Food Control 25 (2012) 380e388

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Food Control
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A novel dispersive liquideliquid microextraction (DLLME) gas chromatography-

mass spectrometry (GCeMS) method for the determination of eighteen biogenic
amines in beer
C. Almeida, J.O. Fernandes, S.C. Cunha*
REQUIMTE, Department of Chemical Sciences, Laboratory of Bromatology and Hydrology, Faculty of Pharmacy, University of Porto, Rua Anbal Cunha 164, 4099-030 Porto, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: A novel dispersive liquideliquid microextraction (DLLME) gas chromatography mass-spectrometry
Received 29 June 2011 (GCeMS) method was developed for the determination of 18 biogenic amines in beers. The
Received in revised form method features the simultaneous extraction/derivatization of the amines providing a simple and
15 October 2011
fast mode of extract enrichment. A mixture of acetonitrile (dispersive solvent; 1.0 mL), toluene
Accepted 22 October 2011
(extractive solvent; 325 mL), and isobutyl choloroformate (derivatizing reagent; 25 mL) was used as
extractive/derivatizing reagent, added to 5 mL of sample. The proposed method showed good
Keywords:
linearity (correlation coefcients > 0.997), good recoveries (from 72 to 113%), and good intra-day
DLLME
GCeMS
precision (below 13%) and inter-day precision (below 14%). Moreover, detection limits were never
Isobutyl chloroformate over 2.9 mg L
1. The developed method was successfully applied to the analysis of 22 beer samples
Biogenic amines commercialized in Portugal. Fourteen of the eighteen biogenic amines analyzed were found in most
Beer of the beers, with predominance of putrescine, tyramine, dimethylamine, cadaverine, pyrrolidine
and 1,3-diaminopropane.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
A signicant amount of food products are usually processed,
prepared or preserved before eating. Food fermentation is an
ancient conservation method which can greatly contribute to
improve its nutritional value, tastiness and texture besides assuring
a longer and safer use. Fermented beverages, as wine and beer, are
often fermented by various microorganisms. Beer, in particular, is
made from brewed and fermented cereals, usually malted barley,
and avoured with hops. During beer production, alcoholic
fermentation takes place by the action of selected yeast strains,
such as Saccharomyces cerevisiae (top fermenting), Saccharomyces
carlsbergensis (bottom fermenting), together with wild yeast and
lactic acid bacteria (LAB) (Sarkadi, 2009). In addition to the
conversion of carbohydrates to alcohols and carbon dioxide wild
yeasts and LAB often produce other compounds, such as biogenic
amines, through decarboxylation of amino acids. The occurrence of
biogenic amines in alcoholic beverages has received special public
attention due to the fact that alcohol may enhance its effects by
directly or indirectly inhibiting amine oxidases (Maynard &
Schenker, 1996). Low concentration of biogenic amines can be
* Corresponding author. Tel.: 351222078910; fax: 351222003977.
E-mail address: sara.cunha@ff.up.pt (S.C. Cunha).
easily tolerated by the human body if they are efciently detoxied
by mono and diamine oxidase in the intestinal tract. However, if
either an excessive amount of biogenic amines is ingested or the
normal catabolic routes are inhibited or genetically decient,
several physiological disorders can take place. Among biogenic
amines found in alcoholic beverages, histamine and tyramine are
often described as the more harmful due to their potential allergic
or immune response, induction of neurological disorders (hista-
mine), migraines, hypertension (tyramine), Parkinsons disease,
schizophrenia and mood disorders (tyramine) (Medina, Quesada,
Castro, & Snchez-Jimnez, 1999; Silla Santos, 1996; Smith, 1980;
Ten Brink, Damink, Joosten, & Huis in t Veld, 1990). Despite
controversy over exposure levels, no ofcial limits have been set for
histamine or tyramine concentrations in beers. According to a rec-
ommended guideline, 6 mg of tyramine ingestion within a 4 h
period is considered a safe amount for beers (Izquierdo-Pulido,
Hernndez-Jover, Marin-Font, & Carmen Vidal-Carou, 1996).
Biogenic amines in beer are of great interest not only for their
potential risk to human health but also because they could have
a role as chemical indicators of unwanted microbial contamination
or decient processing conditions (Sarkadi, 2009). The presence of
higher amounts of histamine and tyramine in beers has been
associated with microbial contamination during brewing. In
contrast, putrescine, agmatine, spermidine, and spermine are

0956-7135/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2011.10.052
 C. Almeida et al. / Food Control 25 (2012) 380e388
considered natural beer constituents, that primarily originate from
malt (Kalac & Krizek, 2003; Romero, Bagur, Snchez-Vinas, &
Gzques, 2003), while tyramine and 2-phenylethylamine could be
present in hop (Slomkowska & Ambroziak, 2002). Besides these
biogenic amines, other amines often neglected by researchers can
be found in beers as result of the reductive amination or trans-
amination of the corresponding aldehyde or ketone, such as eth-
ylamine, methylamine, dimethylamine, and pyrrolidine (Ough,
Daudt, & Crowell, 1981; Smith, 1980). These short-chain aliphatic
or alicyclic amines may react with nitrosating agents, leading to the
formation of potentially carcinogenic N-nitrosamines (Tricker,
Pfundstein, Theobald, Preussmann, & Spiegelhalder, 1991).
The simultaneous and rapid analysis of several biogenic amines
in beers is of utmost importance, requiring inexpensive, reliable,
rapid and simple methods. In this regard, a wide variety of methods
for the determination of these compounds in food have been re-
ported (Ancn-Azpilicueta, Gonzlez-Marco, & Jimnez-Moreno,
2008; Karovicov & Kohajdov, 2005; nal, 2007; Smit, du Toit, &
du Toit, 2008). Methodologies based on colorimetric detection,
thin-layer chromatography or enzymatic approaches generally
require both inexpensive equipment and reagents; however they
lack accuracy being characterized for providing qualitative or at
best semi-quantitative results. High performance liquid chroma-
tography (HPLC), capillary electrophoresis and gas chromatography
(GC), in combination with various detectors are the most usefulness
and popular methods for precise quantitative analysis of biogenic
amines. However, to obtain optimal analysis conditions, an
appropriate sample preparation that could include an extraction-
cleanup step or concentration and derivatization procedures, are
usually mandatory. Regarding the extraction-cleanup step several
solvents have been applied including 0.6 M-perchloric acid, 5e10 %
trichloroacetic acid, 0.1 M-hydrochloric acid, butanol or butanol-
chloroform at basic pH (Karovicov & Kohajdov, 2005). However,
these liquideliquid procedures are generally time-consuming and
require large amounts of solvents. To overcome these problems,
alternative methods have been applied, such as solid-phase
extraction (Gianotti et al., 2008; Gosetti, Mazzucco, Gianotti,
Polati, & Gennaro, 2007; Zotou, Loukou, Soueros, & Stratis,
2003), solid-phase microextraction (Awan, Fleet, & Thomas,
2008), hollow bre liquid-phase microextraction (Saaid et al.,
2009) or more recently dispersive liquideliquid microextraction
(DLLME) (Huang et al., 2011). Among these, the most simple, rapid
and environmental friendly approach seems to be the DLLME
procedure, as Cunha, Fernandes, and Oliveira (2011, chp 1) explains
in a comprehensive review. Basically, DLLME consists in the
formation of a cloudy solution promoted by the fast addition of
a mixture of extractive and dispersive solvents to an aqueous
sample. The tiny droplets formed and dispersed among the
aqueous sample solution are further joined by centrifugation,
allowing great enrichment factors and good yields. This procedure
has been successfully applied for extraction of compounds with
high or moderate lipophilic properties such as aromatic hydrocar-
bons (Rezaee, Yamini, & Faraji, 2010), aromatic amines (Farajzadeh,
Bahram, & Jnsson, 2007) and pesticide residues (Cunha,
Fernandes, Alves, & Oliveira, 2009). For high hydrophilic
compounds, hardly extracted with the water-immiscible solvents
used in DLLME, in situ derivatization is required to increase the
yields. This type of one step DLLME/derivatization approach has
been restricted until now to few compounds such as bisphenol A
and B (Cunha & Fernandes, 2010; Cunha, Almeida, Mendes,
Fernandes, 2011), chlorophenols (Fattahi, Assadi, Milani Hosseini,
& Jahromi, 2007) and anilines (Chiang & Huang, 2008). To the
best of our knowledge the use of a DLLME procedure for the
simultaneous extraction and derivatization of biogenic amines in
beers was not yet reported.
381
Recently, Huang et al. (2011) associated the ultrasound-assisted
DLLME extraction (UDLLME) with HPLC for the determination of
some biogenic amines (octopamine, tyramine and 2-
phenylethylamine) in beers, but their method includes a pre-
extraction and a lengthy pre- derivatization step before UDLLME
procedure. From the viewpoint of laboratory efciency (time and
consumables) it is desirable to invest in sample preparations that
involve simultaneously performed extraction and derivatization
operations.
The main objective of this study was to develop and validate
a reliable DLLME/GCeMS method for simultaneous determination
of aliphatic, heterocyclic, and aromatic biogenic amines in beers.
Special attention was given on the optimization of the DLLME
procedure, by careful evaluation of the nature and amount of
extractive and dispersive solvents as well as the amount of deriv-
ative reagent and the reaction time. The developed method was
used to assess the occurrence of 18 biogenic amines in beer samples
commercialized in Portugal.
2. Experimental
2.1. Reagents and materials
The amine standards were obtained, mostly as hydrochloride
salts, from Sigma (St Louis, MO, USA), Aldrich (Milwaukee, WI,
USA), and Fluka (Buchs, Switzerland). The deuterated internal
standards (IS), ethyl[2H5]amine HCl, a,a,b.b-[2H4]histamine.2HCl,
methyl[2H3]amine.HCl, 1,4-butane[2H8]diamine.2HCl (or [2H8]
putrescine.2HCl), and 2,2,3,3,4,4,5,5[2H8]pyrrolidine were supplied
by CDN isotopes (Qubec, Canada) through Regie (Montlugon,
France). The internal standards, amphetamine and hydrox-
yamphetamine sulphate were from Sigma. Stock standard solutions
(2.0 mg mL
1) of each free compound were prepared by weighing
and dissolving in 0.1 M HCl; the solutions were stored at 4  C in
silanized screw-capped vials with solid PTFE-lined caps (Supelco,
Bellefonte, PA, USA). Working standard solutions were prepared by
dilution and mixing of these solutions with 0.1 M HCl.
The derivatizing reagent isobutyl chloroformate (IBCF) was
supplied by Sigma. Dispersive solvents acetonitrile (MeCN),
acetone (AC) and methanol (MeOH) were high purity grade
solvents for pesticide residue analysis obtained all from Fluka.
Extractive solvents isooctane, carbon tetrachloride, chloroform,
tetrachloroethylene, trichloroethylene and toluene were high
purity solvents for HPLC analysis obtained from Sigma. HCl 0.1 M
and NaOH 10 M were also obtained from Fluka; the latter was
diluted with water at the time of analysis. Other chemicals were of
analytical grade. The solution of alkaline methanol was prepared by
dissolving KOH in methanol until saturation.
Ultrahigh purity He (helium) for GCeMS and N2 (nitrogen) for
solvent evaporation were obtained from Gasin (Maia, Portugal).
2.2. Sampling
A total of 22 samples of beers comprising 2 weissbier dark, 3 ales
trappist, 7 lager dark and 10 ales stout were purchased in local
supermarkets. All the samples were stored at room temperature
(20  C) protected from light and opened on the moment of analysis.
2.3. Sample preparation optimized
Five millilitres of sample were placed into a 25 mL screw cap
plastic, spiked with IS (50 mL of an HCl 0.1 M solution containing all
the internal standards at 100 mg L
1), NaOH 2.0 M was added until
the pH reaches 12 and buffered with phosphate buffer 0.5 M. A
mixture of MeCN (1 mL), toluene (325 mL) and IBCF (25 mL) was
382 C. Almeida et al. / Food Control 25 (2012) 380e388

rapidly injected into the sample tube. The tube was closed and
shaken gently by hand for 5 min. After that it was centrifuged at
5000 rpm for 2 min and 150 mL of the upper phase was transferred
for a vial and 75 mL of alkaline methanol was added. The tube was
shaken by hand 1 min, then 450 mL of NaOH 5 M was added and the
mixture was shaken for another 1 min. After centrifuging at
5000 rpm, the toluene layer was used for analyzing all amines
except tyramine and histamine.
A minor portion of the toluene layer (50 mL) was transferred
to an identical vial and evaporated to dryness under a stream of
nitrogen. The dry residue was redissolved in 25 mL of toluene
and the solution was used for analysis of tyramine and
histamine.
2.4. GCeMS equipment and conditions
The gas chromatograph 6890 (Agilent, Little Falls, DE, USA)
equipped with an electronically controlled split/splitless injection
port was interfaced to a single quadrupole inert mass selective
detector (5973N, Agilent) with electron impact ionization chamber.
GC separation was performed on DB-5MS capillary column
(20 m  0.18 mm I.D., 0.18 mm lm thickness) (J&W Scientic,
Folsom, CA, USA). Helium was the carrier gas with a constant
pressure of 30 psi. The injection was made in pulsed splitless mode
(injection pulse pressure 32 ps) at 280  C. The oven temperature
program was as follows: 100  C held for 1.2 min, ramped to 160  C
at 10  C min
1; then ramped to 280  C at 25  C min
1 and held for
13.3 min. Total run time was 25 min. The MS transfer line
temperature was held at 280  C.
Mass spectrometric parameters were set as follows: electron
impact ionization with 70 eV energy; ion source temperature,
230  C and MS quadrupole temperature, 150  C. The MS system
was routinely set in selective ion monitoring (SIM) mode and
each analyte was quantied based on peak area using one target
and one or more qualier ion(s). Complete SIM parameters and
retention times of the analytes are shown in Table 1. Agilent
Chemstation was used for data collection/processing and GCeMS
control.
3. Results and discussion
3.1. Optimization of extraction and derivatization conditions
Since the extractive and dispersive solvents nature, solvents
volume and derivatizing reagent volume can affect the DLLME
efciencies, these parameters were systematically studied in order
to achieve a good sensitivity, precision, and selectivity for all
biogenic amines in study.
3.1.1. Selection of extractive and dispersive solvents
In a rst stage, the selection of the optimal extractive solvent
was carried out. The extractive solvent was appraised aiming at
satisfying the following four requirements: higher or lower density
than water, (2) immiscibility with water, (3) good solubility for
derivatives and compatibility with derivatizing reagent, and (4)
good chromatographic behaviour. On the basis of these consider-
ations, isooctane (density: 0.83 g mL
1) and toluene (density:
0.87 g mL
1) with density lower than water and trichloroethylene
(density: 1.46 g mL
1) carbon tetrachloride (density: 1.59 g mL
1),
chloroform (density: 1.48 g mL
1), and tetrachloroethylene
(density: 1.62 g mL
1) with density higher than water, were
investigated in the preliminary experiments. Extractions were
carried out for 10 min from 5 mL of beer sample (with pH adjusted
to 12) spiked with 2.5 mg L
1 of all the biogenic amines and 25 mL of
IBCF with a combination of 250 mL of each extractive solvent with
1 mL of MeCN as dispersive solvent. The experiments in GCeMS
responses were signicant different for these solvents. Most of
derivative compounds were not efciently extracted by chloroform.
Trichloroethylene and carbon tetrachloride gave asymmetric peaks
with tailing, probably because the poor solubility of derivatives in
these solvents. Toluene shows a higher peak response for all
derivatives than those presented by isooctane and tetrachloro-
ethylene. It seems that the ring aromatic structure of toluene
benet the extraction of chloroformate derivatives. Thus, toluene
was selected as the extractive solvent.
The miscibility of the dispersive solvent in the extractive solvent
and in the aqueous phase (sample solution) is the main point for

Table 1
MS conditions for GCeMS analysis of biogenic amines and I.S. derivatized (time windows and ions selected in SIM mode, quantication ions in bold).

Injection Compounds Start window tR (min) m/z SIM ions (abundance)

time (min)
First [2H3] Methylamine (IS) 1.2 1.98 79 (92), 61 (100)
Methylamine 1.99 76 (100), 58 (99), 88 (8)
Dimethylamine 2.07 72 (86), 90 (100), 145 (2)
[2H5] Ethylamine (IS) 2.2 2.38 95 (100), 77 (78)
Ethylamine 2.40 90 (100), 72 (74), 130 (6)
Isopropylamine 2.58 144 (100), 86 (57), 104 (60)
Diethylamine 2.75 3.02 118 (58), 72 (32), 102 (46), 158 (24), 173 (4)
Isobutylamine 3.25 3.78 130 (63), 118 (34), 100 (11), 158 (3), 173 (5)
2-Methylbutylamine 3.95 4.97 130 (89), 187 (8), 114 (10), 132 (31)
Pyrrolidine 4.98 98 (53), 114 (48), 116 (100)
[2H8] Pyrrolidine (IS) 4.99 106 (50), 124 (100)
Isoamylamine 5.05 132 (56), 114 (17), 130 (56), 187 (8)
Morfoline 5.2 5.38 116 (48), 114 (42), 130 (34), 187 (12)
Piperidine 5.49 128 (100), 112 (30), 130 (56)
Amylamine 5.50 132 (99), 114 (18), 130 (80), 187 (5)
Amphetamine (IS) 8.8 9.17 144 (100), 162 (5), 91 (63)
2-Phenylethylamine 9.20 130 (100), 221 (35), 91 (59), 104 (70), 148 (18)
1,3-Diaminopropane 10.1 10.78 101 (86), 144 (62), 201 (16), 274 (8)
[2H8] Putrescine (IS) 11.25 11.34 176 (36), 296 (9)
Putrescine 11.37 170 (71), 130 (46), 288 (8)
Cadaverine 11.7 11.78 130 (76), 84 (78), 129 (72), 302 (9)
Second [2H4] Histamine (IS) 1.2 11.95 197 (65), 242 (7), 128 (15)
Histamine 11.96 194 (93), 238 (9), 138 (22)
Hydroxyamphetamine (IS) 12.52 144 (100), 107 (20)
Tyramine 12.62 120 (100), 107 (55), 176 (12), 237 (5), 337 (2)
 C. Almeida et al. / Food Control 25 (2012) 380e388
the selection of the dispersive solvent Cunha, Fernandes et al.
(2011), chp 1. In the preliminary experiments MeCN, MeOH and
AC were selected as dispersive solvents. The extraction was carried
out from 5 mL beer sample (with pH adjusted to 12) spiked with
2.5 mg L
1 of all the biogenic amines and 25 mL of IBCF with
a combination of 1 mL of each dispersive solvent with the extractive
solvent (toluene). The volume of extractive solvent was changed
simultaneously with the dispersive solvent in order to obtain
a constant volume of the upper phase (150 mL). Thus, 325, 345 and
330 mL of toluene was used with 1 mL MeCN, MeOH and AC,
respectively. The extraction efcient for most of the derivatives was
higher using acetonitrile as the dispersive solvent compared to
other solvents, as can be seen in Fig. 1. Therefore, acetonitrile was
selected as dispersive solvent.
3.1.2. Volume of extractive and dispersive solvents
To evaluate the effect of the extractive solvent on the extraction
efciency, a constant volume of dispersive (MeCN, 1 mL) containing
different volumes of toluene (250 mL, 325 mL, 500 mL and 750 mL)
was subjected to the same DLLME procedure. The low volume of
upper phase (85 mL) obtained with 250 mL of toluene makes repli-
cates impracticable, with consequently problems of reproducibility.
By increasing the volume of toluene from 325 mL to 750 mL the
volume of upper phase increased from 150 mL to 600 mL. However,
the enrichment factors [(%Recover  (Vaq/Vsed))/100; Vaq e
volume aqueous Vsed- volume sedimented phase] decreased from
39 to 5 (Fig. 2). Thus, 325 mL was selected in order to obtain high
enrichment factors and low detection limits.
To study the effect of the dispersive solvent volume on extrac-
tion efciency, different volumes of MeCN (from 0.5 to 2 mL with
gaps of 0.5 mL) containing 25 mL of IBCF and 325 mL of extractive
solvent were tested in a beer sample. The results indicate that with
the MeCN volume increment the extraction efciency was higher
(from 0.5 to 1.0 mL), and then decreased (from 1 mL to 2 mL) for all
derivatives. It was observed that with volume of dispersive solvent
above 1.5 mL the upper phase volume also increase (450 mL in the
case of 2 mL), and consequently peak response decreased. Thus,
based in experimental results 1.0 mL of MeCN was chosen as the
optimum volume for the dispersive solvent.
25000000
MeCN
20000000
k response
15000000
Peak
10000000
5000000
Fig. 1. Comparison of average peak area response of different dispersive solvents MeCN, AC and MeOH using toluene as extractive solvent (n 2).
383
3.1.3. Selection of derivatizing reagent, effects of its volume and
reaction time
Alkyl chloroformates constitutes a group of derivatizing
reagents with very favourable characteristics to the gas chro-
matographic determination of compounds with amino groups. In
the present study, IBCF was chosen taking into account its avail-
ability, simplicity of use and time consumed. The derivatization
with IBCF was successfully applied in a previous work for the
determination of biogenic amines in beer (Cunha, Faria, &
Fernandes, 2011; Fernandes, Judas, Oliveira, Ferreira, & Ferreira,
2001).
Hence, the simultaneous DLLME- derivatization procedure was
evaluated in order to select both optimum volume of IBCF and the
reaction time. The inuence of derivatizing reagent volume was
evaluated by using 1 mL of MeCN containing 325 mL of toluene and
different volumes of IBCF (25, 50 and 100 mL) in a sample spiked
with 1 mg L
1 of all biogenic amines in studied. The highest
derivatization reaction yield was observed when the volume of
25 mL of IBCF was used, thus it was chosen as a volume of deriva-
tizing reagent. Because of the high pKa values of the investigated
biogenic amines, an extraction medium at pH 12 was used which
guarantee a good yield derivatization of the amine function (Lundh
& Akesson, 1993).
Under the optimal conditions chosen, experiments were made
with increasing reaction times, from 1 min to 10 min. The experi-
mental results showed an increase of analytical signal, especially
for the diamines putrescine and cadaverine, up to 5 min. Higher
reaction times did not result in the increment of the peak areas of
the analytes. Therefore, 5 min was chosen as the optimum reaction
time.
3.1.4. Other factors
Derivatization with IBCF has many advantageous properties as
above mentioned, one of which is that no standing or heating
period is needed following the derivatization. However, elimina-
tion of excess reagent is considered necessary to prevent degra-
dation of the GC column (Cunha, Faria et al., 2011; Fernandes et al.,
2001). In the procedure described here, the IBCF derivatives
into toluene were washed with an alkaline methanol solution to
MeOH AC
384 C. Almeida et al. / Food Control 25 (2012) 380e388
80
250 L 325 L 500 L
70
60
Enrichment factor
50
40
30
20
10
Fig. 2. Enrichment factor obtanied using different volumes of extractive solvent (toluene).
remove the excess of derivatization reagent. Different volumes of
both alkaline methanol solution (50e500 mL) and 5 M NaOH
(300 mLe1.0 mL) were experimented. The combination of 75 mL of
alkaline methanol solution with 450 mL of 5 M NaOH was chosen
as the best for the elimination of excess derivatization reagent.
The wash step was not applied for quantication of tyramine
and histamine due to its partial or total degradation by the alkaline
solution used. Thus, histamine and tyramine were analysed after
eliminating the excess of IBCF by evaporation, which did not cause
loss of sensitivity.
The great novelty of the method here proposed is the high
enrichment factor achieved in combination with the simplicity of
execution, which allows the processing of a large number of
samples within a working day, with high levels of accuracy and
sensitivity. Further, the use of GCeMS allowing the simultaneous
detection of most of the biogenic amines of interest makes the
developed method a true alternative for the routine determination
of these compounds in beer samples.
3.2. Analytical features of the method
3.2.1. Linearity
Ten aqueous solutions containing all amines under study with
concentrations ranging from 0.010 to 15.0 mg L
1 were submitted to
the whole analytical procedure. The results obtained showed that
linearity were excellent for all the compounds with correlation
coefcients ranging from 0.9976 to 0.9990. Amine concentrations
were measured using the ratio of the peak areas of the target ion
chosen for the amine and those of the corresponding IS. To allow
both an unambiguous identication and quantication of biogenic
amines in study the following IS were used: [2H3] methylamine for
methylamine and dimethylamine, [2H5] ethylamine for ethylamine,
diethylamine, and isopropylamine, 2,2,3,3,4,4,5,5 [2H8] pyrrolidine
for pyrrolidine, piperidine and morpholine, amphetamine for
2-phenylethylamine, [2H8] putrescine for cadaverine, putrescine,
1,3-diaminopropane, amylamine, isoamylamine and 2-methyl-
butylamine, a,a,b.b-[2H4]histamine for histamine, and hydrox-
yamphetamine for tyramine. Most of the IS used were deuterated
750 L
because they possesses virtually the same chromatographic,
desorption, ionization and fragmentation characteristic as the cor-
responding compound, as can be seen in Fig. 3.
3.2.2. Recovery
The recovery was determined by comparing unspiked samples
to spiked samples for two concentration levels (0.05 mg L
1 and
0.25 mg L
1), being each level performed six times. The average
recovery values ranged from 72 to 113% as can be seen in Table 2.
Additionally, it was constructed a calibration curve in a sample with
a known amount of biogenic amines, by adding six levels of
concentrations of all amines (0, 0.05 mg L
1, 0.1 mg L
1, 0.25 mg L
1,
0.50 mg L
1 and 1.0 mg L
1). The samples were treated as described
for the overall method and injected twice. The correlation coef-
cients obtained were higher than 0.995.
3.2.3. Intra-day and inter-day precision
The intra-day precision was determined by analysing in the
same day six replicates of beer samples spiked at two levels
(0.05 mg L
1 and 0.25 mg L
1); each replicate was submitted to the
overall developed method. Inter-day precision was determined by
analysis of samples on three different days over a period of three
weeks. The relative standard deviation (RSD) for inter-day precision
ranged from 1% to 13% and for intra-day precision ranged from 1 to
14% (Table 2).
3.2.4. Limits of detection (LODs) and limits of quantication (LOQs)
The LODs of the proposed method were determined by
successive analyses of chromatographic extracts of aqueous solu-
tion spiked with decreasing amounts of the analytes until a signal-
to-noise ratio 3:1 was reached, whereas the LOQs were determined
considering a signal-to-noise ratio of 10:1. The LODs ranged from
0.3 to 2.9 mg L
1 and the LOQs ranged from 1.0 to 9.5 mg L
1. The
LODs values are lower than those reported in our previous paper
(Fernandes et al., 2001) with 1.0 mg L
1 for most of the biogenic
amines as well as those reported by other authors, e.g. 1e150 mg L
1
(Zotou et al., 2003) or 5.0e126.6 mg L
1 (Cortacero-Ramrez, Arrez-
Romn, Segura-Carretero, & Fernndez-Gutirrez, 2007).
 C. Almeida et al. / Food Control 25 (2012) 380e388 385

Fig. 3. Mass spectrum of A) [2H5] ethylamine and ethylamine B) [2H8] putrescine and putrescine.

3.3. Analysis of biogenic amines in beers
The reliability of the proposed method was evaluated by
analyzing a set of 22 commercial beer samples acquired in Portugal.
As can be seen in Table 3 methylamine, dimethylamine, ethylamine,
diethylamine, isobutylamine, isoamylamine, pyrrolidine, piperi-
dine, 2-phenylethylamine, 1,3-diaminopropane, putrescine, cadav-
erine and tyramine could be detected and quantied in all the
analyzed samples, with the exception of histamine which appear
in only some samples. Isopropylamine, 2-methylbutylamine,
morfoline and amylamine were not found in any of the samples
analyzed. Fig. 4 shows a total ion chromatogram of a beer obtained
using the developed method.
The methylamine was determined as the principal primary
amine in the analyzed beers, with levels ranging from 0.026 to
0.610 mg L
1, following by isobutylamine (from 0.134 to
0.234 mg L
1), isoamylamine (from 0.033 to 0.590 mg L
1) and
ethylamine (from 0.027 to 0.272 mg L
1). A previously published
report (Fernandes et al., 2001) indicates that the primary amines
(ethylamine, methylamine and isoamylamine) have been detected

Table 2
average recoveries (%), intra-day repeatability (%RSD), inter-day repeatability (%RSD) and limits of quantication (LOQ- mg L
1) detection (LOD - mg L
1) obtained with the
DLLME method in spiked beer samples, analyzed by GCeMS (n 6 at each level).

Compounds Concentration levels Interday (% RSD) LOQ (mg L
1) LOD (mg L
1)

0.05 mg L
1 0.25 mg L
1

Recovery (%) Intraday (%RSD) Recovery (%) Intraday (%RSD)

Methylamine 82 11 100 13 6 5.3 1.6
Dimethylamine 72 12 76 12 14 2.5 0.8
Ethylamine 90 5 113 6 6 6.6 2.0
Isopropylamine 73 10 111 6 8 4.6 1.4
Diethylamine 74 9 83 5 6 1.0 0.3
Isobutylamine 79 9 83 1 8 3.0 0.9
2-Methylbutylamine 87 11 94 1 7 4.3 1.3
Pyrrolidine 109 8 106 7 7 7.6 2.3
Isoamylamine 83 3 88 1 2 5.6 1.7
Morfoline 72 11 95 7 10 1.0 0.3
Piperidine 75 12 103 4 7 3.7 1.1
Amylamine 80 10 94 6 10 1.1 0.3
2-Phenylethylamine 80 6 101 2 6 6.6 2.0
1,3-Diaminopropane 76 6 77 1 7 9.0 2.7
Putrescine 73 3 87 1 1 1.7 0.5
Cadaverine 83 8 81 2 6 1.0 0.3
Histamine 75 7 88 5 4 9.5 2.9
Tyramine 95 5 83 2 5 7.0 2.1
 386 C. Almeida et al. / Food Control 25 (2012) 380e388

1,3-Diamino- Putrescine Cadaverine Histamine Tyramine at levels between 0.011 (isoamylamine) and 0.264 mg L
1 (ethyl-
amine) in beers. These differences could be related with the sample
0.800
3.034
0.871
2.196
2.141
0.727
0.884
0.394
0.695
0.752
3.345
4.558
5.916
0.669
0.620
0.968
0.442
0.791
0.790
0.666
0.989
0.538
type analyzed.
The secondary amine, dimethylamine, which is important as
a potential precursor of the carcinogen dimethylnitrosamine, was
found with levels ranging from 0.119 to 1.929 mg L
1. The dime-
0.339
0.312

0.152

0.020

0.140

0.017
0.036
0.160

0.065
0.026
n.d.

n.d.
n.d.

n.d.
n.d.
n.d.

n.d.
n.d.
n.d.

n.d.
n.d.
n.d.
thylamine is probably derived from gramine and hordennine
during the kilning process of malt production (Smith, 1980). The
other secondary amine quantied was diethylamine, which levels
ranged from 0.059 to 0.082 mg L
1.
0.404
0.187
0.396
0.458
0.634
0.552
0.670
0.562
0.375
0.324
0.473
0.354
0.319
0.948
1.381
0.974
0.370
0.438
0.846
0.467
1.059
1.234
Among the heterocyclic amines found in the analyzed beers,
histamine is described as the most toxic for human. Histamine was
present in ten of the twenty two samples analyzed, with levels
6.322
2.097
3.850
4.745
12.777
6.889
10.900
10.177
6.025
6.394
5.202
5.159
4.124
9.016
7.535
7.778
7.969
4.956
6.933
6.940
10.499
9.897
ranging from 0.017 to 0.339 mg L
1. Different values were found in
a previous work from our group in which histamine levels ranged
from 0.03 to 0.143 mg L
1. On the other hand, the determined levels
were slightly lower than those observed by other authors that re-
ethylamine propane

ported the presence of histamine in all the samples analyzed with

0.388
0.153
0.372
0.344
0.226
0.191
0.548
0.470
0.130
0.166
0.474
0.438
0.274
0.350
0.358
0.214
0.239
0.209
0.304
0.289
0.462
0.539
levels ranging from 0.30 to 1.20 mg L
1 (Izquierdo-Pulido, Vidal-
Carou, & Marin-Font, 1989), from 0.63 to 5.80 mg L
1 (Izquierdo-
Pulido et al., 1996), from 0.48 to 2.11 mg L
1 (Zotou et al., 2003)
Methylamine Dimethylamine Ethylamine Diethylamine Isobuthylamine Pyrrolidine Isoamylamine Piperidine 2-phenyl-

or from 0.10 to 0.62 mg L
1 (Cortacero-Ramrez et al., 2007). These

0.045
0.042
0.040
0.054
0.115
0.069
0.059
0.057
0.065
0.074
0.044
0.065
0.050
0.089
0.045
0.154
0.047
0.055
0.060
0.046
0.068
0.065

differences could be related with microbial contamination during

brewing.
Other heterocyclic amines, pyrrolidine and piperidine, were
0.016
0.014
0.015
0.016
0.026
0.021
0.023
0.022
0.019
0.022
0.016
0.025
0.023
0.050
0.025
0.072
0.054
0.019
0.029
0.041
0.023
0.029

found in beers at levels ranging from 0.126 to 1.777 mg L
1 and from
0.014 to 0.072 mg L
1, respectively. The presence of pyrrolidine in
beer samples is scarcely reported in literature, with exception of
Fernandes et al. (2001) and Slomkowska and Ambroziak (2002)
which found levels from 0.04 to 0.186 mg L
1 and from not detec-
0.055
0.043
0.047
0.057
0.073
0.066
0.066
0.057
0.059
0.068
0.051
0.058
0.056
0.033
0.054
0.162
0.076
0.590
0.064
0.050
0.060
0.066

ted to 0.570 mg L
1, respectively. Piperidine, never before reported

in beer samples, was the biogenic amine found at the lower levels in
the samples analyzed. The high sensitivity allowed by the present
method could explain its detection.
0.131
0.128
0.126
0.205
0.542
0.363
0.297
0.273
0.291
0.357
0.185
0.463
0.324
0.845
0.138
1.562
1.777
0.283
0.565
0.176
0.353
0.447

Putrescine, cadaverine and 1,3-diaminopropane were the

diamines found in the analyzed beers. Putrescine levels ranged
from 2.097 to 12.777 mg L
1, which are slightly higher than those
reported in our study ten years ago with levels ranging from 1.353
to 3.355 mg L
1. This amine is the most predominant amine in beers
0.137
0.136
0.138
0.141
0.161
0.149
0.148
0.147
0.147
0.155
0.139
0.161
0.134
0.188
0.141
0.234
0.140
0.150
0.157
0.134
0.152
0.159

as reported in literature; its levels could arrive to 22 mg L
1 as

found in extra beer by Cortacero-Ramrez et al. (2007). Cadaverine
levels ranged from 0.187 to 1.381 mg L
1; similar values were ob-
tained in our previous work, with levels ranging from 0.124 to
0.066
0.063
0.062
0.062
0.064
0.078
0.082
0.063
0.062
0.064
0.063
0.063
0.059
0.070
0.080
0.074
0.069
0.070
0.070
0.077
0.066
0.062

0.811 mg L
1. 1,3-diaminopropane levels ranged from 0.130 to

0.548 mg L
1, which is slightly lower than those reported in our
previous work (Fernandes et al., 2001).
Tyramine was one of the most expressive amine in the analyzed
0.064
0.113
0.116
0.121
0.173
0.028
0.039
0.056
0.125
0.118
0.027
0.096
0.135
0.052
0.109
0.135
0.032
0.245
0.079
0.116
0.272
0.240

beers, ranging from 0.394 to 5.916 mg L
1. These levels were lower
Biogenic amine levels found in different type of beer (n 2).

than those reported in literature which ranged from 0.60 to

17.21 mg L
1 (Buiatti, Boschelle, Mozou, & Battistutta, 1995), from
1.0 to 7.75 mg L
1 (Glria & Izquierdo-Pulido, 1999), from 3.10 to
Biogenic amines (mg L
1)

22.52 mg L
1 (Izquierdo-Pulido et al., 1996), from 0.17 to

0.423
0.119
0.270
0.238
1.735
1.361
1.929
1.858
0.348
0.588
0.287
0.807
0.454
1.580
0.389
1.104
1.225
1.407
1.802
1.426
1.240
0.995

31.60 mg L
1 (Romero et al., 2003) or from 1.42 to 7.120 mg L
1

(Zotou et al., 2003). The presence of this amine in beers could be
related with its presence in malt or with its formation during
mashing and wort boiling.
Beyond tyramine, 2-phenylethylamine is the other aromatic
0.092
0.026
0.051
0.062
0.210
0.610
0.196
0.178
0.103
0.132
0.079
0.183
0.102
0.190
0.320
0.239
0.527
0.169
0.363
0.125
0.186
0.184

amine that was present in all the samples, with levels ranging from
0.040 to 0.154 mg L
1, similar to those reported by Glria and
alcohol

Izquierdo-Pulido (1999), ranging from 0.05 to 0.18 mg L
1, but

n.d. - not detected.
5.2
4.1
4.7
4.1
5.8
5.0
6.6
6.4
5.0
5.0
6.2
4.1
4.1
4.8
5.4
5.5
4.8
6.5
Trappist 9.0
7.0
Weissbier 5.6
7.3
%

slightly higher than those previously reported by our group, with

levels ranging from 0.012 to 0.057 mg L
1 (Fernandes et al., 2001).
Stout

Dark

Although 2-phenylethylamine could be formed by heat decarbox-

Table 3

Beers

Lager
Type

Alles

Alles

ylation of phenylalanine during the severe heat treatment applied

to the malt in the manufacturing of some dark beers (Cerutti, Finoli,
 C. Almeida et al. / Food Control 25 (2012) 380e388 387

Fig. 4. Total ion chromatogram of a weissbier beer with 7.3% of alcohol: 1) [2H3] methylamine (IS, ion 79), 2) methylamine (ion 76), 3) dimethylamine, 4) [2H5] ethylamine (IS; ion
95) 5) ethylamine (ion 90), 6) diethylamine (ion 118), 7) isobutylamine (ion 130), 8) [2H8] pyrrolidine (IS, ion 106), 9) pyrrolidine (ion 98), 10) isoamylamine (ion 132), 11) piperidine
(ion 128), 12) amphetamine (IS; ion 144), 13) 2-phenylethylamine (ion 130), 14) 1,3-diaminopropane, 15) [2H8] putrescine (IS, ion 176), 16) putrescine (ion 170), 17) cadaverine (the
concentration of each biogenic amine is given in Table 3).

Peluzzi, & Vecchio, 1985), particularly high levels of this amine were
not found in dark beers.
The total concentrations of biogenic amines (mg L
1) followed the
order: putrescine > tyramine > dimethylamine > cadaverine>
pyrrolidine>1,3-diaminopropane, similarly to those obtained in our
previous study (Fernandes et al., 2001).
4. Conclusions
A new simultaneous DLLME- derivatization procedure followed
by GCeMS analysis was developed and validated for a reliable
determination of 18 biogenic amines in beers. The main conclu-
sions of the study can be summarized as follows:
- An efcient and high reproducible extraction was achieved by
DLLME- derivatization procedure for all biogenic amines using
1.0 mL of acetonitrile as dispersive solvent, 325 ml of toluene as
extractive solvent and 25 mL IBCF as derivatizing reagent.
- The use of several deuterated internal standards allowed
accurate identication and quantication of all the biogenic
amines, including volatile amines scarcely reported in
literature.
- For all biogenic amines assayed the gures of merit obtained
from validation, namely linearity, recovery, precision and limits
of quantication and detection were very acceptable.
- The concentrations of biogenic amines determined in 22 beers
commercialized in Portugal decreased in the order: putrescine,
tyramine, dimethylamine, cadaverine, pyrrolidine, 1,3-dia-
minopropane. The levels of biogenic amines found in the beers
analyzed were comparable with the previous research studies
carried out in Portugal as well as in Europe.
Acknowledgments
This research was supported by a grant from the FCT project
PTDC/AGR-ALI/101583/2008,COMPETE FSE/FEDER/OE and PEst-
C/EQB/LA0006/2011. S.C.C. is grateful to POPH-QREN- Tipologia
4.2, Fundo Social Europeu e Fundo Nacional MCTES. C. Almeida is
grateful to FCT for a grant under the Project BII/REQUIMTE/Car-
acterizao Qumica de Alimentos/2009.
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