research article spectrophotometric determination of...

Hindawi Publishing CorporationISRN Analytical ChemistryVolume 2013 Article ID 861851 7 pageshttpdxdoiorg1011552013861851

Research ArticleSpectrophotometric Determination of Bromhexine HCl in Pureand Pharmaceutical Forms

K Susmitha1 M Thirumalachary1 and G Venkateshwarlu2

1 Department of Chemistry Jawaharlal Nehru Technology University Hyderabad 500085 India2Department of Chemistry Nizam College Hyderabad 500001 India

Correspondence should be addressed to G Venkateshwarlu venkateshwarlugoudyahoocom

Received 8 June 2013 Accepted 3 July 2013

Academic Editors E Billiot S Gokturk and D Patra

Copyright copy 2013 K Susmitha et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Five spectrophotometric methods for determination of Bromhexine HCl have been developed validated and applied for theassay of the drug in pharmaceuticals Methods A B and C are based on ion pair complexation of drug in acidic buffers withtriphenylmethane dyes namely Bromothymol blue (BTB) Bromophenol blue (BPB) and Bromocresol green (BCG)The complexesare extracted into chloroform and absorbance is measured around at 415 nm as function of concentration of the drug Thestoichiometry of the complex is found 1 1 in each case Method D depends upon charge transfer complexation of neutralizeddrug with iodine which produces iodide ion whose absorbance at 366 nm is measured as function of concentration of the drugThis complex too has 1 1 composition as determined by Jobrsquos method Method E is developed on the basis of oxidation of the drugwith alkaline KMnO

4which generates green colored manganate ion with 120582max 610 nm As the intensity of green color increased

with increasing time kinetics of the reaction is followed and calibration curves are constructed by using initial rate and fixedtime methods Excellent recovery studies with high accuracy and precision indicate that the methods can be successfully usedin industries for the assay of drug in pure form and pharmaceuticals

1 Introduction

Bromhexine HCl (BRH) chemically named 2-amino-35-dibromo-N-cyclohexyl-N-methyl benzenemethanaminehydrochloride is a mucolytic agent used in the treatmentof respiratory disorders associated with viscid or excessivemucus [1 2] The drug is official in IP [3] and BP [4]Because of its physiological importance the drug has beenquantified by exploiting its chemical [5 6] and physicalproperties [7ndash25] The different analytical methods usedto quantify the drug as a single active pharmaceuticalingredient include flow injection analysis with ion-selective electrodes [7] inductively coupled plasma massspectrometry [8] electrokinetic chromatography [9] electro-chemical oxidation at the glassy carbon electrode [10] liquidchromatography [11] liquid gas chromatography [12] GCwith mass detection [13] and voltammetry [14] The drughas also been quantified in its combined formulations usingHPLC [15ndash18] direct and derivative UV spectrophotometry

[19ndash23] These methods involve scarcely available costlyequipment and tedious experimentation Simple accurateand precise methods using spectrophotometry have alsobeen developed based on the production of chromophoreby the interaction of the drug with an analytical reagentas chromogen [24ndash26] Availability of vast number ofanalytical reagents to serve as chromogens always leaves alot of scope for pharmaceutical analysis Thorough surveyof the literature showed that the quantification methods ofour interest are not reported yet and hence in the presentcommunication we report five quantification methodsnamely A B C D and E that have been developed andvalidated for quantification of bromhexine HCl both in pureand pharmaceutical forms

2 Experiment

21 Instruments The UV-Visible spectra of the samples wererecorded on SHIMADZU 140 double beam Thermo Nicolet

2 ISRN Analytical Chemistry

Br

OHO

Br

Br

BrN

NH3

+

SO3

minus

Bromothymol blue R1

R1R1

= isopropyl R2

R2R2

= CH3

Bromophenol blue R1 = Br R2 = HBromocresol green R1 = Br R2 = CH3

Scheme 1 Bromhexine HC1-dye complex

D + fast 1(DI+)Iminus (inner complex)

(DI+)Iminus (inner complex) + slow 2

fast 3(DI+) +I2

I2

I3minus

DI2 (outer complex)DI2 (outer complex)

Scheme 2

1000 and ELICO 159 UV-Visible single beam spectropho-tometers using quartz cells of 10mm path length An Elicomodel Li-120 pH meter was used for pH measurement

22 Materials HPLC grade chloroform 12 Dichloroethane(DCE) Analytical grade (AR) HCl Sodium acetate KMnO

4

Sodium hydroxide and dyes viz (a) BTB (b) BPB (c) BCGsupplied by Sd Fine Chemicals Mumbai were used in thestudy Iodine (BDH Poole UK) was twice sublimed andpreserved in vacuum desiccator (mp 1136ndash3∘C) Iodine in12-dichloroethane (DCE) was freshly prepared (daily) bydissolving 254mg solute in 50mL of solvent (40 times 10minus3M)The drug was procured from Aurobindo PharmaceuticalsHyderabad as a gift sample

23 Methods A B and C The methods A B and C arebased on the interaction of the drug with bromothymolblue (BTB) bromophenol blue (BPB) and bromocresolgreen (BCG) respectively to form chloroform-extractableion pair complexes (Scheme 1) which absorb around 415 nm(Figure 1) The absorbance of this band increases withincreasing the concentration of the drug and formed a basisfor the quantification of the drug The dyestuffs were usedas 0025 solutions in doubly distilled water Sodium acetatehydrochloric acid buffers of pH 28 25 and 35were preparedby mixing 50mL of 10M sodium acetate solution with4950mL 5050mL and 4625mL of 10MHCl solutionrespectively and diluted to 250mL with doubly distilledwaterThe pH of each solution was adjusted to an appropriatevalue with the aid of a pH meter

24 Method D Interaction of Iodine with Drug The methoddepends upon the interaction of neutralized drug with iodinethat generates iodide ion having an absorption band at366 nm (Figure 2) The absorbance of this band increases

300 350 400 450 500 55000

02

04

06

08

10

12

14

Abso

rban

ce

Wavelength (nm)

(a)

(b)(c)

Figure 1 Absorption spectra of bromhexine hydrochloride-dyecomplex extracted into 10mL chloroform (a) drug = 10 120583gmLminus1 +5mL of 0025BTB + 5mL of pH28 buffer (b) drug = 9 120583gmLminus1 +5mL of 0025BPB + 5mL of pH25 buffer and (c) drug =95 120583gmLminus1 + 5mL of 0025BCG + 5mL of pH35 buffer

300 350 400 450 500 550 60000

05

10

15

20Ab

sorb

ance

Wavelength (nm)

(a)

(b)

Figure 2 Absorption spectra of (a) reaction product of 125 120583gmLminus1bromhexine hydrochloride with iodine in 12-dichloroethane and(b) iodine

with increasing the concentration of the drug and formed abasis for the quantification of the drug Mixing the solutionof iodine prepared in DCE with BRH resulted in a change ofviolet color of iodine into light brown to pale yellow color andas a consequence absorption spectra exhibited a new bandof 366 nm This is attributed due to I

3

minus ion formed by theinteraction of iodine with drug (Scheme 2)

25 Method E Kinetic Method The method depends on theoxidation of the drug with alkaline KMnO

4(1 times 10minus2M) to

produce manganate ion which absorbs at 610 nm (Figure 3)and formed a basis for quantification of drug A solution of045MNaOH is used to produce required alkalinity Mixingthe solutions of permanganate and the drug slowly developedgreen colour and hence kinetics of the reaction was followedspectrophotometrically with a view to develop a method forthe quantitative determination of the drug spectrophotomet-rically The initial rate and fixed time methods are followedfor the determination of BRH

ISRN Analytical Chemistry 3

Table 1 Optical characteristics precision and accuracy data

Parameters BTB (A) BPB (B) BCG (C) I2 (D) KMnO4 (E)120582max (nm) 41200 41400 41500 36600 61000Beerrsquos law limit (120583gmLminus1) 25ndash25 25ndash25 20ndash25 5ndash50 125ndash150Sandellrsquos sensitivity (120583g cmminus2) 003 005 001 002 035Slope (specific absorptivity) 119887 003 002 001 006 005Intercept (119886) 016 005 006 001 003Correlation coefficient (119903) 100 100 100 100 100Standard deviation of intercepts( 119899 = 6) 002 001 001 006 001

Limit of detection 120583gmLminus1 212 165 258 353 1130Limit of quantification 120583gmLminus1 637 495 774 1060 3410

Regression equation 119910 = 00311 times

119862 + 01598

119910 = 002 times

119862 + 0047

119910 = 00115 times

119862 + 0059

119910 = 0056 times

119862 + 0009

119910 = 00029 times

119862 + 0027

119884 = 119886 times 119862+ 119887 where 119862 is the concentration in 120583gmLminus1

200 300 400 500 600 700 80000020406081012141618

Abso

rban

ce

Wavelength (nm)

(a)

(b)

(c)

Figure 3 Absorption spectra of (a) drug (b) KMnO4(1 times 10minus2M)

and (c) reaction product of 15120583gmLminus1 bromhexine hydrochloridewith alkaline KMnO

4

26 Calibration Curves for Methods A B and C Differentaliquots of drug solution were transferred into 125mL sep-arating funnel To this 5mL of buffer 5mL of dye was addedand total volume was made up to 20mL with water 10mLof chloroform was added and the contents were shaken for5min The two layers were allowed to separate for 5minThe organic layer was separated and absorbance of yellow-colored solutionwhich is stable at least for 3 hrs ismeasured at415 nm against blank similarly preparedThe same procedureof analysis is followed either for assay of pure drug or fordosage form The calibration graphs are linear for the druganalysed using these dyes (Figure 4)

27 Calibration Curve for Method D Into separate 10mLvolumetric flasks different aliquots of BRH solution wastransferred followed by the addition of 1mL of iodinesolution The volume was completed using the same solventand the absorbance was measured against reagent blank at366 nm

0 5 10 15 20 2500051015202530354045

BTB

BPB

BCGIodine

KMnO4

Abso

rban

ce+

0 BTB

+0

5 fo

r BPB

+1

for

BCG+

15

for i

odin

e+2

0 fo

r KM

nO4

Concentration (120583g mLminus1)

Figure 4 Calibration curves

28 Calibration Curve for Method E

281 Initial Rate Method Aliquots of 1 to 8mL of bromhex-ine HCl solution containing 25mgmLminus1 of drug were pipet-ted into a series of 10mL standard flasks To each flask10mL of 045MNaOH and 1mL of 1 times 10minus2M potassiumpermanganate were added successively and then diluted withdistilled water at 25 plusmn 1∘C The contents of each flask weremixed well and the increase in absorbance as a function oftime was measured at 610 nm The initial rate of the reaction(]) at different concentrations was evaluated by measuringthe slope of the tangent to the absorbance-time plot Thecalibration graphs were obtained by plotting the initial rateof reaction (]) versus concentration of bromhexine HCl

282 FixedTimeMethod Afixed time of 20minwas selectedfor the fixed time method At this time the absorbance ofreaction mixture measured against a reagent blank preparedsimilarly The calibration curve was obtained by plotting theabsorbance against the initial concentration of bromhexineHCl Table 1 summarizes the limits of Beerrsquos law Limitof Detection (LOD) Limit of quantification (LOQ) molar


Table 2 Application of proposed methods for the determination of BRH in pure form

BRH BTB (A) BPB (B) BCG (C) I2 (D) KMnO4 (E)

Taken (120583gmLminus1)

300 300 300 500 2000600 600 600 1000 2500900 900 900 1500 30001200 1200 1200 2000 35001500 1500 1500 2500 4000

Found (120583gmLminus1)

301 304 310 499 2002605 603 609 1005 2495899 908 910 1502 30051198 1206 1208 1995 35061510 1496 1498 2496 3996

Recovery ()

9967 9868 9677 10020 99909917 9950 9852 9950 1002010011 9912 9890 9987 998310017 9950 9934 10025 99839934 10027 10013 10016 10010

Mean 9969 9942 9873 10000 9997SD 150 180 125 160 125Reference method [14] 9977 plusmn 048 9977 plusmn 048 9977 plusmn 048 9977 plusmn 048 9977 plusmn 048

119905-test (110)lowast 011 042 173 031 033119865-test (261)lowast 225 324 156 256 156Each result is the average of five separate determinations lowastValues in paranthesis are thetabulated 119905 and 119865 values at 119875 = 005 [27]

absorptivity regression equation correlation coefficients andrelative standard deviations

29 Assay of Drug in Pharmaceuticals

291 Procedure for the Assay of Pure Drug Five differentsolutions of pure drug in the range of calibration curve wereselected and the recovery experiments were performed Therecoveries and their relative standard deviation are tabulatedin (Table 2)

292 Procedure for the Assay of Dosage Forms Ten tabletsof bromhexine 8mg are powdered and dissolved in doublydistilled water and stirred thoroughly and then filteredthrough aWhatman number 42 filter paperThis solutionwastransferred into 100mL standard volumetric flask and dilutedwith doubly distilled water as required Different solutions ofdrug in the range of calibration curve were chosen and theassaywas estimated using the calibration curve and the resultsof the recovery studies are tabulated in (Table 3)

3 Results and Discussion

31 Methods A B and C BRH forms ion-pair complexes inacidic buffer with dyestuffs viz BTB BPB and BCG Thesecomplexes are quantitatively extracted into chloroform Theion-pair complexes with BTB BPB and BCG absorbed maxi-mally at 415 nm (Figure 1) The reagent blank under similarconditions showed no absorption BRH contains aromaticamine group which is protonated in acidic medium whilesulphonic acid group is present in BTB that is the only

group undergoing dissociation in the PH range 1ndash5 BPBand BCG are examples of sulphonepthalein type of dye Thecolour of such dyes is due to the opening of lactoid ringand subsequent formation of quinoid group It is supposedthat the two tautomers are present in equilibrium but due tostrong acidic nature of the sulphonic acid group the quinoidbody must predominate Finally the protonated BRH formsion-pairswith the dyestuffswhich are quantitatively extractedinto chloroform

32 Stoichiometry In order to establish molar ratio betweenBRHand dyestuffs used Jobrsquosmethod of continuous variation(Figure 5) has been applied [28] In this method solutionsof drug and dyestuff with identical molar concentrations(8 times 10minus5M) were mixed in varying volume ratios in sucha way that the total volume of each mixture was the sameThe absorbance of each solution was measured and plottedagainst the mole fraction of the drug [drug][drug] +[dyestuff]

This measurement showed that 1 1 complex was formedJobrsquos method of stoichiometry is also applied for iodinewith BRH which indicated that the charge-transfer complexformed is of 1 1 composition

The stoichiometric ratio between bromhexine HCl andpotassium permanganate was evaluated by limiting logarith-mic method [29] In this method two sets of experimentswere performed In the first set the concentration of BRHwas varied keeping a constant concentration of KMnO

4

while in the second set the concentration of BRH was keptconstant and the KMnO

4concentration was varied Log

absorbance versus log[BRH] or [KMnO4] was plotted to


Table 3 Application of proposed methods for the determination of BRH in pharmaceutical preparation

BRH BTB (A) BPB (B) BCG ( C) I2 (D) KMnO4 (E)


500 500 500 1000 2000700 700 700 2000 3000900 900 900 3000 40001100 1100 1100 4000 5000


495 504 498 996 2006694 697 706 1994 2995905 897 905 2996 40031094 1107 1108 4002 5009

Recovery ()

10101 9921 10040 10040 997010086 10043 9915 10030 100179945 10033 9945 10013 999310055 9937 9928 9995 9982


119905-test (110)lowast 128 008 019 088 016119865-test (261)lowast 125 240 484 094 289Each result is the average of four separate determinations lowastValues in paranthesis are the tabulated 119905 and 119865 values at 119875 = 005 [27]

00 02 04 06 08 10000204060810121416182022

Mole fraction of the drug

BTBBCG

BPB

Iodine

Abso

rban

ce +

0 fo

r BTB

+ 0

5 fo

r BCG

+ 1

0 fo

r BPB

+ 1

5 fo

r Iod

ine

Figure 5 Jobs plots

evaluate the slopes of the respective lines From evaluationof slopes molar combining ratio is 5 2 between bromhexineHCl and potassium permanganate The literature surveyreveals that bromhexine HCl undergoes oxidation and givesrise to 2-amino-3 5-dibromobenzaldehyde and N-methylcyclohexanamine [14] (Figure 6)

33 Formation Constants The formation constants formethod A B and C are estimated from Jobrsquos plot by thefollowing method described by Likussar and Boltz [30] andMomoki et al [31]Themethod involves drawing the tangentsat the origin of Jobrsquos plot from both sides and the absorbanceat intersection point is taken for 100 complexation Theabsorbance at peak height of Jobrsquos plot is taken for (100 minus119909) where 119909 is the degree of dissociation of the complex

The instability constant 1198701015840 = 119862119909(100 minus 119909) is calculatedwhere 119862 is the concentration of drug used for Jobrsquos methodThe reciprocal of1198701015840 is the required stability constant119870

34 Formation Constant for Method D Formation constant(119870) has been evaluated by using Benesi-Hildebrand equation[32]

[1198600]

119889=119868

119870 [1198630] 120598+1

120598 (1)

where 119889 is absorbance 120598 is molar absorptivity and1198600and119863

0

are initial concentrations of acceptor [I2] and donor [drug]

respectively A plot of [1198600]119889 versus 1[119863

0] yields a straight

line whose slope and intercept give the value of119870

35 Optimization of the Factors Affecting the Absorbance Thefactors effecting the absorbance of ion-pair complexes like pHand volume of the dye in methods A B and C have beenoptimized 18mL of BTB and buffer of pH 28 16mL of BPBand buffer of pH 25 and 20mL of BCG and buffer of pH 35are found to be optimal for methods A B and C respectivelyHowever 5mL of each dye is used at optimal pH in thestudy to ensure complete extraction of the drug Similarly the1mL of iodine for method D and 1mL of KMnO

4 1 mL of

045NaOH for method E are found to be optimal and henceare used in the study

36 Validation of the ProposedMethods Theproposedmeth-ods have been validated in terms of guidelines proposed byICH [33] namely selectivity specificity accuracy precisionlimits of calibration curve LOD LOQ robustness rugged-ness and regression equation Studentrsquos 119905-test and variance119865-test have been performed in comparison with a reference


Br

Br

N

CHOBr

Br

HN

NaOH

Bromhexine HCl N-Methyl cyclohexanamine2-Amino-35-dibromobenzaldehyde

NH2

NH2

+CH3

KMnO4

Figure 6 Oxidation of bromhexine HCl with alkaline KMnO4

method To test the reproducibility of the proposed methodsfive replicate determinations of 150 120583gmL of BRH weremade The coefficient of variation was found to be less than12 for all the procedures

The proposed methods have been successfully applied tothe determination of BRH in pharmaceutical preparationsThe results obtained and shown in Table 1 were comparedto those obtained by a reference method [14] by means of 119905-test at 95 confidence level In all cases the average resultsobtained by proposed methods and reference method werestatistically identical as the difference between the averagevalues had no significance at 95 confidence level

4 Conclusions

The proposed spectrophotometric methods present selectiveand simple specific and inexpensive analytical proceduresfor determination of BRH in pure or in tablet dosage formswithout interference from common excipients Moreover thedeveloped methods are time saving and do not require elab-orate treatments associated with chromatographic methodsThese attributes make them suitable for routine analysis inquality control laboratories

Acknowledgments

The authors are grateful to the Head Department of Chem-istry and Principal Nizam College for providing facilitiesThe support rendered by Department of Chemistry Jawahar-lal Nehru Technology University is duly acknowledged

References

[1] L Parvez M Vaidya A Sakhardande S Subburaj and TG Rajagopalan ldquoEvaluation of antitussive agents in manrdquoPulmonary Pharmacology vol 9 no 5-6 pp 299ndash308 1996

[2] D M Cobbin F M Elliott and A S Rebuck ldquoThe mucolyticagent bromhexine (bisolvon) in chronic lung disease A double-blind crossover trialrdquo Australian and New Zealand Journal ofMedicine vol 1 no 2 pp 137ndash140 1971

[3] Indian Pharmacopoeia 1996 and Addendum 2000 (Govern-ment of India Ministry of Health and Family welfare Con-troller of publications New Delhi India)

[4] British Pharmacopoeia vol 2 HMSO London UK 1996

[5] S S Zarapker R V Rele and V J Doshi ldquoSimple spectropho-tometricmethods for estimation of bromhexine hydrochloriderdquoIndian Drugs vol 26 no 1 pp 38ndash41 1988

[6] M I R M Santoro M M Dos Santos and J F MagalhaesldquoSpectrophotometric determination of bromhexine hydrochlo-ride in pharmaceutical preparationsrdquo Journal of the Associationof Official Analytical Chemists vol 67 no 3 pp 532ndash534 1984

[7] N T Abdel-Ghani YM Issa andHM Ahmed ldquoPotentiomet-ric flow injection analysis of bromhexine hydrochloride and itspharmaceutical preparation using conventional and coatedwireion-selective electrodesrdquo Scientia Pharmaceutica vol 74 no 3pp 121ndash135 2006

[8] B P Jensen B Gammelgaard S H Hansen and J V Ander-sen ldquoHPLC-ICP-MS compared with radiochemical detectionfor metabolite profiling of 3H-bromohexine in rat urine andfaecesrdquo Journal of Analytical Atomic Spectrometry vol 20 no3 pp 204ndash209 2005

[9] H Okamoto T Nakajima Y Ito T Aketo K Shimadaand S Yamato ldquoSimultaneous determination of ingredientsin a cold medicine by cyclodextrin-modified microemulsionelectrokinetic chromatographyrdquo Journal of Pharmaceutical andBiomedical Analysis vol 37 no 3 pp 517ndash528 2005

[10] M Pospısilova M Polasek and V Jokl ldquoDetermination ofambroxol or bromhexine in pharmaceuticals by capillary iso-tachophoresisrdquo Journal of Pharmaceutical and Biomedical Anal-ysis vol 24 no 3 pp 421ndash428 2001

[11] E V Rao G R Rao S Raghuveer and P KhadgapathildquoGas-liquid chromatographic and ion-pair high-performanceliquid chromatographic determination of pseudoephedrinehydrochloride and bromhexine hydrochloride in pharmaceu-ticalsrdquo Analyst vol 112 no 6 pp 871ndash874 1987

[12] O-W Lau and Y-M Cheung ldquoSimultaneous determination ofsome active ingredients in coughmdashcold syrups by gasmdashliquidchromatographyrdquo Analyst vol 115 no 10 pp 1349ndash1353 1990

[13] C E Uboh J A Rudy L R Soma et al ldquoCharacterization ofbromhexine and ambroxol in equine urine effect of furosemideon identification and confirmationrdquo Journal of Pharmaceuticaland Biomedical Analysis vol 9 no 1 pp 33ndash39 1991

[14] M Turchan P Jara-Ulloa S Bollo L J Nunez-Vergara JA Squella and A Alvarez-Lueje ldquoVoltammetric behaviour ofbromhexine and its determination in pharmaceuticalsrdquoTalantavol 73 no 5 pp 913ndash919 2007

[15] J-P Rauha H Salomies and M Aalto ldquoSimultaneous deter-mination of bromhexine hydrochloride and methyl and propylp-hydroxybenzoate and determination of dextromethorphanhydrobromide in cough-cold syrup by high-performance liquidchromatographyrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 15 no 2 pp 287ndash293 1996


[16] M Vasudevan S Ravisankar M George and J Ravi ldquoSimulta-neous estimation of terbutaline bromhexine and guaiphenesinin soft gelatin capsules by HPLCmethodrdquo Indian Drugs vol 37no 10 pp 489ndash492 2000

[17] H N Dave R C Mashru and A K Patel ldquoThin layer chro-matodraphy method for the determination of ternary mixturecontaining salbutamol sulphate bromhexine hydrochloride andEtofyllinerdquo Journal of Pharmaceutical Sciences andResearch vol2 no 3 pp 143ndash148 2010

[18] A El-Gindy S Emara andH Shaaban ldquoDevelopment and vali-dation of chemometrics-assisted spectrophotometric and liquidchromatographic methods for the simultaneous determinationof two multicomponent mixtures containing bronchodilatordrugsrdquo Journal of Pharmaceutical and Biomedical Analysis vol43 no 3 pp 973ndash982 2007

[19] V Gupta M Verma U Misra and R K Nema ldquoSimultaneousspectrophotoetric estimation of bromhexine hydrochloride andpseudoephedrine hydrochloride in tablet dosagesrdquo Asian Jour-nal of Chemistry vol 21 no 2 pp 1633ndash1635 2009

[20] A K Gupta and S G Kaskhedikar ldquoDerivative spectrophoto-metric estimation of amoxycillin and bromhexine hydrochlo-ride in tabletsrdquo Asian Journal of Chemistry vol 15 no 2 pp977ndash980 2003

[21] S K Panda A K Sharma and L K Sahu ldquoSimultane-ous analysis of phenylpropanolamine chlorpheniramine andbromhexine in syrups by derivative spectrophotometryrdquo IndianJournal of Pharmaceutical Sciences vol 64 no 6 pp 540ndash5442002

[22] S Gangwal and P Trivedi ldquoSimultaneous determination ofterbutaline sulphate bromhexine hydrochloride and guaiphen-esin in three-component tablet formulation by UV spectropho-tometryrdquo Indian Journal of Pharmaceutical Sciences vol 61 no1ndash6 pp 128ndash130 1999

[23] X Song XWang and D Ze ldquoDetermination of aminophyllineand bromhexine hydrochloride in Dingchuanzhike tablets byUV spectrophotometryrdquo Zhongguo Yaoke Daxue Xuebao vol30 no 2 pp 100ndash102 1999

[24] S V Murali Mohan Rao I Nageswara Rao T Rama SubbaReddy and C S P Sastry ldquoAssay of bromhexine hydrochloridein pharmaceutical formulations by extraction spectrophotom-etryrdquo Indian Journal of Chemical Technology vol 12 no 2 pp170ndash174 2005

[25] S V Murali Mohan Rao U Viplava Prasad I Nageswara Raoand M Satya Babu ldquoSensitive spectrophotometric methods forthe determination of bromhexine hydrochloriderdquo Acta CienciaIndica Chemistry vol 30 no 2 pp 105ndash108 2004

[26] S Kanna Babu P A V Ganapathi N V S N Raju and GMadhuKumar ldquoSpectrophotometric estimation of bromhexinehydrochloride in pharmaceutical preparationsrdquo Eastern Phar-macist vol 42 no 493 pp 135ndash136 1999

[27] J C Miller and J N Miller Statistics and Chemometrics forAnalytical Chemistry Harlow England 2005

[28] P Job Analytica Chimica Acta 16 1936 in Advanced Physico-chemical Experiments Oliner and Boyd Edinburgh UK 2ndedition

[29] J Rose Advanced Physicochemical Experiments Pitman Lon-don UK 1964

[30] W Likussar and D F Boltz ldquoTheory of continuous variationsplots and a new method for spectrophotometric determinationof extraction and formation constantsrdquo Analytical Chemistryvol 43 no 10 pp 1265ndash1272 1971

[31] K Momoki J Sekino H Sato and N Yamaguchi ldquoTheory ofcurved molar ratio plots and a new linear plotting methodrdquoAnalytical Chemistry vol 41 no 10 pp 1286ndash1299 1969

[32] H A Benesi and J H Hildebrand ldquoA spectrophotometricinvestigation of the interaction of iodine with aromatic hydro-carbonsrdquo Journal of the American Chemical Society vol 71 no8 pp 2703ndash2707 1949

[33] International Conference on Harmonization (ICH) of Techni-cal Requirement for the Registration of Pharmaceuticals forHuman use Validation of analytical procedures definitions andTerminology Genera 1996

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CatalystsJournal of


Br

OHO

Br

Br

BrN

NH3

+

SO3

minus

Bromothymol blue R1

R1R1

= isopropyl R2

R2R2

= CH3

Bromophenol blue R1 = Br R2 = HBromocresol green R1 = Br R2 = CH3

Scheme 1 Bromhexine HC1-dye complex

D + fast 1(DI+)Iminus (inner complex)

(DI+)Iminus (inner complex) + slow 2

fast 3(DI+) +I2

I2

I3minus

DI2 (outer complex)DI2 (outer complex)

Scheme 2

1000 and ELICO 159 UV-Visible single beam spectropho-tometers using quartz cells of 10mm path length An Elicomodel Li-120 pH meter was used for pH measurement

22 Materials HPLC grade chloroform 12 Dichloroethane(DCE) Analytical grade (AR) HCl Sodium acetate KMnO

4

Sodium hydroxide and dyes viz (a) BTB (b) BPB (c) BCGsupplied by Sd Fine Chemicals Mumbai were used in thestudy Iodine (BDH Poole UK) was twice sublimed andpreserved in vacuum desiccator (mp 1136ndash3∘C) Iodine in12-dichloroethane (DCE) was freshly prepared (daily) bydissolving 254mg solute in 50mL of solvent (40 times 10minus3M)The drug was procured from Aurobindo PharmaceuticalsHyderabad as a gift sample

23 Methods A B and C The methods A B and C arebased on the interaction of the drug with bromothymolblue (BTB) bromophenol blue (BPB) and bromocresolgreen (BCG) respectively to form chloroform-extractableion pair complexes (Scheme 1) which absorb around 415 nm(Figure 1) The absorbance of this band increases withincreasing the concentration of the drug and formed a basisfor the quantification of the drug The dyestuffs were usedas 0025 solutions in doubly distilled water Sodium acetatehydrochloric acid buffers of pH 28 25 and 35were preparedby mixing 50mL of 10M sodium acetate solution with4950mL 5050mL and 4625mL of 10MHCl solutionrespectively and diluted to 250mL with doubly distilledwaterThe pH of each solution was adjusted to an appropriatevalue with the aid of a pH meter

24 Method D Interaction of Iodine with Drug The methoddepends upon the interaction of neutralized drug with iodinethat generates iodide ion having an absorption band at366 nm (Figure 2) The absorbance of this band increases

300 350 400 450 500 55000

02

04

06

08

10

12

14

Abso

rban

ce

Wavelength (nm)

(a)

(b)(c)

Figure 1 Absorption spectra of bromhexine hydrochloride-dyecomplex extracted into 10mL chloroform (a) drug = 10 120583gmLminus1 +5mL of 0025BTB + 5mL of pH28 buffer (b) drug = 9 120583gmLminus1 +5mL of 0025BPB + 5mL of pH25 buffer and (c) drug =95 120583gmLminus1 + 5mL of 0025BCG + 5mL of pH35 buffer

300 350 400 450 500 550 60000

05

10

15

20Ab

sorb

ance

Wavelength (nm)

(a)

(b)

Figure 2 Absorption spectra of (a) reaction product of 125 120583gmLminus1bromhexine hydrochloride with iodine in 12-dichloroethane and(b) iodine

with increasing the concentration of the drug and formed abasis for the quantification of the drug Mixing the solutionof iodine prepared in DCE with BRH resulted in a change ofviolet color of iodine into light brown to pale yellow color andas a consequence absorption spectra exhibited a new bandof 366 nm This is attributed due to I

3

minus ion formed by theinteraction of iodine with drug (Scheme 2)

25 Method E Kinetic Method The method depends on theoxidation of the drug with alkaline KMnO

4(1 times 10minus2M) to

produce manganate ion which absorbs at 610 nm (Figure 3)and formed a basis for quantification of drug A solution of045MNaOH is used to produce required alkalinity Mixingthe solutions of permanganate and the drug slowly developedgreen colour and hence kinetics of the reaction was followedspectrophotometrically with a view to develop a method forthe quantitative determination of the drug spectrophotomet-rically The initial rate and fixed time methods are followedfor the determination of BRH






119862 + 01598

119910 = 002 times

119862 + 0047

119910 = 00115 times

119862 + 0059

119910 = 0056 times

119862 + 0009

119910 = 00029 times

119862 + 0027


200 300 400 500 600 700 80000020406081012141618

Abso

rban

ce

Wavelength (nm)

(a)

(b)

(c)



4



0 5 10 15 20 2500051015202530354045

BTB

BPB

BCGIodine

KMnO4

Abso

rban

ce+

0 BTB

+0

5 fo

r BPB

+1

for

BCG+

15

for i

odin

e+2

0 fo

r KM

nO4










300 300 300 500 2000600 600 600 1000 2500900 900 900 1500 30001200 1200 1200 2000 35001500 1500 1500 2500 4000


301 304 310 499 2002605 603 609 1005 2495899 908 910 1502 30051198 1206 1208 1995 35061510 1496 1498 2496 3996

Recovery ()

9967 9868 9677 10020 99909917 9950 9852 9950 1002010011 9912 9890 9987 998310017 9950 9934 10025 99839934 10027 10013 10016 10010













4








500 500 500 1000 2000700 700 700 2000 3000900 900 900 3000 40001100 1100 1100 4000 5000


495 504 498 996 2006694 697 706 1994 2995905 897 905 2996 40031094 1107 1108 4002 5009

Recovery ()

10101 9921 10040 10040 997010086 10043 9915 10030 100179945 10033 9945 10013 999310055 9937 9928 9995 9982



00 02 04 06 08 10000204060810121416182022


BTBBCG

BPB

Iodine

Abso

rban

ce +

0 fo

r BTB

+ 0

5 fo

r BCG

+ 1

0 fo

r BPB

+ 1

5 fo

r Iod

ine

Figure 5 Jobs plots





[1198600]

119889=119868

119870 [1198630] 120598+1

120598 (1)


0






4 1 mL of




Br

Br

N

CHOBr

Br

HN

NaOH


NH2

NH2

+CH3

KMnO4




4 Conclusions


Acknowledgments


References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






119862 + 01598

119910 = 002 times

119862 + 0047

119910 = 00115 times

119862 + 0059

119910 = 0056 times

119862 + 0009

119910 = 00029 times

119862 + 0027


200 300 400 500 600 700 80000020406081012141618

Abso

rban

ce

Wavelength (nm)

(a)

(b)

(c)



4



0 5 10 15 20 2500051015202530354045

BTB

BPB

BCGIodine

KMnO4

Abso

rban

ce+

0 BTB

+0

5 fo

r BPB

+1

for

BCG+

15

for i

odin

e+2

0 fo

r KM

nO4










300 300 300 500 2000600 600 600 1000 2500900 900 900 1500 30001200 1200 1200 2000 35001500 1500 1500 2500 4000


301 304 310 499 2002605 603 609 1005 2495899 908 910 1502 30051198 1206 1208 1995 35061510 1496 1498 2496 3996

Recovery ()

9967 9868 9677 10020 99909917 9950 9852 9950 1002010011 9912 9890 9987 998310017 9950 9934 10025 99839934 10027 10013 10016 10010













4








500 500 500 1000 2000700 700 700 2000 3000900 900 900 3000 40001100 1100 1100 4000 5000


495 504 498 996 2006694 697 706 1994 2995905 897 905 2996 40031094 1107 1108 4002 5009

Recovery ()

10101 9921 10040 10040 997010086 10043 9915 10030 100179945 10033 9945 10013 999310055 9937 9928 9995 9982



00 02 04 06 08 10000204060810121416182022


BTBBCG

BPB

Iodine

Abso

rban

ce +

0 fo

r BTB

+ 0

5 fo

r BCG

+ 1

0 fo

r BPB

+ 1

5 fo

r Iod

ine

Figure 5 Jobs plots





[1198600]

119889=119868

119870 [1198630] 120598+1

120598 (1)


0






4 1 mL of




Br

Br

N

CHOBr

Br

HN

NaOH


NH2

NH2

+CH3

KMnO4




4 Conclusions


Acknowledgments


References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





300 300 300 500 2000600 600 600 1000 2500900 900 900 1500 30001200 1200 1200 2000 35001500 1500 1500 2500 4000


301 304 310 499 2002605 603 609 1005 2495899 908 910 1502 30051198 1206 1208 1995 35061510 1496 1498 2496 3996

Recovery ()

9967 9868 9677 10020 99909917 9950 9852 9950 1002010011 9912 9890 9987 998310017 9950 9934 10025 99839934 10027 10013 10016 10010













4








500 500 500 1000 2000700 700 700 2000 3000900 900 900 3000 40001100 1100 1100 4000 5000


495 504 498 996 2006694 697 706 1994 2995905 897 905 2996 40031094 1107 1108 4002 5009

Recovery ()

10101 9921 10040 10040 997010086 10043 9915 10030 100179945 10033 9945 10013 999310055 9937 9928 9995 9982



00 02 04 06 08 10000204060810121416182022


BTBBCG

BPB

Iodine

Abso

rban

ce +

0 fo

r BTB

+ 0

5 fo

r BCG

+ 1

0 fo

r BPB

+ 1

5 fo

r Iod

ine

Figure 5 Jobs plots





[1198600]

119889=119868

119870 [1198630] 120598+1

120598 (1)


0






4 1 mL of




Br

Br

N

CHOBr

Br

HN

NaOH


NH2

NH2

+CH3

KMnO4




4 Conclusions


Acknowledgments


References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





500 500 500 1000 2000700 700 700 2000 3000900 900 900 3000 40001100 1100 1100 4000 5000


495 504 498 996 2006694 697 706 1994 2995905 897 905 2996 40031094 1107 1108 4002 5009

Recovery ()

10101 9921 10040 10040 997010086 10043 9915 10030 100179945 10033 9945 10013 999310055 9937 9928 9995 9982



00 02 04 06 08 10000204060810121416182022


BTBBCG

BPB

Iodine

Abso

rban

ce +

0 fo

r BTB

+ 0

5 fo

r BCG

+ 1

0 fo

r BPB

+ 1

5 fo

r Iod

ine

Figure 5 Jobs plots





[1198600]

119889=119868

119870 [1198630] 120598+1

120598 (1)


0






4 1 mL of




Br

Br

N

CHOBr

Br

HN

NaOH


NH2

NH2

+CH3

KMnO4




4 Conclusions


Acknowledgments


References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Br

Br

N

CHOBr

Br

HN

NaOH


NH2

NH2

+CH3

KMnO4




4 Conclusions


Acknowledgments


References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

research article spectrophotometric determination of...

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