (Figure 3) and formed a basis for quantification of drug.A solution of 0.45 M NaOH is used to produce required alkalinity.Mixing the solutions of permanganate and the drug slowly developed green colour, and hence kinetics of the reaction was followed spectrophotometrically with a view to develop a method for the quantitative determination of the drug spectrophotometrically.The initial rate and fixed time methods are followed for the determination of BRH.A, B, and C. Different aliquots of drug solution were transferred into 125 mL separating funnel.To this 5 mL of buffer, 5 mL of dye was added, and total volume was made up to 20 mL with water.10 mL of chloroform was added, and the contents were shaken for 5 min.The two layers were allowed to separate for 5 min.

Calibration Curves for Methods
The organic layer was separated, and absorbance of yellowcolored solution which is stable at least for 3 hrs is measured at 415 nm against blank similarly prepared.The same procedure of analysis is followed either for assay of pure drug or for dosage form.The calibration graphs are linear for the drug analysed using these dyes (Figure 4).

Calibration Curve for Method D.
Into separate 10 mL volumetric flasks different aliquots of BRH solution was transferred followed by the addition of 1 mL of iodine solution.The volume was completed using the same solvent and the absorbance was measured against reagent blank at 366 nm.

Fixed Time Method.
A fixed time of 20 min was selected for the fixed time method.At this time the absorbance of reaction mixture measured against a reagent blank prepared similarly.The calibration curve was obtained by plotting the absorbance against the initial concentration of bromhexine HCl.Table 1 summarizes the limits of Beer's law, Limit of Detection (LOD), Limit of quantification (LOQ), molar absorptivity, regression equation, correlation coefficients and relative standard deviations.

Assay of Drug in Pharmaceuticals
2.9.1.Procedure for the Assay of Pure Drug.Five different solutions of pure drug in the range of calibration curve were selected, and the recovery experiments were performed.The recoveries and their relative standard deviation are tabulated in (Table 2).
2.9.2.Procedure for the Assay of Dosage Forms.Ten tablets of bromhexine 8 mg are powdered and dissolved in doubly distilled water and stirred thoroughly and then filtered through a Whatman number 42 filter paper.This solution was transferred into 100 mL standard volumetric flask and diluted with doubly distilled water as required.Different solutions of drug in the range of calibration curve were chosen, and the assay was estimated using the calibration curve and the results of the recovery studies are tabulated in (Table 3).A, B, and C. BRH forms ion-pair complexes in acidic buffer with dyestuffs viz., BTB, BPB and BCG.These complexes are quantitatively extracted into chloroform.The ion-pair complexes with BTB, BPB and BCG absorbed maximally at 415 nm (Figure 1).The reagent blank under similar conditions showed no absorption.BRH contains aromatic amine group which is protonated in acidic medium, while sulphonic acid group is present in BTB: that is the only group undergoing dissociation in the PH range 1-5.BPB and BCG are examples of sulphonepthalein type of dye.The colour of such dyes is due to the opening of lactoid ring and subsequent formation of quinoid group.It is supposed that the two tautomers are present in equilibrium but due to strong acidic nature of the sulphonic acid group, the quinoid body must predominate.Finally the protonated BRH forms ion-pairs with the dyestuffs which are quantitatively extracted into chloroform.

Stoichiometry.
In order to establish molar ratio between BRH and dyestuffs used, Job's method of continuous variation (Figure 5) has been applied [28].In this method, solutions of drug and dyestuff with identical molar concentrations (8 × 10 −5 M) were mixed in varying volume ratios in such a way that the total volume of each mixture was the same.The absorbance of each solution was measured and plotted against the mole fraction of the drug, This measurement showed that 1 : 1 complex was formed.Job's method of stoichiometry is also applied for iodine with BRH which indicated that the charge-transfer complex formed is of 1 : 1 composition.
The stoichiometric ratio between bromhexine HCl and potassium permanganate was evaluated by limiting logarithmic method [29].In this method two sets of experiments were performed.In the first set the concentration of BRH was varied keeping a constant concentration of KMnO 4 , while in the second set the concentration of BRH was kept constant and the KMnO 4 concentration was varied.Log absorbance versus log[BRH] or [ KMnO 4 ] was plotted to  evaluate the slopes of the respective lines.From evaluation of slopes molar combining ratio is 5 : 2 between bromhexine HCl and potassium permanganate.The literature survey reveals that bromhexine HCl undergoes oxidation and gives rise to 2-amino-3, 5-dibromobenzaldehyde and N-methyl cyclohexanamine [14] (Figure 6).

Formation Constants.
The formation constants for method A, B and C are estimated from Job's plot by the following method described by Likussar and Boltz [30] and Momoki et al. [31].The method involves drawing the tangents at the origin of Job's plot from both sides, and the absorbance at intersection point is taken for 100% complexation.The absorbance at peak height of Job's plot is taken for (100 − )%, where  is the % degree of dissociation of the complex.
The instability constant,   = /(100 − ), is calculated, where  is the concentration of drug used for Job's method.The reciprocal of   is the required stability constant .

Formation Constant for Method D.
Formation constant () has been evaluated by using Benesi-Hildebrand equation [32]: where  is absorbance,  is molar absorptivity, and  0 and  0 are initial concentrations of acceptor [I 2 ] and donor [drug], respectively.A plot of [ 0 ]/ versus 1/[ 0 ] yields a straight line whose slope and intercept give the value of .

Optimization of the Factors Affecting the Absorbance.
The factors effecting the absorbance of ion-pair complexes like pH and volume of the dye, in methods A, B, and C, have been optimized.1.8 mL of BTB and buffer of pH 2.8, 1.6 mL of BPB and buffer of pH 2.5, and 2.0 mL of BCG and buffer of pH 3.5 are found to be optimal for methods A, B, and C, respectively.However 5 mL of each dye is used, at optimal pH, in the study to ensure complete extraction of the drug.Similarly the 1 mL of iodine for method D and 1 mL of KMnO 4 , 1 mL of 0.45 NaOH for method E are found to be optimal and hence are used in the study.method.To test the reproducibility of the proposed methods, five replicate determinations of 15.0 g mL of BRH were made.The coefficient of variation was found to be less than 1.2% for all the procedures.

Validation of the
The proposed methods have been successfully applied to the determination of BRH in pharmaceutical preparations.The results obtained and shown in Table 1 were compared to those obtained by a reference method [14] by means of test at 95% confidence level.In all cases, the average results obtained by proposed methods and reference method were statistically identical, as the difference between the average values had no significance at 95% confidence level.

Conclusions
The proposed spectrophotometric methods present selective and simple, specific, and inexpensive analytical procedures for determination of BRH, in pure or in tablet dosage forms without interference from common excipients.Moreover, the developed methods are time saving and do not require elaborate treatments associated with chromatographic methods.These attributes make them suitable for routine analysis in quality control laboratories.

Table 1 :
Optical characteristics, precision, and accuracy data. =  ×  + , where  is the concentration in g mL −1 .

Table 2 :
Application of proposed methods for the determination of BRH in pure form.
[27] result is the average of five separate determinations.*Values in paranthesis are thetabulated  and  values, at  = 0.05[27].

Table 3 :
[27]ication of proposed methods for the determination of BRH in pharmaceutical preparation.Each result is the average of four separate determinations.*Values in paranthesis are the tabulated  and  values, at  = 0.05[27].