Olive Leaves 2009

Talanta 78 (2009) 4048

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Talanta

journa l homepage: www.e lsev ier .com

Fast an coliquid c emonito n

N. Snch uquDepartment of , Crdo

a r t i c l

Article history:Received 30 JuReceived in reAccepted 24 OAvailable onlin

Keywords:Triterpenic compoundsLCMSMSMultiple reaction monitoring (MRM)Natural productsMicrowave-assisted extraction

inatiotriterion oDue td in

solubilisation. Microwave assistance provided a signicant shortening of the leaching time as comparedto conventional procedures by maceration, which usually requires at least 5h. After extraction, determi-nation was carried out by liquid chromatographytandem mass spectrometry (LCMSMS) with a triplequadrupole (qQq) mass detector without any clean-up step prior to chromatographic analysis. Highlyselective identication of triterpenes was conrmed by multiple reaction monitoring (MRM) using themost representative transitions from the precursor ion to the different product ions, while the mostsensitive transitions were used for MSMS quantitation. Total analysis performed in 25min enables the

1. Introdu

Themainaimed at thand diseasesis of new dproperties fties of natutremendoudrugs [1]. Tpromise for[2]. In addiproved, thuisolated fro

The inhethe presentsis methodstep, whichical process

CorresponE-mail add

0039-9140/$ doi:10.1016/j.tcharacterization of a fraction with particular interest in the pharmacological area. 2008 Elsevier B.V. All rights reserved.

ction

endeavourof thepharmaceutical industry is at presente prevention and/or treatment of pathological disorderss affecting our society. This is accomplished by synthe-rugs and isolation of compounds with pharmacologicalrom natural products. Despite pharmacological proper-ral products have for centuries been investigated, theirs diversity makes them an inexhaustible source of newhus, natural products continue at present being a greatdiscovery and development of new pharmaceuticals

tion, new properties of well-known natural drugs ares contributing to the relevance of research on productsm plants.rent diversity and interest on natural products justifydemand for development of fast and selective analy-

s. These methods involve rst an extraction (leaching)is the key for subsequent steps of the overall analyt-leading to identication and/or quantitation of target

ding author. Tel.: +34 957218615; fax: +34 957218615.ress: [email protected] (F. Priego-Capote).

plant constituents. An ideal leaching procedure should be exhaus-tive with respect to the constituents to be analysed, fast, simple,inexpensive, environmental friendly and amenable to automationfor routine analysis. Looking for these characteristics, alternativeleaching techniques such as ultrasound- and microwave-assistedextraction, pressurized liquid extraction and supercritical uidextraction have been applied to this eld [36]. Based on thesetechniques, extraction methods in which both the extract vol-ume and the process time are drastically shortened have beendeveloped.

Concerning individual separation of the target analytes, a pow-erful separation technique such as gas chromatography (GC) has fordecades been applied to the analysis of volatile natural products orderivatives. Despite this technique interfaced tomass spectrometry(MS) is especially effectivewith identicationandquantitationpur-poses, the role recently played by liquid chromatography (LC) foranalysis of natural products is invaluable. This is supported on thefact that approximately 80% of all known natural compounds arenon-volatile or thermally unstable, and thus incompatible with GC[7]. In this sense, although LC has lower resolution capability thanGC (ultra-performance LC has considerably improved this aspect),the former shortens the analysis time because derivatization isnot required. In addition, the LCMS coupling and, particularly

see front matter 2008 Elsevier B.V. All rights reserved.alanta.2008.10.037d selective determination of triterpenichromatographytandem mass spectromring after microwave-assisted extractio

ez-vila, F. Priego-Capote , J. Ruiz-Jimnez, M.D. LAnalytical Chemistry, Annex C-3 Building, Campus of Rabanales, University of Crdoba

e i n f o

ne 2008vised form 20 October 2008ctober 2008e 31 October 2008

a b s t r a c t

Amethod for fast and selective determoleanolic, ursolic andmaslinic acids as is reported here. Quantitative isolatassistance using ethanol as extractant.ferent ethanolwater ratios were teste/ locate / ta lanta

mpounds in olive leaves bytry with multiple reaction

e de Castroba, Spain

n of themain triterpenic compounds present in olive leavespenic acids and, uvaol anderythrodiol as triterpenicdialcoholsf the analytes has been accomplished in 5min by microwaveo the medium polarity of triterpenic acids and dialcohols, dif-order to select the optimum extractant composition for their

N. Snchez-vila et al. / Talanta 78 (2009) 4048 41

Table 1Optimization of the chromatographic conditions for separation of triterpeniccompounds.

Variable Tested range Optimum value

Flow rate (mLmin1) 0.71.2 1.2Temperature (C) 535 15Water content (%) 020 7.9Acetonitrile content (%) 0100 90

Polarity modier Methanol MethanolEthanol2-Propanol

Polarity modier (%) 110 2Ammonia 01 0.05Ammonium formate 01 0.05

LCMSMS, provide powerful tools for quantitative and qualitativeanalysis [8].

One important bioactive fraction isolated from plants is thatcomposedby triterpenic compounds, either in the formof free acidsor aglycones of triterpenoid saponins. The literature abounds ontheir pharmacological propertieswhich include anti-inammatory

[9], hepatoprotective [10], anti-tumour [11], anti-viral [12], anti-HIV [13], anti-microbial [14], anti-fungal [15], anti-diabetic [16],gastroprotective [17], and anti-hyperlipidemic effects [18]. Bioac-tive pentacyclic tritepenoids are widely distributed in medicinalherbs and plants, Olea europaea included [19]. In Spain, approxi-mately 110 tonnes of olive leaves are annually generated both frompruning and after separation of olive fruit in mills prior to olive oilproduction. This residue is apotential source for isolationof oleano-lic and maslinic acid together with other triterpenic compounds.

Due to the low volatility and high molecular weight of triter-penes, a derivatization step is mandatory prior to GC analysis.Derivatization of triterpenes has traditionally been carried outby silylation, a tedious procedure requiring reaction times from30min to 3h [2024]. Recent investigations carried out by ourresearch group have shortened the silylation step by virtue ofthe assistance with an auxiliary energy such as ultrasound [25].One alternative to avoid the derivatization step is the use ofLC separation. Thus, an LCLC coupling to MS with electrosprayionization (ESI) has been used to analyse boswellic acids [26].Triterpene glycosides have been determined by a photoarrayabsorptiometricmass-spectrometricevaporative-light scattering

Fig. 1. Total ion chromatogram obtained with a standard solution of the target triterpenes under the optimum chromatographic conditions (Section 2.5). (1) maslinic acid,15gmL1; (2) betulinic (IS) 5gmL1, (3) oleanolic and (4) ursolic acids, 30gmL1; (5) erythrodiol, 30gmL1 and (6) uvaol, 10gmL1.

Table 2Optimization o ounds

Analyte y (eV)

Maslinic acid

Betulinic acid

Oleanolic acid

Ursolic acid

Erythrodiol

Uvaol

a Voltage off the MSMS step for qualitative and quantitative determination of terpenic comp

Voltage MS1a (V) Precursor ion (m/z) Collision energ

160 409 10160 439 10

160 439 10

160 439 10

140 425 10

140 425 10

the rst quadrupole to focus the precursor ion for each analyte..

Product ions (m/z) Quantitation transition (m/z)

203 409203189

203 439191191 439203203 439191191 439203203 439191191 439203217 425217191 425191217 425217191 425191

42 N. Snchez-vila et al. / Talanta 78 (2009) 4048

Fig. 2. Selected transitions from precursor ions to product ions used for quantitation of the terpenes after optimization by MRM. (1) maslinic acid, 15gmL1; (2) betulinic(IS) 5gmL1, (3) oleanolic and (4) ursolic acids, 30gmL1; (5) erythrodiol, 30gmL1 and (6) uvaol, 10gmL1.


ngerprintiof a photoato LC equip

A metholeaves is hequadrupoleconrmatiotriterpenicstep has beto achieveanalysis ofbeen optimmatographi

2. Experim

2.1. Sample

Olive lealected in Cmilled andFig. 2. (Continued)

ng approach [27] or with a dual system by connectionrray and a mass detector [28], coupled in all instancesment.d for determination of triterpenic compounds in olivere proposed. The coupling LCMSMS with a triple(qQq) mass detector has been used for identication,n (by comparison with standards) and quantitation ofcompounds in extracts from olive leaves. The MSMSen optimized by multiple reaction monitoring (MRM)the demanded levels of sensitivity and selectivity forthis fraction. A microwave-assisted leaching step hasized for isolation of the target analytes prior to chro-c analysis.

ental

s

ves from hojiblanca and acebuche varieties were col-rdoba, dried at 35 C for 24h in an electrical furnace,stored at 4 C until analysis.

2.2. Reagen

The moscompoundsthis researcExtrasynthefrom Sigmaisolated byGarca-Granfrom Sigmadard solutiostored at daily. Analyused in the

Deionizeford, MA, Umethanol oused for prnium formahydroxidephases to ents

t representative and commercially available triterpenicfound in olive leaves [19] were the target analytes ofh. Thus, erythrodiol and uvaol were purchased fromse (Genay, France), oleanolic and ursolic acids wereAldrich (Madrid, Spain). Maslinic acid (>97% purity)a previously reported protocol [29] was a gift from A.ados (University of Granada, Spain). Betulinic acid, also, was used as internal standard. Methanolic stock stan-ns of 1000gmL1 were prepared for each analyte and20 C. Standard dilutions in methanol were preparedtical-grade ethanol fromPanreac (Barcelona, Spain)wasleaching step.d water (18M cm) from a Millipore Milli-Q (Bed-SA) water purication system and acetonitrile andf LCMS grade from Scharlab (Barcelona, Spain) wereeparation of chromatographic mobile phases. Ammo-te fromAgilent (Waldbronn, Germany) and ammoniumfrom Scharlab were used as additives in the mobilehance ionization required for MS detection.


2.3. Apparatus and instruments

A Microdigest 301 microwave-assisted digestor of 200Wmaximum power (Prolabo, Paris, France) furnished with a micro-processor programmer (Prolabo) was used for the leaching step.

Analyses were performed by reversed-phase LC followed by ESIin the positive mode and MSMS detection. Liquid chromatog-raphy was performed with an Agilent (Palo Alto, CA, USA) 1200Series LC system. This consists of a binary pump, vacuum degasser,autosampler and a thermostated column compartment. Detectionwas carried out with an Agilent 6410 Triple Quadrupole LC/MS. Thedata were processed using a MassHunter Workstation Softwarefrom Agilent for qualitative and quantitative analysis. An InertsilODS-2C18 analytical column (4.0mm i.d.250mm; 5m particlesize, GL Sciences Inc., Tokyo, Japan) was used for chromatographicseparation.

2.4. Proposed leaching procedure

Two grams of milled leaves and 16mL of extractant mixture(80/20, ethanolwater) were placed into the quartz extractionvessel. The mixture was spiked with internal standard (50L of100gmL1). This was positioned for 5min in the microwave-

irradiation zone at 180W irradiation power. After extraction, thesuspensionwas centrifuged at 2000 g and the extract 1/10dilutedin water prior to injection into the liquid chromatograph.

2.5. LCMSMS separation-detection

The separation was carried out under isocratic conditions dur-ing the whole chromatographic process. The mobile phase was90% acetonitrile, 7.9% water and 2% methanol with 0.05% ammo-nia and 0.05% ammonium formate (pH 9.1). The chromatographicanalysis took 32min at a ow-rate of 1mLmin1. The tempera-ture of the analytical column was 5 C and the injection volume10L.

The entire eluate was electrosprayed, ionized in positive modeand monitored by MSMS detection in MRM mode, which wascarried out by optimization of the product ions obtained by frag-mentation of the isolated precursor ion for each analyte. This wasachieved by setting the optimum voltage of the rst quadrupole,which operates as a mass lter, and that of the collision cell forfragmentation of precursor ions. The ow-rate and temperature ofthe drying gas (N2) were 13 Lmin1 and 350 C, respectively. Thenebulizer pressure was 35psi and the capillary voltage 4000V. Thedwell time was set at 250s.

mpouFig. 3. Tentative fragmentation pathways for triterpenic co nds by MSMS determination.


3. Results

3.1. Optimiz

Chromatthese compacids on thside, are isomatographiin order toest time. Ththe optimuoptimized wtemperaturthese variawell as thecompositiothe pH of tstudied (5being 5 C tphase, acidwhile basicmise solutioFig. 1 obtainmatographiFig. 3. (Continued)

and discussion

ation of the liquid chromatographic method

ographic separation is a key step for determination ofounds taking into account that ursolic and oleanolice one side and, erythrodiol and uvaol, on the othermers. The main variables with inuence on the chro-c separation were optimized by a univariate approachobtain the best separation of the analytes in the short-is study was carried out with standard solutions; then,m conditions were tested with extracts. The variablesere composition of the mobile phase, ow-rate and

e of the analytical column compartment. Table 1 showsbles, the ranges studied and their optimum values assolvents used in the optimization of the mobile phasen. The most critical variables were the temperature andhe mobile phase. Low temperatures within the range35 C) favoured the separation of the target analyteshe optimum value. Concerning the pH of the mobilepHs favoured the separation of the triterpenic acidspHs improved that of triterpenic dialcohols. A compro-nwas to set thepHat 9.1 as shows the chromatogramofed with a standard solution under the optimum chro-c conditions. The studywas carried out in scanmode, in

which onlya limited mof 500ms. Bphysical anand its absematogramfrom incomin the labor

In additiion for all anall triterpenmaslinic acithe carboxy

3.2. Optimi

The stratwas based omatographistandards atriterpenictions wereselectivity,ysis of comFig. 2 showthe second mass lter is operating to detect ions withinass range (between 50 and 600m/z) with a scan timeetulinic acid was used as internal standard due to its

d chemical behaviour similar that of the target analytesnce in olive leaves. The other peaks present in the chro-were identied as the solvent front and interferencesplete purication of maslinic acid, which was isolatedatory.on, this analysis enables the selection of the precursoralytes, which are listed in Table 2. The precursor ion foreswas [MH2O]+ by loss of awatermolecule, except ford, which was [MH2OCOO]+ by additional cleavage oflic group.

zation of the detection method by MSMS

egy followed in the optimization of theMSMSmethodn multiple reaction monitoring. Similarly to the chro-c performance, this study was carried out both withndextracts. Theoptimumvalues for eachparameter andcompound are shown in Table 2. Two different transi-monitored for each analyte to achieve an extra level ofwhich is of particular interest for conrmatory anal-plex-matrix samples by comparison with standards.s the selected transition used for quantitation of each


triterpene.two transitiproduct ionand 191m/zerythrodioltication wimprove sigsity. The dequantitatioinMRMmosuited for qtatively elucFig. 3 showfrom preculytes is thatsource by ddiol and uvcase of masresulting inuct ions arefragmentatdouble bonare obtaineFig. 3. (Continued)

As can be seen, most of the analytes were quantied byons as fragmentation of their precursor ions led to twos with a similar intensity. These product ions are 203for oleanolic and ursolic acid and 217 and 191m/z forand uvaol. In the case of maslinic acid, additional quan-ith the product ion corresponding to 189m/z did notnicantly in terms of sensitivity due to its low inten-mand for a powerful tool for conrmatory analysis andn of terpenic compounds justies the use of LCMSMSde.Additionally, although theqQq is not amass analyserualitative analysis, fragmentation pathways can be ten-idated to set ngerprints characteristic of each analyte.s the tentative fragmentation scheme for each analytersor to product ions. A common pattern for all ana-their precursor ions are generated in the electrosprayehydration resulting in peaks of 425m/z for erythro-aol and 439m/z for oleanolic and ursolic acids. In thelinic acid, the carboxylic group is additionally cleaveda peak of 409m/z. The schemes for generation of prod-characterized for the target triterpenic compounds by

ion through the oleane structure. As a consequence, thed changes its position and ngerprinting product ionsd for triterpenic dialcohols and acids. For triterpenic

dialcohols,the oleaneof pentacyccharacteristgroup aftertransitionsquantitatiothe ngerprmon for oleacids also ppounds (19similar to thusedwithqferent for m

Table 3Optimizationolive leaves.

Variable

Power (W)Ethanol (%)Irradiation timtwo product ions are generated from fragmentation ofstructure: 191m/z, which is characteristic in generallic triterpenic compounds in olive leaves and, 217m/zic of triterpenic dialcohols that keeps the methylenecleavage of the hydroxyl group. As stated above, the

from precursor ion to these product ions were used forn because of its similar intensity. For triterpenic acids,inting product ion corresponds to 203m/z being com-anolic, ursolic and maslinic acids. Oleanolic and ursolicrovide the product ion characteristic of triterpenic com-1m/z) found in olive leaves, which had an intensityat of the former. For this reason, both transitions were

uantitationpurposes. Theothermajorproduct ion isdif-aslinic acid because of its substitution in two positions

of the microwave-assisted leaching of triterpenic compounds from

Tested range Optimum value

100180 18060100 80

e (min) 115 5


Fig. 4. Total io 2) bet(6) uvaol.

with an alcoto the form189m/z.

Once theof the dwebest value 2improveme

3.3. Optimitriterpenic c

The roleoptimized.irradiation tused in thisratio betwenal standarcarried outtative isolat

A full twdom and inpoints wasvariables prvariableweered in prewith 80% etrequired to

Table 4Concentrationvarieties expre

Triterpene/var

ErythrodiolUvaolOleanolic acidUrsolic acidMaslinic acid

m vexpneticl remwas

ed wd de

ed byons.

aracn chromatogram provided by analysis of the hojiblanca variety. (1) Maslinic acid, (

hol group. Thus, an additional dehydration process ledation of a triple bond resulting in a product peak of

number of transitions was established, the inuencell time on the sensitivity was checked, resulting the50ms. Longer dwell times did not provide signicantnt.

zation of the microwave-assisted leaching ofompounds

of the main variables involved on the leaching step was

optimufurther

A kifor totawhichobtaindetecteprovidconditi

3.4. ChThese potential variables were the irradiation power,ime and extractant composition. The response variablestudy was the extraction efciency expressed as the

en the peak area of each analyte and that of the inter-d. The optimization of the extractant composition wastesting different ethanolwater ratios to obtain quanti-ion of the target compounds [25].o-level factorial design allowing four degrees of free-volving 11 randomized runs including three centre

built for a screening study of the behaviour of the threeeviously cited. Theupper and lower values given to eachre selected from the available data and experience gath-liminary experiments. The best results were obtainedhanol and 180W irradiation power. A 20% of water wassolubilise more polar compounds. The tested and the

s of each triterpene found with the proposed method in two olive leafssed in g/g (n=5).

iety Hojiblanca Acebuche

2653 107 3411 982398 76 2801 60

13028 356 8497 2024861 182 3792 1512997 137 3353 122

Calibratiarea of eachtration of eabuild the ca

The limicalculated wquantitationnoise ratioestimatedband LOQs wvalue of thevalues of LOLOQs the vaume in all0.979and0which was

In orderinter-day animental setday were pexpressed afor the targe4.29 to 7.76posed methextracts in tulinic acid (IS), (3) oleanolic acid, (4) ursolic acid, (5) erythrodiol and

alues obtained for each variable, which were used ineriments, are shown in Table 3.s study was performed to determine the time requiredoval of triterpenic compounds from the olive leaves,obtained after irradiation for 5min. The extracts

ith longer times provided similar results with non-gradation. Fig. 4 shows the chromatogram in TIC modeanalysis of the hojiblanca olive variety under optimal

terization of the method for triterpenes quanticationon plots were run by using the ratios between the peakcompound vs. that of the IS as a function of the concen-ch compound. Seven concentration levels were used tolibration curves.ts of detection (LODs) and quantication (LOQs) wereith the MRM chromatograms from extracts using thetransitions. The peak area-to-averaged background

was calculated, for which the background noise wasy thepeak-to-peakbaselinenear the analytepeak. LODsere then calculated on the basis of a minimal acceptedsignal-to-noise (S/N) ratio of 3 and 10, respectively. TheDs obtained ranged from0.26 to 0.91ng;meanwhile forlues ranged from 0.87 to 3ng, for 10L injection vol-

instances. The regression coefcients ranged between.998 for the lineardynamic range tested for eachanalyte,from the LOQs to 150gmL1.to evaluate the precision of the proposed method,d intra-day variabilitywere evaluated in a single exper--up with duplicates [30]. Two extract analyses pererformed on 7 days. The intra-day assay variability,s relative standard deviation, was from 2.92 to 5.23%t analytes,while inter-dayassayvariability ranged from%. These results demonstrate the suitability of the pro-od for determination of the target analytes in olive leaferms of sensitivity and precision.


3.5. Comparison of the efciency of the microwave-assistedleaching method with the conventional method

The isolation of triterpenic compounds, usually carried out bymaceration with vigorous agitation, requires at least 5h to achievequantitative removal. The use of auxiliary energies to assist theextraction of the target compounds from olive leaves has beenreported in a previous publication [25]. Ultrasonic energy enabledacceleration of the leaching process,whichwas complete in 20min.In the method proposed here, microwaves have provided a shorterleaching time as only 5min is needed to isolate the target com-pounds. The efciency of this alternative has been checked bycomparison with a reference method as conventional macera-tion. Quantitation was performed by the LCMSMS method herereported. The results show that the microwave-assisted methodprovides leaching efciencies similar to those of the conventionalmethod as tvalidation omination ofhojiblanca v

3.6. Applica

The presknown [19commentedquantify thapplied tofeasibility. Tthe arithmeing conditiof triterpenconcentratipounds. Astriterpenicin agreeme

4. Conclus

A methormatory ain olive leaMRM modeby comparilow concendemonstratnation of thchromatogrderivatizati

Quantitamicrowave-analytical p

with conventional protocols based on long sample preparationsteps. Thus, the proposed method can be an interesting tool inthe pharmaceutical industry to take prot from this abundant rawmaterial in Spain for extraction of terpenic valuable compounds orfrom other plants.

Acknowledgement

SpainsMinistry of Science and Innovation is gratefully acknowl-edged for nancial support (Project no. CTQ2006-01614).

References

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under Introduction. LCMSMS was employed toose present in the extracts. The developed method washojiblanca and acebuche variety in order to prove itshe concentration of each triterpene was calculated astic mean of ve experiments under the optimum work-ons. The results obtained show that the compositiones in the target varieties is characterized by the highon of oleanolic acid in comparison to the other com-Table 4 shows, hojiblanca has the highest amount ofacids while dialcohols are more abundant in acebuche,nt with previous studies [25].

ions

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Fast and selective determination of triterpenic compounds in olive leaves by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring after microwave-assisted extractionIntroductionExperimentalSamplesReagentsApparatus and instrumentsProposed leaching procedureLC-MS-MS separation-detection

Results and discussionOptimization of the liquid chromatographic methodOptimization of the detection method by MS-MSOptimization of the microwave-assisted leaching of triterpenic compoundsCharacterization of the method for triterpenes quantificationComparison of the efficiency of the microwave-assisted leaching method with the conventional methodApplicability of the proposed method

ConclusionsAcknowledgementReferences

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Olive Leaves 2009