Hindawi Publishing CorporationJournal of SpectroscopyVolume 2013 Article ID 214270 13 pageshttpdxdoiorg1011552013214270

Research ArticleNew Spectrophotometric and Conductometric Methods forMacrolide Antibiotics Determination in Pure and PharmaceuticalDosage Forms Using Rose Bengal

Rania A Sayed Wafaa S Hassan Magda Y El-Mammli and Abadalla Shalaby

Department of Analytical Chemistry Faculty of Pharmacy Zagazig University Zagazig 44519 Egypt

Correspondence should be addressed to Rania A Sayed raniaadelsayedyahoocom

Received 20 June 2012 Revised 30 November 2012 Accepted 30 November 2012

Academic Editor Mariacutea Carmen Yebra-Biurrun

Copyright copy 2013 Rania A Sayed et alis 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

Two Simple accurate precise and rapid spectrophotometric and conductometric methods were developed for the estimation oferythromycin thiocyanate (I) clarithromycin (II) and azithromycin dihydrate (III) in both pure and pharmaceutical dosage formse spectrophotometric procedure depends on the reaction of rose bengal and copper with the cited drugs to form stable ternarycomplexes which are extractable with methylene chloride and the absorbances were measured at 558 557 and 560 nm for (I) (II)and (III) respectively e conductometric method depends on the formation of an ion-pair complex between the studied drugand rose bengal For the spectrophotometric method Beerrsquos law was obeyed e correlation coefficient (1199031199032) for the studied drugswas found to be 09999emolar absorptivity (120576120576) Sandellrsquos sensitivity limit of detection (LD) and limit of quantication (L)were also calculatede proposedmethodswere successfully applied for the determination of certain pharmaceutical dosage formscontaining the studied drugs

1 Introduction

emacrolides are a large group of antibioticsmainly derivedfrom Streptomyces ey have a common macrocyclic lac-tone ring to which one or more sugars are attached and areall weak bases that are only slightly soluble in water [1]ey are bacteriostatic agents ey inhibit protein synthesisby binding reversibly to 50 S ribosomal subunit of sensitivemicroorganisms [2] Macrolides and related drugs have apostantibiotic effect that is antibacterial activity persists aerconcentrations have dropped below the minimum inhibitoryconcentration [1]

Erythromycin is produced by the actinomycete speciesStreptomyces erythreus Erythromycin is a polyhydroxylac-tone that contains two sugars e aglycone portion of themolecule erythranolide is a 14-membered lactone ringAn amino sugar desosamine is attached through a 120573120573-glycosidic linkage to the C-5 position of the lactone ringe tertiary amine of desosamine confers a basic characterto erythromycin (pKa 88) rough this group a numberof acid salts of the antibiotic have been prepared A second

sugar cladinose which is unique to erythromycin is attachedvia a 120573120573-glycosidic linkage to the C-3 position of the lactonering [3] It is commonly used to treat infections caused bygram-positive organisms Mycoplasma species and certainsusceptible gram-negative and anaerobic bacteria withinrespiratory tract skin so tissues and genital tract [4]

More recently developed macrolides including azithro-mycin and clarithromycin seem to have essentially similarproperties to erythromycin which is a natural macrolidethough they may differ in their pharmacokinetics Clari-thromycin and to a lesser extent azithromycin are moreactive than erythromycin against opportunistic mycobacteriasuch as Mycobacterium avium complex Clarithromycin isalso used in the treatment of leprosy and in regimens forthe eradication of Helicobacter pylori in peptic ulcer diseaseBoth azithromycin and clarithromycin have activity againstprotozoa including Toxoplasma gondii Gastrointestinal dis-turbances are the most frequent adverse effect but are usuallymild and less frequent with clarithromycin and azithromycinthan with erythromycin [1]

2 Journal of Spectroscopy

e literature search revealed different techniques forthe analysis of the studied macrolides e British Pharma-copoeia (BP) stated the liquid chromatography method forthe assay of erythromycin clarithromycin and azithromycin[5] Also the cited drugs have been analyzed spectropho-tometrically in pharmaceutical dosage forms and biologicaluids [6ndash10] and spectrouorimetrically [11 12] Othermethods have been published for the cited drugs eitherin pharmaceutical preparations and biological uids suchas capillary electrophoresis [13 14] HPLC [15ndash17] andvoltametry [18] A microbiological method was reported forthe assay of erythromycin thiocyanate [19] and azithromycin[20]

emajority of the reportedmethods are HPLCmethodswhich require complex and expensive equipment intensivesample preparation and personnel skilled in chromato-graphic techniques Also Due to weak UV absorbanceof macrolide antibiotics it is difficult to develop a sim-ple method for analysis of the drugs in pharmaceuticalapplications and biological uids using conventional UVdetection so there is a need for simple spectrophotometricmethod for the analysis of the cited drugs in pharmaceuticalformulations So the aim of this work is to develop simplesensitive accurate and cheap spectrophotometric (throughternary complex formation with rose bengal and copper) andconductometric mehods (through ion pair formation withrose bengal) for the determination of three macrolide drugsin pure form and in the pharmaceutical preparations

Rose bengal has been used as ion-pair-forming agent fordetermination of chlorphenoxamine hydrcholoride anhy-drous caffeine [21] and oxybuprocaine hydrochloride [22]It was also used for conductometric determination of clin-damycin hydrochloride through ion-pair formation [23] andit was also used for determination of isoniazide nalidixicacid and umequine through formation of ternary complexwith Cd(II) [24] and used for determination of bariumthrough formation of ternary complex with 18-crown-6 [25]Copper was used as a complexing agent for determinationof several drugs for example lincomycin [26] ramiprilperindopril [27] and enalapril maleate [28] lansoprazoleand pantoprazole sodium sesquihydrate [29] and risedronate[30]

2 Experimental

21 Instruments e absorption spectra for all measure-ments were carried out using shimadzu recording spec-trophotometer UV 1201 equipped with 10mm matchedquartz cells Conductometer model 470 portable conductiv-ity TDS meter 25 DEGC-C10 dip-type cell was used with acell constant K cell of 109 Digital analyzer pHmeter (USA)was used

22 Reagents andMaterials All chemicals andmaterialswereof analytical grade and bidistilled water was used throughoutthe work

(i) Erythromycin thiocyanate Erythrocin lm tabletslabelled to contain erythromycin stearate equivalent

to 500mg erythromycin per tablet (Kahira Pharmamp ChemIndCo) and erythrocin pharco powderfor oral suspension labelled to contain erythromycinethylsuccinate equivalent to 200mg erythromycin toeach 5mL reconstituted suspension (Pharco Com-pany For Phrmaceuticals Egypt)

(ii) Clarithromycin Klarimix tablets labelled to contain250mg clarithromycin per tablet and Klarimix pow-der for oral suspension labelled to contain 125mgclarithromycin to each 5mL reconstituted suspension(SIGMA Pharmaceutical Industries Egypt)

(iii) Azithromycin dihydrate Zithrokan capsules Azrolidtablets (Amriya For Phrmaceutical Industries Egypt)labelled to contain 5241mg azithromycin dihydrateper capsuletablet andZithrokan powder for oral sus-pension labelled to contain 200mg azithromycin toeach 5mL reconstituted suspension (Hikma Pharma6th of October city Egypt)

(iv) Rose bengal (Aldrich Germany) was used as 3mgmL in double-distilled water for the spectrophoto-metric method For conductometric method 3 times10minus3M in double distilled water for (I) in 50 (vv)ethanol for (II) and in 50 (vv) methanol for (III)

(v) Copper(II) chloride (El-Nasr chemical pharmaceuti-cal company Egypt) solution was used as 3mgmL indouble-distilled water

(vi) Glucose Sucrose sodium chloride carboxy methylcellulose sodium calcium carbonate magnesiumstearate (El-Nasr chemical pharmaceutical companyEgypt)

All drugs whose purity was found to be 10012 accord-ing to the reported method [6] were used as received

23 Preparation of Sample Solutions

(i) For the spectrophotometric method solution of05mgmL for (I) and (II) and of 02mgmL for(III) was dissolved in low amount of ethanol andcompleted with double distilled water to the mark

(ii) For conductometric method solution of 1mgmLwas dissolved in low amount of methanol for (I) andcompleted with double distilled water to the mark in50 (vv) ethanol for (II) and 50 (vv) methanol for(III)

24 Recommended Procedures and Calibration Curves

241 Spectrophotometric Measurements Into a series of60mL separating funnels aliquots of drug solutionsequivalent to 15ndash6 075ndash4 and 04ndash2mg of erythromycinthiocyanate (I) clarithromycin (II) azithromycin dihydrate(III) respectively were transferred then specic volumes ofCu (II) chloride solution (3mgmL) were added followed byspecied amounts of rose bengal (3mgmL) and le for 5minutes in case of Clarithromycin (II) (Table 1)e complexwas extracted with 3 times 3mL portions of methylene chloride

Journal of Spectroscopy 3

T 1 Quantitative parameters for the spectrophotometric determination of erythromycin thiocyanate (I) clarithromycin (II) andazithromycin dihydrate (III) with rose bengal copper

Parameter (I) (II) (III)120582120582max (nm) 558 557 560Beerrsquos law ranges (120583120583gmL) 15ndash60 75ndash40 4ndash20Regression equationlowastlowast

Slope (119887119887) 00193 00209 00617Intercept (119886119886) minus01413 minus0022 minus01247Correlation coefficient (1199031199032) 09999 09999 09999LOD 120583120583gmL 1 093 031LOQ 120583120583gmL 304 28 095Ringbom optimum concentration range (120583120583gmL) 20ndash50 10ndash35 9ndash16Sandellrsquos sensitivity 120583120583gsdotcmminus2 007 005 002120576120576 (times104) Lsdotmolminus1sdotcmminus1 112 147 378lowastAverage of three experimentslowastlowastA = 119886119886 + 119887119887c

e solutions were vigorously shaken for one minute and theorganic layer ltered over anhydrous sodium sulphate into10mL volumetric asks then the volumes were completedto 10mL with methylene chloride e absorbances of thecolored solutions were measured at 558 557 and 560 nm for(I) (II) and (III) respectively against reagent blank treatedsimilarly (Figure 1) All measurements were made at ambienttemperature

242 Conductometric Titrations Aliquot of sample solutioncontaining 1ndash15mg of the cited drugs was transferred to a50mL calibrated ask and volume was made up to the markusing bidistilled water for (I) 50 (vv) ethanol for (II) and50 (vv) methanol for (III) e contents of the calibratedask were transferred to a beaker and the conductivitycell was immersed 3 times 10minus3M rose bengal was used fortitration e conductance was measured subsequently toeach addition of reagent solution and aer thorough stirringfor twominutes corrected for dilution effect [31] bymeans ofthe following equation assuming that conductivity is a linearfunction of dilution

Ωminus1correct = Ω

minus1obs 10077181007718

100764910076491199071199071 + 1199071199072100766510076651199071199071

10077341007734 (1)

whereΩminus1obs is the observed electrolytic conductivity 1199071199071 is the

initial volume and 1199071199072 is the volume of reagent added A graphof corrected conductivity versus the volume of added titrantwas constructed and the endpoint was determined (Figure 7)

25 Procedure for Pharmaceutical Preparations

251 For Spectrophotometric Method e contents of vecapsules were removed and weighed accurately e com-bined contents were mixed and a quantity of the powderequivalent to 25mg for (I) and (II) and equivalent to 10mgfor (III) was extracted with low amount of ethanol in50mL volumetric ask e volumes were completed to themark with double-distilled water ltered and the assay wascompleted as described under general procedure e same

075

06

04

02

0

minus005500 520 540 560 580 600

(nm)

Absorbance

(a)

(b)

(c)(d)

(e)(f)

F 1 Absorption spectra for (a) the reaction of 13 120583120583gmLazithromycin dihydrate with 05mL Cu(II) chloride 03 wv and02mL 03 wv rose bengal (b) 13 120583120583gmL azithromycin dihydratewith 02mL rose bengal 03 wv (c) 13 120583120583gmL azithromycindihydrate with 05mL Cu(II) chloride 03 wv (d) 02mL rosebengal 03 wv and 05mL Cu(II) chloride 03 wv (e) 02mLrose bengal 03 wv and (f) 05mL Cu(II) chloride 03 wv

procedures were followed with tablets taking an accuratelyweighed amount of ten combined powdered tabletse sameprocedures were followed with oral suspension taking anaccurately measured volume of the freshly reconstituted oralsuspension equivalent to the specied concentration

252 For Conductometric Method e same procedure wasfollowed as in the spectrophotometric method but the quan-tity of the powder taken from tablets or capsules or theaccurately measured volume of the freshly reconstituted oralsuspension is equivalent to 100mg for all drugs and wasextracted by least amount of methanol for (I) 50 (vv)

4 Journal of Spectroscopy

ethanol for (II) and 50 (vv) methanol for (III) lteredand transferred to a 100mL volumetric ask completed tothe mark using distilled water for (I) 50 (vv) ethanol for(II) and in 50 (vv) methanol for (III) and the assay wascompleted as described under general procedure

3 Results and Discussion

Optimization of Variables Ternary complexes have beenwidely used in spectrophotometric analysis e absorptionspectrum in aqueous medium of rose bengal shows anabsorption peak with 120582120582max 540 nm Upon addition of anyof the three cited drugs to a mixture of Cu(II) and rosebengal solution it was found that a pink soluble complex wasformed and the absorbance maximum of Cu(II) rose bengaloriginally at 540 nm was abridged to a shoulder with a newabsorption maxima at 558 557 and 560 nm for (I) (II) and(III) respectively e absorption spectrum shows that theternary complexes formed between rose bengal and copperwith the cited drugs have higher values of molar extinctioncoefficient than the binary complexes for the spectrophoto-metric method (Figure 1) Investigations were carried outto establish the most favorable conditions for the ternarycomplex formation for the spectrophotometric method ofthe cited drugs with rose bengal and copper to achievemaximum color development and for ion-pair formation forthe conductometric method to achieve the endpoint

31 Conditions for Spectrophotometric Method

311 Effect of pH In a trial to elucidate the optimummedium for the quantitative determination of the studieddrugs the effect of different acetate and phosphate buffers ofpH of range 2ndash10 was examined It was found that buffer hadno effect on the reaction

312 Effect of Cu(II) Chloride Volume e effect of Cu(II)chloride volume was also tested by using varying amounts(01ndash2mL) of Cu(II) chloride (3mgmL)e results showedthat 02mL 15mL and 05mL of Cu(II) chloride (3mgmL)for (I) (II) and (III) respectively were sufficient for theproduction of maximum and reproducible colour intensity(Figure 2)

313 Effect of Rose Bengal Volume Highest constantabsorbance was obtained on using 15mL 05mL and02mL of rose bengal (3mgmL) for (I) (II) and (III)respectively (Figure 3)

314 Effect of Order of Addition emost suitable sequencewas drug Cu(II) chloride and then rose bengal for all drugsfor the production of the highest colour intensity while theother sequences produced lower absorbance values

315 Effect of Reaction Time and Stability Maximum colourintensity was attained immediately for the studied drugs

06

05

04

03

02

01

00 05 1 15 2

Volume (mL)

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin dihydrate

F 2 Effect of volume of 03 wv Cu(II) chloride on theternary complex formed with 40 120583120583gmL erythromycin thiocyanateand 15mL rose bengal 03 wv 25 120583120583gmL clarithromycin and05mL rose bengal 03wv 13 120583120583gmL azithromycin dihydrate and02mL rose bengal 03 wv

06

05

04

03

02

01

00 05 1 15 2 25

Volume (mL)

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin dihydrate

F 3 Effect of volume of 03 wv rose bengal on the ternarycomplex formed with 40 120583120583gmL erythromycin thiocyanate and02mL Cu(II) chloride 03 wv 25 120583120583gmL clarithromycin and15mL Cu(II) chloride 03 wv 13 120583120583gmL azithromycin dihydrateand 05mL Cu(II) chloride 03 wv

except for (II) it needs 5 minutes to give complete com-plexation and increasing time beyond this had no effect onabsorptione obtained colour was stable for at least 1 hourfor all drugs at room temperature (25∘C plusmn 2) e formationconstants have been calculated by using the molar ratiomethod [32] e formation constants (Kf ) of the reactionproduct were found to be 713 times 104 76 times 105 and 15 times106 Lsdotmolminus1 for (I) (II) and (III) respectively is highvalue indicates a very stable reaction product e Gibbsfree energy change of the reaction (ΔG) was also calculated

Journal of Spectroscopy 5

06

07

08

05

04

03

02

01

00 05 1 15 2 25 3 35 454

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin

VrVd

F 4 Molar ratio method plot for 25 times 10minus4M erythromycinthiocyanate and 25 times 10minus4M Cu(II) chloride in presence of excessrose bengal (15mL 03) 25 times 10minus4M clarithromycin and 25 times10minus4M Cu(II) chloride in presence of excess rose bengal (05mL03) 125 times 10minus4 M azithromycin dihydrate and 125 times 10minus4MCu(II) chloride in presence of excess rose bengal (02mL 03)VR = Cu(II) chloride and Vd = Drug

and was found to be minus278 times 104 minus335 times 104 and minus352times 104 KsdotJsdotmoleminus1 for (I) (II) and (III) respectively enegative value of ΔG points out to the spontaneous natureof the reaction [33]

316 Effect of the Extraction Time and Number of ExtractionsShaking the reaction mixture with the extracting organicsolvent is required for complete extraction of the ternarycomplex formed It was found that 1 minute is enough timefor complete colour extraction Complete extraction wasattained by triple extraction with 3 times 3mL of methylenechloride for all drugs

317 Effect of Solvent Many organic solvents were tried toextract the ternary complexes formed (chloroform methy-lene chloride ethylene chloride benzene toluene and ethylacetate) e results obtained indicated that methylene chlo-ride is the appropriate solvent for extraction for all drugs

318 Determination of Stoichiometric Balance Using MolarRatio Method [34] e reaction stoichiometry between ery-thromycin thiocyanate (I) clarithromycin (II) azithromycindihydrate (III) with Cu(II) chloride and rose bengal was stud-ied by molar ratio method using an equimolar (25 times 10minus4M)solution of erythromycin thiocyanate (I) clarithromycin (II)and (125times 10minus4M) solutions of azithromycin dihydrate (III)Cu(II) chloride and rose bengal of the same molarities eobtained results showed that the composition of the ternarycomplexes was 1 1 2 for erythromycin thiocyanate (I) and

06

07

05

04

03

02

01

00 05 1 15 2 25 3 35 454

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin

VrVd

F 5 Molar ratio method plot for 25 times 10minus4M erythromycinthiocyanate with 25 times 10minus4M rose bengal in presence of excessCu(II) chloride (02 mL 03) 25 times 10minus4M clarithromycin with25times10minus4Mrose bengal in presence of excessCu(II) chloride (15mL03) 125times 10minus4Mazithromycin dihydratewith 125times 10minus4Mrosebengal in presence of excess Cu(II) chloride (05mL 03) Vr = rosebengal and Vd = Drug

06

05

04

03

02

01

00 05 1 15 2 25 3 35 4

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin

VrVd

F 6 Molar ratio method plot for 25 times 10minus4M rose bengalwith 25 times 10minus4M Cu(II) chloride in presence of excess ery-thromycin thiocyanate (30 120583120583gsdotmLminus1) 25 times 10minus4M rose bengal with25 times 10minus4M Cu(II) chloride in presence of excess clarithromycin(30 120583120583gsdotmLminus1) 125 times 10minus4M rose bengal with 125 times 10minus4M Cu(II)chloride in presence of excess azithromycin dihydrate (10 120583120583gsdotmLminus1)Vr = rose bengal and VR = Cu(II) chloride

1 2 2 for clarithromycin (II) and azithromycin dihydrate(III) Cu(II) chloride rose bengal (Figures 4 5 and 6)

Scheme 1 illustrates the mechanism of the reaction ofclarithromycin rose bengal and copper(II) chloride elactone ring in clarithromycin is substituted with a numberof hydroxyl functional groups ese groups are positionedin suitable conguration for interaction with metal ions So

6 Journal of Spectroscopy

O

O

O

OO

O O

O

O

O

O

O

O

O

O

O

O

O

O CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

OH

OH

Cu

Cu

N(CH3)2

N(CH3)2

OMe

HOHO

HO

HOHO

H3C

H3C

H3C

H3C

H3C

H3C

HO

I I

II

I I

II

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

COO

2

minus4+4

Rose bengalClarithromycin

2Cu+2

COOminus

Ominus

2 ++

OMe

2Na+

OMe

OMe

S 1 ProposalMechanism for the reaction between clarithromycin rose bengal and copper(II) chloride clarithromycin-Cu-rose bengalcomplex

copper cation forms a chelatewith the lone pair of electrons ofthe two hydroxyl groups at C11 andC12 Also copper can forma chelate with the lone pair of electrons of the tertiary aminegroup nitrogen and the oxygen of the 120573120573-glycosidic linkageSo the azithromycin-copper chelate bears an overall positivecharge of the original two metal ions that is four positivechargesis cationic complex associates with two moleculesof rose bengal anions to form the ternary complex

32 Conditions for Conductometric Method Investigationswere carried out to establish the most suitable conditionsfor the ion associates formation of the cited drugs with rosebengal to attain sharp endpoint e optimum conditionsfor performing the titration in a quantitative manner wereelucidated as described later

321 Reaction Medium Titrations in different media wereattempted to obtain the best results Preliminary experimentswere in

(i) aqueous drug solution for (I) only (prepared bydissolving 100mg drug in 2mL methanol then

completing to 100mL with bidistilled water) withaqueous reagent solution

(ii) ethanol drug solution with ethanol reagent solution(iii) drug solution with reagent solution both in ethanol-

water (50 vv) mixture(iv) methanol drug solution with methanol reagent solu-

tion (except for (II) as it is insoluble in methanol)(v) drug solution with reagent solution both in

methanol-water mixture (50 vv) (except for (II) asit is insoluble in methanol)

(vi) drug solution with reagent solution both in acetone-water (50 vv) mixture

Preliminary experiments showed that procedure in aque-ous media was the most suitable for successful results for (I)50 (vv) ethanol for (II) and 50 (vv) methanol for (III)

322 Reagentrsquos Concentration Different concentrations ofrose bengal solution were tried ranging from 2 times 10minus2 to5 times 10minus4 molar solutions e optimum concentration of thereagent was 3 times 10minus3M in titration of the studied drug to

Journal of Spectroscopy 7

80

70

60

50

40

30

20

10

00 2 4 6 8

Volume of rose bengal (mL)

Co

nd

uct

ivit

y (

S cm

minus1)

F 7 Conductometric titration curve of 15mg clarithromycinversus 3 times 10 3M rose bengal

achieve a constant and highly stable conductance readingwithin 1-2min of mixing Concentrations less than theselimits led to unstable readings and more time was neededto obtain constant conductance values

Representative titration curve is shown in (Figure 7)Two straight lines are obtained intersecting at the endpointthe rst branch ascending and the second has conductancevalues that would slightly increase aer the equivalencepoint e increase of conductance may be attributed tothe formation of ion pair in solution as a result of thecomplexation reaction Aer the end-point the titrationcurves indicate a slightly increased value of conductancedespite the excess of the reagent is may be due tofurther ionic condensation leading to species of lowermobility

e conductometric titrations of different volumes of3 times 10minus3 M rose bengal solution were performed e resultsshow anobviousmaximum in the conductance curve at drug-reagent molar ratio of 2 1 for (I) and (II) and 1 1 for (III)e reactions may be represented by (for (I))

[RB]minus2+210077131007713Eryth N (Me)210077291007729H+

⟶[RB]minus210077131007713Eryth NH(Me)2100772910077292+2

(2)

where [RB]minus2 rose bengal anion [Eryth N(Me)2] ery-thromycin thiocyanate [ErythNH(Me)2]

+2 protonated ery-thromycin thiocyanate

Erythromycin thiocyanate tertiary amine nitrogen isprotonated so attraction occurs between it and rose bengalanion forming ion-pair complex

e conductance measured before the addition of thetitrant (volume of rose bengal equals zero) is mainly due tothe hydrogen ions mobility ese hydrogen ions result fromtwo sources

(1) the active methylene group in the drug structureas in Scheme 2 which has the ability to make selfprotonation to the drug

O

CH3

lowast

S 2 lowaste active methylene group in macrolide structure

(2) the protons coming from the alcohols used in solubil-ity as these alcohols have certain acidity character socan protonate the amino group in drug structure

So there is no need for acidic buffer use for drug pro-tonation It was expected that the conductance values wouldremain constant or slightly increase aer the equivalencepoint However the conductance of the solution decreasedis may be due to interaction of the protons available inthe titration medium with the added reagent e resultsfrom the conductometic titrations are summarized in Table 4e data show that accurate results were obtained with goodrecoveries and low standard deviation values e optimumconcentration ranges for determination of the cited drugswere in the range of 1ndash15mg mLminus1 for all drugs At suchranges sharp inections (Figure 7) and stable conductancereading were obtained

33 Method Validation

331 For the Spectrophotometric Method

Linearity Under the optimumconditions described standardcalibration curves for erythromycin thiocyanate (I) clar-ithromycin (II) and azithromycin dihydrate (III) with rosebengal and copper were constructed by plotting absorbanceagainst concentration Conformity with Beerrsquos law was evi-dent in the concentration range of the nal dilution citedin Table 1 Beerrsquos law holds over the concentration rangesof 15ndash60 75ndash40 and 4ndash20 120583120583gsdotmLminus1 for (I) (II) and (III)respectively e linear regression equation for each drugwas listed in Table 1 e correlation coefficient was 09999indicating good linearity over the working concentrationrangeSensitivity e detection limit (LOD) for the proposedmethod was calculated using the following equation accord-ing to the ICH [35]

LOD = 33120590120590S (3)

where 120590120590 = the standard deviation of replicate blank responses(under the same conditions as for sample analysis) and S =the slope of the calibration curve

e limits of quantication LOQ is dened as

LOQ = 10120590120590S (4)

8 Journal of Spectroscopy

04

03

02

01

0430 500 550 600 650

(nm)

Absorbance

(a)

(b)

F 8Absorption spectra of (a) 25times 10minus2Mglucosewith 15mLCu(II) chloride 03 wv and 05mL 03 wv rose bengal (b) 5 times10minus2M sucrose with 15mL Cu(II) chloride 03 wv and 05mL03 wv rose bengal

According to the previous equations the LODs and LOQswere calculated as in Table 1 eir values conrm thesensitivity of the proposed methodAccuracy and Precision In order to determine the accuracyand precision of the proposed method solutions containingone concentration of each drug were prepared and analysedin seven replicates e relative standard deviation (RSD)as precision and percentage relative error (Er) as accuracyof the suggested method were calculated at 95 condencelevels and can be considered satisfactory e percent-age relative error was calculated according the followingequation

Er = 10077171007717found minus added

added10077331007733 times 100 (5)

e inter- and intraday precisions and accuracy resultsare shown in Table 5 e analytical results for accuracyand precision show that the proposed method has goodrepeatability and reproducibilityInterference e effect of presence of common excipi-ents such as glucose sucrose sodium chloride carboxymethyl cellulose sodium calcium carbonate andmagnesiumstearate was studied Table 6 presents the results of determi-nation of clarithromycin as a representative example usingthe proposed procedure in the presence of aforementionedingredients e tolerance ratio was dened as the concen-tration which gave an error of plusmn30 in the determinationof drugs Results indicate that the tolerance ratio of drugexcipient (M M) is 1 50 for glucose and 1 100 for sucroseWhile there were no interference from the presence ofsodium chloride carboxy methyl cellulose sodium calcium

carbonate and magnesium stearate Interference due toglucose and sucrosewas further investigated by studying theirreaction with Cu(II) chloride and rose bengal e studyrevealed that no interference could be observed at 557 nmand an interaction product was obtained at 120582120582max 498 intolerance ratios 1 1 and 1 10 for glucose and 1 1 1 101 50 for sucrose However interference starts to appear at120582120582max 557 upon using 50 times 100-fold excess of the molarconcentration of clarithromycin for glucose and sucroserespectively as shown in Figure 8

332 For the Conductometric Method In order to addressthe validity of the proposed method statistical analysis of thedata obtained from its application on the cited drugs in thepure form and in formulations was performed e resultsrevealed in Tables 5 and 6 showed that the proposed methodis satisfactorily accurate precise and reproducible over aconcentration range of 1ndash15mg Results for the determina-tion of the studied drugs using the previously mentionedmethod were compared with results from reported one [6]Table 7

34 Analytical Applications e proposed methods wereapplied to determine the studied drugs in their pharma-ceutical dosage forms Satisfactory results were obtainede recovery of each drug was calculated by comparing theconcentration obtained from the spiked mixtures with thoseof the drug e results of analysis of the commercial dosageforms and the recovery study are shown in Tables 2 3 and4 e results obtained were compared with the reportedmethod [6] No signicant differences were found betweenthe proposed methods and reported method Statisticalcomparison of the results was performed using Studentrsquos t-test and variance ratio F-test at 95 condence level (Table7)

4 Conclusions

e proposed methods are advantageous when compared tomany of the reported spectrophotometric methods in havinghigher sensitivity e data given previously reveal that theproposed methods are simple accurate and sensitive withgood precision and accuracyWith thesemethods one can dothe analysis at low cost without losing accuracy No interfer-ence from excipients was encountered Also In comparisonwith the chromatographic methods the proposed methodsare advantageous as HPLC and GLC are not available espe-cially in developing countries and they require complexand expensive equipment intensive sample preparation andpersonnel skilled in chromatographic techniques So theproposed methods can be used as alternative methods to thereported ones for the routine determination of erythromycinthiocyanate (I) clarithromycin (II) azithromycin dihydrate(III) in the pure form and in pharmaceutical formulationsdepending upon the availability of chemicals and the equip-ment

Journal of Spectroscopy 9

T2

Determination

oferythrom

ycin

thiocyanateand

clarithrom

ycin

intheirph

armaceutic

aldo

sage

form

sthroug

hcomplexation

with

Cu(II)

chlorid

eand

rose

beng

alby

spectro

photom

etric

procedurelowast

Erythrocin

tablets

Erythrocin

Pharco

suspensio

nKlarim

ixtablets

Klarim

ixsuspensio

nClaimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

151499

9993

151494

9959

75

756

10080

101005

10048

151489

9924

151504

10028

75

746

9952

101014

10144

201986

9930

202002

10008

101010

10096

125

1239

9914

225

2250

10001

252504

10017

151512

10080

151498

9984

252520

10079

302991

9971

175

1727

9870

175

1746

9979

275

2774

10086

325

3261

10033

202014

10072

202005

10024

302976

9919

353483

9953

225

2244

9973

225

2268

10080

353515

10041

404012

10030

252517

10067

275

2756

10022

375

3758

10021

425

4255

10013

275

2756

10022

303005

10016

403965

9919

302995

9984

Mean

9991

10001

10020

10023

Varia

nce

044

010

054

042

SD066

032

073

065

SE015

011

024

023

lowast Averageo

fthree

experim

ents

10 Journal of Spectroscopy

T3Determinationof

azith

romycin

dihydrateinits

pharmaceutic

aldo

sage

form

sthrou

ghcomplexationwith

Cu(II)chlorid

eand

rose

beng

alby

spectro

photom

etric

procedurelowast

Azrolid

tablets

Zithrokancapsules

Zithrokansuspensio

n

Claimed

tobe

taken120583120583g

mL

Authentic

added120583120583g

mL

Foun

dconc

120583120583gm

LRe

covery

Claimed

tobe

taken

120583120583gm

L

Authentic

added120583120583g

mL

Foun

dconc

120583120583gm

LRe

covery

Claimed

tobe

taken120583120583g

mL

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

LRe

covery

4403

10077

4401

10036

4405

10118

4401

10036

4408

10199

4405

10118

7701

10019

5497

9942

6606

10095

8800

10002

6602

10041

8802

10022

9896

9953

8797

9962

101006

10060

101003

10028

9906

10061

121199

9991

111111

10103

101000

9995

141401

10010

121197

9977

111098

9985

161589

9934

141403

10022

121202

10018

161589

9934

131299

9995

Mean

10015

10023

10043

Varia

nce

027

051

043

SD052

072

066

SE017

024

025

lowast Averageo

fthree

experim

ents

Journal of Spectroscopy 11

T 4 Application of the proposed conductometric method to the determination of erythromycin thiocyanate in dosage formslowast

Erythrocin tablets Erythrocin Pharco suspensionTaken (mg) Found (mg) Recovery Taken (mg) Found (mg) Recovery 3 305 10158 2 2 1004 4 100 4 405 101196 607 10119 7 705 100688 81 10119 8 805 1006010 10 100 12 1190 992012 1191 9921 14 1395 996614 1405 10034Mean 10050 10022SD 085 073SE 032 030RSD 084 073Variance 072 054lowastMean of three different experiments

T 5 e intraday and interday precision and accuracy data for the studied drugs using the spectrophotometric method

Intraday Interday

Drug Taken(120583120583gmLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSDbAccuracyEr c

Taken(120583120583g mLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSD bAccuracyEr c

(I) 50 5028 10056 029 056 50 5034 10068 039 068(II) 30 3033 10109 056 109 30 3017 10057 046 057(III) 16 1599 9995 019 minus005 16 1601 10007 025 007aAverage of seven determinationsbRSD percentage relative standard deviationcEr percentage relative error

T 6 Analysis of clarithromycin 28 120583120583gmLminus1 (5times 10minus4 M) by the spectrophotometric method in the presence of some common excipients

Tolerance molar ratio ( M M)lowast Glucose Sucrose Carboxy methyl cellulose sodium Sodium chlorideRecovery lowastlowast

1 1 10124 10107 10089 99821 10 10036 10124 9911 99821 50 11545 10213 10035 99471 100 10657 10746 9822 9929lowastDrug excipientlowastlowastMean of three determinations

T 7 Determination of the studied drugs by the proposed spectrophotometric and conductmetric methods compared with the reportedmethod [6]

(I) (II) (III)Spectrophotometric method

Mean plusmn RSD 10005 plusmn 054 10001 plusmn 047 9987 plusmn 050Variance 029 022 025Studentrsquos 119905119905-test 005 (217)lowast 04 (131)lowast 006 (216)lowast119865119865-test 238 (428)lowast 391 (360)lowast 268 (389)lowast

119899119899 7 9 9Conductometric method

Mean plusmn RSD 10066 plusmn 093 10065 plusmn 063 10062 plusmn 062Variance 087 040 038Studentrsquos 119905119905-test 131 (217)lowast 113 (217)lowast 210 (2228)lowast119865119865-test 126 (387)lowast 215 (397)lowast 176 (805)lowast

119899119899 7 6 6Reported method [6]

Mean plusmn RSD 10004 plusmn 083 10015 plusmn 093 9989 plusmn 082Variance 069 086 067Studentrsquos 119905119905-test mdash mdash mdash119865119865-test mdash mdash mdash119899119899 7 8 6lowaste gures in parenthesis are the theoretical values for 119905119905 and 119865119865-tests (119875119875 119875 005)

12 Journal of Spectroscopy

Acknowledgments

e authors wish to express their great thanks to Dr SoadSelim Abd El Hay and Dr Omnia Ahmed Emam Ismaiel atthe Analytical Chemistry Department Zagazig Universityfor their great help and advices during this work Also theywould like to point out that the authors do not have anyconict of interests with any company or institution

References

[1] S C Sweetman Martindale the Complete Drug ReferencePharmaceutical Press London UK 35th edition 2007

[2] S OmuraMacrolide Antibiotics Academic Press London UK1984

[3] J N Delgado and W A RemersWilson and Gisvoldrsquos Textbookof Organic Medecinal and Pharmaceutical Chemistry J BLippincott Company London UK 1998

[4] H A Krisr Progress inMedicinal Chemistry vol 30 of Edited byG P Ellis and D K Luscombe 1993

[5] British Pharmacopoeia Her Majestyrsquos Stationery Office Lon-don UK 2007

[6] M I Walash M S Rizk M I Eid and M E Fathy ldquoSpec-trophotometric determination of four macrolide antibiotics inpharmaceutical formulations and biological uids via binarycomplex formation with eosin and spectrophotometryrdquo Journalof AOAC International vol 90 no 6 pp 1579ndash1587 2007

[7] J Shah M R Jan and S Manzoor ldquoExtractive spectrophoto-metric methods for determination of clarithromycin in phar-maceutical formulations using bromothymol blue and cresolredrdquo Journal of the Chinese Chemical Society vol 55 no 5 pp1107ndash1112 2008

[8] C E R De Paula V G K Almeida and R J CassellaldquoNovel spectrophotometric method for the determination ofazithromycin in pharmaceutical formulations based on itscharge transfer reaction with quinalizarinrdquo Journal of theBrazilian Chemical Society vol 21 no 9 pp 1664ndash1671 2010

[9] S Ashour and R Bayram ldquoNovel spectrophotometric methodfor determination of somemacrolide antibiotics in pharmaceu-tical formulations using 1 2-naphthoquinone-4-sulphonaterdquoSpectrochim Acta Part A vol 99 pp 74ndash80 2012

[10] S D Magar A P Tupe P Y Pawar and B Y ManeldquoSimultaneous spectrophotometric estimation of ceximeand azithrhomycin in tablet dosage formrdquo Current PharmaResearch vol 2 no 3 pp 535ndash538 2012

[11] P Y Khashaba ldquoSpectrouorimetric analysis of certain mac-rolide antibiotics in bulk and pharmaceutical formulationsrdquoJournal of Pharmaceutical and Biomedical Analysis vol 27 no6 pp 923ndash932 2002

[12] N El-Rabbat H F Askal P Y Khashaba and N N AttialdquoA validated spectrouorometric assay for the determinationof certainmacrolide antibiotics in pharmaceutical formulationsand spiked biological uidsrdquo Journal of AOAC International vol89 no 5 pp 1276ndash1287 2006

[13] C L Flurer ldquoAnalysis of macrolide antibiotics by capillaryelectrophoresisrdquo Electrophoresis vol 17 no 2 pp 359ndash3661996

[14] A K Lalloo S C Chattaraj and I Kanfer ldquoDevelopmentof a capillary electrophoretic method for the separation ofthe macrolide antibiotics erythromycin josamycin and olean-domycinrdquo Journal of Chromatography B vol 704 no 1-2 pp333ndash341 1997

[15] M Hedenmo and B M Eriksson ldquoLiquid chromatographicdetermination of the macrolide antibiotics roxithromycin andclarithromycin in plasma by automated solid-phase extractionand electrochemical detectionrdquo Journal of Chromatography Avol 692 no 1-2 pp 161ndash166 1995

[16] M Dubois D Fluchard E Sior and P Delahaut ldquoIdenticationand quantication of ve macrolide antibiotics in severaltissues eggs and milk by liquid chromatography-electrospraytandem mass spectrometryrdquo Journal of Chromatography B vol753 no 2 pp 189ndash202 2001

[17] R V S Nirogi V N Kandikere M Shukla et al ldquoSensitiveand selective liquid chromatography-tandem mass spectrom-etry method for the quantication of azithromycin in humanplasmardquo Analytica Chimica Acta vol 553 no 1-2 pp 1ndash82005

[18] O A E M Farghaly and N A L Mohamed ldquoVoltammetricdetermination of azithromycin at the carbon paste electroderdquoTalanta vol 62 no 3 pp 531ndash538 2004

[19] J A Bernabeacuteu M A Camacho M E Gil-Alegre V Ruz andA I Torres-Suaacuterez ldquoMicrobiological bioassay of erythromycinthiocyanate optimisation and validationrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 21 no 2 pp 347ndash3531999

[20] H R N Salgado and A F F Roncari ldquoMicrobiological assayfor the determination of azithromycin in ophthalmic solutionsrdquoYaoxue Xuebao vol 40 no 6 pp 544ndash549 2005

[21] A S Amin and M M El-Henawee ldquoColorimetric methodfor the simultaneous determination of chlorphenoxaminehydrochloride and anhydrous caffeine in pure and dosage formswith rose bengalrdquo Mikrochimica Acta vol 118 no 3-4 pp177ndash183 1995

[22] F M Abdel-Gawad ldquoSpectrophotometric determination ofoxybuprocaine hydrochloride with halouorescein derivativesrdquoFarmaco vol 50 no 3 pp 197ndash200 1995

[23] A S Amin ldquoSpectrophotometric and conductometric deter-mination of clindamycin hydrochloride in pure form andin pharmaceutical preparationsrdquo Analusis vol 23 no 8 pp415ndash417 1995

[24] SM Amer Z El-Sherif andMM Amer ldquoSpectrophotometricdetermination of isoniazid nalidixic acid and umequinethrough ternary complex-formation with Cd (II) and rosebengalrdquo Egyptian Journal of Pharmaceutical Sciences vol 35 no1ndash6 pp 627ndash642 1994

[25] H Parham and A G Fazeli ldquoExtraction-spectrophotometricdetermination of trace amounts of barium by 18-crown-6 androse bengalrdquo Analytical Sciences vol 16 no 6 pp 575ndash5772000

[26] M A El Ries ldquoSpectrophotometric and indirect determinationof lincomycin by atomic absorption spectroscopyrdquo AnalyticalLetters vol 27 no 8 pp 1517ndash1531 1994

[27] H E Abdellatef M M Ayad and E A Taha ldquoSpectrophoto-metric and atomic absorption spectrometric determination oframipril and perindopril through ternary complex formationwith eosin and Cu(II)rdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 18 no 6 pp 1021ndash1027 1999

[28] M M Ayad A A Shalaby H E Abdellatef and M M HosnyldquoSpectrophotometric and AAS determination of ramipril andenalapril through ternary complex formationrdquo Journal of Phar-maceutical and Biomedical Analysis vol 28 no 2 pp 311ndash3212002

Journal of Spectroscopy 13

[29] A AMMoustafa ldquoSpectrophotometric methods for the deter-mination of lansoprazole and pantoprazole sodium sesquihy-draterdquo Journal of Pharmaceutical and Biomedical Analysis vol22 no 1 pp 45ndash58 2000

[30] M I Walash M E S Metwally M Eid and R N El-ShahenyldquoSpectrophotometric determination of risedronate in pharma-ceutical formulations via complex formation with Cu(II) ionsapplication to content uniformity testingrdquo International Journalof Biomedical Science vol 4 no 4 pp 303ndash309 2008

[31] J J Lingane Electroanalytical Chemistry Interscience NewYork NY USA 2nd edition 1958

[32] S A Tirmizi F H Wattoo M H S Wattoo S Sarwar AN Memon and A B Ghangro ldquoSpectrophotometric studyof stability constants of cimetidine-Ni(II) complex at differenttemperaturesrdquo Arabian Journal of Chemistry vol 35 no 1 pp93ndash100 2010

[33] J Inczedy Analytical Application of Complex EquilibriiaBudapest John Wiley amp Sons 1976

[34] J H Yoe and A L Jones ldquoCcrforimetric determination of ironwith disodium-12-dlhydroxybenzene-35-disuffonaterdquo Indus-trial and Engineering Chemistry vol 16 pp 111ndash115 1944

[35] International Conference on Harmonisation ICH of TechnicalRequirments for Registeration of Pharmaceuticals for HumanUse 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

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Journal of

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Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

2 Journal of Spectroscopy

e literature search revealed different techniques forthe analysis of the studied macrolides e British Pharma-copoeia (BP) stated the liquid chromatography method forthe assay of erythromycin clarithromycin and azithromycin[5] Also the cited drugs have been analyzed spectropho-tometrically in pharmaceutical dosage forms and biologicaluids [6ndash10] and spectrouorimetrically [11 12] Othermethods have been published for the cited drugs eitherin pharmaceutical preparations and biological uids suchas capillary electrophoresis [13 14] HPLC [15ndash17] andvoltametry [18] A microbiological method was reported forthe assay of erythromycin thiocyanate [19] and azithromycin[20]

emajority of the reportedmethods are HPLCmethodswhich require complex and expensive equipment intensivesample preparation and personnel skilled in chromato-graphic techniques Also Due to weak UV absorbanceof macrolide antibiotics it is difficult to develop a sim-ple method for analysis of the drugs in pharmaceuticalapplications and biological uids using conventional UVdetection so there is a need for simple spectrophotometricmethod for the analysis of the cited drugs in pharmaceuticalformulations So the aim of this work is to develop simplesensitive accurate and cheap spectrophotometric (throughternary complex formation with rose bengal and copper) andconductometric mehods (through ion pair formation withrose bengal) for the determination of three macrolide drugsin pure form and in the pharmaceutical preparations

Rose bengal has been used as ion-pair-forming agent fordetermination of chlorphenoxamine hydrcholoride anhy-drous caffeine [21] and oxybuprocaine hydrochloride [22]It was also used for conductometric determination of clin-damycin hydrochloride through ion-pair formation [23] andit was also used for determination of isoniazide nalidixicacid and umequine through formation of ternary complexwith Cd(II) [24] and used for determination of bariumthrough formation of ternary complex with 18-crown-6 [25]Copper was used as a complexing agent for determinationof several drugs for example lincomycin [26] ramiprilperindopril [27] and enalapril maleate [28] lansoprazoleand pantoprazole sodium sesquihydrate [29] and risedronate[30]

2 Experimental

21 Instruments e absorption spectra for all measure-ments were carried out using shimadzu recording spec-trophotometer UV 1201 equipped with 10mm matchedquartz cells Conductometer model 470 portable conductiv-ity TDS meter 25 DEGC-C10 dip-type cell was used with acell constant K cell of 109 Digital analyzer pHmeter (USA)was used

22 Reagents andMaterials All chemicals andmaterialswereof analytical grade and bidistilled water was used throughoutthe work

(i) Erythromycin thiocyanate Erythrocin lm tabletslabelled to contain erythromycin stearate equivalent

to 500mg erythromycin per tablet (Kahira Pharmamp ChemIndCo) and erythrocin pharco powderfor oral suspension labelled to contain erythromycinethylsuccinate equivalent to 200mg erythromycin toeach 5mL reconstituted suspension (Pharco Com-pany For Phrmaceuticals Egypt)

(ii) Clarithromycin Klarimix tablets labelled to contain250mg clarithromycin per tablet and Klarimix pow-der for oral suspension labelled to contain 125mgclarithromycin to each 5mL reconstituted suspension(SIGMA Pharmaceutical Industries Egypt)

(iii) Azithromycin dihydrate Zithrokan capsules Azrolidtablets (Amriya For Phrmaceutical Industries Egypt)labelled to contain 5241mg azithromycin dihydrateper capsuletablet andZithrokan powder for oral sus-pension labelled to contain 200mg azithromycin toeach 5mL reconstituted suspension (Hikma Pharma6th of October city Egypt)

(iv) Rose bengal (Aldrich Germany) was used as 3mgmL in double-distilled water for the spectrophoto-metric method For conductometric method 3 times10minus3M in double distilled water for (I) in 50 (vv)ethanol for (II) and in 50 (vv) methanol for (III)

(v) Copper(II) chloride (El-Nasr chemical pharmaceuti-cal company Egypt) solution was used as 3mgmL indouble-distilled water

(vi) Glucose Sucrose sodium chloride carboxy methylcellulose sodium calcium carbonate magnesiumstearate (El-Nasr chemical pharmaceutical companyEgypt)

All drugs whose purity was found to be 10012 accord-ing to the reported method [6] were used as received

23 Preparation of Sample Solutions

(i) For the spectrophotometric method solution of05mgmL for (I) and (II) and of 02mgmL for(III) was dissolved in low amount of ethanol andcompleted with double distilled water to the mark

(ii) For conductometric method solution of 1mgmLwas dissolved in low amount of methanol for (I) andcompleted with double distilled water to the mark in50 (vv) ethanol for (II) and 50 (vv) methanol for(III)

24 Recommended Procedures and Calibration Curves

241 Spectrophotometric Measurements Into a series of60mL separating funnels aliquots of drug solutionsequivalent to 15ndash6 075ndash4 and 04ndash2mg of erythromycinthiocyanate (I) clarithromycin (II) azithromycin dihydrate(III) respectively were transferred then specic volumes ofCu (II) chloride solution (3mgmL) were added followed byspecied amounts of rose bengal (3mgmL) and le for 5minutes in case of Clarithromycin (II) (Table 1)e complexwas extracted with 3 times 3mL portions of methylene chloride

Journal of Spectroscopy 3

T 1 Quantitative parameters for the spectrophotometric determination of erythromycin thiocyanate (I) clarithromycin (II) andazithromycin dihydrate (III) with rose bengal copper

Parameter (I) (II) (III)120582120582max (nm) 558 557 560Beerrsquos law ranges (120583120583gmL) 15ndash60 75ndash40 4ndash20Regression equationlowastlowast

Slope (119887119887) 00193 00209 00617Intercept (119886119886) minus01413 minus0022 minus01247Correlation coefficient (1199031199032) 09999 09999 09999LOD 120583120583gmL 1 093 031LOQ 120583120583gmL 304 28 095Ringbom optimum concentration range (120583120583gmL) 20ndash50 10ndash35 9ndash16Sandellrsquos sensitivity 120583120583gsdotcmminus2 007 005 002120576120576 (times104) Lsdotmolminus1sdotcmminus1 112 147 378lowastAverage of three experimentslowastlowastA = 119886119886 + 119887119887c

e solutions were vigorously shaken for one minute and theorganic layer ltered over anhydrous sodium sulphate into10mL volumetric asks then the volumes were completedto 10mL with methylene chloride e absorbances of thecolored solutions were measured at 558 557 and 560 nm for(I) (II) and (III) respectively against reagent blank treatedsimilarly (Figure 1) All measurements were made at ambienttemperature

242 Conductometric Titrations Aliquot of sample solutioncontaining 1ndash15mg of the cited drugs was transferred to a50mL calibrated ask and volume was made up to the markusing bidistilled water for (I) 50 (vv) ethanol for (II) and50 (vv) methanol for (III) e contents of the calibratedask were transferred to a beaker and the conductivitycell was immersed 3 times 10minus3M rose bengal was used fortitration e conductance was measured subsequently toeach addition of reagent solution and aer thorough stirringfor twominutes corrected for dilution effect [31] bymeans ofthe following equation assuming that conductivity is a linearfunction of dilution

Ωminus1correct = Ω

minus1obs 10077181007718

100764910076491199071199071 + 1199071199072100766510076651199071199071

10077341007734 (1)

whereΩminus1obs is the observed electrolytic conductivity 1199071199071 is the

initial volume and 1199071199072 is the volume of reagent added A graphof corrected conductivity versus the volume of added titrantwas constructed and the endpoint was determined (Figure 7)

25 Procedure for Pharmaceutical Preparations

251 For Spectrophotometric Method e contents of vecapsules were removed and weighed accurately e com-bined contents were mixed and a quantity of the powderequivalent to 25mg for (I) and (II) and equivalent to 10mgfor (III) was extracted with low amount of ethanol in50mL volumetric ask e volumes were completed to themark with double-distilled water ltered and the assay wascompleted as described under general procedure e same

075

06

04

02

0

minus005500 520 540 560 580 600

(nm)

Absorbance

(a)

(b)

(c)(d)

(e)(f)

F 1 Absorption spectra for (a) the reaction of 13 120583120583gmLazithromycin dihydrate with 05mL Cu(II) chloride 03 wv and02mL 03 wv rose bengal (b) 13 120583120583gmL azithromycin dihydratewith 02mL rose bengal 03 wv (c) 13 120583120583gmL azithromycindihydrate with 05mL Cu(II) chloride 03 wv (d) 02mL rosebengal 03 wv and 05mL Cu(II) chloride 03 wv (e) 02mLrose bengal 03 wv and (f) 05mL Cu(II) chloride 03 wv

procedures were followed with tablets taking an accuratelyweighed amount of ten combined powdered tabletse sameprocedures were followed with oral suspension taking anaccurately measured volume of the freshly reconstituted oralsuspension equivalent to the specied concentration

252 For Conductometric Method e same procedure wasfollowed as in the spectrophotometric method but the quan-tity of the powder taken from tablets or capsules or theaccurately measured volume of the freshly reconstituted oralsuspension is equivalent to 100mg for all drugs and wasextracted by least amount of methanol for (I) 50 (vv)

4 Journal of Spectroscopy

ethanol for (II) and 50 (vv) methanol for (III) lteredand transferred to a 100mL volumetric ask completed tothe mark using distilled water for (I) 50 (vv) ethanol for(II) and in 50 (vv) methanol for (III) and the assay wascompleted as described under general procedure

3 Results and Discussion

Optimization of Variables Ternary complexes have beenwidely used in spectrophotometric analysis e absorptionspectrum in aqueous medium of rose bengal shows anabsorption peak with 120582120582max 540 nm Upon addition of anyof the three cited drugs to a mixture of Cu(II) and rosebengal solution it was found that a pink soluble complex wasformed and the absorbance maximum of Cu(II) rose bengaloriginally at 540 nm was abridged to a shoulder with a newabsorption maxima at 558 557 and 560 nm for (I) (II) and(III) respectively e absorption spectrum shows that theternary complexes formed between rose bengal and copperwith the cited drugs have higher values of molar extinctioncoefficient than the binary complexes for the spectrophoto-metric method (Figure 1) Investigations were carried outto establish the most favorable conditions for the ternarycomplex formation for the spectrophotometric method ofthe cited drugs with rose bengal and copper to achievemaximum color development and for ion-pair formation forthe conductometric method to achieve the endpoint

31 Conditions for Spectrophotometric Method

311 Effect of pH In a trial to elucidate the optimummedium for the quantitative determination of the studieddrugs the effect of different acetate and phosphate buffers ofpH of range 2ndash10 was examined It was found that buffer hadno effect on the reaction

312 Effect of Cu(II) Chloride Volume e effect of Cu(II)chloride volume was also tested by using varying amounts(01ndash2mL) of Cu(II) chloride (3mgmL)e results showedthat 02mL 15mL and 05mL of Cu(II) chloride (3mgmL)for (I) (II) and (III) respectively were sufficient for theproduction of maximum and reproducible colour intensity(Figure 2)

313 Effect of Rose Bengal Volume Highest constantabsorbance was obtained on using 15mL 05mL and02mL of rose bengal (3mgmL) for (I) (II) and (III)respectively (Figure 3)

314 Effect of Order of Addition emost suitable sequencewas drug Cu(II) chloride and then rose bengal for all drugsfor the production of the highest colour intensity while theother sequences produced lower absorbance values

315 Effect of Reaction Time and Stability Maximum colourintensity was attained immediately for the studied drugs

06

05

04

03

02

01

00 05 1 15 2

Volume (mL)

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin dihydrate

F 2 Effect of volume of 03 wv Cu(II) chloride on theternary complex formed with 40 120583120583gmL erythromycin thiocyanateand 15mL rose bengal 03 wv 25 120583120583gmL clarithromycin and05mL rose bengal 03wv 13 120583120583gmL azithromycin dihydrate and02mL rose bengal 03 wv

06

05

04

03

02

01

00 05 1 15 2 25

Volume (mL)

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin dihydrate

F 3 Effect of volume of 03 wv rose bengal on the ternarycomplex formed with 40 120583120583gmL erythromycin thiocyanate and02mL Cu(II) chloride 03 wv 25 120583120583gmL clarithromycin and15mL Cu(II) chloride 03 wv 13 120583120583gmL azithromycin dihydrateand 05mL Cu(II) chloride 03 wv

except for (II) it needs 5 minutes to give complete com-plexation and increasing time beyond this had no effect onabsorptione obtained colour was stable for at least 1 hourfor all drugs at room temperature (25∘C plusmn 2) e formationconstants have been calculated by using the molar ratiomethod [32] e formation constants (Kf ) of the reactionproduct were found to be 713 times 104 76 times 105 and 15 times106 Lsdotmolminus1 for (I) (II) and (III) respectively is highvalue indicates a very stable reaction product e Gibbsfree energy change of the reaction (ΔG) was also calculated

Journal of Spectroscopy 5

06

07

08

05

04

03

02

01

00 05 1 15 2 25 3 35 454

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin

VrVd

F 4 Molar ratio method plot for 25 times 10minus4M erythromycinthiocyanate and 25 times 10minus4M Cu(II) chloride in presence of excessrose bengal (15mL 03) 25 times 10minus4M clarithromycin and 25 times10minus4M Cu(II) chloride in presence of excess rose bengal (05mL03) 125 times 10minus4 M azithromycin dihydrate and 125 times 10minus4MCu(II) chloride in presence of excess rose bengal (02mL 03)VR = Cu(II) chloride and Vd = Drug

and was found to be minus278 times 104 minus335 times 104 and minus352times 104 KsdotJsdotmoleminus1 for (I) (II) and (III) respectively enegative value of ΔG points out to the spontaneous natureof the reaction [33]

316 Effect of the Extraction Time and Number of ExtractionsShaking the reaction mixture with the extracting organicsolvent is required for complete extraction of the ternarycomplex formed It was found that 1 minute is enough timefor complete colour extraction Complete extraction wasattained by triple extraction with 3 times 3mL of methylenechloride for all drugs

317 Effect of Solvent Many organic solvents were tried toextract the ternary complexes formed (chloroform methy-lene chloride ethylene chloride benzene toluene and ethylacetate) e results obtained indicated that methylene chlo-ride is the appropriate solvent for extraction for all drugs

318 Determination of Stoichiometric Balance Using MolarRatio Method [34] e reaction stoichiometry between ery-thromycin thiocyanate (I) clarithromycin (II) azithromycindihydrate (III) with Cu(II) chloride and rose bengal was stud-ied by molar ratio method using an equimolar (25 times 10minus4M)solution of erythromycin thiocyanate (I) clarithromycin (II)and (125times 10minus4M) solutions of azithromycin dihydrate (III)Cu(II) chloride and rose bengal of the same molarities eobtained results showed that the composition of the ternarycomplexes was 1 1 2 for erythromycin thiocyanate (I) and

06

07

05

04

03

02

01

00 05 1 15 2 25 3 35 454

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin

VrVd

F 5 Molar ratio method plot for 25 times 10minus4M erythromycinthiocyanate with 25 times 10minus4M rose bengal in presence of excessCu(II) chloride (02 mL 03) 25 times 10minus4M clarithromycin with25times10minus4Mrose bengal in presence of excessCu(II) chloride (15mL03) 125times 10minus4Mazithromycin dihydratewith 125times 10minus4Mrosebengal in presence of excess Cu(II) chloride (05mL 03) Vr = rosebengal and Vd = Drug

06

05

04

03

02

01

00 05 1 15 2 25 3 35 4

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin

VrVd

F 6 Molar ratio method plot for 25 times 10minus4M rose bengalwith 25 times 10minus4M Cu(II) chloride in presence of excess ery-thromycin thiocyanate (30 120583120583gsdotmLminus1) 25 times 10minus4M rose bengal with25 times 10minus4M Cu(II) chloride in presence of excess clarithromycin(30 120583120583gsdotmLminus1) 125 times 10minus4M rose bengal with 125 times 10minus4M Cu(II)chloride in presence of excess azithromycin dihydrate (10 120583120583gsdotmLminus1)Vr = rose bengal and VR = Cu(II) chloride

1 2 2 for clarithromycin (II) and azithromycin dihydrate(III) Cu(II) chloride rose bengal (Figures 4 5 and 6)

Scheme 1 illustrates the mechanism of the reaction ofclarithromycin rose bengal and copper(II) chloride elactone ring in clarithromycin is substituted with a numberof hydroxyl functional groups ese groups are positionedin suitable conguration for interaction with metal ions So

6 Journal of Spectroscopy

O

O

O

OO

O O

O

O

O

O

O

O

O

O

O

O

O

O CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

OH

OH

Cu

Cu

N(CH3)2

N(CH3)2

OMe

HOHO

HO

HOHO

H3C

H3C

H3C

H3C

H3C

H3C

HO

I I

II

I I

II

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

COO

2

minus4+4

Rose bengalClarithromycin

2Cu+2

COOminus

Ominus

2 ++

OMe

2Na+

OMe

OMe

S 1 ProposalMechanism for the reaction between clarithromycin rose bengal and copper(II) chloride clarithromycin-Cu-rose bengalcomplex

copper cation forms a chelatewith the lone pair of electrons ofthe two hydroxyl groups at C11 andC12 Also copper can forma chelate with the lone pair of electrons of the tertiary aminegroup nitrogen and the oxygen of the 120573120573-glycosidic linkageSo the azithromycin-copper chelate bears an overall positivecharge of the original two metal ions that is four positivechargesis cationic complex associates with two moleculesof rose bengal anions to form the ternary complex

32 Conditions for Conductometric Method Investigationswere carried out to establish the most suitable conditionsfor the ion associates formation of the cited drugs with rosebengal to attain sharp endpoint e optimum conditionsfor performing the titration in a quantitative manner wereelucidated as described later

321 Reaction Medium Titrations in different media wereattempted to obtain the best results Preliminary experimentswere in

(i) aqueous drug solution for (I) only (prepared bydissolving 100mg drug in 2mL methanol then

completing to 100mL with bidistilled water) withaqueous reagent solution

(ii) ethanol drug solution with ethanol reagent solution(iii) drug solution with reagent solution both in ethanol-

water (50 vv) mixture(iv) methanol drug solution with methanol reagent solu-

tion (except for (II) as it is insoluble in methanol)(v) drug solution with reagent solution both in

methanol-water mixture (50 vv) (except for (II) asit is insoluble in methanol)

(vi) drug solution with reagent solution both in acetone-water (50 vv) mixture

Preliminary experiments showed that procedure in aque-ous media was the most suitable for successful results for (I)50 (vv) ethanol for (II) and 50 (vv) methanol for (III)

322 Reagentrsquos Concentration Different concentrations ofrose bengal solution were tried ranging from 2 times 10minus2 to5 times 10minus4 molar solutions e optimum concentration of thereagent was 3 times 10minus3M in titration of the studied drug to

Journal of Spectroscopy 7

80

70

60

50

40

30

20

10

00 2 4 6 8

Volume of rose bengal (mL)

Co

nd

uct

ivit

y (

S cm

minus1)

F 7 Conductometric titration curve of 15mg clarithromycinversus 3 times 10 3M rose bengal

achieve a constant and highly stable conductance readingwithin 1-2min of mixing Concentrations less than theselimits led to unstable readings and more time was neededto obtain constant conductance values

Representative titration curve is shown in (Figure 7)Two straight lines are obtained intersecting at the endpointthe rst branch ascending and the second has conductancevalues that would slightly increase aer the equivalencepoint e increase of conductance may be attributed tothe formation of ion pair in solution as a result of thecomplexation reaction Aer the end-point the titrationcurves indicate a slightly increased value of conductancedespite the excess of the reagent is may be due tofurther ionic condensation leading to species of lowermobility

e conductometric titrations of different volumes of3 times 10minus3 M rose bengal solution were performed e resultsshow anobviousmaximum in the conductance curve at drug-reagent molar ratio of 2 1 for (I) and (II) and 1 1 for (III)e reactions may be represented by (for (I))

[RB]minus2+210077131007713Eryth N (Me)210077291007729H+

⟶[RB]minus210077131007713Eryth NH(Me)2100772910077292+2

(2)

where [RB]minus2 rose bengal anion [Eryth N(Me)2] ery-thromycin thiocyanate [ErythNH(Me)2]

+2 protonated ery-thromycin thiocyanate

Erythromycin thiocyanate tertiary amine nitrogen isprotonated so attraction occurs between it and rose bengalanion forming ion-pair complex

e conductance measured before the addition of thetitrant (volume of rose bengal equals zero) is mainly due tothe hydrogen ions mobility ese hydrogen ions result fromtwo sources

(1) the active methylene group in the drug structureas in Scheme 2 which has the ability to make selfprotonation to the drug

O

CH3

lowast

S 2 lowaste active methylene group in macrolide structure

(2) the protons coming from the alcohols used in solubil-ity as these alcohols have certain acidity character socan protonate the amino group in drug structure

So there is no need for acidic buffer use for drug pro-tonation It was expected that the conductance values wouldremain constant or slightly increase aer the equivalencepoint However the conductance of the solution decreasedis may be due to interaction of the protons available inthe titration medium with the added reagent e resultsfrom the conductometic titrations are summarized in Table 4e data show that accurate results were obtained with goodrecoveries and low standard deviation values e optimumconcentration ranges for determination of the cited drugswere in the range of 1ndash15mg mLminus1 for all drugs At suchranges sharp inections (Figure 7) and stable conductancereading were obtained

33 Method Validation

331 For the Spectrophotometric Method

Linearity Under the optimumconditions described standardcalibration curves for erythromycin thiocyanate (I) clar-ithromycin (II) and azithromycin dihydrate (III) with rosebengal and copper were constructed by plotting absorbanceagainst concentration Conformity with Beerrsquos law was evi-dent in the concentration range of the nal dilution citedin Table 1 Beerrsquos law holds over the concentration rangesof 15ndash60 75ndash40 and 4ndash20 120583120583gsdotmLminus1 for (I) (II) and (III)respectively e linear regression equation for each drugwas listed in Table 1 e correlation coefficient was 09999indicating good linearity over the working concentrationrangeSensitivity e detection limit (LOD) for the proposedmethod was calculated using the following equation accord-ing to the ICH [35]

LOD = 33120590120590S (3)

where 120590120590 = the standard deviation of replicate blank responses(under the same conditions as for sample analysis) and S =the slope of the calibration curve

e limits of quantication LOQ is dened as

LOQ = 10120590120590S (4)

8 Journal of Spectroscopy

04

03

02

01

0430 500 550 600 650

(nm)

Absorbance

(a)

(b)

F 8Absorption spectra of (a) 25times 10minus2Mglucosewith 15mLCu(II) chloride 03 wv and 05mL 03 wv rose bengal (b) 5 times10minus2M sucrose with 15mL Cu(II) chloride 03 wv and 05mL03 wv rose bengal

According to the previous equations the LODs and LOQswere calculated as in Table 1 eir values conrm thesensitivity of the proposed methodAccuracy and Precision In order to determine the accuracyand precision of the proposed method solutions containingone concentration of each drug were prepared and analysedin seven replicates e relative standard deviation (RSD)as precision and percentage relative error (Er) as accuracyof the suggested method were calculated at 95 condencelevels and can be considered satisfactory e percent-age relative error was calculated according the followingequation

Er = 10077171007717found minus added

added10077331007733 times 100 (5)

e inter- and intraday precisions and accuracy resultsare shown in Table 5 e analytical results for accuracyand precision show that the proposed method has goodrepeatability and reproducibilityInterference e effect of presence of common excipi-ents such as glucose sucrose sodium chloride carboxymethyl cellulose sodium calcium carbonate andmagnesiumstearate was studied Table 6 presents the results of determi-nation of clarithromycin as a representative example usingthe proposed procedure in the presence of aforementionedingredients e tolerance ratio was dened as the concen-tration which gave an error of plusmn30 in the determinationof drugs Results indicate that the tolerance ratio of drugexcipient (M M) is 1 50 for glucose and 1 100 for sucroseWhile there were no interference from the presence ofsodium chloride carboxy methyl cellulose sodium calcium

carbonate and magnesium stearate Interference due toglucose and sucrosewas further investigated by studying theirreaction with Cu(II) chloride and rose bengal e studyrevealed that no interference could be observed at 557 nmand an interaction product was obtained at 120582120582max 498 intolerance ratios 1 1 and 1 10 for glucose and 1 1 1 101 50 for sucrose However interference starts to appear at120582120582max 557 upon using 50 times 100-fold excess of the molarconcentration of clarithromycin for glucose and sucroserespectively as shown in Figure 8

332 For the Conductometric Method In order to addressthe validity of the proposed method statistical analysis of thedata obtained from its application on the cited drugs in thepure form and in formulations was performed e resultsrevealed in Tables 5 and 6 showed that the proposed methodis satisfactorily accurate precise and reproducible over aconcentration range of 1ndash15mg Results for the determina-tion of the studied drugs using the previously mentionedmethod were compared with results from reported one [6]Table 7

34 Analytical Applications e proposed methods wereapplied to determine the studied drugs in their pharma-ceutical dosage forms Satisfactory results were obtainede recovery of each drug was calculated by comparing theconcentration obtained from the spiked mixtures with thoseof the drug e results of analysis of the commercial dosageforms and the recovery study are shown in Tables 2 3 and4 e results obtained were compared with the reportedmethod [6] No signicant differences were found betweenthe proposed methods and reported method Statisticalcomparison of the results was performed using Studentrsquos t-test and variance ratio F-test at 95 condence level (Table7)

4 Conclusions

e proposed methods are advantageous when compared tomany of the reported spectrophotometric methods in havinghigher sensitivity e data given previously reveal that theproposed methods are simple accurate and sensitive withgood precision and accuracyWith thesemethods one can dothe analysis at low cost without losing accuracy No interfer-ence from excipients was encountered Also In comparisonwith the chromatographic methods the proposed methodsare advantageous as HPLC and GLC are not available espe-cially in developing countries and they require complexand expensive equipment intensive sample preparation andpersonnel skilled in chromatographic techniques So theproposed methods can be used as alternative methods to thereported ones for the routine determination of erythromycinthiocyanate (I) clarithromycin (II) azithromycin dihydrate(III) in the pure form and in pharmaceutical formulationsdepending upon the availability of chemicals and the equip-ment

Journal of Spectroscopy 9

T2

Determination

oferythrom

ycin

thiocyanateand

clarithrom

ycin

intheirph

armaceutic

aldo

sage

form

sthroug

hcomplexation

with

Cu(II)

chlorid

eand

rose

beng

alby

spectro

photom

etric

procedurelowast

Erythrocin

tablets

Erythrocin

Pharco

suspensio

nKlarim

ixtablets

Klarim

ixsuspensio

nClaimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

151499

9993

151494

9959

75

756

10080

101005

10048

151489

9924

151504

10028

75

746

9952

101014

10144

201986

9930

202002

10008

101010

10096

125

1239

9914

225

2250

10001

252504

10017

151512

10080

151498

9984

252520

10079

302991

9971

175

1727

9870

175

1746

9979

275

2774

10086

325

3261

10033

202014

10072

202005

10024

302976

9919

353483

9953

225

2244

9973

225

2268

10080

353515

10041

404012

10030

252517

10067

275

2756

10022

375

3758

10021

425

4255

10013

275

2756

10022

303005

10016

403965

9919

302995

9984

Mean

9991

10001

10020

10023

Varia

nce

044

010

054

042

SD066

032

073

065

SE015

011

024

023

lowast Averageo

fthree

experim

ents

10 Journal of Spectroscopy

T3Determinationof

azith

romycin

dihydrateinits

pharmaceutic

aldo

sage

form

sthrou

ghcomplexationwith

Cu(II)chlorid

eand

rose

beng

alby

spectro

photom

etric

procedurelowast

Azrolid

tablets

Zithrokancapsules

Zithrokansuspensio

n

Claimed

tobe

taken120583120583g

mL

Authentic

added120583120583g

mL

Foun

dconc

120583120583gm

LRe

covery

Claimed

tobe

taken

120583120583gm

L

Authentic

added120583120583g

mL

Foun

dconc

120583120583gm

LRe

covery

Claimed

tobe

taken120583120583g

mL

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

LRe

covery

4403

10077

4401

10036

4405

10118

4401

10036

4408

10199

4405

10118

7701

10019

5497

9942

6606

10095

8800

10002

6602

10041

8802

10022

9896

9953

8797

9962

101006

10060

101003

10028

9906

10061

121199

9991

111111

10103

101000

9995

141401

10010

121197

9977

111098

9985

161589

9934

141403

10022

121202

10018

161589

9934

131299

9995

Mean

10015

10023

10043

Varia

nce

027

051

043

SD052

072

066

SE017

024

025

lowast Averageo

fthree

experim

ents

Journal of Spectroscopy 11

T 4 Application of the proposed conductometric method to the determination of erythromycin thiocyanate in dosage formslowast

Erythrocin tablets Erythrocin Pharco suspensionTaken (mg) Found (mg) Recovery Taken (mg) Found (mg) Recovery 3 305 10158 2 2 1004 4 100 4 405 101196 607 10119 7 705 100688 81 10119 8 805 1006010 10 100 12 1190 992012 1191 9921 14 1395 996614 1405 10034Mean 10050 10022SD 085 073SE 032 030RSD 084 073Variance 072 054lowastMean of three different experiments

T 5 e intraday and interday precision and accuracy data for the studied drugs using the spectrophotometric method

Intraday Interday

Drug Taken(120583120583gmLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSDbAccuracyEr c

Taken(120583120583g mLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSD bAccuracyEr c

(I) 50 5028 10056 029 056 50 5034 10068 039 068(II) 30 3033 10109 056 109 30 3017 10057 046 057(III) 16 1599 9995 019 minus005 16 1601 10007 025 007aAverage of seven determinationsbRSD percentage relative standard deviationcEr percentage relative error

T 6 Analysis of clarithromycin 28 120583120583gmLminus1 (5times 10minus4 M) by the spectrophotometric method in the presence of some common excipients

Tolerance molar ratio ( M M)lowast Glucose Sucrose Carboxy methyl cellulose sodium Sodium chlorideRecovery lowastlowast

1 1 10124 10107 10089 99821 10 10036 10124 9911 99821 50 11545 10213 10035 99471 100 10657 10746 9822 9929lowastDrug excipientlowastlowastMean of three determinations

T 7 Determination of the studied drugs by the proposed spectrophotometric and conductmetric methods compared with the reportedmethod [6]

(I) (II) (III)Spectrophotometric method

Mean plusmn RSD 10005 plusmn 054 10001 plusmn 047 9987 plusmn 050Variance 029 022 025Studentrsquos 119905119905-test 005 (217)lowast 04 (131)lowast 006 (216)lowast119865119865-test 238 (428)lowast 391 (360)lowast 268 (389)lowast

119899119899 7 9 9Conductometric method

Mean plusmn RSD 10066 plusmn 093 10065 plusmn 063 10062 plusmn 062Variance 087 040 038Studentrsquos 119905119905-test 131 (217)lowast 113 (217)lowast 210 (2228)lowast119865119865-test 126 (387)lowast 215 (397)lowast 176 (805)lowast

119899119899 7 6 6Reported method [6]

Mean plusmn RSD 10004 plusmn 083 10015 plusmn 093 9989 plusmn 082Variance 069 086 067Studentrsquos 119905119905-test mdash mdash mdash119865119865-test mdash mdash mdash119899119899 7 8 6lowaste gures in parenthesis are the theoretical values for 119905119905 and 119865119865-tests (119875119875 119875 005)

12 Journal of Spectroscopy

Acknowledgments

e authors wish to express their great thanks to Dr SoadSelim Abd El Hay and Dr Omnia Ahmed Emam Ismaiel atthe Analytical Chemistry Department Zagazig Universityfor their great help and advices during this work Also theywould like to point out that the authors do not have anyconict of interests with any company or institution

References

[1] S C Sweetman Martindale the Complete Drug ReferencePharmaceutical Press London UK 35th edition 2007

[2] S OmuraMacrolide Antibiotics Academic Press London UK1984

[3] J N Delgado and W A RemersWilson and Gisvoldrsquos Textbookof Organic Medecinal and Pharmaceutical Chemistry J BLippincott Company London UK 1998

[4] H A Krisr Progress inMedicinal Chemistry vol 30 of Edited byG P Ellis and D K Luscombe 1993

[5] British Pharmacopoeia Her Majestyrsquos Stationery Office Lon-don UK 2007

[6] M I Walash M S Rizk M I Eid and M E Fathy ldquoSpec-trophotometric determination of four macrolide antibiotics inpharmaceutical formulations and biological uids via binarycomplex formation with eosin and spectrophotometryrdquo Journalof AOAC International vol 90 no 6 pp 1579ndash1587 2007

[7] J Shah M R Jan and S Manzoor ldquoExtractive spectrophoto-metric methods for determination of clarithromycin in phar-maceutical formulations using bromothymol blue and cresolredrdquo Journal of the Chinese Chemical Society vol 55 no 5 pp1107ndash1112 2008

[8] C E R De Paula V G K Almeida and R J CassellaldquoNovel spectrophotometric method for the determination ofazithromycin in pharmaceutical formulations based on itscharge transfer reaction with quinalizarinrdquo Journal of theBrazilian Chemical Society vol 21 no 9 pp 1664ndash1671 2010

[9] S Ashour and R Bayram ldquoNovel spectrophotometric methodfor determination of somemacrolide antibiotics in pharmaceu-tical formulations using 1 2-naphthoquinone-4-sulphonaterdquoSpectrochim Acta Part A vol 99 pp 74ndash80 2012

[10] S D Magar A P Tupe P Y Pawar and B Y ManeldquoSimultaneous spectrophotometric estimation of ceximeand azithrhomycin in tablet dosage formrdquo Current PharmaResearch vol 2 no 3 pp 535ndash538 2012

[11] P Y Khashaba ldquoSpectrouorimetric analysis of certain mac-rolide antibiotics in bulk and pharmaceutical formulationsrdquoJournal of Pharmaceutical and Biomedical Analysis vol 27 no6 pp 923ndash932 2002

[12] N El-Rabbat H F Askal P Y Khashaba and N N AttialdquoA validated spectrouorometric assay for the determinationof certainmacrolide antibiotics in pharmaceutical formulationsand spiked biological uidsrdquo Journal of AOAC International vol89 no 5 pp 1276ndash1287 2006

[13] C L Flurer ldquoAnalysis of macrolide antibiotics by capillaryelectrophoresisrdquo Electrophoresis vol 17 no 2 pp 359ndash3661996

[14] A K Lalloo S C Chattaraj and I Kanfer ldquoDevelopmentof a capillary electrophoretic method for the separation ofthe macrolide antibiotics erythromycin josamycin and olean-domycinrdquo Journal of Chromatography B vol 704 no 1-2 pp333ndash341 1997

[15] M Hedenmo and B M Eriksson ldquoLiquid chromatographicdetermination of the macrolide antibiotics roxithromycin andclarithromycin in plasma by automated solid-phase extractionand electrochemical detectionrdquo Journal of Chromatography Avol 692 no 1-2 pp 161ndash166 1995

[16] M Dubois D Fluchard E Sior and P Delahaut ldquoIdenticationand quantication of ve macrolide antibiotics in severaltissues eggs and milk by liquid chromatography-electrospraytandem mass spectrometryrdquo Journal of Chromatography B vol753 no 2 pp 189ndash202 2001

[17] R V S Nirogi V N Kandikere M Shukla et al ldquoSensitiveand selective liquid chromatography-tandem mass spectrom-etry method for the quantication of azithromycin in humanplasmardquo Analytica Chimica Acta vol 553 no 1-2 pp 1ndash82005

[18] O A E M Farghaly and N A L Mohamed ldquoVoltammetricdetermination of azithromycin at the carbon paste electroderdquoTalanta vol 62 no 3 pp 531ndash538 2004

[19] J A Bernabeacuteu M A Camacho M E Gil-Alegre V Ruz andA I Torres-Suaacuterez ldquoMicrobiological bioassay of erythromycinthiocyanate optimisation and validationrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 21 no 2 pp 347ndash3531999

[20] H R N Salgado and A F F Roncari ldquoMicrobiological assayfor the determination of azithromycin in ophthalmic solutionsrdquoYaoxue Xuebao vol 40 no 6 pp 544ndash549 2005

[21] A S Amin and M M El-Henawee ldquoColorimetric methodfor the simultaneous determination of chlorphenoxaminehydrochloride and anhydrous caffeine in pure and dosage formswith rose bengalrdquo Mikrochimica Acta vol 118 no 3-4 pp177ndash183 1995

[22] F M Abdel-Gawad ldquoSpectrophotometric determination ofoxybuprocaine hydrochloride with halouorescein derivativesrdquoFarmaco vol 50 no 3 pp 197ndash200 1995

[23] A S Amin ldquoSpectrophotometric and conductometric deter-mination of clindamycin hydrochloride in pure form andin pharmaceutical preparationsrdquo Analusis vol 23 no 8 pp415ndash417 1995

[24] SM Amer Z El-Sherif andMM Amer ldquoSpectrophotometricdetermination of isoniazid nalidixic acid and umequinethrough ternary complex-formation with Cd (II) and rosebengalrdquo Egyptian Journal of Pharmaceutical Sciences vol 35 no1ndash6 pp 627ndash642 1994

[25] H Parham and A G Fazeli ldquoExtraction-spectrophotometricdetermination of trace amounts of barium by 18-crown-6 androse bengalrdquo Analytical Sciences vol 16 no 6 pp 575ndash5772000

[26] M A El Ries ldquoSpectrophotometric and indirect determinationof lincomycin by atomic absorption spectroscopyrdquo AnalyticalLetters vol 27 no 8 pp 1517ndash1531 1994

[27] H E Abdellatef M M Ayad and E A Taha ldquoSpectrophoto-metric and atomic absorption spectrometric determination oframipril and perindopril through ternary complex formationwith eosin and Cu(II)rdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 18 no 6 pp 1021ndash1027 1999

[28] M M Ayad A A Shalaby H E Abdellatef and M M HosnyldquoSpectrophotometric and AAS determination of ramipril andenalapril through ternary complex formationrdquo Journal of Phar-maceutical and Biomedical Analysis vol 28 no 2 pp 311ndash3212002

Journal of Spectroscopy 13

[29] A AMMoustafa ldquoSpectrophotometric methods for the deter-mination of lansoprazole and pantoprazole sodium sesquihy-draterdquo Journal of Pharmaceutical and Biomedical Analysis vol22 no 1 pp 45ndash58 2000

[30] M I Walash M E S Metwally M Eid and R N El-ShahenyldquoSpectrophotometric determination of risedronate in pharma-ceutical formulations via complex formation with Cu(II) ionsapplication to content uniformity testingrdquo International Journalof Biomedical Science vol 4 no 4 pp 303ndash309 2008

[31] J J Lingane Electroanalytical Chemistry Interscience NewYork NY USA 2nd edition 1958

[32] S A Tirmizi F H Wattoo M H S Wattoo S Sarwar AN Memon and A B Ghangro ldquoSpectrophotometric studyof stability constants of cimetidine-Ni(II) complex at differenttemperaturesrdquo Arabian Journal of Chemistry vol 35 no 1 pp93ndash100 2010

[33] J Inczedy Analytical Application of Complex EquilibriiaBudapest John Wiley amp Sons 1976

[34] J H Yoe and A L Jones ldquoCcrforimetric determination of ironwith disodium-12-dlhydroxybenzene-35-disuffonaterdquo Indus-trial and Engineering Chemistry vol 16 pp 111ndash115 1944

[35] International Conference on Harmonisation ICH of TechnicalRequirments for Registeration of Pharmaceuticals for HumanUse 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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CatalystsJournal of

Journal of Spectroscopy 3

T 1 Quantitative parameters for the spectrophotometric determination of erythromycin thiocyanate (I) clarithromycin (II) andazithromycin dihydrate (III) with rose bengal copper

Parameter (I) (II) (III)120582120582max (nm) 558 557 560Beerrsquos law ranges (120583120583gmL) 15ndash60 75ndash40 4ndash20Regression equationlowastlowast

Slope (119887119887) 00193 00209 00617Intercept (119886119886) minus01413 minus0022 minus01247Correlation coefficient (1199031199032) 09999 09999 09999LOD 120583120583gmL 1 093 031LOQ 120583120583gmL 304 28 095Ringbom optimum concentration range (120583120583gmL) 20ndash50 10ndash35 9ndash16Sandellrsquos sensitivity 120583120583gsdotcmminus2 007 005 002120576120576 (times104) Lsdotmolminus1sdotcmminus1 112 147 378lowastAverage of three experimentslowastlowastA = 119886119886 + 119887119887c

e solutions were vigorously shaken for one minute and theorganic layer ltered over anhydrous sodium sulphate into10mL volumetric asks then the volumes were completedto 10mL with methylene chloride e absorbances of thecolored solutions were measured at 558 557 and 560 nm for(I) (II) and (III) respectively against reagent blank treatedsimilarly (Figure 1) All measurements were made at ambienttemperature

242 Conductometric Titrations Aliquot of sample solutioncontaining 1ndash15mg of the cited drugs was transferred to a50mL calibrated ask and volume was made up to the markusing bidistilled water for (I) 50 (vv) ethanol for (II) and50 (vv) methanol for (III) e contents of the calibratedask were transferred to a beaker and the conductivitycell was immersed 3 times 10minus3M rose bengal was used fortitration e conductance was measured subsequently toeach addition of reagent solution and aer thorough stirringfor twominutes corrected for dilution effect [31] bymeans ofthe following equation assuming that conductivity is a linearfunction of dilution

Ωminus1correct = Ω

minus1obs 10077181007718

100764910076491199071199071 + 1199071199072100766510076651199071199071

10077341007734 (1)

whereΩminus1obs is the observed electrolytic conductivity 1199071199071 is the

initial volume and 1199071199072 is the volume of reagent added A graphof corrected conductivity versus the volume of added titrantwas constructed and the endpoint was determined (Figure 7)

25 Procedure for Pharmaceutical Preparations

251 For Spectrophotometric Method e contents of vecapsules were removed and weighed accurately e com-bined contents were mixed and a quantity of the powderequivalent to 25mg for (I) and (II) and equivalent to 10mgfor (III) was extracted with low amount of ethanol in50mL volumetric ask e volumes were completed to themark with double-distilled water ltered and the assay wascompleted as described under general procedure e same

075

06

04

02

0

minus005500 520 540 560 580 600

(nm)

Absorbance

(a)

(b)

(c)(d)

(e)(f)

F 1 Absorption spectra for (a) the reaction of 13 120583120583gmLazithromycin dihydrate with 05mL Cu(II) chloride 03 wv and02mL 03 wv rose bengal (b) 13 120583120583gmL azithromycin dihydratewith 02mL rose bengal 03 wv (c) 13 120583120583gmL azithromycindihydrate with 05mL Cu(II) chloride 03 wv (d) 02mL rosebengal 03 wv and 05mL Cu(II) chloride 03 wv (e) 02mLrose bengal 03 wv and (f) 05mL Cu(II) chloride 03 wv

procedures were followed with tablets taking an accuratelyweighed amount of ten combined powdered tabletse sameprocedures were followed with oral suspension taking anaccurately measured volume of the freshly reconstituted oralsuspension equivalent to the specied concentration

252 For Conductometric Method e same procedure wasfollowed as in the spectrophotometric method but the quan-tity of the powder taken from tablets or capsules or theaccurately measured volume of the freshly reconstituted oralsuspension is equivalent to 100mg for all drugs and wasextracted by least amount of methanol for (I) 50 (vv)

4 Journal of Spectroscopy

ethanol for (II) and 50 (vv) methanol for (III) lteredand transferred to a 100mL volumetric ask completed tothe mark using distilled water for (I) 50 (vv) ethanol for(II) and in 50 (vv) methanol for (III) and the assay wascompleted as described under general procedure

3 Results and Discussion

Optimization of Variables Ternary complexes have beenwidely used in spectrophotometric analysis e absorptionspectrum in aqueous medium of rose bengal shows anabsorption peak with 120582120582max 540 nm Upon addition of anyof the three cited drugs to a mixture of Cu(II) and rosebengal solution it was found that a pink soluble complex wasformed and the absorbance maximum of Cu(II) rose bengaloriginally at 540 nm was abridged to a shoulder with a newabsorption maxima at 558 557 and 560 nm for (I) (II) and(III) respectively e absorption spectrum shows that theternary complexes formed between rose bengal and copperwith the cited drugs have higher values of molar extinctioncoefficient than the binary complexes for the spectrophoto-metric method (Figure 1) Investigations were carried outto establish the most favorable conditions for the ternarycomplex formation for the spectrophotometric method ofthe cited drugs with rose bengal and copper to achievemaximum color development and for ion-pair formation forthe conductometric method to achieve the endpoint

31 Conditions for Spectrophotometric Method

311 Effect of pH In a trial to elucidate the optimummedium for the quantitative determination of the studieddrugs the effect of different acetate and phosphate buffers ofpH of range 2ndash10 was examined It was found that buffer hadno effect on the reaction

312 Effect of Cu(II) Chloride Volume e effect of Cu(II)chloride volume was also tested by using varying amounts(01ndash2mL) of Cu(II) chloride (3mgmL)e results showedthat 02mL 15mL and 05mL of Cu(II) chloride (3mgmL)for (I) (II) and (III) respectively were sufficient for theproduction of maximum and reproducible colour intensity(Figure 2)

313 Effect of Rose Bengal Volume Highest constantabsorbance was obtained on using 15mL 05mL and02mL of rose bengal (3mgmL) for (I) (II) and (III)respectively (Figure 3)

314 Effect of Order of Addition emost suitable sequencewas drug Cu(II) chloride and then rose bengal for all drugsfor the production of the highest colour intensity while theother sequences produced lower absorbance values

315 Effect of Reaction Time and Stability Maximum colourintensity was attained immediately for the studied drugs

06

05

04

03

02

01

00 05 1 15 2

Volume (mL)

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin dihydrate

F 2 Effect of volume of 03 wv Cu(II) chloride on theternary complex formed with 40 120583120583gmL erythromycin thiocyanateand 15mL rose bengal 03 wv 25 120583120583gmL clarithromycin and05mL rose bengal 03wv 13 120583120583gmL azithromycin dihydrate and02mL rose bengal 03 wv

06

05

04

03

02

01

00 05 1 15 2 25

Volume (mL)

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin dihydrate

F 3 Effect of volume of 03 wv rose bengal on the ternarycomplex formed with 40 120583120583gmL erythromycin thiocyanate and02mL Cu(II) chloride 03 wv 25 120583120583gmL clarithromycin and15mL Cu(II) chloride 03 wv 13 120583120583gmL azithromycin dihydrateand 05mL Cu(II) chloride 03 wv

except for (II) it needs 5 minutes to give complete com-plexation and increasing time beyond this had no effect onabsorptione obtained colour was stable for at least 1 hourfor all drugs at room temperature (25∘C plusmn 2) e formationconstants have been calculated by using the molar ratiomethod [32] e formation constants (Kf ) of the reactionproduct were found to be 713 times 104 76 times 105 and 15 times106 Lsdotmolminus1 for (I) (II) and (III) respectively is highvalue indicates a very stable reaction product e Gibbsfree energy change of the reaction (ΔG) was also calculated

Journal of Spectroscopy 5

06

07

08

05

04

03

02

01

00 05 1 15 2 25 3 35 454

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin

VrVd

F 4 Molar ratio method plot for 25 times 10minus4M erythromycinthiocyanate and 25 times 10minus4M Cu(II) chloride in presence of excessrose bengal (15mL 03) 25 times 10minus4M clarithromycin and 25 times10minus4M Cu(II) chloride in presence of excess rose bengal (05mL03) 125 times 10minus4 M azithromycin dihydrate and 125 times 10minus4MCu(II) chloride in presence of excess rose bengal (02mL 03)VR = Cu(II) chloride and Vd = Drug

and was found to be minus278 times 104 minus335 times 104 and minus352times 104 KsdotJsdotmoleminus1 for (I) (II) and (III) respectively enegative value of ΔG points out to the spontaneous natureof the reaction [33]

316 Effect of the Extraction Time and Number of ExtractionsShaking the reaction mixture with the extracting organicsolvent is required for complete extraction of the ternarycomplex formed It was found that 1 minute is enough timefor complete colour extraction Complete extraction wasattained by triple extraction with 3 times 3mL of methylenechloride for all drugs

317 Effect of Solvent Many organic solvents were tried toextract the ternary complexes formed (chloroform methy-lene chloride ethylene chloride benzene toluene and ethylacetate) e results obtained indicated that methylene chlo-ride is the appropriate solvent for extraction for all drugs

318 Determination of Stoichiometric Balance Using MolarRatio Method [34] e reaction stoichiometry between ery-thromycin thiocyanate (I) clarithromycin (II) azithromycindihydrate (III) with Cu(II) chloride and rose bengal was stud-ied by molar ratio method using an equimolar (25 times 10minus4M)solution of erythromycin thiocyanate (I) clarithromycin (II)and (125times 10minus4M) solutions of azithromycin dihydrate (III)Cu(II) chloride and rose bengal of the same molarities eobtained results showed that the composition of the ternarycomplexes was 1 1 2 for erythromycin thiocyanate (I) and

06

07

05

04

03

02

01

00 05 1 15 2 25 3 35 454

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin

VrVd

F 5 Molar ratio method plot for 25 times 10minus4M erythromycinthiocyanate with 25 times 10minus4M rose bengal in presence of excessCu(II) chloride (02 mL 03) 25 times 10minus4M clarithromycin with25times10minus4Mrose bengal in presence of excessCu(II) chloride (15mL03) 125times 10minus4Mazithromycin dihydratewith 125times 10minus4Mrosebengal in presence of excess Cu(II) chloride (05mL 03) Vr = rosebengal and Vd = Drug

06

05

04

03

02

01

00 05 1 15 2 25 3 35 4

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin

VrVd

F 6 Molar ratio method plot for 25 times 10minus4M rose bengalwith 25 times 10minus4M Cu(II) chloride in presence of excess ery-thromycin thiocyanate (30 120583120583gsdotmLminus1) 25 times 10minus4M rose bengal with25 times 10minus4M Cu(II) chloride in presence of excess clarithromycin(30 120583120583gsdotmLminus1) 125 times 10minus4M rose bengal with 125 times 10minus4M Cu(II)chloride in presence of excess azithromycin dihydrate (10 120583120583gsdotmLminus1)Vr = rose bengal and VR = Cu(II) chloride

1 2 2 for clarithromycin (II) and azithromycin dihydrate(III) Cu(II) chloride rose bengal (Figures 4 5 and 6)

Scheme 1 illustrates the mechanism of the reaction ofclarithromycin rose bengal and copper(II) chloride elactone ring in clarithromycin is substituted with a numberof hydroxyl functional groups ese groups are positionedin suitable conguration for interaction with metal ions So

6 Journal of Spectroscopy

O

O

O

OO

O O

O

O

O

O

O

O

O

O

O

O

O

O CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

OH

OH

Cu

Cu

N(CH3)2

N(CH3)2

OMe

HOHO

HO

HOHO

H3C

H3C

H3C

H3C

H3C

H3C

HO

I I

II

I I

II

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

COO

2

minus4+4

Rose bengalClarithromycin

2Cu+2

COOminus

Ominus

2 ++

OMe

2Na+

OMe

OMe

S 1 ProposalMechanism for the reaction between clarithromycin rose bengal and copper(II) chloride clarithromycin-Cu-rose bengalcomplex

copper cation forms a chelatewith the lone pair of electrons ofthe two hydroxyl groups at C11 andC12 Also copper can forma chelate with the lone pair of electrons of the tertiary aminegroup nitrogen and the oxygen of the 120573120573-glycosidic linkageSo the azithromycin-copper chelate bears an overall positivecharge of the original two metal ions that is four positivechargesis cationic complex associates with two moleculesof rose bengal anions to form the ternary complex

32 Conditions for Conductometric Method Investigationswere carried out to establish the most suitable conditionsfor the ion associates formation of the cited drugs with rosebengal to attain sharp endpoint e optimum conditionsfor performing the titration in a quantitative manner wereelucidated as described later

321 Reaction Medium Titrations in different media wereattempted to obtain the best results Preliminary experimentswere in

(i) aqueous drug solution for (I) only (prepared bydissolving 100mg drug in 2mL methanol then

completing to 100mL with bidistilled water) withaqueous reagent solution

(ii) ethanol drug solution with ethanol reagent solution(iii) drug solution with reagent solution both in ethanol-

water (50 vv) mixture(iv) methanol drug solution with methanol reagent solu-

tion (except for (II) as it is insoluble in methanol)(v) drug solution with reagent solution both in

methanol-water mixture (50 vv) (except for (II) asit is insoluble in methanol)

(vi) drug solution with reagent solution both in acetone-water (50 vv) mixture

Preliminary experiments showed that procedure in aque-ous media was the most suitable for successful results for (I)50 (vv) ethanol for (II) and 50 (vv) methanol for (III)

322 Reagentrsquos Concentration Different concentrations ofrose bengal solution were tried ranging from 2 times 10minus2 to5 times 10minus4 molar solutions e optimum concentration of thereagent was 3 times 10minus3M in titration of the studied drug to

Journal of Spectroscopy 7

80

70

60

50

40

30

20

10

00 2 4 6 8

Volume of rose bengal (mL)

Co

nd

uct

ivit

y (

S cm

minus1)

F 7 Conductometric titration curve of 15mg clarithromycinversus 3 times 10 3M rose bengal

achieve a constant and highly stable conductance readingwithin 1-2min of mixing Concentrations less than theselimits led to unstable readings and more time was neededto obtain constant conductance values

Representative titration curve is shown in (Figure 7)Two straight lines are obtained intersecting at the endpointthe rst branch ascending and the second has conductancevalues that would slightly increase aer the equivalencepoint e increase of conductance may be attributed tothe formation of ion pair in solution as a result of thecomplexation reaction Aer the end-point the titrationcurves indicate a slightly increased value of conductancedespite the excess of the reagent is may be due tofurther ionic condensation leading to species of lowermobility

e conductometric titrations of different volumes of3 times 10minus3 M rose bengal solution were performed e resultsshow anobviousmaximum in the conductance curve at drug-reagent molar ratio of 2 1 for (I) and (II) and 1 1 for (III)e reactions may be represented by (for (I))

[RB]minus2+210077131007713Eryth N (Me)210077291007729H+

⟶[RB]minus210077131007713Eryth NH(Me)2100772910077292+2

(2)

where [RB]minus2 rose bengal anion [Eryth N(Me)2] ery-thromycin thiocyanate [ErythNH(Me)2]

+2 protonated ery-thromycin thiocyanate

Erythromycin thiocyanate tertiary amine nitrogen isprotonated so attraction occurs between it and rose bengalanion forming ion-pair complex

e conductance measured before the addition of thetitrant (volume of rose bengal equals zero) is mainly due tothe hydrogen ions mobility ese hydrogen ions result fromtwo sources

(1) the active methylene group in the drug structureas in Scheme 2 which has the ability to make selfprotonation to the drug

O

CH3

lowast

S 2 lowaste active methylene group in macrolide structure

(2) the protons coming from the alcohols used in solubil-ity as these alcohols have certain acidity character socan protonate the amino group in drug structure

So there is no need for acidic buffer use for drug pro-tonation It was expected that the conductance values wouldremain constant or slightly increase aer the equivalencepoint However the conductance of the solution decreasedis may be due to interaction of the protons available inthe titration medium with the added reagent e resultsfrom the conductometic titrations are summarized in Table 4e data show that accurate results were obtained with goodrecoveries and low standard deviation values e optimumconcentration ranges for determination of the cited drugswere in the range of 1ndash15mg mLminus1 for all drugs At suchranges sharp inections (Figure 7) and stable conductancereading were obtained

33 Method Validation

331 For the Spectrophotometric Method

Linearity Under the optimumconditions described standardcalibration curves for erythromycin thiocyanate (I) clar-ithromycin (II) and azithromycin dihydrate (III) with rosebengal and copper were constructed by plotting absorbanceagainst concentration Conformity with Beerrsquos law was evi-dent in the concentration range of the nal dilution citedin Table 1 Beerrsquos law holds over the concentration rangesof 15ndash60 75ndash40 and 4ndash20 120583120583gsdotmLminus1 for (I) (II) and (III)respectively e linear regression equation for each drugwas listed in Table 1 e correlation coefficient was 09999indicating good linearity over the working concentrationrangeSensitivity e detection limit (LOD) for the proposedmethod was calculated using the following equation accord-ing to the ICH [35]

LOD = 33120590120590S (3)

where 120590120590 = the standard deviation of replicate blank responses(under the same conditions as for sample analysis) and S =the slope of the calibration curve

e limits of quantication LOQ is dened as

LOQ = 10120590120590S (4)

8 Journal of Spectroscopy

04

03

02

01

0430 500 550 600 650

(nm)

Absorbance

(a)

(b)

F 8Absorption spectra of (a) 25times 10minus2Mglucosewith 15mLCu(II) chloride 03 wv and 05mL 03 wv rose bengal (b) 5 times10minus2M sucrose with 15mL Cu(II) chloride 03 wv and 05mL03 wv rose bengal

According to the previous equations the LODs and LOQswere calculated as in Table 1 eir values conrm thesensitivity of the proposed methodAccuracy and Precision In order to determine the accuracyand precision of the proposed method solutions containingone concentration of each drug were prepared and analysedin seven replicates e relative standard deviation (RSD)as precision and percentage relative error (Er) as accuracyof the suggested method were calculated at 95 condencelevels and can be considered satisfactory e percent-age relative error was calculated according the followingequation

Er = 10077171007717found minus added

added10077331007733 times 100 (5)

e inter- and intraday precisions and accuracy resultsare shown in Table 5 e analytical results for accuracyand precision show that the proposed method has goodrepeatability and reproducibilityInterference e effect of presence of common excipi-ents such as glucose sucrose sodium chloride carboxymethyl cellulose sodium calcium carbonate andmagnesiumstearate was studied Table 6 presents the results of determi-nation of clarithromycin as a representative example usingthe proposed procedure in the presence of aforementionedingredients e tolerance ratio was dened as the concen-tration which gave an error of plusmn30 in the determinationof drugs Results indicate that the tolerance ratio of drugexcipient (M M) is 1 50 for glucose and 1 100 for sucroseWhile there were no interference from the presence ofsodium chloride carboxy methyl cellulose sodium calcium

carbonate and magnesium stearate Interference due toglucose and sucrosewas further investigated by studying theirreaction with Cu(II) chloride and rose bengal e studyrevealed that no interference could be observed at 557 nmand an interaction product was obtained at 120582120582max 498 intolerance ratios 1 1 and 1 10 for glucose and 1 1 1 101 50 for sucrose However interference starts to appear at120582120582max 557 upon using 50 times 100-fold excess of the molarconcentration of clarithromycin for glucose and sucroserespectively as shown in Figure 8

332 For the Conductometric Method In order to addressthe validity of the proposed method statistical analysis of thedata obtained from its application on the cited drugs in thepure form and in formulations was performed e resultsrevealed in Tables 5 and 6 showed that the proposed methodis satisfactorily accurate precise and reproducible over aconcentration range of 1ndash15mg Results for the determina-tion of the studied drugs using the previously mentionedmethod were compared with results from reported one [6]Table 7

34 Analytical Applications e proposed methods wereapplied to determine the studied drugs in their pharma-ceutical dosage forms Satisfactory results were obtainede recovery of each drug was calculated by comparing theconcentration obtained from the spiked mixtures with thoseof the drug e results of analysis of the commercial dosageforms and the recovery study are shown in Tables 2 3 and4 e results obtained were compared with the reportedmethod [6] No signicant differences were found betweenthe proposed methods and reported method Statisticalcomparison of the results was performed using Studentrsquos t-test and variance ratio F-test at 95 condence level (Table7)

4 Conclusions

e proposed methods are advantageous when compared tomany of the reported spectrophotometric methods in havinghigher sensitivity e data given previously reveal that theproposed methods are simple accurate and sensitive withgood precision and accuracyWith thesemethods one can dothe analysis at low cost without losing accuracy No interfer-ence from excipients was encountered Also In comparisonwith the chromatographic methods the proposed methodsare advantageous as HPLC and GLC are not available espe-cially in developing countries and they require complexand expensive equipment intensive sample preparation andpersonnel skilled in chromatographic techniques So theproposed methods can be used as alternative methods to thereported ones for the routine determination of erythromycinthiocyanate (I) clarithromycin (II) azithromycin dihydrate(III) in the pure form and in pharmaceutical formulationsdepending upon the availability of chemicals and the equip-ment

Journal of Spectroscopy 9

T2

Determination

oferythrom

ycin

thiocyanateand

clarithrom

ycin

intheirph

armaceutic

aldo

sage

form

sthroug

hcomplexation

with

Cu(II)

chlorid

eand

rose

beng

alby

spectro

photom

etric

procedurelowast

Erythrocin

tablets

Erythrocin

Pharco

suspensio

nKlarim

ixtablets

Klarim

ixsuspensio

nClaimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

151499

9993

151494

9959

75

756

10080

101005

10048

151489

9924

151504

10028

75

746

9952

101014

10144

201986

9930

202002

10008

101010

10096

125

1239

9914

225

2250

10001

252504

10017

151512

10080

151498

9984

252520

10079

302991

9971

175

1727

9870

175

1746

9979

275

2774

10086

325

3261

10033

202014

10072

202005

10024

302976

9919

353483

9953

225

2244

9973

225

2268

10080

353515

10041

404012

10030

252517

10067

275

2756

10022

375

3758

10021

425

4255

10013

275

2756

10022

303005

10016

403965

9919

302995

9984

Mean

9991

10001

10020

10023

Varia

nce

044

010

054

042

SD066

032

073

065

SE015

011

024

023

lowast Averageo

fthree

experim

ents

10 Journal of Spectroscopy

T3Determinationof

azith

romycin

dihydrateinits

pharmaceutic

aldo

sage

form

sthrou

ghcomplexationwith

Cu(II)chlorid

eand

rose

beng

alby

spectro

photom

etric

procedurelowast

Azrolid

tablets

Zithrokancapsules

Zithrokansuspensio

n

Claimed

tobe

taken120583120583g

mL

Authentic

added120583120583g

mL

Foun

dconc

120583120583gm

LRe

covery

Claimed

tobe

taken

120583120583gm

L

Authentic

added120583120583g

mL

Foun

dconc

120583120583gm

LRe

covery

Claimed

tobe

taken120583120583g

mL

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

LRe

covery

4403

10077

4401

10036

4405

10118

4401

10036

4408

10199

4405

10118

7701

10019

5497

9942

6606

10095

8800

10002

6602

10041

8802

10022

9896

9953

8797

9962

101006

10060

101003

10028

9906

10061

121199

9991

111111

10103

101000

9995

141401

10010

121197

9977

111098

9985

161589

9934

141403

10022

121202

10018

161589

9934

131299

9995

Mean

10015

10023

10043

Varia

nce

027

051

043

SD052

072

066

SE017

024

025

lowast Averageo

fthree

experim

ents

Journal of Spectroscopy 11

T 4 Application of the proposed conductometric method to the determination of erythromycin thiocyanate in dosage formslowast

Erythrocin tablets Erythrocin Pharco suspensionTaken (mg) Found (mg) Recovery Taken (mg) Found (mg) Recovery 3 305 10158 2 2 1004 4 100 4 405 101196 607 10119 7 705 100688 81 10119 8 805 1006010 10 100 12 1190 992012 1191 9921 14 1395 996614 1405 10034Mean 10050 10022SD 085 073SE 032 030RSD 084 073Variance 072 054lowastMean of three different experiments

T 5 e intraday and interday precision and accuracy data for the studied drugs using the spectrophotometric method

Intraday Interday

Drug Taken(120583120583gmLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSDbAccuracyEr c

Taken(120583120583g mLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSD bAccuracyEr c

(I) 50 5028 10056 029 056 50 5034 10068 039 068(II) 30 3033 10109 056 109 30 3017 10057 046 057(III) 16 1599 9995 019 minus005 16 1601 10007 025 007aAverage of seven determinationsbRSD percentage relative standard deviationcEr percentage relative error

T 6 Analysis of clarithromycin 28 120583120583gmLminus1 (5times 10minus4 M) by the spectrophotometric method in the presence of some common excipients

Tolerance molar ratio ( M M)lowast Glucose Sucrose Carboxy methyl cellulose sodium Sodium chlorideRecovery lowastlowast

1 1 10124 10107 10089 99821 10 10036 10124 9911 99821 50 11545 10213 10035 99471 100 10657 10746 9822 9929lowastDrug excipientlowastlowastMean of three determinations

T 7 Determination of the studied drugs by the proposed spectrophotometric and conductmetric methods compared with the reportedmethod [6]

(I) (II) (III)Spectrophotometric method

Mean plusmn RSD 10005 plusmn 054 10001 plusmn 047 9987 plusmn 050Variance 029 022 025Studentrsquos 119905119905-test 005 (217)lowast 04 (131)lowast 006 (216)lowast119865119865-test 238 (428)lowast 391 (360)lowast 268 (389)lowast

119899119899 7 9 9Conductometric method

Mean plusmn RSD 10066 plusmn 093 10065 plusmn 063 10062 plusmn 062Variance 087 040 038Studentrsquos 119905119905-test 131 (217)lowast 113 (217)lowast 210 (2228)lowast119865119865-test 126 (387)lowast 215 (397)lowast 176 (805)lowast

119899119899 7 6 6Reported method [6]

Mean plusmn RSD 10004 plusmn 083 10015 plusmn 093 9989 plusmn 082Variance 069 086 067Studentrsquos 119905119905-test mdash mdash mdash119865119865-test mdash mdash mdash119899119899 7 8 6lowaste gures in parenthesis are the theoretical values for 119905119905 and 119865119865-tests (119875119875 119875 005)

12 Journal of Spectroscopy

Acknowledgments

e authors wish to express their great thanks to Dr SoadSelim Abd El Hay and Dr Omnia Ahmed Emam Ismaiel atthe Analytical Chemistry Department Zagazig Universityfor their great help and advices during this work Also theywould like to point out that the authors do not have anyconict of interests with any company or institution

References

[1] S C Sweetman Martindale the Complete Drug ReferencePharmaceutical Press London UK 35th edition 2007

[2] S OmuraMacrolide Antibiotics Academic Press London UK1984

[3] J N Delgado and W A RemersWilson and Gisvoldrsquos Textbookof Organic Medecinal and Pharmaceutical Chemistry J BLippincott Company London UK 1998

[4] H A Krisr Progress inMedicinal Chemistry vol 30 of Edited byG P Ellis and D K Luscombe 1993

[5] British Pharmacopoeia Her Majestyrsquos Stationery Office Lon-don UK 2007

[6] M I Walash M S Rizk M I Eid and M E Fathy ldquoSpec-trophotometric determination of four macrolide antibiotics inpharmaceutical formulations and biological uids via binarycomplex formation with eosin and spectrophotometryrdquo Journalof AOAC International vol 90 no 6 pp 1579ndash1587 2007

[7] J Shah M R Jan and S Manzoor ldquoExtractive spectrophoto-metric methods for determination of clarithromycin in phar-maceutical formulations using bromothymol blue and cresolredrdquo Journal of the Chinese Chemical Society vol 55 no 5 pp1107ndash1112 2008

[8] C E R De Paula V G K Almeida and R J CassellaldquoNovel spectrophotometric method for the determination ofazithromycin in pharmaceutical formulations based on itscharge transfer reaction with quinalizarinrdquo Journal of theBrazilian Chemical Society vol 21 no 9 pp 1664ndash1671 2010

[9] S Ashour and R Bayram ldquoNovel spectrophotometric methodfor determination of somemacrolide antibiotics in pharmaceu-tical formulations using 1 2-naphthoquinone-4-sulphonaterdquoSpectrochim Acta Part A vol 99 pp 74ndash80 2012

[10] S D Magar A P Tupe P Y Pawar and B Y ManeldquoSimultaneous spectrophotometric estimation of ceximeand azithrhomycin in tablet dosage formrdquo Current PharmaResearch vol 2 no 3 pp 535ndash538 2012

[11] P Y Khashaba ldquoSpectrouorimetric analysis of certain mac-rolide antibiotics in bulk and pharmaceutical formulationsrdquoJournal of Pharmaceutical and Biomedical Analysis vol 27 no6 pp 923ndash932 2002

[12] N El-Rabbat H F Askal P Y Khashaba and N N AttialdquoA validated spectrouorometric assay for the determinationof certainmacrolide antibiotics in pharmaceutical formulationsand spiked biological uidsrdquo Journal of AOAC International vol89 no 5 pp 1276ndash1287 2006

[13] C L Flurer ldquoAnalysis of macrolide antibiotics by capillaryelectrophoresisrdquo Electrophoresis vol 17 no 2 pp 359ndash3661996

[14] A K Lalloo S C Chattaraj and I Kanfer ldquoDevelopmentof a capillary electrophoretic method for the separation ofthe macrolide antibiotics erythromycin josamycin and olean-domycinrdquo Journal of Chromatography B vol 704 no 1-2 pp333ndash341 1997

[15] M Hedenmo and B M Eriksson ldquoLiquid chromatographicdetermination of the macrolide antibiotics roxithromycin andclarithromycin in plasma by automated solid-phase extractionand electrochemical detectionrdquo Journal of Chromatography Avol 692 no 1-2 pp 161ndash166 1995

[16] M Dubois D Fluchard E Sior and P Delahaut ldquoIdenticationand quantication of ve macrolide antibiotics in severaltissues eggs and milk by liquid chromatography-electrospraytandem mass spectrometryrdquo Journal of Chromatography B vol753 no 2 pp 189ndash202 2001

[17] R V S Nirogi V N Kandikere M Shukla et al ldquoSensitiveand selective liquid chromatography-tandem mass spectrom-etry method for the quantication of azithromycin in humanplasmardquo Analytica Chimica Acta vol 553 no 1-2 pp 1ndash82005

[18] O A E M Farghaly and N A L Mohamed ldquoVoltammetricdetermination of azithromycin at the carbon paste electroderdquoTalanta vol 62 no 3 pp 531ndash538 2004

[19] J A Bernabeacuteu M A Camacho M E Gil-Alegre V Ruz andA I Torres-Suaacuterez ldquoMicrobiological bioassay of erythromycinthiocyanate optimisation and validationrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 21 no 2 pp 347ndash3531999

[20] H R N Salgado and A F F Roncari ldquoMicrobiological assayfor the determination of azithromycin in ophthalmic solutionsrdquoYaoxue Xuebao vol 40 no 6 pp 544ndash549 2005

[21] A S Amin and M M El-Henawee ldquoColorimetric methodfor the simultaneous determination of chlorphenoxaminehydrochloride and anhydrous caffeine in pure and dosage formswith rose bengalrdquo Mikrochimica Acta vol 118 no 3-4 pp177ndash183 1995

[22] F M Abdel-Gawad ldquoSpectrophotometric determination ofoxybuprocaine hydrochloride with halouorescein derivativesrdquoFarmaco vol 50 no 3 pp 197ndash200 1995

[23] A S Amin ldquoSpectrophotometric and conductometric deter-mination of clindamycin hydrochloride in pure form andin pharmaceutical preparationsrdquo Analusis vol 23 no 8 pp415ndash417 1995

[24] SM Amer Z El-Sherif andMM Amer ldquoSpectrophotometricdetermination of isoniazid nalidixic acid and umequinethrough ternary complex-formation with Cd (II) and rosebengalrdquo Egyptian Journal of Pharmaceutical Sciences vol 35 no1ndash6 pp 627ndash642 1994

[25] H Parham and A G Fazeli ldquoExtraction-spectrophotometricdetermination of trace amounts of barium by 18-crown-6 androse bengalrdquo Analytical Sciences vol 16 no 6 pp 575ndash5772000

[26] M A El Ries ldquoSpectrophotometric and indirect determinationof lincomycin by atomic absorption spectroscopyrdquo AnalyticalLetters vol 27 no 8 pp 1517ndash1531 1994

[27] H E Abdellatef M M Ayad and E A Taha ldquoSpectrophoto-metric and atomic absorption spectrometric determination oframipril and perindopril through ternary complex formationwith eosin and Cu(II)rdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 18 no 6 pp 1021ndash1027 1999

[28] M M Ayad A A Shalaby H E Abdellatef and M M HosnyldquoSpectrophotometric and AAS determination of ramipril andenalapril through ternary complex formationrdquo Journal of Phar-maceutical and Biomedical Analysis vol 28 no 2 pp 311ndash3212002

Journal of Spectroscopy 13

[29] A AMMoustafa ldquoSpectrophotometric methods for the deter-mination of lansoprazole and pantoprazole sodium sesquihy-draterdquo Journal of Pharmaceutical and Biomedical Analysis vol22 no 1 pp 45ndash58 2000

[30] M I Walash M E S Metwally M Eid and R N El-ShahenyldquoSpectrophotometric determination of risedronate in pharma-ceutical formulations via complex formation with Cu(II) ionsapplication to content uniformity testingrdquo International Journalof Biomedical Science vol 4 no 4 pp 303ndash309 2008

[31] J J Lingane Electroanalytical Chemistry Interscience NewYork NY USA 2nd edition 1958

[32] S A Tirmizi F H Wattoo M H S Wattoo S Sarwar AN Memon and A B Ghangro ldquoSpectrophotometric studyof stability constants of cimetidine-Ni(II) complex at differenttemperaturesrdquo Arabian Journal of Chemistry vol 35 no 1 pp93ndash100 2010

[33] J Inczedy Analytical Application of Complex EquilibriiaBudapest John Wiley amp Sons 1976

[34] J H Yoe and A L Jones ldquoCcrforimetric determination of ironwith disodium-12-dlhydroxybenzene-35-disuffonaterdquo Indus-trial and Engineering Chemistry vol 16 pp 111ndash115 1944

[35] International Conference on Harmonisation ICH of TechnicalRequirments for Registeration of Pharmaceuticals for HumanUse 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

4 Journal of Spectroscopy

ethanol for (II) and 50 (vv) methanol for (III) lteredand transferred to a 100mL volumetric ask completed tothe mark using distilled water for (I) 50 (vv) ethanol for(II) and in 50 (vv) methanol for (III) and the assay wascompleted as described under general procedure

3 Results and Discussion

Optimization of Variables Ternary complexes have beenwidely used in spectrophotometric analysis e absorptionspectrum in aqueous medium of rose bengal shows anabsorption peak with 120582120582max 540 nm Upon addition of anyof the three cited drugs to a mixture of Cu(II) and rosebengal solution it was found that a pink soluble complex wasformed and the absorbance maximum of Cu(II) rose bengaloriginally at 540 nm was abridged to a shoulder with a newabsorption maxima at 558 557 and 560 nm for (I) (II) and(III) respectively e absorption spectrum shows that theternary complexes formed between rose bengal and copperwith the cited drugs have higher values of molar extinctioncoefficient than the binary complexes for the spectrophoto-metric method (Figure 1) Investigations were carried outto establish the most favorable conditions for the ternarycomplex formation for the spectrophotometric method ofthe cited drugs with rose bengal and copper to achievemaximum color development and for ion-pair formation forthe conductometric method to achieve the endpoint

31 Conditions for Spectrophotometric Method

311 Effect of pH In a trial to elucidate the optimummedium for the quantitative determination of the studieddrugs the effect of different acetate and phosphate buffers ofpH of range 2ndash10 was examined It was found that buffer hadno effect on the reaction

312 Effect of Cu(II) Chloride Volume e effect of Cu(II)chloride volume was also tested by using varying amounts(01ndash2mL) of Cu(II) chloride (3mgmL)e results showedthat 02mL 15mL and 05mL of Cu(II) chloride (3mgmL)for (I) (II) and (III) respectively were sufficient for theproduction of maximum and reproducible colour intensity(Figure 2)

313 Effect of Rose Bengal Volume Highest constantabsorbance was obtained on using 15mL 05mL and02mL of rose bengal (3mgmL) for (I) (II) and (III)respectively (Figure 3)

314 Effect of Order of Addition emost suitable sequencewas drug Cu(II) chloride and then rose bengal for all drugsfor the production of the highest colour intensity while theother sequences produced lower absorbance values

315 Effect of Reaction Time and Stability Maximum colourintensity was attained immediately for the studied drugs

06

05

04

03

02

01

00 05 1 15 2

Volume (mL)

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin dihydrate

F 2 Effect of volume of 03 wv Cu(II) chloride on theternary complex formed with 40 120583120583gmL erythromycin thiocyanateand 15mL rose bengal 03 wv 25 120583120583gmL clarithromycin and05mL rose bengal 03wv 13 120583120583gmL azithromycin dihydrate and02mL rose bengal 03 wv

06

05

04

03

02

01

00 05 1 15 2 25

Volume (mL)

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin dihydrate

F 3 Effect of volume of 03 wv rose bengal on the ternarycomplex formed with 40 120583120583gmL erythromycin thiocyanate and02mL Cu(II) chloride 03 wv 25 120583120583gmL clarithromycin and15mL Cu(II) chloride 03 wv 13 120583120583gmL azithromycin dihydrateand 05mL Cu(II) chloride 03 wv

except for (II) it needs 5 minutes to give complete com-plexation and increasing time beyond this had no effect onabsorptione obtained colour was stable for at least 1 hourfor all drugs at room temperature (25∘C plusmn 2) e formationconstants have been calculated by using the molar ratiomethod [32] e formation constants (Kf ) of the reactionproduct were found to be 713 times 104 76 times 105 and 15 times106 Lsdotmolminus1 for (I) (II) and (III) respectively is highvalue indicates a very stable reaction product e Gibbsfree energy change of the reaction (ΔG) was also calculated

Journal of Spectroscopy 5

06

07

08

05

04

03

02

01

00 05 1 15 2 25 3 35 454

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin

VrVd

F 4 Molar ratio method plot for 25 times 10minus4M erythromycinthiocyanate and 25 times 10minus4M Cu(II) chloride in presence of excessrose bengal (15mL 03) 25 times 10minus4M clarithromycin and 25 times10minus4M Cu(II) chloride in presence of excess rose bengal (05mL03) 125 times 10minus4 M azithromycin dihydrate and 125 times 10minus4MCu(II) chloride in presence of excess rose bengal (02mL 03)VR = Cu(II) chloride and Vd = Drug

and was found to be minus278 times 104 minus335 times 104 and minus352times 104 KsdotJsdotmoleminus1 for (I) (II) and (III) respectively enegative value of ΔG points out to the spontaneous natureof the reaction [33]

316 Effect of the Extraction Time and Number of ExtractionsShaking the reaction mixture with the extracting organicsolvent is required for complete extraction of the ternarycomplex formed It was found that 1 minute is enough timefor complete colour extraction Complete extraction wasattained by triple extraction with 3 times 3mL of methylenechloride for all drugs

317 Effect of Solvent Many organic solvents were tried toextract the ternary complexes formed (chloroform methy-lene chloride ethylene chloride benzene toluene and ethylacetate) e results obtained indicated that methylene chlo-ride is the appropriate solvent for extraction for all drugs

318 Determination of Stoichiometric Balance Using MolarRatio Method [34] e reaction stoichiometry between ery-thromycin thiocyanate (I) clarithromycin (II) azithromycindihydrate (III) with Cu(II) chloride and rose bengal was stud-ied by molar ratio method using an equimolar (25 times 10minus4M)solution of erythromycin thiocyanate (I) clarithromycin (II)and (125times 10minus4M) solutions of azithromycin dihydrate (III)Cu(II) chloride and rose bengal of the same molarities eobtained results showed that the composition of the ternarycomplexes was 1 1 2 for erythromycin thiocyanate (I) and

06

07

05

04

03

02

01

00 05 1 15 2 25 3 35 454

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin

VrVd

F 5 Molar ratio method plot for 25 times 10minus4M erythromycinthiocyanate with 25 times 10minus4M rose bengal in presence of excessCu(II) chloride (02 mL 03) 25 times 10minus4M clarithromycin with25times10minus4Mrose bengal in presence of excessCu(II) chloride (15mL03) 125times 10minus4Mazithromycin dihydratewith 125times 10minus4Mrosebengal in presence of excess Cu(II) chloride (05mL 03) Vr = rosebengal and Vd = Drug

06

05

04

03

02

01

00 05 1 15 2 25 3 35 4

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin

VrVd

F 6 Molar ratio method plot for 25 times 10minus4M rose bengalwith 25 times 10minus4M Cu(II) chloride in presence of excess ery-thromycin thiocyanate (30 120583120583gsdotmLminus1) 25 times 10minus4M rose bengal with25 times 10minus4M Cu(II) chloride in presence of excess clarithromycin(30 120583120583gsdotmLminus1) 125 times 10minus4M rose bengal with 125 times 10minus4M Cu(II)chloride in presence of excess azithromycin dihydrate (10 120583120583gsdotmLminus1)Vr = rose bengal and VR = Cu(II) chloride

1 2 2 for clarithromycin (II) and azithromycin dihydrate(III) Cu(II) chloride rose bengal (Figures 4 5 and 6)

Scheme 1 illustrates the mechanism of the reaction ofclarithromycin rose bengal and copper(II) chloride elactone ring in clarithromycin is substituted with a numberof hydroxyl functional groups ese groups are positionedin suitable conguration for interaction with metal ions So

6 Journal of Spectroscopy

O

O

O

OO

O O

O

O

O

O

O

O

O

O

O

O

O

O CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

OH

OH

Cu

Cu

N(CH3)2

N(CH3)2

OMe

HOHO

HO

HOHO

H3C

H3C

H3C

H3C

H3C

H3C

HO

I I

II

I I

II

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

COO

2

minus4+4

Rose bengalClarithromycin

2Cu+2

COOminus

Ominus

2 ++

OMe

2Na+

OMe

OMe

S 1 ProposalMechanism for the reaction between clarithromycin rose bengal and copper(II) chloride clarithromycin-Cu-rose bengalcomplex

copper cation forms a chelatewith the lone pair of electrons ofthe two hydroxyl groups at C11 andC12 Also copper can forma chelate with the lone pair of electrons of the tertiary aminegroup nitrogen and the oxygen of the 120573120573-glycosidic linkageSo the azithromycin-copper chelate bears an overall positivecharge of the original two metal ions that is four positivechargesis cationic complex associates with two moleculesof rose bengal anions to form the ternary complex

32 Conditions for Conductometric Method Investigationswere carried out to establish the most suitable conditionsfor the ion associates formation of the cited drugs with rosebengal to attain sharp endpoint e optimum conditionsfor performing the titration in a quantitative manner wereelucidated as described later

321 Reaction Medium Titrations in different media wereattempted to obtain the best results Preliminary experimentswere in

(i) aqueous drug solution for (I) only (prepared bydissolving 100mg drug in 2mL methanol then

completing to 100mL with bidistilled water) withaqueous reagent solution

(ii) ethanol drug solution with ethanol reagent solution(iii) drug solution with reagent solution both in ethanol-

water (50 vv) mixture(iv) methanol drug solution with methanol reagent solu-

tion (except for (II) as it is insoluble in methanol)(v) drug solution with reagent solution both in

methanol-water mixture (50 vv) (except for (II) asit is insoluble in methanol)

(vi) drug solution with reagent solution both in acetone-water (50 vv) mixture

Preliminary experiments showed that procedure in aque-ous media was the most suitable for successful results for (I)50 (vv) ethanol for (II) and 50 (vv) methanol for (III)

322 Reagentrsquos Concentration Different concentrations ofrose bengal solution were tried ranging from 2 times 10minus2 to5 times 10minus4 molar solutions e optimum concentration of thereagent was 3 times 10minus3M in titration of the studied drug to

Journal of Spectroscopy 7

80

70

60

50

40

30

20

10

00 2 4 6 8

Volume of rose bengal (mL)

Co

nd

uct

ivit

y (

S cm

minus1)

F 7 Conductometric titration curve of 15mg clarithromycinversus 3 times 10 3M rose bengal

achieve a constant and highly stable conductance readingwithin 1-2min of mixing Concentrations less than theselimits led to unstable readings and more time was neededto obtain constant conductance values

Representative titration curve is shown in (Figure 7)Two straight lines are obtained intersecting at the endpointthe rst branch ascending and the second has conductancevalues that would slightly increase aer the equivalencepoint e increase of conductance may be attributed tothe formation of ion pair in solution as a result of thecomplexation reaction Aer the end-point the titrationcurves indicate a slightly increased value of conductancedespite the excess of the reagent is may be due tofurther ionic condensation leading to species of lowermobility

e conductometric titrations of different volumes of3 times 10minus3 M rose bengal solution were performed e resultsshow anobviousmaximum in the conductance curve at drug-reagent molar ratio of 2 1 for (I) and (II) and 1 1 for (III)e reactions may be represented by (for (I))

[RB]minus2+210077131007713Eryth N (Me)210077291007729H+

⟶[RB]minus210077131007713Eryth NH(Me)2100772910077292+2

(2)

where [RB]minus2 rose bengal anion [Eryth N(Me)2] ery-thromycin thiocyanate [ErythNH(Me)2]

+2 protonated ery-thromycin thiocyanate

Erythromycin thiocyanate tertiary amine nitrogen isprotonated so attraction occurs between it and rose bengalanion forming ion-pair complex

e conductance measured before the addition of thetitrant (volume of rose bengal equals zero) is mainly due tothe hydrogen ions mobility ese hydrogen ions result fromtwo sources

(1) the active methylene group in the drug structureas in Scheme 2 which has the ability to make selfprotonation to the drug

O

CH3

lowast

S 2 lowaste active methylene group in macrolide structure

(2) the protons coming from the alcohols used in solubil-ity as these alcohols have certain acidity character socan protonate the amino group in drug structure

So there is no need for acidic buffer use for drug pro-tonation It was expected that the conductance values wouldremain constant or slightly increase aer the equivalencepoint However the conductance of the solution decreasedis may be due to interaction of the protons available inthe titration medium with the added reagent e resultsfrom the conductometic titrations are summarized in Table 4e data show that accurate results were obtained with goodrecoveries and low standard deviation values e optimumconcentration ranges for determination of the cited drugswere in the range of 1ndash15mg mLminus1 for all drugs At suchranges sharp inections (Figure 7) and stable conductancereading were obtained

33 Method Validation

331 For the Spectrophotometric Method

Linearity Under the optimumconditions described standardcalibration curves for erythromycin thiocyanate (I) clar-ithromycin (II) and azithromycin dihydrate (III) with rosebengal and copper were constructed by plotting absorbanceagainst concentration Conformity with Beerrsquos law was evi-dent in the concentration range of the nal dilution citedin Table 1 Beerrsquos law holds over the concentration rangesof 15ndash60 75ndash40 and 4ndash20 120583120583gsdotmLminus1 for (I) (II) and (III)respectively e linear regression equation for each drugwas listed in Table 1 e correlation coefficient was 09999indicating good linearity over the working concentrationrangeSensitivity e detection limit (LOD) for the proposedmethod was calculated using the following equation accord-ing to the ICH [35]

LOD = 33120590120590S (3)

where 120590120590 = the standard deviation of replicate blank responses(under the same conditions as for sample analysis) and S =the slope of the calibration curve

e limits of quantication LOQ is dened as

LOQ = 10120590120590S (4)

8 Journal of Spectroscopy

04

03

02

01

0430 500 550 600 650

(nm)

Absorbance

(a)

(b)

F 8Absorption spectra of (a) 25times 10minus2Mglucosewith 15mLCu(II) chloride 03 wv and 05mL 03 wv rose bengal (b) 5 times10minus2M sucrose with 15mL Cu(II) chloride 03 wv and 05mL03 wv rose bengal

According to the previous equations the LODs and LOQswere calculated as in Table 1 eir values conrm thesensitivity of the proposed methodAccuracy and Precision In order to determine the accuracyand precision of the proposed method solutions containingone concentration of each drug were prepared and analysedin seven replicates e relative standard deviation (RSD)as precision and percentage relative error (Er) as accuracyof the suggested method were calculated at 95 condencelevels and can be considered satisfactory e percent-age relative error was calculated according the followingequation

Er = 10077171007717found minus added

added10077331007733 times 100 (5)

e inter- and intraday precisions and accuracy resultsare shown in Table 5 e analytical results for accuracyand precision show that the proposed method has goodrepeatability and reproducibilityInterference e effect of presence of common excipi-ents such as glucose sucrose sodium chloride carboxymethyl cellulose sodium calcium carbonate andmagnesiumstearate was studied Table 6 presents the results of determi-nation of clarithromycin as a representative example usingthe proposed procedure in the presence of aforementionedingredients e tolerance ratio was dened as the concen-tration which gave an error of plusmn30 in the determinationof drugs Results indicate that the tolerance ratio of drugexcipient (M M) is 1 50 for glucose and 1 100 for sucroseWhile there were no interference from the presence ofsodium chloride carboxy methyl cellulose sodium calcium

carbonate and magnesium stearate Interference due toglucose and sucrosewas further investigated by studying theirreaction with Cu(II) chloride and rose bengal e studyrevealed that no interference could be observed at 557 nmand an interaction product was obtained at 120582120582max 498 intolerance ratios 1 1 and 1 10 for glucose and 1 1 1 101 50 for sucrose However interference starts to appear at120582120582max 557 upon using 50 times 100-fold excess of the molarconcentration of clarithromycin for glucose and sucroserespectively as shown in Figure 8

332 For the Conductometric Method In order to addressthe validity of the proposed method statistical analysis of thedata obtained from its application on the cited drugs in thepure form and in formulations was performed e resultsrevealed in Tables 5 and 6 showed that the proposed methodis satisfactorily accurate precise and reproducible over aconcentration range of 1ndash15mg Results for the determina-tion of the studied drugs using the previously mentionedmethod were compared with results from reported one [6]Table 7

34 Analytical Applications e proposed methods wereapplied to determine the studied drugs in their pharma-ceutical dosage forms Satisfactory results were obtainede recovery of each drug was calculated by comparing theconcentration obtained from the spiked mixtures with thoseof the drug e results of analysis of the commercial dosageforms and the recovery study are shown in Tables 2 3 and4 e results obtained were compared with the reportedmethod [6] No signicant differences were found betweenthe proposed methods and reported method Statisticalcomparison of the results was performed using Studentrsquos t-test and variance ratio F-test at 95 condence level (Table7)

4 Conclusions

e proposed methods are advantageous when compared tomany of the reported spectrophotometric methods in havinghigher sensitivity e data given previously reveal that theproposed methods are simple accurate and sensitive withgood precision and accuracyWith thesemethods one can dothe analysis at low cost without losing accuracy No interfer-ence from excipients was encountered Also In comparisonwith the chromatographic methods the proposed methodsare advantageous as HPLC and GLC are not available espe-cially in developing countries and they require complexand expensive equipment intensive sample preparation andpersonnel skilled in chromatographic techniques So theproposed methods can be used as alternative methods to thereported ones for the routine determination of erythromycinthiocyanate (I) clarithromycin (II) azithromycin dihydrate(III) in the pure form and in pharmaceutical formulationsdepending upon the availability of chemicals and the equip-ment

Journal of Spectroscopy 9

T2

Determination

oferythrom

ycin

thiocyanateand

clarithrom

ycin

intheirph

armaceutic

aldo

sage

form

sthroug

hcomplexation

with

Cu(II)

chlorid

eand

rose

beng

alby

spectro

photom

etric

procedurelowast

Erythrocin

tablets

Erythrocin

Pharco

suspensio

nKlarim

ixtablets

Klarim

ixsuspensio

nClaimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

151499

9993

151494

9959

75

756

10080

101005

10048

151489

9924

151504

10028

75

746

9952

101014

10144

201986

9930

202002

10008

101010

10096

125

1239

9914

225

2250

10001

252504

10017

151512

10080

151498

9984

252520

10079

302991

9971

175

1727

9870

175

1746

9979

275

2774

10086

325

3261

10033

202014

10072

202005

10024

302976

9919

353483

9953

225

2244

9973

225

2268

10080

353515

10041

404012

10030

252517

10067

275

2756

10022

375

3758

10021

425

4255

10013

275

2756

10022

303005

10016

403965

9919

302995

9984

Mean

9991

10001

10020

10023

Varia

nce

044

010

054

042

SD066

032

073

065

SE015

011

024

023

lowast Averageo

fthree

experim

ents

10 Journal of Spectroscopy

T3Determinationof

azith

romycin

dihydrateinits

pharmaceutic

aldo

sage

form

sthrou

ghcomplexationwith

Cu(II)chlorid

eand

rose

beng

alby

spectro

photom

etric

procedurelowast

Azrolid

tablets

Zithrokancapsules

Zithrokansuspensio

n

Claimed

tobe

taken120583120583g

mL

Authentic

added120583120583g

mL

Foun

dconc

120583120583gm

LRe

covery

Claimed

tobe

taken

120583120583gm

L

Authentic

added120583120583g

mL

Foun

dconc

120583120583gm

LRe

covery

Claimed

tobe

taken120583120583g

mL

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

LRe

covery

4403

10077

4401

10036

4405

10118

4401

10036

4408

10199

4405

10118

7701

10019

5497

9942

6606

10095

8800

10002

6602

10041

8802

10022

9896

9953

8797

9962

101006

10060

101003

10028

9906

10061

121199

9991

111111

10103

101000

9995

141401

10010

121197

9977

111098

9985

161589

9934

141403

10022

121202

10018

161589

9934

131299

9995

Mean

10015

10023

10043

Varia

nce

027

051

043

SD052

072

066

SE017

024

025

lowast Averageo

fthree

experim

ents

Journal of Spectroscopy 11

T 4 Application of the proposed conductometric method to the determination of erythromycin thiocyanate in dosage formslowast

Erythrocin tablets Erythrocin Pharco suspensionTaken (mg) Found (mg) Recovery Taken (mg) Found (mg) Recovery 3 305 10158 2 2 1004 4 100 4 405 101196 607 10119 7 705 100688 81 10119 8 805 1006010 10 100 12 1190 992012 1191 9921 14 1395 996614 1405 10034Mean 10050 10022SD 085 073SE 032 030RSD 084 073Variance 072 054lowastMean of three different experiments

T 5 e intraday and interday precision and accuracy data for the studied drugs using the spectrophotometric method

Intraday Interday

Drug Taken(120583120583gmLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSDbAccuracyEr c

Taken(120583120583g mLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSD bAccuracyEr c

(I) 50 5028 10056 029 056 50 5034 10068 039 068(II) 30 3033 10109 056 109 30 3017 10057 046 057(III) 16 1599 9995 019 minus005 16 1601 10007 025 007aAverage of seven determinationsbRSD percentage relative standard deviationcEr percentage relative error

T 6 Analysis of clarithromycin 28 120583120583gmLminus1 (5times 10minus4 M) by the spectrophotometric method in the presence of some common excipients

Tolerance molar ratio ( M M)lowast Glucose Sucrose Carboxy methyl cellulose sodium Sodium chlorideRecovery lowastlowast

1 1 10124 10107 10089 99821 10 10036 10124 9911 99821 50 11545 10213 10035 99471 100 10657 10746 9822 9929lowastDrug excipientlowastlowastMean of three determinations

T 7 Determination of the studied drugs by the proposed spectrophotometric and conductmetric methods compared with the reportedmethod [6]

(I) (II) (III)Spectrophotometric method

Mean plusmn RSD 10005 plusmn 054 10001 plusmn 047 9987 plusmn 050Variance 029 022 025Studentrsquos 119905119905-test 005 (217)lowast 04 (131)lowast 006 (216)lowast119865119865-test 238 (428)lowast 391 (360)lowast 268 (389)lowast

119899119899 7 9 9Conductometric method

Mean plusmn RSD 10066 plusmn 093 10065 plusmn 063 10062 plusmn 062Variance 087 040 038Studentrsquos 119905119905-test 131 (217)lowast 113 (217)lowast 210 (2228)lowast119865119865-test 126 (387)lowast 215 (397)lowast 176 (805)lowast

119899119899 7 6 6Reported method [6]

Mean plusmn RSD 10004 plusmn 083 10015 plusmn 093 9989 plusmn 082Variance 069 086 067Studentrsquos 119905119905-test mdash mdash mdash119865119865-test mdash mdash mdash119899119899 7 8 6lowaste gures in parenthesis are the theoretical values for 119905119905 and 119865119865-tests (119875119875 119875 005)

12 Journal of Spectroscopy

Acknowledgments

e authors wish to express their great thanks to Dr SoadSelim Abd El Hay and Dr Omnia Ahmed Emam Ismaiel atthe Analytical Chemistry Department Zagazig Universityfor their great help and advices during this work Also theywould like to point out that the authors do not have anyconict of interests with any company or institution

References

[1] S C Sweetman Martindale the Complete Drug ReferencePharmaceutical Press London UK 35th edition 2007

[2] S OmuraMacrolide Antibiotics Academic Press London UK1984

[3] J N Delgado and W A RemersWilson and Gisvoldrsquos Textbookof Organic Medecinal and Pharmaceutical Chemistry J BLippincott Company London UK 1998

[4] H A Krisr Progress inMedicinal Chemistry vol 30 of Edited byG P Ellis and D K Luscombe 1993

[5] British Pharmacopoeia Her Majestyrsquos Stationery Office Lon-don UK 2007

[6] M I Walash M S Rizk M I Eid and M E Fathy ldquoSpec-trophotometric determination of four macrolide antibiotics inpharmaceutical formulations and biological uids via binarycomplex formation with eosin and spectrophotometryrdquo Journalof AOAC International vol 90 no 6 pp 1579ndash1587 2007

[7] J Shah M R Jan and S Manzoor ldquoExtractive spectrophoto-metric methods for determination of clarithromycin in phar-maceutical formulations using bromothymol blue and cresolredrdquo Journal of the Chinese Chemical Society vol 55 no 5 pp1107ndash1112 2008

[8] C E R De Paula V G K Almeida and R J CassellaldquoNovel spectrophotometric method for the determination ofazithromycin in pharmaceutical formulations based on itscharge transfer reaction with quinalizarinrdquo Journal of theBrazilian Chemical Society vol 21 no 9 pp 1664ndash1671 2010

[9] S Ashour and R Bayram ldquoNovel spectrophotometric methodfor determination of somemacrolide antibiotics in pharmaceu-tical formulations using 1 2-naphthoquinone-4-sulphonaterdquoSpectrochim Acta Part A vol 99 pp 74ndash80 2012

[10] S D Magar A P Tupe P Y Pawar and B Y ManeldquoSimultaneous spectrophotometric estimation of ceximeand azithrhomycin in tablet dosage formrdquo Current PharmaResearch vol 2 no 3 pp 535ndash538 2012

[11] P Y Khashaba ldquoSpectrouorimetric analysis of certain mac-rolide antibiotics in bulk and pharmaceutical formulationsrdquoJournal of Pharmaceutical and Biomedical Analysis vol 27 no6 pp 923ndash932 2002

[12] N El-Rabbat H F Askal P Y Khashaba and N N AttialdquoA validated spectrouorometric assay for the determinationof certainmacrolide antibiotics in pharmaceutical formulationsand spiked biological uidsrdquo Journal of AOAC International vol89 no 5 pp 1276ndash1287 2006

[13] C L Flurer ldquoAnalysis of macrolide antibiotics by capillaryelectrophoresisrdquo Electrophoresis vol 17 no 2 pp 359ndash3661996

[14] A K Lalloo S C Chattaraj and I Kanfer ldquoDevelopmentof a capillary electrophoretic method for the separation ofthe macrolide antibiotics erythromycin josamycin and olean-domycinrdquo Journal of Chromatography B vol 704 no 1-2 pp333ndash341 1997

[15] M Hedenmo and B M Eriksson ldquoLiquid chromatographicdetermination of the macrolide antibiotics roxithromycin andclarithromycin in plasma by automated solid-phase extractionand electrochemical detectionrdquo Journal of Chromatography Avol 692 no 1-2 pp 161ndash166 1995

[16] M Dubois D Fluchard E Sior and P Delahaut ldquoIdenticationand quantication of ve macrolide antibiotics in severaltissues eggs and milk by liquid chromatography-electrospraytandem mass spectrometryrdquo Journal of Chromatography B vol753 no 2 pp 189ndash202 2001

[17] R V S Nirogi V N Kandikere M Shukla et al ldquoSensitiveand selective liquid chromatography-tandem mass spectrom-etry method for the quantication of azithromycin in humanplasmardquo Analytica Chimica Acta vol 553 no 1-2 pp 1ndash82005

[18] O A E M Farghaly and N A L Mohamed ldquoVoltammetricdetermination of azithromycin at the carbon paste electroderdquoTalanta vol 62 no 3 pp 531ndash538 2004

[19] J A Bernabeacuteu M A Camacho M E Gil-Alegre V Ruz andA I Torres-Suaacuterez ldquoMicrobiological bioassay of erythromycinthiocyanate optimisation and validationrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 21 no 2 pp 347ndash3531999

[20] H R N Salgado and A F F Roncari ldquoMicrobiological assayfor the determination of azithromycin in ophthalmic solutionsrdquoYaoxue Xuebao vol 40 no 6 pp 544ndash549 2005

[21] A S Amin and M M El-Henawee ldquoColorimetric methodfor the simultaneous determination of chlorphenoxaminehydrochloride and anhydrous caffeine in pure and dosage formswith rose bengalrdquo Mikrochimica Acta vol 118 no 3-4 pp177ndash183 1995

[22] F M Abdel-Gawad ldquoSpectrophotometric determination ofoxybuprocaine hydrochloride with halouorescein derivativesrdquoFarmaco vol 50 no 3 pp 197ndash200 1995

[23] A S Amin ldquoSpectrophotometric and conductometric deter-mination of clindamycin hydrochloride in pure form andin pharmaceutical preparationsrdquo Analusis vol 23 no 8 pp415ndash417 1995

[24] SM Amer Z El-Sherif andMM Amer ldquoSpectrophotometricdetermination of isoniazid nalidixic acid and umequinethrough ternary complex-formation with Cd (II) and rosebengalrdquo Egyptian Journal of Pharmaceutical Sciences vol 35 no1ndash6 pp 627ndash642 1994

[25] H Parham and A G Fazeli ldquoExtraction-spectrophotometricdetermination of trace amounts of barium by 18-crown-6 androse bengalrdquo Analytical Sciences vol 16 no 6 pp 575ndash5772000

[26] M A El Ries ldquoSpectrophotometric and indirect determinationof lincomycin by atomic absorption spectroscopyrdquo AnalyticalLetters vol 27 no 8 pp 1517ndash1531 1994

[27] H E Abdellatef M M Ayad and E A Taha ldquoSpectrophoto-metric and atomic absorption spectrometric determination oframipril and perindopril through ternary complex formationwith eosin and Cu(II)rdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 18 no 6 pp 1021ndash1027 1999

[28] M M Ayad A A Shalaby H E Abdellatef and M M HosnyldquoSpectrophotometric and AAS determination of ramipril andenalapril through ternary complex formationrdquo Journal of Phar-maceutical and Biomedical Analysis vol 28 no 2 pp 311ndash3212002

Journal of Spectroscopy 13

[29] A AMMoustafa ldquoSpectrophotometric methods for the deter-mination of lansoprazole and pantoprazole sodium sesquihy-draterdquo Journal of Pharmaceutical and Biomedical Analysis vol22 no 1 pp 45ndash58 2000

[30] M I Walash M E S Metwally M Eid and R N El-ShahenyldquoSpectrophotometric determination of risedronate in pharma-ceutical formulations via complex formation with Cu(II) ionsapplication to content uniformity testingrdquo International Journalof Biomedical Science vol 4 no 4 pp 303ndash309 2008

[31] J J Lingane Electroanalytical Chemistry Interscience NewYork NY USA 2nd edition 1958

[32] S A Tirmizi F H Wattoo M H S Wattoo S Sarwar AN Memon and A B Ghangro ldquoSpectrophotometric studyof stability constants of cimetidine-Ni(II) complex at differenttemperaturesrdquo Arabian Journal of Chemistry vol 35 no 1 pp93ndash100 2010

[33] J Inczedy Analytical Application of Complex EquilibriiaBudapest John Wiley amp Sons 1976

[34] J H Yoe and A L Jones ldquoCcrforimetric determination of ironwith disodium-12-dlhydroxybenzene-35-disuffonaterdquo Indus-trial and Engineering Chemistry vol 16 pp 111ndash115 1944

[35] International Conference on Harmonisation ICH of TechnicalRequirments for Registeration of Pharmaceuticals for HumanUse 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Spectroscopy 5

06

07

08

05

04

03

02

01

00 05 1 15 2 25 3 35 454

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin

VrVd

F 4 Molar ratio method plot for 25 times 10minus4M erythromycinthiocyanate and 25 times 10minus4M Cu(II) chloride in presence of excessrose bengal (15mL 03) 25 times 10minus4M clarithromycin and 25 times10minus4M Cu(II) chloride in presence of excess rose bengal (05mL03) 125 times 10minus4 M azithromycin dihydrate and 125 times 10minus4MCu(II) chloride in presence of excess rose bengal (02mL 03)VR = Cu(II) chloride and Vd = Drug

and was found to be minus278 times 104 minus335 times 104 and minus352times 104 KsdotJsdotmoleminus1 for (I) (II) and (III) respectively enegative value of ΔG points out to the spontaneous natureof the reaction [33]

316 Effect of the Extraction Time and Number of ExtractionsShaking the reaction mixture with the extracting organicsolvent is required for complete extraction of the ternarycomplex formed It was found that 1 minute is enough timefor complete colour extraction Complete extraction wasattained by triple extraction with 3 times 3mL of methylenechloride for all drugs

317 Effect of Solvent Many organic solvents were tried toextract the ternary complexes formed (chloroform methy-lene chloride ethylene chloride benzene toluene and ethylacetate) e results obtained indicated that methylene chlo-ride is the appropriate solvent for extraction for all drugs

318 Determination of Stoichiometric Balance Using MolarRatio Method [34] e reaction stoichiometry between ery-thromycin thiocyanate (I) clarithromycin (II) azithromycindihydrate (III) with Cu(II) chloride and rose bengal was stud-ied by molar ratio method using an equimolar (25 times 10minus4M)solution of erythromycin thiocyanate (I) clarithromycin (II)and (125times 10minus4M) solutions of azithromycin dihydrate (III)Cu(II) chloride and rose bengal of the same molarities eobtained results showed that the composition of the ternarycomplexes was 1 1 2 for erythromycin thiocyanate (I) and

06

07

05

04

03

02

01

00 05 1 15 2 25 3 35 454

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin

VrVd

F 5 Molar ratio method plot for 25 times 10minus4M erythromycinthiocyanate with 25 times 10minus4M rose bengal in presence of excessCu(II) chloride (02 mL 03) 25 times 10minus4M clarithromycin with25times10minus4Mrose bengal in presence of excessCu(II) chloride (15mL03) 125times 10minus4Mazithromycin dihydratewith 125times 10minus4Mrosebengal in presence of excess Cu(II) chloride (05mL 03) Vr = rosebengal and Vd = Drug

06

05

04

03

02

01

00 05 1 15 2 25 3 35 4

Ab

sorb

ance

Erythromycin thiocyanateClarithromycinAzithromycin

VrVd

F 6 Molar ratio method plot for 25 times 10minus4M rose bengalwith 25 times 10minus4M Cu(II) chloride in presence of excess ery-thromycin thiocyanate (30 120583120583gsdotmLminus1) 25 times 10minus4M rose bengal with25 times 10minus4M Cu(II) chloride in presence of excess clarithromycin(30 120583120583gsdotmLminus1) 125 times 10minus4M rose bengal with 125 times 10minus4M Cu(II)chloride in presence of excess azithromycin dihydrate (10 120583120583gsdotmLminus1)Vr = rose bengal and VR = Cu(II) chloride

1 2 2 for clarithromycin (II) and azithromycin dihydrate(III) Cu(II) chloride rose bengal (Figures 4 5 and 6)

Scheme 1 illustrates the mechanism of the reaction ofclarithromycin rose bengal and copper(II) chloride elactone ring in clarithromycin is substituted with a numberof hydroxyl functional groups ese groups are positionedin suitable conguration for interaction with metal ions So

6 Journal of Spectroscopy

O

O

O

OO

O O

O

O

O

O

O

O

O

O

O

O

O

O CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

OH

OH

Cu

Cu

N(CH3)2

N(CH3)2

OMe

HOHO

HO

HOHO

H3C

H3C

H3C

H3C

H3C

H3C

HO

I I

II

I I

II

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

COO

2

minus4+4

Rose bengalClarithromycin

2Cu+2

COOminus

Ominus

2 ++

OMe

2Na+

OMe

OMe

S 1 ProposalMechanism for the reaction between clarithromycin rose bengal and copper(II) chloride clarithromycin-Cu-rose bengalcomplex

copper cation forms a chelatewith the lone pair of electrons ofthe two hydroxyl groups at C11 andC12 Also copper can forma chelate with the lone pair of electrons of the tertiary aminegroup nitrogen and the oxygen of the 120573120573-glycosidic linkageSo the azithromycin-copper chelate bears an overall positivecharge of the original two metal ions that is four positivechargesis cationic complex associates with two moleculesof rose bengal anions to form the ternary complex

32 Conditions for Conductometric Method Investigationswere carried out to establish the most suitable conditionsfor the ion associates formation of the cited drugs with rosebengal to attain sharp endpoint e optimum conditionsfor performing the titration in a quantitative manner wereelucidated as described later

321 Reaction Medium Titrations in different media wereattempted to obtain the best results Preliminary experimentswere in

(i) aqueous drug solution for (I) only (prepared bydissolving 100mg drug in 2mL methanol then

completing to 100mL with bidistilled water) withaqueous reagent solution

(ii) ethanol drug solution with ethanol reagent solution(iii) drug solution with reagent solution both in ethanol-

water (50 vv) mixture(iv) methanol drug solution with methanol reagent solu-

tion (except for (II) as it is insoluble in methanol)(v) drug solution with reagent solution both in

methanol-water mixture (50 vv) (except for (II) asit is insoluble in methanol)

(vi) drug solution with reagent solution both in acetone-water (50 vv) mixture

Preliminary experiments showed that procedure in aque-ous media was the most suitable for successful results for (I)50 (vv) ethanol for (II) and 50 (vv) methanol for (III)

322 Reagentrsquos Concentration Different concentrations ofrose bengal solution were tried ranging from 2 times 10minus2 to5 times 10minus4 molar solutions e optimum concentration of thereagent was 3 times 10minus3M in titration of the studied drug to

Journal of Spectroscopy 7

80

70

60

50

40

30

20

10

00 2 4 6 8

Volume of rose bengal (mL)

Co

nd

uct

ivit

y (

S cm

minus1)

F 7 Conductometric titration curve of 15mg clarithromycinversus 3 times 10 3M rose bengal

achieve a constant and highly stable conductance readingwithin 1-2min of mixing Concentrations less than theselimits led to unstable readings and more time was neededto obtain constant conductance values

Representative titration curve is shown in (Figure 7)Two straight lines are obtained intersecting at the endpointthe rst branch ascending and the second has conductancevalues that would slightly increase aer the equivalencepoint e increase of conductance may be attributed tothe formation of ion pair in solution as a result of thecomplexation reaction Aer the end-point the titrationcurves indicate a slightly increased value of conductancedespite the excess of the reagent is may be due tofurther ionic condensation leading to species of lowermobility

e conductometric titrations of different volumes of3 times 10minus3 M rose bengal solution were performed e resultsshow anobviousmaximum in the conductance curve at drug-reagent molar ratio of 2 1 for (I) and (II) and 1 1 for (III)e reactions may be represented by (for (I))

[RB]minus2+210077131007713Eryth N (Me)210077291007729H+

⟶[RB]minus210077131007713Eryth NH(Me)2100772910077292+2

(2)

where [RB]minus2 rose bengal anion [Eryth N(Me)2] ery-thromycin thiocyanate [ErythNH(Me)2]

+2 protonated ery-thromycin thiocyanate

Erythromycin thiocyanate tertiary amine nitrogen isprotonated so attraction occurs between it and rose bengalanion forming ion-pair complex

e conductance measured before the addition of thetitrant (volume of rose bengal equals zero) is mainly due tothe hydrogen ions mobility ese hydrogen ions result fromtwo sources

(1) the active methylene group in the drug structureas in Scheme 2 which has the ability to make selfprotonation to the drug

O

CH3

lowast

S 2 lowaste active methylene group in macrolide structure

(2) the protons coming from the alcohols used in solubil-ity as these alcohols have certain acidity character socan protonate the amino group in drug structure

So there is no need for acidic buffer use for drug pro-tonation It was expected that the conductance values wouldremain constant or slightly increase aer the equivalencepoint However the conductance of the solution decreasedis may be due to interaction of the protons available inthe titration medium with the added reagent e resultsfrom the conductometic titrations are summarized in Table 4e data show that accurate results were obtained with goodrecoveries and low standard deviation values e optimumconcentration ranges for determination of the cited drugswere in the range of 1ndash15mg mLminus1 for all drugs At suchranges sharp inections (Figure 7) and stable conductancereading were obtained

33 Method Validation

331 For the Spectrophotometric Method

Linearity Under the optimumconditions described standardcalibration curves for erythromycin thiocyanate (I) clar-ithromycin (II) and azithromycin dihydrate (III) with rosebengal and copper were constructed by plotting absorbanceagainst concentration Conformity with Beerrsquos law was evi-dent in the concentration range of the nal dilution citedin Table 1 Beerrsquos law holds over the concentration rangesof 15ndash60 75ndash40 and 4ndash20 120583120583gsdotmLminus1 for (I) (II) and (III)respectively e linear regression equation for each drugwas listed in Table 1 e correlation coefficient was 09999indicating good linearity over the working concentrationrangeSensitivity e detection limit (LOD) for the proposedmethod was calculated using the following equation accord-ing to the ICH [35]

LOD = 33120590120590S (3)

where 120590120590 = the standard deviation of replicate blank responses(under the same conditions as for sample analysis) and S =the slope of the calibration curve

e limits of quantication LOQ is dened as

LOQ = 10120590120590S (4)

8 Journal of Spectroscopy

04

03

02

01

0430 500 550 600 650

(nm)

Absorbance

(a)

(b)

F 8Absorption spectra of (a) 25times 10minus2Mglucosewith 15mLCu(II) chloride 03 wv and 05mL 03 wv rose bengal (b) 5 times10minus2M sucrose with 15mL Cu(II) chloride 03 wv and 05mL03 wv rose bengal

According to the previous equations the LODs and LOQswere calculated as in Table 1 eir values conrm thesensitivity of the proposed methodAccuracy and Precision In order to determine the accuracyand precision of the proposed method solutions containingone concentration of each drug were prepared and analysedin seven replicates e relative standard deviation (RSD)as precision and percentage relative error (Er) as accuracyof the suggested method were calculated at 95 condencelevels and can be considered satisfactory e percent-age relative error was calculated according the followingequation

Er = 10077171007717found minus added

added10077331007733 times 100 (5)

e inter- and intraday precisions and accuracy resultsare shown in Table 5 e analytical results for accuracyand precision show that the proposed method has goodrepeatability and reproducibilityInterference e effect of presence of common excipi-ents such as glucose sucrose sodium chloride carboxymethyl cellulose sodium calcium carbonate andmagnesiumstearate was studied Table 6 presents the results of determi-nation of clarithromycin as a representative example usingthe proposed procedure in the presence of aforementionedingredients e tolerance ratio was dened as the concen-tration which gave an error of plusmn30 in the determinationof drugs Results indicate that the tolerance ratio of drugexcipient (M M) is 1 50 for glucose and 1 100 for sucroseWhile there were no interference from the presence ofsodium chloride carboxy methyl cellulose sodium calcium

carbonate and magnesium stearate Interference due toglucose and sucrosewas further investigated by studying theirreaction with Cu(II) chloride and rose bengal e studyrevealed that no interference could be observed at 557 nmand an interaction product was obtained at 120582120582max 498 intolerance ratios 1 1 and 1 10 for glucose and 1 1 1 101 50 for sucrose However interference starts to appear at120582120582max 557 upon using 50 times 100-fold excess of the molarconcentration of clarithromycin for glucose and sucroserespectively as shown in Figure 8

332 For the Conductometric Method In order to addressthe validity of the proposed method statistical analysis of thedata obtained from its application on the cited drugs in thepure form and in formulations was performed e resultsrevealed in Tables 5 and 6 showed that the proposed methodis satisfactorily accurate precise and reproducible over aconcentration range of 1ndash15mg Results for the determina-tion of the studied drugs using the previously mentionedmethod were compared with results from reported one [6]Table 7

34 Analytical Applications e proposed methods wereapplied to determine the studied drugs in their pharma-ceutical dosage forms Satisfactory results were obtainede recovery of each drug was calculated by comparing theconcentration obtained from the spiked mixtures with thoseof the drug e results of analysis of the commercial dosageforms and the recovery study are shown in Tables 2 3 and4 e results obtained were compared with the reportedmethod [6] No signicant differences were found betweenthe proposed methods and reported method Statisticalcomparison of the results was performed using Studentrsquos t-test and variance ratio F-test at 95 condence level (Table7)

4 Conclusions

e proposed methods are advantageous when compared tomany of the reported spectrophotometric methods in havinghigher sensitivity e data given previously reveal that theproposed methods are simple accurate and sensitive withgood precision and accuracyWith thesemethods one can dothe analysis at low cost without losing accuracy No interfer-ence from excipients was encountered Also In comparisonwith the chromatographic methods the proposed methodsare advantageous as HPLC and GLC are not available espe-cially in developing countries and they require complexand expensive equipment intensive sample preparation andpersonnel skilled in chromatographic techniques So theproposed methods can be used as alternative methods to thereported ones for the routine determination of erythromycinthiocyanate (I) clarithromycin (II) azithromycin dihydrate(III) in the pure form and in pharmaceutical formulationsdepending upon the availability of chemicals and the equip-ment

Journal of Spectroscopy 9

T2

Determination

oferythrom

ycin

thiocyanateand

clarithrom

ycin

intheirph

armaceutic

aldo

sage

form

sthroug

hcomplexation

with

Cu(II)

chlorid

eand

rose

beng

alby

spectro

photom

etric

procedurelowast

Erythrocin

tablets

Erythrocin

Pharco

suspensio

nKlarim

ixtablets

Klarim

ixsuspensio

nClaimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

151499

9993

151494

9959

75

756

10080

101005

10048

151489

9924

151504

10028

75

746

9952

101014

10144

201986

9930

202002

10008

101010

10096

125

1239

9914

225

2250

10001

252504

10017

151512

10080

151498

9984

252520

10079

302991

9971

175

1727

9870

175

1746

9979

275

2774

10086

325

3261

10033

202014

10072

202005

10024

302976

9919

353483

9953

225

2244

9973

225

2268

10080

353515

10041

404012

10030

252517

10067

275

2756

10022

375

3758

10021

425

4255

10013

275

2756

10022

303005

10016

403965

9919

302995

9984

Mean

9991

10001

10020

10023

Varia

nce

044

010

054

042

SD066

032

073

065

SE015

011

024

023

lowast Averageo

fthree

experim

ents

10 Journal of Spectroscopy

T3Determinationof

azith

romycin

dihydrateinits

pharmaceutic

aldo

sage

form

sthrou

ghcomplexationwith

Cu(II)chlorid

eand

rose

beng

alby

spectro

photom

etric

procedurelowast

Azrolid

tablets

Zithrokancapsules

Zithrokansuspensio

n

Claimed

tobe

taken120583120583g

mL

Authentic

added120583120583g

mL

Foun

dconc

120583120583gm

LRe

covery

Claimed

tobe

taken

120583120583gm

L

Authentic

added120583120583g

mL

Foun

dconc

120583120583gm

LRe

covery

Claimed

tobe

taken120583120583g

mL

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

LRe

covery

4403

10077

4401

10036

4405

10118

4401

10036

4408

10199

4405

10118

7701

10019

5497

9942

6606

10095

8800

10002

6602

10041

8802

10022

9896

9953

8797

9962

101006

10060

101003

10028

9906

10061

121199

9991

111111

10103

101000

9995

141401

10010

121197

9977

111098

9985

161589

9934

141403

10022

121202

10018

161589

9934

131299

9995

Mean

10015

10023

10043

Varia

nce

027

051

043

SD052

072

066

SE017

024

025

lowast Averageo

fthree

experim

ents

Journal of Spectroscopy 11

T 4 Application of the proposed conductometric method to the determination of erythromycin thiocyanate in dosage formslowast

Erythrocin tablets Erythrocin Pharco suspensionTaken (mg) Found (mg) Recovery Taken (mg) Found (mg) Recovery 3 305 10158 2 2 1004 4 100 4 405 101196 607 10119 7 705 100688 81 10119 8 805 1006010 10 100 12 1190 992012 1191 9921 14 1395 996614 1405 10034Mean 10050 10022SD 085 073SE 032 030RSD 084 073Variance 072 054lowastMean of three different experiments

T 5 e intraday and interday precision and accuracy data for the studied drugs using the spectrophotometric method

Intraday Interday

Drug Taken(120583120583gmLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSDbAccuracyEr c

Taken(120583120583g mLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSD bAccuracyEr c

(I) 50 5028 10056 029 056 50 5034 10068 039 068(II) 30 3033 10109 056 109 30 3017 10057 046 057(III) 16 1599 9995 019 minus005 16 1601 10007 025 007aAverage of seven determinationsbRSD percentage relative standard deviationcEr percentage relative error

T 6 Analysis of clarithromycin 28 120583120583gmLminus1 (5times 10minus4 M) by the spectrophotometric method in the presence of some common excipients

Tolerance molar ratio ( M M)lowast Glucose Sucrose Carboxy methyl cellulose sodium Sodium chlorideRecovery lowastlowast

1 1 10124 10107 10089 99821 10 10036 10124 9911 99821 50 11545 10213 10035 99471 100 10657 10746 9822 9929lowastDrug excipientlowastlowastMean of three determinations

T 7 Determination of the studied drugs by the proposed spectrophotometric and conductmetric methods compared with the reportedmethod [6]

(I) (II) (III)Spectrophotometric method

Mean plusmn RSD 10005 plusmn 054 10001 plusmn 047 9987 plusmn 050Variance 029 022 025Studentrsquos 119905119905-test 005 (217)lowast 04 (131)lowast 006 (216)lowast119865119865-test 238 (428)lowast 391 (360)lowast 268 (389)lowast

119899119899 7 9 9Conductometric method

Mean plusmn RSD 10066 plusmn 093 10065 plusmn 063 10062 plusmn 062Variance 087 040 038Studentrsquos 119905119905-test 131 (217)lowast 113 (217)lowast 210 (2228)lowast119865119865-test 126 (387)lowast 215 (397)lowast 176 (805)lowast

119899119899 7 6 6Reported method [6]

Mean plusmn RSD 10004 plusmn 083 10015 plusmn 093 9989 plusmn 082Variance 069 086 067Studentrsquos 119905119905-test mdash mdash mdash119865119865-test mdash mdash mdash119899119899 7 8 6lowaste gures in parenthesis are the theoretical values for 119905119905 and 119865119865-tests (119875119875 119875 005)

12 Journal of Spectroscopy

Acknowledgments

e authors wish to express their great thanks to Dr SoadSelim Abd El Hay and Dr Omnia Ahmed Emam Ismaiel atthe Analytical Chemistry Department Zagazig Universityfor their great help and advices during this work Also theywould like to point out that the authors do not have anyconict of interests with any company or institution

References

[1] S C Sweetman Martindale the Complete Drug ReferencePharmaceutical Press London UK 35th edition 2007

[2] S OmuraMacrolide Antibiotics Academic Press London UK1984

[3] J N Delgado and W A RemersWilson and Gisvoldrsquos Textbookof Organic Medecinal and Pharmaceutical Chemistry J BLippincott Company London UK 1998

[4] H A Krisr Progress inMedicinal Chemistry vol 30 of Edited byG P Ellis and D K Luscombe 1993

[5] British Pharmacopoeia Her Majestyrsquos Stationery Office Lon-don UK 2007

[6] M I Walash M S Rizk M I Eid and M E Fathy ldquoSpec-trophotometric determination of four macrolide antibiotics inpharmaceutical formulations and biological uids via binarycomplex formation with eosin and spectrophotometryrdquo Journalof AOAC International vol 90 no 6 pp 1579ndash1587 2007

[7] J Shah M R Jan and S Manzoor ldquoExtractive spectrophoto-metric methods for determination of clarithromycin in phar-maceutical formulations using bromothymol blue and cresolredrdquo Journal of the Chinese Chemical Society vol 55 no 5 pp1107ndash1112 2008

[8] C E R De Paula V G K Almeida and R J CassellaldquoNovel spectrophotometric method for the determination ofazithromycin in pharmaceutical formulations based on itscharge transfer reaction with quinalizarinrdquo Journal of theBrazilian Chemical Society vol 21 no 9 pp 1664ndash1671 2010

[9] S Ashour and R Bayram ldquoNovel spectrophotometric methodfor determination of somemacrolide antibiotics in pharmaceu-tical formulations using 1 2-naphthoquinone-4-sulphonaterdquoSpectrochim Acta Part A vol 99 pp 74ndash80 2012

[10] S D Magar A P Tupe P Y Pawar and B Y ManeldquoSimultaneous spectrophotometric estimation of ceximeand azithrhomycin in tablet dosage formrdquo Current PharmaResearch vol 2 no 3 pp 535ndash538 2012

[11] P Y Khashaba ldquoSpectrouorimetric analysis of certain mac-rolide antibiotics in bulk and pharmaceutical formulationsrdquoJournal of Pharmaceutical and Biomedical Analysis vol 27 no6 pp 923ndash932 2002

[12] N El-Rabbat H F Askal P Y Khashaba and N N AttialdquoA validated spectrouorometric assay for the determinationof certainmacrolide antibiotics in pharmaceutical formulationsand spiked biological uidsrdquo Journal of AOAC International vol89 no 5 pp 1276ndash1287 2006

[13] C L Flurer ldquoAnalysis of macrolide antibiotics by capillaryelectrophoresisrdquo Electrophoresis vol 17 no 2 pp 359ndash3661996

[14] A K Lalloo S C Chattaraj and I Kanfer ldquoDevelopmentof a capillary electrophoretic method for the separation ofthe macrolide antibiotics erythromycin josamycin and olean-domycinrdquo Journal of Chromatography B vol 704 no 1-2 pp333ndash341 1997

[15] M Hedenmo and B M Eriksson ldquoLiquid chromatographicdetermination of the macrolide antibiotics roxithromycin andclarithromycin in plasma by automated solid-phase extractionand electrochemical detectionrdquo Journal of Chromatography Avol 692 no 1-2 pp 161ndash166 1995

[16] M Dubois D Fluchard E Sior and P Delahaut ldquoIdenticationand quantication of ve macrolide antibiotics in severaltissues eggs and milk by liquid chromatography-electrospraytandem mass spectrometryrdquo Journal of Chromatography B vol753 no 2 pp 189ndash202 2001

[17] R V S Nirogi V N Kandikere M Shukla et al ldquoSensitiveand selective liquid chromatography-tandem mass spectrom-etry method for the quantication of azithromycin in humanplasmardquo Analytica Chimica Acta vol 553 no 1-2 pp 1ndash82005

[18] O A E M Farghaly and N A L Mohamed ldquoVoltammetricdetermination of azithromycin at the carbon paste electroderdquoTalanta vol 62 no 3 pp 531ndash538 2004

[19] J A Bernabeacuteu M A Camacho M E Gil-Alegre V Ruz andA I Torres-Suaacuterez ldquoMicrobiological bioassay of erythromycinthiocyanate optimisation and validationrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 21 no 2 pp 347ndash3531999

[20] H R N Salgado and A F F Roncari ldquoMicrobiological assayfor the determination of azithromycin in ophthalmic solutionsrdquoYaoxue Xuebao vol 40 no 6 pp 544ndash549 2005

[21] A S Amin and M M El-Henawee ldquoColorimetric methodfor the simultaneous determination of chlorphenoxaminehydrochloride and anhydrous caffeine in pure and dosage formswith rose bengalrdquo Mikrochimica Acta vol 118 no 3-4 pp177ndash183 1995

[22] F M Abdel-Gawad ldquoSpectrophotometric determination ofoxybuprocaine hydrochloride with halouorescein derivativesrdquoFarmaco vol 50 no 3 pp 197ndash200 1995

[23] A S Amin ldquoSpectrophotometric and conductometric deter-mination of clindamycin hydrochloride in pure form andin pharmaceutical preparationsrdquo Analusis vol 23 no 8 pp415ndash417 1995

[24] SM Amer Z El-Sherif andMM Amer ldquoSpectrophotometricdetermination of isoniazid nalidixic acid and umequinethrough ternary complex-formation with Cd (II) and rosebengalrdquo Egyptian Journal of Pharmaceutical Sciences vol 35 no1ndash6 pp 627ndash642 1994

[25] H Parham and A G Fazeli ldquoExtraction-spectrophotometricdetermination of trace amounts of barium by 18-crown-6 androse bengalrdquo Analytical Sciences vol 16 no 6 pp 575ndash5772000

[26] M A El Ries ldquoSpectrophotometric and indirect determinationof lincomycin by atomic absorption spectroscopyrdquo AnalyticalLetters vol 27 no 8 pp 1517ndash1531 1994

[27] H E Abdellatef M M Ayad and E A Taha ldquoSpectrophoto-metric and atomic absorption spectrometric determination oframipril and perindopril through ternary complex formationwith eosin and Cu(II)rdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 18 no 6 pp 1021ndash1027 1999

[28] M M Ayad A A Shalaby H E Abdellatef and M M HosnyldquoSpectrophotometric and AAS determination of ramipril andenalapril through ternary complex formationrdquo Journal of Phar-maceutical and Biomedical Analysis vol 28 no 2 pp 311ndash3212002

Journal of Spectroscopy 13

[29] A AMMoustafa ldquoSpectrophotometric methods for the deter-mination of lansoprazole and pantoprazole sodium sesquihy-draterdquo Journal of Pharmaceutical and Biomedical Analysis vol22 no 1 pp 45ndash58 2000

[30] M I Walash M E S Metwally M Eid and R N El-ShahenyldquoSpectrophotometric determination of risedronate in pharma-ceutical formulations via complex formation with Cu(II) ionsapplication to content uniformity testingrdquo International Journalof Biomedical Science vol 4 no 4 pp 303ndash309 2008

[31] J J Lingane Electroanalytical Chemistry Interscience NewYork NY USA 2nd edition 1958

[32] S A Tirmizi F H Wattoo M H S Wattoo S Sarwar AN Memon and A B Ghangro ldquoSpectrophotometric studyof stability constants of cimetidine-Ni(II) complex at differenttemperaturesrdquo Arabian Journal of Chemistry vol 35 no 1 pp93ndash100 2010

[33] J Inczedy Analytical Application of Complex EquilibriiaBudapest John Wiley amp Sons 1976

[34] J H Yoe and A L Jones ldquoCcrforimetric determination of ironwith disodium-12-dlhydroxybenzene-35-disuffonaterdquo Indus-trial and Engineering Chemistry vol 16 pp 111ndash115 1944

[35] International Conference on Harmonisation ICH of TechnicalRequirments for Registeration of Pharmaceuticals for HumanUse 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

6 Journal of Spectroscopy

O

O

O

OO

O O

O

O

O

O

O

O

O

O

O

O

O

O CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

OH

OH

Cu

Cu

N(CH3)2

N(CH3)2

OMe

HOHO

HO

HOHO

H3C

H3C

H3C

H3C

H3C

H3C

HO

I I

II

I I

II

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

COO

2

minus4+4

Rose bengalClarithromycin

2Cu+2

COOminus

Ominus

2 ++

OMe

2Na+

OMe

OMe

S 1 ProposalMechanism for the reaction between clarithromycin rose bengal and copper(II) chloride clarithromycin-Cu-rose bengalcomplex

copper cation forms a chelatewith the lone pair of electrons ofthe two hydroxyl groups at C11 andC12 Also copper can forma chelate with the lone pair of electrons of the tertiary aminegroup nitrogen and the oxygen of the 120573120573-glycosidic linkageSo the azithromycin-copper chelate bears an overall positivecharge of the original two metal ions that is four positivechargesis cationic complex associates with two moleculesof rose bengal anions to form the ternary complex

32 Conditions for Conductometric Method Investigationswere carried out to establish the most suitable conditionsfor the ion associates formation of the cited drugs with rosebengal to attain sharp endpoint e optimum conditionsfor performing the titration in a quantitative manner wereelucidated as described later

321 Reaction Medium Titrations in different media wereattempted to obtain the best results Preliminary experimentswere in

(i) aqueous drug solution for (I) only (prepared bydissolving 100mg drug in 2mL methanol then

completing to 100mL with bidistilled water) withaqueous reagent solution

(ii) ethanol drug solution with ethanol reagent solution(iii) drug solution with reagent solution both in ethanol-

water (50 vv) mixture(iv) methanol drug solution with methanol reagent solu-

tion (except for (II) as it is insoluble in methanol)(v) drug solution with reagent solution both in

methanol-water mixture (50 vv) (except for (II) asit is insoluble in methanol)

(vi) drug solution with reagent solution both in acetone-water (50 vv) mixture

Preliminary experiments showed that procedure in aque-ous media was the most suitable for successful results for (I)50 (vv) ethanol for (II) and 50 (vv) methanol for (III)

322 Reagentrsquos Concentration Different concentrations ofrose bengal solution were tried ranging from 2 times 10minus2 to5 times 10minus4 molar solutions e optimum concentration of thereagent was 3 times 10minus3M in titration of the studied drug to

Journal of Spectroscopy 7

80

70

60

50

40

30

20

10

00 2 4 6 8

Volume of rose bengal (mL)

Co

nd

uct

ivit

y (

S cm

minus1)

F 7 Conductometric titration curve of 15mg clarithromycinversus 3 times 10 3M rose bengal

achieve a constant and highly stable conductance readingwithin 1-2min of mixing Concentrations less than theselimits led to unstable readings and more time was neededto obtain constant conductance values

Representative titration curve is shown in (Figure 7)Two straight lines are obtained intersecting at the endpointthe rst branch ascending and the second has conductancevalues that would slightly increase aer the equivalencepoint e increase of conductance may be attributed tothe formation of ion pair in solution as a result of thecomplexation reaction Aer the end-point the titrationcurves indicate a slightly increased value of conductancedespite the excess of the reagent is may be due tofurther ionic condensation leading to species of lowermobility

e conductometric titrations of different volumes of3 times 10minus3 M rose bengal solution were performed e resultsshow anobviousmaximum in the conductance curve at drug-reagent molar ratio of 2 1 for (I) and (II) and 1 1 for (III)e reactions may be represented by (for (I))

[RB]minus2+210077131007713Eryth N (Me)210077291007729H+

⟶[RB]minus210077131007713Eryth NH(Me)2100772910077292+2

(2)

where [RB]minus2 rose bengal anion [Eryth N(Me)2] ery-thromycin thiocyanate [ErythNH(Me)2]

+2 protonated ery-thromycin thiocyanate

Erythromycin thiocyanate tertiary amine nitrogen isprotonated so attraction occurs between it and rose bengalanion forming ion-pair complex

e conductance measured before the addition of thetitrant (volume of rose bengal equals zero) is mainly due tothe hydrogen ions mobility ese hydrogen ions result fromtwo sources

(1) the active methylene group in the drug structureas in Scheme 2 which has the ability to make selfprotonation to the drug

O

CH3

lowast

S 2 lowaste active methylene group in macrolide structure

(2) the protons coming from the alcohols used in solubil-ity as these alcohols have certain acidity character socan protonate the amino group in drug structure

So there is no need for acidic buffer use for drug pro-tonation It was expected that the conductance values wouldremain constant or slightly increase aer the equivalencepoint However the conductance of the solution decreasedis may be due to interaction of the protons available inthe titration medium with the added reagent e resultsfrom the conductometic titrations are summarized in Table 4e data show that accurate results were obtained with goodrecoveries and low standard deviation values e optimumconcentration ranges for determination of the cited drugswere in the range of 1ndash15mg mLminus1 for all drugs At suchranges sharp inections (Figure 7) and stable conductancereading were obtained

33 Method Validation

331 For the Spectrophotometric Method

Linearity Under the optimumconditions described standardcalibration curves for erythromycin thiocyanate (I) clar-ithromycin (II) and azithromycin dihydrate (III) with rosebengal and copper were constructed by plotting absorbanceagainst concentration Conformity with Beerrsquos law was evi-dent in the concentration range of the nal dilution citedin Table 1 Beerrsquos law holds over the concentration rangesof 15ndash60 75ndash40 and 4ndash20 120583120583gsdotmLminus1 for (I) (II) and (III)respectively e linear regression equation for each drugwas listed in Table 1 e correlation coefficient was 09999indicating good linearity over the working concentrationrangeSensitivity e detection limit (LOD) for the proposedmethod was calculated using the following equation accord-ing to the ICH [35]

LOD = 33120590120590S (3)

where 120590120590 = the standard deviation of replicate blank responses(under the same conditions as for sample analysis) and S =the slope of the calibration curve

e limits of quantication LOQ is dened as

LOQ = 10120590120590S (4)

8 Journal of Spectroscopy

04

03

02

01

0430 500 550 600 650

(nm)

Absorbance

(a)

(b)

F 8Absorption spectra of (a) 25times 10minus2Mglucosewith 15mLCu(II) chloride 03 wv and 05mL 03 wv rose bengal (b) 5 times10minus2M sucrose with 15mL Cu(II) chloride 03 wv and 05mL03 wv rose bengal

According to the previous equations the LODs and LOQswere calculated as in Table 1 eir values conrm thesensitivity of the proposed methodAccuracy and Precision In order to determine the accuracyand precision of the proposed method solutions containingone concentration of each drug were prepared and analysedin seven replicates e relative standard deviation (RSD)as precision and percentage relative error (Er) as accuracyof the suggested method were calculated at 95 condencelevels and can be considered satisfactory e percent-age relative error was calculated according the followingequation

Er = 10077171007717found minus added

added10077331007733 times 100 (5)

e inter- and intraday precisions and accuracy resultsare shown in Table 5 e analytical results for accuracyand precision show that the proposed method has goodrepeatability and reproducibilityInterference e effect of presence of common excipi-ents such as glucose sucrose sodium chloride carboxymethyl cellulose sodium calcium carbonate andmagnesiumstearate was studied Table 6 presents the results of determi-nation of clarithromycin as a representative example usingthe proposed procedure in the presence of aforementionedingredients e tolerance ratio was dened as the concen-tration which gave an error of plusmn30 in the determinationof drugs Results indicate that the tolerance ratio of drugexcipient (M M) is 1 50 for glucose and 1 100 for sucroseWhile there were no interference from the presence ofsodium chloride carboxy methyl cellulose sodium calcium

carbonate and magnesium stearate Interference due toglucose and sucrosewas further investigated by studying theirreaction with Cu(II) chloride and rose bengal e studyrevealed that no interference could be observed at 557 nmand an interaction product was obtained at 120582120582max 498 intolerance ratios 1 1 and 1 10 for glucose and 1 1 1 101 50 for sucrose However interference starts to appear at120582120582max 557 upon using 50 times 100-fold excess of the molarconcentration of clarithromycin for glucose and sucroserespectively as shown in Figure 8

332 For the Conductometric Method In order to addressthe validity of the proposed method statistical analysis of thedata obtained from its application on the cited drugs in thepure form and in formulations was performed e resultsrevealed in Tables 5 and 6 showed that the proposed methodis satisfactorily accurate precise and reproducible over aconcentration range of 1ndash15mg Results for the determina-tion of the studied drugs using the previously mentionedmethod were compared with results from reported one [6]Table 7

34 Analytical Applications e proposed methods wereapplied to determine the studied drugs in their pharma-ceutical dosage forms Satisfactory results were obtainede recovery of each drug was calculated by comparing theconcentration obtained from the spiked mixtures with thoseof the drug e results of analysis of the commercial dosageforms and the recovery study are shown in Tables 2 3 and4 e results obtained were compared with the reportedmethod [6] No signicant differences were found betweenthe proposed methods and reported method Statisticalcomparison of the results was performed using Studentrsquos t-test and variance ratio F-test at 95 condence level (Table7)

4 Conclusions

e proposed methods are advantageous when compared tomany of the reported spectrophotometric methods in havinghigher sensitivity e data given previously reveal that theproposed methods are simple accurate and sensitive withgood precision and accuracyWith thesemethods one can dothe analysis at low cost without losing accuracy No interfer-ence from excipients was encountered Also In comparisonwith the chromatographic methods the proposed methodsare advantageous as HPLC and GLC are not available espe-cially in developing countries and they require complexand expensive equipment intensive sample preparation andpersonnel skilled in chromatographic techniques So theproposed methods can be used as alternative methods to thereported ones for the routine determination of erythromycinthiocyanate (I) clarithromycin (II) azithromycin dihydrate(III) in the pure form and in pharmaceutical formulationsdepending upon the availability of chemicals and the equip-ment

Journal of Spectroscopy 9

T2

Determination

oferythrom

ycin

thiocyanateand

clarithrom

ycin

intheirph

armaceutic

aldo

sage

form

sthroug

hcomplexation

with

Cu(II)

chlorid

eand

rose

beng

alby

spectro

photom

etric

procedurelowast

Erythrocin

tablets

Erythrocin

Pharco

suspensio

nKlarim

ixtablets

Klarim

ixsuspensio

nClaimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

151499

9993

151494

9959

75

756

10080

101005

10048

151489

9924

151504

10028

75

746

9952

101014

10144

201986

9930

202002

10008

101010

10096

125

1239

9914

225

2250

10001

252504

10017

151512

10080

151498

9984

252520

10079

302991

9971

175

1727

9870

175

1746

9979

275

2774

10086

325

3261

10033

202014

10072

202005

10024

302976

9919

353483

9953

225

2244

9973

225

2268

10080

353515

10041

404012

10030

252517

10067

275

2756

10022

375

3758

10021

425

4255

10013

275

2756

10022

303005

10016

403965

9919

302995

9984

Mean

9991

10001

10020

10023

Varia

nce

044

010

054

042

SD066

032

073

065

SE015

011

024

023

lowast Averageo

fthree

experim

ents

10 Journal of Spectroscopy

T3Determinationof

azith

romycin

dihydrateinits

pharmaceutic

aldo

sage

form

sthrou

ghcomplexationwith

Cu(II)chlorid

eand

rose

beng

alby

spectro

photom

etric

procedurelowast

Azrolid

tablets

Zithrokancapsules

Zithrokansuspensio

n

Claimed

tobe

taken120583120583g

mL

Authentic

added120583120583g

mL

Foun

dconc

120583120583gm

LRe

covery

Claimed

tobe

taken

120583120583gm

L

Authentic

added120583120583g

mL

Foun

dconc

120583120583gm

LRe

covery

Claimed

tobe

taken120583120583g

mL

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

LRe

covery

4403

10077

4401

10036

4405

10118

4401

10036

4408

10199

4405

10118

7701

10019

5497

9942

6606

10095

8800

10002

6602

10041

8802

10022

9896

9953

8797

9962

101006

10060

101003

10028

9906

10061

121199

9991

111111

10103

101000

9995

141401

10010

121197

9977

111098

9985

161589

9934

141403

10022

121202

10018

161589

9934

131299

9995

Mean

10015

10023

10043

Varia

nce

027

051

043

SD052

072

066

SE017

024

025

lowast Averageo

fthree

experim

ents

Journal of Spectroscopy 11

T 4 Application of the proposed conductometric method to the determination of erythromycin thiocyanate in dosage formslowast

Erythrocin tablets Erythrocin Pharco suspensionTaken (mg) Found (mg) Recovery Taken (mg) Found (mg) Recovery 3 305 10158 2 2 1004 4 100 4 405 101196 607 10119 7 705 100688 81 10119 8 805 1006010 10 100 12 1190 992012 1191 9921 14 1395 996614 1405 10034Mean 10050 10022SD 085 073SE 032 030RSD 084 073Variance 072 054lowastMean of three different experiments

T 5 e intraday and interday precision and accuracy data for the studied drugs using the spectrophotometric method

Intraday Interday

Drug Taken(120583120583gmLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSDbAccuracyEr c

Taken(120583120583g mLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSD bAccuracyEr c

(I) 50 5028 10056 029 056 50 5034 10068 039 068(II) 30 3033 10109 056 109 30 3017 10057 046 057(III) 16 1599 9995 019 minus005 16 1601 10007 025 007aAverage of seven determinationsbRSD percentage relative standard deviationcEr percentage relative error

T 6 Analysis of clarithromycin 28 120583120583gmLminus1 (5times 10minus4 M) by the spectrophotometric method in the presence of some common excipients

Tolerance molar ratio ( M M)lowast Glucose Sucrose Carboxy methyl cellulose sodium Sodium chlorideRecovery lowastlowast

1 1 10124 10107 10089 99821 10 10036 10124 9911 99821 50 11545 10213 10035 99471 100 10657 10746 9822 9929lowastDrug excipientlowastlowastMean of three determinations

T 7 Determination of the studied drugs by the proposed spectrophotometric and conductmetric methods compared with the reportedmethod [6]

(I) (II) (III)Spectrophotometric method

Mean plusmn RSD 10005 plusmn 054 10001 plusmn 047 9987 plusmn 050Variance 029 022 025Studentrsquos 119905119905-test 005 (217)lowast 04 (131)lowast 006 (216)lowast119865119865-test 238 (428)lowast 391 (360)lowast 268 (389)lowast

119899119899 7 9 9Conductometric method

Mean plusmn RSD 10066 plusmn 093 10065 plusmn 063 10062 plusmn 062Variance 087 040 038Studentrsquos 119905119905-test 131 (217)lowast 113 (217)lowast 210 (2228)lowast119865119865-test 126 (387)lowast 215 (397)lowast 176 (805)lowast

119899119899 7 6 6Reported method [6]

Mean plusmn RSD 10004 plusmn 083 10015 plusmn 093 9989 plusmn 082Variance 069 086 067Studentrsquos 119905119905-test mdash mdash mdash119865119865-test mdash mdash mdash119899119899 7 8 6lowaste gures in parenthesis are the theoretical values for 119905119905 and 119865119865-tests (119875119875 119875 005)

12 Journal of Spectroscopy

Acknowledgments

e authors wish to express their great thanks to Dr SoadSelim Abd El Hay and Dr Omnia Ahmed Emam Ismaiel atthe Analytical Chemistry Department Zagazig Universityfor their great help and advices during this work Also theywould like to point out that the authors do not have anyconict of interests with any company or institution

References

[1] S C Sweetman Martindale the Complete Drug ReferencePharmaceutical Press London UK 35th edition 2007

[2] S OmuraMacrolide Antibiotics Academic Press London UK1984

[3] J N Delgado and W A RemersWilson and Gisvoldrsquos Textbookof Organic Medecinal and Pharmaceutical Chemistry J BLippincott Company London UK 1998

[4] H A Krisr Progress inMedicinal Chemistry vol 30 of Edited byG P Ellis and D K Luscombe 1993

[5] British Pharmacopoeia Her Majestyrsquos Stationery Office Lon-don UK 2007

[6] M I Walash M S Rizk M I Eid and M E Fathy ldquoSpec-trophotometric determination of four macrolide antibiotics inpharmaceutical formulations and biological uids via binarycomplex formation with eosin and spectrophotometryrdquo Journalof AOAC International vol 90 no 6 pp 1579ndash1587 2007

[7] J Shah M R Jan and S Manzoor ldquoExtractive spectrophoto-metric methods for determination of clarithromycin in phar-maceutical formulations using bromothymol blue and cresolredrdquo Journal of the Chinese Chemical Society vol 55 no 5 pp1107ndash1112 2008

[8] C E R De Paula V G K Almeida and R J CassellaldquoNovel spectrophotometric method for the determination ofazithromycin in pharmaceutical formulations based on itscharge transfer reaction with quinalizarinrdquo Journal of theBrazilian Chemical Society vol 21 no 9 pp 1664ndash1671 2010

[9] S Ashour and R Bayram ldquoNovel spectrophotometric methodfor determination of somemacrolide antibiotics in pharmaceu-tical formulations using 1 2-naphthoquinone-4-sulphonaterdquoSpectrochim Acta Part A vol 99 pp 74ndash80 2012

[10] S D Magar A P Tupe P Y Pawar and B Y ManeldquoSimultaneous spectrophotometric estimation of ceximeand azithrhomycin in tablet dosage formrdquo Current PharmaResearch vol 2 no 3 pp 535ndash538 2012

[11] P Y Khashaba ldquoSpectrouorimetric analysis of certain mac-rolide antibiotics in bulk and pharmaceutical formulationsrdquoJournal of Pharmaceutical and Biomedical Analysis vol 27 no6 pp 923ndash932 2002

[12] N El-Rabbat H F Askal P Y Khashaba and N N AttialdquoA validated spectrouorometric assay for the determinationof certainmacrolide antibiotics in pharmaceutical formulationsand spiked biological uidsrdquo Journal of AOAC International vol89 no 5 pp 1276ndash1287 2006

[13] C L Flurer ldquoAnalysis of macrolide antibiotics by capillaryelectrophoresisrdquo Electrophoresis vol 17 no 2 pp 359ndash3661996

[14] A K Lalloo S C Chattaraj and I Kanfer ldquoDevelopmentof a capillary electrophoretic method for the separation ofthe macrolide antibiotics erythromycin josamycin and olean-domycinrdquo Journal of Chromatography B vol 704 no 1-2 pp333ndash341 1997

[15] M Hedenmo and B M Eriksson ldquoLiquid chromatographicdetermination of the macrolide antibiotics roxithromycin andclarithromycin in plasma by automated solid-phase extractionand electrochemical detectionrdquo Journal of Chromatography Avol 692 no 1-2 pp 161ndash166 1995

[16] M Dubois D Fluchard E Sior and P Delahaut ldquoIdenticationand quantication of ve macrolide antibiotics in severaltissues eggs and milk by liquid chromatography-electrospraytandem mass spectrometryrdquo Journal of Chromatography B vol753 no 2 pp 189ndash202 2001

[17] R V S Nirogi V N Kandikere M Shukla et al ldquoSensitiveand selective liquid chromatography-tandem mass spectrom-etry method for the quantication of azithromycin in humanplasmardquo Analytica Chimica Acta vol 553 no 1-2 pp 1ndash82005

[18] O A E M Farghaly and N A L Mohamed ldquoVoltammetricdetermination of azithromycin at the carbon paste electroderdquoTalanta vol 62 no 3 pp 531ndash538 2004

[19] J A Bernabeacuteu M A Camacho M E Gil-Alegre V Ruz andA I Torres-Suaacuterez ldquoMicrobiological bioassay of erythromycinthiocyanate optimisation and validationrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 21 no 2 pp 347ndash3531999

[20] H R N Salgado and A F F Roncari ldquoMicrobiological assayfor the determination of azithromycin in ophthalmic solutionsrdquoYaoxue Xuebao vol 40 no 6 pp 544ndash549 2005

[21] A S Amin and M M El-Henawee ldquoColorimetric methodfor the simultaneous determination of chlorphenoxaminehydrochloride and anhydrous caffeine in pure and dosage formswith rose bengalrdquo Mikrochimica Acta vol 118 no 3-4 pp177ndash183 1995

[22] F M Abdel-Gawad ldquoSpectrophotometric determination ofoxybuprocaine hydrochloride with halouorescein derivativesrdquoFarmaco vol 50 no 3 pp 197ndash200 1995

[23] A S Amin ldquoSpectrophotometric and conductometric deter-mination of clindamycin hydrochloride in pure form andin pharmaceutical preparationsrdquo Analusis vol 23 no 8 pp415ndash417 1995

[24] SM Amer Z El-Sherif andMM Amer ldquoSpectrophotometricdetermination of isoniazid nalidixic acid and umequinethrough ternary complex-formation with Cd (II) and rosebengalrdquo Egyptian Journal of Pharmaceutical Sciences vol 35 no1ndash6 pp 627ndash642 1994

[25] H Parham and A G Fazeli ldquoExtraction-spectrophotometricdetermination of trace amounts of barium by 18-crown-6 androse bengalrdquo Analytical Sciences vol 16 no 6 pp 575ndash5772000

[26] M A El Ries ldquoSpectrophotometric and indirect determinationof lincomycin by atomic absorption spectroscopyrdquo AnalyticalLetters vol 27 no 8 pp 1517ndash1531 1994

[27] H E Abdellatef M M Ayad and E A Taha ldquoSpectrophoto-metric and atomic absorption spectrometric determination oframipril and perindopril through ternary complex formationwith eosin and Cu(II)rdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 18 no 6 pp 1021ndash1027 1999

[28] M M Ayad A A Shalaby H E Abdellatef and M M HosnyldquoSpectrophotometric and AAS determination of ramipril andenalapril through ternary complex formationrdquo Journal of Phar-maceutical and Biomedical Analysis vol 28 no 2 pp 311ndash3212002

Journal of Spectroscopy 13

[29] A AMMoustafa ldquoSpectrophotometric methods for the deter-mination of lansoprazole and pantoprazole sodium sesquihy-draterdquo Journal of Pharmaceutical and Biomedical Analysis vol22 no 1 pp 45ndash58 2000

[30] M I Walash M E S Metwally M Eid and R N El-ShahenyldquoSpectrophotometric determination of risedronate in pharma-ceutical formulations via complex formation with Cu(II) ionsapplication to content uniformity testingrdquo International Journalof Biomedical Science vol 4 no 4 pp 303ndash309 2008

[31] J J Lingane Electroanalytical Chemistry Interscience NewYork NY USA 2nd edition 1958

[32] S A Tirmizi F H Wattoo M H S Wattoo S Sarwar AN Memon and A B Ghangro ldquoSpectrophotometric studyof stability constants of cimetidine-Ni(II) complex at differenttemperaturesrdquo Arabian Journal of Chemistry vol 35 no 1 pp93ndash100 2010

[33] J Inczedy Analytical Application of Complex EquilibriiaBudapest John Wiley amp Sons 1976

[34] J H Yoe and A L Jones ldquoCcrforimetric determination of ironwith disodium-12-dlhydroxybenzene-35-disuffonaterdquo Indus-trial and Engineering Chemistry vol 16 pp 111ndash115 1944

[35] International Conference on Harmonisation ICH of TechnicalRequirments for Registeration of Pharmaceuticals for HumanUse 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Spectroscopy 7

80

70

60

50

40

30

20

10

00 2 4 6 8

Volume of rose bengal (mL)

Co

nd

uct

ivit

y (

S cm

minus1)

F 7 Conductometric titration curve of 15mg clarithromycinversus 3 times 10 3M rose bengal

achieve a constant and highly stable conductance readingwithin 1-2min of mixing Concentrations less than theselimits led to unstable readings and more time was neededto obtain constant conductance values

Representative titration curve is shown in (Figure 7)Two straight lines are obtained intersecting at the endpointthe rst branch ascending and the second has conductancevalues that would slightly increase aer the equivalencepoint e increase of conductance may be attributed tothe formation of ion pair in solution as a result of thecomplexation reaction Aer the end-point the titrationcurves indicate a slightly increased value of conductancedespite the excess of the reagent is may be due tofurther ionic condensation leading to species of lowermobility

e conductometric titrations of different volumes of3 times 10minus3 M rose bengal solution were performed e resultsshow anobviousmaximum in the conductance curve at drug-reagent molar ratio of 2 1 for (I) and (II) and 1 1 for (III)e reactions may be represented by (for (I))

[RB]minus2+210077131007713Eryth N (Me)210077291007729H+

⟶[RB]minus210077131007713Eryth NH(Me)2100772910077292+2

(2)

where [RB]minus2 rose bengal anion [Eryth N(Me)2] ery-thromycin thiocyanate [ErythNH(Me)2]

+2 protonated ery-thromycin thiocyanate

Erythromycin thiocyanate tertiary amine nitrogen isprotonated so attraction occurs between it and rose bengalanion forming ion-pair complex

e conductance measured before the addition of thetitrant (volume of rose bengal equals zero) is mainly due tothe hydrogen ions mobility ese hydrogen ions result fromtwo sources

(1) the active methylene group in the drug structureas in Scheme 2 which has the ability to make selfprotonation to the drug

O

CH3

lowast

S 2 lowaste active methylene group in macrolide structure

(2) the protons coming from the alcohols used in solubil-ity as these alcohols have certain acidity character socan protonate the amino group in drug structure

So there is no need for acidic buffer use for drug pro-tonation It was expected that the conductance values wouldremain constant or slightly increase aer the equivalencepoint However the conductance of the solution decreasedis may be due to interaction of the protons available inthe titration medium with the added reagent e resultsfrom the conductometic titrations are summarized in Table 4e data show that accurate results were obtained with goodrecoveries and low standard deviation values e optimumconcentration ranges for determination of the cited drugswere in the range of 1ndash15mg mLminus1 for all drugs At suchranges sharp inections (Figure 7) and stable conductancereading were obtained

33 Method Validation

331 For the Spectrophotometric Method

Linearity Under the optimumconditions described standardcalibration curves for erythromycin thiocyanate (I) clar-ithromycin (II) and azithromycin dihydrate (III) with rosebengal and copper were constructed by plotting absorbanceagainst concentration Conformity with Beerrsquos law was evi-dent in the concentration range of the nal dilution citedin Table 1 Beerrsquos law holds over the concentration rangesof 15ndash60 75ndash40 and 4ndash20 120583120583gsdotmLminus1 for (I) (II) and (III)respectively e linear regression equation for each drugwas listed in Table 1 e correlation coefficient was 09999indicating good linearity over the working concentrationrangeSensitivity e detection limit (LOD) for the proposedmethod was calculated using the following equation accord-ing to the ICH [35]

LOD = 33120590120590S (3)

where 120590120590 = the standard deviation of replicate blank responses(under the same conditions as for sample analysis) and S =the slope of the calibration curve

e limits of quantication LOQ is dened as

LOQ = 10120590120590S (4)

8 Journal of Spectroscopy

04

03

02

01

0430 500 550 600 650

(nm)

Absorbance

(a)

(b)

F 8Absorption spectra of (a) 25times 10minus2Mglucosewith 15mLCu(II) chloride 03 wv and 05mL 03 wv rose bengal (b) 5 times10minus2M sucrose with 15mL Cu(II) chloride 03 wv and 05mL03 wv rose bengal

According to the previous equations the LODs and LOQswere calculated as in Table 1 eir values conrm thesensitivity of the proposed methodAccuracy and Precision In order to determine the accuracyand precision of the proposed method solutions containingone concentration of each drug were prepared and analysedin seven replicates e relative standard deviation (RSD)as precision and percentage relative error (Er) as accuracyof the suggested method were calculated at 95 condencelevels and can be considered satisfactory e percent-age relative error was calculated according the followingequation

Er = 10077171007717found minus added

added10077331007733 times 100 (5)

e inter- and intraday precisions and accuracy resultsare shown in Table 5 e analytical results for accuracyand precision show that the proposed method has goodrepeatability and reproducibilityInterference e effect of presence of common excipi-ents such as glucose sucrose sodium chloride carboxymethyl cellulose sodium calcium carbonate andmagnesiumstearate was studied Table 6 presents the results of determi-nation of clarithromycin as a representative example usingthe proposed procedure in the presence of aforementionedingredients e tolerance ratio was dened as the concen-tration which gave an error of plusmn30 in the determinationof drugs Results indicate that the tolerance ratio of drugexcipient (M M) is 1 50 for glucose and 1 100 for sucroseWhile there were no interference from the presence ofsodium chloride carboxy methyl cellulose sodium calcium

carbonate and magnesium stearate Interference due toglucose and sucrosewas further investigated by studying theirreaction with Cu(II) chloride and rose bengal e studyrevealed that no interference could be observed at 557 nmand an interaction product was obtained at 120582120582max 498 intolerance ratios 1 1 and 1 10 for glucose and 1 1 1 101 50 for sucrose However interference starts to appear at120582120582max 557 upon using 50 times 100-fold excess of the molarconcentration of clarithromycin for glucose and sucroserespectively as shown in Figure 8

332 For the Conductometric Method In order to addressthe validity of the proposed method statistical analysis of thedata obtained from its application on the cited drugs in thepure form and in formulations was performed e resultsrevealed in Tables 5 and 6 showed that the proposed methodis satisfactorily accurate precise and reproducible over aconcentration range of 1ndash15mg Results for the determina-tion of the studied drugs using the previously mentionedmethod were compared with results from reported one [6]Table 7

34 Analytical Applications e proposed methods wereapplied to determine the studied drugs in their pharma-ceutical dosage forms Satisfactory results were obtainede recovery of each drug was calculated by comparing theconcentration obtained from the spiked mixtures with thoseof the drug e results of analysis of the commercial dosageforms and the recovery study are shown in Tables 2 3 and4 e results obtained were compared with the reportedmethod [6] No signicant differences were found betweenthe proposed methods and reported method Statisticalcomparison of the results was performed using Studentrsquos t-test and variance ratio F-test at 95 condence level (Table7)

4 Conclusions

e proposed methods are advantageous when compared tomany of the reported spectrophotometric methods in havinghigher sensitivity e data given previously reveal that theproposed methods are simple accurate and sensitive withgood precision and accuracyWith thesemethods one can dothe analysis at low cost without losing accuracy No interfer-ence from excipients was encountered Also In comparisonwith the chromatographic methods the proposed methodsare advantageous as HPLC and GLC are not available espe-cially in developing countries and they require complexand expensive equipment intensive sample preparation andpersonnel skilled in chromatographic techniques So theproposed methods can be used as alternative methods to thereported ones for the routine determination of erythromycinthiocyanate (I) clarithromycin (II) azithromycin dihydrate(III) in the pure form and in pharmaceutical formulationsdepending upon the availability of chemicals and the equip-ment

Journal of Spectroscopy 9

T2

Determination

oferythrom

ycin

thiocyanateand

clarithrom

ycin

intheirph

armaceutic

aldo

sage

form

sthroug

hcomplexation

with

Cu(II)

chlorid

eand

rose

beng

alby

spectro

photom

etric

procedurelowast

Erythrocin

tablets

Erythrocin

Pharco

suspensio

nKlarim

ixtablets

Klarim

ixsuspensio

nClaimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

151499

9993

151494

9959

75

756

10080

101005

10048

151489

9924

151504

10028

75

746

9952

101014

10144

201986

9930

202002

10008

101010

10096

125

1239

9914

225

2250

10001

252504

10017

151512

10080

151498

9984

252520

10079

302991

9971

175

1727

9870

175

1746

9979

275

2774

10086

325

3261

10033

202014

10072

202005

10024

302976

9919

353483

9953

225

2244

9973

225

2268

10080

353515

10041

404012

10030

252517

10067

275

2756

10022

375

3758

10021

425

4255

10013

275

2756

10022

303005

10016

403965

9919

302995

9984

Mean

9991

10001

10020

10023

Varia

nce

044

010

054

042

SD066

032

073

065

SE015

011

024

023

lowast Averageo

fthree

experim

ents

10 Journal of Spectroscopy

T3Determinationof

azith

romycin

dihydrateinits

pharmaceutic

aldo

sage

form

sthrou

ghcomplexationwith

Cu(II)chlorid

eand

rose

beng

alby

spectro

photom

etric

procedurelowast

Azrolid

tablets

Zithrokancapsules

Zithrokansuspensio

n

Claimed

tobe

taken120583120583g

mL

Authentic

added120583120583g

mL

Foun

dconc

120583120583gm

LRe

covery

Claimed

tobe

taken

120583120583gm

L

Authentic

added120583120583g

mL

Foun

dconc

120583120583gm

LRe

covery

Claimed

tobe

taken120583120583g

mL

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

LRe

covery

4403

10077

4401

10036

4405

10118

4401

10036

4408

10199

4405

10118

7701

10019

5497

9942

6606

10095

8800

10002

6602

10041

8802

10022

9896

9953

8797

9962

101006

10060

101003

10028

9906

10061

121199

9991

111111

10103

101000

9995

141401

10010

121197

9977

111098

9985

161589

9934

141403

10022

121202

10018

161589

9934

131299

9995

Mean

10015

10023

10043

Varia

nce

027

051

043

SD052

072

066

SE017

024

025

lowast Averageo

fthree

experim

ents

Journal of Spectroscopy 11

T 4 Application of the proposed conductometric method to the determination of erythromycin thiocyanate in dosage formslowast

Erythrocin tablets Erythrocin Pharco suspensionTaken (mg) Found (mg) Recovery Taken (mg) Found (mg) Recovery 3 305 10158 2 2 1004 4 100 4 405 101196 607 10119 7 705 100688 81 10119 8 805 1006010 10 100 12 1190 992012 1191 9921 14 1395 996614 1405 10034Mean 10050 10022SD 085 073SE 032 030RSD 084 073Variance 072 054lowastMean of three different experiments

T 5 e intraday and interday precision and accuracy data for the studied drugs using the spectrophotometric method

Intraday Interday

Drug Taken(120583120583gmLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSDbAccuracyEr c

Taken(120583120583g mLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSD bAccuracyEr c

(I) 50 5028 10056 029 056 50 5034 10068 039 068(II) 30 3033 10109 056 109 30 3017 10057 046 057(III) 16 1599 9995 019 minus005 16 1601 10007 025 007aAverage of seven determinationsbRSD percentage relative standard deviationcEr percentage relative error

T 6 Analysis of clarithromycin 28 120583120583gmLminus1 (5times 10minus4 M) by the spectrophotometric method in the presence of some common excipients

Tolerance molar ratio ( M M)lowast Glucose Sucrose Carboxy methyl cellulose sodium Sodium chlorideRecovery lowastlowast

1 1 10124 10107 10089 99821 10 10036 10124 9911 99821 50 11545 10213 10035 99471 100 10657 10746 9822 9929lowastDrug excipientlowastlowastMean of three determinations

T 7 Determination of the studied drugs by the proposed spectrophotometric and conductmetric methods compared with the reportedmethod [6]

(I) (II) (III)Spectrophotometric method

Mean plusmn RSD 10005 plusmn 054 10001 plusmn 047 9987 plusmn 050Variance 029 022 025Studentrsquos 119905119905-test 005 (217)lowast 04 (131)lowast 006 (216)lowast119865119865-test 238 (428)lowast 391 (360)lowast 268 (389)lowast

119899119899 7 9 9Conductometric method

Mean plusmn RSD 10066 plusmn 093 10065 plusmn 063 10062 plusmn 062Variance 087 040 038Studentrsquos 119905119905-test 131 (217)lowast 113 (217)lowast 210 (2228)lowast119865119865-test 126 (387)lowast 215 (397)lowast 176 (805)lowast

119899119899 7 6 6Reported method [6]

Mean plusmn RSD 10004 plusmn 083 10015 plusmn 093 9989 plusmn 082Variance 069 086 067Studentrsquos 119905119905-test mdash mdash mdash119865119865-test mdash mdash mdash119899119899 7 8 6lowaste gures in parenthesis are the theoretical values for 119905119905 and 119865119865-tests (119875119875 119875 005)

12 Journal of Spectroscopy

Acknowledgments

e authors wish to express their great thanks to Dr SoadSelim Abd El Hay and Dr Omnia Ahmed Emam Ismaiel atthe Analytical Chemistry Department Zagazig Universityfor their great help and advices during this work Also theywould like to point out that the authors do not have anyconict of interests with any company or institution

References

[1] S C Sweetman Martindale the Complete Drug ReferencePharmaceutical Press London UK 35th edition 2007

[2] S OmuraMacrolide Antibiotics Academic Press London UK1984

[3] J N Delgado and W A RemersWilson and Gisvoldrsquos Textbookof Organic Medecinal and Pharmaceutical Chemistry J BLippincott Company London UK 1998

[4] H A Krisr Progress inMedicinal Chemistry vol 30 of Edited byG P Ellis and D K Luscombe 1993

[5] British Pharmacopoeia Her Majestyrsquos Stationery Office Lon-don UK 2007

[6] M I Walash M S Rizk M I Eid and M E Fathy ldquoSpec-trophotometric determination of four macrolide antibiotics inpharmaceutical formulations and biological uids via binarycomplex formation with eosin and spectrophotometryrdquo Journalof AOAC International vol 90 no 6 pp 1579ndash1587 2007

[7] J Shah M R Jan and S Manzoor ldquoExtractive spectrophoto-metric methods for determination of clarithromycin in phar-maceutical formulations using bromothymol blue and cresolredrdquo Journal of the Chinese Chemical Society vol 55 no 5 pp1107ndash1112 2008

[8] C E R De Paula V G K Almeida and R J CassellaldquoNovel spectrophotometric method for the determination ofazithromycin in pharmaceutical formulations based on itscharge transfer reaction with quinalizarinrdquo Journal of theBrazilian Chemical Society vol 21 no 9 pp 1664ndash1671 2010

[9] S Ashour and R Bayram ldquoNovel spectrophotometric methodfor determination of somemacrolide antibiotics in pharmaceu-tical formulations using 1 2-naphthoquinone-4-sulphonaterdquoSpectrochim Acta Part A vol 99 pp 74ndash80 2012

[10] S D Magar A P Tupe P Y Pawar and B Y ManeldquoSimultaneous spectrophotometric estimation of ceximeand azithrhomycin in tablet dosage formrdquo Current PharmaResearch vol 2 no 3 pp 535ndash538 2012

[11] P Y Khashaba ldquoSpectrouorimetric analysis of certain mac-rolide antibiotics in bulk and pharmaceutical formulationsrdquoJournal of Pharmaceutical and Biomedical Analysis vol 27 no6 pp 923ndash932 2002

[12] N El-Rabbat H F Askal P Y Khashaba and N N AttialdquoA validated spectrouorometric assay for the determinationof certainmacrolide antibiotics in pharmaceutical formulationsand spiked biological uidsrdquo Journal of AOAC International vol89 no 5 pp 1276ndash1287 2006

[13] C L Flurer ldquoAnalysis of macrolide antibiotics by capillaryelectrophoresisrdquo Electrophoresis vol 17 no 2 pp 359ndash3661996

[14] A K Lalloo S C Chattaraj and I Kanfer ldquoDevelopmentof a capillary electrophoretic method for the separation ofthe macrolide antibiotics erythromycin josamycin and olean-domycinrdquo Journal of Chromatography B vol 704 no 1-2 pp333ndash341 1997

[15] M Hedenmo and B M Eriksson ldquoLiquid chromatographicdetermination of the macrolide antibiotics roxithromycin andclarithromycin in plasma by automated solid-phase extractionand electrochemical detectionrdquo Journal of Chromatography Avol 692 no 1-2 pp 161ndash166 1995

[16] M Dubois D Fluchard E Sior and P Delahaut ldquoIdenticationand quantication of ve macrolide antibiotics in severaltissues eggs and milk by liquid chromatography-electrospraytandem mass spectrometryrdquo Journal of Chromatography B vol753 no 2 pp 189ndash202 2001

[17] R V S Nirogi V N Kandikere M Shukla et al ldquoSensitiveand selective liquid chromatography-tandem mass spectrom-etry method for the quantication of azithromycin in humanplasmardquo Analytica Chimica Acta vol 553 no 1-2 pp 1ndash82005

[18] O A E M Farghaly and N A L Mohamed ldquoVoltammetricdetermination of azithromycin at the carbon paste electroderdquoTalanta vol 62 no 3 pp 531ndash538 2004

[19] J A Bernabeacuteu M A Camacho M E Gil-Alegre V Ruz andA I Torres-Suaacuterez ldquoMicrobiological bioassay of erythromycinthiocyanate optimisation and validationrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 21 no 2 pp 347ndash3531999

[20] H R N Salgado and A F F Roncari ldquoMicrobiological assayfor the determination of azithromycin in ophthalmic solutionsrdquoYaoxue Xuebao vol 40 no 6 pp 544ndash549 2005

[21] A S Amin and M M El-Henawee ldquoColorimetric methodfor the simultaneous determination of chlorphenoxaminehydrochloride and anhydrous caffeine in pure and dosage formswith rose bengalrdquo Mikrochimica Acta vol 118 no 3-4 pp177ndash183 1995

[22] F M Abdel-Gawad ldquoSpectrophotometric determination ofoxybuprocaine hydrochloride with halouorescein derivativesrdquoFarmaco vol 50 no 3 pp 197ndash200 1995

[23] A S Amin ldquoSpectrophotometric and conductometric deter-mination of clindamycin hydrochloride in pure form andin pharmaceutical preparationsrdquo Analusis vol 23 no 8 pp415ndash417 1995

[24] SM Amer Z El-Sherif andMM Amer ldquoSpectrophotometricdetermination of isoniazid nalidixic acid and umequinethrough ternary complex-formation with Cd (II) and rosebengalrdquo Egyptian Journal of Pharmaceutical Sciences vol 35 no1ndash6 pp 627ndash642 1994

[25] H Parham and A G Fazeli ldquoExtraction-spectrophotometricdetermination of trace amounts of barium by 18-crown-6 androse bengalrdquo Analytical Sciences vol 16 no 6 pp 575ndash5772000

[26] M A El Ries ldquoSpectrophotometric and indirect determinationof lincomycin by atomic absorption spectroscopyrdquo AnalyticalLetters vol 27 no 8 pp 1517ndash1531 1994

[27] H E Abdellatef M M Ayad and E A Taha ldquoSpectrophoto-metric and atomic absorption spectrometric determination oframipril and perindopril through ternary complex formationwith eosin and Cu(II)rdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 18 no 6 pp 1021ndash1027 1999

[28] M M Ayad A A Shalaby H E Abdellatef and M M HosnyldquoSpectrophotometric and AAS determination of ramipril andenalapril through ternary complex formationrdquo Journal of Phar-maceutical and Biomedical Analysis vol 28 no 2 pp 311ndash3212002

Journal of Spectroscopy 13

[29] A AMMoustafa ldquoSpectrophotometric methods for the deter-mination of lansoprazole and pantoprazole sodium sesquihy-draterdquo Journal of Pharmaceutical and Biomedical Analysis vol22 no 1 pp 45ndash58 2000

[30] M I Walash M E S Metwally M Eid and R N El-ShahenyldquoSpectrophotometric determination of risedronate in pharma-ceutical formulations via complex formation with Cu(II) ionsapplication to content uniformity testingrdquo International Journalof Biomedical Science vol 4 no 4 pp 303ndash309 2008

[31] J J Lingane Electroanalytical Chemistry Interscience NewYork NY USA 2nd edition 1958

[32] S A Tirmizi F H Wattoo M H S Wattoo S Sarwar AN Memon and A B Ghangro ldquoSpectrophotometric studyof stability constants of cimetidine-Ni(II) complex at differenttemperaturesrdquo Arabian Journal of Chemistry vol 35 no 1 pp93ndash100 2010

[33] J Inczedy Analytical Application of Complex EquilibriiaBudapest John Wiley amp Sons 1976

[34] J H Yoe and A L Jones ldquoCcrforimetric determination of ironwith disodium-12-dlhydroxybenzene-35-disuffonaterdquo Indus-trial and Engineering Chemistry vol 16 pp 111ndash115 1944

[35] International Conference on Harmonisation ICH of TechnicalRequirments for Registeration of Pharmaceuticals for HumanUse 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

8 Journal of Spectroscopy

04

03

02

01

0430 500 550 600 650

(nm)

Absorbance

(a)

(b)

F 8Absorption spectra of (a) 25times 10minus2Mglucosewith 15mLCu(II) chloride 03 wv and 05mL 03 wv rose bengal (b) 5 times10minus2M sucrose with 15mL Cu(II) chloride 03 wv and 05mL03 wv rose bengal

According to the previous equations the LODs and LOQswere calculated as in Table 1 eir values conrm thesensitivity of the proposed methodAccuracy and Precision In order to determine the accuracyand precision of the proposed method solutions containingone concentration of each drug were prepared and analysedin seven replicates e relative standard deviation (RSD)as precision and percentage relative error (Er) as accuracyof the suggested method were calculated at 95 condencelevels and can be considered satisfactory e percent-age relative error was calculated according the followingequation

Er = 10077171007717found minus added

added10077331007733 times 100 (5)

e inter- and intraday precisions and accuracy resultsare shown in Table 5 e analytical results for accuracyand precision show that the proposed method has goodrepeatability and reproducibilityInterference e effect of presence of common excipi-ents such as glucose sucrose sodium chloride carboxymethyl cellulose sodium calcium carbonate andmagnesiumstearate was studied Table 6 presents the results of determi-nation of clarithromycin as a representative example usingthe proposed procedure in the presence of aforementionedingredients e tolerance ratio was dened as the concen-tration which gave an error of plusmn30 in the determinationof drugs Results indicate that the tolerance ratio of drugexcipient (M M) is 1 50 for glucose and 1 100 for sucroseWhile there were no interference from the presence ofsodium chloride carboxy methyl cellulose sodium calcium

carbonate and magnesium stearate Interference due toglucose and sucrosewas further investigated by studying theirreaction with Cu(II) chloride and rose bengal e studyrevealed that no interference could be observed at 557 nmand an interaction product was obtained at 120582120582max 498 intolerance ratios 1 1 and 1 10 for glucose and 1 1 1 101 50 for sucrose However interference starts to appear at120582120582max 557 upon using 50 times 100-fold excess of the molarconcentration of clarithromycin for glucose and sucroserespectively as shown in Figure 8

332 For the Conductometric Method In order to addressthe validity of the proposed method statistical analysis of thedata obtained from its application on the cited drugs in thepure form and in formulations was performed e resultsrevealed in Tables 5 and 6 showed that the proposed methodis satisfactorily accurate precise and reproducible over aconcentration range of 1ndash15mg Results for the determina-tion of the studied drugs using the previously mentionedmethod were compared with results from reported one [6]Table 7

34 Analytical Applications e proposed methods wereapplied to determine the studied drugs in their pharma-ceutical dosage forms Satisfactory results were obtainede recovery of each drug was calculated by comparing theconcentration obtained from the spiked mixtures with thoseof the drug e results of analysis of the commercial dosageforms and the recovery study are shown in Tables 2 3 and4 e results obtained were compared with the reportedmethod [6] No signicant differences were found betweenthe proposed methods and reported method Statisticalcomparison of the results was performed using Studentrsquos t-test and variance ratio F-test at 95 condence level (Table7)

4 Conclusions

e proposed methods are advantageous when compared tomany of the reported spectrophotometric methods in havinghigher sensitivity e data given previously reveal that theproposed methods are simple accurate and sensitive withgood precision and accuracyWith thesemethods one can dothe analysis at low cost without losing accuracy No interfer-ence from excipients was encountered Also In comparisonwith the chromatographic methods the proposed methodsare advantageous as HPLC and GLC are not available espe-cially in developing countries and they require complexand expensive equipment intensive sample preparation andpersonnel skilled in chromatographic techniques So theproposed methods can be used as alternative methods to thereported ones for the routine determination of erythromycinthiocyanate (I) clarithromycin (II) azithromycin dihydrate(III) in the pure form and in pharmaceutical formulationsdepending upon the availability of chemicals and the equip-ment

Journal of Spectroscopy 9

T2

Determination

oferythrom

ycin

thiocyanateand

clarithrom

ycin

intheirph

armaceutic

aldo

sage

form

sthroug

hcomplexation

with

Cu(II)

chlorid

eand

rose

beng

alby

spectro

photom

etric

procedurelowast

Erythrocin

tablets

Erythrocin

Pharco

suspensio

nKlarim

ixtablets

Klarim

ixsuspensio

nClaimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

151499

9993

151494

9959

75

756

10080

101005

10048

151489

9924

151504

10028

75

746

9952

101014

10144

201986

9930

202002

10008

101010

10096

125

1239

9914

225

2250

10001

252504

10017

151512

10080

151498

9984

252520

10079

302991

9971

175

1727

9870

175

1746

9979

275

2774

10086

325

3261

10033

202014

10072

202005

10024

302976

9919

353483

9953

225

2244

9973

225

2268

10080

353515

10041

404012

10030

252517

10067

275

2756

10022

375

3758

10021

425

4255

10013

275

2756

10022

303005

10016

403965

9919

302995

9984

Mean

9991

10001

10020

10023

Varia

nce

044

010

054

042

SD066

032

073

065

SE015

011

024

023

lowast Averageo

fthree

experim

ents

10 Journal of Spectroscopy

T3Determinationof

azith

romycin

dihydrateinits

pharmaceutic

aldo

sage

form

sthrou

ghcomplexationwith

Cu(II)chlorid

eand

rose

beng

alby

spectro

photom

etric

procedurelowast

Azrolid

tablets

Zithrokancapsules

Zithrokansuspensio

n

Claimed

tobe

taken120583120583g

mL

Authentic

added120583120583g

mL

Foun

dconc

120583120583gm

LRe

covery

Claimed

tobe

taken

120583120583gm

L

Authentic

added120583120583g

mL

Foun

dconc

120583120583gm

LRe

covery

Claimed

tobe

taken120583120583g

mL

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

LRe

covery

4403

10077

4401

10036

4405

10118

4401

10036

4408

10199

4405

10118

7701

10019

5497

9942

6606

10095

8800

10002

6602

10041

8802

10022

9896

9953

8797

9962

101006

10060

101003

10028

9906

10061

121199

9991

111111

10103

101000

9995

141401

10010

121197

9977

111098

9985

161589

9934

141403

10022

121202

10018

161589

9934

131299

9995

Mean

10015

10023

10043

Varia

nce

027

051

043

SD052

072

066

SE017

024

025

lowast Averageo

fthree

experim

ents

Journal of Spectroscopy 11

T 4 Application of the proposed conductometric method to the determination of erythromycin thiocyanate in dosage formslowast

Erythrocin tablets Erythrocin Pharco suspensionTaken (mg) Found (mg) Recovery Taken (mg) Found (mg) Recovery 3 305 10158 2 2 1004 4 100 4 405 101196 607 10119 7 705 100688 81 10119 8 805 1006010 10 100 12 1190 992012 1191 9921 14 1395 996614 1405 10034Mean 10050 10022SD 085 073SE 032 030RSD 084 073Variance 072 054lowastMean of three different experiments

T 5 e intraday and interday precision and accuracy data for the studied drugs using the spectrophotometric method

Intraday Interday

Drug Taken(120583120583gmLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSDbAccuracyEr c

Taken(120583120583g mLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSD bAccuracyEr c

(I) 50 5028 10056 029 056 50 5034 10068 039 068(II) 30 3033 10109 056 109 30 3017 10057 046 057(III) 16 1599 9995 019 minus005 16 1601 10007 025 007aAverage of seven determinationsbRSD percentage relative standard deviationcEr percentage relative error

T 6 Analysis of clarithromycin 28 120583120583gmLminus1 (5times 10minus4 M) by the spectrophotometric method in the presence of some common excipients

Tolerance molar ratio ( M M)lowast Glucose Sucrose Carboxy methyl cellulose sodium Sodium chlorideRecovery lowastlowast

1 1 10124 10107 10089 99821 10 10036 10124 9911 99821 50 11545 10213 10035 99471 100 10657 10746 9822 9929lowastDrug excipientlowastlowastMean of three determinations

T 7 Determination of the studied drugs by the proposed spectrophotometric and conductmetric methods compared with the reportedmethod [6]

(I) (II) (III)Spectrophotometric method

Mean plusmn RSD 10005 plusmn 054 10001 plusmn 047 9987 plusmn 050Variance 029 022 025Studentrsquos 119905119905-test 005 (217)lowast 04 (131)lowast 006 (216)lowast119865119865-test 238 (428)lowast 391 (360)lowast 268 (389)lowast

119899119899 7 9 9Conductometric method

Mean plusmn RSD 10066 plusmn 093 10065 plusmn 063 10062 plusmn 062Variance 087 040 038Studentrsquos 119905119905-test 131 (217)lowast 113 (217)lowast 210 (2228)lowast119865119865-test 126 (387)lowast 215 (397)lowast 176 (805)lowast

119899119899 7 6 6Reported method [6]

Mean plusmn RSD 10004 plusmn 083 10015 plusmn 093 9989 plusmn 082Variance 069 086 067Studentrsquos 119905119905-test mdash mdash mdash119865119865-test mdash mdash mdash119899119899 7 8 6lowaste gures in parenthesis are the theoretical values for 119905119905 and 119865119865-tests (119875119875 119875 005)

12 Journal of Spectroscopy

Acknowledgments

e authors wish to express their great thanks to Dr SoadSelim Abd El Hay and Dr Omnia Ahmed Emam Ismaiel atthe Analytical Chemistry Department Zagazig Universityfor their great help and advices during this work Also theywould like to point out that the authors do not have anyconict of interests with any company or institution

References

[1] S C Sweetman Martindale the Complete Drug ReferencePharmaceutical Press London UK 35th edition 2007

[2] S OmuraMacrolide Antibiotics Academic Press London UK1984

[3] J N Delgado and W A RemersWilson and Gisvoldrsquos Textbookof Organic Medecinal and Pharmaceutical Chemistry J BLippincott Company London UK 1998

[4] H A Krisr Progress inMedicinal Chemistry vol 30 of Edited byG P Ellis and D K Luscombe 1993

[5] British Pharmacopoeia Her Majestyrsquos Stationery Office Lon-don UK 2007

[6] M I Walash M S Rizk M I Eid and M E Fathy ldquoSpec-trophotometric determination of four macrolide antibiotics inpharmaceutical formulations and biological uids via binarycomplex formation with eosin and spectrophotometryrdquo Journalof AOAC International vol 90 no 6 pp 1579ndash1587 2007

[7] J Shah M R Jan and S Manzoor ldquoExtractive spectrophoto-metric methods for determination of clarithromycin in phar-maceutical formulations using bromothymol blue and cresolredrdquo Journal of the Chinese Chemical Society vol 55 no 5 pp1107ndash1112 2008

[8] C E R De Paula V G K Almeida and R J CassellaldquoNovel spectrophotometric method for the determination ofazithromycin in pharmaceutical formulations based on itscharge transfer reaction with quinalizarinrdquo Journal of theBrazilian Chemical Society vol 21 no 9 pp 1664ndash1671 2010

[9] S Ashour and R Bayram ldquoNovel spectrophotometric methodfor determination of somemacrolide antibiotics in pharmaceu-tical formulations using 1 2-naphthoquinone-4-sulphonaterdquoSpectrochim Acta Part A vol 99 pp 74ndash80 2012

[10] S D Magar A P Tupe P Y Pawar and B Y ManeldquoSimultaneous spectrophotometric estimation of ceximeand azithrhomycin in tablet dosage formrdquo Current PharmaResearch vol 2 no 3 pp 535ndash538 2012

[11] P Y Khashaba ldquoSpectrouorimetric analysis of certain mac-rolide antibiotics in bulk and pharmaceutical formulationsrdquoJournal of Pharmaceutical and Biomedical Analysis vol 27 no6 pp 923ndash932 2002

[12] N El-Rabbat H F Askal P Y Khashaba and N N AttialdquoA validated spectrouorometric assay for the determinationof certainmacrolide antibiotics in pharmaceutical formulationsand spiked biological uidsrdquo Journal of AOAC International vol89 no 5 pp 1276ndash1287 2006

[13] C L Flurer ldquoAnalysis of macrolide antibiotics by capillaryelectrophoresisrdquo Electrophoresis vol 17 no 2 pp 359ndash3661996

[14] A K Lalloo S C Chattaraj and I Kanfer ldquoDevelopmentof a capillary electrophoretic method for the separation ofthe macrolide antibiotics erythromycin josamycin and olean-domycinrdquo Journal of Chromatography B vol 704 no 1-2 pp333ndash341 1997

[15] M Hedenmo and B M Eriksson ldquoLiquid chromatographicdetermination of the macrolide antibiotics roxithromycin andclarithromycin in plasma by automated solid-phase extractionand electrochemical detectionrdquo Journal of Chromatography Avol 692 no 1-2 pp 161ndash166 1995

[16] M Dubois D Fluchard E Sior and P Delahaut ldquoIdenticationand quantication of ve macrolide antibiotics in severaltissues eggs and milk by liquid chromatography-electrospraytandem mass spectrometryrdquo Journal of Chromatography B vol753 no 2 pp 189ndash202 2001

[17] R V S Nirogi V N Kandikere M Shukla et al ldquoSensitiveand selective liquid chromatography-tandem mass spectrom-etry method for the quantication of azithromycin in humanplasmardquo Analytica Chimica Acta vol 553 no 1-2 pp 1ndash82005

[18] O A E M Farghaly and N A L Mohamed ldquoVoltammetricdetermination of azithromycin at the carbon paste electroderdquoTalanta vol 62 no 3 pp 531ndash538 2004

[19] J A Bernabeacuteu M A Camacho M E Gil-Alegre V Ruz andA I Torres-Suaacuterez ldquoMicrobiological bioassay of erythromycinthiocyanate optimisation and validationrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 21 no 2 pp 347ndash3531999

[20] H R N Salgado and A F F Roncari ldquoMicrobiological assayfor the determination of azithromycin in ophthalmic solutionsrdquoYaoxue Xuebao vol 40 no 6 pp 544ndash549 2005

[21] A S Amin and M M El-Henawee ldquoColorimetric methodfor the simultaneous determination of chlorphenoxaminehydrochloride and anhydrous caffeine in pure and dosage formswith rose bengalrdquo Mikrochimica Acta vol 118 no 3-4 pp177ndash183 1995

[22] F M Abdel-Gawad ldquoSpectrophotometric determination ofoxybuprocaine hydrochloride with halouorescein derivativesrdquoFarmaco vol 50 no 3 pp 197ndash200 1995

[23] A S Amin ldquoSpectrophotometric and conductometric deter-mination of clindamycin hydrochloride in pure form andin pharmaceutical preparationsrdquo Analusis vol 23 no 8 pp415ndash417 1995

[24] SM Amer Z El-Sherif andMM Amer ldquoSpectrophotometricdetermination of isoniazid nalidixic acid and umequinethrough ternary complex-formation with Cd (II) and rosebengalrdquo Egyptian Journal of Pharmaceutical Sciences vol 35 no1ndash6 pp 627ndash642 1994

[25] H Parham and A G Fazeli ldquoExtraction-spectrophotometricdetermination of trace amounts of barium by 18-crown-6 androse bengalrdquo Analytical Sciences vol 16 no 6 pp 575ndash5772000

[26] M A El Ries ldquoSpectrophotometric and indirect determinationof lincomycin by atomic absorption spectroscopyrdquo AnalyticalLetters vol 27 no 8 pp 1517ndash1531 1994

[27] H E Abdellatef M M Ayad and E A Taha ldquoSpectrophoto-metric and atomic absorption spectrometric determination oframipril and perindopril through ternary complex formationwith eosin and Cu(II)rdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 18 no 6 pp 1021ndash1027 1999

[28] M M Ayad A A Shalaby H E Abdellatef and M M HosnyldquoSpectrophotometric and AAS determination of ramipril andenalapril through ternary complex formationrdquo Journal of Phar-maceutical and Biomedical Analysis vol 28 no 2 pp 311ndash3212002

Journal of Spectroscopy 13

[29] A AMMoustafa ldquoSpectrophotometric methods for the deter-mination of lansoprazole and pantoprazole sodium sesquihy-draterdquo Journal of Pharmaceutical and Biomedical Analysis vol22 no 1 pp 45ndash58 2000

[30] M I Walash M E S Metwally M Eid and R N El-ShahenyldquoSpectrophotometric determination of risedronate in pharma-ceutical formulations via complex formation with Cu(II) ionsapplication to content uniformity testingrdquo International Journalof Biomedical Science vol 4 no 4 pp 303ndash309 2008

[31] J J Lingane Electroanalytical Chemistry Interscience NewYork NY USA 2nd edition 1958

[32] S A Tirmizi F H Wattoo M H S Wattoo S Sarwar AN Memon and A B Ghangro ldquoSpectrophotometric studyof stability constants of cimetidine-Ni(II) complex at differenttemperaturesrdquo Arabian Journal of Chemistry vol 35 no 1 pp93ndash100 2010

[33] J Inczedy Analytical Application of Complex EquilibriiaBudapest John Wiley amp Sons 1976

[34] J H Yoe and A L Jones ldquoCcrforimetric determination of ironwith disodium-12-dlhydroxybenzene-35-disuffonaterdquo Indus-trial and Engineering Chemistry vol 16 pp 111ndash115 1944

[35] International Conference on Harmonisation ICH of TechnicalRequirments for Registeration of Pharmaceuticals for HumanUse 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Spectroscopy 9

T2

Determination

oferythrom

ycin

thiocyanateand

clarithrom

ycin

intheirph

armaceutic

aldo

sage

form

sthroug

hcomplexation

with

Cu(II)

chlorid

eand

rose

beng

alby

spectro

photom

etric

procedurelowast

Erythrocin

tablets

Erythrocin

Pharco

suspensio

nKlarim

ixtablets

Klarim

ixsuspensio

nClaimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

Claimed

tobe

taken

120583120583gm

L

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

L

Recovery

151499

9993

151494

9959

75

756

10080

101005

10048

151489

9924

151504

10028

75

746

9952

101014

10144

201986

9930

202002

10008

101010

10096

125

1239

9914

225

2250

10001

252504

10017

151512

10080

151498

9984

252520

10079

302991

9971

175

1727

9870

175

1746

9979

275

2774

10086

325

3261

10033

202014

10072

202005

10024

302976

9919

353483

9953

225

2244

9973

225

2268

10080

353515

10041

404012

10030

252517

10067

275

2756

10022

375

3758

10021

425

4255

10013

275

2756

10022

303005

10016

403965

9919

302995

9984

Mean

9991

10001

10020

10023

Varia

nce

044

010

054

042

SD066

032

073

065

SE015

011

024

023

lowast Averageo

fthree

experim

ents

10 Journal of Spectroscopy

T3Determinationof

azith

romycin

dihydrateinits

pharmaceutic

aldo

sage

form

sthrou

ghcomplexationwith

Cu(II)chlorid

eand

rose

beng

alby

spectro

photom

etric

procedurelowast

Azrolid

tablets

Zithrokancapsules

Zithrokansuspensio

n

Claimed

tobe

taken120583120583g

mL

Authentic

added120583120583g

mL

Foun

dconc

120583120583gm

LRe

covery

Claimed

tobe

taken

120583120583gm

L

Authentic

added120583120583g

mL

Foun

dconc

120583120583gm

LRe

covery

Claimed

tobe

taken120583120583g

mL

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

LRe

covery

4403

10077

4401

10036

4405

10118

4401

10036

4408

10199

4405

10118

7701

10019

5497

9942

6606

10095

8800

10002

6602

10041

8802

10022

9896

9953

8797

9962

101006

10060

101003

10028

9906

10061

121199

9991

111111

10103

101000

9995

141401

10010

121197

9977

111098

9985

161589

9934

141403

10022

121202

10018

161589

9934

131299

9995

Mean

10015

10023

10043

Varia

nce

027

051

043

SD052

072

066

SE017

024

025

lowast Averageo

fthree

experim

ents

Journal of Spectroscopy 11

T 4 Application of the proposed conductometric method to the determination of erythromycin thiocyanate in dosage formslowast

Erythrocin tablets Erythrocin Pharco suspensionTaken (mg) Found (mg) Recovery Taken (mg) Found (mg) Recovery 3 305 10158 2 2 1004 4 100 4 405 101196 607 10119 7 705 100688 81 10119 8 805 1006010 10 100 12 1190 992012 1191 9921 14 1395 996614 1405 10034Mean 10050 10022SD 085 073SE 032 030RSD 084 073Variance 072 054lowastMean of three different experiments

T 5 e intraday and interday precision and accuracy data for the studied drugs using the spectrophotometric method

Intraday Interday

Drug Taken(120583120583gmLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSDbAccuracyEr c

Taken(120583120583g mLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSD bAccuracyEr c

(I) 50 5028 10056 029 056 50 5034 10068 039 068(II) 30 3033 10109 056 109 30 3017 10057 046 057(III) 16 1599 9995 019 minus005 16 1601 10007 025 007aAverage of seven determinationsbRSD percentage relative standard deviationcEr percentage relative error

T 6 Analysis of clarithromycin 28 120583120583gmLminus1 (5times 10minus4 M) by the spectrophotometric method in the presence of some common excipients

Tolerance molar ratio ( M M)lowast Glucose Sucrose Carboxy methyl cellulose sodium Sodium chlorideRecovery lowastlowast

1 1 10124 10107 10089 99821 10 10036 10124 9911 99821 50 11545 10213 10035 99471 100 10657 10746 9822 9929lowastDrug excipientlowastlowastMean of three determinations

T 7 Determination of the studied drugs by the proposed spectrophotometric and conductmetric methods compared with the reportedmethod [6]

(I) (II) (III)Spectrophotometric method

Mean plusmn RSD 10005 plusmn 054 10001 plusmn 047 9987 plusmn 050Variance 029 022 025Studentrsquos 119905119905-test 005 (217)lowast 04 (131)lowast 006 (216)lowast119865119865-test 238 (428)lowast 391 (360)lowast 268 (389)lowast

119899119899 7 9 9Conductometric method

Mean plusmn RSD 10066 plusmn 093 10065 plusmn 063 10062 plusmn 062Variance 087 040 038Studentrsquos 119905119905-test 131 (217)lowast 113 (217)lowast 210 (2228)lowast119865119865-test 126 (387)lowast 215 (397)lowast 176 (805)lowast

119899119899 7 6 6Reported method [6]

Mean plusmn RSD 10004 plusmn 083 10015 plusmn 093 9989 plusmn 082Variance 069 086 067Studentrsquos 119905119905-test mdash mdash mdash119865119865-test mdash mdash mdash119899119899 7 8 6lowaste gures in parenthesis are the theoretical values for 119905119905 and 119865119865-tests (119875119875 119875 005)

12 Journal of Spectroscopy

Acknowledgments

e authors wish to express their great thanks to Dr SoadSelim Abd El Hay and Dr Omnia Ahmed Emam Ismaiel atthe Analytical Chemistry Department Zagazig Universityfor their great help and advices during this work Also theywould like to point out that the authors do not have anyconict of interests with any company or institution

References

[1] S C Sweetman Martindale the Complete Drug ReferencePharmaceutical Press London UK 35th edition 2007

[2] S OmuraMacrolide Antibiotics Academic Press London UK1984

[3] J N Delgado and W A RemersWilson and Gisvoldrsquos Textbookof Organic Medecinal and Pharmaceutical Chemistry J BLippincott Company London UK 1998

[4] H A Krisr Progress inMedicinal Chemistry vol 30 of Edited byG P Ellis and D K Luscombe 1993

[5] British Pharmacopoeia Her Majestyrsquos Stationery Office Lon-don UK 2007

[6] M I Walash M S Rizk M I Eid and M E Fathy ldquoSpec-trophotometric determination of four macrolide antibiotics inpharmaceutical formulations and biological uids via binarycomplex formation with eosin and spectrophotometryrdquo Journalof AOAC International vol 90 no 6 pp 1579ndash1587 2007

[7] J Shah M R Jan and S Manzoor ldquoExtractive spectrophoto-metric methods for determination of clarithromycin in phar-maceutical formulations using bromothymol blue and cresolredrdquo Journal of the Chinese Chemical Society vol 55 no 5 pp1107ndash1112 2008

[8] C E R De Paula V G K Almeida and R J CassellaldquoNovel spectrophotometric method for the determination ofazithromycin in pharmaceutical formulations based on itscharge transfer reaction with quinalizarinrdquo Journal of theBrazilian Chemical Society vol 21 no 9 pp 1664ndash1671 2010

[9] S Ashour and R Bayram ldquoNovel spectrophotometric methodfor determination of somemacrolide antibiotics in pharmaceu-tical formulations using 1 2-naphthoquinone-4-sulphonaterdquoSpectrochim Acta Part A vol 99 pp 74ndash80 2012

[10] S D Magar A P Tupe P Y Pawar and B Y ManeldquoSimultaneous spectrophotometric estimation of ceximeand azithrhomycin in tablet dosage formrdquo Current PharmaResearch vol 2 no 3 pp 535ndash538 2012

[11] P Y Khashaba ldquoSpectrouorimetric analysis of certain mac-rolide antibiotics in bulk and pharmaceutical formulationsrdquoJournal of Pharmaceutical and Biomedical Analysis vol 27 no6 pp 923ndash932 2002

[12] N El-Rabbat H F Askal P Y Khashaba and N N AttialdquoA validated spectrouorometric assay for the determinationof certainmacrolide antibiotics in pharmaceutical formulationsand spiked biological uidsrdquo Journal of AOAC International vol89 no 5 pp 1276ndash1287 2006

[13] C L Flurer ldquoAnalysis of macrolide antibiotics by capillaryelectrophoresisrdquo Electrophoresis vol 17 no 2 pp 359ndash3661996

[14] A K Lalloo S C Chattaraj and I Kanfer ldquoDevelopmentof a capillary electrophoretic method for the separation ofthe macrolide antibiotics erythromycin josamycin and olean-domycinrdquo Journal of Chromatography B vol 704 no 1-2 pp333ndash341 1997

[15] M Hedenmo and B M Eriksson ldquoLiquid chromatographicdetermination of the macrolide antibiotics roxithromycin andclarithromycin in plasma by automated solid-phase extractionand electrochemical detectionrdquo Journal of Chromatography Avol 692 no 1-2 pp 161ndash166 1995

[16] M Dubois D Fluchard E Sior and P Delahaut ldquoIdenticationand quantication of ve macrolide antibiotics in severaltissues eggs and milk by liquid chromatography-electrospraytandem mass spectrometryrdquo Journal of Chromatography B vol753 no 2 pp 189ndash202 2001

[17] R V S Nirogi V N Kandikere M Shukla et al ldquoSensitiveand selective liquid chromatography-tandem mass spectrom-etry method for the quantication of azithromycin in humanplasmardquo Analytica Chimica Acta vol 553 no 1-2 pp 1ndash82005

[18] O A E M Farghaly and N A L Mohamed ldquoVoltammetricdetermination of azithromycin at the carbon paste electroderdquoTalanta vol 62 no 3 pp 531ndash538 2004

[19] J A Bernabeacuteu M A Camacho M E Gil-Alegre V Ruz andA I Torres-Suaacuterez ldquoMicrobiological bioassay of erythromycinthiocyanate optimisation and validationrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 21 no 2 pp 347ndash3531999

[20] H R N Salgado and A F F Roncari ldquoMicrobiological assayfor the determination of azithromycin in ophthalmic solutionsrdquoYaoxue Xuebao vol 40 no 6 pp 544ndash549 2005

[21] A S Amin and M M El-Henawee ldquoColorimetric methodfor the simultaneous determination of chlorphenoxaminehydrochloride and anhydrous caffeine in pure and dosage formswith rose bengalrdquo Mikrochimica Acta vol 118 no 3-4 pp177ndash183 1995

[22] F M Abdel-Gawad ldquoSpectrophotometric determination ofoxybuprocaine hydrochloride with halouorescein derivativesrdquoFarmaco vol 50 no 3 pp 197ndash200 1995

[23] A S Amin ldquoSpectrophotometric and conductometric deter-mination of clindamycin hydrochloride in pure form andin pharmaceutical preparationsrdquo Analusis vol 23 no 8 pp415ndash417 1995

[24] SM Amer Z El-Sherif andMM Amer ldquoSpectrophotometricdetermination of isoniazid nalidixic acid and umequinethrough ternary complex-formation with Cd (II) and rosebengalrdquo Egyptian Journal of Pharmaceutical Sciences vol 35 no1ndash6 pp 627ndash642 1994

[25] H Parham and A G Fazeli ldquoExtraction-spectrophotometricdetermination of trace amounts of barium by 18-crown-6 androse bengalrdquo Analytical Sciences vol 16 no 6 pp 575ndash5772000

[26] M A El Ries ldquoSpectrophotometric and indirect determinationof lincomycin by atomic absorption spectroscopyrdquo AnalyticalLetters vol 27 no 8 pp 1517ndash1531 1994

[27] H E Abdellatef M M Ayad and E A Taha ldquoSpectrophoto-metric and atomic absorption spectrometric determination oframipril and perindopril through ternary complex formationwith eosin and Cu(II)rdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 18 no 6 pp 1021ndash1027 1999

[28] M M Ayad A A Shalaby H E Abdellatef and M M HosnyldquoSpectrophotometric and AAS determination of ramipril andenalapril through ternary complex formationrdquo Journal of Phar-maceutical and Biomedical Analysis vol 28 no 2 pp 311ndash3212002

Journal of Spectroscopy 13

[29] A AMMoustafa ldquoSpectrophotometric methods for the deter-mination of lansoprazole and pantoprazole sodium sesquihy-draterdquo Journal of Pharmaceutical and Biomedical Analysis vol22 no 1 pp 45ndash58 2000

[30] M I Walash M E S Metwally M Eid and R N El-ShahenyldquoSpectrophotometric determination of risedronate in pharma-ceutical formulations via complex formation with Cu(II) ionsapplication to content uniformity testingrdquo International Journalof Biomedical Science vol 4 no 4 pp 303ndash309 2008

[31] J J Lingane Electroanalytical Chemistry Interscience NewYork NY USA 2nd edition 1958

[32] S A Tirmizi F H Wattoo M H S Wattoo S Sarwar AN Memon and A B Ghangro ldquoSpectrophotometric studyof stability constants of cimetidine-Ni(II) complex at differenttemperaturesrdquo Arabian Journal of Chemistry vol 35 no 1 pp93ndash100 2010

[33] J Inczedy Analytical Application of Complex EquilibriiaBudapest John Wiley amp Sons 1976

[34] J H Yoe and A L Jones ldquoCcrforimetric determination of ironwith disodium-12-dlhydroxybenzene-35-disuffonaterdquo Indus-trial and Engineering Chemistry vol 16 pp 111ndash115 1944

[35] International Conference on Harmonisation ICH of TechnicalRequirments for Registeration of Pharmaceuticals for HumanUse 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

10 Journal of Spectroscopy

T3Determinationof

azith

romycin

dihydrateinits

pharmaceutic

aldo

sage

form

sthrou

ghcomplexationwith

Cu(II)chlorid

eand

rose

beng

alby

spectro

photom

etric

procedurelowast

Azrolid

tablets

Zithrokancapsules

Zithrokansuspensio

n

Claimed

tobe

taken120583120583g

mL

Authentic

added120583120583g

mL

Foun

dconc

120583120583gm

LRe

covery

Claimed

tobe

taken

120583120583gm

L

Authentic

added120583120583g

mL

Foun

dconc

120583120583gm

LRe

covery

Claimed

tobe

taken120583120583g

mL

Authentic

added

120583120583gm

L

Foun

dconc

120583120583gm

LRe

covery

4403

10077

4401

10036

4405

10118

4401

10036

4408

10199

4405

10118

7701

10019

5497

9942

6606

10095

8800

10002

6602

10041

8802

10022

9896

9953

8797

9962

101006

10060

101003

10028

9906

10061

121199

9991

111111

10103

101000

9995

141401

10010

121197

9977

111098

9985

161589

9934

141403

10022

121202

10018

161589

9934

131299

9995

Mean

10015

10023

10043

Varia

nce

027

051

043

SD052

072

066

SE017

024

025

lowast Averageo

fthree

experim

ents

Journal of Spectroscopy 11

T 4 Application of the proposed conductometric method to the determination of erythromycin thiocyanate in dosage formslowast

Erythrocin tablets Erythrocin Pharco suspensionTaken (mg) Found (mg) Recovery Taken (mg) Found (mg) Recovery 3 305 10158 2 2 1004 4 100 4 405 101196 607 10119 7 705 100688 81 10119 8 805 1006010 10 100 12 1190 992012 1191 9921 14 1395 996614 1405 10034Mean 10050 10022SD 085 073SE 032 030RSD 084 073Variance 072 054lowastMean of three different experiments

T 5 e intraday and interday precision and accuracy data for the studied drugs using the spectrophotometric method

Intraday Interday

Drug Taken(120583120583gmLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSDbAccuracyEr c

Taken(120583120583g mLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSD bAccuracyEr c

(I) 50 5028 10056 029 056 50 5034 10068 039 068(II) 30 3033 10109 056 109 30 3017 10057 046 057(III) 16 1599 9995 019 minus005 16 1601 10007 025 007aAverage of seven determinationsbRSD percentage relative standard deviationcEr percentage relative error

T 6 Analysis of clarithromycin 28 120583120583gmLminus1 (5times 10minus4 M) by the spectrophotometric method in the presence of some common excipients

Tolerance molar ratio ( M M)lowast Glucose Sucrose Carboxy methyl cellulose sodium Sodium chlorideRecovery lowastlowast

1 1 10124 10107 10089 99821 10 10036 10124 9911 99821 50 11545 10213 10035 99471 100 10657 10746 9822 9929lowastDrug excipientlowastlowastMean of three determinations

T 7 Determination of the studied drugs by the proposed spectrophotometric and conductmetric methods compared with the reportedmethod [6]

(I) (II) (III)Spectrophotometric method

Mean plusmn RSD 10005 plusmn 054 10001 plusmn 047 9987 plusmn 050Variance 029 022 025Studentrsquos 119905119905-test 005 (217)lowast 04 (131)lowast 006 (216)lowast119865119865-test 238 (428)lowast 391 (360)lowast 268 (389)lowast

119899119899 7 9 9Conductometric method

Mean plusmn RSD 10066 plusmn 093 10065 plusmn 063 10062 plusmn 062Variance 087 040 038Studentrsquos 119905119905-test 131 (217)lowast 113 (217)lowast 210 (2228)lowast119865119865-test 126 (387)lowast 215 (397)lowast 176 (805)lowast

119899119899 7 6 6Reported method [6]

Mean plusmn RSD 10004 plusmn 083 10015 plusmn 093 9989 plusmn 082Variance 069 086 067Studentrsquos 119905119905-test mdash mdash mdash119865119865-test mdash mdash mdash119899119899 7 8 6lowaste gures in parenthesis are the theoretical values for 119905119905 and 119865119865-tests (119875119875 119875 005)

12 Journal of Spectroscopy

Acknowledgments

e authors wish to express their great thanks to Dr SoadSelim Abd El Hay and Dr Omnia Ahmed Emam Ismaiel atthe Analytical Chemistry Department Zagazig Universityfor their great help and advices during this work Also theywould like to point out that the authors do not have anyconict of interests with any company or institution

References

[1] S C Sweetman Martindale the Complete Drug ReferencePharmaceutical Press London UK 35th edition 2007

[2] S OmuraMacrolide Antibiotics Academic Press London UK1984

[3] J N Delgado and W A RemersWilson and Gisvoldrsquos Textbookof Organic Medecinal and Pharmaceutical Chemistry J BLippincott Company London UK 1998

[4] H A Krisr Progress inMedicinal Chemistry vol 30 of Edited byG P Ellis and D K Luscombe 1993

[5] British Pharmacopoeia Her Majestyrsquos Stationery Office Lon-don UK 2007

[6] M I Walash M S Rizk M I Eid and M E Fathy ldquoSpec-trophotometric determination of four macrolide antibiotics inpharmaceutical formulations and biological uids via binarycomplex formation with eosin and spectrophotometryrdquo Journalof AOAC International vol 90 no 6 pp 1579ndash1587 2007

[7] J Shah M R Jan and S Manzoor ldquoExtractive spectrophoto-metric methods for determination of clarithromycin in phar-maceutical formulations using bromothymol blue and cresolredrdquo Journal of the Chinese Chemical Society vol 55 no 5 pp1107ndash1112 2008

[8] C E R De Paula V G K Almeida and R J CassellaldquoNovel spectrophotometric method for the determination ofazithromycin in pharmaceutical formulations based on itscharge transfer reaction with quinalizarinrdquo Journal of theBrazilian Chemical Society vol 21 no 9 pp 1664ndash1671 2010

[9] S Ashour and R Bayram ldquoNovel spectrophotometric methodfor determination of somemacrolide antibiotics in pharmaceu-tical formulations using 1 2-naphthoquinone-4-sulphonaterdquoSpectrochim Acta Part A vol 99 pp 74ndash80 2012

[10] S D Magar A P Tupe P Y Pawar and B Y ManeldquoSimultaneous spectrophotometric estimation of ceximeand azithrhomycin in tablet dosage formrdquo Current PharmaResearch vol 2 no 3 pp 535ndash538 2012

[11] P Y Khashaba ldquoSpectrouorimetric analysis of certain mac-rolide antibiotics in bulk and pharmaceutical formulationsrdquoJournal of Pharmaceutical and Biomedical Analysis vol 27 no6 pp 923ndash932 2002

[12] N El-Rabbat H F Askal P Y Khashaba and N N AttialdquoA validated spectrouorometric assay for the determinationof certainmacrolide antibiotics in pharmaceutical formulationsand spiked biological uidsrdquo Journal of AOAC International vol89 no 5 pp 1276ndash1287 2006

[13] C L Flurer ldquoAnalysis of macrolide antibiotics by capillaryelectrophoresisrdquo Electrophoresis vol 17 no 2 pp 359ndash3661996

[14] A K Lalloo S C Chattaraj and I Kanfer ldquoDevelopmentof a capillary electrophoretic method for the separation ofthe macrolide antibiotics erythromycin josamycin and olean-domycinrdquo Journal of Chromatography B vol 704 no 1-2 pp333ndash341 1997

[15] M Hedenmo and B M Eriksson ldquoLiquid chromatographicdetermination of the macrolide antibiotics roxithromycin andclarithromycin in plasma by automated solid-phase extractionand electrochemical detectionrdquo Journal of Chromatography Avol 692 no 1-2 pp 161ndash166 1995

[16] M Dubois D Fluchard E Sior and P Delahaut ldquoIdenticationand quantication of ve macrolide antibiotics in severaltissues eggs and milk by liquid chromatography-electrospraytandem mass spectrometryrdquo Journal of Chromatography B vol753 no 2 pp 189ndash202 2001

[17] R V S Nirogi V N Kandikere M Shukla et al ldquoSensitiveand selective liquid chromatography-tandem mass spectrom-etry method for the quantication of azithromycin in humanplasmardquo Analytica Chimica Acta vol 553 no 1-2 pp 1ndash82005

[18] O A E M Farghaly and N A L Mohamed ldquoVoltammetricdetermination of azithromycin at the carbon paste electroderdquoTalanta vol 62 no 3 pp 531ndash538 2004

[19] J A Bernabeacuteu M A Camacho M E Gil-Alegre V Ruz andA I Torres-Suaacuterez ldquoMicrobiological bioassay of erythromycinthiocyanate optimisation and validationrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 21 no 2 pp 347ndash3531999

[20] H R N Salgado and A F F Roncari ldquoMicrobiological assayfor the determination of azithromycin in ophthalmic solutionsrdquoYaoxue Xuebao vol 40 no 6 pp 544ndash549 2005

[21] A S Amin and M M El-Henawee ldquoColorimetric methodfor the simultaneous determination of chlorphenoxaminehydrochloride and anhydrous caffeine in pure and dosage formswith rose bengalrdquo Mikrochimica Acta vol 118 no 3-4 pp177ndash183 1995

[22] F M Abdel-Gawad ldquoSpectrophotometric determination ofoxybuprocaine hydrochloride with halouorescein derivativesrdquoFarmaco vol 50 no 3 pp 197ndash200 1995

[23] A S Amin ldquoSpectrophotometric and conductometric deter-mination of clindamycin hydrochloride in pure form andin pharmaceutical preparationsrdquo Analusis vol 23 no 8 pp415ndash417 1995

[24] SM Amer Z El-Sherif andMM Amer ldquoSpectrophotometricdetermination of isoniazid nalidixic acid and umequinethrough ternary complex-formation with Cd (II) and rosebengalrdquo Egyptian Journal of Pharmaceutical Sciences vol 35 no1ndash6 pp 627ndash642 1994

[25] H Parham and A G Fazeli ldquoExtraction-spectrophotometricdetermination of trace amounts of barium by 18-crown-6 androse bengalrdquo Analytical Sciences vol 16 no 6 pp 575ndash5772000

[26] M A El Ries ldquoSpectrophotometric and indirect determinationof lincomycin by atomic absorption spectroscopyrdquo AnalyticalLetters vol 27 no 8 pp 1517ndash1531 1994

[27] H E Abdellatef M M Ayad and E A Taha ldquoSpectrophoto-metric and atomic absorption spectrometric determination oframipril and perindopril through ternary complex formationwith eosin and Cu(II)rdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 18 no 6 pp 1021ndash1027 1999

[28] M M Ayad A A Shalaby H E Abdellatef and M M HosnyldquoSpectrophotometric and AAS determination of ramipril andenalapril through ternary complex formationrdquo Journal of Phar-maceutical and Biomedical Analysis vol 28 no 2 pp 311ndash3212002

Journal of Spectroscopy 13

[29] A AMMoustafa ldquoSpectrophotometric methods for the deter-mination of lansoprazole and pantoprazole sodium sesquihy-draterdquo Journal of Pharmaceutical and Biomedical Analysis vol22 no 1 pp 45ndash58 2000

[30] M I Walash M E S Metwally M Eid and R N El-ShahenyldquoSpectrophotometric determination of risedronate in pharma-ceutical formulations via complex formation with Cu(II) ionsapplication to content uniformity testingrdquo International Journalof Biomedical Science vol 4 no 4 pp 303ndash309 2008

[31] J J Lingane Electroanalytical Chemistry Interscience NewYork NY USA 2nd edition 1958

[32] S A Tirmizi F H Wattoo M H S Wattoo S Sarwar AN Memon and A B Ghangro ldquoSpectrophotometric studyof stability constants of cimetidine-Ni(II) complex at differenttemperaturesrdquo Arabian Journal of Chemistry vol 35 no 1 pp93ndash100 2010

[33] J Inczedy Analytical Application of Complex EquilibriiaBudapest John Wiley amp Sons 1976

[34] J H Yoe and A L Jones ldquoCcrforimetric determination of ironwith disodium-12-dlhydroxybenzene-35-disuffonaterdquo Indus-trial and Engineering Chemistry vol 16 pp 111ndash115 1944

[35] International Conference on Harmonisation ICH of TechnicalRequirments for Registeration of Pharmaceuticals for HumanUse 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Spectroscopy 11

T 4 Application of the proposed conductometric method to the determination of erythromycin thiocyanate in dosage formslowast

Erythrocin tablets Erythrocin Pharco suspensionTaken (mg) Found (mg) Recovery Taken (mg) Found (mg) Recovery 3 305 10158 2 2 1004 4 100 4 405 101196 607 10119 7 705 100688 81 10119 8 805 1006010 10 100 12 1190 992012 1191 9921 14 1395 996614 1405 10034Mean 10050 10022SD 085 073SE 032 030RSD 084 073Variance 072 054lowastMean of three different experiments

T 5 e intraday and interday precision and accuracy data for the studied drugs using the spectrophotometric method

Intraday Interday

Drug Taken(120583120583gmLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSDbAccuracyEr c

Taken(120583120583g mLminus1)

Found(120583120583gmLminus1)a Recovery Precision

RSD bAccuracyEr c

(I) 50 5028 10056 029 056 50 5034 10068 039 068(II) 30 3033 10109 056 109 30 3017 10057 046 057(III) 16 1599 9995 019 minus005 16 1601 10007 025 007aAverage of seven determinationsbRSD percentage relative standard deviationcEr percentage relative error

T 6 Analysis of clarithromycin 28 120583120583gmLminus1 (5times 10minus4 M) by the spectrophotometric method in the presence of some common excipients

Tolerance molar ratio ( M M)lowast Glucose Sucrose Carboxy methyl cellulose sodium Sodium chlorideRecovery lowastlowast

1 1 10124 10107 10089 99821 10 10036 10124 9911 99821 50 11545 10213 10035 99471 100 10657 10746 9822 9929lowastDrug excipientlowastlowastMean of three determinations

T 7 Determination of the studied drugs by the proposed spectrophotometric and conductmetric methods compared with the reportedmethod [6]

(I) (II) (III)Spectrophotometric method

Mean plusmn RSD 10005 plusmn 054 10001 plusmn 047 9987 plusmn 050Variance 029 022 025Studentrsquos 119905119905-test 005 (217)lowast 04 (131)lowast 006 (216)lowast119865119865-test 238 (428)lowast 391 (360)lowast 268 (389)lowast

119899119899 7 9 9Conductometric method

Mean plusmn RSD 10066 plusmn 093 10065 plusmn 063 10062 plusmn 062Variance 087 040 038Studentrsquos 119905119905-test 131 (217)lowast 113 (217)lowast 210 (2228)lowast119865119865-test 126 (387)lowast 215 (397)lowast 176 (805)lowast

119899119899 7 6 6Reported method [6]

Mean plusmn RSD 10004 plusmn 083 10015 plusmn 093 9989 plusmn 082Variance 069 086 067Studentrsquos 119905119905-test mdash mdash mdash119865119865-test mdash mdash mdash119899119899 7 8 6lowaste gures in parenthesis are the theoretical values for 119905119905 and 119865119865-tests (119875119875 119875 005)

12 Journal of Spectroscopy

Acknowledgments

e authors wish to express their great thanks to Dr SoadSelim Abd El Hay and Dr Omnia Ahmed Emam Ismaiel atthe Analytical Chemistry Department Zagazig Universityfor their great help and advices during this work Also theywould like to point out that the authors do not have anyconict of interests with any company or institution

References

[1] S C Sweetman Martindale the Complete Drug ReferencePharmaceutical Press London UK 35th edition 2007

[2] S OmuraMacrolide Antibiotics Academic Press London UK1984

[3] J N Delgado and W A RemersWilson and Gisvoldrsquos Textbookof Organic Medecinal and Pharmaceutical Chemistry J BLippincott Company London UK 1998

[4] H A Krisr Progress inMedicinal Chemistry vol 30 of Edited byG P Ellis and D K Luscombe 1993

[5] British Pharmacopoeia Her Majestyrsquos Stationery Office Lon-don UK 2007

[6] M I Walash M S Rizk M I Eid and M E Fathy ldquoSpec-trophotometric determination of four macrolide antibiotics inpharmaceutical formulations and biological uids via binarycomplex formation with eosin and spectrophotometryrdquo Journalof AOAC International vol 90 no 6 pp 1579ndash1587 2007

[7] J Shah M R Jan and S Manzoor ldquoExtractive spectrophoto-metric methods for determination of clarithromycin in phar-maceutical formulations using bromothymol blue and cresolredrdquo Journal of the Chinese Chemical Society vol 55 no 5 pp1107ndash1112 2008

[8] C E R De Paula V G K Almeida and R J CassellaldquoNovel spectrophotometric method for the determination ofazithromycin in pharmaceutical formulations based on itscharge transfer reaction with quinalizarinrdquo Journal of theBrazilian Chemical Society vol 21 no 9 pp 1664ndash1671 2010

[9] S Ashour and R Bayram ldquoNovel spectrophotometric methodfor determination of somemacrolide antibiotics in pharmaceu-tical formulations using 1 2-naphthoquinone-4-sulphonaterdquoSpectrochim Acta Part A vol 99 pp 74ndash80 2012

[10] S D Magar A P Tupe P Y Pawar and B Y ManeldquoSimultaneous spectrophotometric estimation of ceximeand azithrhomycin in tablet dosage formrdquo Current PharmaResearch vol 2 no 3 pp 535ndash538 2012

[11] P Y Khashaba ldquoSpectrouorimetric analysis of certain mac-rolide antibiotics in bulk and pharmaceutical formulationsrdquoJournal of Pharmaceutical and Biomedical Analysis vol 27 no6 pp 923ndash932 2002

[12] N El-Rabbat H F Askal P Y Khashaba and N N AttialdquoA validated spectrouorometric assay for the determinationof certainmacrolide antibiotics in pharmaceutical formulationsand spiked biological uidsrdquo Journal of AOAC International vol89 no 5 pp 1276ndash1287 2006

[13] C L Flurer ldquoAnalysis of macrolide antibiotics by capillaryelectrophoresisrdquo Electrophoresis vol 17 no 2 pp 359ndash3661996

[14] A K Lalloo S C Chattaraj and I Kanfer ldquoDevelopmentof a capillary electrophoretic method for the separation ofthe macrolide antibiotics erythromycin josamycin and olean-domycinrdquo Journal of Chromatography B vol 704 no 1-2 pp333ndash341 1997

[15] M Hedenmo and B M Eriksson ldquoLiquid chromatographicdetermination of the macrolide antibiotics roxithromycin andclarithromycin in plasma by automated solid-phase extractionand electrochemical detectionrdquo Journal of Chromatography Avol 692 no 1-2 pp 161ndash166 1995

[16] M Dubois D Fluchard E Sior and P Delahaut ldquoIdenticationand quantication of ve macrolide antibiotics in severaltissues eggs and milk by liquid chromatography-electrospraytandem mass spectrometryrdquo Journal of Chromatography B vol753 no 2 pp 189ndash202 2001

[17] R V S Nirogi V N Kandikere M Shukla et al ldquoSensitiveand selective liquid chromatography-tandem mass spectrom-etry method for the quantication of azithromycin in humanplasmardquo Analytica Chimica Acta vol 553 no 1-2 pp 1ndash82005

[18] O A E M Farghaly and N A L Mohamed ldquoVoltammetricdetermination of azithromycin at the carbon paste electroderdquoTalanta vol 62 no 3 pp 531ndash538 2004

[19] J A Bernabeacuteu M A Camacho M E Gil-Alegre V Ruz andA I Torres-Suaacuterez ldquoMicrobiological bioassay of erythromycinthiocyanate optimisation and validationrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 21 no 2 pp 347ndash3531999

[20] H R N Salgado and A F F Roncari ldquoMicrobiological assayfor the determination of azithromycin in ophthalmic solutionsrdquoYaoxue Xuebao vol 40 no 6 pp 544ndash549 2005

[21] A S Amin and M M El-Henawee ldquoColorimetric methodfor the simultaneous determination of chlorphenoxaminehydrochloride and anhydrous caffeine in pure and dosage formswith rose bengalrdquo Mikrochimica Acta vol 118 no 3-4 pp177ndash183 1995

[22] F M Abdel-Gawad ldquoSpectrophotometric determination ofoxybuprocaine hydrochloride with halouorescein derivativesrdquoFarmaco vol 50 no 3 pp 197ndash200 1995

[23] A S Amin ldquoSpectrophotometric and conductometric deter-mination of clindamycin hydrochloride in pure form andin pharmaceutical preparationsrdquo Analusis vol 23 no 8 pp415ndash417 1995

[24] SM Amer Z El-Sherif andMM Amer ldquoSpectrophotometricdetermination of isoniazid nalidixic acid and umequinethrough ternary complex-formation with Cd (II) and rosebengalrdquo Egyptian Journal of Pharmaceutical Sciences vol 35 no1ndash6 pp 627ndash642 1994

[25] H Parham and A G Fazeli ldquoExtraction-spectrophotometricdetermination of trace amounts of barium by 18-crown-6 androse bengalrdquo Analytical Sciences vol 16 no 6 pp 575ndash5772000

[26] M A El Ries ldquoSpectrophotometric and indirect determinationof lincomycin by atomic absorption spectroscopyrdquo AnalyticalLetters vol 27 no 8 pp 1517ndash1531 1994

[27] H E Abdellatef M M Ayad and E A Taha ldquoSpectrophoto-metric and atomic absorption spectrometric determination oframipril and perindopril through ternary complex formationwith eosin and Cu(II)rdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 18 no 6 pp 1021ndash1027 1999

[28] M M Ayad A A Shalaby H E Abdellatef and M M HosnyldquoSpectrophotometric and AAS determination of ramipril andenalapril through ternary complex formationrdquo Journal of Phar-maceutical and Biomedical Analysis vol 28 no 2 pp 311ndash3212002

Journal of Spectroscopy 13

[29] A AMMoustafa ldquoSpectrophotometric methods for the deter-mination of lansoprazole and pantoprazole sodium sesquihy-draterdquo Journal of Pharmaceutical and Biomedical Analysis vol22 no 1 pp 45ndash58 2000

[30] M I Walash M E S Metwally M Eid and R N El-ShahenyldquoSpectrophotometric determination of risedronate in pharma-ceutical formulations via complex formation with Cu(II) ionsapplication to content uniformity testingrdquo International Journalof Biomedical Science vol 4 no 4 pp 303ndash309 2008

[31] J J Lingane Electroanalytical Chemistry Interscience NewYork NY USA 2nd edition 1958

[32] S A Tirmizi F H Wattoo M H S Wattoo S Sarwar AN Memon and A B Ghangro ldquoSpectrophotometric studyof stability constants of cimetidine-Ni(II) complex at differenttemperaturesrdquo Arabian Journal of Chemistry vol 35 no 1 pp93ndash100 2010

[33] J Inczedy Analytical Application of Complex EquilibriiaBudapest John Wiley amp Sons 1976

[34] J H Yoe and A L Jones ldquoCcrforimetric determination of ironwith disodium-12-dlhydroxybenzene-35-disuffonaterdquo Indus-trial and Engineering Chemistry vol 16 pp 111ndash115 1944

[35] International Conference on Harmonisation ICH of TechnicalRequirments for Registeration of Pharmaceuticals for HumanUse 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

12 Journal of Spectroscopy

Acknowledgments

e authors wish to express their great thanks to Dr SoadSelim Abd El Hay and Dr Omnia Ahmed Emam Ismaiel atthe Analytical Chemistry Department Zagazig Universityfor their great help and advices during this work Also theywould like to point out that the authors do not have anyconict of interests with any company or institution

References

[1] S C Sweetman Martindale the Complete Drug ReferencePharmaceutical Press London UK 35th edition 2007

[2] S OmuraMacrolide Antibiotics Academic Press London UK1984

[3] J N Delgado and W A RemersWilson and Gisvoldrsquos Textbookof Organic Medecinal and Pharmaceutical Chemistry J BLippincott Company London UK 1998

[4] H A Krisr Progress inMedicinal Chemistry vol 30 of Edited byG P Ellis and D K Luscombe 1993

[5] British Pharmacopoeia Her Majestyrsquos Stationery Office Lon-don UK 2007

[6] M I Walash M S Rizk M I Eid and M E Fathy ldquoSpec-trophotometric determination of four macrolide antibiotics inpharmaceutical formulations and biological uids via binarycomplex formation with eosin and spectrophotometryrdquo Journalof AOAC International vol 90 no 6 pp 1579ndash1587 2007

[7] J Shah M R Jan and S Manzoor ldquoExtractive spectrophoto-metric methods for determination of clarithromycin in phar-maceutical formulations using bromothymol blue and cresolredrdquo Journal of the Chinese Chemical Society vol 55 no 5 pp1107ndash1112 2008

[8] C E R De Paula V G K Almeida and R J CassellaldquoNovel spectrophotometric method for the determination ofazithromycin in pharmaceutical formulations based on itscharge transfer reaction with quinalizarinrdquo Journal of theBrazilian Chemical Society vol 21 no 9 pp 1664ndash1671 2010

[9] S Ashour and R Bayram ldquoNovel spectrophotometric methodfor determination of somemacrolide antibiotics in pharmaceu-tical formulations using 1 2-naphthoquinone-4-sulphonaterdquoSpectrochim Acta Part A vol 99 pp 74ndash80 2012

[10] S D Magar A P Tupe P Y Pawar and B Y ManeldquoSimultaneous spectrophotometric estimation of ceximeand azithrhomycin in tablet dosage formrdquo Current PharmaResearch vol 2 no 3 pp 535ndash538 2012

[11] P Y Khashaba ldquoSpectrouorimetric analysis of certain mac-rolide antibiotics in bulk and pharmaceutical formulationsrdquoJournal of Pharmaceutical and Biomedical Analysis vol 27 no6 pp 923ndash932 2002

[12] N El-Rabbat H F Askal P Y Khashaba and N N AttialdquoA validated spectrouorometric assay for the determinationof certainmacrolide antibiotics in pharmaceutical formulationsand spiked biological uidsrdquo Journal of AOAC International vol89 no 5 pp 1276ndash1287 2006

[13] C L Flurer ldquoAnalysis of macrolide antibiotics by capillaryelectrophoresisrdquo Electrophoresis vol 17 no 2 pp 359ndash3661996

[14] A K Lalloo S C Chattaraj and I Kanfer ldquoDevelopmentof a capillary electrophoretic method for the separation ofthe macrolide antibiotics erythromycin josamycin and olean-domycinrdquo Journal of Chromatography B vol 704 no 1-2 pp333ndash341 1997

[15] M Hedenmo and B M Eriksson ldquoLiquid chromatographicdetermination of the macrolide antibiotics roxithromycin andclarithromycin in plasma by automated solid-phase extractionand electrochemical detectionrdquo Journal of Chromatography Avol 692 no 1-2 pp 161ndash166 1995

[16] M Dubois D Fluchard E Sior and P Delahaut ldquoIdenticationand quantication of ve macrolide antibiotics in severaltissues eggs and milk by liquid chromatography-electrospraytandem mass spectrometryrdquo Journal of Chromatography B vol753 no 2 pp 189ndash202 2001

[17] R V S Nirogi V N Kandikere M Shukla et al ldquoSensitiveand selective liquid chromatography-tandem mass spectrom-etry method for the quantication of azithromycin in humanplasmardquo Analytica Chimica Acta vol 553 no 1-2 pp 1ndash82005

[18] O A E M Farghaly and N A L Mohamed ldquoVoltammetricdetermination of azithromycin at the carbon paste electroderdquoTalanta vol 62 no 3 pp 531ndash538 2004

[19] J A Bernabeacuteu M A Camacho M E Gil-Alegre V Ruz andA I Torres-Suaacuterez ldquoMicrobiological bioassay of erythromycinthiocyanate optimisation and validationrdquo Journal of Pharma-ceutical and Biomedical Analysis vol 21 no 2 pp 347ndash3531999

[20] H R N Salgado and A F F Roncari ldquoMicrobiological assayfor the determination of azithromycin in ophthalmic solutionsrdquoYaoxue Xuebao vol 40 no 6 pp 544ndash549 2005

[21] A S Amin and M M El-Henawee ldquoColorimetric methodfor the simultaneous determination of chlorphenoxaminehydrochloride and anhydrous caffeine in pure and dosage formswith rose bengalrdquo Mikrochimica Acta vol 118 no 3-4 pp177ndash183 1995

[22] F M Abdel-Gawad ldquoSpectrophotometric determination ofoxybuprocaine hydrochloride with halouorescein derivativesrdquoFarmaco vol 50 no 3 pp 197ndash200 1995

[23] A S Amin ldquoSpectrophotometric and conductometric deter-mination of clindamycin hydrochloride in pure form andin pharmaceutical preparationsrdquo Analusis vol 23 no 8 pp415ndash417 1995

[24] SM Amer Z El-Sherif andMM Amer ldquoSpectrophotometricdetermination of isoniazid nalidixic acid and umequinethrough ternary complex-formation with Cd (II) and rosebengalrdquo Egyptian Journal of Pharmaceutical Sciences vol 35 no1ndash6 pp 627ndash642 1994

[25] H Parham and A G Fazeli ldquoExtraction-spectrophotometricdetermination of trace amounts of barium by 18-crown-6 androse bengalrdquo Analytical Sciences vol 16 no 6 pp 575ndash5772000

[26] M A El Ries ldquoSpectrophotometric and indirect determinationof lincomycin by atomic absorption spectroscopyrdquo AnalyticalLetters vol 27 no 8 pp 1517ndash1531 1994

[27] H E Abdellatef M M Ayad and E A Taha ldquoSpectrophoto-metric and atomic absorption spectrometric determination oframipril and perindopril through ternary complex formationwith eosin and Cu(II)rdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 18 no 6 pp 1021ndash1027 1999

[28] M M Ayad A A Shalaby H E Abdellatef and M M HosnyldquoSpectrophotometric and AAS determination of ramipril andenalapril through ternary complex formationrdquo Journal of Phar-maceutical and Biomedical Analysis vol 28 no 2 pp 311ndash3212002

Journal of Spectroscopy 13

[29] A AMMoustafa ldquoSpectrophotometric methods for the deter-mination of lansoprazole and pantoprazole sodium sesquihy-draterdquo Journal of Pharmaceutical and Biomedical Analysis vol22 no 1 pp 45ndash58 2000

[30] M I Walash M E S Metwally M Eid and R N El-ShahenyldquoSpectrophotometric determination of risedronate in pharma-ceutical formulations via complex formation with Cu(II) ionsapplication to content uniformity testingrdquo International Journalof Biomedical Science vol 4 no 4 pp 303ndash309 2008

[31] J J Lingane Electroanalytical Chemistry Interscience NewYork NY USA 2nd edition 1958

[32] S A Tirmizi F H Wattoo M H S Wattoo S Sarwar AN Memon and A B Ghangro ldquoSpectrophotometric studyof stability constants of cimetidine-Ni(II) complex at differenttemperaturesrdquo Arabian Journal of Chemistry vol 35 no 1 pp93ndash100 2010

[33] J Inczedy Analytical Application of Complex EquilibriiaBudapest John Wiley amp Sons 1976

[34] J H Yoe and A L Jones ldquoCcrforimetric determination of ironwith disodium-12-dlhydroxybenzene-35-disuffonaterdquo Indus-trial and Engineering Chemistry vol 16 pp 111ndash115 1944

[35] International Conference on Harmonisation ICH of TechnicalRequirments for Registeration of Pharmaceuticals for HumanUse 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Spectroscopy 13

[29] A AMMoustafa ldquoSpectrophotometric methods for the deter-mination of lansoprazole and pantoprazole sodium sesquihy-draterdquo Journal of Pharmaceutical and Biomedical Analysis vol22 no 1 pp 45ndash58 2000

[30] M I Walash M E S Metwally M Eid and R N El-ShahenyldquoSpectrophotometric determination of risedronate in pharma-ceutical formulations via complex formation with Cu(II) ionsapplication to content uniformity testingrdquo International Journalof Biomedical Science vol 4 no 4 pp 303ndash309 2008

[31] J J Lingane Electroanalytical Chemistry Interscience NewYork NY USA 2nd edition 1958

[32] S A Tirmizi F H Wattoo M H S Wattoo S Sarwar AN Memon and A B Ghangro ldquoSpectrophotometric studyof stability constants of cimetidine-Ni(II) complex at differenttemperaturesrdquo Arabian Journal of Chemistry vol 35 no 1 pp93ndash100 2010

[33] J Inczedy Analytical Application of Complex EquilibriiaBudapest John Wiley amp Sons 1976

[34] J H Yoe and A L Jones ldquoCcrforimetric determination of ironwith disodium-12-dlhydroxybenzene-35-disuffonaterdquo Indus-trial and Engineering Chemistry vol 16 pp 111ndash115 1944

[35] International Conference on Harmonisation ICH of TechnicalRequirments for Registeration of Pharmaceuticals for HumanUse 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

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