Analytica Chimica Acta 556 (2006) 462468

Quantitative analysis of malic and citric acids in fruit juices using

proton nuclear magnetic resonance spectroscopy
Gloria del Campo , Inaki Berregi, Raul Caracena, J. Ignacio Santos
Department of Applied Chemistry, Faculty of Chemistry, University of the Basque Country, Manuel Lardizabal 3,
P.O. Box 1072, E-20018 San Sebastian, Spain
Received 5 August 2005; received in revised form 14 September 2005; accepted 15 September 2005
Available online 20 October 2005
This article is dedicated to Prof. Cecelia Sarasola.

Abstract
1
H NMR spectroscopy was applied to the quantitative determination of malic and citric acids in apple, apricot, pear, kiwi, orange, strawberry
and pineapple juices. Aspartic acid was studied as a potential interference. The effect of the sample pH on the chemical shifts of signals from malic,
citric and aspartic acids was examined and a value of 1.0 was selected to carry out the determination. Integration of NMR signals at 2.892.95
and 3.003.04 ppm were used for calculating the concentration of malic and citric acids, respectively. At this pH the integrated signals were
not overlapped. Sodium 3-(trimethylsilyl)tetradeuteropropionate (TSP) was used as an internal reference. The obtained results applying NMR
procedures to analyze the juices from different fruits were compared to those obtained using enzymatic methods and both were in close agreement.
The intra- and inter-day repeatability was tested for apple juice (7.86 g l1 malic acid, 0.32 g l1 citric acid) and apricot juice (5.06 g l1 malic
acid, 4.79 g l1 citric acid) obtaining coefficients of variation lower than 3.4% for intra-day measures (n = 10) and lower than 3.8% for inter-day
measures (n = 20).
2005 Elsevier B.V. All rights reserved.

Keywords: Quantitative NMR spectroscopy; 1 H NMR; Malic acid; Citric acid; Fruit juices

1. Introduction
High-resolution NMR spectroscopy is commonly recognized
as a reliable technique for quantification of natural or synthetic
samples [1,2] and is widely used in pharmaceutical analysis.
Recent reviews [3,4] show the increasing use that International Pharmacopoeias make of qualitative and quantitative
NMR spectroscopy. At present, the availability of high-field
instruments in conjunction with improvements in probe design,
electronic performance and efficient signal treatments have
considerably increased the sensitivity, precision and resolution
of this technique.
1 H NMR has also shown to be a valuable technique for the
identification of major compounds (sugars and organic acids)
as well as minor compounds (amino acids and phenolic compounds) in fruit juices [58]. Besides qualitative information,

Corresponding author. Tel.: +34 43 018213; fax: +34 43 015270.

E-mail address: qppcamag@sc.ehu.es (G. del Campo).

0003-2670/$ see front matter 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2005.09.039

NMR can provide quantitative information about the sample

since the intensity (or the area) of a signal is directly proportional
to the number of nuclei producing the signal. The precision of
the integrals determines the accuracy of quantification, which
depends on: (a) the noise level of the spectrum, (b) the line
shape, (c) quality of shimming, (d) choice of the window function and (e) phase-, baseline- and drift corrections [9]. However,
as the complexity of samples increases, the resonance overlaps
form a serious problem that easily degrades the accuracy of the
analysis. Sometimes, by changing the kind of solvent, the pH
value or by adding shift reagents, a separation of signals can be
achieved.
l-Malic (MA) and citric acids (CA) are the major organic
acids of fruits [10]. MA is predominant in apple, pear and stone
fruits, while CA is most abundant in berries, citrus and tropical
fruits. Different techniques can be used for the determination
of acids in fruit juices, among them, HPLC is more extensively used, permitting the quantification of different acids in
1020 min [11]. Enzymatic methods to determine MA, CA and
other organic acids are also used [12], their main advantages

G. del Campo et al. / Analytica Chimica Acta 556 (2006) 462468

are having a high selectivity and sensitivity. However, each acid

must be determined separately, hence increasing the analysis
time. Although MA and CA show well-defined signals in 1 H
NMR spectrum, they are partially overlapped and no procedure
has been reported for their quantification.
In this work, a new 1 H NMR method was evaluated for the
simultaneous determination of malic and citric acids in fruit
juices. The proposed method was successfully applied in apple,
apricot, pear, kiwi, orange, strawberry and pineapple juices.

463

Citric acid was determined using the enzyme citrate-lyase to

produce oxaloacetate and acetate. In the presence of l-malate
dehydrogenase and l-lactate dehydrogenase enzymes, oxaloacetate and its decarboxylation product (pyruvate) are respectively
reduced to l-malate and l-lactate, by means of NADH. The
amount of NAD+ formed in these reactions is stoichiometric to
the amount of citrate and is measured by means of its absorbance
at 340 nm [14].
2.5. 1 H NMR apparatus and parameters

2. Experimental
2.1. Reagents
All reagents were analytical grade unless stated otherwise.
dl-Malic and dl-aspartic acids (minimum 99%) were supplied by AldrichSigma, citric acid (minimum 99%), sodium 3(trimethylsilyl)tetradeuteropropionate (TSP) (deuterium degree
minimum 98% for the calibration of NMR spectra), HCl and
NaOH by Merck. Deuterated oxide (>99%) by Aldrich and lmalic acid and citric enzymatic UV-test kits for food analysis
were from Boehringer Mannheim.
2.2. Standards preparation
Standards of malic, citric and aspartic acids were prepared
at pH 1.00 by dissolving 3.5000 g of the product in about 30 ml
water. After adjusting the pH by addition of diluted HCl (2.0 and
0.1 M) the solution was made up to 50 ml with water at equal pH.
Subsequent standards of all used concentrations were prepared
by diluting the main standards with water at pH 1.00 adjusted
with HCl.
2.3. Samples
Apple, apricot, pear, kiwi, orange, strawberry and pineapple
fruits were purchased from local markets. The juice of each fruit
was obtained using an electric liquidizer and it was clarified by
centrifugation (12,000 g, 20 min). The pH of 30.0 ml of fruit
juice was adjusted at 1.00 with 0.1 M HCl and the solution was
made up to 50 ml with water at equal pH. Prior to measuring, the
samples were microfiltered through a 0.45 m pore size filter.
2.4. Enzymatic analysis
The procedure for l-malic determination utilized the enzyme
l-malic dehydrogenase to catalyze the reaction between lmalate and nicotinamide adenine dinucleotide (NAD+ ) forming
oxaloacetate and the reduced form of dinucleotide (NADH). The
equilibrium of this reaction lies on the side of l-malate and, in
order to displace the equilibrium in favour of oxaloacetate, it
is removed by conversion to l-aspartate in the presence of lglutamate and the enzyme glutamate-oxaloacetate transaminase.
The amount of NADH formed is stoichiometric to the amount of
l-malate and is measured by means of its absorbance at 340 nm
[13].

Six hundred microlitres of the sample (standard or fruit

juice) and 100 L of a solution containing 70% D2 O and
10.5 g l1 sodium 3-(trimethylsilyl)tetradeuteropropionate were
placed into an NMR tube of 5 mm outer diameter; the final
concentrations were 10% D2 O and 1.50 g l1 of TSP. Onedimensional spectra were recorded on a Bruker Avance-500
spectrometer (Karlsruhe, Germany). To measure the longitudinal relaxation time (T1 ) of malic and citric acids, the longitudinal
relaxation delays of the selected protons were determined by
the inversion recovery pulse sequence method, using T1 cal
Bruker program which fitted the data to the exponential equation I = I0 + P exp(/T1 ), where I is the intensity of each acid
resonance at inversion delay and I0 at the equilibrium state
and P a constant. Inversion delays used were 0.25, 0.50, 1.00,
2.00, 4.00, 8.00, 15.00, 30.00 and 60.00 s. To obtain the spectra of the samples, 128 scans of 32 K data points were acquired
at 300 K using a spectral width of 8012 Hz (16 ppm), acquisition time of 4.09 s, recycle delay of 3.40 s, flip angle of 90
and a constant gain of 28.5. Solvent suppression was achieved
using the Watergate pulse sequence [15]. FIDs were processed
before Fourier transformation by means of an exponential filter of function f(t) = exp(Rt) using XWIN-NMR (version 3.1,
Bruker GmbH, Germany). The broad-line is of R/Hz in the corresponding transformed spectrum. A positive R value increases
the sensitivity (i.e. signal/noise) but it increases the line width
(i.e. reduces the resolution), and R value of 1.0 Hz was selected
as a compromise between both factors. To attain reliable results
the phasing and the baseline correction over the entire spectral
range are critical, so these processes were carried out manually.
In all instances, the baseline was additionally corrected over
the integrated regions. The spectra were referenced to the TSP
singlet peak at 0.0 ppm. Areas of the peak were measured by
electronic integration of expanded regions around selected resonances. Total measurement time, from sample preparation in
the NMR tube through obtaining and integrating the spectrum,
was about 20 min.
3. Results and discussion
3.1. Optimization of NMR acquisition conditions and the
sample pH
To obtain a high accuracy in quantitative analysis the elapsed
time between the successive acquisitions of the spectra must be
three times T1 for a maximal error of 5% and five times T1 for
a maximal error of 1% [16]. T1 values were measured using the

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G. del Campo et al. / Analytica Chimica Acta 556 (2006) 462468

inversion recovery pulse sequence for frequencies offset centred

on the signals of MA and CA. The T1 values for signal of MA
at 2.90 ppm was 1.502 0.005 s and for CA at 3.01 ppm was
1.310 0.017 s, therefore, by using a delay time of 3.40 s and
an acquisition time of 4.09 s, the total relaxation of the protons
involved in the quantitative measure was achieved.
It is well known that the shifts of some resonances observed in
the 1 H NMR spectra depend on the pH of the sample. Molecules
whose ionization state changes with changing pH show different
shifts at different pHs. It is evidenced in the literature comparing the shifts for MA and CA reported by a number of authors.
For protons -CH2 (dddd) of the MA, the reported shift values were 2.372.66 ppm in tomato juice [8], 2.682.85 ppm in
orange juice [17] and 2.772.89 ppm in Boscop apple juice [18].
For protons CH2 (dd) of the CA the reported shift values were
2.532.66 ppm in tomato juice [8], 2.762.82 ppm in orange
juice [17] and 2.832.88 ppm in Boscop apple juice [18]. Moreover, aspartic acid is also present in the fruits and, although its
concentration is very low compared to those of MA and CA,
the potential interference from aspartic acid was considered
in this study. For protons -CH2 (dddd) of the aspartic acid
the reported shift values were 2.682.80 ppm in tomato juice
[8], 2.852.95 ppm in orange juice [17] and 2.922.97 ppm in
Boscop apple juice [18]. Regarding these data one can conclude
that the resonances corresponding to the three acids are partially
overlapped. Protons -CH of the MA show resonance signals
(dd) at about 4.8 ppm, but the Watergate pulse sequence used to
suppress the water signal causes distortion in this region, therefore this signal was discarded to determine MA.
To know the influence of pH on the shifts of MA, CA and
aspartic acid and to optimize the pH for measuring, seven spectra
of each acid at pH values of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 were
obtained. As can be seen in Fig. 1, for the three acids the shifts
decreased with increasing pH and these changes were higher
at pH from 1.0 to 2.5. Moreover, increasing pH increased the
separation among the double doublets from MA and decreased
the separation among the doublets from CA. As a consequence,
at pH 1.00 the overlapping between the signals from both acids
was only produced at shift values higher than 2.90 ppm, permitting the determinations of MA and CA over the intervals
2.892.95 and 3.003.04 ppm, respectively. At pH 1.00 the interference from aspartic acid was also minimized because its triplet
is located at shifts higher than 3.10 ppm.
3.2. Quantitative analysis
Many factors which influence absolute intensities, such as
variable sample volumes, spectrometer performance and B1
inhomogeneity over the sample, will be compensated for by the
use of relative quantification. It is usually for this reason that
quantification in NMR spectroscopy is performed with the aid
of an internal standard. In this work we used TSP as an internal
standard and the areas from MA and CA signals were normalized
with respect to that of TSP for each spectrum (A/ATSP ). TSP was
chosen as a suitable internal standard because it is freely soluble in water and stable in the sample media, does not show any
interaction with sample compounds, is available in high purity

and has a very simple NMR spectrum consisting of a single nine

proton singlet. Moreover, it also was used as standard reference
to fix the chemical shift at 0.0 ppm in the NMR spectra and its
use as internal standard avoids the need for addition of a second
standard to the sample [19].
3.2.1. Linearity and limit of detection
To determine the linear range for malic and citric acids, a
series of 11 synthetic samples containing both acids over the
range 0.1012.00 g l1 was prepared. The calibration data are
summarized in Table 1. Correlation coefficients (R) were >0.999
for both analytes, which indicates a good linearity response
within the concentration range studied.
The limit of detection (LOD) is not applicable to major component methods. However, the range of MA and CA content
varies in the different fruits and the estimation of this limit permits the quantitative NMR method to be applied in those fruit
juices where one of the acids becomes a minor component. There
are various definitions of LOD [2022] and we calculated it from
3Sy/x + intercept [22]. The slope for MA is higher than that for
CA and, consequently, the LOD calculated for MA is slightly
lower than that for CA. The main cause of this difference is the
lower molecular weight of MA with respect to that of CA.
3.2.2. Accuracy and precision
The intra- and inter-day repeatability were performed by
analyzing two juices of different fruits (apple and apricot) containing their natural and different levels of MA and CA. For
the intra-day assay, 10 aliquots of each fruit juice recently
obtained were taken, treated according to the general procedure, and their spectra was obtained and processed for each
sample. After measurements, the 10 NMR tubes containing the
samples were kept in a refrigerator at about 5 C. For inter-day
assay, the same determinations were carried out after three days.
For intra-day measures (n = 10) the acid contents determined
were 7.86 0.21 g l1 of MA and 0.32 0.01 g l1 of CA for
apple juice and 5.06 0.11 g l1 of MA and 4.79 0.05 g l1
of CA for apricot juice. Coefficients of variation were 2.7%
(apple juice) and 2.2% (apricot juice) for MA and 3.1% (apple
juice) and 1.0% (apricot juice) for CA. When the measures
were repeated after three days, a decrease in the contents of
both acids was observed, probably due to instability of the
samples even at pH 1.00. The acid contents determined were
7.54 0.19 g l1 of MA and 0.30 0.01 g l1 of CA for apple
juice and 4.82 0.16 g l1 of MA and 4.56 0.05 g l1 of CA
for apricot juice. Coefficients of variation were 2.6% (apple
juice) and 3.3% (apricot juice) for MA and 3.3% (apple juice)
and 1.2% (apricot juice) for CA. To calculate the inter-day
repeatability all measures were considered (n = 20), coefficients
of variation so obtained were 3.4% (apple juice) and 3.7% (apricot juice) for MA and 3.2% (apple juice) and 2.8% (apricot juice)
for CA.
The above precision study was performed by a single analyst and, because the manual processing of the spectra is an area
of potential experimental error, the effect of the analyst factor
on the A/ATSP measurement was evaluated. Seven FIDs were
selected, each one corresponding to a different fruit juice spec-

G. del Campo et al. / Analytica Chimica Acta 556 (2006) 462468

Fig. 1. Structures and 1 H NMR spectra of malic, citric and aspartic acids at different pH values: (a) 1.0; (b) 1.5; (c) 2.0; (d) 2.5; (e) 3.0; (f) 3.5; and (g) 4.0.

465

G. del Campo et al. / Analytica Chimica Acta 556 (2006) 462468

466
Table 1
Calibration data
Analyte

Signal

a Sa a

b Sb b

R2 (n = 11)

Sy/x c

LODd (mg l1 )

Malic acid
Citric acid

A/ATSP
A/ATSP

0.1234 0.0003
0.0927 0.0001

0.0004 0.0004
0.0005 0.0006

1.0000
0.9998

0.0016
0.0014

39
46

a
b
c
d

Slope standard error for slope.

Intercept standard error for intercept.
Standard error for regression line.
Limit of detection (3 Sy/x + b).

Table 2
Results obtained in the determination of malic and citric acids in 11 fruit juices measured in triplicate, by the 1 H NMR and the enzymatic methods (EM)
Sample

Fruit

Malic acid (g l1 )
1H

1
2
3
4
5
6
7
8
9
10
11

Apple 1
Apple 2
Apple 3
Apricot
Pear 1
Pear 2
Kiwi
Orange
Strawberry
Pineapple 1
Pineapple 2

NMR

10.12
7.64
3.42
4.59
2.49
2.61
2.66
2.13
1.74
1.43
1.23

0.23
0.05
0.02
0.04
0.09
0.03
0.13
0.01
0.10
0.09
0.05

trum, and each FID was processed three times by three different
analysts. Analysis of variance and Scheffe test for means comparison were used to determine the statistical significance for
processing spectra (significance level: 0.05). The results showed
significant differences only for an analyst in the case of malic
acid in orange juice and citric acid in pineapple juice. Although
in these cases the analyst factor was statistically significant, its
effect on the contents of malic and citric acids was small, these
contents being about a 7% lower than those obtained by the other
two analysts.
3.2.3. Application
To validate the NMR procedures, 11 samples corresponding to juices of seven diverse fruits were analysed in triplicate. The fruits were chosen for their different contents in MA
and CA: apples have a high content in MA, but low CA content, apricots and pears have a medium content in both acids
and kiwis, oranges, strawberries and pineapples have higher
CA content than MA. Fig. 2 shows a representative spectrum of each fruit juice. The peaks used for integration were
2.892.95 and 3.003.04 ppm for MA and CA, respectively.
These spectra display that at adjusted pH for sample measurements, the selected peaks are well resolved and no overlapping
occurs.
Fig. 2 also shows the presence of peaks right between the
doublet peaks from CA, at chemical shifts higher than those
used for quantification of CA. These resonance signals were
tentatively assigned to asparagine amino acid by comparison
with chemical shifts reported in the literature [8,17,18]. To

Citric acid (g l1 )
EM

1H

9.92 0.33
7.85 0.03
3.47 0.07
4.51 0.17
2.62 0.06
2.72 0.10
2.54 0.02
1.94 0.03
1.65 0.08
1.49 0.09
1.17 0.05

0.36
0.60
0.09
4.13
1.64
0.45
11.00
11.71
7.13
6.52
5.49

NMR

EM
0.02
0.03
0.01
0.05
0.07
0.03
0.14
0.17
0.34
0.18
0.15

0.33
0.62
0.08
4.19
1.78
0.44
11.22
11.25
7.33
6.77
5.46

0.01
0.02
0.01
0.00
0.01
0.03
0.25
0.12
0.18
0.10
0.07

confirm this assignment, the spectrum of an asparagine solution

at pH 1.00 was measured, showing that the chemical shifts of
the resonance signals appeared at values higher than 3.04 ppm,
coinciding with the peaks observed in the spectra from fruit
juices.
In Table 2, the results obtained by applying the proposed
NMR method with those obtained by enzymatic methods (EM)
are compared. As can be seen there is a good agreement between
the contents measured by both methods, the differences ranging
from 1.4% (apple 3) to 9.8% (orange) for malic acid and from
0.6% (pineapple 2) to 12.5% (apple 3). The results of a paired
samples comparison t-test are shown in Table 3. The t calculated
did not exceed the theoretical value, which indicates the absence
of any significant difference between NMR and enzymatic methods. In addition, the regression lines were obtained from Table 2
by plotting the concentration data obtained from NMR method
versus EM for malic and citric acids (Table 3). Because the
confidence interval (p < 0.05) for the intercept includes zero
and the confidence interval for the slope includes 1, neither
Table 3
Results of the statistical treatment performed on the pairs of data shown in
Table 2
Analyte

Malic acid
Citric acid

t-test

0.562
0.791

Linear regression (NMR vs. EM)

Slope

Intercept

R2

1.000 0.010
1.004 0.011

0.017 0.047
0.050 0.065

0.997
0.996

t tabulated at the 95% confidence level and 32 degrees of freedom: 2.037.

G. del Campo et al. / Analytica Chimica Acta 556 (2006) 462468

467

background nor systematic errors were detected and the high

values of the correlation coefficients (R) indicate low random
errors.
4. Conclusion
This study demonstrated that using an appropriate choice of
experimental conditions, the 1 H NMR spectroscopy is a suitable
method for the quantitative determination of malic and citric
acids in different fruit juices. This method offers advantages
in terms of rapidity and simplicity of sample preparation. A
further gain is the possibility of the simultaneous determination of other components in the same sample, in particular
polyphenolic compounds as described earlier for apple juice
[23,24].
Acknowledgements
We are grateful to the Gipuzkoako Foru Aldundia for
its financial support and the NMR Service of the Faculty
of Chemistry of Donostia, UPV/EHU, for its professional
work.
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Fig. 2. The 1 H NMR spectrum (2.703.10 ppm) of the different fruit juices: (a)
apple; (b) apricot; (c) pear; (d) kiwi; (e) orange; (f) strawberry; and (g) pineapple.

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