Analytica Chimica Acta 513 (2004) 247–256

Differentiation of proanthocyanidin tannins from seeds, skins andstems of grapes (Vitis vinifera) and heartwood of Quebracho

(Schinopsis balansae) by matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry and thioacidolysis/liquid

chromatography/electrospray ionization mass spectrometry

Nicolas Vivasa,∗, Marie-Françoise Noniera, Nathalie Vivas de Gaulejaca,Christelle Absalonb, Alain Bertrandc, Marie Mirabelc

a Demptos Cooperage Posted to CESAMO (Centre d’Etudes Structurales et d’Analyses des Molécules Organiques),Université Bordeaux I-351, Cours de la Libération, 33405 Talence, France

b CESAMO, Université Bordeaux I, Talence, Francec Faculté d’œnologie, Université Victor Segalen Bordeaux II, Talence, France

Received 30 October 2003; accepted 14 November 2003

Available online 4 February 2004

Abstract

To control and identify with confidence the principal enological tannins (ETs), we have devised a specific method based on the char-acterization of proanthocyanidin composition. We started by controlling the red colouring produced by hydrochloric acid butanolysis (e.g.Bate–Smith reaction), due to the formation of anthyocyanidins, typical of proanthocyanidin tannins. Using thioacidolysis/liquid chromatogra-phy/electrospray ionization mass spectrometry we were able to identify: (i) the nature of the flavan-3-ols (catechin, epicatechin for procyanidinstannins; gallocatechin, epigallocatechin for prodelphinidins tannins), (ii) the degree of galloylation, (iii) the average degree of polymeriza-tion (mDP). We also performed a complete structural study by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry(MALDI-TOF-MS). By comparing the chromatographic profiles of standard proanthocyanidins prepared in our laboratory with those of avariety of commercial enological tannins, we were able to identify their origin: seed proanthocyanidins (PA): only procyanidins, high levelof galloylation, with a large amount of epicatechin, and a low mDP corresponding to a majority of oligomeric tannins; skins PA: a mixture ofprocyanidins and prodelphinidins, with a predominance of procyanidins, a low level of galloylation, with a large amount of epicatechin, anda very variable mDP; stems PA, mixture of procyanidins and prodelphinidins, little galloylation, with a very low level of epicatechin in theterminal unit, and a medium value for mDP (>5); Quebracho PA: results show no known flavan-3-ols, and according to MALDI-TOF-MS,the main structure is attributed to a large amount of profisetinidin, corresponding to a resorcinol proanthocyanidin.© 2003 Elsevier B.V. All rights reserved.

Keywords:Enological tannins; Grapes tannins; Quebracho tannins; Proanthocyanidins; Thioacidolysis; MALDI-TOFMS

1. Introduction

Enological tannins (ETs) have been used for a very longtime. Originally, the treatment often consisted of usingwood or galls tannins (e.g. proanthocyanidins, ellagitanninsand gallotannins)[1–3]. But to really improve the polyphe-nolic structure and stability of wine, grape tannins are the

∗ Corresponding author. Tel.:+33-5-40-00-25-78;fax: +33-5-40-00-26-23.

E-mail address:n.vivas@cesamo.u-bordeaux1.fr (N. Vivas).

best, mainly because wine and grape ET proanthocyanidins(PAs) have a similar structure. Chemical reactions occurringduring ageing, particularly oxidation, produce combina-tions of PA/PA or PA/anthocyanidins monoglucosideviaacetaldehyde bridges and vinyllinkage[4,5]. Grape ETs arebeing used more and more, their market share increasingsignificantly every year, although they are more expensivethan other conventional ETs like ellagitannins, gallotanninsor Quebracho tannins. Higher to lower values are respec-tively skins > seeds> stems≥ Quebracho tannins. Thisis why it is absolutely necessary to distinguish between

0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved.doi:10.1016/j.aca.2003.11.085

248 N. Vivas et al. / Analytica Chimica Acta 513 (2004) 247–256

these different ET sources and verify the information andinstructions on the labels.

The chemical composition of PA changes notably withits botanical origin and the nature of the tissues[6–9]. PAsare polymers of flavan-3-ol units with a phloroglucinol orresorcinol A ring: catechin and epicatechin for procyanidins(PAc); gallocatechin and epigallocatechin for prodelphini-dins (PAd); fisetinidol and epifisetinidol for profisetinidin(PAf); robinetinidol and epirobinetinidol for prorobine-tinidin (PAr). In the case of PAc and PA, an additionalvariable can occur due to possible galloylation. Thioacidol-ysis is a selective acidic depolymerization method using athiol as a nucleophilic agent[10,11]. With this method themonomeric composition and discrimination of polymericPA was easily obtained. To confirm identification and com-plete the structural information we used matrix-assistedlaser desorption ionization (MALDI) time-of-flight massspectrometry (TOF-MS), which particularly well sunitedfor large molecules[6]. This study focuses on the maincommercial products made from the skins, seeds and stemsof Vitis vinifera sp.and Quebracho (Schinopsis balansa)heartwood.

2. Experimental

2.1. Reagents

(+)-Catechin; 2,5-dihydroxybenzoic acid (DHB); benzylmercaptan 99% (toluene-�-thiol); sodium iodide were pur-chased from Aldrich. NH4Fe(SO4)2·12H2O was purchasedfrom Prolabo. Polystyrene standards were purchased fromPolymer Laboratories.

Hydrochloric acid (37%) and pyridine were purchasedfrom Riedel-de Haën; methanol (HPLC grade), orthophos-phoric acid (84%), and acetic anhydride were purchasedfrom Prolabo; butanol was purchased from Acros; chloro-form (sds).

Different samples of industrial tannins were provided di-rectly by producers and bought on the market: skins tannins(6); seeds (14); stems (4); Quebracho (8).

2.2. Proanthocyanidins isolation for reference samples

Grapes (V. vinifera) were collected at commercial matu-rity. Seeds, skins and stems were separated manually. Iso-lated seeds, skins and stems were lyophilised, powdered andfrozen at−18◦C until used.

Portions (4 g) of seeds, skins and stems were extractedin to 20 ml of EtOH/acidified water (1:1; v:v) under nitro-gen with mechanical agitation for 12 h. The same volume ofchloroform was added to eliminate lipids and pigments. Themixture was centrifuged for 10 min. The lower green chlo-roform phase was eliminated and the upper yellow phasecorresponding to the hydroalcoholic (HA) component wasrecovered. Extraction was repeated three times and all the

HA extracts of the same sample were collected together,evaporated to dryness, and frozen at−18◦C until analysis.

The Quebracho tannins (Schinopsis balansae) were ex-tracted differently: sawdust (60 mesh) was extracted withacetone/water (7:3; v:v) at room temperature during 12 h.The extract was filtered, acetone and water were removedunder reduced pressure and the final extract was lyophilized.

2.3. Polyphenols index

Total phenolic compounds of tannins was calculated bymeasuring the absorption at 280 nm of the extracts diluted inwater at 10 mg l−1 [12]. The total phenolic index (IPT) wascalculated from the formula: IPT= absorbance at 280 nm×100. The results can also be expressed as a percent in relationto different standards; the choice of the standard depends onthe nature of the tannins ((+)-catechin for PA).

2.4. Bate–Smith reaction

The method is based on the oxidative depolymerizationwith heat, in a mineral acidic medium of proanthocyanidinsand on the formation of anthocyanins absorbing at 550 nm(red color). The method used is butanolysis in a hydrochloricmedium with ferrous salts (FeSO4) as catalyst.

In a glass tube (10 ml) sealed with a PTFE screwcap, weintroduced 1 ml of a methanolic solution containing 0.01%(w:v) tannins, 6 ml of butanol–HCl (95:5, v:v) and 0.2 mlof a iron(II) sulfate solution at 2% (w:v). The tube was her-metically sealed, shaken and the reaction developed within40 min in a water-bath at 95◦C. The solution was cooledin ice water. The intensity (absorbance) of the colour whichdeveloped was measured at 550 nm (Anthelie SecomamTM

spectrophotometer). The absorbance of the colour formedwas subtracted from that of the unheated reference tube. Re-sults are given according to a calibration plot for a solutionof procyanidins prepared in the laboratory (hydroalcoholicmaceration of seeds extracted with ethyl acetate).

2.5. Thioacidolysis/liquid chromatography/electrosprayionisation

2.5.1. Thioacidolysis0.5 ml of the tannins solution (1 g l−1) was placed in a

glass ampoule with an equal volume of reagent (5% solutiontoluene-�-thiol in MeOH containing 1.7% HCl). After seal-ing the ampoule, the mixture was shaken and heated at 60◦Cfor 10 min. 0.5 ml of water was added to the hydrolyzed so-lution to avoid the formation of asymmetric peaks and toimprove chromatographic resolution. The solution obtainedwas analyzed directly by HPLC.

2.5.2. LC analysisThe extract was analyzed by reversed phase LC on

a WatersTM system 600 E, equipped with a WatersTM

600 E pump. The column was an InterchromTM C18

N. Vivas et al. / Analytica Chimica Acta 513 (2004) 247–256 249

10�m (250 mm× 4.6 mm) with a guard column of thesame material. The eluant was a mixture of two solventsfiltered through 0.45�m membrane filters; solvent A:MeOH/H3PO4 999:1, solvent B: H2O/H3PO4 999:1. Theinjection volume was 20�l (RheodyneTM 7725i; manualinjection). The elution programme was performed at aconstant flow of 1 ml min−1, at a temperature of 20◦C,passing from 70% of B (for 5 min) to 10% of B in 40 min,and then rising to 70% of B in 10 min for 5 min. Theprogram included washing and reconditioning the column.Detection was by a variable-wavelength spectrophotometric(WatersTM 486 MS) at 280 nm.

2.5.3. Purification of productsPurification was realized by LC using the aforementioned

equipment and the same analysis conditions. Fractions werecollected using a FRAC-100-type fraction collector (Amer-sham Pharmacia BiotechTM) connected to a UV detector. Tooptimize fractionation, collector parameters were adjusted.The principal programming characteristics were: collectionmode: 0, fraction size: 5.5 min, peak threshold: 2%.

2.5.4. Mass spectrometric analysisA Platform II mass spectrometer (Micromass, Manch-

ester, UK) with electrospray injection (ESI) was used, cou-pled to the LC apparatus. Proanthocyanidins and flavan-3-olscorresponding thioethers can easily shed a proton, generat-ing intense negative ions [M–H]−, detection was thereforeperformed in the negative ion mode. A low voltage was usedto avoid fragmentation; the products were identified by theirmolecular peaks.

2.6. MALDI-TOF-MS analysis

2.6.1. MALDI-TOF-MSMALDI-MS spectra were obtained using a TofSpec

Maldi-Tof mass spectrometer from Micromass (Manchester,UK). The instrument is equipped with a pulsed nitrogenlaser (337 nm, 4 ns pulse width) and a time-delayed ex-tracted ion source. Spectra were recorded in the positive-ionmode using the reflectron and with a 20 kV acceleratingvoltage.

2.6.2. MALDI-TOF sample preparationSamples were dissolved in methanol at 10 mg-ml−1. The

DHB matrix solution was prepared by dissolving 10 mg

Table 1Total phenol index and PA content for reference and commercial tannins

Reference tannins Commercial tannins

Seeds Skins Stems Quebracho Seeds Skins Stems Quebracho

Total Phenol Indexa (IPT) 24 14 10 31 16.5 13 7.5 20PA contenta (in g g−1 PAc oligomers) 1.8 1.02 0.5 0.8 1.55 0.85 0.3 0.48

a Standard deviation for reference tannins 14± 2.5% and for commercial tannins 38± 6%.

in 1 ml of methanol. A methanol solution of sodium io-dide was prepared at 10 mg-ml−1. The solutions werecombined in a 10:1:1 volume ratio of matrix to polymerto cationisation agent. One to two of the obtained solu-tion were deposited onto the sample target and vacuumdried.

2.7. Size exclusion chromatography (SEC) analysis

The study of theMp distribution of tannins was per-formed using the acetyl derivatives. Samples (10 mg) offreeze-dried material were acetylated with pyridine-aceticanhydride (1:1; v:v) for three days at room temperature.The precipitate obtained by pouring the mixture into coolwater was recovered by centrifugation. This precipitate waswashed with distilled water, methanol and finally chloro-form. It was dried and dissolved in 0.5 ml of tetrahydrogenfuran (THF) and filtered before analysis by SEC (gel per-meation chromatography).

SEC analysis was performed using a Thermo QuestTM

Spectra Series P100 instrument equipped with an isocraticThermo QuestTM pump, with three columns (300 mm×7.8 mm): TSKTM Gel G 1000 HXL, TSKTM Gel G 2000HXL, TSKTM Gel G 2500 HXL, in series, protected witha guard column of the same material. The analysis condi-tions were: THF as the eluent, flow-rate: 1 ml min−1, injec-tion volume: 20�l (RheodyneTM 7725i; manual injection),analysis time: 45 min. The calibration graph was obtainedwith polystyrene, and (+)-catechin was utilized as the stan-dard. Detection was at 280 nm with a UV absorbance detec-tor (Spectra SeriesTM UV-150). PL CaliberTM software wasused for data acquisition.

3. Results and discussion

3.1. Polyphenolic composition

Firstly, we controlled the production of the red colour af-ter hydrochloric acid butanolysis (e.g. Bate–Smith reaction),due to the formation of anthocyanidins, typical of proan-thocyanidins tannins. Then, to have a pre-indication of thepolyphenolic composition of ET and reference preparationswe estimated the total polyphenols index on the one hand andthe PA content measured by Bate–Smith reaction (Table 1)on the other.

250 N. Vivas et al. / Analytica Chimica Acta 513 (2004) 247–256

Fig. 1. MALDI mass spectrum of: (a) grape seed tannin extract and (b) grape skin tannin extract.

3.2. Partial structural characterizationby MALDI-TOF-MS

All the samples were analyzed by MALDI-TOF MS andprovide interesting information on the nature of the PAs(Figs. 1 and 2).

3.2.1. Seeds PA present the characteristic profile of PActannins

Each main signal was incremented by 288 Da correspond-ing to a catechin and epicatechin involved C–C linkage inthe polymer (M − 2H) whereM is the molecular mass ofa flavan-3-ol, as in accordance to previous observations, thepolymerization, level wasM + n (M − 2H) wheren is thedegree of polymerization. The spectra revealed a second in-crement of 152 Da, of the galloyl group; this means we needto add 152g to the previous equation, whereg is the numberof galloyl unit. Finally, in our experimental conditions, themass of the molecules is increased by a Na+ adduct (m/z:

23 Da), giving the following general equation for measuredmass:

M + (n − 1)(M − 2H) + 152g + 23[E]

Application of the formula was helpful for the MALDI spec-tral interpretation. For seeds PAc, the equation, after numer-ical application, became: 290+ 288(n − 1) + 152g + 23.In all samples we obtained signals of dimeric to octamericPAc with partial galloylation (g = 0–4), (Table 2).

3.2.2. Skins PA present PAc and PAd profilesTo analyze MALDI spectra, we can apply the general

equation described above for seed PAc. We also noted thateach mean signal was incremented by 16 Da, correspondingto the presence of an OH group added to flavan-3-ols units.This additional information, supplied by MALDI analysis,made it possible to reveal the presence of PAd in skin tan-nins. To take account of the presence ofn′ trihydroxylated

N. Vivas et al. / Analytica Chimica Acta 513 (2004) 247–256 251

Fig. 2. MALDI mass spectrum of: (a) grape stem tannin extract and (b) Quebracho tannin extract.

flavan-3-ols (gallocatechin or epigallocatechin) the equation[E] became:(

M + n′

np

)+ (n − 1)

[(M − 2H) + n′

np

]+ 152g+23

wherep = 16 Da (from the OH group)After numerical application, the modified equation was:

(290+ 16

n′

n

)+ (n − 1)

[288+ 16

n′

n

]+ 152g+23

The results are grouped inTable 3. We note that in fact thetrihydroxylated flavan-3-ols were associated with dihydrox-ylated flavan-3-ols to form a hetero-PA (hPA).

3.2.3. Stems PA present PAc and PAd profilesTo analyze the MALDI spectra, we also applied the equa-

tion established previously for the skins. The results aresummarized inTable 4. As in skins, we identified hPA in

stems, that presented a different number of trihydroxylatedflavan-3-ols ranging from 0 to 3.

3.2.4. Quebracho PA present PAf and PAr profilesAs can be seen in the spectra, there are more peak series,

due to different end groups. They have the same repeat units,for example 1401–1673 Da and 1673–1945 Da inFig. 2. Theincrement of 272 Da corresponds to fisetinidol and epifise-tinidol (PAf) involved C–C linkage in the polymer [274-2H].There are other repeat units, corresponding to 288 Da, char-acterizing robinetinidol, and epirobinetinidol (PAr) involvedC–C linkage in the polymer [290-2H]. The PAr present thesamem/zvalue for the flavan-3-ols units as PAc; fortunately,characterization of the PAf made it possible to confirm theattribution of the signal to prorobinetididins structures. Themain reason was the biochemistry of PA synthesis: the planttissues were able to produce either phloroglucinol PAs orresorcinol PAs families. Like the skins, the 16 Da incrementcorresponds to trihydroxylated flavan-3ols. In our analysis

252 N. Vivas et al. / Analytica Chimica Acta 513 (2004) 247–256

Table 2Observed and calculated masses of grape seed PA by MALDI-TOF-MS

Polymer Numberof galloylunits (g)

CalculatedM + Na+

ObservedM + Na+

(positive reflectron)

Trimer 0 889.8 889.41 1041.9 1041.52 1194 1193.73 1346.1 1346

Tetramer 0 1178 1177.71 1330.1 1330.12 1482.2 1482.33 1634.4 1634.7

Pentamer 0 1466.3 1466.41 1618.4 1618.52 1770.5 1770.63 1922.6 1922.74 2074.7 2074.8

Hexamer 0 1754.5 1754.81 1906.7 1906.92 2058.8 2059.13 2210.9 2211.44 2363 2361.6

Heptamer 0 2042.8 20431 2194.9 2194.72 2347 2345.93 2499.1 2500.44 2651.2 2652.4

Octamer 0 2331.1 23301 2483.2 2480.52 2635.3 2637.23 2787.4 2790.24 2939.5 2943

Nanamer 0 2619.3 2618.11 2771.4 2774.22 2923.5 2927.23 3075.6 3080.8

Decamer 0 2907.6 29101 3059.7 3065.1

Table 3Observed and calculated masses of grape skin PA by MALDI-TOF-MS

Polymer Numberof galloylunits (g)

Number of tri-hydroxylatedflavan-3-ols(n’)

CalculatedM + Na+

ObservedM + Na+,(positivereflectron)

Trimer 0 0 889.8 889.61 0 1041.9 1042.30 1 905.7 905

Tetramer 0 0 1178 1177.71 0 1330.1 1330.80 1 1194 1194

Pentamer 0 0 1466.3 1465.90 1 1482.3 1482

Table 4Observed and calculated masses of grape stem PA by MALDI-TOF-MS

Polymer Numberof units(g)

Number of tri-hydroxylatedflavan-3-ols(n′)

CalculatedM + Na+

ObservedM + Na+,(positivereflectron)

Trimer 0 0 889.8 889.31 0 1041.9 1041.5

Tetramer 0 0 1178.0 1177.40 1 1194.0 1193.40 2 1210.0 1209.31 0 1330.1 1329.4

Pentamer 0 0 1466.3 1465.30 1 1482.3 1482.50 2 1498.2 1497.60 3 1514.3 1513.5

Hexamer 0 0 1754.5 1754.10 1 1770.5 1770.40 2 1786.5 1785.5

conditions, the mass of the molecules were increased by anNa+ adduct (23 Da). For this specific type of PA the equa-tion was:(

274+ 16n′

n

)+ (n − 1)

[272+ 16

n′

n

]+ 152g+23

Observed and calculated masses of Quebracho PA byMALDI are grouped inTable 5. Numerical values for masscalculations by each general equation are presented inTable 6.

Table 5Observed and calculated masses of Quebracho PA by MALDI-TOF-MS

Polymer CalculatedM + Na+

ObservedM + Na+

Unit type

Positivereflectron

Fisetinidol Robinetinetinidol

Trimer 841.9 841.4 3 –857.9 857.4 2 1873.9 873.4 1 2

Tetramer 1114.2 1113.5 4 –1130.2 1129.5 3 11146.2 1145.5 2 2

Pentamer 1386.5 1385.6 5 –1402.5 1401.5 4 11418.5 1418.6 3 21434.5 2 3

Hexamer 1658.8 – 6 –1674.8 1673.6 5 11690.8 1691.5 4 21706.8 1707.5 3 3

Heptamer 1947.1 1945.6 6 11963.1 1965.4 5 21979.1 1979.6 4 3

Octamer 2235.4 2236.7 6 2

N. Vivas et al. / Analytica Chimica Acta 513 (2004) 247–256 253

Table 6Numerical values for masses calculations by general equations

M (g mol−1) n n′ p g

Phlorogucinol PAPAc 290 2–. . . 0 – 0–· · ·PAd 290 2–. . . 1−n 16 0–· · ·

Resorcinol PAPAf 274 2–. . . 0 – 0–· · ·PAr 274 2–. . . 1–n 16 0–· · ·

Fig. 3. Liquid chromatogram of grape skin proanthocyanidins and ESI mass spectrum of catechin and catechin benzylthioether (R∗ = reagent reside).

Table 7Characteristics of compounds identified by HPLC following thioacidolysis degradation

Number of chromatography peak Compound Molecular mass(g mol−1)

Maximum wavelengthof absorbance (nm)

Response factor(mmol l−1)

Terminal units1 Epigallocatechin 306 280 17619302 Catechin 290 280 37241703 Epicatechin 290 280 40158704 Epicatechin-3-O-gallate 442 280 18146500

Extension units5 Catechin benzylthioether I 412 280 53069406 Epigallocatechin benzylthioether 428 280 17674007 Catechin benzylthioether II 412 280 53069408 Epicatechin benzylthioether 412 280 52317009 Epicatechin-3-O-gallate benzylthioether 564 280 14009500

3.3. Thioacidolysis method for rapid discrimination of PAtannins of different origins

Firstly, we identified the main thiolysis-released prod-ucts by ESI-MS. The corresponding spectra are shownin Fig. 3, making it possible to attribute each one to thechromatograms. In agreement with the chemical reactionof thioacidolysis (Fig. 4), two classes of compounds canbe characterized; each was collected by the LC fractioncollector (Table 7).

254N

.V

ivas

et

al./A

na

lyticaC

him

icaA

cta5

13

(20

04

)2

47

–2

56

Table 8Composition of seeds, skins, stems and Quebracho PA determined by LC following thioacidolysis degradation

Terminal units Extension units

EGC Cat EC ECG EGC Cat EC ECG

a b c a b c a b c a b c a b c a b c a b c a b c

Reference tanninsSeeds – – – 0.24 69.3 12.9 0.14 42 7.8 0.05 23.3 4.3 – – – 0.14 59.3 11.1 0.54 223.2 41.7 0.21 118.6 22.1Skins Tr Tr Tr 0.33 95 29 0.18 52 15.9 Tr Tr Tr 0.04 18 5.5 0.08 32 9.8 0.28 115 35.2 0.03 15 4.6Stems – – – 0.07 21 11.2 0.01 2.9 1.5 0.01 1.2 0.6 0.05 19.7 10.5 0.02 10.5 5.6 0.24 100.6 53.8 0.05 30.9 16.5Quebracho – – – – – – – – – – – – – – – – – – – – – – – –

Commercial tanninsSeeds – – – 0.22 65.2 10.7 0.23 66.5 10.9 0.03 12.7 2.1 – – – 0.13 55.8 9.1 0.75 306.7 50.3 0.18 102.9 16.9Skins Tr Tr Tr 0.33 97.9 30.3 0.17 50.3 15.5 Tr Tr Tr 0.04 16.8 5.2 0.07 29.5 9.1 0.28 117.7 36.4 0.02 11.1 3.4Stems – – – 0.05 15.4 11.8 – – – – – – 0.03 11.6 8.9 0.04 15.5 11.9 0.19 77.1 59.3 0.02 10.4 8Quebracho – – – – – – – – – – – – – – – – – – – – – – – –

Standard deviation: 4± 1.5% for reference tannins/6± 0.5% for commercial tannins.a mmol l−1.b mg g−1.c Relative percentage.

N. Vivas et al. / Analytica Chimica Acta 513 (2004) 247–256 255

OHO

OH

OH

OH

OH

OHO

OH

OH

OH

OH

OHO

OH

OH

OH

OH

OHO

OH

OH

OH

OH

OO

OH

OH

OH

OH

catechin carbocation (+)-catechin

H+, T

and

Nucleophile(Ethanethiol)

OHO

OH

OH

OH

OH

SCH2

CH3

4a-ethylthiocatechin

˚

Fig. 4. Principle of thioacidolysis reaction for PA tannins.

• Terminal units of flavan-3-ols, catechin and epicatechinand epicatechin-3-O-gallate for PAc, gallocatechin andepigallocatechin for PAd.

• Extension units linking by C4–C6 or C4–C8: catechinbenzylthioether I, II (isomeric forms), epicatechin ben-zylthioether, epicatechin-3-O-gallate benzylthioether forPAc and epigallocatechin benzylthioether for PAd.

The thioacidolysis reaction was recorded for severalsamples, some of these prepared in our laboratories (asreferences), others supplied in their commercial form. All

Table 9Characteristics of grape seed, skin, stem and Quebracho for reference and commercial tannins

Reference tannins Commercial tannins

Seeds Skins Stems Quebracho Seeds Skins Stems Quebracho

mDPa (thioacidolysis) 3.1 1.8 5 – 3.2 1.8 6.6 –mDPa (SEC) 4.9 3.5 7 4.8 3.3 6.9 2.7Galloylation (%) 0.2 0.03 0.13 – 0.14 0.02 0.06 –Prodelphinidins (%) 0 0.04 0.11 – 0 0.04 0.09 –

a Average degree of polymerization.

the analyses are summarized inTables 8 and 9. We notedthat seed proanthocyanidins (PA) are composed only ofprocyanidins, with a high level of galloylation, a largeamount of epicatechin, and a low mDP correspondingto a majority of oligomeric tannins. Skin PAs are char-acterized by a mixture of procyanidins and prodelphini-dins, with a predominance of procyanidins, a low levelof galloylation, a large amount of epicatechin, and a veryvariable mDP. Stem PAs are a mixture of procyanidinsand prodelphinidins, with a low level of galloylation, avery low level of epicatechin in the terminal unit, and a

256 N. Vivas et al. / Analytica Chimica Acta 513 (2004) 247–256

medium value for mDP (>5). Quebracho PA gave no knownflavan-3-ols.

The mDP was calculated by SEC analysis of each tan-nin. Results (Table 9) were compared to those obtained bythioacidolysis. SEC values were superior but the overall ten-dency was the same.

4. Conclusion

The thioacidolysis/LC/ESI/MS method ensured iden-tification of: (i) the nature of flavan-3-ols (catechin,epicatechin for procyanidins tannins; gallocatechin, epi-gallocatechin for prodelphinidins tannins), (ii) the degreeof galloylation, (iii) the average degree of polymerization(mDP). Also, we performed a complete structural study byMALDI-TOFMS spectrometry. By comparing the differ-ent commercial enological tannins chromatographic pro-files with those obtained with standard proanthocyanidinsprepared to laboratories, we were able to identify theirorigin.

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