Journal of Chromatography A, 1103 (2006) 2228

Comparison between sample disruption methos fr

D. M Comaoicas

niversE), Per 20

er 200

Abstract

Sea sand d erephenolic com ingleby these met ghersolid disrupt d wawas also the orogemethod. 2005 Else

Keywords: Sea sand disruption method; SSDM; MSPD; Ficus carica; Phenolic acid; Flavonol; Coumarin

1. Introd

The figthe Mediteclimate likfor humantive and pthat their cits high cinhibits thother partpharmacogated. Inaction of2000, Canfrom a declevels ofties are pcompound

CorrespoE-mail a

0021-9673/$doi:10.1016uction

tree (Ficus carica L., Moraceae) is very common inrranean and in countries with dry and warm-temperatee Portugal. Since ancient times the figs have been usedconsumption, but it was only recently that their nutri-

harmacological value has been investigated. It seemsonsumption helps in the prevention of vein blockage,

ontent in fibers has laxative effects, and the fig latexe growth of carcinoma cells [1]. Despite the fact thats of the fig tree, like the fig leaves, have also reportedlogical properties they have been much less investi-1998, Serraclara et al. [2] reported the hypoglycemica fig leaf decoction in type-I diabetic patients, and inal et al. [3] used a chloroform extract, obtained alsooction of F. carica leaves, to decrease the cholesterolrats with diabetes. These pharmacological proper-robably in part due to the high content of phenolics in these plant extracts. Some phenolic compounds,

nding author. Tel.: +351 266745343; fax: +351 266745349.ddress: cmtc@uevora.pt (C.T. da Costa).

with reported pharmacological properties have already been iso-lated from fig leaves, namely furanocoumarins like psoralen andbergapten [4], flavonoids like rutin [5], phenolic acids like fer-rulic acid [6], and also phytosterols like taraxasterol [7].

The extraction of phenolic compounds from plants hasbeen traditionally performed using solvent extraction or steamdistillation techniques. Traditional methods of extraction arelabour-intensive, time consuming, and require large volumes ofsolvents. Following the rapid development of analytical tech-niques, trends in analytical extraction have been a movementtoward less (organic) solvent consumption, faster extractiontime and improved quantification [8]. In the last years, severalnew methods have been applied for plant phenolic extractionsuch as supercritical fluid extraction (SFE) [912], pressurizedfluid extraction (PFE) [9,13,14] and matrix solid-phase disper-sion (MSPD) [1517], which are less labour intensive and moreenvironmentally friendly. Despite the use of this new extractiontechniques solidliquid extraction (SLE) is still commonly used[1820].

MSPD is a patented process [21] that permits simultane-ous disruption and extraction of semi-solid and solid samples.This technique is based on the blending of a viscous, solid orsemi-solid sample with an abrasive solid support material. This

see front matter 2005 Elsevier B.V. All rights reserved./j.chroma.2005.11.047(SLE) to extract phenolic compoundartins Teixeira a,b, R. Ferreira Patao a, A. Varela

a Departamento de Qumica da Universidade de Evora, CLAV Rua Rob Instituto de Ciencias Agrarias e Mediterran

c Instituto de Tecnologia Qumica e Biologica (ITQB) da Ud Centro de Qumica de Evora (CQ

Received 23 May 2005; received in revised form 29 OctobAvailable online 15 Decemb

isruption method (SSDM) and matrix solid phase disruption (MSPD) wpounds from the Ficus carica leaves. Statistical treatment, ANOVA-s

hods, and for the majority of the extracted compounds, significantly hiion methods are faster and ecologically friendly, but the sea sand metholeast expensive method. Recoveries above 85% were obtained for chl

vier B.V. All rights reserved.ds and solidliquid extractionom Ficus carica leaveselho a,c, C. Teixeira da Costa a,d,

Ramalho no 59, 7000-617 Evora, Portugal(ICAM), Portugalidade Nova de Lisboa, Portugalortugal05; accepted 3 November 20055

compared to solidliquid extraction (SLE) for extraction offactor, was used to compare the extraction yields obtained

yields were obtained by the solid disruption methods. Boths more reproducible (RSD < 5% for most compounds), andnic acid, rutin, and psoralen using the sea sand extraction

D.M. Teixeira et al. / J. Chromatogr. A 1103 (2006) 2228 23

method has been applied mainly to the analysis of herbicides,pesticides and pollutants from animal tissues, fruits, vegeta-bles and alreports havphenolic exnolic compnamely, phflavanonesuse of a newfrom plantsimilar to ting media.(SSDM), cthones andpomifera [1sample dismethod ma

The aimfamilies ofextracted bwere evalurutin (flavonolic acid)tested, andson of the pLC analysition was acby LC-DADof the sea sand the deMSPD was

2. Experim

2.1. Mater

Acetonireagent) we(HPLC graGermany; nscan (Dublobtained frLIPORE Sand LC ana

The soliC18, 40mmany). Seasand particdiameter.

The F.the Springweek; a foparticles, afground barStandardsAgros Orgafrom Fluka

2.2. Preparation of standards

.0 moraleetrictockns w0.0,n wns wuse.

xtrac

Solimgol:w, re

h a 0

Mattionh C118 waasheetha00 mh 20of ng a

lumninto. Th

apere walutednderteredny).

. Opof ouppoiffere weriede meMach

epro

reprf thk areincluere

C-DAso from other matrices [2227]. So far, only a fewe been published using MSPD technique for planttraction [1517]. Only a few families of plant phe-ounds have been extracted by the MSPD techniqueenolic acids [15], isoflavonoids [16], xanthones and[17]. In 2005, Teixeira and da Costa [17] reported the

method for extraction of xanthones and flavanonesmaterial, which evolve an experimental procedurehe MSPD, but uses sea sand as the sample disrupt-This new procedure, the sea sand disruption methodompared favorably with MSPD and SLE for xan-flavanones extraction from the root bark of Maclura7]. Higher yields were obtained by the more expedite

ruption methods, but the lower cost of the sea sandkes it a very promising extraction procedure.of this work is to evaluate the use of SSDM to other

plant phenolics. In order to do it, F. carica leaves werey MSPD, SSDM, and SLE, and the extraction yieldsated for several extracted compounds which includenol), psoralen (coumarin) and chlorogenic acid (phe-. Several elution media and elution volumes werethe extraction efficiency was evaluated by compari-eak areas of the individual compounds obtained by

s using diode array detection. Compound identifica-hieved by their UV and mass spectra obtained on-line

and LC-ESI-MS, respectively. The chemical natureand used was determined by X-ray diffraction (XRD)gree of sample disruption attained by SSDM andevaluated by optical microscopy (OM).

ental

ials and reagents

trile (HPLC gradient grade) and methanol (analyticalre purchased from SDS (Peypin, France); methanoldient grade) was purchase from Merck (Darmstadt,-hexane (analytical reagent) was obtained from Lab-

in, Ireland); formic acid (HPLC gradient grade) wasom Merck (Darmstadt, Germany). Water from a MIL-implicityTM system was used for sample preparationlysis.d support material used for MSPD was Polygoprep, non-end-capped 14% C, (Macherey-Nagel, Ger-sand was collected in Faro Beach, Portugal. The

les size was homogenised with a sieve for 1 mm

carica leaves were collected in Evora, Portugal inof 2004. The green leaves were air-dried for one

od processor was used to grind the leaves into fineter which they were stored at 4 C. The same batch ofk was used with the different extraction techniques.of chlorogenic acid and rutin were purchased fromnics (New Jersey, USA), and psoralen was obtained(Madrid, Spain).

A 5and psvolumthree ssolutio80.0, 6psoralesolutiobefore

2.3. E

2.3.1.500

methanvacuum

throug

2.3.2.disrup

Botuse: Cwas w

with mA 5

tar wit2.0 mLtar usinfor coferredbottomfilter psyringwere e

dried uand filGerma

2.3.2.1nationsolid sFour dmixturwere dmixturfilter (

2.4. R

Thebility othe peawhichcates wcate Lg amount of each standard (chlorogenic acid, rutinn) was weighed, dissolved and transferred to 5 mLflasks with methanol (HPLC gradient grade) to yieldsolutions (1000g/mL). By serial dilution of thoseith methanol, calibration standards at levels of 100.0,40.0 and 20.0g/mL of chlorogenic acid, rutin andere obtained. All the stock solutions and workingere stored at 4 C, and brought to room temperature

tion procedures

dliquid extractionsamples of dry leaves were soaked in 20.0 mL ofater (7:3, v/v) for 24 h. All extracts were dried under

dissolved in 5.0 mL of the same mixture, and filtered.45-m PTFE filter (Macherey-Nagel, Germany).

rix solid phase dispersion (MSPD) and sea sandmethod (SSDM)8 solid support material and sand were cleaned befores washed three times with methanol and the sea sand

d several times with deionised water and three timesnol. Both materials were air dried before use.g sample of dried leaves was placed in a glass mor-

00 mg of the previously cleaned C18 or sea sand and-hexane. The materials were mixed in the glass mor-

glass pestle to obtain a homogenous material suitablepacking. The blend was then quantitatively trans-

a 5 mL syringe with three circles of filter paper on thee packing material was covered with another circle ofand compressed using the syringe plunger. The filleds then dried under vacuum. The phenolic compounds

with methanol:water (7:3, v/v). All extracts werevacuum, redissolved in 5.0 mL of the same mixture,through a 0.45m PTFE filter (Macherey-Nagel,

timal elution volume determination. The determi-ptimal elution volume was done using sea sand asrt and methanol:water (7:3, v/v) as elution media.

ent elution volumes of the methanol:water (7:3, v/v)re tested: 5.0, 10.0, 15.0 and 20.0 mL. All extractsunder vacuum, redissolved in 5.0 mL of the samethanol:water, and filtered through a 0.45m PTFEerey-Nagel, Germany).

ducibility and recovery

oducibility of the analytical methods and the repeata-e extraction procedures were assessed by evaluatinga variation of eight compounds present in the extractsde chlorogenic acid, rutin and psoralen. Five repli-

performed for each extraction assay, and three repli-D analyses were performed on each filtrate.

24 D.M. Teixeira et al. / J. Chromatogr. A 1103 (2006) 2228

Statistical treatment (ANOVA-single factor, p < 0.001,Microsoft Excel 2000) was performed to the data to determinesignificant

The recwas assess

rutin and pto 300.0ggenic acidwith 500 mperformedwas dried,for the ext(Machereyand three r

2.5. LC-D

An Agilwith a diodlent TechnThe analytXDB-C18,cle size (Agcolumn wa(length IGermany).vent B: waGradient prof solvent20 to 40%solvent Atemperaturrate was 1.to 500 nm,254 nm.

2.6. LC-ES

LC-ESItage Thermtrospray ionwas controcoupled to(DAD) (Suveyor Therillary temp80.0A, avoltage 4.545.0 V into those us

3. Results

3.1. Comp

The F. cflavonols, c

ferent extraction procedures, namely MSPD, SSDM, and SLE,in order to evaluate their extraction efficiency.

eralphenixturerin

nol:ractinolicelue, likcom

eenndedv) asracting mves

and-DAtractare n

unds-DADmedcatipouchlo

28,2lic acver bnd 8tivel[4].

mariherentsed uso thagm

+ Hthe

r 7)tternpso

t = 37. M O

entifionlym ofor3) is

APCrder

, thewn,differences whenever they occurred.overy of the sea sand disruption method (SSDM)ed by measuring the recovery of chlorogenic acid,soralen. 300.0L of rutin and psoralen (equivalent), and 400.0L (equivalent to 400.0g) of chloro-standard stock solutions were added to the mortarg of plant and 2000 mg of sand. The extraction waswith 10.0 mL methanol:water (7:3, v/v). The extractrecovered in 5.0 mL of the solvent mixture used

raction, and filtered through a 0.45m PTFE filter-Nagel, Germany). This assay was repeated five timeseplica analyses were performed on each extract.

AD

lent 1100 system (Agilent Technologies, Germany)e-array detector (DAD) and an HP ChemStation (Agi-ologies, Germany) was used for LC-DAD analyses.ical column was a reversed-phase Zorbax Eclipse250 mm 4.6 mm (length I.D.) and 5m parti-ilent Technologies, Germany). The analytical guards a Zorbax Eclipse XDB-C18, 12.5 mm 4.6 mm

.D.) and 5m particle size (Agilent Technologies,The mobile phase was: solvent A: acetonitrile; sol-ter with acetonitrile (2.5%) and formic acid (0.5%).ogram was adopted as follows: linear from 0 to 15%A (05 min), 15 to 20% of solvent A (525 min),of solvent A (2530 min) and from 40 to 45% of

(3040 min). LC analyses were performed at roome; the injection volume was 20L, and the flow-0 mL/min; the DAD detector was scanned from 200and the chromatographic profile was recorded at

I-MS/MS

-MS/MS analyses were carried out in a LCQ Advan-oFinnigan mass spectrometer equipped with an elec-ization source and using an ion trap mass analyser. It

lled by Xcalibur software (ThermoFinnigan). It wasan HPLC system with a photodiode array detectorrveyor ThermoFinnigan) and an autosampler (Sur-moFinnigan). The conditions of analyses were: cap-erature 250 C; source voltage 4.0 kV, source currentnd capillary voltage 7.0 V in positive mode; sourcekV, source current 80.0A, and capillary voltagenegative mode. The elution conditions were similar

ed for the LC-DAD analysis.

and discussion

arison of extraction procedures

arica leaves, rich in plant phenolics families, namelyoumarins and phenolic acids, were extracted by dif-

Sevleavesand mConsidmethaall extmethaferredarationmatrixhave bcyl bo9:1 (v/the ext

Usiica leasilica,HPLCent exand 5compo

LCperforidentififor comfied asrutin [phenohas neber 7 arespecleavesnocou

whilefragmeanalyzand alther fr143 [Mpeak innumbetion pafor thewith rm/z 21[M + Hwas idralen,spectruerature(m/z 20under

In ociencyunknoelution media were initially evaluated for the figolic extraction: dichloromethane, ethanol, methanoles of methanol:water (9:1, 7:3, 4:6, and 1:1, v/v).g the yields and number of compounds extracted,water mixtures were the most efficient eluents foron methods tested. Among the different aqueoussolutions, methanol:water (7:3, v/v) was the pre-

nt because it originated better chromatographic sep-ely because the extracts contained less unwantedponents (data not shown). Unlike to what might

expected due to the chemical properties of octade-silica (C18), the use of methanol or methanol:watereluents for the MSPD procedure, did not increased

on yields of the non-polar compounds.ethanol:water (7:3, v/v) as elution media, the F. car-

were extracted by SLE, MSPD with C18 derivatizedSSDM, and the different extracts were analyzed byD (see Fig. 1). A careful examination of the differ-s chromatograms reveals that compounds number 2ot extracted by SLE, and higher amounts of thoseseam to have been removed when SSDM is used.and LC-DAD-MS/MS analysis of the extracts was

and the mass and UV spectra obtained enabled theon of some of the extracted compounds (see Table 1nd identification). Compound number 1 was identi-rogenic acid [2830], and compound number 4 as

9,31]. Chlorogenic acid (5-caffeoylquinic acid) is aid very common in plants but, as far as we know, iteen identified in F. carica leaves. Compounds num-were identified as psoralen [32] and bergapten [33],

y, and these had already been identified in F. caricaThe reported on-line mass spectra for these fura-

ns has been obtained by the APCI interface [32,33],we used an ESI interface, and as expected, more

were observed. When a standard of psoralen wasnder ESI conditions a fragment at m/z 187 [M + H+]e acetonitrile adduct at m/z 228 were observed. Fur-entation of parent ion at m/z 187, yield ions at m/z CO2+], and m/z 115 [M + H CO2 CO+]. The

extracts chromatograms with rt = 34 min (compoundwas identified as psoralen as it yield a fragmenta-, an UV spectra, and a rt similar to those obtainedralen standard under similar conditions. The peak

8 min (compound number 8) yield a parent ion atS2 of the parent ion yielded fragments at m/z 203Me+] and m/z 173 [M + H CO2+]. This compounded as bergapten, a furanocoumarin similar to pso-with an extra methoxi group on carbon 5. The UVf this compound is similar to that reported on the lit-bergapten, and a fragment loss of a methoxy groupalso observed when the mass spectrum was obtained

I conditions [33].to access the different extraction procedures effi-

peak areas for the different compounds, known andwere evaluated (see Table 2 and Fig. 2). As it was

D.M. Teixeira et al. / J. Chromatogr. A 1103 (2006) 2228 25

Fig. 1. LC-DAD chromatograms of methanol:water (7:3, v/v) extracts of Ficus carica leaves samples, using SLE (A), MSPD (B) and sea sand extraction method (C).Column: Zorbax Eclipse XBD-C18. Elution conditions: Solvent A, acetonitrile; Solvent B water with acetonitrile (2.5%) and formic acid (0.5%). Gradient program:linear from 0 to 15% of solvent A (05 min), 15 to 20% of solvent A (525 min), 20 to 40% of solvent A (2530 min) and from 40 to 45% of solvent A (3040 min).Peak identification: chlorogenic acid (1); rutin (4); psoralen (7); bergapten (8); unknown (2, 3, 5, and 6).

Table 1Identification of known compounds in the leaves of Ficus carica by HPLC-DAD and HPLC-ESI-MS

Compd Identity HPLC-DADmax

ESI full scanMS () m/z

ESI-MS2 () m/z ESI full scanMS (+) m/z

ESI-MS2 (+) m/z

1 Chlorogenic acid(5-O-caffeoylquinicacid)

241 353 [M H] 191 [M C9H6O3 H] 355 [M + H]+ 163 [C9H6O3]+305sh 707 [2M H]326

4 Rutin (quercetin-3-O-rutinoside)

254 609 [M H] 301 [M rutinoside H] 611 [M + H]+ 465 [M rhamnose + H]+355 303 [M rutinoside + H]+

7 Psoralen 252 187 [M + H]+ 143 [M + H CO2]+300 228 [M + H + CH3CN]+ 115 [M + H CO2 CO]+332sh

8 Bergapten(5-Methoxypsoralen)

252 217 [M + H]+ 203 [M + H OMe]+256 173 [M + H CO2]+268312

26 D.M. Teixeira et al. / J. Chromatogr. A 1103 (2006) 2228

Table 2Evaluation of the precision on the SLE, MSPD and sea sand method extraction and LC analysis of plant phenolic compounds from the leaves of Ficus carica

Compound (peak number) SLE: peak areaa (mAUs) MSPD C18: peak areaa (mA a

Meanb (SD)c % RSDd Meanb (SD)c %Chlorogenic acid (1) 2896.101 (104.72) 3.62 3353.392 (143.68) 4.Unknown (2) not extracted 10727.221 (1360.30) 12.Unknown (3) 12049.211 (493.37) 4.09 16581.062 (844.34) 5.Rutin (4) 26165.621 (1117.55) 4.27 22392.372 (1994.97) 8.Unknown (5) not extracted 5592.161 (869.91) 15.Unknown (6) 3135.771 (271.74) 8.67 4380.252 (942.75) 21.Psoralen (7) 21516.191 (454.34) 2.11 15117.562 (2913.06) 19.Bergapten (8) 6348.991 (441.13) 6.95 2770.762 (361.68) 13.

a Normalized to 500 mg of leaves extracted, sample dried and redissolved in 5 mL of methanol:wb The values represent the mean of three replicate measurements on the five different extract

significantly different (ANOVA: single factor Microsoft Excel 2000, P < 0.001).c Standard deviation of a single measurement.d Relative standard deviation.

stated before, unknowns 2 and 5 are only extracted by the disrup-tion methods, and statistical analysis by ANOVA-single factorof the peak areas of these compounds indicate that efficiency ofthe two methods was not significantly different. This is likelydue to the fact that, for these compounds, the difference betweenaverage peak areas obtained with the two extraction proceduresis smaller tmuch lesscompoundsthis methodods were mand unknowicantly hig

For rutimethod gavefficiency wthese comp

Fig. 2. Compcarica leavesplant; agitatio2000 mg of Cextraction me(7:3, v/v) and

time with tarchitecturMSPD metthe non-po

The higthe most p

andtionend tDMtionesisple dche

by phe md w

lluscis cois mawn)

SPD). Thhen their replicate error. The MSPD extractions werereproducible and the average extraction yields for2 and 5 were affected by the large RSD values for. When compared with SLE, both disruption meth-ore efficient in the extraction of chlorogenic acidns 3 and 6, with their extraction yields being signif-

her when the sea sand method was used.n, psoralen and bergapten the conventional SLEe similar results to SSDM, whereas the MSPD C18as significantly lower. The extraction efficiency for

ounds is probably controlled by the higher contact

MSPDdisrupvent, tthe SSinterachypothof sam

Theminedysis. Tthe sanof moanalyswhichnot shoand Mshownarison between SLE, MSPD (C18) and sea sand extraction of Ficussamples with methanol:water (7:3, v/v). Conditions: 500 mg ofn at room temperature with 20 mL of solvent for 24 h in SLE;

18 or sea sand eluted with 20 mL of solvent in MSPD and sea sandthod; all extracts dried, redissolved in 5 mL of methanol:wateranalyzed by LC-DAD. Compound identification: see Table 1.

efficient dipieces andthe cell placorroboratewith the SSfactors: veinteraction

3.2. Deterextraction

The asselution volSSDM extsolvent. Thtically evalUs) Sea sand extraction method: peak area (mAUs)RSDd Meanb (SD)c % RSDd

28 3781.763 (89.81) 2.3768 11724.301 (676.39) 5.7709 18803.663 (266.74) 1.4291 26395.221 (1523.60) 5.7756 6043.681 (157.70) 2.6152 6402.023 (178.54) 2.7927 20050.651 (1054.80) 5.2605 5721.951 (428.21) 7.48ater (7:3, v/v); 20L injection.

s. For each compound means with different index numbers are

he solvent (in SLE), or by a more effective samplee disruption (in SSDM). The lower efficiency of thehod might also be due to strong interactions betweenlar furanocoumarins with the C18 materials.her extraction efficiency observed for the majority ofolar compounds (with the exception of rutin) whenSSDM methods are used is likely due to the sample

which, by exposing the cell components to the sol-o yield richer extracts. Another important factor forextraction efficiency is probably the lack of chemicals between the sand and the analytes. To verify thisthe chemical composition of the sand and the degreeisruption were analyzed.mical composition of the sea sand used was deter-etrographic microscopy and X-ray diffraction anal-icroscopic pictures (data not shown) indicated that

as mainly composed by quartz, with minor amountsshells and sandstone aggregates. X-ray diffraction

nfirmed the mineralogical composition of this sand,inly quartz with traces of orthoclase and calcite (data

. The degree of sample disruption attained by SSDMwas evaluated by optical microscopy (OM) (data note abrasive properties of sand seem to provide a more

sruption of the plant material, breaking it in smallerin this way exposing, in a more efficiently manner,nt components to the eluents. These results seem tothe idea that the high extraction efficiency observed

DM method is probably due to a combination of twory effective sample disruption and lack of chemicals between the analytes and the solid support.

mination of optimum elution volume for sea sand

ays performed for the determination of the optimumume were done using the optimized conditions forraction using methanol:water (7:3, v/v) as elutione peak areas of all analyzed compounds were statis-uated for various volumes of the elution solvent. The

D.M. Teixeira et al. / J. Chromatogr. A 1103 (2006) 2228 27

Fig. 3. LC peak area variation of the compounds extracted from Ficus caricaleaves samples by sea sand method with increasing methanol:water (7:3, v/v)volume. Conditions: 500 mg of plant; 2000 mg of sea sand eluted with 5.0, 10.0,15.0 and 20.0 mL of methanol:water (7:3, v/v); all extracts dried, redissolved in5 mL of methanol:water (7:3, v/v) and analyzed by LC-DAD.

data presented in Fig. 3 and Table 3 show that maximum yieldsfor all compounds, except unknown 2, were obtained with 10 mLof solvent. Maximum yields of unknown 2 was obtained withonly 5 mL. Extraction of this compound is only accomplished bythe disruption methods, and maximum yields are obtained withless solvent. Probably, this due to the fact that unknown 2 is oneof the most polar compounds extracted, and it is likely to have ahigh affinity with the chosen eluent. Chlorogenic acid is anotherpolar extracted compound, and a similar behavior should also beexpected. In fact, when elution was performed with 5 and 10 mL

of eluent, the difference in peak areas for the chlorogenic acidwas so small that could only be recognized when the data wassubjected to statistical analysis.

3.3. Validation: reproducibility and recovery

The data presented in Table 2 demonstrate that SLE andSSDM procedures are reproducible as RSD values were lessthan 5% for almost all compounds. However, when MSPDC18 was used the RSD values were higher. The small repro-ducibility of the MSPD method together with the cost of theC18 materials makes it much less attractive than the sea sandmethod.

To evaluate the sea sand disruption method recovery, spik-ing experiments were performed for three known compounds,chlorogenic acid, rutin and psoralen. The mean peak area ofthe three spiked compounds was calculated by subtracting thetotal peak area after spiking from the mean peak area in theextract of the plant before spiking. Calibration curves for thecompounds were constructed using the standard solutions pre-pared. The characteristic data, the correlation coefficients andthe errors of estimation of slope and intercept parameters arelisted in Table 4. The recoveries were 97.3, 85.7 and 86.2% forchlorogenic acid, rutin and psoralen, respectively.

The limits of detection (LOD) were estimated as 0.0515,0.0310 and 0.0342 mg/g for chlorogenic acid, rutin and pso-ralen, respectively and corresponding to the analyte concentra-tion giving a signal equal to the blank signal plus three standarddeviations of the blank [34]. The limits of quantification (LOQ)

Table 3Evaluation of ethaleaves of Ficu

Compound (p1

d M

Chlorogenic aUnknown (2) 1Unknown (3) 1Rutin (4)Unknown (5)Unknown (6)Psoralen (7)Bergapten (8)

a Normalizeb The value

significantly dc Standard dd Relative s

Table 4Calibration cu

Compound

Chlorogenic aRutinPsoralen

a Upper andthe precision on the optimal volume determination in SSDM extraction with ms carica

eak number) Volume of elution media (mL): peak areaa (mAUs)5.0 10.0

Meanb (SD)c % RSDd Meanb (SD)c % RSDcid (1) 2423.261 (82.01) 3.38 3691.462 (147.98) 4.01

9414.861 (704.70) 7.49 10653.301 (1002.62) 9.4113642.981 (213.66) 1.57 18926.542 (655.80) 3.47

1 218928.77 (262.77) 1.39 28016.93 (1355.05) 4.84 24958.771 (229.36) 4.63 6152.112 (286.39) 4.663090.461 (185.07) 5.99 5518.762 (231.15) 4.19

11918.901 (899.85) 7.55 18043.822 (919.82) 5.10 12217.651 (242.48) 10.93 4366.322 (353.75) 8.10

d to 500 mg of fig leaves extracted, sample dried and redissolved in 5 mL of methans represent the mean of three replicate measurements on the five different extractifferent (ANOVA: single factor Microsoft Excel 2000, P < 0.001).eviation of a single measurement.

tandard deviation.

rves, recovery, LOD and LOQ of spiking experiments of phenolic compounds in thea (estimation error)a b (estimation error)a R2

cid 41.744 (123.770; 40.283) 17.205 (15.851; 18.560) 0.919.945 (60.753; 100.642) 28.127 (26.795; 29.460) 0.929.301 (92.209; 150.812) 38.419 (36.412; 40.426) 0.9

lower 95%confidence limits (regression statistics Microsoft Excel 2000).nol:water (7:3, v/v), and LC analysis of plant phenolics from the

5.0 20.0

eanb (SD)c % RSDd Meanb (SD)c % RSDd

3581.782 (351.14) 9.80 3781.762 (89.81) 2.371487.661 (937.86) 8.16 11724.301 (676.39) 5.779442.282 (1246.36) 6.41 18803.662 (266.74) 1.429318.382 (1735.65) 5.92 26395.222 (1523.60) 5.776439.382 (555.75) 8.63 6043.682 (157.70) 2.615926.722 (834.53) 14.08 6402.022(178.54) 2.798817.752 (2083.83) 11.07 20050.652 (1054.80) 5.264866.852 (772.74) 15.88 5721.952 (428.21) 7.48ol:water (7:3, v/v); 20L injection.s. For each compound means with different index numbers are

leaves of Ficus carica

Recovery (%) LOD (mg/g) LOQ (mg/g)97 97.3 0.0515 0.188899 85.7 0.0310 0.113799 86.2 0.0342 0.1253

28 D.M. Teixeira et al. / J. Chromatogr. A 1103 (2006) 2228

were estimated as the analyte concentration giving a signalequal to the blank signal plus eleven standard deviations of theblank [35]. The corresponding LOQ values were 0.1888 mg/gfor chlorogenic acid, 0.1137 mg/g for rutin and 0.1253 mg/g forpsoralen.

4. Conclusions

The data presented here show that the solid disruption meth-ods compare favorably to SLE in the extraction of severalphenolic compounds belonging to different families, namely,phenolic acids, flavonols and coumarins, from the leaves of F.carica. More compounds and higher yields were obtained bythese methods, using smaller amounts of solvents, and less sam-ple preparation time.

The optimized extraction procedure involves the use of seasand as solid support, and methanol:water (7:3, v/v) as elutionmedia. Higher extraction yields and smaller RSD values wereobtained with SSDM when compared with MSPD.

Acknowledgements

The auth(FCT)-Proj(FEDER) fthe Opticalrott for the

Reference

[1] S. RubnJ. Nat. P

[2] A. SerraTorres, D

[3] J.R. Can(1) (200

[4] A. Dam[5] I.S. El K[6] J.A. Du

and Oth

[7] A.K. Athnasios, I.E. El Kholy, G. Soliman, M.A.M. Shaban, Int. J.Chem. Soc. 62 (1962) 4253.

[8] D.E. Raynie, Anal Chem. 76 (2004) 4659.[9] C.T. da Costa, S.A. Margolis, B.A. Benner Jr., D. Horton, J. Chromatogr.

A 831 (1999) 167.[10] P. Castioni, P. Christen, J.L. Veuthey, Analusis 23 (1995) 95.[11] R.M. Smith, LC-GC 13 (1995) 930.[12] C.D. Bevan, P.S. Marshall, Nat. Prod. Rep. 11 (1994) 451.[13] T.S. Reighard, S.V. Olesik, Crit. Rev. Anal. Chem. 26 (1996) 61.[14] M. Schantz, J.J. Nichols, S.A. Wise, Anal. Chem. 69 (1997) 4210.[15] A. Ziakova, E. Brandsteterova, E. Blahova, J. Chromatogr. A 983 (2003)

271.[16] H.B. Xiao, M. Krucker, K. Albert, X.M. Liang, J. Chromatogr. A 1032

(2004) 117.[17] D.M. Teixeira, C.T. da Costa, J. Chromatogr. A 1062 (2005) 175.[18] U. Jin, J. Lee, S. Kang, J. Kim, W. Park, J. Kim, S. Moon, C. Kim,

Life Sci. 77 (2005) 2760.[19] N.A. Ayoub, Phytochemistry 63 (2003) 433.[20] U. Justesen, J. Chromatogr. A 902 (2000) 369.[21] S.A. Barker, A.R. Long, C.R. Short, J. Chromatogr. 475 (1989) 353.[22] S.A. Barker, J. Chromatogr. A 885 (2000) 115.[23] C. Blasco, Y. Pico, J. Manes, G. Font, J. Chromatogr. A 947 (2002)

227.[24] M. Navarro, Y. Pico, R. Marn, J. Manes, J. Chromatogr. A 968 (2002)

201.BlascBlasc

. Gatogr.. Tomrce, A

ParejoCodinSchu(2004Wangric

Wang1055Dugomed.. Miemist.

Prags01) 7ors thank the Fundacao para a Ciencia e Tecnologiaect POCTI/QUI/41839 and Fundo Social Europeuor the financial support, Prof. Antonio Candeias forMicroscopy analysis and Prof. Manuela Ribeiro Car-XRD analysis of sea sand.

s

ov, Y. Kashman, R. Rabinowitz, M. Schlesinger, R. Mechoulam,rod. 64 (2001) 993.

clara, F. Hawkins, C. Perez, E. Dominguez, J.E. Campillo, M.D.iabetes Res. Clin. Pract. 39 (1998) 19.

al, M.D. Torres, A. Romero, C. Perez, Acta Physiol. Hung. 870) 71.janic, B. Akacie, Planta Med. 26 (2) (1974) 119.holy, M.A. Shaban, J. Chem. Soc. Perkin1 13 (1966) 1140.

ke, Handbook of Phytochemical Constituents of GRAS Herbser Economic Plants, CRC Press, Boca Raton, FL, 1992.

[25] C.[26] C.[27] R.M

ma

[28] F.APie

[29] I.C.

[30] K.52

[31] M.J. A

[32] X.A

[33] P.Bio

[34] J.NCh121

[35] F.(20o, G. Font, Y. Pico, J. Chromatogr. A 970 (2002) 201.o, G. Font, Y. Pico, J. Chromatogr. A 1028 (2004) 267.rcinuno, L. Ramos, P. Fernandez-Hernando, C. Camara, J. Chro-A 1041 (2004) 35.

as-Barberan, M.I. Gil, P. Cremin, A.L. Waterhouse, B. Hess-.A. Kader, J. Agric. Food Chem. 49 (2001) 4748.

, O. Jauregui, F. Sanchez-Rabaneda, F. Viladomat, J. Bastida,a, J. Agric. Food Chem. 52 (2004) 3679.

tz, D. Kammerer, R. Carle, A. Schieber, J. Agric. Food Chem.) 4090.

g, Y. Tadmor, Q.L. Wu, C.K. Chin, S.A. Garrison, J.E. Simon,. Food Chem. 51 (2003) 6132., Y. Wang, J. Yuan, Q. Sun, J. Liu, C. Zheng, J. Chromatogr.(2004) 135., L. Mondello, L. Dugo, R. Stancanelli, G. Dugo, J. Pharm.Anal. 24 (2000) 147.

ller, J.C. Miller, Statistics and Chemometrics for Analyticalry, fourth ed., Pearson Education Limited, London, 2000, p.

t, V. Auwaerter, F. Sporkert, K. Spiegel, Forensic Sci. Int. 1216.

Comparison between sample disruption methods and solid-liquid extraction (SLE) to extract phenolic compounds from Ficus carica leavesIntroductionExperimentalMaterials and reagentsPreparation of standardsExtraction proceduresSolid-liquid extractionMatrix solid phase dispersion (MSPD) and sea sand disruption method (SSDM)Optimal elution volume determination

Reproducibility and recoveryLC-DADLC-ESI-MS/MS

Results and discussionComparison of extraction proceduresDetermination of optimum elution volume for sea sand extractionValidation: reproducibility and recovery

ConclusionsAcknowledgementsReferences