Research Article

[Nagaraja ET

AL.,

3(10): Oct., 2012]

CODEN (USA): IJPLCP

ISSN: 0976-7126

INTERNATIONAL JOURNAL OF PHARMACY & LIFE SCIENCES

Formulation and evaluation of ofloxacin aqueous injection

Y.S.Nagaraja1,T.S.Nagaraja2.,D.R.Bharathi2andT.O.Manjunatha2
S.J.M.CollegeofPharmacy,Chitradurga,(Karnataka)India

Abstract

Ofloxacinisasyntheticfluoroquinolonebroadspectrumantimicrobialagentusedinthetreatmentofbacterial
infectionsanditispresentlyavailableinthemarketonlyastabletdosageform.Itispreferredinthetreatmentof
adultswithcommunityacquiredpneumoniaandacutebacterialexacerbationsofchronicbronchitiscausedby
susceptibleorganism.Thepresentstudywasundertakenwithanintentiontodevelopastableandeffective
parenteralformulation,containingthedrugOfloxacin.Ofloxacinisalightsensitiveandwatersolubledrugbut
unstableathighertemperatureinwater.SotheeffectsofvariouscosolventsinthesolubilityofOfloxacinhavebeen
evaluated.OfloxacinwastriedwithcosolventssuchasPEG200,Span20andGlycerin.Thedrugwasmadeinto
injectionformulationforadministeredasaSVP.VariousbatchesofOfloxacininjectionformulationwereprepared
inordertoassesstheinfluenceofheat,light,atmosphericoxygenandantioxidantonthestabilityofthedrugandthe
formulationswerealsosubjectedtoacceleratedstabilitytest.Outofalltrials,formulationcontaining45%ofPEG
200wasfoundtobemorestableandpassedalltestssatisfactorily.

KeyWords:Ofloxacin,Fluoroquinolone,Parenteralformulation,SVP,Acceleratedstability

Introduction

Injectionsincludeawidevarietyoftherapeuticagents,
e.g.,forthetreatmentofcancer,infections,
cardiovasculardiseases,arthritis,inflammatory
diseases,diabetes,hormonaldeficienciesandmany
otherdiseasestatesincludinglifethreatening

emergencyconditions.Therearemorethan400
injectionsproductslistedintheUSPand,becauseof
thehugenumberofbiotechnologymoleculesin
clinicalstudy,thisnumberwillcontinuetogrow
rapidlyoverthenextseveralyears.About80%or
greaterofallSVPscommerciallyavailableare
preparedbyasepticprocessing.LVPsusuallyinvolve
intravenousinfusion,dialysis,orirrigationfluids
containingelectrolytes,sugar,aminoacids,blood,
bloodproducts,andfattylipidemulsions.SVP
formulationsaresimpleformulationscomparedwith
otherpharmaceuticaldosageforms,composedof
activeingredients,solventsystem(preferably
aqueous),minimalnumberofexcipients,inthe
appropriatecontainerandclosurepackagingsystem.
Formulationscientistshavesevererestrictionsin
numberandchoiceofaddedsubstancesbecauseof
safetyconsiderations1.

Ontheotherhand,ifthefishfarmerscanproducean
Ofloxacinismemberoffluoroquinoloneclassof
antimicrobialdrugs.Itisactiveagainstawiderangeof
Gram+veandGramveorganisms.Ofloxacinis
*CorrespondingAuthor
E.mail:nagraj.ys@gmail.com

preferredinthetreatmentofadultswithacute
exacerbationsofchronicbronchitisandcommunity
acquiredpneumoniacausedbysusceptibleorganisms.
ItisthemostpotentfluoroquinoloneagainstS.
pneumoniaeanddemonstratesexcellentinvitro
activityversuspenicillin,macrolide,cephalosporin,
andquinoloneresistantstrains.Ofloxacinretainsgood
activityagainstGramnegativeorganismsandisactive
againstatypicalpathogensalthoughithasshowntobe
effectiveintreatmentofRTIs.

Theaimofthepresentstudyistoformulateand
evaluatetheparenteraldosageformcontaining
Ofloxacin.Theobjectivesofthestudyare,tostudythe
solubilitybehaviorofthedrugindifferentsolvents,to
developananalyticalmethodforassayofOfloxacin,to
designandformulateastableparenteralformulationof
Ofloxacin,toevaluatepreparedparenteralformulations
ofOfloxacin.

MaterialandMethods25
PreformulationStudies

SolubilitystudiesofOfloxacinindifferentsolvents:

Excessofdrugwasaddedtodifferentsolventsin10ml
stopperedvolumetricflasks.ThenDrugwasmadeto
dissolveinthesolventbyplacingthevolumetricflask
intheshakerbathat25Cfor6hours.Thevolumetric

Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 3, Issue 10: October: 2012, 2015-2020
2015
Research Article

dilutionsweremadetomeasureabsorbancesat300nm
usingUVvisiblespectrophotometer,andwateras
blank.ThedataaregiveninTable2.

CODEN (USA): IJPLCP

flaskswerethenplacedatroomtemperaturefor24
hours.Thesolutionswerefilteredandappropriate

EffectofTemperatureonStabilityofDrug:1%
OfloxacinsolutioninPEG200isfilledintovials.The
vialsweresealedandplacedatrefrigeration,room

temperature,50C,75Cand95Cfor1weekand
observedforcolourchangeandcrystalgrowth.The
samplesplacedatrefrigerationandroomtemperature
servedascontrols.ThedataaregiveninTable3.Light
StabilityofDrug:1%ofOfloxacinsolutionin20%
PEG200isfilledinto20mlglassvials(amberand
clear).Alsosamplesofdrugsubstanceareplacedinan
openPetridishtoexposealargesurface.Drugand
dilutionsplacedinalightresistantambercoloured
glassvials,foilwrappedandinacardboardboxas
controls.Thisiscarriedoutfor4weekswithweekly
examinationsforvisiblecolourchangeorprecipitation
insolutioninclearvials,thecompoundcanbe
consideredaspotentiallylightsensitiveandshouldbe
handledaccordingly.ThedataaregiveninTable4.

C2
C3
C4
C5
C6

Ofloxacin
25
25
25

EffectofOxygenonDrug:1%ofOfloxacininPEG
200isfilledintovialsandplacedat30Cand40C.
Onegroupispurgedandanothergroupissealedwith
air.Solutionsareobservedforcolourchangeanddrug
content.ThedataaregiveninTable5and6.

25
25
25

Formulationdevelopment

Attemptsweremadetodevelopastableparenteral
formulationusingcosolvent/salongwithother
excipients.Thedoseselectedforformulationwas250
mgofOfloxacinin1mlsolvent.Theprepared
formulationscontainthefollowingingredientsalong
withtheirconcentrationsaregiveninTable1.

Table1:Concentrationofdifferentingredientsused
invarioustrialformulations

PEG200

Ingredien

45

Formulation(%)ingrams

ts

45

C1

Glycerin

Resorcinol

4
4

Benzyl

1.5

0.1

1.5
1.5

Paraben

1.5

98

1.5

98

1.5

98
98

alcohol

98
98

Propyl
0.0
0.0
0.0
0.0
0.0
0.0

Paraben
22
22
22
22
Methyl
0.1

22
22

0.1
0.1
0.1
0.1

[Nagaraja ET

AL.,

3(10): Oct., 2012]

ISSN: 0976-7126

qs
Sodium

qs
qs

Injection

metabisulp
0.1
0.1
0.1
0.1
0.1
0.1

hite

Thuspreparedformulationswereassayedfordrug
contentrespectivelyand10mlofthesewereplacedat
5C,roomtemperature(RT),37C,40Cand45Cfor
sixweeksandobservedforcrystalgrowth,clarity,pH
change,anddrugcontent.

Waterfor
qs
qs
qs

PostformulationevaluationsAssayofFormulations
ReferenceSolutionPreparation

100mlofstockreferencesolutionsforeach
formulationwasprepared.Thecompositionofthe
referencestocksolutionwassimilartothatofthe
respectiveformulationsexcludingthedrugandalso
theyweredilutedsimilarlyastheformulationswere
dilutedusingwater.Thisresultingsolutionisusedas
referencesolution(blank)incomparisonwiththe
preparedformulationstomeasurethe%drugcontent
bymeasuringtheabsorbenciesusingShimadzuUV
Visiblespectrophotometer.TheamountofOfloxacin
wasdeterminedfromstandardcalibrationcurve.The
dataaregiveninTable7.

factor,frompointofviewofsafetyofthepatientbeing
treatedwithandtogetasafeandmaximumtherapeutic
responseofthedrug.

Theprovisionofrapidmeansofqualitycontrol,which
ensuresthatnounexpectedchangesinthestored
productareoccurredlike:Crystalgrowth,pHchanges,
Clarityand%Drugcontent.Thedataaregivenin
Table9to12.

CrystalGrowth
SterilizationStudies:Theinjectionsampleswere
takeninglasssyringe,themembranefilterholderwas
attachedtothesyringe.Aprefilterof1.5micrometers
wasplacedinthisholder,afterwhichfiltersof0.22,
0.45,1.2and1.5micrometerswereplacedsuccessively
andtestedwhethertheinjectionsamplecouldpass
throughthesemembraneornot.Thedataaregivenin
Table8and13.

10mloftheeachpreparedformulationsC1,C5were
placedatrefrigeration,roomtemperature,37C,40C
and45Crespectivelyforsixweeksandobservedfor
crystalgrowth.ThedataaregiveninTable15.

pHChanges
StabilityStudies
10mloftheeachpreparedformulationsC1,C5werekept
atdifferenttemperatures/conditionssuchasrefrigeration,

Foranypharmaceuticaldosageformstabilityofthe
preparedformulationisaverybasicandimportant

roomtemperature,37C,40C,45Cand

Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 3, Issue 10: October: 2012, 2015-2020
2016
Research Article

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underlight.Atregulartimeintervalsthesampleswere
examinedforpHchangesforsixweeksusingadigital
pHmeter.ThedataaregiveninTable14.

Clarity

10mloftheformulationswereplacedatrefrigeration,
roomtemperature,37C,40Cand45Cforsixweeks
andobservedforcolourchangeorturbidity.Thedataare
giveninTable16.

%DrugContent

ThedrugcontentoftheformulationsC1,C5were
determinedbyfollowingthesameproceduresas

mentionedinassay.Theestimatesweredoneatintervals
ofoneweekuptosixweeks.ThedataaregiveninTable
17and18.
at270nm

ResultsandDiscussion

mg/ml
deviation

FTIRspectrumofpureOfloxacin
DMWater
0.736
92.1439
0.108

0.1NaoH
0.292
36.5571
0.003

Ascorbic
0.279
34.9295
0.026

acid

Table2:SolubilityprofileofOfloxacinindifferent
solvents

Solvents
Absorbance*
Conc.
Standard

37.0579
0.008
Nicotinamide
0.058

Span80

7.2613

0.264

0.009

33.0516
0.006

20%PEG
0.295

Urea

36.9327

0.385

0.004

48.20031
0.007

400
20%
0.317
39.6870
0.003

Glycerine

Resorcinol
0.322
40.3129
0.006

Tween80
0.296

Polysarbate

0.744
93.1455

Duration(weeks)

0.003

80

[Nagaraja ET

AL.,

3(10): Oct., 2012]

ISSN: 0976-7126

Stabilityevaluation

Variousstresstestsareperformedonsolidandsolution
samplestoestablishtheeffectofheat,lightandoxygen
onthedrugsubstancestability.

Heatstability

Table3:HeatstabilityprofileofOfloxacin

Temperature(C)

Refrigeration

40

50

Roomtemperature

75

+
1

+Colourchange,Nocolourchange

Lightstability

Table4:LightstabilitystudyofOfloxacin

Observations

Withdrawalweek

Clear
+
Amber

Concentration
Concentration
Clear,+Turbidity

at270nm
ing/ml
inmg/ml

Effectofoxygen

Table5:Testforcolourchangeafteraweek

0.794
9940.53

Temperature

9.9405

Airsealed
Perged
(C)
vials

Formulationdevelopment

vials
25
+

Astableparenteralformulationofwatersolubledrug
Ofloxacinwasformulatedafterperformingtrialswith
varioussolvents.Thuspreparedformulationswere
subjectedforvarioustestsandresultsarediscussedin
thefollowingsection.

30
+
+

Table7:%Drugcontentofvariousformulation
trialscontainingOfloxacin

+colourchange,nocolourchange
Absorbance*
Estimationofdrugcontent

Drug
%Drug

Table6:Drugcontentinfreshlyprepareddrug
solution
Absorbance

Formulation

content

at286nm

C1
0.202

content

252.890
101.156

C2
(mg/ml)

0.209
261.658
104.663

Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 3, Issue 10: October: 2012, 2015-2020
2017
Research Article

C5
0.204

CODEN (USA): IJPLCP

255.398
102.159
C6

C3
0.200
250.390
100.156

0.200
250.390
100.156
*Eachvalueisanaverageofthreedeterminations

C4
0.205
256.651
102.660

Table8:Filterporesizeandfilterabilityofthe
formulationsofOfloxacin

Formulation

Filter
pore
Observation

size(m)

1.5

0.22

+
0.22
C2
+

0.45

C1

0.45

1.2

+
1.2

1.5

+
1.5

0.22

+
0.22
C4
+

0.45

C3

0.45

1.2

+
1.2

1.5

0.22

+
0.22
C6
+

0.45

C5

0.45

1.2

+
1.2

1.5

+
1.5

+Injectionpassesthrough.Injectiondoesnotpass
through

Alltheformulationswerefoundtobeeasilypassing
throughalltheporesizefiltersandhence0.22mpore
sizefilterwasselectedtofilteralltheprepared
formulationsseparately.

ISSN: 0976-7126

C3
+

Noneoftheformulationsshowedturbidityorsignsof
microbialgrowth(exceptthepositivecontrol)attheend
ofincubationperiod,indicatingalltheformulationswere
sterileandthusalltheformulationsaresubjectedto
furtherevaluations.

+
+
C4

PostFormulationStudies

EffectofdifferenttemperatureoncrystalgrowthTable
9:Effectofdifferenttemperatureoncrystal

growth

C5

Formulation

RT

C6

40C

Light

C1

+Crystalgrowth,Nocrystalgrowth

IntheformulationsC1,C5,C4andC6nocrystalswere
developedaftertwoweeks.SoC1,C5,C4andC6are
stableattemperaturesstudied.

C2

Effectofdifferenttemperatureonclarity

Table10:Effectofdifferenttemperatureonclarity

Formulation
RT

[Nagaraja ET

AL.,

3(10): Oct., 2012]

40C
Light
C1

C2

C3
+
+

C4

C5

C6

+Turbid,Clear

EffectofdifferenttemperatureoncolourchangeTable
11:Effectofdifferenttemperatureoncolour

change
Formulation
5C
RT
40C
C1

C2

C3
+
+
+
C4

C5

+
+

C1,C2,C4andC6areclearaftertwoweeks.SoC1,C2,
C4andC5arestableattemperaturesstudied.

C6

(mg/ml)

+colourchange,nocolourchange.

C1,C5andC5shownocolourchangeupto400Cafter
twoweeks.SoC1,C5andC5arestableattemperatures
studied.

Scaleupstudies
Assayoftheformulations
C1
Table12:DrugcontentofC1,C5

0.202
252.89
101.156

Absorbance*
Drug
%Drug

Formulation

content

C5
0.209
261.658
104.663

at291.5nm
*Eachvalueisanaverageofthreedeterminations
content

SterilizationstudiesandsterilitytestingFiltration

Theresultsoffilterabilityshowthatboththe
formulationsofOfloxacinpassesthroughallthefour

membranefilters.Hencetheycanbesterilizedby
filteration.

Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 3, Issue 10: October: 2012, 2015-2020
2018
Research Article

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1.2
Table13:Filterporesizeandfilterabilityofthe
formulationsofOfloxacin

Formulation
Filterpore
Observation

size(m)

1.5
+

0.22
+

C5
0.22

0.45

C1
0.45

1.2

1.5
+

+Injectionpassesthrough.Injectiondoesnotpass
through.

0
4.18

Theresultsoffilterabilityshowthatboththe
formulationsofOfloxacinpassesthroughallthefour
membranefilters.Hencetheycanbesterilizedby
filteration.

4.18
4.18

AcceleratedstabilitystudiespHChanges
1
Table14:pHchangesofformulationC1,C5atdifferent
temperatures/conditionsonageing

4.25
4.25
4.28

Formulation
Withdrawal
37C

40C

4.25

Light

4.27
4.30

Week

C1

3
4.29

4.29

4.13

4.30

4.12
4.15

4
4.32

4.33

4.19

4.35

4.17
4.19

C5

4.24

4.32

4.25

4.34

4.24
4.27

6
4.20

4.27

4.25

4.29

4.21
4.24

0
4.06

4.06

4.23

4.06

4.21
4.20

0
6

4.15

4.19

4.17

Crystalgrowth

Table15:CrystalgrowthofformulationC1,C5at
differenttemperatures/conditionsonageing

Formulation
Withdrawal
37C

40C

45C

Week

C1
3

C5
0

[Nagaraja ET

AL.,

3(10): Oct., 2012]

ISSN: 0976-7126

+crystalgrowth,nocrystalgrowth

Nocrystalgrowthwasobservedintheformulationsat
differenttemperatures/conditions.

Claritystudies

1
Table16:ClarityofformulationC1,C5atdifferent
temperatures/conditionsonageing

Withdrawal
37C

40C

45C

Formulation
Week

C1
3

+turbid,clear

C5

Alltheformulationswereclearatdifferenttemperatures/
conditions.

Drugcontent

Table17:PercentdrugcontentofformulationC1at
differenttemperatures/conditionsonageing
Sample

%DrugContent

100.973
101.000

withdrawal
37C

2
100.873

40C
Light

100.761
100.728

(week)
3
100.798

100.537
100.569

4
100.569

100.365
100.296

0
101.156

101.156
101.156

1
101.015

5
100.470

100.107
100.100

6
100.017

99.897
99.901

Table18:PercentdrugcontentofformulationC5at
differenttemperatures/conditionsonageing

Sample
0
%DrugContent

withdrawal
37C

40C

104.066

104.066
104.066

Light

103.857

(week)

103.810
103.829

Int. J. of Pharm. & Life Sci. (IJPLS), Vol. 3, Issue 10: October: 2012, 2015-2020
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Research Article

103.628
103.698

CODEN (USA): IJPLCP

103.725
3
103.501

103.427
103.500
4
103.389

GopalKrishna,HodnickWF,LangW,LinX,
KarraS,MaoJ,AlmassianB.Pharmaceutical
DevelopmentandManufacturingofaParenteral
FormulationofaNovelAntitumorAgent.AAPS
PharmSciTech.2001;2(3):14.

103.251
103.317
5
103.109

NaharM,JainNK.Formulationandevaluationof
saquinavirinjection.IndianJPharmSci2006;
68:60814

103.006
103.108
6

[Nagaraja ET

AL.,

3(10): Oct., 2012]

ISSN: 0976-7126

102.961
102.894
102.996

AnupamaB.Formulationandevaluationof
rofecoxibinjection.M.Pharmdissertation:Rajiv
GandhiUniversityofHealthSciences;2007.

Conclusion

Theconceptofparenteralformulationscontaining
Ofloxacinoffersasuitable,practicalapproachtoachieve
desiredstableparenteralpreparationwithsolubilityof
druginsuitablesolventcomposition.Inpresentwork,
parenteralformulationofOfloxacinwasprepared
successfullybyusingdifferentconcentrationsand
combinationsofPEG200informulationdesign.These
formulationswereexpectedtobestableforsufficiently
longtime.Theconclusionsarrivedfromtheaboveresults
indicatedthattheparenteralformulationcontaining
Ofloxacindevelopedwasfoundtobecomplying
satisfactorilywithalltheevaluationtestsperformedand
wasstableforsufficientlylongerdurationoftime.

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