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JournalofPharmaceuticalandBiomedicalAnalysis37 (2005) 597-601

LCdeterminationofcitralinvolatileoilCristianedaS.Rauber un b, , S1lviaS.Guterres una c, ElfridesE.S.Schapoval un b,

unProgramadePos4GraduaBaoemCienBiasFarmaBeutiBasQFaBuldadedeFarmaBiaQUniversidadeFederaldoRioGrandedoSulBUFRGSzQPortoAlegreQ(CEP2KwhK4KKKRSQ(Brasil

b LaboratoriodeEnsinoePesquisaemControledeQualidadeQ(Fa(BuldadedeFarma(BiaQ(PortoAlegreQ(UFRGSQ(BrasilQ(CEP2KwhK4KKKRSc LaboratorioJKfQ(Fa(BuldadedeFarma(BiaQ(PortoAlegreQ(UFRGSQ(BrasilQ(CEP2KwhK4KKKRS

Accepted26October2004Availableonline18December2004

Resumen

ItwastheaimofthisstudytodevelopandvalidateaHPLCmethodforthequantitativedeterminationofcitralinvoltilaceite.TheHPLCassaywasperformedusingaSpherisorb CNcolumn(250mm 4.6 mm, 5 m), a - hexano: ethanol(85:15) mobilephaseandanUVdetector(setat233nm).Thefollowingparameterswereevaluated:linearity, precisin, exactitud, specicity, quanticationanddetectionlimits.Themethodshowedlinearityintherangeof10.0 30.0 - 1of20 GML.Theconcentrationofcitralin /volatileoilobtainedwiththisassaywas75%.TheHPLCmethoddevelopedinthis- 1

GML.Precisionandaccuracyweredeterminedattheconcentrationandcanbeappliedtoassaycitralinvolatileoil

studyshowedanexcellentperformance (linealidad, precisin, ciudad accuracyandspeci). ' 2004ElsevierB.V.Allrightsreserved.

Palabras(clave(Cymbopogon(Bitratus(BMVolatileoilMCitralMNormalJphasechromatography

1gIntroduction

Cymbopogon(Bitratus(BBDCzStapfBGramineaezisanherb(BworldwideknownaslemongrassfTheteamadefromits(BleavesispopularlyusedinBrazilasantispasmodicQanalgesicQ(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-itycontrolofessentialoilsandformulationscontainingveg-etableoilsorextractsfrommedicinalplantsisveryimportant anditshouldinvolveanalyticalmethodsforthequantica-tionoftheconstituentspresentinthesample, themainsubstance particular.

Gaschromatography (GC), tcnicas de orincombinationwithother, suchasmassspectrometry GC/MS; headspace, sobre-lineliquidchromatography GC(LCGC), hasbeenused forseparation, identicationandquanticationofseveral volatilecompounds.TheGCfeaturesareusedinthequal-itycontrolofnaturalmaterialsandproducts, 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-niqueformanydosageforms, includingnaturalproducts. HPLCisthemethodofchoiceintheanalysisoflessvolatile constituentsofessentialoils [16].Adems, thedevelop-mentofquantitativeassays, forterpenoidcompoundsand theirmetabolitesinbiologicalmatricesandpharmaceutical productsisimportant [17].AlthoughGCmethodshavebeenreportedinthelitera-tureasthemainanalyticalmethodforVOanalysis, un andpharmaceuticalproductscanalsobefound examplesoftheuseofHPLCasanalternativemethodforas-sayingterpenoidsandtheirmetabolitesinbiologicalmatrices.Thecompo-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-jectionvalvewitha20SeparationwasachievedusingaSpherisorb

lloop(Shimadzu,Kyoto,Japan). CNcolumn

(250mmUMN(10mm

4.6 mm, 5

4.6 mm, 5

andSpherisorb m) m).Themobilephaseusedcon-

CNguardcol-

sistedof-hexano: ethanol(85:15,v/v), owingatarateof0.3mlmin -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.TheFIDinjectoranddetectortemperatureswere220and250 C, respectivamente.Thecom-posiciones, inpercentage, wereobtainedfromelectronicinte-

grationmeasurementsusingameionizationdetectionwith-outtakingintoaccountresponsefactors.GC MSanalysiswascarriedoutinacapillaryGC quadrupoleMSsystem(QP5000 Shimadzu, Kioto, Japn) operatingat70eV,andheliumascarriergas (2mlmin - 1ditionsasdescribedabove.Theidenticationofthecom-

), inthesamecon -

poundswasperformedbycomparingtheirretentionindexes(determinedrelativelytotheretentiontimesofaseriesof-alcanos) andmassspectratothoseobtainedfromauthenticmuestras.

3gMethods

/5w5CalibrationBurves

Aliquotsof2.0, 3.0, 4.0, 5.0and6.0mlofa125 G M L- 1citralstandardsolutionweretransferredto25mlvolumet-ricasksanddilutedtovolumewith-hexano.The-nalconcentrationsobtainedwere10.0, 15.0, 20.0, 25.0and30.0timesandtriplicateinjectionsofeachsolutionweremade

G M L- 1, respectivamente.Eachsolutionwaspreparedthree

intotheHPLCsystem.

/5:5Samplepreparation

A25.0mgof /VOwasweightedandtransferred toa200mlvolumetricaskanddilutedwith-hexane(theo-reticalconcentrationof125 MADEwith - hexanetogiveanaltheoreticalconcentration

G M L- 1).Furtherdilutionswere

of20.0 G M L- 1

/5/5Methodvalidation

Themethodwasvalidatedbydeterminingtheparameters: linearity,precision,accuracy,specicity,detectionlimit(DL) andquantitationlimit (QL), accordingtoICHguidelinesand USP27recommendations [14,19].

Thelinearityofthemethodwasdeterminedusingcitralas areferencesubstanceatveconcentrationlevels.Threecali-brationcurveswerepreparedasdescribedabove.Theslopes andthestatisticalanalysisofthecalibrationcurveswerecal-culatedbylinearregression.TheDLandQLwerecalculated basedontheS.D.andtheslope (S) ofthecalibrationcurves [14,19].

Method'sprecisionwasstudiedbyrepeatabilityandinter-mediaryprecision.Therepeatabilityofthemethodwaseval-uatedbyeightrepeatedassaysoftheVOatsameconcentra-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 G M L- 1 citralstandardsolutionwereaddedtothreeVOsamplessolutions, preparedascitedinSection 3.2 (correspondingto5.0, 10.0and12.5 G M L- 1agregado, respectivamente).TheamountrecoveredwasdeterminedbyHPLC.Eachsolutionwaspreparedintriplicateandin-jectedthreetimes.

Thespecicityofthemethodinthepresenceofother compoundsoftheVOwasevaluatedbyassayinganamount ofgeraniol(about5%contentsinVO).Thesolutionwasmade con - hexanetogiveanalconcentrationof2 G M L- 1

4gResultsanddiscussion

Thechemicalcompositionofthe /VOconsists ofmonoterpenescompounds, hidrocarburos, cetonas, alde-hydesandesters.TheGCmethodwasusedtoidentifyand quantifythesecompounds.Theoilischaracterizedbyhigh percentagesofcitral (70-85%) accordingtothegeographical rea [2].

Inthiswork, amethodbasedonnormal-phaseHPLCcom-binedtoUVspectroscopicdetectionforassayingcitralin /VOwasdeveloped.Theaimofthisstudywastode - velopandtovalidateasimpleHPLCassayfortheanalysis ofthissubstanceinVO.AHPLCmethodtodeterminecitral en /VOanddosageformshasnotbeenreportedin theliteratureyet.ThecontentofcitralinVOisusuallyde-terminedbyGCanalysisandtheHPLCmethodcouldbean alternativetechniquetoquantifythisdruginvegetablesam-ples, porejemplo, essentialsoilsasproposedinthisstudy.

Thechoiceofthechromatographicconditionswasinu-encedbythenatureofthedrug, 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-cordingtotheICHguidelinesandUSP27 [14,19], algunos validationparametersmustbeevaluated, suchaslinearity, specicity, precisin, exactitud, lmites de detectionandquantitation, androbustness.Sin embargo, thereisnoneedtoevalu-atealltheseparameters, andtheanalystshouldselectthose consideredrelevantforeachtestprocedure.Thelinearityofananalyticalmethodisitsabilitytoelicit testsresultsthataredirectly, orbyawell-denedmathemat-icaltransformation, proportionaltotheconcentrationofana-lyteinsampleswithinagivenrange [14,19].Relaci6n, el linearityofHPLCmethodwasinvestigatedforcitralinthe rangeof10 30

ralretentiontimewasabout13.8min.Thecalibrationcurves

G M L- 1 atveconcentrationlevels.AutobsCIT-

wereconstructedbyplottingconcentrationsversuspeakareaandshowedgoodlinearityintherangeof10 30 G M L- 1,(withexcellentcorrelationcoefcients).Therepresenta-tivelinearequationforcitralwas: = 191154 < +78863 (= 6, = 0.9991).Thedatawerevalidatedbymeansoftheanalysis ofvariance, whichdemonstratedsignicantlinearregression

Fig.1.Chromatogramofcitralat20 G M LSpherisorb CN(250mm 4.6 mm, 5

-andSpherisorb m)

1: (A)referencesubstance;(B) /VO; (C) geraniolat2 G M L- 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 1Results of quantitative determination of citral in / VO by HPLC repeatability test

Sample Concentrationof citral in VO( gml 1

Content(%)

Average(%)

R.S.D.(%)

1 14.737 73.692 14.998 74.993 15.185 75.924 14.769 73.85 75.20 1.375 15.323 76.626 15.083 75.417 15.012 75.068 15.205 76.02

and not-significant 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 canbe detected, but not necessarily quantitated, under the statedexperimental conditions. The quantitation limit (QL) is thelowest amount of analyte in a sample that can be determinedwith acceptable precision and accuracy under the stated ex-perimental conditions. The DL and QL are usually expressedas the concentration of analyte in the sample [14,19]. TheDL and QL determined were 0.36 and 0.12 g ml1, respec-tively. The low values indicated the sensitivity of the HPLCmethod.

The method was validated by evaluating the precision (re peatab ili tyintra- da y)andinter med iaryprecision(inter - day ).Thep rec isionofan an alyticalme tho disthedegreeof agr eement amo ngindivid ua ltestresul tsw henthemethod 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(20g ml1), during the same day. The R.S.D. obtainedwas 1.37%. The concentration of citral in VO obtained in thi sassay was 75.2%(Table1) .T heinte rm ed iaryprec ision 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 tionof20g ml1. The lowsR.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 2Data obtained from intermediary precision of VO samples by HPLC analysis

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 3Recovery of standard solution added to / VO samples

Amount added( gml1

Amount found( gml1

55013 100 27

Percentage ofrecoverya

R.S.D.(%)

.

10 9 811 98 11 1.11.

.

1 2 5 12 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 known 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 .

Specificity is define as the ability to assess unequivocally theanalyte in thepr es enc eofcomp on entsth atmaybeex- pectedtobe pr esent ,s uch asimpur it ies,de gradationproducts, andmatr ix compo ne nts [14,19].Th es pecifi ci tyt estwase va l- uat edbyassayinganamo untofge ra nio l(2g ml1), whichis 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 thedrug 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 firstre 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 tinourlaborat ory.

Acknowledgement

The authors thank Conselho Nacional de Desenvolvi-mento Cientfico e Tecnologico,CNPq, for financial support.

References

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[2]M.S.C.F erreir a,M.C.Fon tele s,Rev.Bra s.Fa rm70( 1989 )9 497. [3]A.K.Mis hra,N. K.Dubey,A ppl. Environ.M icro biol. 60(1 99 4) 11011105.

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[5]E.O .Lima,O.F.Go mper tz,A. M.Gi esbrecht, M.Q. Paulo,Mycos es 36(199 3)333336. [6]G.O .Onawunmi,Le tt.A ppl.M icro biol.9(19 89)1 05108. [7]H.H .El-Kamali,A .H.A hmed, A.S. Mohammed, A.A. M.Yahia,I.H . El-Tay eb,A.A.Ali,F itot erapi a69( 1998)777 8. [8]J.- P.Chaumont,D .Le ger,A nn.P harm.Fr.5 0(19 92)156166. [9]K.C imanga,K.Kam bu,L .Tona ,S.A pers,T.De Bruy ne,N.Her- mans,J .Totte,L.Pi eter s,A.J .Vli etinck,J. Ethn opharmacol. 79 (2002) 213220. [10]G. O.Onawunmi,W .-A. B.Yis ak,E .O.Ogunla na,J .Ethnopharm acol. 12( 1984)279286 . [11]N. Kurita,M.Miy aji, R.Kur ane, Y.Takahar a,Ag ric.Biol.Ch em. 45(198 1)945952.

[12] V.J.A. Schuck, M. Fratini, C.S. Rauber, Rev. Bras. Cienc. Farm 47(2001) 4549.

[13]C. S.Rauber,A. T.He nriques ,S.S .Guterres, E.E. S.Schapov al,INP I (2002) PI0203521-9 . [14]Un itedStatesP harm acopoei a,Va lidationof Comp endialMet hods, 27thed .UnitedStat esPh armacop eial Convention ,Roc kville,20 04.[15] Farmacopeia Brasileira, 4 ed. Atheneu, Sao Paulo, 1988.[16] G.B. Lockwood, J. Chromatogr. A 936 (2001) 2331.[17] K.K. Chan, J. Chromatogr. A 936 (2001) 4757.[18] M. Hudaib, M.G. Bellardi, C. Rubies-Autonell, J. Fiori, V. Cavrini,

Il Farmaco 56 (2001) 219227. [1 9]Inter na tional ConferenceonH armonizationo fTechnical Re quire- me ntsforR eg istrat ionofPharmace uticalsforHum anUse(ICH) Q2 B.Guide li neonVa lidationofAna lyticalProced uresMetho d- ol ogy,199 6.

C. da S. Rauber et al. / Journal of Pharma(euti(al and Biomedi(al Analysis 37 (2005) 597601

Determinacin de LC de citral en Cymbopogon citratus aceite voltilIntroduccinExperimentalProductos qumicos y reactivosAparatos y condiciones cromatogrficasAnlisis de GC

MtodosCurvas de calibracinPreparacin de la muestraValidacin del mtodo

Resultados y discusinConclusinReconocimientoReferencias