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-

Biogenic amines are mainly nitrogenous low molecular
weight compounds with biological activity that may be
formed or metabolised in the cells of living organisms (ani-
mals, plants and micro-organisms). Biogenic amines are
derived mainly from amino acids through substrate-specific
decarboxylase enzymes (Loukou & Zotou, 2003). Amines
may be formed by yeasts during the alcoholic fermentation
(mostly putrazine); by lactic acid bacteria (LAB) during
malolactic fermentation (MLF) and during maturation of
wines (Lonvaud-Funel, 2001). Biogenic amines can also
be present in the must (Arce, Rios, & Valcarel, 1998).
The main biogenic amines in wine are histamine, tyramine,
putrescine, cadaverine and phenylethylamine. Biogenic
amines are important because they contain a health risk
*
Corresponding author. Tel./fax: +30 210 5294186.
E-mail address: achem@aua.gr (M. Komaitis).
atorial discomfort, hot flushes, cold sweat, palpitations,
headaches, red rash, high or low blood pressure (Mo Dugo,
Vilasi, La Torre, & Pellicanò, 2006). Alcohol and acetalde-
hyde have been found to increase the sensitivity to biogenic
amines (Busto, Guasch, & Borrull, 1995). High levels of
biogenic amines correlate fairly well with other wine spoil-
age components for example butyric acid, lactic acid, acetic
acid, ethyl acetate and diethyl succinate, which is why
wines with higher levels usually also have higher levels of
volatile acid. Red wines also have higher levels than white
wines, mainly because of vinification practices and matura-
tion (Bauza, Blaise, Daumas, & Cabanis, 1995). An
increase in the levels of biogenic amines usually occurs
towards the end of the MLF or during maturation, when
lactobacilli and pediococci are the biggest culprits (Soufle-
ros, Barrios, & Bertrand, 1998). Their presence in wines has
been associated with a lack of hygiene during the winemak-
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
2003). Determination of biogenic amines is not simple
because of their structure and because they are usually
present at low levels in a complex matrix such as wine.
High-performance liquid chromatography (HPLC) is the
technique most extensively used due to its high resolution,
sensitivity, great versatility, and simple sample treatment.
Biogenic amines do not exhibit satisfactory absorption at
the visible or ultraviolet wavelengths, nor do they exhibit
fluorescence. Therefore, pre- or post-column chemical
derivatization is usually applied for their analysis by
HPLC, increasing the selectivity and sensitivity of detection
(Vidal-Carou, Lahoz-Portoles, Bover-Cid, & Marine-Font,
2003). Sensitivity is also increased by using fluorescence
instead of diode array detector. The determination of bio-
genic amines in wines and alcoholic beverages generally,
has been carried out mostly by HPLC (Garcia-Villar, Saur-
ina, & Hernandez-Cassou, 2006; Moret & Conte, 1996;
Romero, Gazquez, Bagur, & Sauchez-Vines, 2000) after
derivatization with reagents such as o-phthalaldehyde
(OPA) (Bover-Cid, Iquierdo-Pulido, Marine-Font, &
Vidal-Carou, 2006) dansyl chloride (Busto, Guasch, &
Borrull, 1997; Lasekan & Lasekan, 2000) or 6-aminoquin-
olyl-N-hydroxysuccinimidy carbamate (Busto, Guasch, &
Borrull, 1996). OPA has the disadvantages that it reacts
only with primary amines and that their fluorescent deriv-
atives are unstable, while dabsyl-chloride and dansyl
chloride (Dns-Cl) react with both primary and secondary
amino groups providing very stable derivatives (Loukou &
Zotou, 2003). A very useful review has been published by
Lehtonen (1996) in which he has summarized the features
of 16 methods for measuring biogenic amines in wine. Nine
of these methods employed derivatization with OPA, but in
only one (Lehtonen, Saarinen, Vesanto, & Riekkola, 1992)
was pre-column (as opposed to post-column) derivatiza-
tion used together with automation of the derivatization
procedure.
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).
1219
In this work, nine of the most oenologically important
biogenic amines are determined in red and white wines
after pretreatment with polyvinylpyrrolidone (PVP), which
removes the substances that interfere in the derivatization
and subsequent quantification of the derivatives such as
polyphenols. PVP reacts with polyphenols and create a pre-
cipitate that can be removed by centrifuge or filtration. The
applied post-derivatization SPE procedure increases the
selectivity and sensitivity of the method. The present
method advantages include: the use of a simple gradient
which effectively separates all the amines in a relatively
short time, a simple time- and cost-effective pretreatment
of the samples and a sensitive and selective detection by
using fluorescence detection.
2. Materials and methods
2.1. Standards and reagents
Standard solution including: cadaverine (CAD), putres-
cine (PUT), histamine (HIST), tyramine (TYR), trypt-
amine (TRYP), isoamylamine (ISO), agmatine (AGM)
dansyl chloride and Na2CO3 were obtained from Sigma–
Aldrich; methylamine (MET), ethanolamine (ETA) and
polyvinylpyrrolidone (PVP) were supplied by Fluka. Ace-
tonitrile and water of HPLC grade was Carlo Erba
reagents. A stock of standard solution was prepared by
adding an accurately weighed amount of each amine (ca.
10 mg) to a 10 ml volumetric flask and brought to the mark
with 0.1 M HCl. The standard solutions were stored at
4 °C until the use. A calibration curve for each dansylated
amine was obtained by analyzing the standard solutions
diluted at different concentrations (Fig. 1). A standard mix-
ture of biogenic amines was prepared by adding 0.1 ml of
each amine stock solution in a 10 ml volumetric flask and
making up to the mark with 0.1 M HCl. These solutions
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

The derivatisation reaction was carried out by adding
1.6 ml of dansyl chloride solution to 1.5 ml of amine solu-
tion and adjusted to pH 7.8, with Na2CO3 solution. The
mixture was heated in a water-bath for an hour at 40 °C.
After derivatization, the reaction vials were left to cool at
room temperature and acetone was removed under a
stream of nitrogen. For wine samples dansylation reaction
was performed as described earlier for standard solutions,
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
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)
The remaining aqueous phase of the derivatized stan-
dard solutions or wine samples was then applied to Supelco
Discovery C18 cartridges (500 mg 3 ml 1), which had been
previously activated with two cartridge volumes of metha-
nol followed by two volumes of water. After the samples
had passed through, the cartridges were washed with two
cartridge volumes of water–acetone (80:20, v/v) and taken
to dryness under vacuum. The samples were finally eluted
with 3 ml of acetonitrile and the eluates were evaporated
to dryness under nitrogen. The residues were reconstituted
to 2 ml with acetonitrile and the resulting solutions were fil-
trated through a 0.45 lm membrane millipore filter (Lou-
kou & Zotou, 2003). The resulting sample was injected
three times to HPLC.
2.5. HPLC analysis
HPLC apparatus that was used for the analysis, con-
sisted of a ternary gradient unit (Jasco CG-1580-02), a
pump (Jasco PU-980), a fluorescence detector (Jasco FL-
950) a data processing system (Chrompack), a reversed
phase column PhenomenexÒ C18, Luna 5 lm (250 
4.6 mm) protected by a guard column PhenomenexÒ C18,
(4.0  3.0 mm) and finally, a rheodyne injection system
(model 7725i) with a loop of 20 ll. Gradient elution of
two solvents was used: Solvent A consisted of HPLC grade
water and solvent B was acetonitrile. The gradient pro-
gramme used had as the one used by Soufleros, Bouloump-
asi, Zotou, and Loukou, 2007 with some modifications:
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
finally to reach the initial conditions and achieve mobile
phase stabilization. The flow rate was 1.0 ml min 1and
the temperature was set at 22.5 °C. Quantification was car-
ried out by fluorescence detection at 320 nm (excitation
wavelength, kem) and 523 nm (emission wavelength, kem).
1221
2.6. Method validation
The analytical characteristics of the method are pre-
sented in Table 1. The recovery was calculated by adding
various known amounts of each amine to each of six differ-
ent wines whose amine content had been previously deter-
mined in quadruplicate. After addition, the concentration
of each amine in each sample was measured in duplicate.
The difference between the new and original values was
expressed as a percentage of the amount added, and for
all the values, the mean ± SD (standard deviation) was cal-
culated as the overall recovery. All of the amines had
recoveries over 85%. The highest detection limit was for
agmatine (0.18 mg l 1). To calculate the reproducibility
of the analyses, each biogenic amine was added at different
concentrations (1, 4, 8, and 10 mg l 1) to a simulated wine
preparation. Six replicate assays were performed for each
amine at each concentration level. Means and SDs were
calculated; the latter was expressed as a percentage of the
former (coefficient of variation, CV) and the results for
the three concentrations were averaged. The imprecision
was less than 4.0%, apart from isoamylamine (7.9%).
2.7. Statistical analysis
Statistical analysis was performed using Minitab 13.1
for Windows. One-way analysis of variance (ANOVA)
was used to determine if there were any statistically signif-
icant differences between the mean values for each individ-
ual amine. Tukey’s test was then used to determine which
mean values were different.
3. Results and discussion
The concentrations of the amines detected in the ana-
lysed wines varied over a wide range, probably reflecting
large compositional differences among wine samples and
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
prior to their analyses by HPLC. Wine analyses are most
often done with weak cation-exchange adsorbents formed
by carboxylic groups (CBAs), strong cation exchangers
(SCXs) made up of sulphonic groups and octadecylsilane
(C18). Before the amines are separated, the samples must
be cleaned up in order to ensure adequate selectivity. The
SPE process eliminates matrix interferences and allowed
the amines’ peaks to be identified. Under the aforemen-
tioned experimental conditions, the amines were quantita-
tively retained on the C18 minicolumn and efficiently
eluted from it by using acetonitrile. Methanol was added
in order to decrease hydrophobic interactions between
amines and the column material. All of the amines mea-
sured with recoveries over 85%. The highest detection limit
was for agmatine (0.18 mg l 1). Linearity was above 0.998
for all amines, and linearity range was almost similar for
most of the amines examined. The results are summarised
in Table 3. Putrescine, cadaverine and histamine were the
most abundant amines in the samples analysed. The rela-
tive concentrations of biogenic amines (mg l 1) approxi-
mated the following order: putrescine > histamine >
isoamylamine > ethylamine > methylamine > agmatine =
tyramine = cadaverine = tryptamine. It is observed from
our results that both wine aging and acidity are variables
influencing the content of biogenic amines in wines. Higher
global amounts of biogenic amines are generally found in
older wines such as Mavro Mesenikola and Vartzami,
which have aged for longer periods in barrels or in bottles
after winemaking and most probably underwent the malo-
lactic fermentation. Young wines (from 2005 and 2006 vin-
tages) contain lower amounts of these compounds as they
were directly bottled after winemaking and have not under-
gone further maturation processes. On the other hand, less
acid wines contained higher amounts of histamine. This
can be associated with the development of microorganisms
with decarboxylase activity, which is enhanced at higher
pH, and thus, histidine decarboxylation is enhanced (Gar-
cia-Villar et al., 2006). None of these wines surpass the
toxic levels reported in the literature (Lehtonen, 1996)
and thus, these Greek wine samples analysed do not repre-
sent any toxicological risk for human health. More specif-
ically, histamine was higher in all red wines than white
wines (p < 0.05). Among red wines, Limnio and Kotsifali
wines had the highest mean concentration (p < 0.05) fol-
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
Vostilidi and Muscat Hamburg wines had the highest mean
histamine concentrations (p < 0.05). The concentrations of
putrescine were much higher than those of histamine
(p < 0.05) but, like the latter, higher amounts were present
in red than in white wines, with Agiorgitiko and Kotsifali
wines having the highest concentrations among the former
(p < 0.05). Vostilidi and Muscat Hamburg wines had
higher concentrations of this amine than the other white
ones. The formation of putrescine can be attributed to var-
ious factors. A certain amount might come from raw mate-
rial, e.g., endogenous compounds in grapes. Red wine
vinification is usually carried out in the presence of grape
skin and pulp and thus, putrescine from these parts can
be released into the must. This could explain, at least par-
tially, the higher levels of this diamine in red wine. During
winemaking, putrescine can also originate from the micro-
bial decarboxylation of ornithine, which may also be
formed by the microbial metabolism of amino acids such
as citrulline or arginine. Also, putrescine can be formed
from arginine via agmatine. All these pathways have been
described in several wine lactic acid bacteria which can
develop during malolactic fermentation (Arena & Manca
de Nadra, 2001; Bover-Cid et al., 2006). This offers a good
explanation for the higher concentration of putrescine
found in red wine in comparison with the white ones. This
secondary fermentation is less usual in white wines.
For isoamylamine, the third most abundant amine
detected, the mean concentrations of several of the white
wines surpassed those of most red wines, notably Vostilidi
and Vilana, which had the highest mean concentration
(p < 0.05), followed by those from Malagouzia (p < 0.05).
Those three wines were from older vintages (2001 and
2002).
Although the concentrations of cadaverine were much
lower than those of the three aforementioned amines
(p < 0.05), all red wines had mean values exceeding those
of the whites (p > 0.05), with Moschomauro wine having
the highest, followed by Mandilaria. Cadaverine is usually
associated with decarboxylase activity of contaminant
enterobacteria (Halasz, Barath, Simon-Sarkadi, & Holzap-
fel, 1994), although those kinds of bacteria are not usual
contaminants in wine.
Tyramine was detected in small amounts in most of
the investigated wines. Two red wines Limnio and
Kotsifali + Mantilaria (no. 10) had higher concentrations
than all the other wines, both wine and red (p < 0.05). It
is noteworthy that red wines contained higher amounts
of ethylamine in comparison to white ones (p < 0.05);
Vilana and Malagouzia had marginally the highest levels
among the white wines. Of the red wines, Agiorgitiko,
followed by Kotsifali and Limnio, contained higher
amounts of this amine. Methylamine concentrations of
red and white wines showed almost same values (Table
3). It was mainly detected in white wines rather than
in the red ones. Indeed, Limnio wine showed the highest
mean concentration of all the cultivars (p < 0.05). Next
highest were Vilana and Asyrtiko (p < 0.05). Tryptamine
1223
was detected in very few of the wines examined in this
survey. This amine was not detected in young wines.
Agmatine was only found in some red wine samples,
with a maximum value of 0.63 mg l 1. Little is known
about the significance of this polyamine in food and bev-
erages. It is considered as an intermediate product of the
metabolism of arginine to putrescine. It has also been
related to food spoilage (Halasz et al., 1994; Veciana-
Nogues, Marine-Font, & Vidal-Carou, 1997). However,
the relationship between agmatine and spoilage cannot
be applied to wine on the basis of the results found in
the present investigation.
There are no legal maximum tolerable levels for any bio-
genic amine in wines. However, concentrations of 2–10 mg
ml 1 of histamine in alcoholic beverages, 10–80 mg l 1 of
tyramine and 3 mg l 1 of 2-phenylethylamine have been
suggested as toxic levels depending on the country (Lehto-
nen, 1996; Soufleros et al., 2007). In general, 8–40 mg his-
tamine can cause slight, over 40 mg, moderate, and over
100 mg, severe poisoning, whereas, 1080 mg tyramine can
cause toxic swelling and over 100 mg may cause migraine
(Shalaby, 1996). On the basis of the findings of Bartholo-
mew, Berry, Rodhouse, and Grilbert (1987) that depending
on a survey of scombrotoxic fish poisoning in Britain which
involved 250 suspected incidents from 1976 to 1986, the
guidelines for histamine content of fish are less than 5 mg
100 g 1 (safe for consumption), 5–20 mg 100 g 1 (possibly
toxic), 20–100 mg 100 g 1 (probably toxic), and >100 mg
100 g 1 (toxic and unsafe for consumption). An intake of
more than 40 mg biogenic amines per meal has been con-
sidered potentially toxic (Shalaby, 1996). However, not
all amines are equally toxic, consequently histamine, tyra-
mine, and phenylethylamine are of concern. With regards
to possible health hazards, the biogenic amines should be
seen as an important quality factor. Some countries have
legislated about the maximum amount of histamine in
some products. The permissible limit of histamine in Swit-
zerland is 10 mg l 1 for wines.
4. Conclusions
The method used is suitable for the simultaneous quan-
tification of nine oenologically important biogenic amines
(methylamine, ethylamine, isoamylamine, putrescine,
cadaverine, histamine, tyramine, tryptamine and agma-
tine), by HPLC – fluorescence detection. The separation
is accomplished in a short time with an excellent resolution
for all the biogenic amines peaks. In this work, the combi-
nation of derivatization and solid phase extraction proce-
dures, ensures a quantitative elimination of potential
interferences from other compounds like amino acids. Bio-
genic amines were present in Greek wines in relatively low
quantities. It was observed from the results that both wine
aging and acidity influenced the content of biogenic amines
in wines. Higher amounts of biogenic amines were gener-
ally detected in older wines, which have aged for longer
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
amines as they were directly bottled after winemaking
and have not undergone further maturation processes. It
is noteworthy that less acid wines gave rise to higher hista-
mine contents.
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