J Sci Food Agric 1990,53, 85-92

Separation and Quantitative Determination of Grape and Wine Procyanidins by High Performance Reversed Phase

Liquid Chromatography

Jorge Manuel Ricardo da Silva

Instituto Superior de Agronomia, Laboratorio Ferreira Lapa, Universidade Tecnica de Lisboa, 1399 Lisboa Codex, Portugal

Jean-Philippe Rosec, Michel Bourzeix and Nicolas Heredia

Institut National de la Recherche Agronomique, Institut des Produits de la Vigne, Station de Pech-Rougeparbonne, 11 104 Narbonne Cedex, France

(Received 16 October 1989; revised version received 5 February 1990; accepted 26 February 1990)

ABSTRACT

A method is described for the separation and quantitative analysis of procyanidins in grape and wine. On a polyamide chromatographic column an initial elution with water at pH 70 enabled elimination of interfering phenolic acids. A second elution with acetonitrilelwater (30:70 v ) was performed to extract catechins. A third elution with acetonelwater (75:25 v) was carried out to obtain procyanidins.

The quantitative determination of procyanidins was done by high performance reversed phase liquid chromatography. The HPLC analysis was brought down to -55 min. The method is accurate and reproducible and has an acceptable extraction efjiciency.

Key words: Grape, wine, procyanidins, reversed phase HPLC.

INTRODUCTION

The phenolic compounds of grapes and wines are of undoubted importance and they contribute in wines to the sensory properties-colour, flavour, astringency and bitterness (Timberlake and Bridle 1976; Lea et a1 1979; Ramey et a1 1986), oxidative and enzymatic browning (Oszmianski et a1 1985; Cheynier et a1 1988; Lee and

85

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86 J M Ricardo DQ Silua, J - P Rosec, M Bourzeix, N Heredia

Jaworski 1988), haze formation and interactions with proteins (Vancraenenbroeck et a1 1979; Oh and Hoff 1986; Yokotsuka and Singleton 1987; Powers et a1 1988; Jouve et a1 1988), ageing behaviour of wines (Haslam 1980) and physiological effects (Laparra et a1 1978; Masquelier 1988).

The procyanidins are a class of natural products that arise from flavan-3-01s and exist in a range of forms: dimers, trimers and polymers. Separation, identification and compositional changes have been studied in grapes and wines (Joslyn and Dittmar 1967; Weinges and Piretti 1971; Czochanska et a1 1979; Wulf and Nagel 1980; Bourzeix et al1986; Oszmianski et al 1986; Romeyer et all986; Boukharta et al1988; Etievant et a1 1988). Various authors have proposed analytical methods for the quantitative determination of procyanidins in different natural products: grapes and wines (Lea et al 1979; Lea 1980; Salagoity-Auguste and Bertrand 1984; Bourzeix et a1 1986; Jaworski and Lee 1987; Lunte et al 1988), cider (Lea and Timberlake 1974; Lea 1979, 1980), beer (Vancraenenbroeck et al 1979; Kirby and Wheeler 1980; Delcour et a1 1981; Van de Casteele et a1 1983; Jerumanis 1985), barley (Jende-Strid and Moller 1981; McMurrough 1981; Mulkay et a1 1981; Jerumanis 1985), hops (McMurrough 1981 ; Derdelinckx and Jerumanis 1984; Jerumanis 1985), malt (Derdelinckx and Jerumanis 1984), sorghum (Glennie et a1 1981), apple (Chiavari et al 1987) and apple juice (Wilson 1981; Lea 1982).

The main objective of the present study was to elaborate a simple and reliable method for the quantification of grape and wine procyanidins. The use of polyamide chromatography with three successive elutions to eliminate HPLC-interfering compounds such as phenolic acids and to separate catechins from procyanidins allowed us to obtain a pure procyanidin fraction. This fraction was further analysed by high performance reversed phase liquid chromatography. This was particularly useful in red wines which are complex and rich in phenolics.

EXPERIMENTAL

Materials

Grapes and wines Pinot Noir wine was obtained in 1987 at the Institut National de la Recherche Agronomique-Station experimentale de Pech-Rouge (Gruissan, France) by pressing and fermentation at 25°C with the skins removed after 4 days. After fermentation the wine was racked and bottled, and analysed in October 1988.

The proposed analytical method was also applied to methanol extracts of grape skins, seeds, pulps and stems.

Standards The standards (+ )<atechin and ( - )-epicatechin were purchased from Sarsyntex (Merignac, France).

Procyanidins B,, B,, B,, B,, trimer C, (epicatechin 4p+8 epicatechin 48-8 epicatechin) and a trimer (epicatechin 4 8 4 8 epicatechin 48+8 catechin) named in this work as trimer 2 were isolated from grape seed methanol extracts. Their isolation and identification will be described in a future paper.

Separation and determination of grape and wine procyanidins 87

Purification of the sample and elution of catechin and epicatechin from grapes and wine

A chromatographic column (id 2 cm x 25 cm) filled with 3 g of glass powder (particle size 200-400 pm) and 1.2 g of polyamide for thin layer chromatography, TLC 6, Macherey-Nagel (Duren, FRG) was prepared. A layer of glass powder followed by one of polyamide were added. Between 2 and 5 ml of grape extract or wine (the volume depending on richness of the sample) were mixed with 0 5 g of polyamide TLC 6 and about 10ml of H,O adjusted to pH 7.0 with phosphate buffer: KH,PO, and Na,HPO, in concentrated standard solution purchased from Normadose Prolabo (Paris, France). After 2 min the mixture was slowly poured on to the column. Elution was carried out with 80 ml of H,O adjusted to pH 7.0 with buffer, to eliminate phenolic acids. This 80 ml of neutral water was added in small quantities to avoid disturbing the 0 5 g of polyamide together with the adsorbed sample. If the sample is dispersed in a larger volume, HPLC-interfering compounds are desorbed into the aqueous phase; then again adsorbed on to polyamide before the neutral water really plays an eluant role. The elution was continued with 50 ml of acetonitrile/water (30: 70 v) to desorb catechins.

The flow rate of the elution solvents was about 1.5 ml min-'. The eluant was removed under vacuum at 30°C. The residue was dissolved in 2 ml of methanol/ water (50: 50 v), followed by HPLC analysis.

Elution of procyanidins

This elution was carried out with 50 ml of acetone/water (75:25 v). The flow rate and the treatment of the eluant were as described above.

The appearance of red colour in the eluate showed that anthocyanins were eluted with acetone/water (75:25 v) and also with acetonitrile/water (30: 70 v).

Apparatus and HPLC column

A high performance Waters-Millipore liquid chromatograph (Milford, CT, USA) equipped with 6000 A and EM 45 pumps, UV-V detector with four simultaneous detection channels (model 490) and data module (model 730) was used. The detection wavelength was 280 nm. A second channel at 313 mm was used to observe the presence of phenolic acids. Separations were performed with an Ultrabase column from SFCC (Neuilly-Plaisance, France; id 4.6 mm x 15 cm) packed with 5- pm C-8 reversed phase packing material at 32°C. A column from SFCC (id 4-6 mm x 25 cm) packed with 5-pm C-18 Nucleosil packing material satisfactorily resolved the peaks but retention times were -10min longer. All the chromatographic procedures were the same for the two columns. We will present only the results obtained with the Ultrabase column.

Chromatographic procedures

The detector sensitivity was 0100 aufs. Injection volume was 50 pl. The following mobile phases were used: solvent A, acetic acid/bidistilled water (10:90v) and solvent B, bidistilled water. A linear gradient was run from 10 vol A + 90 vol B to 82

88 J M Ricardo Da Silua, J - P Rosec, M Bourzeix, N Heredia

1,

vol A + 18 vol B during 47 min and then to pure A during 8 min. The flow rate was 0.8 ml min-'.

Finally the column was washed isocratically with a mixture of solvents (methanol/bidistilled water/acetic acid (50:35:15 v) for 40 min at the same flow rate as before.

3,

2, 1

4

RESULTS AND DISCUSSION

Separation

The chromatograms shown in Figs 1 and 2 present the separation performed by the method described above with the Pinot Noir wine. Acetonitrile/water (30:70 v ) (Fig 1) elutes catechin and epicatechin while acetone/water (75:25 v) (Fig 2) elutes pyrocyanidins B1, B2, B3, B4 and trimers C1 and 2. The same separation was tested on standard solutions with similar results.

The peaks were identified by injection of the standards (see 'Experimental' for details).

Effective separation of catechin and trimer 2, which have similar retention times, was also monitored by TLC on DC Alufolien-Kieselgel 60 Merck (Darmstadt, FRG); solvents: toluene/acetone/formic acid (30: 30:lO v); visualised using a solution of l o g vanillin in HCL (1OOg litre-'). This TLC system separates catechins and procyanidin oligomers in order of molecular weight (Lea et a1 1979).

Procyanidin B3 (peak 5) in Fig 2 appears to elute at a retention time similar to that of an unknown peak in Fig 1. Injection of a mixture of the two eluates gave a chromatogram in which peak 5 is a shoulder at the end of the unknown peak and easy to distinguish. The unknown peak is not procyanidin B3.

Fig 1. Chromatogram of acetonitrile/water Fig 2. Chromatogram of acetone/water (75:25 v) (30:70v) eluate. 1, (+) Catechin; 2, (-1 eluate. 3, Procyanidin B,; 4, procyanidin B,; 5,

procyanidin B,; 6, procyanidin B,; 7, procyanidin trimer C,; 8, procyanidin trimer 2.

epicat echin .

Separation and determination of grape and wine procyanidins 89

TABLE 1 Extraction etliciency of catechins and procyanidins using the proposed methods

Compounds Amounts (mg li tre-') Extraction (Means of 4 replications) efficiency

(%I Known added Wine Wine

quantities (measured) + (standards) known quantities

(measured)

(+ )<atechin 500 1100 156.8 98.0

Procyanidin B, 62.0 82.0 144.3 1 0 0 5 Procyanidin B, 45.0 36.5 80.8 95.0 Procyanidin B, 55.0 15.6 69.0 97.0 Procyanidin B, 50.0 1.4 56.4 98.0 Procyanidin trimer C , 500 102 57.8 96.0 Procyanidin trimer 2 50.0 25.5 71.0 94.0

(- )-Epicatechin 500 38.2 89.1 101.0

We checked by simultaneous detection at 313 nm that there was no interference between phenolic acids, catechins and procyanidins. The anthocyanins were eluted with the washing solvent and did not interfere with HPLC analysis of the catechins and procyanidins.

Extraction efficiency

The extraction eficiency was determined by analysis of Pinot Noir wine with and without additions of known amounts of standard solution. The results are shown in Table 1; the recovery was between 94 and 101 % for all tested compounds (four replications each).

Reproducibility

The Pinot Noir wine was analysed ten times to evaluate the reproducibility of procyanidin quantification. The results are listed in Table 2.

Standardisation and detection threshold

Three standard solutions containing increasing concentrations of procyanidins, catechin and epicatechin were directly injected into the HPLC system. Linear correlation coefficients were superior to 0.999 for each compound. The calibration lines used were determined by a least squares regression method. Response factor (mmT2 mg litre-') and detection threshold (mg litre-') for each compound are shown in Table 3.

CONCLUSION

The method proposed for the quantification of procyanidins in grapes and wines, which consists of three successive elutions on a polyamide column using neutral water (PH 7-0), acetonitrile/water (30:70 v) and acetone/water (75: 25 v), followed

90 J M Ricardo Da Silua, J - P Rosec, M Bourzeix, N Heredia

TABLE 2 Reproducibility of catechin and procyanidin quantification in Pinot Noir wine (ten replicate

extractions)

Compounds

(+)-Catechin (- )-Epicatechin Procyanidin IB, Procyanidin B, Procyanidin B, Procyanidin B, Procyanidin trimer C, Procyanidin trimer 2

Average area

(mm2 )

Corresponding amount'

(mg litre- ')

Standard deviation

(mm2)

Variation coeficient

( %)

172 71 168 60 27 1 1 17 49

107.3 52-8 94.8 40.1 17.3 6.2 10.7 36.8

4.86 2-27 4.28 1.56 1.39 0.35 0.60 1.96

2.8 3-2 2.5 2.6 5.2 3.1 3.5 4.0

Dilution factor = 0.4.

TABLE 3 Response factor and detection threshold of phenolic compounds

Compounds Range Response Detection used factor threshold (d )

(mg litre- ') (rnrn-, mg litre-') (mg litre-')

( + )-Catechin ( - )-Epicatechin Procyanidin B, Procyanidin B, Procyanidin B, Procyanidin B, Procyanidin trimer C Procyanidin trimer 2

0-200 s200 0-150 C150 0-200 s200 0-100 0-100

1.56 2 1.86 1<d<2 1.41 2<d<3 1.67 2 1.60 2 1.41 2<d<3 1.57 2 1.88 1<d<2

a In the injected solution.

by high performance reversed phase liquid chromatography, gave good results. Compounds interfering with HPLC analysis were eliminated and the catechins were separated from the procyanidins. This technique has potential practical application in research and industry. Baseline and peak resolution are very acceptable. HPLC analysis is possible in about 55 min and the method proposed is accurate and reproducible and gives acceptable extraction efficiency.

ACKNOWLEDGEMENTS

This work was supported by grants from FundaCBo Calouste Gulbenkian and FundaGiio Luso Americana of Portugal. The authors are grateful to Dr Veronique Cheynier, Dr M Moutounet and Eng J Rigaud, of the Institut des Produits de la

Separation and determination of grape and wine procyanidins 91

Vigne, Laboratoire des Polymeres et des Techniques Physicochimiques (Montpellier, France), for helpful discussions.

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