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JournalofPharmaceuticalandBiomedicalAnalysis37 (2005) 597-601
LCdeterminationofcitralinvolatileoil
CristianedaS.Rauber
un b , ,
S1lviaS.Guterres
una c ,
ElfridesE.S.Schapoval
un b ,
un
ProgramadePos4GraduaB aoemCienBiasFarmaBeutiBasQFaBuldadedeFarmaBiaQUniversidadeFederaldoRioGrandedoSulBUFRGSzQ
PortoAlegreQ(CEP2KwhK4KKKRSQ(Brasil
b
LaboratoriodeEnsinoePesquisaemControledeQualidadeQ(Fa(BuldadedeFarma(BiaQ(PortoAlegreQ(UFRGSQ(BrasilQ(CEP2KwhK4KKKRS
c
LaboratorioJKfQ(Fa(BuldadedeFarma(BiaQ(PortoAlegreQ(UFRGSQ(BrasilQ(CEP2KwhK4KKKRS
Accepted26October2004
Availableonline18December2004
Resumen
ItwastheaimofthisstudytodevelopandvalidateaHPLCmethodforthequantitativedeterminationofcitralinvoltil
aceite.TheHPLCassaywasperformedusingaSpherisorb

CNcolumn(250mm 4.6 mm, 5 m), a - hexano: ethanol(85:15) mobilephase
andanUVdetector(setat233nm).Thefollowingparameterswereevaluated:linearity, precisin, exactitud, speci city, quanti cationand
detectionlimits.Themethodshowedlinearityintherangeof10.0 30.0
1
of20 GML.Theconcentrationofcitralin /volatileoilobtainedwiththisassaywas75%.TheHPLCmethoddevelopedinthis
1
GML.Precisionandaccuracyweredeterminedattheconcentration

Palabras(clave(Cymbopogon(Bitratus(BMVolatileoilMCitralMNormalJphasechromatography
1gIntroduction
Cymbopogon(Bitratus(BBDCzStapfBGramineaezisanherb(
BworldwideknownaslemongrassfTheteamadefromits(
BleavesispopularlyusedinBrazilasantispasmodicQanalgesic
Q(BantiJinSammatoryQantipyreticQdiureticandsedative(
B[w](fThe(
BvolatileoilBVOzobtainedfromfreshleavesofthisplantis(
BwidelyusedbytheperfumeandcosmeticsindustriesfIthas(
BalsobeenusedinchemicalsynthesisQdueitshighcontents(
BofcitralQanaturalmixtureoftwoisomericaldehydesQneral(
Bandgeranial(BfTheantibacterialandantifungalactivitiesof([](
BlemongrassVOanditscomponentshavebeenreportedinthe(
Bliteratura(B[.((:](fMoreoverQtheliteraturepointsthattheVO(
BpropertiesaremainlyduetoitsmajorcomponentQcitral(B[wK](
f(BKuritaetalf(B[ww](Bhaveshownthatcitralactasafungicidal(
Bagentbecauseitisabletoformachargetransfercomplexwith(
BanelectrondonoroffungalcellsQwhichresultinfungaldeathf

Correspondingauthor.Tel.:+555133165214;fax:+555133165378.
E4mailaddresses(Bcristie@farmaciafufrgsfbrQcsrauber@hotmailfcom
(C.da.S.Rauber).
Previousstudieshavereportedagreatantifungalactivity de
los /VOagainst [12].Adems,
semi-solidformulationscontainingVOwerepreparedand
caracteriza [13].
Accordingtovariouspharmacopoeias [14,15],
thequal-itycontrolofessentialoilsandformulationscontainingv
eg-etableoilsorextractsfrommedicinalplantsisveryimportant
anditshouldinvolveanalyticalmethodsforthequantica-tionoft
heconstituentspresentinthesample, themainsubstance
particular.
Gaschromatography (GC), tcnicas de
orincombinationwithother, suchasmassspectrometry
GC/MS; headspace, sobre-lineliquidchromatography
GC(LCGC), hasbeenused forseparation,
identi cationandquanti cationofseveral
volatilecompounds.TheGCfeaturesareusedinthequal-itycontr
olofnaturalmaterialsandproducts, aswellasin
thecharacterizationofnewvolatileoilsandbiotechnological
investigaciones [16].
AnalternativetoGCanalysisishighperformanceliquid
cromatografa (HPLC) withUVdetection, becauseofits
selectividad, sensitivityandoverallversatility.Itisalsoused
0731-7085/$seefrontmatter2004ElsevierB.V.Allrightsreserved.
doi:10.1016/j.jpba.2004.10.042
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C5daS5Rauberetal5.JournalofPharma(BIUTI(BalandBiomedi(BalAnalysis/3(B:KKfz(f234hKw
routinelyinpharmaceuticalindustryasaqualitycontroltech-niq
ueformanydosageforms, includingnaturalproducts.
HPLCisthemethodofchoiceintheanalysisoflessvolatile
constituentsofessentialoils [16].Adems,
thedevelop-mentofquantitativeassays,
forterpenoidcompoundsand
theirmetabolitesinbiologicalmatricesandpharmaceutical
productsisimportant [17].
AlthoughGCmethodshavebeenreportedinthelitera-tureasthe
mainanalyticalmethodforVOanalysis, un
andpharmaceuticalproductscanalsobefound
examplesoftheuseofHPLCasanalternativemethodforas-sayin
gterpenoidsandtheirmetabolitesinbiologicalmatrices.Thecom
po-sitionofessentialoilsfromhealthyandinfected florezca /

plantswasperformedusingGC MSandHPLCanalysis, como
reportedbyHudaibetal. [18].
Por lo tanto, itwastheobjectiveofthisworktodevelopand
tovalidatealiquidchromatographymethodtoquantifythe
citralcontentsin /VO.
2gExperimental
:5w5ChemiBalandreagents
Citral (assignedpurityof95%) wasobtainedfrom Sigma
Aldrich(Darmstadt,Germany) mientras /
VOwasobtainedcommercially(DestilariaMaripa,Parana,
Brazil).Ethanoland wereusedforpreparationofmobilephase -
hexaneLCgrade (Merck, Darmstadt, Alemania).
-SigmaAldrich(Darmstadt,Germany)
Hexanewasusedasoildiluent.Geraniolwasobtainedfrom.
:5:5apparatusandBhromatographiBBonditions
Detector de
TheliquidchromatographysystemconsistedofaSchi-madzuLC
-10AwithaSPD-10Avariable-wavelengthUV (setat233nm),
aSCL-10Asystemcontroller, un C-R6Aintegrator,
aLC-10ASpump, andaRheodynein-jectionvalvewitha20

SeparationwasachievedusingaSpherisorb
lloop(Shimadzu,Kyoto,Japan).

CNcolumn
(250mm
UMN(10mm
4.6 mm, 5

4.6 mm, 5
andSpherisorb m)

m).Themobilephaseusedcon-
CNguardcol-

sistedof-hexano: ethanol(85:15,v/v), owingatarateof

0.3mlmin

atureandthedetectorsensitivitywassetto1.0AUFS.
1
.Theinstrumentwasoperatedatroomtemper-
:5/5GCanalysis
Capillarycolumn(30m usingafusedsilica
GCanalysiswasperformedonaPerkin-Elmerchromato-graph
(AutoSystemXL, Shelton, Estados Unidos)
Andnitrogenascarriergas (1mlmin de lmthickness)
0.25 m m, coatedwithDB-525 m
1
).
Thetemperatureprogrammingrangedfrom60to300 C

at15 Cmin
1
incrementos.TheFIDinjectoranddetector
temperatureswere220and250 C, respectivamente.Thecom-
posiciones, inpercentage, wereobtainedfromelectronicinte-
grationmeasurementsusingameionizationdetectionwith-
outtakingintoaccountresponsefactors.GC MSanalysis
wascarriedoutinacapillaryGC quadrupoleMSsystem
(QP5000 Shimadzu, Kioto, Japn) operatingat70eV,
andheliumascarriergas (2mlmin
1
ditionsasdescribedabove.Theidenticationofthecom-
), inthesamecon -
poundswasperformedbycomparingtheirretentionindexes
(determinedrelativelytotheretentiontimesofaseriesof-
alcanos) andmassspectratothoseobtainedfromauthentic
muestras.
3gMethods
/5w5CalibrationBurves
Aliquotsof2.0, 3.0, 4.0, 5.0and6.0mlofa125 GML
1
citralstandardsolutionweretransferredto25mlvolumet-
ricasksanddilutedtovolumewith-hexano.The-
nalconcentrationsobtainedwere10.0, 15.0, 20.0, 25.0and
30.0
timesandtriplicateinjectionsofeachsolutionweremade
GML
1
, respectivamente.Eachsolutionwaspreparedthree
intotheHPLCsystem.
/5:5Samplepreparation
A25.0mgof /VOwasweightedandtransferred
toa200mlvolumetricaskanddilutedwith-hexane(theo-retical
concentrationof125
MADEwith - hexanetogiveanaltheoreticalconcentration
GML
1
).Furtherdilutionswere
of20.0 GML
1
/5/5Methodvalidation
Themethodwasvalidatedbydeterminingtheparameters:
linearity,precision,accuracy,speci city,detectionlimit(DL)
andquantitationlimit (QL), accordingtoICHguidelinesand
USP27recommendations [14,19].
Thelinearityofthemethodwasdeterminedusingcitralas
areferencesubstanceatveconcentrationlevels.Threecali-brati
oncurveswerepreparedasdescribedabove.Theslopes
andthestatisticalanalysisofthecalibrationcurveswerecal-culat
edbylinearregression.TheDLandQLwerecalculated
basedontheS.D.andtheslope (S) ofthecalibrationcurves
[14,19].
Method'sprecisionwasstudiedbyrepeatabilityandinter-mediar
yprecision.Therepeatabilityofthemethodwaseval-uatedbyeig
htrepeatedassaysoftheVOatsameconcentra-nes,
intriplicateinjections, duringthesamedayunderthe
sameexperimentalconditions.Theintermediaryprecision
wasdemonstratedbyassayingsixsamplesofVO, las
concentraciones de atsame,
duringthreeconsecutivedays.TheS.D.and
R.S.D.werecalculated.Thecitralcontentofthe /
VOwasdeterminedbyreferringtothecalibrationcurveorby
muestra/equivalentreferencesubstancedirectmatching.El
resultswerecomparedtotheGCanalysis.
598
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599
Accuracywasdeterminedbyrecovery, inwhichknown
amountsofcitralwereaddedtoVOsolutions.Therecovery
testwasperformedatthreeconcentrationlevels.Aliquotsof
1.0,2.0and2.5mlofa125.0
GML
1
citralstandardsolution
wereaddedtothreeVOsamplessolutions, preparedascited
inSection 3.2 (correspondingto5.0, 10.0and12.5 GML
1
agregado, respectivamente).Theamountrecoveredwasdetermined
byHPLC.Eachsolutionwaspreparedintriplicateandin-
jectedthreetimes.
Thespecicityofthemethodinthepresenceofother
compoundsoftheVOwasevaluatedbyassayinganamount
ofgeraniol(about5%contentsinVO).Thesolutionwasmade
con - hexanetogiveanalconcentrationof2
GML
1

4gResultsanddiscussion
Thechemicalcompositionofthe /VOconsists
ofmonoterpenescompounds, hidrocarburos, cetonas,
alde-hydesandesters.TheGCmethodwasusedtoidentifyand
quantifythesecompounds.Theoilischaracterizedbyhigh
percentagesofcitral (70-85%) accordingtothegeographical
rea [2].
Inthiswork,
amethodbasedonnormal-phaseHPLCcom-binedtoUVspectro
scopicdetectionforassayingcitralin /
VOwasdeveloped.Theaimofthisstudywastode -
velopandtovalidateasimpleHPLCassayfortheanalysis
ofthissubstanceinVO.AHPLCmethodtodeterminecitral en /
VOanddosageformshasnotbeenreportedin
theliteratureyet.ThecontentofcitralinVOisusuallyde-termined
byGCanalysisandtheHPLCmethodcouldbean
alternativetechniquetoquantifythisdruginvegetablesam-ples,
porejemplo, essentialsoilsasproposedinthisstudy.
Thechoiceofthechromatographicconditionswasin u-encedby
thenatureofthedrug, suchassolubilityandUV de
absorcin.Themobilephase, amixtureof-hexano: etanol
(85:15, v/v), aswellastheotherchromatographicconditions,
showedhighresolutionofthecitralpeak, indicatingthatthe
proposedmethodcouldbeappliedforthedeterminationof
citralinthe /VO.TheUVabsorptionspectrum
ofcitralshowsintenseabsorptionandgoodselectivityat
233nm.La Fig.1 (AandB)
showtheidenticalHPLCpro- lesat233nmforthecitral
(referencesubstance) y /VO.
Fordruganalysisinqualitycontrol, thevalidationofthe
analyticmethodsisnecessaryandthemainobjectiveisto
demonstratethatitissuitableforitsintendedpurpose.AC-cordin
gtotheICHguidelinesandUSP27 [14,19], algunos
validationparametersmustbeevaluated, suchaslinearity,
speci city, precisin, exactitud, lmites de
detectionandquantitation, androbustness.Sin embargo,
thereisnoneedtoevalu-atealltheseparameters,
andtheanalystshouldselectthose
consideredrelevantforeachtestprocedure.
Thelinearityofananalyticalmethodisitsabilitytoelicit
testsresultsthataredirectly,
orbyawell-denedmathemat-icaltransformation,
proportionaltotheconcentrationofana-lyteinsampleswithinagi
venrange [14,19].Relaci6n, el
linearityofHPLCmethodwasinvestigatedforcitralinthe
rangeof10 30

ralretentiontimewasabout13.8min.Thecalibrationcurves
GML
1
at veconcentrationlevels.AutobsCIT-
wereconstructedbyplottingconcentrationsversuspeakarea
andshowedgoodlinearityintherangeof10 30 GML
1
,
(withexcellentcorrelationcoefcients).Therepresenta-
tivelinearequationforcitralwas: = 191154 < +78863 (= 6, =
0.9991).Thedatawerevalidatedbymeansoftheanalysis
ofvariance, whichdemonstratedsignicantlinearregression
Fig.1.Chromatogramofcitralat20 GML
Spherisorb

CN(250mm 4.6 mm, 5

andSpherisorb m)
1
: (A)referencesubstance;(B) /VO; (C) geraniolat2 GML
1

CNguardcolumn(10mm 4.6 mm, 5

.Chromatographicconditions columna:
m); mobilephase:-hexano: ethanol(85:15,v/v); ujo
l; detectionUV233nm(1.0AUFS); retentiontimeabout13.8min. Rate0.3mlmin
1
; injectionvolume:20
C5daS5Rauberetal5.JournalofPharma(BIUTI(BalandBiomedi(BalAnalysis/3(B:KKfz(f234hKw
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)
) )
C. da S. Rauber et al. / Journal of Pharma(euti(al and Biomedi(al Analysis 37 (2005) 597601
Table 1
Results of quantitative determination of citral in / VO by HPLC
repeatability test
Sample Concentration
of citral in VO
( gml
1
Content
(%)
Average
(%)
R.S.D.
(%)
1 14.737 73.69
2 14.998 74.99
3 15.185 75.92
4 14.769 73.85 75.20 1.37
5 15.323 76.62
6 15.083 75.41
7 15.012 75.06
8 15.205 76.02
and not-signicant linearity deviation ( <0.01). The R.S.D.
of the slope of the three lines was 1.78%. The detection limit
(DL) is the lowest amount of analyte in a sample that can
be detected, but not necessarily quantitated, under the stated
experimental conditions. The quantitation limit (QL) is the
lowest amount of analyte in a sample that can be determined
with acceptable precision and accuracy under the stated ex-
perimental conditions. The DL and QL are usually expressed
as the concentration of analyte in the sample [14,19]. The
DL and QL determined were 0.36 and 0.12 g ml
1
, respec-
tively. The low values indicated the sensitivity of the HPLC
method.
The method was validated by evaluating the precision
(re peatab ili tyintra- da y)andinter med iaryprecision(in
ter -
day ).Thep rec isionofan an alyticalme tho disthedegreeo
f
agr eement amo ngindivid ua ltestresul tsw henthemetho
d
isa pplied rep eatedlyto mu ltiplesamp lin gsofahomoge- neoussample [14,19].In thi sstudy,there peatabili tywas studied bya ssayingei gh ts amplesofVO ,atsam ec onc entra- tion(20
g ml
1
), during the same day. The R.S.D. obtained
was 1.37%. The concentration of citral in VO obtained in
thi sassay was 75.2%( Table1) .T heinte rm ed iaryprec isi
on
was demons tra tedbyassaying si xsampl es of VO,durin g
thr eesucc ess ivedays( Table 2) .TheR. S. D. obtained was 1.1 2%for cit ralin thecon cen tra tionof20
g ml
1
. The lows
R.S.D.s values obtained showed the precision of the method,
especia llyfor complexm atrice s,a sVO.There te nti ontime
obtaine dforth esamples wasabo ut1 3.8min.
This method was applied for the determination of citral
cont entin /
VO,sho win gaconte nto f75 %ofcitral ( Ta bl e1 ).Inaddition ,theG C/FIDandGC/M Sanalys i sof
Table 2
Data obtained fromintermediary precision of VOsamples by HPLCanalysis
Sample Citral content (%)
1 Day 2 Day 3 Day Average R.S.D. (%)
1 74.52 74.72 73.69
2 74.68 74.89 74.99
3 74.46 74.99 75.92
4 75.49 74.67 73.8575 .011
. 12
5 75.75 75.43 76.62 6 76.38 73.74 75.41
Table 3
Recovery of standard solution added to / VO samples
Amount added
( gml
1

Amount found
( gml
1

55013 100 27
Percentage of
recovery
a
R.S.D.
(%)
.
10 9 811 98 11 1.11
.
.
1 2 5 1 2 3 5 1 9 8 8 1
.
. .
Each value is the mean of three determinations.
.
a
the / VO revealed a similar citral content (76%),
demonstrating the suitability of proposed HPLC method.
The accuracy of an analytical method is the closeness of
tes tresults ob ta inedbythat method to the truevalue .Itcan
bed etermine db ya pplication ofthea na lyt icalproce dureto
ana nalyteof kn ow npurityorr ecover ys tud ies,where kno
wn amountof st an dardisadde d
[14 ,19].The re co verytestre -
sul tedin99. 06 %o fmeanrecov erywit hl owR .S.D.(les sthan 2 .0%)( Ta ble3),wh ic hsho wtheac cu rac yoft hemeth od .
Specicity is dene as the ability to assess unequivocally
theanalyte in thepr es enc eofcomp on entsth atmaybeex-
pectedtobe pr esent ,s uch asimpur it ies,de gradationproduct
s, andmatr ix compo ne nts
[14,19].Th es peci ci tyt estwase va l- uat edbyassayinganamo untofge ra nio l(2
g ml
1
), which
is also present in VO (in concentrations about 5%). No inter-
ference was observed at the detection wavelength (233 nm).
The chromatogram obtained showed no interference in the
drug peak ( Fig. 1C).
5gConclusion
The HPLC method developed in this study showed excel-
len tper forman ceandshow ed tobe simpl e,line ar,precisio n,
acc urat eandse nsitive.I tc anbe usedt oquant ifycitralin /
VO, giving concentra ti onsi milar tothat obtained
byG C/FI Danaly sis.Moreo ve r,it isthe rstre portonthe
val idat ionofc itralinVO by HPLC metho d.
The proposed method can also be applied to assay citral
ins emi-soli dformu lat ions co ntainin g /
VOinand
sta bilityst udieso fth esen at uralpro du cts,a saccordin gto resear chdevelopmen tinourl aborat ory.
Acknowledgement
The authors thank Conselho Nacional de Desenvolvi-
mento Cientco e Tecnologico,CNPq, for nancial support.
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C. da S. Rauber et al. / Journal of Pharma(euti(al and Biomedi(al Analysis 37 (2005) 597601