simultaneous electrochemical determination of...

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Insights in Analytical ElectrochemistryISSN 2470-9867

2017Vol. 3 No. 1: 1

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Research Article

DOI: 10.21767/2470-9867.100018

Ali Attia K1,Noha Rashed S2,Manal Fouad M2,3 andReem Wasfy A1

1 NationalOrganizationforDrugControland Research,Cairo,Egypt

2 AnalyticalChemistryDepartment,FacultyofPharmacy,Al-AzharUniversity,Cairo,Egypt

3 OctoberUniversityforModernSciencesandArts,Cairo,Egypt

Corresponding author: AliAK

[email protected]

DepartmentofAnalyticalChemistry,NationalOrganizationforDrugControlandResearch,Cairo,Egypt.

Tel:01007618876(or)+20235851278Fax:+20235855582

Citation: AliAttiaK,NohaRashedS,ManalFouadM,etal.SimultaneousElectrochemicalDeterminationofArticaineHClandEpinephrine.InsightsAnalElectrochem.2017,3:1.

IntroductionArticaineHClisawhiteoralmostwhitecrystallinepowder,freelysolubleinwater,methanolandethanol[1].Itisanintermediate-potency, short-acting amide local anesthetic with a fastmetabolismduetoanestergroupinitsstructure.Itiseffectivewithlocalinfiltrationorperipheralnerveblockindentistry,whenadministeredasaspinal,epidural,ocular,orregionalnerveblock,or when injected intravenously for regional anesthesia. Localanestheticdrugsare administeredtotheareasaroundthenervesto be blocked as the skin, subcutaneous tissues, retrobulbar,intrathecal, and epidural spaces. Articaine has a half-life of 60minutesafterenteringthecirculationandisquicklymetabolizedviahydrolysisintoitsinactivemetabolitearticainicacid,whichispartlymetabolizedinthekidneyintoarticainicacidglucuronide.Seventy-fivepercentofarticainicacidisexcretedunchanged;therest is glucuronidatedby the kidneysbefore excretion [2]. It isan amino-amide anesthetic having a thiophene, rather than abenzenering,as wellasanadditionalestergroupthatissubjecttometabolismbyplasmaesterases.Ithasawidespreadpopularityindentalanesthesia,whereitisgenerallyconsideredtobemoreeffective,andpossiblysafer,thanlidocaine,thepriorstandard[3].Epinephrinegreatlyincreasesthe durationofthelocalanesthesia

byproducingvasoconstrictionatthesiteofinjection.Thisallowsthe localanesthetictopersistatthe injectionsitebeforebeingabsorbedintothesystemiccirculation[4].

Literature survey reveals that several different analyticaltechniques have been developed for the determination ofarticaineHClandepinephrineincludinghighperformanceliquidchromatographic methods [5-7], thin layer chromatographicmethods [8], gas chromatographic methods [9] andspectrophotometric methods [10] for articaine HCl and highperformance liquid chromatographic methods [11-15], thinlayer chromatographic methods [16,17], gas chromatographicmethods [18,19], spectrophotometric methods [20-24] andelectrochemicalmethods[25-30]forepinephrine.Carbonbasedelectrodes are currently in widespread use in electroanalyticalchemistry, because of their broad potential window, low cost,rich surface chemistry, low background current and chemicalinertness. Carbon paste electrode (CPE) has some special

Simultaneous Electrochemical Determination of Articaine HCl and Epinephrine

AbstractAsimple,precise,accurateandinexpensivevoltammetricmethodwasdevelopedfor the simultaneous determination of articaine HCl and epinephrine in bulk,pharmaceutical formulations and human urine at carbon paste electrode inBritton-RobinsonbufferofpH7and40µLof10−2molL−1sodiumdodecylsulphate.Several factorswerestudiedsuchasbuffer type,pH, surfactant, scan rate,andaccumulationtime to obtain the optimumconditions for analysis. Themethodshowedlinearityinconcentrationrangeof1.0×10−6-2.6×10-5molL−1.Thelimitsof detection and quantitation were found 2.88 × 10−7 mol L−1 and 8.72 × 10−7 molL−1forarticaineHCland5.10×10−8molL−1and1.56×10-7forepinephrine,respectively. The proposed voltammetric method was successfully applied forsimultaneousdeterminationofarticaineHClandepinephrine inbulk, injectionsand urine. The results obtained from the proposed method were statisticallycomparedwiththoseofareportedmethodandshowednosignificantdifference.Thus,itcanbeusedasqualitycontrolinlaboratories.

Keywords:ArticaineHCl; Epinephrine; Sodiumdodecyl sulphate;Carbonpaste;Voltammetry; Bulk;Urine

Received: April07,2017; Accepted: April22,2017; Published: April24,2017

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characteristicsandbenefitssuchastheeaseofsurfacerenewal,individual polarizability, and easy to apply modifications. Thedisadvantage of CPE is the tendency of the organic binder todissolveinsolutionscontaininganappreciablefractionoforganicsolvent[31].

In this study articaine HCl and epinephrine were determinedsimultaneously using differential pulse voltammetry (DPV) atcarbonpasteelectrodeinbulk,pharmaceuticalformulationsandhumanurine.

Materials and MethodsExperimental preparationsPure and market samples: Articaine HCl and epinephrinebitartrate were kindly supplied by Inibsa Laboratories, Spain.Theirpuritywas100%asstatedbythesupplier.

Artinibsa ampoules; batch no. K-3, labeled to contain 40 mgArticaineHCland0.01mgEpinephrineperampoule,productofInibsaLaboratories,Spain.

Standard solutions: Standard solutions of articaine HCl andepinephrine bitartarate were prepared in methanol. For cyclicvoltammetry(CV)1.0x10−2molL−1solutionswerepreparedbydissolving32.1mgofarticaineHCland33.3mgofepinephrinebitartarate, each in 10 mL volumetric flask and diluted withmethanol.ForDPV,onemLofeach1.0×10−2molL−1solutionwastakenanddilutedto10mLwiththesamesolventtoobtain1.0×10−3molL−1workingsolution.

Chemicals and reagents: All reagents used were of analyticalgradeandsolventswereofspectroscopicgrade.Distilledwaterwasusedthroughoutthework.

(i)Methanol,HPLCgrade(FischerChemical,UK).

(ii)Sodiumhydroxide(QualikemsfinechemicalPvt.Ltd).

(iii)Sodiumdodecylsulphate(SDS)(Sigma-Aldrich,Germany).

(iv) Dioctyl sodium sulfosuccinate (DSS) (Sigma-Aldrich,Germany).

(v) Hexane Sulfonic acid sodium salt monohydrate (HSA)(SharlabS.L.,Spain).

(vi) Potassium dihydrogen o-phosphate (El NasrPharmaceuticalChemicals,Egypt).

(vii)Phosphoricacid(Sigma-Aldrich,Germany).

(viii) Britton-Robinson (BR) buffer was prepared by mixing0.04molL−1ofphosphoricacid(Sigma-Aldrich),aceticacid(LobaChemieCo.,India)andboricacid(Sigma-Aldrich).

Buffersolutionswereadjustedwiththeappropriateamountof0.2molL−1NaOH (QualikemsfinechemicalPvt. Ltd) toget thedesiredpH2-11[32].

Phosphatebufferwaspreparedbyadding34.7mLof0.2molL−1 NaOHto50mLof0.2molL−1monobasicpotassiumphosphatesolution (prepared by dissolving 27.22 gmonobasic potassiumphosphate in water and dilute with water to 1000 mL) andcompleteto200mLwithwater[32].

Instruments: All voltammetricmeasurementswere carriedoutusinga computer-drivenanalyticalelectrochemicalworkstation(modelAEW2)withECProg3electrochemistrysoftware(Sycopel,England) in combinationwith a three-electrode configuredC-3stand. The working electrode was a carbon paste electrode(MF-2010,BASmodel),thereferenceelectrodeAg/AgCl/3molL−1 NaCl (MW-2063, BAS model) and a platinum wire counterelectrode (MW-1032, BAS model). A digital pH-meter (JenwaypHmeter,UK)withcombinedglasselectrodewasusedtocarryoutthepHmeasurements.Allelectrochemicalexperimentswereperformedatanambienttemperatureof25±0.2°C.

ProceduresPreparation of working electrode: In a mortar 0.5 g graphitepowder and 0.3 mL paraffin oil were mixed thoroughly witha pestle. The pastewas packed into the hole of the electrodebody and smoothed on a filter paper until it acquired a shinyappearance;thepastewascarefullyremovedandreplacedbyanewoneaftereachmeasurement[33].

Linearity: Aliquots of working standard articaine HCl andepinephrinesolutions(1.0×10−3)wereaddedseparatelytotheelectrolyticcellcontaining5mLofBritton-RobinsonbufferofpH7and40µLof1.0×10−2molL−1SDS,thesolutionswerestirredfor5satopencircuitconditions,voltammetricanalyseswerecarriedoutandvoltammogramswererecordedatascanrate20mVs−1.

Calibrationcurvewasconstructedbyplottinganodicpeakcurrentagainst drug concentrations in molarity; and the regressionequationwascomputed.

Accuracy and precision: ThreedifferentconcentrationscoveringthelinearityrangeofarticaineHClandepinephrinewereanalyzedin triplicates within the same day for intraday and for threesuccessivedaysforinterdayusingtheproceduredetailedunderlinearity part. Accuracy was calculated as precision (Relativestandarddeviation(%RSD)).

Application to pharmaceutical formulationsAnaliquot(0.8mL)containing32.1mgarticaineHCland0.0144mgepinephrinebitartaratewastransferredto10mLvolumetricflask, 33.286 mg pure standard epinephrine bitartarate wastransferredtothevolumetricflaskandvolumecompletedwithmethanoltoobtain1.0×10−2molL−1solutionofeachdrug.OnemL of the prepared 1.0 × 10−2mol L−1 solutionwas taken anddilutedto10mLwiththesamesolventtoobtain1.0×10−3molL−1 workingsolutiontobeanalyzedbytheproposedelectrochemicalmethodusing theproceduredetailedunder linearity part. Thedrug concentrations were calculated from the correspondingregressionequation;theproposedmethodwasfurthervalidatedbyusingthestandardadditiontechnique.

Analysis of articaine HCl and epinephrine in urine: Fortheanalysisof articaineHCl andepinephrine inurine, 1.0mLof urinewasmixedwith9.0mLBRbufferofpH7.0,thensuccessiveadditionsof 1.0 × 10−3mol L−1 working standard solutions (covering thelinearityrange)wereaddedtothevoltammetriccellcontaining5.0mLofthepreviouslydilutedurine.

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Results and DiscussionElectrochemical oxidation of articaine HClThereversibilityoftheoxidationprocessofarticaineHClatCPEwas studied by CV, the cyclic voltammogram showed onewelldefinedanodicpeakinBRbufferofpH7.0(anodicpeakcurrent(I)=19.58µAat1.095V),andthereversescanshowednocathodicpeakindicatingthatarticaineHCloxidationisirreversible(Figure 1a).TheproposedoxidationmechanismwasshowninFigure 1b.

Optimization of experimental conditionsEffect of pH: CVatCPEandscanrateof100mVs−1wasusedtoinvestigatetheeffectofpHupontheoxidationofarticaineHCl.4.5mLofBRbuffer(2-11)and0.5mLof1.0×10-2molL−1articaineHClstandardsolutionwereaddedtotheelectrochemicalcell,andtherespectivevoltammetricresponsewasrecorded(Figure 2a).Thisexperimentwasrepeatedusingphosphatebuffer(Figure 3a) toobtaintheoptimumbufferwiththeoptimumpHvalue.

Figures 2a and 3a showthattheanodicpeakpotential (E)wasshiftednegativelyby increasingpH indicatingthatarticaineHCloxidationatCPEispHdependantandthoseprotonsareinvolvedinthereaction.Figures 2c and 3cshowthathigherpeakcurrentwasachievedusingBRbufferofpH7.0.

The anodic peak current at pH 7 (BR buffer) was found 19.58µA and at pH6.8 (Phosphatebuffer)was 15.10µA. The linearrelationsbetweenpHandpeakpotential(E)observedinthepHrange7-11 (BR)buffer, and6.4-8.0phosphatebufferwasusedtoapplyNernestequationasshown inFigures 2b and 3bwitha regression equation: E(V)=−0.053 pH+1.465, R2 (correlationcoefficient=0.9974 and E(V)=−0.0695 pH+1.548, R2=0.9977 incaseofBRandphosphatebuffers,respectively.ApplyingNernstequation using the formula ∆Ep/∆pH (Slope)=0.059x/n, wherex and n is the number of protons and electrons involved inthe reaction, respectively. It can be concluded that number ofelectronstransferredisequaltothenumberofprotons,thusn=x[34,35].

Effect of surfactant: A surface-active agent (surfactant) isone which tends to accumulate at a surface or interface. Aprerequisiteforsurfactantstobesurfaceactiveisthepropertyof

l/µA

5

0

-5

-10

-15

-20

-250.4 0.6 0.8 1.0 1.2 1.4

E/V

Figure 1a Cyclicvoltammogramof1.0×10−3 molL−1 articaineHClinBRbufferofpH7.0at CPE,scanrate100mVs−1.

CH3CH3

CH3CH3

CH3CH3

H3CH3C

OO

OO

OO

NHNH

NH

SS

HCl

HCl

H20

N

OH

-2 c, -2 H+

Figure 1b OxidationmechanismofarticaineHCl.

5

0

-5

-10

-15

-20

-25

-300.4 0.6 0.8 1.0 1.2 1.4 1.6

E/V

pH 2pH 3pH 4pH 5pH 6pH 7pH 8pH 9PH 10pH 11

l/µA

Figure 2a Cyclic voltammograms of 1.0 × 10−3 mol L−1 articaineHClinBRbufferpH(2-11)at CPE,scanrate100mVs−1.

1.20

1.15

1.10

1.05

1.00

0.95

0.90

0.85

E/V

6 8 10 12

pH

Figure 2b PlotoftheanodicpeakpotentialasafunctionofpH(BRbuffer).

20

18

16

14

12

10

85 6 7 8 9 10 11 12

pH

l/µA

Figure 2c PlotoftheanodicpeakcurrentasafunctionofpH(BRbuffer).

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thesemoleculestoadsorbattheinterfacebetweenbulkphases,suchasairandwater,oil andwaterorelectrodeand solution.Thedistinct structural featureofa surfactant is thehydrophilicregionof themoleculeor thepolarheadgroupwhichmaybepositive, negative, neutral or zwitterionic and the hydrophobicregion or the tail that consists of one or more hydrocarbonchains,usuallywith6-22carbonatoms,thustheyarealsocalledamphiphiles, i.e., compounds having both polar and nonpolarregionsintheirmolecules.Dependingonthechemicalstructureofthehydrophilicmoietyboundtothehydrophobicportion,thesurfactant may be classified as cationic, anionic, non-ionic orzwitterionic.

Two important properties of surfactants, adsorption atinterface and aggregation into supramolecular structures areadvantageouslyusedinelectrochemistry.Surfactantscanmodifyandcontrolthepropertiesofelectrodesurfaces(Figure 4)[36].

Surfactants were widely used in electroanalytical chemistryto improve sensitivity and selectivity and were applied fordetermination of potassium ferricyanide and dopamine [37],diethylstilbestrol [38], dopamine, uric acid and ascorbic acid[39], uric and ascorbic acids [40], thyroxine [41], moexiprilhydrochloride[33],cefdinir[42],drotaverinehydrochloride[43]andatorvastatin[44].

Theanodicpeakcurrentwasfoundtobe19.58µAat1.095VinBRbufferofpH7.0atCPE(Figure 1a),and39.28µAat1.065V

inpresenceof40µLof1.0x10−2molL−1SDS(Figures 5 and 6) whichenhances thecurrentvalueandshowingcatalyticeffect.SeveralsurfactantsweretriedincludingSDS,DSS,andHSA,whereSDSgavethebestresultswiththehighestanodicpeakcurrentasshowninFigure 6.

Effect of SDS concentration: TheanodicpeakcurrentofarticaineHCl increased gradually upon increasing the concentration ofSDS,howeveritdecreasedwhenSDSconcentrationexceeded8.0×10−5molL−1(40µLof1.0×10−2molL−1SDS),whichwaschosenastheoptimumconcentrationofSDS.TherelationshipbetweenanodicpeakcurrentofarticaineHClandSDSconcentrationwasillustratedinFigures 7a and 7b.

Effect of scan rate: Theeffectofdifferentscanrates(υ)rangingfrom 10 to 250 mV s−1 on the cyclic voltammetric responseof articaine HCl in BR buffer (pH 7.0) was investigated, withincreasing scan rates, the anodic peak was slightly shifted to

5

0

-5

-10

-15

-20

-250.4 0.6 0.8 1.0 1.2 1.4

E/V

pH 6pH 6.4pH 6.8pH 7.2pH 7.4pH 8

l/µA

Figure 3a Cyclic voltammograms of 1.0 × 10−3 mol L−1 articaine HCl in phosphate buffer pH (6-8) atCPE,scanrate100mVs−1.

1.12

1.10

1.08

1.06

1.04

1.02

1.00

0.986.5 7.0 7.5 8.0

pH

E/V

Figure 3b PlotoftheanodicpeakpotentialasafunctionofpH(Phosphatebuffer).

15.5

15.0

14.5

14.0

13.5

13.06.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2

pH

l/µA

Figure 3c PlotoftheanodicpeakcurrentasafunctionofpH(Phosphatebuffer).

electrode

Figure 4 Sideviewsofsurfacemicellesorcylinders.

10

0

-10

-20

-30

-40

-50

0.4 0.6 0.8 1.0 1.2 1.4

E/V

l/µA

Figure 5 Cyclicvoltammogramof1.0×10−3 molL−1 articaineHClinBRbufferofpH7.0with 40µLof1.0×10−2 molL−1SDSatCPE,scanrate100mVs−1.

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the positive potential direction and the peak current increasedremarkablywith increasing scan rates (Figure 8a). Itwas foundthatthe logarithmofanodicpeakcurrent (log I) is lineartothelogarithmofscanrate(logυ),withthelinearregressionequationaslogI=0.59logυ+0.421,R2=0.9966.Fromthevalueoftheslope,0.59,itcanbededucedthattheelectrochemicaloxidationprocessof articaine HCl at CPE is diffusion controlled process with an

adsorptioncontribution(Figure 8b)[45].

Effect of accumulation time: Itwasfoundthatthepeakcurrentdependedontheaccumulationtime(Tacc)of1.0×10−3molL−1 articaineHClatCPE inBRbuffer (pH7.0)and40µLSDS (1.0×10−2 mol L−1). Sharp increase was observed at 5 s reaching itsmaximum value, and then decreasedwith increasing time. 5 swaschosenastheoptimumTaccfordeterminationofarticaineHCl(Figures 9a and 9b).

Simultaneous determination of articaine HCl and epinephrineFigure 10a shows the cyclic voltamogramof 1.0 × 10−3mol L−1 epinephrineatCPEinBRbuffer(pH7.0)containing40µLSDS(1.0×10−2molL−1).Figure 10b exhibitstheoxidationmechanismofepinephrine[34].Figure 10cpresentsthecyclicvoltammogramof1.0×10−3molL−1articaineandepinephrineatCPEusingthesame conditions referring to well defined separate peaks ofarticaineandepinephrinewereobtained,thereforethesedrugscanbedeterminedsimultaneously.

Method validationLinearity: Linear relationships were found between the peakcurrentsandconcentrationsofthetwodrugsintherangeof1.0×10−6-2.6×10−5molL−1(0.320-8.34µgmL−1)forarticaineHCland(0.183-4.763µgmL-1) forepinephrineusingtheproposedmethodasshowninFigure 11.Themeanpercentagerecoverieswereof

10

0

-10

-20

-30

-40

-500.4 0.6 0.8 1.0 1.2 1.4

E/V

40 µL 0.01 mol L-1 SDS40 µL 0.01 mol L-1-1 DSS40 µL 0.01 mol L-1-1 HSA

l/µA

Figure 6 Comparisonofdifferenttypesofsurfactantsused.

10

0

-10

-20

-30

-40

-500.4 0.6 0.8 1.0 1.2 1.4

E/V

0 µL10 µL20 µL30 µL40 µL50 µL70 µL

l/µA

Figure 7a Cyclicvoltammogramsof1.0×10−3 molL−1 articaineHClatCPEinBRbufferofpH 7.0asafunctionofSDSconcentrationatscanrate100mVs−1.

45

40

35

30

25

20

150 20 40 60 80

µL of 0.01 mol L-1 of SDS

l/µA

Figure 7b Plot of the anodic peak current versus SDSconcentration.

20

0

-20

-40

-60

-80

0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3

E/V

10 mV s-1

25 mV s-1

50 mV s-1

75 mV s-1

100 mV s-1

150 mV s-1

200 mV s-1

250 mV s-1

l/µA

Figure 8a Cyclic voltammogramsof 1.0 × 10−3 mol L−1 articaineHClatCPEinBRbuffer(pH 7.0)and40µLSDS(1.0×10−2molL−1)asafunctionofscanrate.

2.0

1.8

1.6

1.4

1.2

1.0

0.80.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6

log υ

log

l

Figure 8b Plotoflog(I)versuslog(υ).

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98.1±1.96and98.2±1.94 for articaineHCl andepinephrine,respectively. The proposed method is more sensitive thanspectrophotmetricmethod for articaineHCl 10-70µg/mL [10],spectrophotometric methods for epinephrine: 2-20 mg mL-1

[20],1.5-30,3.0-30,and1.5-25μmolL-1[21],0.4-12.8mgmL-1 [22],1.143-142.90μgmL-1 [23],and1.8-35.3μgmL-1 [24],andelectrochemical methods for epinephrine: 3.0-175.0 μmol L−1 [26], and 1.0 × 10−5 to 1.0 × 10−3 mol L−1 [29]. The regressionparameterswerecomputedandpresentedinTable 1.

LODandLOQ:Thelimitsofdetection(LOD)andofquantification(LOQ)weredeterminedaccordingtoICH[46]usingthestandarddeviation of multiple blank samples and the slope of the

0

-20

-40

-60

-80

0.4 0.6 0.8 1.0 1.2 1.4

E/V

zero s5s10 s15 s20 s25 s30 s35 s40 s

50 s55 s60 s

45 s

l/µA

Figure 9a Cyclic voltammograms of 1.0 × 10−3 mol L−1 articaineHClatCPEinBRbuffer(pH 7.0)and40µLSDS(1.0×10−2molL−1)atscanrate100mV s−1 as a function of accumulation timefrom0to60s.

65

60

55

50

45

40

350 10 20 30 40 50 60 70

Tacc/s

l/µA

Figure 9b Plot of the anodic peak current versusaccumulationtime.

30

20

10

0

-10

-20

-30

E/V-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8

l/µA

Figure 10a Cyclicvoltammogramof1.0×10−3 molL−1 epinephrineinBRbufferofpH7.0and 40µLSDS(1.0×10−2molL−1)atCPE,scanrate100mVs−1.

HO

OH

OHH H

H HNNCH3

CH3

Epincphrinc

-2 e, -2 H+

+2 e, +2 H+

O

O

OH

Figure 10b OxidationmechanismofepinephrineatCPE

20

10

0

-10

-20

-30

-40

-50

l/µA

-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

E/V

Figure 10c Cyclicvoltammogramof1.0×10−3 molL−1 articaineHClandepinephrineinBR bufferofpH7.0and40µLSDS(1.0×10−2molL−1)atCPE,scanrate100mVs−1.

20

18

16

14

12

10

8

6

4

2

l/µA

0 5 10 15 20 25

Conc/µ mol L-1

Figure 11a Calibration curve of the anodic peak current to thecorrespondingconcentrationofarticaineHCl.

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calibrationcurve.Thelimitsofdetectionandquantificationwerefound2.88×10−7molL−1and8.72×10−7molL−1forarticaineHCland5.10×10−8molL−1and1.56×10−7forepinephrine,respectively(Table 1).

RobustnessTherobustnessofproposedDPVmethodusing6.0×10−6molL−1 ofthetwodrugswasevaluatedbyvariationintheconcentrationofSDSandpHofBRbuffer.Therelativestandarddeviation(%RSD)andRecovery(%R)valueslistedinTable 2indicatethattheDPVmethodwasnotaffectedbydeliberatechangesintheoptimumparametersofthemethod.

Accuracy and precisionIntradayandinterdayaccuracy(%R)rangedfrom99.3%to101.7%andprecision(%RSD)from0.23%to1.49%forarticaineHClandfrom98.3%to101.5%andfrom0.58%to1.78%forepinephrine(Table 3).

Application to pharmaceutical formulationThe proposed method was applied successfully for thedeterminationofarticaineHCLandepinephrineinpharmaceuticaldosage form in thepresenceofexcipientsandadditives in thesame concentration range as in the bulk without interferencewitharecoveryof100.4±0.65and99.2±0.71,respectively.Thestandardadditiontechniquewasfurtherusedtodeterminetherecoveryoftheproposedmethod (Table 4).

25

20

15

10

5

00 5 10 15 20 25

Conc/µ mol L-1

l/µA

Figure 11b Calibration curve of the anodic peak current to thecorrespondingconcentrationofepinephrine.

Parameter Articaine HCl EpinephrineLinearityrange(mol

L−1) 1.0×10−6-2.6×10−5 1.0×10−6-2.6×10−5

Slope±SD* 0.631±0.57 0.834±0.49Intercept±SD* 2.637±0.055 1.370±0.013

R2 0.9998 0.9995LOD(molL−1) 2.88×10−7 5.10×10−8

LOQ(molL−1) 8.72×10−7 1.56×10−7

Table 1 Regression parameters for the determination of articaineHClandepinephrineby proposedelectrochemicalmethod.

*Averageofthreedeterminationsateachlevel

ParameterArticaine HCl Epinephrine

%RSD* %R* %RSD* %R*pH6.8 0.98 100.8 0.53 99.15pH7.2 0.14 98.9 0.38 99.7038μ 0.63 101.3 1.21 100.3542μ 1.52 100.8 0.85 101.6

*Averageofthreedeterminationsateachlevel.

Table 2 RobustnessresultsoftheproposedDPVmethod.

ProcedureTaken (μmol

L−1)

Intraday InterdayFound(μmol

L−1)%R* %RSD*

Found(μmol

L−1)%R* %RSD*

Articaine HCl2 2.03 101.5 1.03 2.01 100.5 1.498 7.94 99.3 1.1 8.07 100.9 1.1

12 12.04 100.3 1.25 12.2 101.7 0.23

Epinephrine1 0.991 99.1 0.86 0.983 98.3 1.128 8.04 100.5 1.41 8.12 101.5 0.68

12 12.15 101.3 0.58 11.8 98.3 1.78

*Averageofthreedeterminationsateachlevel.

Table 3 IntradayandInterdayaccuracyandprecisionforthesimultaneousdetermination of articaine HCl and epinephrine by the proposedelectrochemicalmethod.

Statistical analysis of the results obtained by the proposedmethod compared with a reported method [47] revealed nosignificant difference between the proposed and reportedmethods, where the calculated t- and F- values are less thanthe tabulated ones, confirming accuracy and precision at 95%confidence limit [48]. However, the proposed method allowssimultaneousdeterminationofbothdrugs,thereportedmethoddeterminesbothdrugsseparately(Table 5).

The reported method involved determination of articaine HCland epinephrine by HPLC; articaine HCl was determined usingmobilephase(600mLacetonitrileand400mL[1.3mLfrom1.0mol L−1 sodium phosphate+32.5mL from 0.5mol L−1 disodiumphosphatein1000mLwithdistilledwater(pH8.0)],lichrospher100RP-18(5µm×250mm×4mm)ascolumnandwavelengthof240nm.Epinephrinewasdeterminedusinggradientmobilephase [A: methanol, B: 0.42% w/v tetramethylammoniumhydrogensulphate+0.116%w/vsodiumheptanesulfonate+0.21%v/vdisodiumedetateatpH3.5with40%NaOH],KromacilC185µm×4mm×150mmandwavelengthof205nm.

Simultaneous determination of articaine HCl and epinephrine in spiked urine samplesSuccessiveadditionsofarticaineHClandepinephrinesolutions(1.0×10−3molL−1)coveringthelinearityrangeof(1.0×10−6-2.6×10−5mol L−1)wereadded to thevoltammetric cell containing5mL of diluted urine and 40µL of 1.0 × 10-2mol L−1 SDS, thevoltammogramswererecordedatascanrateof20mVs−1usingDPVatcarbonpasteelectrodewithpercentagerecoverieswerecalculatedfromregressionequation(Table 6).

ConclusionIn the present work a simple, sensitive, accurate and preciseelectroanalytical voltammetric method was developed for

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simultaneous determination of articaine HCl and epinephrinein bulk, pharmaceutical dosage forms and spikedhumanurinebasedontheelectrochemicaloxidationofthesedrugsatcarbonpasteelectrodeinBRbufferofpH7.0inthepresenceofsodiumdodecyl sulphate, which allows to use the proposed methodfor routine quality control applications for articaine HCl andepinephrine.

Articaine HCl Epinephrine

Mean ± RSD Standard addition Mean ± RSD Standard addition

Artinibsa® carpules

Claimed taken (μmol L−1)

Pure added(μmol L−1) %R of added

Artiniba® carpules

Claimed taken(μmol L−1)

Pure added(μmol L−1) %R of added

6.0

2 100.6

1.0

1 99.84 100.6 3 98.86 100.3 5 98.88 101.3 7 98.5

12 99.4 11 99.618 99.9 15 98.2

25 100.2

Mean±RSD 100.4±0.65 Mean±RSD 99.2±0.71

Table 4 ApplicationofstandardadditiontechniqueforthesimultaneousdeterminationofarticaineHClandepinephrineinArtinibsa®carpulesbytheproposedelectrochemicalmethod.

ParametersArticaine HCl Epinephrine

Proposed method Reported method [47] Proposed method Reported method [47]Linearityrange(μgmL−1) 0.32-8.34 20-70 0.183-4.763 1.5-4.0N(numberofreplicates) 9 6 9 6

Mean% 98.1 99.8 98.2 99.5SD* 1.96 1.38 1.94 1.17

Variance 3.84 1.9 3.76 1.37t-test 1.95(2.77) 1.61(2.77)F-test 1.95(6.39) 2.74(6.39)

*Averageofthreedeterminationsateachlevel;FiguresinparenthesisaretheoreticaltandFvaluesat95%confidencelevel.

Table 5 Results obtained by the proposed electrochemical method compared with the reportedmethod for the analysis of articaine HCl andepinephrineinArtinibsa®carpules.

Concentration (μmol L−1) %R Articaine HCl %R Epinephrine2 102.2 98.43 97.3 101.24 99.5 97.26 100.3 99.28 98.5 102.3

12 100.8 101.018 99.7 99.326 98.5 100.8

Mean ±RSD 99.6±1.53 99.9±1.68

Table 6 SimultaneousdeterminationofarticaineHClandepinephrineinspikedurinesamples.

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