International Journal of Chemistry Research

Vol 1, Issue 1, 2010

ResearchArticle

DEVELOPMENTANDVALIDATIONOFAUVVISSPECTROPHOTOMETRICMETHODFORTHE
ESTIMATIONANDDEGRADATIONMONITORINGOFCEFADROXILINBULKAND
PHARMACEUTICALDOSAGEFORMS

*SUDDHASATTYADEY,K.KALYANI,B.SAMYUKTHA,SUDHIRKUMARSAHOO,SUBHASISMOHAPATRA,P.N.

MURTHY,DHIRAJKUMAR

GuruNanakInstituteofPharmacy,Hyderabad,RoyalCollegeofPharmacyandHealthSciences,Berhampur.
Email:kuntal.kuni@gmail.com
Received:28April2010,RevisedandAccepted:19May2010
ABSTRACT
The aim of the present work is to develop a simple accurate, precise and cost effective UVVis spectrophotometric method for the estimation of
cefadroxil, a first generation cephalosporin an antibiotic drug, in bulk and pharmaceutical dosage form. The solvent used was methanol and
distilledwater(50:50)andthemaxortheabsorptionmaximaofthedrugwasfoundtobe264nm.Alinearresponsewasobservedintherangeof
1050g/ml with a regression coefficient of 0.9999. The method was then validated for different parameters as per the I.C.H. (International
ConferenceforHarmonization)guidelines.Thismethodcanbeusedforthedeterminationofcefadroxilinqualitycontrolofformulation without
interference of the excipients. Cefadroxil was subjected to stress degradation under different conditions recommended by ICH. The samples
generatedwereusedfordegradationstudiesusingthedevelopedmethod.
Keywords:Cefadroxil,antibiotic,max,ICH,UVVisspectroscopy

INTRODUCTION
Cefadroxil chemically a 7[[2amino 2(4hydroxyphenyl) acetyl]
amino]3methyl 8oxo5thia1azabicyclo [4.2.0] oct2ene2
carboxylicacid1

water (50:50) which was of AR grade, purchased from SD Fine

ChemicalsLimited,Indiaanddoubledistilledwater.
UVmethoddevelopment
Solubilitytest
Solubility test for the drug cefadroxil was performed by using
various solvents. The solvents include Water, Methanol, Ethanol,
Acetonitrile, Hydrochloric Acid (HCl), Sodium Hydroxide (NaOH)
andChloroform.
Determinationofmax
Preparationofstocksolution

Cefadroxil, a firstgeneration cephalosporin antibiotic, is used to

treat urinary tract infections, skin and skin structure infections,
pharyngitisandtonsillitis.Likeallbetalactamantibiotics,cefadroxil
binds to specific penicillinbinding proteins (PBPs) located inside
the bacterial cell wall, causing the inhibition of the third and last
stage of bacterial cell wall synthesis. Cell lysis is then mediated by
bacterialcellwallautolyticenzymessuchasautolysins;itispossible
that cefadroxil interferes with an autolysin inhibitor. Literature
survey revealed that cefadroxil was qualitatively assayed in
biological fluids either individually or in presence of other
antibacterial drugs using liquid chromatography 5, other new
methodsandusinghydrotopearealsothereforthedeterminationof
cefadroxil 2,6,7. However no UV spectrophotometry method was
proposedforthe estimationofcefadroxilwithoutusinghydrotope2
inbulkandpharmaceuticaldosageforms.Theaimofthisworkisto
develop and validate an analytical method by using UV
spectrophotometry for the estimation of cefadroxil in bulk and
pharmaceuticaldosageformsandalsoperformdegradationstudies
onthedrugasperICHguidelinesusingtheproposedmethod3,4
MATERIALSANDMETHODS
The drug, Cefadroxil is a gift of Cipla, Goa branch. The instrument
used for the present study was a UVVis double beam
spectrophotometer(modelT60,AnalyticalTechnologicalsLtd.)with
1cm matched pair quartz cell. The solvent used was methanol and

Standard stock solution of cefadroxil was prepared by dissolving

10mgofcefadroxilin10mlofmethanolanddistilledwater(50:50)
whichgives1000g/ml.Onemlofthisstocksolutionwastakenand
was diluted up to 10ml by using methanol and distilled water
(50:50)toproduceaconcentrationof100g/mlsolution.
Preparationofworkingsolution
From the above stock solution 2ml was transferred into 10ml
volumetric flask and volume was made up to the mark with
methanoltomake20g/ml.ThenthesamplewasscannedwithUV
Vis Spectrophotometer in the range 200400nm against methanol
and distilled water (50:50) as blank and the wavelength
correspondingtomaximumabsorbancewasnotedwhichisitsmax
i.e.at264nm(fig.1).
Preparationofcalibrationcurve
One ml of this 100g/ml solution was further diluted and the
volumewasmadeupto10mlbyusingmethodtoproduce10g/ml
solution.2ml,3ml,4mland5mlof100g/mlsolutionwerediluted
and the volume was made up to 10ml using methanol to produce
20g/ml, 30g/ml, 40g/ml, 50g/ml solutions respectively. Then
the constructionofcalibrationcurvewas done bytakingtheabove
prepared solutions of different concentration ranging from 10
50g/ml.Thentakingtheabsorbancecalibrationcurvewasplotted
taking concentration on xaxis and absorbance on yaxis which
showedastraightline(infig.2).Thisstraightlineobeyedlinearityin
theconcentrationrangeof1050g/ml.Thecorrelation coefficient
wasfoundtobe0.9999.

IntJChemRes,Vol1,issue1(2010),2934

Methodvalidation

AssayofCefadroxiltablet(DROXIL250mg)

Validationisaprocess ofestablishing documentedevidence,which

provides a high degree of assurance that a specific activity will
consistently produce a desired result or product meeting its
predeterminedspecificationsandqualitycharacteristics 3.

Aquantityofpowderequivalentto50mgofcefadroxilwastakenin
a 50ml volumetric flask and it was dissolved and diluted upto the
mark with methanol and water. The resultant solution was ultra
sonicated for 5 minutes. The solution was then filtered using
Whatmann filter paper No. 40. From the filtrate, appropriate
dilutions were made in methanol and water (50:50) to obtain the
desired concentration (50 g/ml). This solution was then analysed
inUVandtheresultwasindicatedby%recoverygivenintable1.

The validation for UV method development was performed using

parameters like Linearity, Accuracy, Precision, Robustness,
Ruggedness, and Limit of detection (LOD), Limit of quantification
(LOQ).
Linearity

Degradationstudies

Various aliquots were prepared from the secondary stock solution

(100g/ml)rangingfrom1050g/ml.Thesampleswerescanned
in UVVis Spectrophotometer against methanol and distilled water
(50:50)asblank.Itwasfoundthattheselecteddrugshowslinearity
betweentherangesof1050g/ml(table1&5).

The International Conference on Harmonization (ICH) guideline

entitled stability testing of new drug substances and products
requiresthat stresstesting becarriedout toelucidate theinherent
stabilitycharacteristicsoftheactivesubstance.Theaimofthiswork
was to perform the stress degradation studies on the cefadroxil
usingtheproposedmethod4.

Accuracy

Hydrolyticdegradationunderacidiccondition

Solutionswerepreparedintriplicateatlevels80%,100%and120%
of test concentration using cefadroxil working Standard as per the
testmethodandtakenabsorbanceofeachsolutionintriplicate.

To2mlofstocksolution(1000g/ml)ofCefadroxil,1mlof3NHCl
wasaddedin10mlofvolumetricflaskandthevolumewasmade
up to the mark with methanol and water (50:50). Then, the
volumetricflaskwaskeptatnormalconditionfor90minutes.After
90 min. time interval, 1 ml of solution was pipetted out from this
flask, neutralized and diluted with methanol and water (50:50) in
ordertomakethevolumeupto10mlandthedilutionwascarried
out to achieve the appropriate concentration (20 g/ml). This
solution was taken in cuvette. For the blank, 0.5 ml solution of 3N
HCland0.5mlsolutionof3NNaOHweredilutedwithmethanolin
10mlofvolumetricflaskwasrepeated(tableno.2&fig.no.3).

The recovery results showed that the proposed method has an

acceptable level of accuracy for cefadroxil which is from 80%
120%oftestconcentrationisform99.51%100.01%(table1&5).
Precision
Precisionofthemethodwasdemonstratedbyintradayandinterday
variationstudies.Inintradayvariationstudyninedifferentsolutions
ofsameconcentration20g/mlwereanalyzedthreetimesinaday
i.e. from morning, afternoon and evening and the absorbance is
noted. From the absorbance result mean, standard deviation and
%RSDwascalculatedandgivenintable6&7.
In the interday variation studies, solution of same concentration
20g/ml were analyzed three times for the three consecutive days
andtheabsorbanceresultmean,standarddeviationand%RSDwas
calculatedandgivenintable8.
Specificity
10mgofCefadroxilwasspikedwith50%(5mg),100%(10mg),and
150% (15 mg) of excipient mix (Magnesium Stearate) and the
samplewasanalysedfor%recoveryofCefadroxil(tableno.9).
Robustness
Robustness of the method was determined by carrying out the
analysis under different temperature condition i.e. at room
temperature and at 180c. The respective absorbances of 20g/ml
werenotedandtheresultwasindicatedas%RSDandgivenintable
10.
Ruggedness
Ruggedness of the method was determined by carrying out the
analysis by different analyst and the respective absorbance of 20
g/ml was noted. The result was indicated as %RSD and given in
table10.
LimitofDetection(LOD)
Thelimitofdetection(LOD)wasdeterminedbypreparingsolutions
of different concentrations ranging from 0.10.5 g/ml. The
detection limit of an individual analytical procedure is the lowest
amount of analyte in a sample, which can be detected but not
necessarilyquantificationasanexactvalue(tableno.1).
LimitofQuantification(LOQ)
TheLOQistheconcentrationthatcanbequantificationreliablywith
aspecifiedlevelofaccuracyandprecision.TheLOQwascalculated
using the formula involving standard deviation of response and
slopeofcalibrationcurve(tableno.1).

Hydrolyticdegradationunderalkalinecondition
To2mlofstocksolutionofcefadroxil1mlof0.1NNaOHwasadded
in 10 ml of volumetric flask and made up the volume to the mark
with methanol and water (50:50). Volumetric flask was kept at
normal condition for 90 min. After 90 min time interval, 1 ml of
solution was pipetted out from this flask, neutralized and diluted
withmethanolandwater(50:50)inordertomakethevolumeupto
10mlandthedilutionswerecarriedouttoachievetheappropriate
concentration (20 g/ml). The solution was then taken in cuvette.
Fortheblank,0.5mlsolutionof0.1NHCland0.5mlsolutionof0.1N
NaOHdilutedwithmethanolin10mlofvolumetricflask(tableno.2
&fig.no.4).
Dryheatinduceddegradation
Cefadroxil sample was taken in a petriplate and exposed to a
temperatureof70cfor48hoursinanoven.After48hours,10mg
of the sample was diluted with methanol and water (50:50) in
ordertomakethevolumeupto10ml.Fromthissolution,dilutions
were carried out to achieve the appropriate concentration (20
g/ml)andthesolutionwastakenincuvettefortheUVVisAnalysis
(tableno.2&fig.no.5).
Oxidativedegradation
To1.5mlofthestocksolutionofcefadroxil(1000g/ml),1mlof30
%w/vofhydrogenperoxideaddedin10mlofvolumetricflaskand
the volume was made up to the mark with methanol and water
(50:50).Thevolumetricflaskwasthenkeptatroomtemperaturefor
15 min.Forthe blank, 1 mlofthe 30 %w/v ofhydrogenperoxide
was kept at normal condition for overnight in 10 ml of volumetric
flask. Both solutions were heated on boiling water bath to remove
the excess of hydrogen peroxide. Finally after 15 minutes dilutions
were made from the stock solution to achieve the required
concentration(30g/ml).Thesolutionwasthentakeninacuvette
andanalyzed(tableno.2&fig.no.6).
Photolyticdegradation
Sample of cefadroxil was exposed to near ultra violet lamp in
photostablity chamber providing illumination of not less than 1.2
million lux hours. Ten milligrams sample was dissolved methanol
2
30

IntJChemRes,Vol1,issue1(2010),2934

andwater(50:50)andvolumemadeupto10ml.Fromthissolution
appropriatedilution(20g/ml)wasmadeusingmethanolandwater
(50:50)andtakenincuvettefortheUVanalysis(tableno.2&fig.no.7)

insubmicrogramlevelindicatingthesensitivityofthemethod.The
methodwasalsofoundtoberobustandruggedasindicatedbythe
% RSD values which are less than 2 %. The results of assay show
thattheamountofdrugwasingoodagreementwiththelabelclaim
oftheformulationasindicatedby%recovery(101.8%).Summary
ofvalidationparametersofproposedspectrophotometricmethodis
shown in table 1. The stress degradation studies showed that
Cefadroxil undergoes degradation in acidic, oxidation and alkaline
conditionswhereasitisrelatively stablewhenexposed todry heat
and photolytic conditions. Summary of the results of stress
degradationstudiesofCefadroxilareshowninthetable2.

RESULTSANDDISCUSSION
Thedevelopedmethodwasfoundtobepreciseasthe%RSDvalues
for intraday and interday were found to be less than 2%. Good
recoveries(99.97%to101.4%)ofthedrugwereobtainedateach
added concentration, indicating that the method was accurate. The
methodwasalsofoundtobespecificindicatedbythe%recoveries
rangingfrom98.2%101.2%.TheLODandLOQwerefoundtobe

Table1:Summaryofvalidation
Parameter
Linearityindicatedbycorrelationcoefficient
Precisionindicatedby%RSD
Accuracyindicatedby%recovery
Specificityindicatedby%recovery
LimitofDetection
LimitofQuantification
Range
Linearregressionequation
Robustnessindicatedby%RSD
Assayindicatedby%recovery

Result
0.9999
0.26%
99.9044%
100%
0.25g/ml
0.825g/ml
1050g/ml
0.033x0.004
0.162%
99.98%

Table2:Summaryofresultofstressdegradationstudies
R4b
AcidicHydrolysis
AlkalineHydrolysis
30%HydrogenPeroxide(1ml)
DryHeat70
Photolytic

Time
90min
90min
15min
48hr
3hr

%Degradation
maxshifted
maxshifted
maxshifted
8.9%
5.15%

Table3:LinearityTableofCefadroxilinWorkingStandard
Absorbance
0.338
0.663
0.989
1.378
1.672

Concentration(g/ml)
10
20
30
40
50

Fig.1:maxofCefadroxilshowingat264nm,Peak1264.000.660

conc.
0
10
20
30
40
50

Abs
0
0.338
0.663
0.989
1.378
1.672

3
31

IntJChemRes,Vol1,issue1(2010),2934

Fig.2:CalibrationcurveofCefadroxil

Table4:Opticalcharacteristics
Beerslawlimit(g/mL)
Molarextinctioncoefficient(1mole1c.m1)
Correlationcoefficient
Regressionequation(Y*)
Slope(a)
Intercept(b)

1050g/ml
338
0.9999
0.033x0.004
0.033x
0.004

Table5:AccuracyreadingsofCefadroxil

No.ofpreparations
S1:80%
S2:80%
S3:80%
S4:100%
S5:100%
S6:100%
S7:120%
S8:120%
S9:120%

Concentration(g/lml)
Formulation
Puredrug
10
8
10
8
10
8
10
10
10
10
10
10
10
12
10
12
10
12

%Recovery

Statisticalresults
Mean
SD

99.58
1.04

100.67
0.66

99.46
0.90

99.24
98.75
100.75
100.9
99.93
101.18
98.98
98.91
100.5

%RSD
1.04

0.65

0.90

Table6:PrecisionresultsshowingrepeatabilityofCefadroxil
Absorbance
0.660
0.661
0.662
0.660
0.661
0.661
0.661
0.661
0.660
0.661

Concentrations(g/ml)
20
20
20
20
20
20
20
20
20
20

Statisticalanalysis

Mean=0.6608
SD=0.000632
%RSD=0.095

Table7:Intraassayprecision
Concentrations(g/ml)
20
20
20
20
20
20
20
20
20
%RSD

Absorbance1
0.660
0.658
0.661
0.660
0.663
0.661
0.661
0.663
0.659
0.25

Absorbance2
0.661
0.661
0.660
0.658
0.658
0.662
0.660
0.660
0.659
0.20

Absorbance3
0.661
0.661
0.663
0.658
0.661
0.660
0.660
0.661
0.661
0.20

Average%RSD

0.21%

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IntJChemRes,Vol1,issue1(2010),2934

Table8:Interassayprecision
Concentrations
(g/ml)
20

Average%RSD

%RSD
Day1
0.165%

Day2
0.28%

Day3
0.32%

0.255%

Table9:Testforspecificityshowingnoeffectofexcipient.
Sample
No.
1
2
3

Excipient
conc.(%)
100%
50%
150%

Cefadroxil
input(mg)
10
10
10

Cefadroxil
recovered(mg)
9.8
10.4
10.12

Cefadroxil
recovered(%)
98.4
100.4
101.2

Meanrecovered
(%)

100%

S.D.

1.44

%R.S.D.

1.44

Table10:Resultsshowingrobustness&ruggednessofmethodforCefadroxil
Analyst1
Conc.
Abs.
(g/ml)
20
0.661
20
0.661
20
0.660
20
0.660
20
0.663
20
0.661
RoomTemperature
Conc.
Abs.
(g/ml)
20
0.661
20
0.661
20
0.660
20
0.658
20
0.659
20
0.660

StatisticalAnalysis
Mean=0.661

SD=0.0011

%RSD=0.16
StatisticalAnalysis
Mean=0.6598

SD=0.0012

%RSD=0.17

Analyst2
Conc.
Abs.
(g/ml)
20
0.661
20
0.661
20
0.662
20
0.662
20
0.661
20
0.663
Temp.18oC
Conc.
Abs.
(g/ml)
20
0.661
20
0.663
20
0.661
20
0.659
20
0.661
20
0.660

StatisticalAnalysis
Mean=0.6617

SD=0.0008

%RSD=0.12
StatisticalAnalysis
Mean=0.6608

SD=0.0013

%RSD=0.20

Fig.3:ComparisonbetweenstandardCefadroxil(20g/ml)&acidDegradedsampleofCefadroxil(20g/ml)after90minutes.Druggot
degradedanditsmaxshifted

Fig.4:ComparisonbetweenstandardCefadroxil(20g/ml)&alkalidegradedsampleofCefadroxil(20g/ml)after90minutes.Druggot
degradedanditsmaxshifted.
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IntJChemRes,Vol1,issue1(2010),2934

Fig.5:ComparisonbetweenstandardCefadroxil(20g/ml)&temperaturedegradedsampleofCefadroxil(20g/ml).Druggotdegraded
by8.9%whenexposedtoatempof70cfor48hours

Fig.6:ComparisonbetweenstandardCefadroxil(20g/ml)&OxidisedsampleofCefadroxil(20g/ml).Druggottotallydegradedandits
maxshifted

Fig.7:ComparisonbetweenstandardCefadroxil(20g/ml)&UVdegradedsampleofCefadroxil(20g/ml)after3hoursDrugwhen
exposedtoUVlightfor3hrs,gotdegradedby5.15%
CONCLUSION

2.

All the above factors lead to the conclusion that the proposed
method is accurate, precise, simple, sensitive, robust and cost
effective and can be applied successfully for the estimation of
cefadroxil in bulk and pharmaceutical formulation. The proposed
method is also useful for determination of cefadroxil stability in
sampleofpharmaceuticaldosageforms.

3.
4.

ACKNOWLEDGEMENT
I Mr.Suddhasattya Dey is very much thankfull to Prof. Vivek V.
Bhayatti, Principal, Guru Nanak Institute of Pharmacy, Hyderabad
for providing the necessary chemicals for our work. I am also
thankful to Associate Prof. S.A.Srinivas and Asst. Prof.
S.Vaidiyanathanforgivingvaluablesuggestionstome.
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Blanchin MD, Kok WT and Fabre H. New detection modes for
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