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Ciencia y Tecnología Alimentaria ISSN: 1135-8122 [email protected] Sociedad Mexicana de Nutrición y Tecnología de Alimentos México

Giménez, R.; Villalón, M.; López, H.; Navarro, M.; Cabrera, C.; Olalla, M.; Quesada, J.J.; López, M.C. Determination of gallic acid in commercial brandies using high performance liquid chromatography Ciencia y Tecnología Alimentaria, vol. 3, núm. 1, diciembre, 2000, pp. 13-20 Sociedad Mexicana de Nutrición y Tecnología de Alimentos Reynosa, México

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DETERMINATION OF GALLIC ACID IN COMMERCIAL BRANDIES USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

DETERMINACIÓN DE ÁCIDO GÁLICO EN BRANDIES COMERCIALES MEDIANTE CROMATOGRAFÍA LÍQUIDA DE ALTA EFICACIA

DETERMINACIÓN DE ÁCIDO GALICO EN BRANDIES COMERCIAIS MEDIANTE CROMATOGRAFÍA LÍQUIDA DE ALTA EFICACIA

Giménez, R.*; Villalón, M.; López, H.; Navarro, M.; Cabrera, C.; Olalla, M.; Quesada, J.J.; López, M.C.

Departamento de Nutrición y Bromatología, Facultad de Farmacia, Universidad de Granada, Campus Universitario de Cartuja sn. 18012 Granada (España)

* Autor para la correspondencia. E-mail: [email protected]

Recibido: 10 de Octubre de 2000; recibida versión revisada: 22 de Octubre de 2000; aceptado: 23 de Octubre de 2000 Received: 10 October 2000; revised version received: 22 October 2000; accepted: 23 October 2000

Abstract One of the components presents in oak wood is gallic acid, which brandies extract during their aging process. In this study, a HPLC technique for the separation and identification of gallic acid by direct injection, has been performed. Studies, which tested the accuracy of the method, showed a 99.59% recovery percentage for this acid. This technique has been applied to the analysis of commercial brandies, thus revealing the existing differences in the gallic acid concentration according to the aging system followed: Static or traditional and dynamic or criaderas and soleras.

Key words: gallic acid, HPLC, brandies.

Resumen El ácido gálico es uno de los componentes presentes en la madera de roble que es extraido por los aguardientes durante el proceso de envejecimiento de los mismos. En el presente trabajo se ha puesto a punto la técnica por HPLC para la separación e identificación del ácido gálico mediante inyección directa de la muestra. Estudios de precisión del método indicaron un porcentaje de recuperación del 99,59%. La técnica se ha aplicado al análisis de brandies comerciales, lo que ha permitido conocer las diferencias existentes en las concentraciones de dicho ácido en función del sistema de envejecimiento seguido: Estático o tradicional y dinámico o de criaderas y soleras.

Palabras clave: ácido gálico, HPLC, brandies.

Resumo O ácido gálico é un dos compostos presentes na madeira de carballo que é extraido polos aguardentes durante o proceso de envellecemento dos mesmos. No presente traballo púxose a punto a técnica por HPLC para a separación e identificación do ácido gálico mediante inxección directa da mostra. Estudos de precisión do método indicaron unha porcentaxe de recuperación do 99,59%. A técnica aplicouse á análise de brandies comerciais, o que permitiu coñece-las diferencias existentes nas concentracións deste ácido en función do sistema de envellecemento seguido: Estático ou tradicional e dinámico ou de criadeiras e soleras.

Palabras chave: ácido gálico, HPLC, brandies.

INTRODUCTION substances extracted from the wood of the barrel by ethanol, lignin in particular. Lignin is of great importance For many centuries oak wood has been used in the mainly because its degradation products (aromatic storage of alcoholic beverages such as wine, whisky, rum, aldehydes) have a substantial influence on the organoleptic brandy, etc. and has come to have a decisive influence on characteristics of aged brandies (Guymon and Crowell, such beverages. 1968; Bricout, 1971; Marche and Joseph 1975; Puech and Jouret, 1982; Caumeil, 1983). The mechanisms, by which For brandy, the oak containers are of such these benzoic and cinnamic aldehydes are formed and importance in the aging process that Spanish legislation diffused into the hydroalcoholic mixture, are not well stipulates “the brandy compound obtained from wine known. They may be of a biochemical type (Chen and distillates, spirits or wine holands must be stored in suitable Chang, 1985), a physical type (hydrothermolysis when the atmospheric conditions and in oak containers for a staves are bound to give the barrel its shape or direct sufficient period of time for the organoleptic features pyrolysis during the burning process) and a chemical type peculiar to each production system to be acquired” (hydroalcoholysis and acidolysis). During the aging period, (DECRETO 2484/1974). the chemical mechanisms are most predominant, as a very slight acidolysis of the lignin, favoured by the conditions Furthermore, the European Community regulations in the cellar, may occur. Other authors (Baldwind et al., (REGLAMENTO 1576/89) specify the minimum storage time 1967; Nishimura et al., 1983) suggest hydrolytic for brandy in an oak barrel to be one year, or six months if degradation of the lignin, which is formed from phenyl- the capacity of the barrel is less than a thousand litres. propan derivatives by action of the alcohol. This hydrolysis is followed by the formation of benzoic and cinnamic During the storage period, a series of reactions and aldehydes and acids through oxidisation reactions. transfers occur between the wood and the beverage with which it is in contact, leading to the aged state associated For Puech (1984), the process, which occurs during with an objective notion of quality. However, the higher or aging, is hydroalcoholysis at room temperature, as it is lower content of extractable components in brandy is impossible to speak strictly of hydrolysis, alcoholysis or conditioned by numerous factors, amongst which the ethanolysis. Moreover, this process is favoured by an variety of oak used in the assembling of the barrel is notable increase in the acidity in the atmosphere, due to the as the proportion of tannins, lignin and other constituents formation of acetic acid from xylan degradation (Puech, in wood vary from one variety to another and, even within 1987-a; Puech, 1987-b). one variety depending on which part of the forest it comes from (Puech, 1984; Wiesental, 1987; Puech, 1987-a; Puech, Likewise, Puech and Jouret (1982), after studying 1987-b); treatment of the wood during the barrel assembling the evolution of aromatic aldehydes in whiskies and process (securing and drying of staves, binding of the brandies, showed the principle aldehyde to be staves by direct heat and burning of the cask), which syringaldehyde, representing 50% of the total aromatic permits a greater or lesser degradation of lignin and the aldehydes, followed by vanillin, 24%, and coniferaldehyde formation of aromatic aldehydes which give these and sinapaldehyde with approximately 12% each. Thus, beverages certain organoleptic properties (Joseph and the relative proportions between syringaldehyde and Marche, 1972; Pontallier et al., 1982; Nomdedeu et al., 1988; vanillin should be maintained within narrow margins, which Chatonnet et al., 1989; Artajona ,1991; Nivas and Glories, range between 1.4 and 2.6. Other authors (Delgado and 1993; Ruiz Hernández, 1994); and the aging characteristics, Gómez-Cordovés, 1987) found 1 to be the lower limit but such as the conditions in the cellar, the degree of alcohol did not mention an upper limit. in the sample, the length of the aging process, etc. (Casares and Olmedo, 1949; Reazin, 1981; Puech et al., 1984; Joumier, This, together with the fact that vanillin is one of 1988). the aldehydes which contributes most to the aroma of these beverages and is also easy to obtain at an industrial This latter point (length of aging process) is of great level, may encourage producers to add it to brandies, importance as, during this period the initial chemical simulating an aged character. Thus, the time spent in the characteristics of the brandy start to change from the very barrels can be reduced and consequently costs lowered. first moment. According to Nilov and Skourikhin (1957), in In this case the Sy/Va ratio will be less than unity. cognacs submitted to an aging process, a vanilla bouquet, a sweet, pleasant taste and a pale yellow colour can be Such an increase in the vanillin concentration may detected after 5 months of maturation and exposure to air. also result from maceration of the brandy with termical treated almond shells. To detect this process, Delgado and According to Jouret and Puech (1975), brandies Gómez-Cordovés (1987) propose the simultaneous aged for a certain length of time can be distinguished initially consideration of Gal. Ac/Vanillin and Sy/Va ratios. In the from those that have not, by their aroma. This flavour may case of brandies aged in oak wood barrels, the Gal.Ac/Va be labelled “terciary aroma” or “bouquet” as opposed to ratio should be greater than 0.5, whilst the ratio for those “primary aroma” which is due to the grape, or “secondary” macerated in almond shells is close to zero, as this variety which is the result of microorganism metabolism during of wood is almost completely lacking in gallic acid. fermentation. A large part of the tertiary aroma is due to 14 ALTAGA ©2000 Giménez et al.: Determination of gallic acid in commercial brandies ...

The objective of this study was firstly to develop s ×t an HPLC technique, which allowed rapid, simple R.E. = m ×100 identification and quantification of gallic acid in brandies Mean peak area in order to determine the Gal.Ac/Va and Sy/Va ratios in commercial brandies and thus, judge their quality. The results from this study were expressed by the Mean Area, which was 138943; the Standard Deviation

(sn-1) which was 765.9133 and the Relative Error (R.E.), MATERIALS AND METHODS whose value was 0.3943.

A chromatographic technique enabling separation, Calibration Curves identification and quantification of gallic acid was To determine the intervals in which the analytical developed. The following chromatographic equipment was method presented linear proportionality between the gallic used: acid concentration and the peak area found, hydroalcoholic solutions at 40% V/V of increasing concentrations of this - Liquid chromatograph KONIK-Instrument, model KNK- compound were prepared. 500-A, equipped with 4 pumps allowing the use of an elution gradient, and an injector model Rheodyne In accordance with studies carried out by Lindsay 7125. The chromatograph included an oven, which (1987) and Quesada (1993) a Response Factor (R.F) for allowed the column to be heated to 25º C. gallic acid may be obtained at the intervals in the curve - KONIK ultraviolet-visible detector, model Uvis 200 which where linearity existed. allowed absorbance at 280 nm to be measured. - Hewlett-Packard Integrator, model 3394A. Figure 1 represents the calibration curve for gallic - Column SPHERISORB CH-18 reverse phase, 20 cm long acid, presenting linearity between 0.5 mg/L and 35 mg/L. with an internal diameter of 4.6 mm and particle size The mean response factor, at the interval where gallic acid 5 mm. presented linearity, was 7.1680 x 10-5 (Table 1).

The separation required the use of an elution Accuracy of Technique gradient. Thus, the mobile phase used involved two The accuracy of the HPLC analytical technique was solvents: confirmed by applying the statistical calculation proposed by Martin and Luna (1990), to a hydroalcoholic solutions Solvent A: (bidistilled water-trifluroacetic acid: standard at 40 % (V/V), which contained a 10 mg/L 1000:1.5 V/V) concentration of gallic acid. The accuracy of this method Solvent B: (bidistilled water-methanol-trifluroacetic was studied from 10 injections under identical conditions acid: 700:300:1.5 V/V) and expressed as a percentage of recovery. These Flow: 1 ml/min.

Separation was conducted in accordance with the elution program proposed by Giménez et al. (1993). Table 1.- Determination of the average responses factor from the solutions standard of increasing concentrations. The gallic acid concentration of the initial standard solution was 5 g/l in 40º G.L. ethanol. From this, different dilutions at increasing concentrations (of gallic acid) were Concentration Peak Area Response Factor prepared, ranging from 0.25 mg/L to 80 mg/L. They were (mg/L) x 104 x 10-5 degassed in an ultrasonic bath, filtered through Millipore filters (0.45 um pore diameter) and directly injected into the 0.5 0.6977 7.1660 chromatograph. 1 1.3991 7.1475 The statistical study was carried out using 2 2.7859 7.1790 Statgraphic Plus Statistical Software version 2.0, STSC Inc. 5 6.9809 7.1624 10 13.9520 7.1674 Reproducibility of Chromatographic Method A reproducibility study was carried out by direct 15 21.0320 7.1320 injection of 10 samples of a hydroalcoholic mixture at 40% 20 27.7492 7.2074 (V/V), with a 10 mg/L concentration of gallic acid. The accuracy was expressed by the Relative Error (R.E.), 25 34.8080 7.1823 calculated from Student’s factor t , whose value for n=10 0.05 35 48.8282 7.1680 was 2.262.

1200000

1000000

800000

600000

400000

Response at 280 nm, area 200000

0 0 10 20 30 40 50 60 70 80 90 Gallic acid conc. (mg/L)

Figure 1. Calibration curve of gallic acid. concentrations were calculated from the peak area, which Table 2.- Confirmation of accuracy of the analytical technique by appeared in the chromatogram, and from the mean HPLC in the determination of gallic acid. response factor using the following equation:

Concentration (mg/L) = Peak Area x absolute R.F. GALLIC ACID Real Concentration (mg/L) 10 The results obtained appear in Table 2. They confirm that the proposed method for the determination Mean Concentration Found (mg/L) 9.9594 of gallic acid using HPLC presented great accuracy with a Recovery (%) 99.59 recovery percentage of 99.59%. Other representative parameters used to determine the accuracy of the method Standard Deviation (Sd) 0.0548 appears in Table 2. Coefficient Variant (C.V.%) 0.5511

Standard Error (Sm) 0.01375 RESULTS AND DISCUSSION Relative Error (R.E.) 0.3942

Application of HPLC Technique to the Determination of Xm ± Sm x t 9.9594 ± 0.03926 Gallic Acid in Commercial Brandies This technique was applied to the determination of gallic acid in commercial brandies. Figure 2 corresponds The gallic acid concentrations found in these to the chromatogram of the commercial brandy after direct samples analysed (appear in Figure 3) varied from 0 mg/L injection of the sample. This chromatogram shows to 17.43 mg/L. In some samples, the concentration of this separation of gallic acid and its retention time, and others acid was very low. This may be due to two factors: either phenolic compounds such as vanillin and syringaldehyde, the brandy had been in the barrel for a very short period which are usually in brandies and others alcoholic of time or the barrels used were too old and overused and, beverages. consequently the wood may have been exhausted.

38 samples of commercial brandies, produced both A difference in the gallic acid content between in Spain and other European Union countries with a strong brandies aged along the traditional system and the soleras vitivinicultural tradition such as France, were acquired at system was appreciated. random. The samples were classified according to the aging process followed: Thus, the concentration of this acid in traditional brandies ranged from 0 to 12.62 mg/L. In 10% of the - Brandies aged along the traditional or static system. samples, the concentration was found to be below the detection limits of the technique, and in a further 15% it - Brandies aged along the dynamic or criaderas and soleras was not detected. In the remaining samples of traditional system. brandies (75% of the samples) the gallic acid concentration was above 1.5 mg/L.

For brandies aged along the soleras system, the gallic acid content ranged between 0 and 17.43 mg/L. In 11.11% of this group, gallic acid was not detected, while 27.78% were below the detection limits of the technique. The concentration of the remaining samples was above 1 mg/L. Nevertheless, the soleras aged brandies contained a lower average concentration of gallic acid than that found in the traditionally aged brandies.

1- Gallic acid 2- Vanillin The difference in the gallic acid concentrations 3- Syringaldehyde between traditional and soleras brandies may be due to many factors, amongst which the variety of oak used in the construction of the barrel (French oak in the traditional method and American oak in the soleras system) is of note; the age and life of the barrel used in the aging process (in the soleras method, barrels which previously contained wine or which were used to age sherry are employed so a lower gallic acid content is perfectly normal). This difference could also be influenced by the length of time the brandy is kept in the barrel, as this conditions the greater or lesser extraction of phenolic compounds from the wood.

To discover whether differences existed in the gallic acid content according to the aging system used, a statistical study using variance analysis was carried out. This involved the use of statgraphycs. Firstly, we determined:

Figure 2. Chromatogram of a commercial brandy.

20 Soleras Traditionally 15

5 Gallic acid conc., (mg/L) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Brandy samples

Table 3.- Kolmogorov-Smirnov test for the distribution normality Table 4.- Bartlett test for variance homogeneity study of the gallic study of the gallic acid in the sample population. acid concentration in the samples studied.

Kolmogorov Test Variance Homogeneity Test Estimated Kolmogorov statistic DPLUS 0.203604 COCHRAN`S test: P = 0.85359 Estimated Kolmogorov statistic 0.210410 0.529421 DMINUS BARTLETT`S test: P(0.0606813) = 0.805422 Estimated overall statistic DN 0.210410 B = 1.00173 Approximate Significance level 0.069142 HARTLEY`S test 1.12504

Table 5.- One-way variance analysis (ANOVA I) of the gallic acid concentration according to the aging system followed.

Source of Variation Sum of Degree of Mean of Scuares F-ratio Significance Scuares Freedom Level Between Groups 33.53526 1 33.535262 1.920 0.1744 Within Groups 628.75826 36 17.465507 Total 662.29353 37

Table 6.-Multiple range test for the homogeneity study of the gallic acid concentration in brandies according to aging system followed.

Method Level Count Average Homogeneous Groups 95 18 2.4155556 X 20 4.2970000 X Contrast Difference +/- Limits t - s 1.88144 2.75436

- Whether the data obtained from the gallic acid randomness condition was fulfilled, we proceeded with concentrations followed a normal distribution. The the variance analysis. A variance analysis parametric test KOLMOGOROV-SMIRNOV test was performed. was required, such as ANOVA 1 or a one factor or one As reflected in Table 3, the significance level way variance analysis, where the response variable was obtained from this test was 0.0691415, which was the gallic acid concentration and the aging system was

higher than a (a = 0.05), and led us to accept Ho, the factor. Table 5 shows the results. As can be that is to say these samples follow a normal appreciated, the significance level was 0.1744, which distribution. means that for a confidence level of 95% (a = 0.05), the

null hypothesis (Ho) was accepted and thus, no significant - The variance homogeneity required confirmation and statistical differences existed in the gallic acid content this was done using the planting of the BARTLETT with respect to the aging method followed. This was test. In this case, P = 0.805422, which indicated confirmed by the multiple range analysis (Table 6). that variance homogeneity existed. This was confirmed by applying other tests such as the Figure 4 shows the mean gallic acid concentrations COCHRANE or HARTLEY tests. The results appear according to the aging system followed (soleras and in Table 4. traditional). As you can see it exists an overlaying between the means of both groups; this fact allows us to graphically When a normal distribution was confirmed, confirm that there are not significant differences among homogeneity between the variances existed and the brandies according to their ageing system.

Figure 4. Graphical representation of variance analysis (mean values) in the gallic acid concentration study according to aging system followed.

CONCLUSION due to the particular characteristics of each aging system (variety of wood, how the barrel is assembled, etc.). High performance liquid chromatography with However, when a statistical study was carried out using direct injection of the samples, is a rapid and effective one way variance analysis (ANOVA I), no significant technique for the separation, identification and statistical differences in the gallic acid content existed quantification of gallic acid in commercial brandies. between commercial brandies with respect to the aging Furthermore, this technique allows the simultaneous system followed. separation and quantification of benzoic aldehydes, vanillin and syringaldehyde in this type of beverage. ACKNOWLEDGEMENTS The gallic acid concentration in brandies aged along the traditional system was somewhat higher than in The authors would like to thank A.J. Macmichael brandies matured along the soleras method. This may be for translating this manuscript.

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