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Food

Chemistry
Food Chemistry 106 (2008) 1218–1224
www.elsevier.com/locate/foodchem

Analytical, Nutritional and Clinical Methods

Determination of biogenic amines in wines by HPLC


with precolumn dansylation and fluorimetric detection
Charalampos Proestos, Paul Loukatos, Michael Komaitis *
Laboratory of Food Chemistry, Department of Food Science and Technology, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece

Received 29 March 2007; received in revised form 19 June 2007; accepted 23 June 2007

Abstract

Thirty-two samples of commercially available Greek wines were analysed in order to determine the content of biogenic amines. The
method involves pre-column dansylation of the amines and subsequent solid phase extraction (SPE) of the derivatives through C18 car-
tridges. For the analysis, RP-HPLC (reversed phase-high performance liquid chromatography) coupled with fluorimetric detection at
excitation and emission wavelengths of 320 and 523 nm, respectively was used. All amines measured had recoveries over 85%. The high-
est detection limit was for agmatine (0.18 mg l 1). Putrescine, cadaverine, histamine and isoamylamine are the most abundant amines in
the samples analysed. The relative concentrations of biogenic amines expressed in mg l 1 had as follows: putrescine > histamine > iso-
amylamine > ethylamine > methylamine > cadaverine = tyramine = agmatine = tryptamine. Higher amounts of biogenic amines were
generally detected in wines, aged for long periods in barrels or in bottles. However, young wines contained lower amounts of these com-
pounds as they were directly bottled after winemaking and have not undergone any further maturation processing. Moreover, less acid
wines gave rise to higher histamine contents.
Ó 2007 Elsevier Ltd. All rights reserved.

Keywords: Biogenic amines; Greek wines; RP-HPLC; SPE; Dansyl derivatives

1. Introduction for sensitive individuals. Symptoms include nausea, respir-


atorial discomfort, hot flushes, cold sweat, palpitations,
Biogenic amines are mainly nitrogenous low molecular headaches, red rash, high or low blood pressure (Mo Dugo,
weight compounds with biological activity that may be Vilasi, La Torre, & Pellicanò, 2006). Alcohol and acetalde-
formed or metabolised in the cells of living organisms (ani- hyde have been found to increase the sensitivity to biogenic
mals, plants and micro-organisms). Biogenic amines are amines (Busto, Guasch, & Borrull, 1995). High levels of
derived mainly from amino acids through substrate-specific biogenic amines correlate fairly well with other wine spoil-
decarboxylase enzymes (Loukou & Zotou, 2003). Amines age components for example butyric acid, lactic acid, acetic
may be formed by yeasts during the alcoholic fermentation acid, ethyl acetate and diethyl succinate, which is why
(mostly putrazine); by lactic acid bacteria (LAB) during wines with higher levels usually also have higher levels of
malolactic fermentation (MLF) and during maturation of volatile acid. Red wines also have higher levels than white
wines (Lonvaud-Funel, 2001). Biogenic amines can also wines, mainly because of vinification practices and matura-
be present in the must (Arce, Rios, & Valcarel, 1998). tion (Bauza, Blaise, Daumas, & Cabanis, 1995). An
The main biogenic amines in wine are histamine, tyramine, increase in the levels of biogenic amines usually occurs
putrescine, cadaverine and phenylethylamine. Biogenic towards the end of the MLF or during maturation, when
amines are important because they contain a health risk lactobacilli and pediococci are the biggest culprits (Soufle-
ros, Barrios, & Bertrand, 1998). Their presence in wines has
*
Corresponding author. Tel./fax: +30 210 5294186. been associated with a lack of hygiene during the winemak-
E-mail address: achem@aua.gr (M. Komaitis). ing process (Moreno-Arribas, Polo, Jorganes, & Munoz,

0308-8146/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2007.06.048
C. Proestos et al. / Food Chemistry 106 (2008) 1218–1224 1219

2003). Determination of biogenic amines is not simple In this work, nine of the most oenologically important
because of their structure and because they are usually biogenic amines are determined in red and white wines
present at low levels in a complex matrix such as wine. after pretreatment with polyvinylpyrrolidone (PVP), which
High-performance liquid chromatography (HPLC) is the removes the substances that interfere in the derivatization
technique most extensively used due to its high resolution, and subsequent quantification of the derivatives such as
sensitivity, great versatility, and simple sample treatment. polyphenols. PVP reacts with polyphenols and create a pre-
Biogenic amines do not exhibit satisfactory absorption at cipitate that can be removed by centrifuge or filtration. The
the visible or ultraviolet wavelengths, nor do they exhibit applied post-derivatization SPE procedure increases the
fluorescence. Therefore, pre- or post-column chemical selectivity and sensitivity of the method. The present
derivatization is usually applied for their analysis by method advantages include: the use of a simple gradient
HPLC, increasing the selectivity and sensitivity of detection which effectively separates all the amines in a relatively
(Vidal-Carou, Lahoz-Portoles, Bover-Cid, & Marine-Font, short time, a simple time- and cost-effective pretreatment
2003). Sensitivity is also increased by using fluorescence of the samples and a sensitive and selective detection by
instead of diode array detector. The determination of bio- using fluorescence detection.
genic amines in wines and alcoholic beverages generally,
has been carried out mostly by HPLC (Garcia-Villar, Saur- 2. Materials and methods
ina, & Hernandez-Cassou, 2006; Moret & Conte, 1996;
Romero, Gazquez, Bagur, & Sauchez-Vines, 2000) after 2.1. Standards and reagents
derivatization with reagents such as o-phthalaldehyde
(OPA) (Bover-Cid, Iquierdo-Pulido, Marine-Font, & Standard solution including: cadaverine (CAD), putres-
Vidal-Carou, 2006) dansyl chloride (Busto, Guasch, & cine (PUT), histamine (HIST), tyramine (TYR), trypt-
Borrull, 1997; Lasekan & Lasekan, 2000) or 6-aminoquin- amine (TRYP), isoamylamine (ISO), agmatine (AGM)
olyl-N-hydroxysuccinimidy carbamate (Busto, Guasch, & dansyl chloride and Na2CO3 were obtained from Sigma–
Borrull, 1996). OPA has the disadvantages that it reacts Aldrich; methylamine (MET), ethanolamine (ETA) and
only with primary amines and that their fluorescent deriv- polyvinylpyrrolidone (PVP) were supplied by Fluka. Ace-
atives are unstable, while dabsyl-chloride and dansyl tonitrile and water of HPLC grade was Carlo Erba
chloride (Dns-Cl) react with both primary and secondary reagents. A stock of standard solution was prepared by
amino groups providing very stable derivatives (Loukou & adding an accurately weighed amount of each amine (ca.
Zotou, 2003). A very useful review has been published by 10 mg) to a 10 ml volumetric flask and brought to the mark
Lehtonen (1996) in which he has summarized the features with 0.1 M HCl. The standard solutions were stored at
of 16 methods for measuring biogenic amines in wine. Nine 4 °C until the use. A calibration curve for each dansylated
of these methods employed derivatization with OPA, but in amine was obtained by analyzing the standard solutions
only one (Lehtonen, Saarinen, Vesanto, & Riekkola, 1992) diluted at different concentrations (Fig. 1). A standard mix-
was pre-column (as opposed to post-column) derivatiza- ture of biogenic amines was prepared by adding 0.1 ml of
tion used together with automation of the derivatization each amine stock solution in a 10 ml volumetric flask and
procedure. making up to the mark with 0.1 M HCl. These solutions

Fig. 1. Chromatogram of a standard solution of nine biogenic amines. Peaks number: 1: histamine (4 mg l 1), 2: methylamine (10 mg l 1), 3: ethylamine
(4 mg l 1), 4: tyramine (8 mg l 1), 5: agmatine (1 mg l 1), 6: tryptamine (8 mg l 1), 7: isoamylamine (10 mg l 1), 8: putrescine (4 mg l 1) and 9: cadaverine
(1 mg l 1).
1220 C. Proestos et al. / Food Chemistry 106 (2008) 1218–1224

were stored at 4 °C until further use. Dansyl chloride solu- Table 1


tion (10 g l 1) was prepared dissolving 100 mg in 10 ml of Analytical characteristics of method
acetone, which was stored at 4 °C until use. A solution Biogenic Recoverya Linearity Linearity Detection %
of 40 g l 1 Na2CO3 was prepared for the derivatisation amines (%) (R2)b range limitc CVa
(mg l 1) (mg l 1)
reaction.
Methylamine 89.9 ± 2.6 0.999 0.03–5.97 0.04 3.2
Ethylamine 93.2 ± 2.7 0.998 0.03–6.75 0.03 4.1
2.2. Samples and pretreatment Isoamylamine 98.8 ± 2.9 1.000 0.05–10.12 0.02 3.4
Histamine 104.3 ± 11.2 0.999 0.05–6.13 0.02 3.6
A total of 32 commercial wine samples were analysed. A Tryptamine 101.2 ± 12.5 0.999 0.09–7.21 0.05 3.5
list of all wines analysed in this study is presented in Table Tyramine 97.5 ± 3.1 0.999 0.09–8.93 0.04 4.6
2. Alcoholic content and pH are presented in the same Putrescine 87.3 ± 1.6 0.999 0.06–6.45 0.03 3.8
Cadaverine 92.5 ± 2.3 0.999 0.07–6.55 0.04 4.4
table. A 0.5 g quantity of polyvinylpyrrolidone (PVP) was Agmatine 86.2 ± 2.9 0.998 0.10–6.33 0.18 6.9
added to 10 ml of standard solutions (mixture of the a
Average of six replicates at different concentrations; CV: coefficient of
amines) or real samples in a beaker. The resulting mixtures variation.
were stirred for 15 min on a magnetic stirrer and were then b
Square of regression coefficient.
centrifuged for 15 min at 3500g. The supernatants were c
Three times the noise level.
finally filtered through 0.2 lm filters.

2.3. Derivatisation reaction


After derivatization, the reaction vials were left to cool at
The derivatisation reaction was carried out by adding room temperature and acetone was removed under a
1.6 ml of dansyl chloride solution to 1.5 ml of amine solu- stream of nitrogen. For wine samples dansylation reaction
tion and adjusted to pH 7.8, with Na2CO3 solution. The was performed as described earlier for standard solutions,
mixture was heated in a water-bath for an hour at 40 °C. using 1.5 ml of wine (e.g. Fig. 2).

Table 2
List of the analyzed wine samples
Sample no. Cultivar(s) Year Type Location Alcoholic contenta pH
1 Limnia + Kalampaki 2006 Red Limnos island 14.0 3.68
2 Xinomauro 2004 Red Naoussa 12.5 3.60
3 Agiorgitiko 2003 Red Nemea 13.5 3.66
4 Vartzami 2006 Red Lefkada island 12.5 3.87
5 Agiomavritiko‘Vardea’ 2005 White Lefkada island 11.5 3.46
6 Asirtiko 2004 White Santorini island 12.0 3.48
7 Muscat d’Alexandrie 2005 White Limnos island 12.5 3.57
8 Debina 2005 White Zitsa Ioannina 12.0 3.42
9 Athiri 2004 White Rhodes island 12.0 3.30
10 Kotsifali + Mantilaria 2004 Red Crete 12.5 3.58
11 Romeiko 2005 Red Chania Crete 13.0 3.44
12 Roditis 2005 White Nemea 11.8 3.18
13 Moschofilero 2006 White Mantinia 12.5 3.16
14 Vilana 2002 White Crete 11.5 3.39
15 Robola 2005 White Kefallonia island 12.0 3.17
16 Monemvasia 2005 White Paros island 12.0 3.46
17 Tsaousi 2005 White Kefallonia island 11.5 3.32
18 Moschomauro 2005 Red Macedonia 12.5 3.48
19 Limnio 2003 Red Limnos island 13.0 3.70
20 Kotsifali 2003 Red Crete 12.5 3.51
21 Mandilaria 2005 Red Paros island 12.5 3.59
22 Mavrodafni 2004 Red Kefallonia island 12.9 3.82
23 Vostilidi 2001 White Kefallonia island 13.8 3.64
24 Savatiano 2005 White Spata Attica 12.0 3.45
25 Priknadi 2005 White Naoussa 12.8 3.49
26 Liatiko 2006 Red Crete 12.5 3.62
27 Aidani 2004 White Santorini island 12.8 3.00
28 Malagouzia 2002 White Macedonia 12.0 3.39
29 Muscat Hamburg 2003 White Tyrnavos 12.0 3.92
30 Mavro Mesenikola 2002 Red Karditsa 11.5 3.82
31 Arikaras 2005 Red Kithira island 12.5 3.65
32 Mantilaria 2006 Red Chios island 12.5 3.32
a
Expressed in % volume.
C. Proestos et al. / Food Chemistry 106 (2008) 1218–1224 1221

Fig. 2. Chromatogram of an analyzed red wine (sample no. 30). Peak numbers as in Fig. 1. UK stands for unknown peak eluted at 10.4 min.

2.4. Solid phase extraction (SPE) 2.6. Method validation

The remaining aqueous phase of the derivatized stan- The analytical characteristics of the method are pre-
dard solutions or wine samples was then applied to Supelco sented in Table 1. The recovery was calculated by adding
Discovery C18 cartridges (500 mg 3 ml 1), which had been various known amounts of each amine to each of six differ-
previously activated with two cartridge volumes of metha- ent wines whose amine content had been previously deter-
nol followed by two volumes of water. After the samples mined in quadruplicate. After addition, the concentration
had passed through, the cartridges were washed with two of each amine in each sample was measured in duplicate.
cartridge volumes of water–acetone (80:20, v/v) and taken The difference between the new and original values was
to dryness under vacuum. The samples were finally eluted expressed as a percentage of the amount added, and for
with 3 ml of acetonitrile and the eluates were evaporated all the values, the mean ± SD (standard deviation) was cal-
to dryness under nitrogen. The residues were reconstituted culated as the overall recovery. All of the amines had
to 2 ml with acetonitrile and the resulting solutions were fil- recoveries over 85%. The highest detection limit was for
trated through a 0.45 lm membrane millipore filter (Lou- agmatine (0.18 mg l 1). To calculate the reproducibility
kou & Zotou, 2003). The resulting sample was injected of the analyses, each biogenic amine was added at different
three times to HPLC. concentrations (1, 4, 8, and 10 mg l 1) to a simulated wine
preparation. Six replicate assays were performed for each
2.5. HPLC analysis amine at each concentration level. Means and SDs were
calculated; the latter was expressed as a percentage of the
HPLC apparatus that was used for the analysis, con- former (coefficient of variation, CV) and the results for
sisted of a ternary gradient unit (Jasco CG-1580-02), a the three concentrations were averaged. The imprecision
pump (Jasco PU-980), a fluorescence detector (Jasco FL- was less than 4.0%, apart from isoamylamine (7.9%).
950) a data processing system (Chrompack), a reversed
phase column PhenomenexÒ C18, Luna 5 lm (250  2.7. Statistical analysis
4.6 mm) protected by a guard column PhenomenexÒ C18,
(4.0  3.0 mm) and finally, a rheodyne injection system Statistical analysis was performed using Minitab 13.1
(model 7725i) with a loop of 20 ll. Gradient elution of for Windows. One-way analysis of variance (ANOVA)
two solvents was used: Solvent A consisted of HPLC grade was used to determine if there were any statistically signif-
water and solvent B was acetonitrile. The gradient pro- icant differences between the mean values for each individ-
gramme used had as the one used by Soufleros, Bouloump- ual amine. Tukey’s test was then used to determine which
asi, Zotou, and Loukou, 2007 with some modifications: mean values were different.
65% A initially, 35% A at 10 min, 20% A at 20 min, and
100% B at 30 min. A 5 min additional step was included 3. Results and discussion
finally to reach the initial conditions and achieve mobile
phase stabilization. The flow rate was 1.0 ml min 1and The concentrations of the amines detected in the ana-
the temperature was set at 22.5 °C. Quantification was car- lysed wines varied over a wide range, probably reflecting
ried out by fluorescence detection at 320 nm (excitation large compositional differences among wine samples and
wavelength, kem) and 523 nm (emission wavelength, kem). variation of the viticultural and enological practices. SPE
1222 C. Proestos et al. / Food Chemistry 106 (2008) 1218–1224

is used to simultaneously clean up and concentrate samples tyramine = cadaverine = tryptamine. It is observed from
prior to their analyses by HPLC. Wine analyses are most our results that both wine aging and acidity are variables
often done with weak cation-exchange adsorbents formed influencing the content of biogenic amines in wines. Higher
by carboxylic groups (CBAs), strong cation exchangers global amounts of biogenic amines are generally found in
(SCXs) made up of sulphonic groups and octadecylsilane older wines such as Mavro Mesenikola and Vartzami,
(C18). Before the amines are separated, the samples must which have aged for longer periods in barrels or in bottles
be cleaned up in order to ensure adequate selectivity. The after winemaking and most probably underwent the malo-
SPE process eliminates matrix interferences and allowed lactic fermentation. Young wines (from 2005 and 2006 vin-
the amines’ peaks to be identified. Under the aforemen- tages) contain lower amounts of these compounds as they
tioned experimental conditions, the amines were quantita- were directly bottled after winemaking and have not under-
tively retained on the C18 minicolumn and efficiently gone further maturation processes. On the other hand, less
eluted from it by using acetonitrile. Methanol was added acid wines contained higher amounts of histamine. This
in order to decrease hydrophobic interactions between can be associated with the development of microorganisms
amines and the column material. All of the amines mea- with decarboxylase activity, which is enhanced at higher
sured with recoveries over 85%. The highest detection limit pH, and thus, histidine decarboxylation is enhanced (Gar-
was for agmatine (0.18 mg l 1). Linearity was above 0.998 cia-Villar et al., 2006). None of these wines surpass the
for all amines, and linearity range was almost similar for toxic levels reported in the literature (Lehtonen, 1996)
most of the amines examined. The results are summarised and thus, these Greek wine samples analysed do not repre-
in Table 3. Putrescine, cadaverine and histamine were the sent any toxicological risk for human health. More specif-
most abundant amines in the samples analysed. The rela- ically, histamine was higher in all red wines than white
tive concentrations of biogenic amines (mg l 1) approxi- wines (p < 0.05). Among red wines, Limnio and Kotsifali
mated the following order: putrescine > histamine > wines had the highest mean concentration (p < 0.05) fol-
isoamylamine > ethylamine > methylamine > agmatine = lowed by Mavro Mesenikola, whereas, among white wines,

Table 3
Biogenic amine contenta of the analysed Greek wines
Sample no. CAD PUT HIST TYR TRYP AGM ISO MET ETA
b
1 ND 2.10 ± 0.02 1.05 ± 0.05 0.09 ± 0.03 ND ND 0.95 ± 0.02 ND ND
2 0.17 ± 0.02 2.11 ± 0.03 1.30 ± 0.03 0.26 ± 0.03 ND ND 0.15 ± 0.03 ND 0.42 ± 0.03
3 ND 2.70 ± 0.03 1.25 ± 0.03 ND 1.10 ± 0.02 ND ND 0.38 ± 0.03 0.71 ± 0.04
4 0.14 ± 0.01 1.86 ± 0.02 1.14 ± 0.03 0.26 ± 0.03 ND 0.21 ± 0.03 0.81 ± 0.02 ND 0.28 ± 0.03
5 0.08 ± 0.02 0.83 ± 0.03 0.40 ± 0.02 ND ND ND 1.92 ± 0.03 0.28 ± 0.05 0.11 ± 0.03
6 0.12 ± 0.02 9.07 ± 0.02 0.83 ± 0.03 1.16 ± 0.03 ND ND 1.93 ± 0.02 0.47 ± 0.03 0.14 ± 0.04
7 0.07 ± 0.01 1.02 ± 0.03 1.09 ± 0.02 0.36 ± 0.02 ND ND 0.42 ± 0.02 0.36 ± 0.02 0.13 ± 0.04
8 ND 0.72 ± 0.02 0.55 ± 0.02 0.13 ± 0.02 ND ND 0.99 ± 0.02 0.22 ± 0.01 0.09 ± 0.02
9 ND 1.14 ± 0.02 1.05 ± 0.03 ND ND ND 0.78 ± 0.02 ND ND
10 0.17 ± 0.03 1.71 ± 0.03 1.31 ± 0.03 0.44 ± 0.02 0.11 ± 0.03 ND ND ND ND
11 ND 1.35 ± 0.02 1.08 ± 0.04 ND ND ND ND ND ND
12 0.10 ± 0.02 0.42 ± 0.03 0.36 ± 0.02 ND ND ND 1.11 ± 0.03 0.24 ± 0.03 0.14 ± 0.03
13 ND ND 0.34 ± 0.02 0.16 ± 0.02 ND ND 0.94 ± 0.02 ND ND
14 0.10 ± 0.02 1.00 ± 0.03 0.65 ± 0.03 ND 0.23 ± 0.03 ND 2.26 ± 0.03 0.49 ± 0.05 0.17 ± 0.02
15 0.11 ± 0.01 0.26 ± 0.02 0.18 ± 0.02 0.09 ± 0.01 ND ND 1.15 ± 0.03 0.38 ± 0.02 0.13 ± 0.02
16 ND 1.16 ± 0.03 1.03 ± 0.03 ND ND ND 0.89 ± 0.02 0.28 ± 0.03 0.12 ± 0.02
17 0.12 ± 0.01 0.81 ± 0.02 0.49 ± 0.02 0.17 ± 0.02 ND ND 1.91 ± 0.03 ND ND
18 0.21 ± 0.02 1.26 ± 0.02 0.98 ± 0.02 ND ND 0.33 ± 0.02 ND ND 0.31 ± 0.03
19 0.14 ± 0.02 2.01 ± 0.03 1.65 ± 0.03 0.46 ± 0.02 0.31 ± 0.02 0.51 ± 0.03 0.61 ± 0.02 0.59 ± 0.03 0.61 ± 0.02
20 0.17 ± 0.01 2.31 ± 0.03 1.54 ± 0.03 0.38 ± 0.02 ND 0.63 ± 0.04 0.45 ± 0.02 0.39 ± 0.03 0.63 ± 0.02
21 0.19 ± 0.03 1.82 ± 0.02 1.03 ± 0.02 ND ND ND 0.50 ± 0.03 ND 0.44 ± 0.03
22 0.15 ± 0.01 1.95 ± 0.03 1.33 ± 0.03 0.18 ± 0.02 ND 0.41 ± 0.02 0.47 ± 0.02 ND 0.34 ± 0.04
23 0.11 ± 0.02 1.16 ± 0.02 1.13 ± 0.03 0.26 ± 0.03 0.51 ± 0.02 ND 2.41 ± 0.03 ND 0.11 ± 0.02
24 ND 0.94 ± 0.02 0.85 ± 0.02 ND ND ND 0.71 ± 0.02 ND ND
25 ND ND 0.45 ± 0.02 ND ND ND 1.22 ± 0.03 ND ND
26 0.16 ± 0.02 1.81 ± 0.02 1.36 ± 0.02 0.26 ± 0.03 ND 0.31 ± 0.02 ND ND ND
27 0.10 ± 0.02 0.51 ± 0.02 0.35 ± 0.03 0.14 ± 0.03 ND ND 0.88 ± 0.02 ND ND
28 0.13 ± 0.02 1.09 ± 0.02 1.01 ± 0.02 0.36 ± 0.03 ND ND 2.02 ± 0.03 0.39 ± 0.02 0.16 ± 0.02
29 0.10 ± 0.02 1.21 ± 0.02 1.10 ± 0.02 0.17 ± 0.02 ND ND 0.82 ± 0.02 ND 0.12 ± 0.04
30 0.15 ± 0.02 ND 1.45 ± 0.02 0.16 ± 0.03 1.32 ± 0.04 ND 0.91 ± 0.02 0.38 ± 0.05 0.22 ± 0.02
31 0.17 ± 0.03 1.31 ± 0.02 1.09 ± 0.03 ND ND 0.23 ± 0.01 0.50 ± 0.02 ND ND
32 0.16 ± 0.02 1.29 ± 0.02 1.11 ± 0.03 ND ND 0.31 ± 0.01 ND ND ND
a 1
Results are given in mg l ± standard deviation.
b
ND: not detected.
C. Proestos et al. / Food Chemistry 106 (2008) 1218–1224 1223

Vostilidi and Muscat Hamburg wines had the highest mean was detected in very few of the wines examined in this
histamine concentrations (p < 0.05). The concentrations of survey. This amine was not detected in young wines.
putrescine were much higher than those of histamine Agmatine was only found in some red wine samples,
(p < 0.05) but, like the latter, higher amounts were present with a maximum value of 0.63 mg l 1. Little is known
in red than in white wines, with Agiorgitiko and Kotsifali about the significance of this polyamine in food and bev-
wines having the highest concentrations among the former erages. It is considered as an intermediate product of the
(p < 0.05). Vostilidi and Muscat Hamburg wines had metabolism of arginine to putrescine. It has also been
higher concentrations of this amine than the other white related to food spoilage (Halasz et al., 1994; Veciana-
ones. The formation of putrescine can be attributed to var- Nogues, Marine-Font, & Vidal-Carou, 1997). However,
ious factors. A certain amount might come from raw mate- the relationship between agmatine and spoilage cannot
rial, e.g., endogenous compounds in grapes. Red wine be applied to wine on the basis of the results found in
vinification is usually carried out in the presence of grape the present investigation.
skin and pulp and thus, putrescine from these parts can There are no legal maximum tolerable levels for any bio-
be released into the must. This could explain, at least par- genic amine in wines. However, concentrations of 2–10 mg
tially, the higher levels of this diamine in red wine. During ml 1 of histamine in alcoholic beverages, 10–80 mg l 1 of
winemaking, putrescine can also originate from the micro- tyramine and 3 mg l 1 of 2-phenylethylamine have been
bial decarboxylation of ornithine, which may also be suggested as toxic levels depending on the country (Lehto-
formed by the microbial metabolism of amino acids such nen, 1996; Soufleros et al., 2007). In general, 8–40 mg his-
as citrulline or arginine. Also, putrescine can be formed tamine can cause slight, over 40 mg, moderate, and over
from arginine via agmatine. All these pathways have been 100 mg, severe poisoning, whereas, 1080 mg tyramine can
described in several wine lactic acid bacteria which can cause toxic swelling and over 100 mg may cause migraine
develop during malolactic fermentation (Arena & Manca (Shalaby, 1996). On the basis of the findings of Bartholo-
de Nadra, 2001; Bover-Cid et al., 2006). This offers a good mew, Berry, Rodhouse, and Grilbert (1987) that depending
explanation for the higher concentration of putrescine on a survey of scombrotoxic fish poisoning in Britain which
found in red wine in comparison with the white ones. This involved 250 suspected incidents from 1976 to 1986, the
secondary fermentation is less usual in white wines. guidelines for histamine content of fish are less than 5 mg
For isoamylamine, the third most abundant amine 100 g 1 (safe for consumption), 5–20 mg 100 g 1 (possibly
detected, the mean concentrations of several of the white toxic), 20–100 mg 100 g 1 (probably toxic), and >100 mg
wines surpassed those of most red wines, notably Vostilidi 100 g 1 (toxic and unsafe for consumption). An intake of
and Vilana, which had the highest mean concentration more than 40 mg biogenic amines per meal has been con-
(p < 0.05), followed by those from Malagouzia (p < 0.05). sidered potentially toxic (Shalaby, 1996). However, not
Those three wines were from older vintages (2001 and all amines are equally toxic, consequently histamine, tyra-
2002). mine, and phenylethylamine are of concern. With regards
Although the concentrations of cadaverine were much to possible health hazards, the biogenic amines should be
lower than those of the three aforementioned amines seen as an important quality factor. Some countries have
(p < 0.05), all red wines had mean values exceeding those legislated about the maximum amount of histamine in
of the whites (p > 0.05), with Moschomauro wine having some products. The permissible limit of histamine in Swit-
the highest, followed by Mandilaria. Cadaverine is usually zerland is 10 mg l 1 for wines.
associated with decarboxylase activity of contaminant
enterobacteria (Halasz, Barath, Simon-Sarkadi, & Holzap- 4. Conclusions
fel, 1994), although those kinds of bacteria are not usual
contaminants in wine. The method used is suitable for the simultaneous quan-
Tyramine was detected in small amounts in most of tification of nine oenologically important biogenic amines
the investigated wines. Two red wines Limnio and (methylamine, ethylamine, isoamylamine, putrescine,
Kotsifali + Mantilaria (no. 10) had higher concentrations cadaverine, histamine, tyramine, tryptamine and agma-
than all the other wines, both wine and red (p < 0.05). It tine), by HPLC – fluorescence detection. The separation
is noteworthy that red wines contained higher amounts is accomplished in a short time with an excellent resolution
of ethylamine in comparison to white ones (p < 0.05); for all the biogenic amines peaks. In this work, the combi-
Vilana and Malagouzia had marginally the highest levels nation of derivatization and solid phase extraction proce-
among the white wines. Of the red wines, Agiorgitiko, dures, ensures a quantitative elimination of potential
followed by Kotsifali and Limnio, contained higher interferences from other compounds like amino acids. Bio-
amounts of this amine. Methylamine concentrations of genic amines were present in Greek wines in relatively low
red and white wines showed almost same values (Table quantities. It was observed from the results that both wine
3). It was mainly detected in white wines rather than aging and acidity influenced the content of biogenic amines
in the red ones. Indeed, Limnio wine showed the highest in wines. Higher amounts of biogenic amines were gener-
mean concentration of all the cultivars (p < 0.05). Next ally detected in older wines, which have aged for longer
highest were Vilana and Asyrtiko (p < 0.05). Tryptamine periods after winemaking. Young wines (from 2005 and
1224 C. Proestos et al. / Food Chemistry 106 (2008) 1218–1224

2006 vintages) contained lower amounts of biogenic Lehtonen, P. (1996). Determination of amines and amino acids in wine – a
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is noteworthy that less acid wines gave rise to higher hista- derivatization with phthalaldehyde and fluorescence detection. Zeits-
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Lonvaud-Funel, A. (2001). Biogenic amines in wines: Role of lactic acid
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