JOURNAL OF MASS SPECTROMETRY J. Mass Spectrom. 2003; 38: 3542 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.

395

Liquid chromatographic/electrospray ionization tandem mass spectrometric study of the phenolic composition of cocoa (Theobroma cacao)
1 Olga Jauregui, 2 Isidre Casals,2 Cristina Andres-Lacueva, 3 Ferran Sanchez-Rabaneda, 3 Maria Izquierdo-Pulido3 and Rosa M. Lamuela-Raventos
1 2 3

Department of Natural Products, University of Barcelona, 08028 Barcelona, Spain Scientic and Technical Services, University of Barcelona, 08028 Barcelona, Spain Department of Nutrition and Bromatology, University of Barcelona, 08028 Barcelona, Spain

Received 21 May 2002; Accepted 11 October 2002

Liquid chromatography coupled with ionspray mass spectrometry in the tandem mode (LC/MS/MS) with negative ion detection was used for the identication of a variety of phenolic compounds in a cocoa sample. Gradient elution with water and acetonitrile, both containing 0.1% HCOOH, was used. Standard solutions of 31 phenolic compounds, including benzoic and cinnamic acids and avonoid compounds, were studied in the negative ion mode using MS/MS product ion scans. At low collisional activation, the deprotonated molecule [M H] was observed for all the compounds studied. For cinnamic and benzoic acids, losses of CO2 or formation of [M CH3 ] in the case of methoxylated compounds were observed. However, for avonol and avone glycosides, the spectra present both the deprotonated molecule [M H] of the glycoside and the ion corresponding to the deprotonated aglycone [A H] . The latter ion is formed by loss of the rhamnose, glucose, galactose or arabinose residue from the glycosides. Different fragmentation patterns were observed in MS/MS experiments for avone-C-glycosides which showed fragmentation in the sugar part. Fragmentation of aglycones provided characteristic ions for each family of avonoids. The optimum LC/MS/MS conditions were applied to the characterization of a cocoa sample that had been subjected to an extraction/clean-up procedure which involved chromatography on Sephadex LH20 and thin-layer chromatographic monitoring. In addition to compounds described in the literature, such as epicatechin and catechin, quercetin, isoquercitrin (quercetin-3-O-glucoside) and quercetin-3-O-arabinose, other compounds were identied for the rst time in cocoa samples, such as hyperoside (quercetin-3-Ogalactoside), naringenin, luteolin, apigenin and some O-glucosides and C-glucosides of these compounds. Copyright 2003 John Wiley & Sons, Ltd.

KEYWORDS: avonoids; cocoa; liquid chromatography; mass spectrometry; tandem mass spectrometry

INTRODUCTION
Foods derived from cocoa beans (Theobroma cacao) have been consumed by the preclassic Maya since 600 BC. The earliest chemical evidence of the use of cocoa is from 2600year-old pottery.1 The dried, roasted cocoa beans from Theobroma cacao L. contain about 300 chemicals, including phenols. Vinson et al.2 reported that the total phenol level measured in cocoa powder is 224 66.4 mol g 1 . Phenols have attracted interest because of their perceived health-benecial effects. They have been reported to have physiological properties that could protect the body from chronic diseases, such as cardiovascular diseases3 and cancer.4 In addition, cocoa procyanidins are potent inhibitors of inammation.5 The various phenols that have been identied in cocoa beans or cocoa products have
Correspondence to: Rosa M. Lamuela-Raventos, Department of Nutrition and Bromatology, University of Barcelona, Joan XXIII s/n, E-8028 Barcelona, Spain. E-mail: lamuela@farmacia.far.ub.es

been reviewed recently by Wollgast and Anklam6 and include phenolic compounds of the avan-3-ol group (catechin, epicatechin, gallocatechin and epigallocatechin) and their dimers or trimers, the procyanidins.7 Also, some anthocyanins (cyanidin glycosides) and avonol glycosides such as quercetin-3-O-arabinose, isoquercitrin, quercetin3-O-glucuronide and quercetin8,9 have been reported in cocoa samples. Among the methods used for the determination of phenols, the coupling of liquid chromatography/mass spectrometry (LC/MS) with atmospheric pressure ionization techniques, i.e., electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), has been demonstrated to be a powerful tool for the identication of natural products in crude plant extracts owing to their soft ionization, which favors the analysis of avonoids, a polar, non-volatile, and thermally labile class of compounds.10 12 For instance, procyanidins were detected in cocoa by Hammerstone et al.13 using a normal-phase (NP) LC/MS method, and by

Copyright 2003 John Wiley & Sons, Ltd.

36

F. S anchez-Rabaneda et al.

Wollgast et al.14 and Natsume et al.15 using reversed-phase (RP) LC/MS methods. Here, we developed an LC/MS/MS method for the identication of polyphenolic compounds in cocoa powder after suitable sample work-up. Some of these compounds are described for the rst time.

EXPERIMENTAL Reagents
Reagents were obtained from the following sources: methanol, ethyl acetate and acetonitrile (HPLC grade) from SDS (Peypin, France), hexane (HPLC grade) from Scharlau (Barcelona, Spain), formic acid from Probus (Badalona, Spain) and acetic acid from Merck (Darmstadt, Germany). Ultrapure water (Milli-Q) was used. Phenolic standards were obtained as follows: protocatechuic acid, coumaric acid and quercetin from Sigma (St. Louis, MO, USA), epicatechin, catechin, gallic acid, ferulic acid and caffeic acid from Fluka (Buchs, Switzerland) and chlorogenic acid, hyperoside, kaempferol, kaempferol-3-O-glucoside, kaempferol-3-Orutinoside, kaempferol-7-O-neohesperidoside, quercitrin, isoquercitrin, isorhamnetin, luteolin, luteolin-6-C-glucoside, luteolin-8-C-glucoside, luteolin-7-O-glucoside, apigenin, apigenin-7-O-glucoside, apigenin-6-C-glucoside, apigenin8-C-glucoside, amentoavone, naringin, naringenin, naringenin-7-O-glucoside, isorhoifolin and rutin from Extrasynthese (Genay, France). The purity of the standards was 98%, and all were prepared as stock solutions at 1 mg l 1 in methanol. Working standard solutions were made by diluting the stock solutions with the LC mobile phase.

following proportions (v/v) of solvent B was applied (t(min), %B): (0, 6), (14, 16.5), (16, 17), (18, 17.5), (20, 17.5), (22, 18.5), (24, 18.5), (27, 20), (46, 100), (48, 6). The compounds described were monitored at 280, 320 and 365 nm. An API 3000 triple-quadrupole mass spectrometer (Perkin-Elmer Sciex, Concord, ON, Canada) was used to obtain the MS and MS/MS data. All the analyses were performed using a Turbo Ionspray source with the following settings: capillary voltage 3500 V, nebulizer gas (N2 ) 10 (arbitrary units), curtain gas (N2 ) 12 (arbitrary units), collision gas (N2 ) 10 (arbitrary units), focusing potential 200 V, entrance potential 10 V, drying gas (N2 ) heated to 400 C and introduced at a owrate of 8000 cm3 min 1 . The declustering potential (DP) and the collision energy (CE) were optimized for each compound in infusion experiments: individual standard solutions (10 ng l 1 ) dissolved in 80 : 20 mobile phase (A : B) were infused at a constant ow-rate of 5 l min 1 into the mass spectrometer using a Model 11 syringe pump (Harvard Apparatus, Holliston, MA, USA). Full-scan data acquisition was performed, scanning from m/z 100 to 800 in prole mode and using a cycle time of 2 s with a step size of 0.1 u and a pause between each scan of 2 ms. MS/MS product ions were produced by collision-activated dissociation (CAD) of selected precursor ions in the collision cell of the triplequadrupole mass spectrometer and mass analyzed using the second analyzer of the instrument. Additional experimental conditions for MS/MS included collision energy (depending on the compound), CAD gas (nitrogen) at 6 (arbitrary units), and scan range, as necessary for the precursor selected. In all the experiments, both quadrupoles (Q1 and Q3) were operated at unit resolution.

Extraction and fractionation of phenols

Natural Forastero cocoa powder from Malaysia provided by Nutrexpa (Barcelona, Spain) was employed. An amount of 20.0 g of cocoa was extracted three times with 500 ml of methanolwater (80 : 20, v/v). The pooled supernatant phases were ltered and concentrated under vacuum to dryness. The dry powder was re-extracted twice with hexane (500 ml each). The two solid phases (fraction nonsoluble in hexane) were pooled and dried. A 2.0 g amount of this fraction was chromatographied on a Sephadex LH-20 (Amersham Pharmacia Biotech, Amersham, UK) column at a ow-rate of 10 ml min 1 using methanol as solvent to give 54 fractions. The fractions were monitored by thin-layer chromatography (TLC) (Alugram Sil G/UV, MachereyNagel, Duren, Germany). Similar fractions were combined to give seven nal fractions (AG).

RESULTS AND DISCUSSION Optimization of LC/MS/MS conditions

The LC/DAD method previously used9 was modied to be compatible with the LC/MS system; acetic acid was replaced by the more volatile formic acid and its concentration was reduced to 0.1%. As a consequence, the ionic strength decreased and the signal-to-noise ratio increased in the negative ion mode. The gradient prole used in this work allowed the separation of all the compounds studied with a retention that, in general, followed the expected reversed-phase pattern of avonoid aglycones > avonoid O-glycosides > avonoid C-glucosides > cinnamic acids > benzoic acids16 (as can be seen in Table 1, where the retention time and the ions for each compound are given). Infusion of individual standard solutions was performed in order to establish the optimum MS and MS/MS conditions. The declustering potential was varied from 5 to 120 V and the collision energy from 5 to 45 V. The product ion scan mass spectra were recorded at ve CE values (up to 40 V) and once the two ions m/z (Q1) ! m/z (Q3) had been chosen, the CE value was optimized in such a way that the sensitivity of the multiple reaction monitoring (MRM) signal was maximum. The optimum CE and DP values for each compound are given in Table 1.

Instrumentation
LC analyses were performed using an Agilent (Waldbronn, Germany) Model 1100 quaternary pump equipped with an autosampler and a diode-array detector (DAD). A Chemstation HP Rev. A.08.03 was used for data analysis. A Luna C18 column (150 2.1 mm i.d., 5 m) (Phenomenex, Torrance, CA, USA) was used. Gradient elution was carried out with water0.1% formic acid (solvent A) and acetonitrile0.1% formic acid (solvent B) at a constant owrate of 400 l min 1 . A linear gradient prole with the

Copyright 2003 John Wiley & Sons, Ltd.

J. Mass Spectrom. 2003; 38: 3542

LC/ESI-MS/MS identication of avonoids in cocoa

37

Table 1. LC/MS/MS characteristics of phenols in the negative mode Peak No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 tr (min) 2.24 4.44 8.48 8.91 9.92 11.81 14.00 15.35 15.72 16.24 17.75 18.11 18.15 18.39 18.95 19.15 21.32 21.99 22.36 22.40 22.56 22.59 23.12 23.32 33.00 33.10 34.53 34.84 35.10 35.40 36.34 MS/MS ions (m/z (relative abundance, %)) 169 (100), 125 (80) 153 (35), 109 (100) 289 (40), 245 (100) 353 (35), 191 (100) 179 (25), 135 (100) 289 (40), 245 (100) 163 (15), 119 (100) 447 (65), 429 (65), 357 (100), 327 (100), 285 (50) 447 (30), 357 (70), 327 (100), 285 (20) 193 (30), 149 (55), 178 (20), 134 (100) 431 (35), 341 (30), 311 (100), 269 (<5) 431 (15), 353 (<5) 341 (40), 311 (100), 269 (<5) 609 (100), 301 (40) 463 (5), 301 (100) 463 (35), 301 (100) 447 (100), 285 (100) 593 (70), 285 (100) 577 (20), 269 (100) 433 (70), 271 (100) 447 (90), 285 (100) 447 (25), 301 (100) 579 (100), 459 (20), 271 (40) 593 (20), 327 (5), 285 (100) 431 (100), 269 (75) 301 (60), 151 (100) 285 (100), 217 (10), 199 (20), 175 (20), 151 (85), 133 (50), 107 (10) 271 (25), 177 (20), 151 (100), 119 (75), 107 (35), 93 (15), 83 (10) 269 (60), 151 (100) 285 (100), 217 (40), 151 (85), 133 (75) 315 (60), 300 (100), 151 (10) 537 (100), 375 (65) DP (V) 40 40 50 50 40 50 40 60 60 40 55 55 60 60 60 60 65 60 60 60 60 80 70 60 60 60 60 60 60 60 CE (V) 20 20 20 20 20 20 20 30 30 20 30 30 30 38 32 30 45 40 20 30 30 35 40 35 35 30 35 35 30 40

Compound Gallic acid Protocatechuic acid Catechin Chlorogenic acid Caffeic acid Epicatechin Coumaric acid Luteolin-6-C-glucoside (isoorientin) Luteolin-8-C-glucoside (orientin) Ferulic acid Apigenin-8-C-glucoside (vitexin) Apigenin-6-C-glucoside (isovitexin) Quercetin-3-O-rutinoside (rutin) Quercetin-3-O-galactoside (hyperoside) Quercetin-3-O-glucoside (isoquercitrin) Luteolin-7-O-glucoside Kaempferol-3-O-rutinoside Apigenin-7-O-rutinoside (isorhoifolin) Naringenin-7-O-glucoside (prunin) Kaempferol-3-O-glucoside Quercetin-3-O-rhamnoside (quercitrin) Naringenin-7-O-neohesperidoside (naringin) Kaempferol-7-O-neohesperidoside Apigenin-7-O-glucoside Quercetin Luteolin Naringenin Apigenin Kaempferol Isorhamnetin Amentoavone

Mr 170 154 290 354 180 290 164 448 448 194 432 432 610 464 464 448 594 578 434 448 448 580 594 432 302 286 272 270 286 316 538

Study of MS/MS for standard solutions

The spectra generated for cinnamic and benzoic acids by ionspray in the negative ion mode gave the deprotonated molecule [M H] and some fragments even at relatively low declustering potentials (up to 20 V). For instance, loss of CO2 was observed for caffeic, ferulic, protocatechuic and gallic acids, giving the [M H 44] as a characteristic ion. Ferulic acid also showed the loss of the CH3 group, providing an [M 15] anion radical at m/z 178. Catechin and epicatechin yielded the deprotonated molecule [M H] (m/z 289) and a loss of a CH2 CHOH group (m/z 245), as described P erez-Magarino et al.17 Chlorogenic acid showed the deprotonated molecule [M H] (m/z 353) and the ion corresponding to the deprotonated quinic acid (m/z 191). For avonol O-glycosides such as hyperoside, isoquercitrin, quercitrin and kaempferol-3-O-glucoside, the spectra generated at a DP of 60 V and a CE between 30 and 40 V showed both the deprotonated molecule

[M H] of the glycoside and the ion corresponding to the deprotonated aglycone [A H] . The latter ion is formed by loss of the rhamnose, glucose or galactose moiety from the glycosides. No ions characteristic of the sugar part were observed in the negative ion mode. The same behavior was observed for avone O-glycosides such as luteolin-7-Oglucoside (m/z 447 ! m/z 285) and apigenin-7-O-glucoside (m/z 431 ! m/z 269) and for the avanone-O-glucoside, namely, naringenin-7-O-glucoside (m/z 433 ! m/z 271) with loss of the glucose residue. The identity of compounds of the same molecular mass (e.g. hyperoside and isoquercitrin) was established based on the relative abundance of the ions from the product ion mass spectra and the retention time compared with those of standards. Flavonol diglycosides such as kaempferol-7-O-neohesperidoside (kaempferol-rhamnosyl-(1 ! 2)-glucoside) and kaempferol-3-rutinoside (kaempferol-rhamnosyl-(1 ! 6)glucoside) were also studied; both have the same molecular

Copyright 2003 John Wiley & Sons, Ltd.

J. Mass Spectrom. 2003; 38: 3542

38

F. S anchez-Rabaneda et al.

mass, Mr 594, and differ only in the position of the sugar substituent and in the interglycosidic linkage type between the two monosaccharides. In both cases, loss of the rhamnoseglucose unit was observed, giving m/z 593 ! m/z 285 for both compounds. They could be differentiated by the relative abundances of the ions and also by the fact that kaempferol-7-O-neohesperidoside shows more pronounced fragmentation than its rutinose analogue with a product ion mass spectrum revealing ions at m/z 473, 429, 387 and 327 in the negative ion mode. These results are consistent with those described by Cuyckens et al.,18 who determined avonoid O-diglycosides by nanoelectrospray ionization mass spectrometry (ESI-ITMS) in the negative ion mode. In our case, the relative abundances of the fragments are always lower than 5% whereas Cuyckens et al.18 reported more pronounced fragmentation using the ion-trap system. Rutin (quercetin-rhamnosyl-(1 ! 6)-glucoside) shows the loss of the rhamnoseglucose unit (m/z 609 ! m/z 301). Other O-diglycosidic compounds studied here were isorhoifolin (apigenin-7-O-rutinoside) and naringin (naringenin-7-Oneohesperidoside), which gave m/z 577 ! m/z 269 and m/z 579 ! m/z 459, 271, respectively. These results are also in agreement with those of Cuyckens et al.18 Flavone C-glucosides such as isoorientin (luteolin-6C-glucoside) and orientin (luteolin-8-C-glucoside) showed

a fragmentation pattern different from those of the Oglucosides, as reported by Becchi and Fraisse,19 Waridel et al.20 and Li et al.,21 who demonstrated that characteristic fragment ions of [M H] ions allow the differentiation between C-glycosylation at the 6- and 8-positions. Using a triple-quadrupole mass spectrometer, the tandem mass spectra of C-glycosides did not reveal abundant [M H] ions but characteristic ions due to fragmentation in the C-glycosidic unit. Losses of 120 and 90 u were observed, corresponding to cross-ring cleavages in the sugar unit. The product ion spectra of the ion at m/z 447 of both compounds differ in the relative abundance of the m/z 357 (loss of 90 u) and m/z 327 (loss of 120 u) ions (see Table 1). Moreover, the ion at m/z 429 in the spectrum of isorientin, which could be assigned to the [M H 18] ion, was not present in that of orientin. Vitexin (apigenin-8-C-glucoside) and isovitexin (apigenin-6-C-glucoside) show the ions at m/z 431 (deprotonated molecule), 341 (loss of 90 u) and 311 (loss of 120 u) as characteristic ions in the MS/MS mode. The product ion spectrum of isovitexin reveals some ions that could help mass spectral differentiation (m/z 413, 353) with vitexin and, in addition at a CE of 40 V, the deprotonated aglycone (m/z 269) shows a relative abundance of 10% for isovitexin whereas it is less than 5% for vitexin.

Table 2. Compounds identied in the present work in Theobroma cacao by LC-MS/MS Compared with standard Yes Yes No Yes Yes Yes Yes Yes Yes Yes No Yes

Compound Catechin Epicatechin Procyanidins Luteolin-8-C-glucoside (orientin) Luteolin-6-C-glucoside (isoorientin) Apigenin-8-C-glucoside (vitexin) Apigenin-6-C-glucoside (isovitexin) Quercetin-3-O-galactoside (hyperoside) Quercetin-3-O-glucoside (isoquercitrin) Luteolin-7-O-glucoside Quercetin-3-O-arabinoside Naringenin-7-O-glucoside (prunin)

MS/MS approach Full Scan Full Scan Full Scan Product ion scan (m/z 427) Product ion scan (m/z 427) Product ion scan (m/z 431) Product ion scan (m/z 431) Product ion scan (m/z 463) Product ion scan (m/z 463) Product ion scan (m/z 427) Product ion scan (m/z 433) MRM

Cocoa fraction D, E, F D, E, F D, E, F E E D, E, F D, E, F E E E F E

Plants Theobroma cacao6 Theobroma cacao6 Theobroma cacao7 Polygonum Orientale, Passiora Incarnata24 Polygonum Orientale, Passiora Incarnata24 Vitex lucens, Pennisetum americanum24 Polygonum Orientale24 Hypericum perforatum24 Theobroma cacao9 Humulus japonicus, Salix spp.24 Theobroma cacao8 Abies spp., Pinus spp., Pocarpus spp., Lycopersicon esculentum, bark of Prunus persica24 Leaf exudates of Scrophulariaceae, Cruciferae, Labiatae and Leguminoseae24 Artemisia spp., Baccharis spp., Centaurea spp., Dahlia spp.24 Surface of Labiatae, and in the farinose exudate of fern fronds24

Luteolin

MRM, Product ion scan (m/z 285)

Yes

E, F

Naringenin Apigenin

MRM, Product ion scan (m/z 271) MRM, Product ion scan (m/z 269)

Yes Yes

E, F E, F

Copyright 2003 John Wiley & Sons, Ltd.

J. Mass Spectrom. 2003; 38: 3542

LC/ESI-MS/MS identication of avonoids in cocoa

39

Finally, the aglycones gave retro-DielsAlder fragmentation as described by Fabre et al.22 For instance, the m/z 151 ion is common for all the aglycones studied, the m/z 117 ion is characteristic for apigenin and its derivatives whereas the m/z 119 ion is characteristic for naringenin and derivatives. Isorhamnetin exhibits specic fragmenta tion with the loss of CH3 radical from the deprotonated aglycone molecule, thus giving m/z 315 ! m/z 300 as described by Justesen.23 The last-eluting compound is a biavone, namely amentoavone. Its tandem mass spectrum reveals the deprotonated molecule [M H] ion at m/z 537 and a fragment ion at m/z 375.

Identication of phenolic compounds in cocoa samples

Once the optimum LC/MS/MS conditions had been established for the compounds studied using standard solutions, cocoa fractions were analyzed by LC/MS/MS in the fullscan mode. Protocatechuic acid was identied in the UV chromatogram and conrmed by comparison with the retention time of the standard and the MS/MS chromatogram in the MRM mode, showing m/z 153 ! m/z 109. Table 2 lists the compounds identied in cocoa extracts, the MS/MS approach used for this purpose and some references to the presence of these compounds in other plants.6,7,9,24 Preliminary examination of the chromatograms in the full-scan mode revealed the presence of catechin, epicatechin (both showing
17.65 3.0e5 2.8e5 2.6e5 2.4e5 2.2e5 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 0.0 0

m/z 289) and some dimers of these compounds, the procyanidins showing the m/z 577 ion (the deprotonated dimer) and m/z 289 ion (the deprotonated catechin or epicatechin unit). The latter compounds have been identied in cocoa by other authors13,15,22 using LC/MS, but their presence was not conrmed in this study because procyanidin standards were not available. Using the m/z 463 mass chromatogram, the presence of isoquercitrin (tr 18.9 min), previously identied in cocoa samples,8,9 could be conrmed. Another peak at tr 18.9 min was characterized as hyperoside on the basis of the m/z 463 product ion spectrum and comparison of its retention time with that of a standard. Another trace investigated in the full-scan chromatogram was m/z 447, where a peak with a retention time of 19.02 min was present. The product ion spectrum of this peak at a CE of 35 V gave a product ion at m/z 285, which suggested a kaempferol or a luteolin glycoside, but the comparison of the retention time with standards conrmed the presence of luteolin-7-O-glucoside. In the m/z 447 mass chromatogram, two other peaks are visible (retention times 15.25 and 15.71 min) whose product ion mass spectra show ions at m/z 327 (loss of 120 u) and m/z 357 (loss of 90 u), which are characteristic of avone C-glucosides, as mentioned earlier. Moreover, the rst peak also showed the m/z 429 ion. Hence the presence of isoorientin and orientin in cocoa extracts was conrmed by comparison of the product ion spectra and the retention times of these peaks with those of authentic standards (see Table 2).

A
11 17.99 12

Intensity, cps

0.87

1.37 8.74 16.48 6.14 8.14 12.38 9.2811.18 5.57 12.68 1.97 4.30 15.95 2 4 6 8 10 12 14 16 18

20.19

20.72 23.26 24.66 22 24 26 28 30

31.58 34.22 34.76 36.60 38.54 32 34 36 38

20

Time, min
8000 7000 6000 HO O O [M-H-90] [M-H-120]-

311.2

HO

B
OH CH2OH OH

Intensity, cps

5000 HO 4000 3000 HO 2000 1000

341.0 O 430.9

268.8 0 60 80 100 120 140 160 180 200 220 240 260 280

296.7 300

323.0 343.2 353.2 320 340 360 380

413.2 400 420 440 460 480 500

m/z, amu

Figure 1. (A) TIC of cocoa extract (fraction D) in product ion scan mode using m/z 431 (up to minute 25) and of m/z 269 (up to minute 50). (B) MS/MS of peak 11 in cocoa extract. Peak assignment: (11) vitexin; (12) isovitexin. For experimental conditions, see text.

Copyright 2003 John Wiley & Sons, Ltd.

J. Mass Spectrom. 2003; 38: 3542

40

F. S anchez-Rabaneda et al.

The presence of apigenin C-glucosides was also investigated by MS/MS. Injection of cocoa extracts in the product ion scan mode of m/z 431 gave as a result the chromatogram shown in Fig. 1(A), where two peaks (peak 11, tr = 17.65 min; peak 12, tr D 17.99 min) show a mass spectrum with typical fragmentation of C-glycosides (loss of 90 and 120 u). These peaks are identied as vitexin and isovitexin by comparison of the product ion spectra and the retention times with those of the authentic standards. Figure 1(B) shows the product ion spectrum of vitexin in the cocoa extract. The presence of naringenin glucosides was evaluated by analysis of cocoa extracts in the product-ion scan mode using m/z 433. The m/z 433 mass chromatogram showed only one peak, at 20.45 min, the mass spectrum of which revealed an ion at m/z 301 (characteristic of a quercetin derivative) and not an ion at m/z 271 (characteristic of a naringenin derivative). This peak could be tentatively assigned to quercetin-3-arabinose (m/z 433 ! m/z 301) previously described in cocoa by Sanbongi et al.8 In order to screen for naringenin derivatives that could
11.65

be present at low concentrations, the MRM approach was used. Naringenin-7-O-glucoside (prunin), showing a retention time of 22.40 min, was then identied (m/z 433 ! m/z 271) and conrmed by comparison of the retention time with that of the standard. The product ion spectrum for this compound could not be acquired, probably because its concentration was not sufcient for this acquisition mode. Concerning the presence of aglycones in cocoa, up to now only quercetin has been described in the literature.8,9 Here, we studied the product ion spectra of m/z 285, 269 and 271 ions corresponding to kaempferol or luteolin, apigenin and naringenin, respectively. Comparison of the product ion spectra and retention times with those of standards provided a useful tool for the conrmation of the presence of these three aglycones in cocoa extracts for the rst time. As an example, Fig. 2(A) shows the chromatogram of fraction E of cocoa extract injected in the product ion scan mode using m/z 285 and the product-ion spectra for the sample (Fig. 2(B)) and for the standard (Fig. 2(C)) in order to illustrate the presence
33.01

3.0e5 2.8e5 2.6e5 2.4e5 2.2e5 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 0.0

26 7.21 8.31

Intensity, cps

1.40

4.77 4.44

10.48

17.39

33.57 35.04

10

12

14

16

18

20

22

24

26

28

30

32

34

36

38

40

42

44

46

48

Time, min
6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 100

284.9

Intensity, cps

B
133.0 151.0

286.0 106.9 174.9 149.2 199.3 201.0 217.0 197.2 222.9 190 200 210 220 230 243.0 240 257.1 250 260 270 280 290 3

110

120

130

140

150

160

170

180

m/z, amu
285.1 2.0e6 1.8e6 1.6e6
HO A
1,3

HO AB O OH OH OH A

m/z 151
C O

Intensity, cps

1.4e6 1.2e6 1.0e6 8.0e5 6.0e5 4.0e5 2.0e5 0.0 100 107.0 175.1 149.1 199.2 201.0 120 130 140 150 160 170 180 190 200 217.0 241.0 243.1 230 240 250 260 132.9 151.1
OH O
1,3

C
m/z 133

OH B

B-

m/z 285
CH

OH

110

210

220

270

280

290

m/z, amu

Figure 2. (A) TIC of cocoa extract (fraction E) in product ion scan mode using m/z 285; (B) MS/MS of peak 26 in cocoa extract; (C) MS/MS of luteolin standard in infusion mode (see text for experimental conditions). Peak 26: luteolin.

Copyright 2003 John Wiley & Sons, Ltd.

J. Mass Spectrom. 2003; 38: 3542

LC/ESI-MS/MS identication of avonoids in cocoa

41

3673 3000 2000 1000 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32

34.82

28

m/z 269m/z 151

34 36 38

Time, min 6893 6000 4000 2000 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

27

34.59

m/z 271m/z 151

Time, min 33.10 4000 3000 2000 1000 0

26

m/z 285m/z 151

0 2 4 6 8 10 12 14 16 18 17.98 20 22 24 26 28 30 32 34 36 38

Time, min 7060 6000 4000 2000 0 0 2 4 6 8 10 12 14 16 18 20 19.80 22 24 26 28 30 32 34 36 38

11

17.62

12

m/z 431m/z 311

Time, min 36.43 24.21 25.00 23.12 0 2 4 6 8 10 12 14 16 15.71 2500 2000 1500 1000 500 0 0 2 4 6 8 10 12 15.25 18 20 22 24 26 28 30 32 34 36 38 2000 1500 1000 500 0

24

Intensity (cps)

m/z 431m/z 269

Time, min

9
19.07 19.24 19.86

m/z 447m/z 327

22 24 26 28 30 32 34 36 38

14

16

18

20

Time, min 1.3e4 1.0e4 5000.0 0.0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Time, min 6.8e4 6.0e4 4.0e4 2.0e4 0.0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Time, min 18.79 3.0e5 2.0e5 1.0e5 0.0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 Time, min 4.5e6 4.0e6 3.0e6 2.0e6 1.0e6 0.0 0 2 4 19.07 6.09 20.56

m/z 433m/z 301

16

m/z 447m/z 285

14
18.26

15

m/z 463m/z 301

11.59 8.26

m/z 289m/z 245

6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

Time, min

Figure 3. Traces for polyphenols in fractions E and F of cocoa extracts in MRM mode. Peaks: (3) catechin; (6) epicatechin; (8) isorientin; (9) orientin; (11) vitexin; (12) isovitexin; (14) hyperoside; (15) isoquercetrin, (16) luteolin-7-O-glucoside; (24) apigenin-7-O-glucoside; (26) luteolin; (27) naringenin; (28) apigenin; (A) quercetin-3-O-arabinoside. LC/MS/MS conditions as described in the text.
Copyright 2003 John Wiley & Sons, Ltd. J. Mass Spectrom. 2003; 38: 3542

42

F. S anchez-Rabaneda et al.

of luteolin. Figure 3 shows the traces for avonoids found in cocoa extracts in the MRM mode.

CONCLUSIONS
The method for extraction, clean-up and analysis by LC/MS/MS presented here allowed the identication of avonols, avones and avanones in cocoa samples. The extraction and fractionation stage described in this work is a critical part of the method, because it removes potential interfering components. This sample clean-up stage includes solidliquid partitioning and column chromatography on Sephadex LH-20, which resulted in partial purication and concentration of the phenolic compounds in a few fractions. The optimum LC/ESI-MS/MS parameters using a triplequadrupole mass spectrometer for 31 phenolic compounds including avonoids and some cinnamic and benzoic acids are presented. LC/ESI-MS/MS has been shown to be an excellent tool for the screening of the avonoid composition. This method has been applied to the screening of cocoa samples, allowing the identication of hyperoside, luteolin-7-O-glucoside, isoorientin, orientin, vitexin, isovitexin, naringenin-7-O-glucoside, naringenin, apigenin and luteolin in cocoa samples for the rst time. To date, cocoa derivative consumption has been based on the pleasant smell and taste of the product. The identication of these compounds opens a new door to an increased understanding of the different health benets brought about by the consumption of cocoa and its derivatives.

REFERENCES
1. Hurst WJ, Tarka SM, Powis TG, Valdez F, Hester TR. Cacao usage by the earliest Maya civilization. Nature (London) 2002; 418: 289. 2. Vinson JA, Proch J, Zubik L. Phenol antioxidant quantity and quality in foods: cocoa, dark chocolate, and mild chocolate. J. Agric. Food Chem. 1999; 47: 4821. 3. Rein D, Lotito S, Holt RR, Keen CL, Schmitz HH, Fraga CG. Epicatechin in human plasma: in vivo determination and effects of chocolate consumption on plasma oxidation. J. Nutr. 2000; 130: 2109S. 4. Stoner GD, Mukhtar H. Polyphenols as cancer chemopreventive agents. J. Cell. Biochem. 1995; 22: 169. 5. Sanbongi C, Suzuki N, Sakane T. Polyphenols in chocolate, which have antioxidant activity, modulate immune functions in humans in vitro. Cell. Immunol. 1997; 177: 129. 6. Wollgast J, Anklam E. Review on polyphenols in Theobroma cacao: changes in composition during the manufacture of chocolate and methodology for identication and quantication. Food Res. Int. 2000; 33: 423. 7. Porter LJ, Ma Z, Chan BG. Cacao Procyanidins: major avonoids and identication of some minor metabolites. Phytochemistry 1991; 30: 1657. 8. Sanbongi C, Osakabe N, Natsume M, Takizawa T, Shuichi G, Osawa T. Antioxidative polyphenols isolated from Theobroma cacao. J. Agric. Food Chem. 1998; 46: 454.

9. Andres-Lacueva C, Lamuela-Raventos auregui O, Casals I, RM, J Izquierdo-Pulido M, Permanyer J. An LC method for the analysis of cocoa phenolics. LC-GC Eur. 2000; 12: 902. 10. Justesen U, Knuthsen P, Torben L. Quantitative analysis of avonols, avones, and avanones in fruits, vegetables and beverages by high-performance liquid chromatography with photo-diode array and mass spectrometric detection. J. Chromatogr. A 1998; 799: 101. 11. Swatsitang P, Tucker G, Robards K, Jardine D. Isolation and identication of phenolic compounds in Citrus sinensis. Anal. Chim. Acta 2000; 417: 231. 12. Justesen U. Negative atmospheric pressure chemical ionisation low-energy collision activation mass spectrometry fo the characterisation of avonoids in extracts of fresh herbs. J. Chromatogr. A 2000; 902: 369. 13. Hammerstone JF, Lazarus SA, Mitchell AE, Rucker R, Schmitz HH. Identication of procyanidins in cocoa (Theobroma cacao) and chocolate using high-performance liquid chromatography/mass spectrometry. J. Agric.Food Chem. 1999; 47: 490. 14. Wollgast J, Pallaroni L, Agazzi ME, Anklam E. Analysis of procyanidins in chocolate by reversed-phase high-performance liquid chromatography with electrospray ionisation mass spectrometric and tandem mass spectrometric detection. J. Chromatogr. A 2001; 926: 211. 15. Natsume M, Osakabe N, Yamagishi M, Takizawa T, Nakamura T, Miyatake H, Hatano T, Yoshida T. Analyses of phenols in cacao liquor, cocoa, and chocolate by normal-phase and reversed-phase HPLC. Biosci. Biotechnol. Biochem. 2000; 64: 2581. 16. Swasitang P, Tucker G, Robards K, Jardine D. Isolation and identication of phenolic compounds in Citrus sinensis. Anal. Chim. Acta 2000; 417: 231. 17. P erez-Magarino alez-Sanjos e ML, Beltr an S. S, Revilla I, Gonz Various applications of liquid chromatographymass spectrometry to the analysis of phenolic compounds. J. Chromatogr. A. 1999; 847: 75. 18. Cuyckens F, Rozenberg R, de Hoffmann E, Claeys M. Structure characterization of avonoid O-diglycosides by positive and negative nano-electrospray ionization ion trap mas spectrometry. J. Mass Spectrom. 2001; 36: 1203. 19. Becchi M, Fraisse D. Fast atom bombardment and fast atom bombardment collision-activated dissociation/mass-analysed ion kinetic energy analysis of C-glycosidic avonoids. Biomed. Environ. Mass Spectrom. 1989; 18: 122. 20. Waridel P, Wolfender JL, Ndjoko K, Hobby KR, Major HJ, Hostettmann K. Evaluation of quadrupole time-of-ight tandem mass spectrometry and ion-trap multiple-stage mass spectrometry for the differentiation of C-glycosidic avonoid isomers. J. Chromatogr. A 2001; 926: 29. 21. Li Q, Van den Heuvel H, Delorenzo O, Corthout J, Pieters AC, Vlietinck AJ, Claeys M. Mass spectral characterization of Cglycosidic avonoids isolated from a medicinal plant (Passiora incarnata). J. Chromatogr. 1991; 562: 435. 22. Fabre N, Rustan I, de Hoffmann E, Quetin-Leclerq J. Determination of avone, avonol, and avanones aglycones by negative ion liquid chromatography electrospray ion trap mass spectrometry. J. Am. Soc. Mass Spectrom. 2001; 12: 707. 23. Justesen U. Collision-induced fragmentation of deprotonated methoxylated avonoids, obtained by electrospray ionization mass spectrometry. J. Mass Spectrom. 2001; 36: 169. 24. Harborne JB, Baxter H. Phytochemical Dictionary. Taylor and Francis: London, 1993.

Copyright 2003 John Wiley & Sons, Ltd.

J. Mass Spectrom. 2003; 38: 3542