Ciencia y Tecnología Alimentaria

Sociedad Mexicana de Nutrición y Tecnología de Alimentos

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ISSN (Versión impresa): 1135-8122
ISSN (Versión en línea): 1696-2443
MÉXICO

2002
S. C. Cunha / J. O. Fernandes / M.A. Faria / I.M.P.L.V.O. Ferreira / M. A. Ferreira
QUANTIFICATION OF ORGANIC ACIDS IN GRAPE MUSTS AND PORT WINES
Ciencia y Tecnología Alimentaria, julio, año/vol. 3, número 004
Sociedad Mexicana de Nutrición y Tecnología de Alimentos
Reynosa, México
pp. 212-216

Red de Revistas Científicas de América Latina y el Caribe, España y Portugal

Universidad Autónoma del Estado de México

http://redalyc.uaemex.mx
Cienc. Tecnol. Aliment. Vol. 3, No. 4, pp. 212-216, 2002
Copyright 2002 Asociación de Licenciados en Ciencia y Tecnología de los Alimentos de Galicia (ALTAGA). ISSN 1135-8122

QUANTIFICATION OF ORGANIC ACIDS

IN GRAPE MUSTS AND PORT WINES

CUANTIFICACIÓN DE ÁCIDOS ORGÁNICOS EN MOSTOS Y VINOS DE OPORTO

CUANTIFICACIÓN DE ÁCIDOS ORGÁNICOS EN MOSTOS E VIÑOS DE PORTO

Cunha, S. C.1; Fernandes, J. O.; Faria, M.A.; Ferreira, I.M.P.L.V.O.*; Ferreira, M. A.

1
Present adress: Instituto Jean Piaget de Mirandela, Av. 25 de Abril, 5730 Mirandela, Portugal
*
Author to whom correspondence should be addressed. E-mail: bromato@ff.up.pt

Recibido: 4 de Junio de 2001; recibida versión revisada: 25 de Septiembre de 2001; aceptado: 4 de Octubre de 2001
Received: 4 June 2001; revised version received: 25 September 2001; accepted: 4 October 2001

Abstract
A simple, rapid and accurate method for derivatization and quantitative HPLC analysis of organic acids, was optimised in
order to provide the determination of the most important acids found in Port wines and grape musts used for their elaboration. The
method was based on the use of O-(4-nitrobenzyl)-N,N’-diisopropylisourea (NBDI) as derivatizing reagent. Samples were primarily
treated 15 min with a strong cation-exchange resin (Dowex 50W-X8) to free the organic acids. HPLC determination was accomplished
by using a RP-18 (3 µm) column, a mobile phase composed of water and acetonitrile (non-linear gradient) at a flow-rate of 1 mL min-
1
, and a UV detector set at 265 nm. Sensitive and well-resolved peaks were obtained from the main organic acids (lactic, acetic,
succinic, tartaric, malic and citric acids) in less than 30 min, either with Port wines or grape musts. Benzylmalonic acid was used as
internal standard. Precision and recovery assays were performed with good results for all of 6 acids considered. The major differences
between grape musts and Port wines were the greater overall amounts of organic acids in the former and the higher content of lactic,
acetic and succinic acids in the latter. © 2002 Altaga. All rights reserved.

Key words: Port wines, organic acids, HPLC.

Resumen
Se optimizó un simple, rápido y exacto método para la derivatización y análisis por HPLC de ácidos orgánicos, con el objeto
de permitir la determinación de los principales ácidos encontrados en mostos y vinos de Oporto. El método se basa en el uso de O-(4-
nitrobenzil)-N,N’-diisopropilisourea (NBDI) como agente de derivatización. Las muestras se pasaron en primer lugar por una resina
de intercambio catiónico fuerte (Dowex 50W-X8) durante 15 min para liberar los ácidos orgánicos. La determinación por HPLC se
desarrolló usando una columna RP-18 (3 µm), una fase móvil compuesta de agua y acetonitrilo (gradiente no-lineal) con un caudal de
1 mL min-1. Se usó un detector UV ajustado a 265 nm. Analizando muestras de mostos y vinos de Oporto se obtuvieron picos resueltos
de los principales ácidos orgánicos (ácido láctico, acético, succinico, tartárico, málico y cítrico) en menos de 30 min. Se usó como
patrón interno ácido benzilmalónico. Los ensayos de precisión y recuperación se realizaron con buenos resultados para los 6 ácidos
considerados. La mayor diferencia entre los mostos y vinos de Oporto fue la mayor cantidad global de ácidos organicos en el primero
y de ácido láctico, acético y succinico en el segundo. © 2002 Altaga. Todos los derechos reservados.

Palabras clave: Vinos de Oporto, ácidos orgánicos, HPLC.

Resumo
Optimizouse un simple, rápido e exacto método para a derivatización e análise por HPLC de ácidos orgánicos, co obxecto de
permiti-la determinación dos principais ácidos atopados nos mostos e viños de Porto. O método basease no uso de O-(4-nitrobenzil)-
N,N'-diisopropilisourea (NBDI) como axente de derivatización. As mostras pasáronse nun primeiro lugar por unha resina de intercambio
catiónico forte (Dowex 50W-X8) durante 15 min para libera-los ácidos orgánicos. A determinación por HPLC desenvolveuse usando
unha columna RP-18 (3 µm), unha fase móbil composta de auga e acetonitrilo (gradiente non-lineal) cun caudal de 1 mL min-1.
Usouse un detector UV axustado a 265 nm. Analizando mostras de mostos e viños de Porto obtivéronse picos resoltos dos principais
ácidos orgánicos (ácido láctico, acético, succínico, tartárico, málico e cítrico) en menos de 30 min. Usouse como patrón interno ácido
benzilmalónico. Os ensaios de precisión e recuperación realizáronse con bos resultados para os 6 ácidos considerados. A maior
diferencia entre os mostos e viños de Porto foi a maior cantidade global de ácidos orgánicos nos primeiros e de ácido láctico, acético
e succínico nos segundos. © 2002 Altaga. Tódolos dereitos reservados.

Palabras chave: Viños de Porto, ácidos orgánicos, HPLC.

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ALTAGA ©2002 Cunha et al.: Quantification of organic acids in grape musts and port wines

INTRODUCTION and acetonitrile (B). The gradient was as follows: 30 to

45% B whitin 10 min, 45 to 55% B whitin 10 min, 55 to
Port is a fortified dessert wine traditionally 80% B whitin 10 min and returning to initial conditions
manufactured in the Douro Valley in Northern Portugal, within 2 min. The flow rate was 1 mL min-1. Detection
using a mixture of grape varieties. In spite of its great was accomplished with UV detector set at 265 nm.
economical significance for Portugal, little information
is available on the organic acid contents of grape musts Reagents and solutions
and Port wines, mainly tartaric (TA), malic (MA), citric All reagents used were (p.a) from Merck.
(CA), succinic (SA), lactic (LA) and acetic (AA) acids. Derivatizing reagent O-(4-nitrobenzyl)-N,N’-
Their origins are diverse, the most important being diisopropylisourea (NBDI) was from Sigma Chemicals
biosynthesised by the wine, metabolic pathways related Co.
to sugar fermentation and malolactic fermentation.
Conjointly, they are of critical importance for the chemical Sample Preparation and Derivatization Procedure
and microbiological stabilisation and they deeply affect A 5 mL aliquot of the samples or standard solutions
the colour and taste of the final product. were added with 100 µL of aqueous benzylmalonic acid
Attending their importance, an accurate and solution 2 gL-1 (I.S.) and 0.5 g of activated strong cation-
reproducible method, involving simple sample preparation exchange resin (Dowex 50W-X8). The mixture was
and suitable for the routine analysis, is therefore desirable. shaking for 15 min. Afterwards, a 50 µL aliquot was mixed
The official method actually adopted by the International with 500 µL of a solution of NBDI in dioxane (10 mgmL-
Office of Wine (OIV) does not fulfil these requirements: 1
). The mixture was heated at 80 ºC for 60 min. After
it makes use of two C8 columns for the separation of TA, cooling, the solution was diluted by addition of 2 mL of
MA and SA and implies the use of a different column acetonitrile and cleaned-up with another portion of the
(Aminex, HPX 87 H) for separating the LA, AA and same ion-exchange resin, to remove the excess of reagent.
CA acids. The detection is achieved at 214 nm, a Finally, the mixture was filtered through 0.22 µm filters
wavelenght at which many other compounds (sugars, e.g.) and an aliquot of 10 µL was injected into the
absorve and can interfere in the separation of the acids chromatograph.
(OIV, 1990). However, other HPLC/UV methods also
without derivatization, but with complex purification Sampling
techniques to eliminate matrix interferences, are described Seven samples of Port wines from Portuguese
for the determination of organic acids in grape juices and market and five samples of grape musts obtained directly
wines, namely by Herrera et al. (1993), Romero et al. from the producer were analysed.
(1993), Radin et al. (1994) Escobal et al. (1997) and
Castellari et al. (2000)
A simple, rapid and accurate method for RESULTS AND DISCUSSION
derivatization and quantitative HPLC analysis of organic
acids in samples of roasted coffee was proposed a few Figure 1 and Figure 2 show chromatograms of a
years ago by Badoud and Pratz (1986). The method was typical grape juice and a Port wine, respectively.
based on the use of O-(4-nitrobenzyl)-N,N’-
diisopropylisourea (NBDI) as derivatizing reagent. This Linearity
is a strong chromophoric labelling agent that readily reacts Calibration curves (6 points) were constructed by
with free carboxylic acids to form the corresponding p- analysing aqueous standard solutions of the 6 organic acids
nitrobenzyl esters in high yields and offers better studied with increasing amounts of each acid, treated in
sensitivity and selectivity. exactly the same way as the samples. The range of
Taking into account the simplicity and the excellent concentrations was 0.1-6 g.L-1 for MA and TA, 0.1-2 g.L-1
chromatographic performance obtained when applied to for LA and AA, and 0.1-2g.L-1 for SA and CA. Correlation
samples of Port wine and grape musts (characterised by coefficients higher than 0.998 were obtained for all the 6
their high sugar contents) we decided to undertake a study acids (Table 1).
in order to validate the method for determination of
organic acids in this kind of samples. The final objective Limits of detection
was to apply the optimised method to the determination The detection limits for each acid, based on a
of quantitative profiles of organic acids in grape musts signal-to-noise ratio of 3, were 0.005 g.L-1 for TA, 0.038
and Port wines. g.L-1 for MA, 0.046 g.L-1 for SA, 0.041 g.L-1 for AA, 0.056
g.L-1 for LA and 0.098 g.L-1 for CA.

MATERIAL AND METHODS Precision

The validity of the method was verified by
Apparatus and operating conditions evaluation of precision and accuracy. Precision was
The separation of the 6 organic acids PNB ester evaluated by assaying six times a Port wine sample and a
derivatives was achieved on a Waters Spherisorb 3 µm, grape must sample. Good results were obtained with
C18 (4.6 x 150 mm) with a Nucleosil guard-column C 18 (4 coefficients of variation being less than 2.9 % for all 6
x 30 mm). The mobile phase was composed of water (A) acids studied (Table 2).

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Cienc. Tecnol. Aliment. Vol. 3, No. 4, pp. 212-216, 2002 ISSN 1135-8122 ©2002 ALTAGA

AUFS 3
6

1
2

10 20 30
Time, min

Figure 1. Chromatogram of a grape must sample with: 1-lactic acid (0.201 g L-1 ), 2-acetic acid (0.070 g L-1 ), 3-tartaric acid
(3.034 g L-1 ), 4-malic acid (1.971 g L-1 ), 5-citric acid (0.500 g L-1 ) and 6-I.S (2 g L-1 ).

7
AUFS

4
3

6
2
1

10 20 30
Ti me, min

Figure 2. Chromatogram of a Port wine sample with: 1-lactic acid (0.294 g L-1), 2-acetic acid (0.231 g L-1), 3-tartaric acid (1.204 g L-1),
4-malic acid (1.082 g L-1), 5- succinic acid (0.348 g L-1), 6-citric acid (0.270 g L-1) and 7-I.S (2 g.L-1).

Table 1.- Calibration curve parameters (y = ax + b), correlation Table 2.- Evaluation of precision of six replicate analyses of grape
coefficients (r) of calibration plots and retention time for the organic musts and Port wine. Legend: n.d.= not detected.
acids under study.
Carboxylic Grape must Port wine
Carboxylic a b r RT acids
acids mean mean (min) Mean ±S.D. C.V. Mean ±S.D. C.V.
Lactic 0.287 0.005 0.9997 7.37 (g L-1) (%) (g L-1) (%)
Acetic 0.429 0.034 0.9994 12.53 Lactic 0.337±0.010 2.9 0.301±0.007 2.3
Tartaric 0.367 0.034 0.9983 14.97 Acetic 0.308±0.005 1.7 0.260±0.005 1.9
Malic 0.382 0.065 0.9982 17.30 Tartaric 2.503±0.039 1.6 0.941±0.014 1.5
Succinic 0.502 -0.002 0.9998 24.00 Malic 1.446±0.014 0.9 1.046±0.007 0.6
Citric 0.402 -0.029 0.9992 26.76
Succinic n.d. - 0.303±0.001 0.4
Citric 0.543±0.009 1.6 0.164±0.004 2.3

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ALTAGA ©2002 Cunha et al.: Quantification of organic acids in grape musts and port wines

Table 3.- Recovery of organic acids from spiked grape musts and Port wine samples.

Carboxylic Grape juice Port wine

acids
Initial Added Recovery Initial Added Recovery
amount (g L-1) (%) amount (g L-1) (%)
(g L -1) (g L -1)
Lactic 0.337 0.301
0.250 102.3 0.250 106.6
0.500 102.3 0.500 94.9
1.000 113.5 1.000 98.6

Acetic 0.308 0.260

0.250 117.3 0.250 109.9
0.500 113.2 0.500 118.9
1.000 106.1 1.000 108.5

Tartaric 2.503 0.941

1.250 112.9 1.250 108.8
2.500 104.4 2.500 106.4
3.750 100.1 3.750 105.9

Malic 1.446 1.046

1.250 101.3 1.250 101.1
2.500 98.2 2.500 102.6
3.750 104.1 3.750 105.7

Succinic n.d. 0.303

0.250 100.9 0.250 103.1
0.500 104.2 0.500 106.7
1.000 114.4 1.000 105.4

Citric 0.543 0.164

0.250 101.9 0.250 105.6
0.500 113.2 0.500 105.2
1.000 99.7 1.000 105.5

Recovery these acids are products of alcoholic fermentation.

The reliability of the method was confirmed by Significant variation between samples was observed
two recovery experiments. A Port wine and of a grape (p<0.05).
must were analysed before and after the addition of known On the other hand, tartaric acid, the most abundant
amounts of mixtures of the organic acids and analysed in organic acid of grape musts, decreases during alcoholic
the same way as the samples. Table 3 presents the results fermentation owing to the precipitation of calcium tartrate
for recovery studies. Recoveries varied between 94.9- and potassium bitartrate. Thus, significantly lower
118.9% for Port wine and between 98.2-117.3% for grape amounts of this organic acid were observed in Port wine
must. samples. Malic and citric acids were also lower in Port
wines owing to their decrease during fermentation process.
Analytical Results for grape musts and Port wines The results obtained were compared with those
The method was used for determining the organic refereed in literature for other types of wines, it is
acid contents of some grape musts and Port wines. Results interesting to note that similar profiles were obtained
obtained there of, are listed in Table 4. (Ryan and Dupont, 1973).
As apparent from Table 4, there are significant
differences between organic acid contents from grape
musts and Port wines. As expected Port wines contain CONCLUSIONS
lower amounts of organic acids when compared with grape
musts, as a result of fermentation and metabolic processes. Analysis of organic acids as their p-nitrobenzyl
However, all Port wines contain larger amounts of esters seems to be a valuable alternative to other current
succinic, lactic and acetic acids, whereas in grape musts available methods for a rapid and very accurate
the former is lacking in all samples, only traces of acetic quantification of this kind of compounds in Port wines
acid were detected in most samples and a few amount of and grape musts. The method is simple and allows for
lactic acid was present. This, results from the fact that well-resolved peaks of the main carboxylic acids in less

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Cienc. Tecnol. Aliment. Vol. 3, No. 4, pp. 212-216, 2002 ISSN 1135-8122 ©2002 ALTAGA

Table 4.- Organic acid contents in grape musts and Port wine samples. Legend: n.d.= not detected.

Carboxylic Acids (g L-1)

Samples Lactic Acetic Tartaric Malic Succinic Citric
0.414 0.282 1.310 1.352 0.751 0.288
0.219 0.320 1.178 1.442 0.238 0.260
0.286 0.221 1.195 1.084 0.978 0.312
Port
0.497 0.222 1.077 0.764 0.245 0.235
Wines
0.421 0.251 1.133 1.158 0.288 0.260
0.294 0.231 1.204 1.082 0.348 0.270
0.350 0.283 1.105 1.180 0.284 0.278

0.256 0.095 4.278 0.361 n.d. 0.581

0.201 n.d. 3.034 1.971 n.d. 0.500
Grape
0.184 0.301 4.752 2.957 n.d. 0.510
juices
0.191 n.d. 2.918 3.696 n.d. 0.512
0.137 0.209 2.176 1.428 n.d. 0.284

than 30 min, either with Port wines or grape juices, with Herrera, M. O.; García, H. L.; Mir, M. V., Martínez, M.
very good precision and enough sensitivity. Thus, it makes C. L. 1993. Determination by high performance
possible to study the evolution of this compounds during liquid chromatography of organic acids in Spanish
fermentation and metabolic processes that occur on Port rosé wines from the alpujarra-contraviesa region
wines production. of Granada. Journal of liquid chromatography &
related technologies, 16, 3101-3112.
Office International de la Vigne et du Vin. Recueil des
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the analysis of organic acids, carbohydrates, and M.D.C. 1993. Determination of organic acids in
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