characterisation by liquid chromatography-electrospray tandem mass spectrometry of anthocyanins in...

Journal of Chromatography A, 1112 (2006) 232–240

Characterisation by liquid chromatography-electrospray tandem massspectrometry of anthocyanins in extracts of Myrtus communis

L. berries used for the preparation of myrtle liqueur

Paola Montoro a,∗, Carlo I.G. Tuberoso b, Angela Perrone a, Sonia Piacente a,Paolo Cabras b, Cosimo Pizza a

a Dipartimento di Scienze Farmaceutiche, Universita di Salerno, Via Ponte don Melillo, 84084 Fisciano (SA), Italyb Dipartimento di Tossicologia - Sez. Alimenti e Ambiente, Universita degli Studi di Cagliari Via Ospedale, 72, 09124 Cagliari (CA), Italy

Abstract

Anthocyanins in extracts of berries of Myrtus communis, prepared following a typical Sardinia myrtle liqueur recipe, were identified andquantified by HPLC coupled with electrospray/tandem mass spectrometry using, respectively, an ion trap and a triple quadrupole mass analyser.Trsi©

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he fragmentation patterns of the anthocyanidins were dependent on the MS technique employed, and differed considerably from those previouslyeported. The anthocyanin profile of five anthocyanin glucosides and four anthocyanin arabinosides, the latter not previously identified in thispecie, was specific for myrtle berry extracts. The quantitative compositions of extracts of myrtle berries derived from different geographical areasn Italy were compared.

2005 Elsevier B.V. All rights reserved.

eywords: Myrtus communis; Anthocyanins; Anthocyanidins; Fragmentation patterns; Quantitative determination; LC–MS; ES/MS/MS

. Introduction

Anthocyanins are C15 phenolic glycosides and belong to thelass of plant pigments generally known as flavonoids. In planta,he anthocyanins can act as insect attractants for the purposesf pollination and, since their synthesis is often induced byV radiation, may also serve as UV screens thereby protect-

ng plant DNA from damage by sunlight. The pigments alsoossess antifeedant properties, since their disagreeable taste isble to deter predatory animals.

In recent years, increased interest has been shown in explor-ng the benefits to human health occasioned by the free-radicalcavenging and antioxidant capacities of those anthocyanins nat-rally available in fruits and vegetables [1]. Thus, anthocyaninsn isolated form or as enriched mixtures, may enhance antiin-ammatory and estrogenic activities, inhibit lipid peroxidationnd enzymes associated with tumour formation, provide pro-ection from DNA cleavage, boost the production of cytokines,

∗ Corresponding author. Tel.: +39 089 964384; fax: +39 089 962828.

decrease capillary permeability and fragility and maintain mem-brane integrity [2–6].

The profiles of the anthocyanins present in fruits and vegeta-bles can be used as fingerprints through which the authenticityof raw materials, products, juices and extracts may be assessed.A number of methodologies are available for the analysis ofanthocyanins, but those most commonly employed are HPLC,CE and LC coupled with MS (for a review, see [7]). Althoughthere are various reports of the use of HPLC with UV detection at520 nm (corresponding to the red colour of the pigments) for thedetermination of anthocyanins in mixtures, such methods cannotproduce very accurate results, since the colour of an anthocyaninis dependent upon the incident light, and on the pH and pres-ence of metal ions [8]. On the other hand, LC coupled with MSdetection permits more accurate analyses and may reveal datathat could be useful in the identification of oxidation productsor equilibrium forms of the pigments that exhibit no absorbanceat 520 nm. Moreover, application of on-line tandem mass spec-trometry (MS/MS) greatly simplifies the procedure, typicallyenabling the establishment of pigment structure through molec-ular mass determination and subsequent selective isolation and

E-mail address: [email protected] (P. Montoro). targeted fragmentation of the ions of interest.

021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2005.11.055

P. Montoro et al. / J. Chromatogr. A 1112 (2006) 232–240 233

The metabolomic characterisation of species of Vitis and ofwines has been achieved through the assay of anthocyanins [9];being one of the main effectors of the grape phenotype, knowl-edge of pigment profile provides information that complementsgene expression and proteome analysis. Numerous other fruits,also employed as food materials or as ingredients for the foodindustry, are rich in anthocyanins. The berries of Myrtus commu-nis L., for example, are used to produce the characteristic myrtleliqueur typical of Sardinia, Italy. The fruits are spherical in shape,dark red to violet in colour, and are reported to contain delphini-din, petunidin, malvidin, peonidin and cyanidin-3-mono- and-3,5-diglucosides [10]. Since the organoleptic properties of theliqueur are strongly associated with the anthocyanin content,and since these pigments show only limited stability [11,12],the objective of the present work was to define an anthocyaninprofile for M. communis, and to use this profile in order to verifythe origin of the berries. We have thus developed an LC–MSmethod for the qualitative and quantitative determination ofanthocyanins and anthocyanidins from myrtle berry extracts. Inthis technique, electrospray (ES) ionisation combined with anion trap (IT) mass analyser was used together with low energyMS/MS for fragmentation studies. A mechanism of fragmen-tation for the anthocyanidins was proposed and compared withliterature data obtained by high-energy collision induced dis-sociation (CID) [13]. Anthocyanin arabinosides, not reportedpreviously in M. communis, were identified, and a pigment pro-fido

tion was investigated by comparison of myrtle extracts obtainedfrom berries collected in different regions of Italy.

2. Experimental

2.1. Reagents and standards

HPLC grade methanol, acetonitrile and trifluoroacetic acidwere purchased from J.T. Baker (Baker Mallinckrodt, Phillips-burg, NJ, USA). HPLC grade water (18 m�) was prepared usinga Millipore (Bedford, MA, USA) Milli-Q purification system.Standards of anthocyanidins (delphinidin, malvidin, peonidinand cyanidin chloride) and of the anthocyanins 1, 2, 4 and5 and cyanidin-3-O-galactopyranoside were purchased fromExtrasynthese (Lyon, France). Standards of the anthocyanins3 and 6–9 (Fig. 1) were isolated in our laboratories using thesemi-preparative procedures described below. Standard stocksolutions (1 mg/mL in methanol) of each of 1–9 were prepared.

2.2. Preparation and analyses of myrtle extracts

Berries of M. communis L. were collected in December 2002from the Italian regions of Sardinia, Campania and Calabria.The Sardinia berries were supplied by Zedda Piras Company(Alghero (SS), Italy), whilst the Campania and Calabria berrieswcsU

le established by which the quality of the product might beefined. Further, the application of the method for the validationf the Sardinian origin of M. communis used for extract prepara-

Fig. 1. ES(IT)/MS/MS of delphinidin. ES/MS/MS work

ere kindly supplied and identified by, respectively, Prof. Vin-enzo De Feo (Dipartimento di Scienze Farmaceutiche, Univer-ita di Salerno, Italy), and Prof. Mariateresa Russo (DISTAA,niversita di Reggio Calabria, Italy). Extracts were obtained

ing in positive ion mode. Collision Energy 30%.

234 P. Montoro et al. / J. Chromatogr. A 1112 (2006) 232–240

using the traditional recipe for the preparation of the liqueurby macerating fresh berries (130 g) in ethanol:water (70:30;250 mL) for 40 days. For qualitative analysis, each extract wasdiluted 1:10 with ethanol:water (70:30) and a 20 �L aliquotinjected into the analytical system. For quantitative determina-tion, each extract was diluted 1:10 with ethanol:water (70:30),an appropriate volume of the internal standard (IS) cyanidin-3-O-galactopyranoside was added to give a final concentrationof 25 �g/mL, and a 20 �L aliquot injected into the analyticalsystem; determinations were replicated five times each.

2.3. HPLC-UV–vis analysis

An Agilent (Palo Alto, CA, USA) 1100 series system con-sisting of a G-1312 binary pump, a G-1328A Rheodyne injector(20 �L injection loop), a G-1322A degasser and a G-1315Aphotodiode array detector was employed. Semi-preparative anal-yses were carried out using a Waters �-Bondapack C18 column(300 mm × 7.8 mm i.d.; particle size 10 �m) eluted with mix-tures of 0.1% trifluoroacetic acid (TFA; solvent A) and acetoni-trile:water (1:1) containing 0.1% TFA (solvent B) at a flow rateof 2.5 mL/min. Elution was by step gradient from 80:20 (A:B)to 67:33 in 18 min, then from 67:33 to 60:40 in 12 min, and sub-sequently isocratic with 60:40 for 10 min; the column effluentwas monitored at 520 nm. Under these chromatographic condi-tions, compounds 1–9 [retention times (R ) 37.10, 41.46, 43.75,4co

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and the activation time 30 ms. In order to tune the LCQ foranthocyanins, the voltages on the lenses were optimised usingthe TunePlus function of the Xcalibur software in the posi-tive ion mode whilst infusing a standard solution of delphinidin(1 �g/mL in methanol) at a flow rate of 3 �L/min. Anthocyani-dins were analysed under the same conditions and with the sameoptimised parameters.

Full ES/MS and CID ES/MS/MS analyses of standardswere performed on an Applied Biosystems (Foster City,CA, USA) API2000 ES mass spectrometer equipped with atriple quadrupole (QqQ) analyser. Parameters were optimisedby infusing a standard solution of delphinidin (1 �g/mL inmethanol) into the source at a flow rate of 5 �L/min. The opti-mised parameters were: declustering potential 80 eV; focusingpotential 400 eV; and entrance potential 10 eV. Experimentswere run in the Q1 MS mode in order to obtain ES/MS spectra,and in the product ion scan mode in order to carry out MS/MSexperiments; in the product ion scan mode, the collision energywas 50 eV, and the collision cell exit potential was 3 eV.

Qualitative on-line LC-ES/MS analyses of extracts wereperformed using the Thermo Finnigan Spectra System HPLCcoupled with the LCQ Deca IT with the chromatographic condi-tions as described above. The flow from the chromatograph wasinjected directly into the ES ion source and MS were acquiredand elaborated using the software provided by the manufacturer.In LC-ES/MS/MS experiments, data were acquired using thedtomw

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t8.32, 50.04, 46.10, 52.22, 53.76 and 61.95 min, respectively]ould be isolated. Compounds 6–9 were examined by NMR inrder to characterise the pentose sugar unit.

.4. NMR analysis

NMR experiments were performed at 300 K on a BrukerBruker BioSpin, Rheinstetten, Germany) DRX-600 spectrom-ter operating at 600 and 150 MHz, respectively, for 1H- and3C-spectra; data were processed using UXNMR software. TheH and 13C NMR data of 6–9 were identical with those reported14] for delphinidin-3-O-�-arabinopyranoside, cyanidin-3-O--arabinopyranoside, petunidin-3-O-�-arabinopyranoside andalvidin-3-O-�-arabinopyranoside, respectively.

.5. ES/MS, LC-ES/MS and LC-ES/MS/MS analyses

ES/MS and ES/MS/MS analyses of standards were per-ormed using a Finnigan (Thermo Finnigan, San Jose, CA, USA)CQ Deca IT instrument equipped with Xcalibur software. Sam-les were dissolved in methanol (1 �g/mL) and the solutionsnfused into the ES ionisation source using a syringe pumpt a flow rate of 5 �L/min. For the analysis of anthocyanins,he instrument was operated in the positive ion mode underhe following conditions: capillary voltage 3 V; spray voltagekV; tube lens offset 40 V; capillary temperature 260 ◦C; and

heath gas (nitrogen) flow rate 60 arbitrary units. Data werecquired in the MS1 and MS/MS scanning modes with scananges of, respectively, 150–900 and 50–500 m/z: the maximumnjection time was 50 ms, and the number of microscans was. For MS/MS, the scanning mode collision energy was 30%,

ependent scanning mode in which the MS software selectedhe ion to fragment on the basis of charge and intensity, andptimised all parameters in order to improve ionisation and frag-entation. In the analysis of anthocyanins, the precursor ionsere, typically [M]+ and [M–sugar]+.Quantitative on-line LC-ES/MS/MS analyses of extracts

ere performed using the Agilent 1100 HPLC system interfacedo the Applied Biosystems API2000 instrument with chromato-raphic conditions as described for the LC-ES/MS experiments.he instrument was used in the tandem MS mode with multi-le reaction monitoring (MRM). For all anthocyanins analysed,he selected fragmentation reaction was the loss of the glyco-ide moiety. The API 2000 ES source was tuned by infusing atandard solution of delphinidin (1 �g/mL in methanol) into theource at a flow rate of 10 �L/min. The optimised parametersere: declustering potential 80 eV; focusing potential 400 eV;

ntrance potential 10 eV; collision energy 50 eV; and collisionell exit potential 3 eV.

.6. Calibration and quantification

A sample (ca. 10 mg) of each of the anthocyanin standardsas weighed accurately into a 10 mL volumetric flask, dissolved

n methanol and the volume made up to the mark with methanol.he resulting stock solutions were diluted with methanol inrder to obtain reference solutions containing 5, 25, 50 and25 �g/mL, and to each reference standard solution was addedn appropriate amount of IS to give a final concentration of5 �g/mL. Calibration curves for each of the reference standardsere constructed by injecting the standard solutions at each con-

entration level in triplicate. The ratios of the peak areas of the


external standard (at each concentration) to those of the IS werecalculated and plotted against the corresponding standard con-centration using weighted linear regression to generate standardcurves.

3. Results and discussion

3.1. ES/MS and ES/MS/MS analyses of standards

Direct flow injection ES/MS analyses, in the positive ionmode, of standard anthocyanins (cyanidin-3-O-galactoside,cyanidin-3-O-glucoside, peonidin-3-O-glucoside, malvidin-3-O-glucoside and delphinidin-3-O-glucoside) and anthocyani-dins (cyanidin, peonidin, malvidin and delphinidin) were ini-tially performed using two different electrospray mass spectrom-eters, one equipped with an IT analyser and the other with a QqQanalyser.

The ES(IT)/MS/MS spectrum of a typical anthocyanidin (del-phinidin) is shown in Fig. 1. The flavylium cation is very stableand does not possess a site of facile rupture, thus the positiveES/MS of the anthocyanidins (analysed in this study as thecommercially available chlorides) showed an intense pseudo-molecular ion [M–Cl]+ owing to the dissociation of the salt, andpoor fragmentation independent of the equipment used for the

analysis. The product ion spectrum (Fig. 1) of the pseudomolec-ular ion [M–Cl]+ of delphinidin (m/z 303) showed fragment ionsat m/z 284.9 [M–Cl-18]+ (loss of water), 274.8 [M–Cl-28]+ (lossof CO), 257.0 [M–Cl-46]+ (loss of CO and water), 247.0 [M–Cl-56]+ (loss of two CO), 228.9 [M–Cl-74]+ (loss of two CO andwater), 218.8 [M–Cl-84]+ (loss of three CO) and 148.9 [M–Cl-154]+ (loss of an aldehyde unit corresponding to ring B of theanthocyanidin).

Whilst the fragmentation pattern observed confirmed theknown structure of delphinidin, it was very different from thatdescribed by Oliveira et al. [13] obtained with high-energy levelCID using a hybrid ES mass spectrometer equipped with twoin-series analysers (quadrupole and time of flight). The pub-lished MS/MS data for this anthocyanidin (see Fig. 2) refersto a fragmentation mechanism that produces a series of rad-ical intermediates and losses of neutral radical fragments orneutral molecules. When a neutral molecule, such as CO, islost it is often followed by radical losses (cf. Fig. 2A) [13].Fig. 2B shows the reported fragmentation pattern subsequent tothe cleavage of the C–C bond in the aglycone [13]. However,when delphinidin was analysed by ES(IT)/MS/MS in the presentstudy, the loss of neutral molecules occurred without the for-mation of radical species. Similar fragmentation patterns wereobserved for peonidin, malvidin and cyanidin. Interestingly, all

Fa

ig. 2. Anthocyanidin fragmentation mechanism reported by Oliveira [13]. Delphinind CO neutral molecules, (B) Aglycon fragmentation.

din analysed by ES-Q-TOF with high collision energy. (A) Losses of radicals


of these anthocyanidins presented an ion at m/z 149, independentof differences involving the B ring. The ion at m/z 149 can thusbe considered a specific ion for the identification of the antho-cyanidin nature of a molecule when detected in ES(IT)/MS/MS.Fig. 3 summarises the fragmentation mechanism proposed onthe basis of the MS/MS behaviour observed in the IT MS ofdelphinidin.

When delphinidin was analysed by ES(QqQ)/MS/MS, a dif-ferent fragmentation pattern was observed. In this case, theprincipal daughter ions were at m/z 257.4 [M–Cl-46]+ (loss ofCO and water), and 229.1 [M–Cl-74]+ (loss of two CO andwater), together with a series of ions of low molecular weight,possibly including an ion at m/z 153.0 arising from the breakageof a C–C bond of the aglycone as described by Oliveira et al.[13] (Fig. 2B). Peonidin, malvidin and cyanidin presented simi-lar fragmentation patterns when analysed by ES(QqQ)/MS/MS;it is noteworthy that the ion at m/z 149.0, which is charac-teristic of these anthocyanidins in the ES(IT)/MS/MS, wasabsent in the ES(QqQ)/MS/MS. Table 1 presents the ES/MSand ES/MS/MS data obtained using the ion trap and thetriple quadrupole mass analyser for each of the anthocyanidinstudied.

3.2. LC–MS of anthocyanins in myrtle extracts

Since the anthocyanin profile of the berries of M. commu-nis has received little attention in comparison with other fruits[15,16], a complete definition of the total anthocyanin patternhad to be determined by LC–MS analysis. When analysed byES/MS, anthocyanins rapidly lose the sugar moiety, and thepseudomolecular ion [M]+ occurs together with the somewhatless-intense aglycone ion [M–sugar moiety]+. Hence, it is pos-sible to identify the aglycone directly in MS1 experiments, andtherefore a complete anthocyanin profile may be obtained byLC-ES/MS experiments in the absence of MS/MS data.

The total ion current (TIC) profile and reconstructed ionchromatograms (RIC) of an extract of M. communis subjectedto positive ion electrospray LC-(IT)MS analysis are shown inFig. 4A. Compounds were identified by comparison of their Rtand m/z values in the TIC profile with those of the selectedstandards. RICs were obtained at each m/z value correspond-ing to a standard compound in order to separate and identifyindividual components. Only one peak appeared in the RICs form/z 465.0 (delphinidin-3-O-glucoside [M]+), 479.0 (petunidin-3-O-glucoside [M]+) and 493.0 (malvidin-3-O-glucoside [M]+),

Fig. 3. Hypothesis for the anthocyanidin fragmentation m
echanism. Delphinidin analysed by ES(IT)/MS/MS.


Table 1MS/MS analyses of anthocyanidins by ES(IT)/MS/MS and ES(QqQ)/MS/MS

Compounds Mr MS1 [M–Cl]+ (IT)/MS/MS (m/z) (QqQ)/MS/MS (m/z)

Delphinidin 303 303.1 284.9 [M–Cl–H2O]+ 303.0 [M–Cl]+

274.8 [M–Cl–CO]+ 257.4 [M–Cl–H2O–CO]+

257.0 [M–Cl–H2O–CO]+ 229.1 [M–Cl–H2O–2CO]+

247.0 [M–Cl–2CO]+ 153.0228.9 [M–Cl–H2O–2CO]+

218.8 [M–Cl–3CO]+

148.9 [C8H5O3]+

Malvidin 331 331.0 313.1 [M–Cl–H2O]+ 331.0 [M–Cl]+

299.0 [M–Cl–CH3OH]+ 303.1 [M–Cl–CO]+

269.9 [M–Cl–CH3OH–CO]+ 285.2 [M–Cl–H2O–CO]+

149.8 [C8H5O3]+ 257.1 [M–Cl–H2O–2CO]+

125.1

Peonidin 301 301.1 286.0 [M–Cl–CH3]+ 285.6 [M–Cl–CH3]+

268.8 [M–Cl–CH3OH]+ 257.1 [M–Cl–CH3–CO]+

257.9 [M–Cl–CH3–CO]+ 201201.0149.1 [C8H5O3]+

Cyanidin 287 287.2 268.8 [M–Cl–H2O]+ 287.0 [M–Cl]+

259.1 [M–Cl–CO]+ 241.3 [M–Cl–H2O–CO]+

240.9 [M–Cl–H2O–CO]+ 213.1 [M–Cl–H2O–2CO]+

231.0 [M–Cl–2CO]+ 136.8148.9 [C8H5O3]+

Fa

ig. 4. LC/ES/MS analysis of anthocyanins in myrtle extract. (A) Reconstructed ionppearing in the RIC at m/z 448.9.

chromatograms (RICs) for compounds 1–9. (B) ES/MS spectra of two peaks


whilst the RIC for m/z 448.9 (cyanidin-3-O-glucoside [M]+)showed two peaks, one at Rt 13.47 min exhibiting a daughterion at m/z 287.0 thus confirming its identity as cyanidin-3-O-glucoside, and the other at Rt 22.45 min presenting a daughterion at m/z 317.1 suggesting the presence of a pentose derivativeof petunidin (Fig. 4B). The RIC for m/z 463.0 [peonidin-3-O-glucoside M]+ also showed two peaks, one at Rt 18.38 minexhibiting a daughter ion at m/z 301 thus confirming its identityas peonidin-3-O-glucoside, and the other at Rt 27.94 present-ing a daughter ion at m/z 331.0 suggesting the presence of apentose derivative of malvidin. Peaks at Rt values of 17.15 and20.51 suggested the presence of, respectively, a delphinidin pen-tose derivative (m/z 435) and a cyanidin pentose derivative (m/z419). MS/MS experiments performed on the [M–sugar]+ ionsof all compounds that were suspected of being anthocyaninsconjugated with pentose sugar, produced daughter ions at m/z149 providing preliminary confirmation of their anthocyaninnature.

In order to establish the identities of the anthocyaninsassociated with the occurrence of pentose units, the fractionsobtained from semi-preparative LC-UV (detection at 520 nm)corresponding to these peaks were analysed by NMR. Com-pounds 6–9 were thus identified as delphinidin-3-O-�-arabino-pyranoside, cyanidin-3-O-�-arabinopyranoside, petunidin-3-O-�-arabinopyranoside and malvidin-3-O-�-arabinopyranoside,respectively, by comparison of their 1H and 13C NMR datawm

3e

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thus be sufficiently specific to identify M. communis from otherberries producing anthocyanins. In order to investigate the appli-cability of the specific anthocyanin pattern in respect of theidentification of the origin of the plant material, the LC-ES/MSprofiles of extracts of M. communis berries collected in Sardinia,Campania and Calabria were compared on a quantitative basis.

In order to provide better accuracy for quantitative analysis,an LC-MS/MS method for the determination of anthocyaninsin myrtle extracts was developed using a mass spectrometerequipped with a triple quadrupole analyser. Since the fragmention at m/z 149.0, which is specific for all anthocyanins, was notobserved in QqQ/MS analyses, it was not possible to use theprecursor ion scan. Instead, an MRM method was developedin which the loss of sugar was selected as the specific reactionthrough which to monitor all anthocyanin derivatives and theIS (cyanidin-3-O-galactoside). Fig. 6 shows the MRM analysisof a sample of Sardinia myrtle spiked with IS at the standardconcentration. The chromatographic profile exhibited all of thepeaks corresponding to the compounds under investigation, andwith sufficient intensity for quantitative purpose.

The calibration graphs, obtained by plotting area ratiobetween external and internal standard against the known con-centration of each anthocyanin, were all linear in the rangeof 5–125 �g/mL with regression coefficients in the range0.995–1.000. The precision of the method was determined byintra- and inter-day assays at each of the four concentration levelsfoissoifiep

F ) SchH ; time

ith literature values [14]. All the anthocyans characterized inyrtle berry extracts are reported in Fig. 5.

.3. Quantitative LC-ES(QqQ)/MS/MS analyses of myrtlextracts from different geographical areas

LC-ES/MS has been shown previously to be a powerful toolor the characterisation of anthocyanins in fruit and vegetables,ffording specific profiles enabling the rapid identification of theource of plant material [17–19]. The reported profile should

ig. 5. (A) LC/ES/MS of hydroalcholic extract of M. communis L. berries. (B

2O:CH3CN 1:1 TFA 0.1%; Gradient: time 0 min, 20% B; time 18 min, 35% B

or each compound, and the RSD% values were ≤2.0%. Basedn a signal-to-noise ratio, the limits of detection of anthocyaninsn a sample were 20 ng/mL. Cyanidin-3-O-galactoside was con-idered to be a suitable IS for the assay, since it is structurallyimilar to the analytes, was not present in any of the profilesbtained and, although its MRM transition was the same as 2,ts retention time was significantly different. These results con-rm that the MRM method described is sensitive and selectiveven when the analysis is conducted on a crude extract withoutre-purification steps.

eme of the anthocyanins in M. communis extracts. (A) H2O TFA 0.1%, (B)30 min, 40% B; time 40 min, 100% B.


Fig. 6. LC/ES(QqQ)/MS/MS XIC (extracted ion chromatograms) of MRM anal-ysis of compounds 1–9. 1 delphinidin-3-O-glc; 2 cyanidin-3-O-glc; 3 petunidin-3-O-glc; 4 peonidin-3-O-glc; 5 malvidin-3-O-glc; 6 delphinidin-3-O-ara; 7cyanidin-3-O-ara; 8 petunidin-3-O-ara; 9 malvidin-3-O-ara.

Each extract of M. communis obtained following the tra-ditional recipe (diluted 1:10 with ethanol:water (70:30) andcontaining IS at the standard concentration) was analysedfive times in order to quantify the anthocyanins present, andthe mean contents determined for compounds 1–9 are shownin Table 2. The homogeneity of the results allowed us todeduce that in the Sardinian extract 5 was the major antho-cyanin (1.48 mg/mL), followed by 3 (0.938 mg/mL) and 1(0.936 mg/mL), with smaller amounts of 2 (0.686 mg/mL)and 4 (0.671 mg/mL) also being present. Amongst the ara-binose derivatives, the major compounds were 8 and 9(0.273 and 0.201 mg/mL, respectively), with 6 and 7 beingpresent in smaller amounts (0.132 and 0.176 mg/mL, respecti-vely).

With respect to samples collected in different geographicalareas, the most consistent differences were observed in the con-tent of the arabinose derivatives. Samples collected in Campaniacontained only about half of the quantities of these compoundscompared with the Sardinian samples, whilst samples collectedin Calabria exhibited lower amounts still. It would thus appearthat myrtles grown in Sardinia are characterised by a muchhigher content of arabinose derivatives. At this stage, it is notpossible to confirm that these differences are necessarily geo-

Table 2Anthocyanin content of extracts of myrtle berries collected in different geo-graphical areas of Italy

Anthocyanina Anthocyanin content (�g/mL of extract)b

Sardinia berries Calabria berries Campania berries

1 935.7 961.3 845.82 685.6 650.1 471.33 938.4 821.7 858.24 670.8 661.7 578.35 1480.6 1431.7 1391.86 132.3 34.2 101.47 175.6 12.1 62.18 273.2 41.6 136.49 201.1 65.4 160.8

a Key to compound numbering: delphinidin-3-O-�-glucopyranoside (1);cyanidin-3-O-�-glucopyranoside (2); petunidin-3-O-�-glucopyranoside (3);peonidin-3-O-�-glucopyranoside (4); malvidin-3-O-�-glucopyranoside (5);delphinidin-3-O-�-arabinopyranoside (6); cyanidin-3-O-�-arabinopyranoside(7); petunidin-3-O-�-arabinopyranoside (8); malvidin -3-O-�-arabinopyr-anoside (9).

b Values are means (n = 5): the relative standard deviations for all compoundswere <2%.

climatic, since they could also be caused by genetic and/orenvironmental factors.

4. Conclusion

This study has demonstrated that the presence of the nineanthocyanin derivatives, including the anthocyanin arabinosidesthe occurrence of which is reported here for the first time, inextracts of berries of M. communis provides a profile that maybe used to identify extracts from this plant and to monitor theirquality with respect to anthocyanin content. Whilst the smallnumber of samples analysed does not permit conclusions to bedrawn concerning the quantitative variability of the compoundsin samples of different geographical origin, the preliminary datasuggest that the anthocyanin profiles may be potential markersof the origin of myrtle berries.

The LC-MS/MS method developed for the quantitative anal-ysis of anthocyanins in crude extracts of M. communis pro-duced good results in terms of precision and accuracy, and wasalso straightforward and convenient to use, since it requiresno expensive and time-consuming sample preparation proce-dures. For these reasons, it may have potential value in industrialquality control in order to quantify marker compounds in rawmaterials and in final products. Furthermore, the preliminaryES(IT)/MS/MS and ES(QqQ)/MS/MS experiments describedppumhcauco

rovided valuable information concerning the fragmentationatterns of the anthocyanins, an area that has been little exploredntil the present. Whilst experiments employing the ion trapass analyser were very informative for qualitative analyses,

igher accuracy, sufficient for the quantitative analysis of antho-yanins present in myrtle extracts, could be attained using MRMnd the triple quadrupole analyser. The information gleanedsing both techniques will be useful in confirming the antho-yanin nature of compounds detected in a mixture when specificn-line LC-MS/MS experiments are developed.


Acknowledgements

This study was supported by a grant from the Italian Min-istry of University and Scientific Research (MIUR; project PON12930). The authors offer their sincere thanks to Prof. VincenzoDe Feo and Prof. Mariateresa Russo for supplying the berries ofM. communis from Campania and Calabria, respectively.

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characterisation by liquid chromatography-electrospray tandem mass spectrometry of anthocyanins in...

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