Food Chemistry 167 (2015) 71–77

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Method development by GC–ECD and HS-SPME–GC–MS for beer volatile

analysis
Gisele C. da Silva ⇑, Abner A.S. da Silva, Larissa S.N. da Silva, Ronoel Luiz de O. Godoy,
Luciana C. Nogueira, Simone L. Quitério, Renata S.L. Raices
Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro (IFRJ), Mestrado Profissional em Ciência e Tecnologia de Alimentos (PCTA), Rua Senador Furtado, 121,
20270-021 Rio de Janeiro, RJ, Brazil

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

Article history: Two methods for the extraction, identification and quantification of the highest occurrence and lowest
Received 14 June 2013 perception threshold off-flavours in fifteen different samples of Brazilian Pilsner beers were developed.
Received in revised form 18 February 2014 For this purpose, headspace solid phase microextraction in combination with a gas chromatography
Accepted 8 June 2014
coupled to a mass spectrometric detection (HS-SPME–GC–MS) as well as headspace extraction in combi-
Available online 16 June 2014
nation with a gas chromatography coupled to electron capture detection (HS–GC–ECD) were evaluated.
The first and the second methods were used for esters and vicinal diketones analysis, respectively. All
Keywords:
data were comprehended below the taster‘s threshold detection limit: ethyl acetate 39.48 ng mL 1
Beer
Off-flavours
(RSD mean value 4.2%), isoamyl acetate 3.88 ng mL 1 (RSD mean value 3.4%), ethyl hexanoate
SPME (solid-phase microextraction) 0.61 ng mL 1 (RSD mean value 3.1%) and 2,3-butanedione 0.10 ng mL 1 (RSD mean value 2.9%). The
HS (headspace) validated method demonstrated to be useful for the analysis of highest incidence beer off-flavours.
Gas chromatography Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction is possible to assume there is a problem in the fermentation

The beer consumer has increasingly demanded products with
sensory appeal. Moreover, the search for the product which has
the best quality and lowest price has been increasing among those
consumers. For these reasons, breweries are concerned with the
market, and it has motivated the adoption of a more strict quality
control (sensory and physicochemical characteristics) of the final
products.
Beer is an alcoholic beverage obtained by fermentation pro-
cesses and it is a very complex mixture. Therefore, the chemical
composition of different beers available varies considerably. The
presence of aromatic substances in beer is very important because
it contributes to the quality of the final product (Silva et al., 2012).
The balance of volatile and non-volatile compounds (esters, alde-
hydes, vicinal diketones, organic acids, higher alcohols, phenols,
iso-alpha-acids and other compounds) is closely related to accep-
tance and quality of the beverage (Siqueira, Bolini, & Macedo,
2008). Esters and diketones, which are responsible for off-flavours,
are normally present in beer and when their contents are higher it
⇑ Corresponding author. Address: Jose Luiz Ferraz Avenue, 550/apt 605. Recreio
dos Bandeirantes, CEP 22790-587, Rio de Janeiro, RJ, Brazil. Tel.: +55 21 7515 7632;
fax: + 55 21 3346 1813.
E-mail address: gisele.dasilva@ig.com.br (G.C. da Silva).
process (Araújo, Silva, & Minim, 2003). The sensory analysis is per-
formed by a tasting panel, which consists of brewers with experience
in the type of sensory tests as well as regular consumers (no experi-
ence in sensory testing) that can be selected and trained for tasting
beers. The training of this panel is accomplished through periodic
training using both commercial beers as test samples as well as beers
added with possible off-flavours. These flavours or odors are often
associated with beer deterioration (Hughes & Baxter, 2001). The
concentration thresholds for sensory detection of the off-flavours
normally present in beer are: isoamyl acetate (banana flavour) –
1400 ng mL 1, ethyl acetate (vernish aroma) – 15,000 ng mL 1, ethyl
hexanoate (apple flavour) – 200 ng mL 1 and 2,3-butanedione
(butter flavouring) – 40 ng mL 1 (Flavouractiv, 2005).
Because of concern about the health of the panelist, the number
of tests to be performed must be limited, requiring thus a greater
number of panelists to take part of the study. This fact is important
once the development of an analytical methodology as an alterna-
tive to identify and quantify off-flavours is required (Rudnitskaya
et al, 2009).
A recent review, in a search for years 2006–2011, has shown
that the most analyzed foods with respect to flavour/volatile
compounds by means of solid phase microextraction are wines fol-
lowed by fruits/vegetables, dairy products, beverages, meat, spice
and herbs, cereals/bakery, snacks, fats and oils, seafood/fish and

http://dx.doi.org/10.1016/j.foodchem.2014.06.033
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
72 G.C. da Silva et al. / Food Chemistry 167 (2015) 71–77
honey. HS-SPME, a solvent-free sample preparation technique,
allows simiultaneous sampling, extraction, pre-concentration and
addition of analytes into a gas chromaographer in a single step,
extending thus the column life and preventing contamination dur-
ing sample injection. The technique can be easily automated with a
XYZ type autosampler or it can also be used in a manual form with
a reusable holder in which the fibres can be replaced. According to
the review, the fibre coating in which the adsorption process dom-
inates is known as Divinylbenzene/Carboxen/Polydimethylsilox-
ane (DVB/CAR/PDMS) and manual or automated procedure offers
high reproducibility (Jelén, Majcher, & Dziadas, 2012).
In a study of flavour fingerprinting of beer by means of a flame
ionization detection, HS-SPME–GC–FID, using divinylbenzene/
Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS), a total of 28
samples of four brands were analyzed and classified into three
main types: chopp, traditional beer and dark beer. A total of 18
volatile compounds were identified by GC-MS (Jiao et al., 2011).
Brazilian beer samples were used for the determination of
(E)-2-nonenal using HS-SPME–GC–MS with Carboxen/Poly-
dimethylsiloxane (CAR/PDMS) fibre (Scherer, Roger, Kowalski, &
Teixeira, 2010). E-2-nonenal is an off-flavour (cardboard flavour)
from the fermentation process of beer. The developed method
proved to be simple, efficient and sensitive for the determination
of this analyte, which can also be easily applied in quality control
programs. (E)-2-nonenal was found in all the beer samples, with
concentrations ranging from 0.17 to 0.42 mg L 1.
Headspace solid phase microextraction coupled to a chromato-
graphic process is a very well-established procedure for analysis of
volatile off-flavours in beverages. Research has shown the applica-
tion of HS–GC–MS (Charry-Parra, DeJesus-Echevarria, & Perez,
2011; Tian, 2010) and HS-SPME coupled to gas chromatography/
tandem mass spectrometry (GC-MS/MS) using fibres recovered
with Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/
PDMS) for the determination of volatile compounds in beers, such
as: monophenols, aldehydes, alcohols, 4-ethylguaiacol, 4-ethyl-
phenol, 4-vinylguaiacol and 4-vinylphenol (Jelén et al., 2012;
Pizarro, Pérez-del-Notario, & González-Sáiz, 2010; Sterckx,
Saidson, & Delvaux, 2010).
The objective of this study was to validate methods for use in
quality control programs for the identification and quantification
of off-flavours in beer. The esters were analyzed using the standard
addition method by HS-SPME–GC–MS, with SPME fibre containing
divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS)
film thickness. The 2,3-butanedione was analyzed by internal stan-
dardization (2,3-pentanedione) using headspace extraction in
combination with a gas chromatography coupled to electron
capture detection (HS–GC–ECD).
The accurate quantification of specific off- flavours in a complex
matrix like beer is a difficult process, especially when we consider
the competition phenomena caused by many volatiles compounds
(Hafez & Wenclawiak, 2013; Jelén et al., 2012). The methods were
applied to 15 beers marketed in the southeastern Brazil.
2. Materials and methods
2.1. Samples and chemicals
Samples from 15 different brands of Pilsner beer known in the
domestic market of southeastern Brazil were obtained at local
supermarkets. Ethyl acetate, ethyl hexanoate and 2,3-butanedione
were purchased from Sigma Aldrich (USA), while 2,3-pentanedione
and isoamyl acetate were purchased from Merck (Germany);
HPLC-grade sodium chloride, methanol and ethanol were from
J.T. Baker (Brazil). Water was obtained from a Millipore Milli-Q
water purification system.
Three stock solutions were prepared. The first solution was a
mixture of esters at a concentration of 0.09 g mL 1 of ethyl acetate,
0.0006 g mL 1 of ethyl hexanoate and 0.0042 g mL 1 of isoamyl
acetate. The second solution was composed of 0.001 g mL 1 of
2,3-butanedione. The third solution was composed of 0.0005 g mL 1
of 2,3-pentanedione (internal standard).
Calibration curves were prepared from dilutions of the stock
solutions using a 10 mL volumetric flask. The concentration range
of esters curve was from 0.1 to 108,000 ng mL 1 (18 to
108,000 ng mL 1 for ethyl acetate, 0.1 to 720 ng mL 1 for ethyl
hexanoate and 0.8 to 5040 ng mL 1 for isoamyl acetate). The range
of concentration of 2,3-butanedione curve was 0.06 to 1200 ng mL 1
(128 ng mL 1 internal standard).
Quantitation was performed using a calibration curve prepared
on the day of the assay. Quantification of the esters was performed
by using the spiking method in beer (standard addition method).
The quantification of 2,3-butenodione was made by internal stan-
dardization using 2,3-pentanedione (internal standard).
2.2. HS-SPME procedure and GC-MS analysis
Before analysis, beer bottles were cooled at 5 °C to prevent loss
of volatiles.
A total of 10 mL of beer and aliquots of the stock solution of
ester patterns were added to 25 mL flasks. The volume was trans-
ferred to headspace vials (40 mL) that contained 3.0 g of sodium
chloride and magnetic stirrers. The vials were sealed with polyeth-
ylene and silicone septum cap.
The samples were conditioned in thermostatic bath at
70 ± 1.0 °C for 1 h. After this sample/headspace equilibration per-
iod, the septum covering the vial headspace was pierced with the
needle containing the fibre retracted and then the fibre was
exposed to the headspace for 30 min at 60 ± 1.0 °C. SPME Fibre
50/30 lm Diviniylbenzene Carboxen/Polydimethylsiloxane (DVB/
CAR/PDMS) and manual holder were purchased from Supelco Park
(USA). The extracted analytes were desorbed in the injection port
of GC–MS.
The analyses were performed using a gas chromatographer
(Hewlett-Packard model 7890A) fitted with a splitless injection
with a liner suitable for SPME analysis and an Agilent 5975C mass
spectrometer (MS) detector in Full-Scan mode. Helium was used as
the carrier gas at a flow rate of 1 mL min 1.
Samples and chemical standards we analyzed on a HP-5 capil-
lary column 30 m  250 lm  1.0 lm film thickness (19091J-
413-Agilent Technologies, USA). The injector temperature was
260 °C. The oven temperature was programmed as follows: 2 min
at 35 °C ? 8 °C min 1 ? 100 °C ? 20 °C min 1 ? 160 °C ? 30 °C
min 1 ? 220 °C. The MS transfer line was kept at 250 °C.
2.3. HS procedure and GC–ECD analysis
A total of 10 mL of cold beer at 5 °C was added into a 25 mL vol-
umetric flask and aliquots of the stock solution of 2,3-butanedione
and 2,3-pentanedione standards and water were added to complete
the volume. The volume was transferred to vials (40 mL of head-
space) containing 3.0 g of sodium chloride and magnetic stirrers.
The vials were sealed with silicone septum and aluminium seal.
The samples were conditioned in a thermostatic bath at
70 ± 1.0 °C for 1 hour. Vials were transferred to an auto sampler
(model Agilent 7697A) of the chromatographic system and main-
tained for 15 min at 60 °C to establish themselves equilibrium
between headspace and sample.
The analyses were performed using a gas chromatographer
(Hewlett-Packard model 7890A) fitted with a splitless injection
with a liner suitable for HS analysis and Agilent electron capture
detector (ECD). The injector temperature was 250 °C. The
 G.C. da Silva et al. / Food Chemistry 167 (2015) 71–77 73

Abundance a

ethyl hexanoate
isoamyl acetate
ethyl

Time (mim)

Abundance b
2,3- butanedione

2,3- pentanedione

Time (mim)

Fig. 1. (a) Chromatogram obtained from beer sample spiked with (a) ethyl acetate (Rt: 2.275 min–90,000 ng mL 1), isoamyl acetate (Rt: 6.783 min–4200 ng mL 1), ethyl
hexanoate (Rt: 9.410 min–600 ng mL 1), 2,3-butanedione (not detected – 1200 ng mL 1) and 2,3-pentanedione (Rt: not detected – 200 ng mL 1). (b) Chromatogram obtained
from beer sample spiked with 2,3-butanedione (Rt: 2.595 min–1200 ng mL 1) and 2,3-pentanedione (Rt: 4.527 min–200 ng mL 1).

Table 1
1
Repeatability in beer, at taster’s threshold detection limit (ng mL ): 2,3-butenodione by HS–GC–ECD and isoamyl acetate, ethyl acetate and ethyl hexanoate by HS-SPME–GC–
MS.

Parameter 2,3-Butanodione Isoamyl acetate Ethyl acetate Ethyl hexanoate

day 1 day 2 day 3 day 1 day 2 day 3 day 1 day 2 day 3 day 1 day 2 day 3
38.2 40.3 41.7 1366.9 1387.2 1305.0 14,067.7 14,410.0 14,180.9 187.6 192.1 189.1
38.2 39.4 39.3 1434.3 1391.5 1364.3 14,855.5 15,591.4 14,329.2 198.1 207.9 191.1
40.5 42.1 40.8 1482.4 1416.7 1364.3 14,868.0 15,719.4 14,730.3 198.2 209.6 196.4
40.5 38.8 39.8 1366.9 1418.3 1412.4 14,868.0 14,279.2 14,986.5 198.2 190.4 199.8
40.9 39.7 40.1 1316.5 1373.6 1458.9 15,408.6 14,410.0 15,954.0 205.4 192.1 212.7
Average 39.6 40.1 40.3 1393.4 1397.4 1381.0 14,813.5 14,882.0 14,836.2 197.5 198.4 197.8
RSDa (%) 3.3 3.1 2.3 4.7 1.4 4.2 3.2 4.8 4.7 3.2 4.8 4.7
a
Relative standard deviation.

components were separated on a DB-5 capillary column 2.4. Statistical analysis

60 m  530 lm  1.5 lm film thickness (125-5062-Agilent Tech-
nologies, USA). Nitrogen was used as the carrier gas at flow rate Linear regression was applied for the working range, and mean
of 1 mL min 1. The analysis time was 10 min with isotherm at values, standard deviations, and variances were computed using
50 °C. Microsoft Excel 2010 for Windows.
74 G.C. da Silva et al. / Food Chemistry 167 (2015) 71–77

Table 2
Precision and Accuracy calculations.

Parameter 2,3-Butanedione Isoamyl acetate Ethyl acetate Ethyl hexanoate

Added Observed Added Observed Added Observed Added Observed
1
(ng mL ) (ng mL 1) (ng mL 1
) (ng mL 1) (ng mL 1
) (ng mL 1) (ng mL 1
) (ng mL 1)
38.8 40.0 1426.6 1326.4 15,180.0 15,180.0 210.6 238.5
37.7 1330.5 16,424.6 229.0
39.1 1354.6 16,559.3 238.2
38.2 1356.1 15,042.2 200.6
38.5 1313.4 15,180.0 238.2
Average 38.7 1336.2 15,677.2 228.9
Precision (%) 2.3 1.4 4.8 7.1
Accuracy (%) 96.8 95.4 104.5 108.7

Table 3 Table 4
Ruggedness: 2,3-butanedione (HS–GC–ECD) and isoamyl acetate, ethyl acetate and Concentration of the main off-flavors found in 15 known-brand beers analyzed by HS-
ethyl hexanoate (HS-SPME–GC–MS). SPME–GC–MS and HS–GC–ECD techniques.
1
Name Time(min) Concentration (ng mL ) RSDa Beers brand recognized Concentration (ng mL 1
)
in the market
2.3-Butanedione 56.4 41.2 39.5 40.3 2.1 HS–GC–ECD HS-SPME–GC–MS
60.0 38.8 40.0 40.7 2.4
2.3- Isoamyl Ethyl Ethyl
63.6 41.0 39.6 39.5 2.1
Butanedione acetate acetate hexanoate
b
RSD – 3.2 0.7 1.5 –
A1 0.10 4.03 41.67 0.62
Isoamyl acetate 56.4 121.6 112.6 114.0 4.1 A2 0.09 4.54 39.43 0.72
60 1512.7 1400.0 1424.4 4.1 A3 0.10 4.11 43.51 0.64
63.6 2567.3 2414.7 2342.5 4.7 A4 0.09 3.69 44.04 0.60
RSDb – 87.6 88.1 86.6 – A5 0.09 4.43 46.11 0.75
A6 0.09 4.05 42.72 0.62
Ethyl acetate 56.4 6199.0 6063.0 5868.7 2.7 A7 0.09 4.55 40.77 0.63
60 10,094.4 10,148.4 9757.2 2.1 A8 0.10 4.09 44.76 0.53
63.6 19,319.0 18,673.0 17,710.5 4.4 A9 0.10 3.98 37.33 0.60
RSDb – 56.8 55.3 54.3 – A10 0.10 2.60 35.43 0.38
A11 0.10 4.05 36.01 0.70
Ethyl hexanoate 56.4 9298.4 9094.5 8803.0 2.7 A12 0.10 3.46 33.86 0.63
60 15,141.6 15,222.6 14,635.8 2.1 A13 0.10 3.57 20.62 0.57
63.6 28,978.5 28,009.5 26,565.7 4.4 A14 0.10 3.97 39.63 0.83
RSDb – 56.8 55.3 54.3 – A15 0.10 3.04 46.28 0.38
a
Relative standard deviation of triplicate under the same conditions.
b
Relative standard deviation of triplicates between the three variations of the
extraction time. remains in the GC injector for another 15 min. Thus, the fibre was
activated for 30 min. To expedite the routine, this time can be opti-
mized with the use of more than one fibre or by using an autosam-
3. Results and discussion pler (Ashraf, Linforth, Bealin-Kelly, Smart & Taylor, 2010; Hill &
Smith, 2000).
A mixture containing off-flavour esters (ethyl acetate, isoamyl After preparing the samples for SPME analysis followed by GC-
acetate, and ethyl hexanoate) and vicinal diketones (2,3-pentane- MS, saturation of the samples with 3.0 g of sodium chloride was
dione and 2,3-butanedione) was tested in water and Pilsner beer. performed, once it is readily soluble in water and influences the
In determining the method for SPME analysis of off-flavours, extraction efficiency by changing the polarity of the medium, forc-
several tests were performed for the following parameters: expo- ing the passage of off-flavours to the headspace. This technique is
sure time and extraction temperature. Charry-Parra et al. (2011) known as salting-out (Cajka, Riddellova, Tomaniova, & Hajslova,
evaluated these parameters for nine volatile compounds (alcohols 2010; Giordano, Calabrese, Davoli, & Rotilio, 2003; Gómez-Ariza,
and esters) in beer samples. Using fibre DVB/CAR/PDMS with García-Barrera, Lorenzo, & Beltrán, 2006; Hafez & Wenclawiak,
extraction time of 45 min and temperature of 24 °C showed greater 2013; Saison, De Schutter, Delvaux, & Delvaux, 2008; Sterckx
sensitivity to ethyl acetate and isoamyl acetate. The best response et al., 2010).
in terms of number of constituents and chromatographic separa- During the preparation of samples and standards added to beer,
tion was obtained in the current work using DVB/CAR/PDMS fibre, foam was produced, hindering the preparation of solutions. To
whose application is suggested for low to high polarity volatile and minimize this effect, samples of cold beer were used, and a low
non-volatile compounds (Jelén et al., 2012; Zapata, Mateo- temperature was found to be important for obtaining linearity
Vivaracho, Lopez, & Ferreira, 2012). (correlation coefficient of 0.99) as well as to avoid undesirable loss
After evaluating the area of the analytes for some variations in of volatile compounds.
the experimental conditions of time and temperature of extraction, Fig. 1a shows the chromatogram of volatile compounds present
an extraction time of 30 min at a temperature of 60 °C was in beer obtained by using a mixture of off-flavour esters and
determined. ketones as analyzed by SPME followed by GC-MS techniques.
The chromatographic conditions of the GC were optimized. In applying the SPME technique, individual solutions for each
However, despite the analytical run time was 15 min, the total ester and ketone in water were extracted successfully with DVB/
analysis time for each analytical run was one hour because the CAR/PDMS fibre. For the beer samples spiked with esters and
extraction time lasted 30 min, and after the analytical run the fibre ketones, only the chromatographic signals concerning esters were
 G.C. da Silva et al. / Food Chemistry 167 (2015) 71–77 75

Fig. 2. Chromatograms refering to the identification of (a) esters: ethyl acetate (Rt = 2.265 min), isoamyl acetate (Rt = 6.775 min) and ethyl hexanoate (Rt = 9.396 min) by HS-
SPME coupled to HRGC-MS and (b) vicinal diketones: 2,3-butanedione (Rt = 2.595 min) and 2,3-pentanedione (Rt = 4.527) by HS-HRGC–ECD in one of the beer samples.

identified (Fig. 1a) due to a competition between the esters and
vicinal diketones (Valente & Augusto, 2000).
The electron capture detector is selective and presents high sen-
sitivity even for trace analysis of halogenated organic compounds,
nitroaromatic compounds and conjugated systems. Compounds of
high electron affinity can be determined at sub-picogram amounts
facilitating trace analysis (Poole, 2013). The HS–GC–ECD technique
was then adopted for the analysis of vicinal diketones in beer.
(Fig. 1b).
3.1. Validation of the method
In order to verify the analytical performance of the developed
methodology, the validation parameters were evaluated in accor-
dance with ISO standards (INMETRO, 2011).
The calibration curves obtained from the off-flavours in beer
and water were used to evaluate selectivity. These curves
presented a concentration interval which encompasses not only
the taster’s perception threshold but also the off-flavours
concentration usually found in beers which are proper for con-
sumption. For the beer samples, the presence of interfering signals
in the retention time of the analytes were not observed. Esters ana-
lyzed by mass spectrometry were identified by the NIST standard
reference database with a probability value greater than 96%.
The effect of the sharp and matrix-induced chromatographic
response is one of the performance parameters that must be
evaluated, since this can markedly affect sample concentrations.
In order to evaluate the matrix effect, the F test is used to assess
whether the residual variances of the two curves (matrix and sol-
vent) are significantly different. If the experimental F-value is
greater than the theoretical F-value, it is possible to state that there
is a significant difference between the residual variances and that
they are not statistically similar. In this case, the matrix has an
effect on the accuracy of the range evaluated in the study
(Cardoso, Nobrega & Abrantes, 2008). Then, Student-t test must
be applied (Snedecor & Cochran, 1989; INMETRO, 2011). According
to Thompson, Ellison, and Wood (2002), it is essential to assess
whether the slopes of the analytical curves for the solvent and
76 G.C. da Silva et al. / Food Chemistry 167 (2015) 71–77
matrix are significantly different by means of the Student-t test,
and this can be carried out by comparing the slopes of the two
curves. The t-test is applied after calculating the pooled standard
deviation. When the experimental t-value is higher than the theo-
retical t-value, it is possible to assume that the matrix affects the
method.
Comparing the slopes as well as the highest and lowest variance
from the concentration levels of the calibration curve made in
water and beer, a matrix effect was observed due to a significant
difference in the slope between curves. All calculated values were
higher than the tabulated value. The lowest calculated F-value
(F = 89.6) obtained for isoamyl acetate was higher than the tabu-
lated F-value (F = 19.0). The lowest calculated t-value (t = 21.6)
obtained for isoamyl acetate was higher than the tabulated t-value
(t = 4.303) (level of significance, a = 0.05). The volatile compounds
of beer certainly compete with the analytes under study. The con-
centration of the analytes was calculated using the standard addi-
tion method, which was used in the validation and quantification
of the test samples (commercial beers).
The fibres used after the analysis of the higher concentration of
the calibration curve were used for injections of blank samples
aiming to assess contamination between analyzes. No signal was
observed in previous injections for blank samples. The exposure
time of the fibre in the injector (30 min) was sufficient to activate
the fibre for a new injection.
With regards to the calibration curve, the homogeneity of vari-
ances was checked by the Cochran’s test (Ctest), and results showed
a calculated value (Ccal = 0.4611) lower than the critical value
(Ccrit = 0.5612) for seven concentration levels (a = 0.05) analyzed
in triplicate (INMETRO, 2011). Linearity was established by using
the least squares method. All curves presented correlation coeffi-
cients higher than 0.99.
Repeatibility or intra-assay precision of the method measures
the variation around the mean for a series of samples prepared
by the same analyst in the shortest time possible using the same
concentration, and it evaluates the efficiency of both analyst and
equipment. The repeatability (Table 1) was assessed in quintupli-
cate using samples spiked at a concentration equivalent to sensory
detection threshold on three consecutive days and results were
expressed by the relative standard deviation (RSD). In both meth-
odologies, HS–GC–MS and HS–GC–ECD, satisfactory RSD values
(less than 5%) were obtained.
The inter-assay precisions were determined as the RSD (%) and
accuracies were expressed as a percentage of the nominal concen-
tration, wherby accuracy (%) = (found concentration/nominal
concentration)  100. Inter-assay precision and accuracy were cal-
culated using the first replicate obtained in five different assays.
The results for inter-assay precisions and accuracy are presented
in Table 2. It is possible to observe that satisfactory results
(RSD < 5%) were obtained for precision, while accuracy results
were between 90% and 110%.
The ruggedness (Table 3) was evaluated from the comparison
between variations of 5% in extraction times (56.4 and 63.6 min)
with the initial condition (60 min). All tests showed repeatability
and RSD remained between 2.1% and 4.7%. The comparison showed
a RSD between 54.3% and 88.1% for esters, indicating the impor-
tance of controlling the extraction time for the SPME technique.
For the extraction by using the headspace technique, the RSD
was less than 5% (0.7–3.2%), demonstrating the ruggedness of this
method.
Table 4 shows the concentrations of esters and 2,3-butenedione
in all 15 beers as analyzed by means of the developed methodolo-
gies. All beer samples had concentrations of off-flavours below the
detection threshold. Fig. 2 shows the chromatographic profile of
beer A1.
4. Conclusion
The techniques were developed and validated using HS-SPME–
GC–MS and HS–GC–ECD for identification and quantification of
off-flavours in beer. Both techniques were shown to be precise,
accurate and with suitable sensitivity for quality control purposes.
The techniques were applied to 15 different beer brands in the
marketed in Brazil, enabling rapid identification and quantification
of compounds of interest in beer industry.
References
Araújo, F. B., Silva, P. H. A., & Minim, V. P. R. (2003). Perfil sensorial e composição
físico-química de cervejas provenientes de dois segmentos do mercado
brasileiro. Revista Ciência Tecnologia de Alimentos, 23(1), 121–128.
Ashraf, N., Linforth, R. S. T., Bealin-Kelly, F., Smart, K., & Taylor, A. J. (2010). Rapid
analysis of selected beer volatiles by atmospheric pressure chemical ionisation-
mass spectrometry. International Journal of Mass Spectrometry, 294, 47–53.
Cajka, T., Riddellova, K., Tomaniova, M., & Hajslova, J. (2010). Recognition of beer
brand based on multivariate analysis of volatile fingerprint. Journal of
Chromatography A, 1217, 4195–4203.
Cardoso, M. H. W. M., Nóbrega, A. W., & Abrantes, S. (2008). Efeito da resposta
cromatográfica acentuada e induzida pela matriz: estudo de caso em tomates.
Revista Analytica, 34, 48–55.
Charry-Parra, G., DeJesus-Echevarria & Perez, F. J. (2011). Beer volatile analysis:
Optimization of HS/SPME coupled to GC/MS/FID. Journal of Food Science, 76(2),
205–211.
Flavouractiv. (2005). The beer flavour handbook (Vol. 2.1). Chinnor, Flavour Limited.
Giordano, L., Calabrese, R., Davoli, E., & Rotilio, D. (2003). Quantitative analysis of 2-
furfural and 5-methylfurfural in different Italian vinegars by headspace solid-
phase microextraction coupled to gas chromatography–mass spectrometry
using isotope dilution. Journal of Chromatography A, 1017, 141–149.
Gómez-Ariza, J. L., García-Barrera, T., Lorenzo, F., & Beltrán, R. (2006). Use of
multiple headspace solid-phase microextraction and pervaporation for the
determination of off-flavours in wine. Journal of Chromatography A, 1112,
133–140.
Hafez, A. M., & Wenclawiak, B. W. (2013). Development of a new porous gold SPME
fiber for selective and efficient extraction of dodecanethiol followed by GC–MS
analysis. Analytical and Bioanalytical Chemistry, 405, 1753–1758.
Hughes, P. S., Baxter, E. D. (2001). Quality, safety and nutritional aspects. Cambridge
CB4 0WF, The Royal Society of Chemistry.
INMETRO. (2011). DOQ-CGCRE-008. Orientação sobre validação de métodos
analíticos. Documento de caráter orientativo.
Jelén, H. H., Majcher, M., & Dziadas, M. (2012). Microextration techiniques in the
analysis of food flavor compounds: A review. Analytica Chimica Acta, 738, 13–26.
Jiao, J., Ding, N., Shi, T., Chai, X., Cong, P., & Zhu, Z. (2011). Study of Chromatographic
Fingerprint of the flavor in beer by HS-SPME–GC. Analytical Letters, 44, 648–655.
Peter, G., Hill, P. G., & Smith, R. M. (2000). Determination of sulphur compounds in
beer using headspace solidphase microextraction and gas chromatographic
analysis with pulsed flame photometric detection. Journal of Chromatography A,
872, 203–213.
Pizarro, C., Pérez-del-Notario, N., & González-Sáiz, J. M. (2010). Optimisation of a
sample and reliable based on headspace solid-phase microextraction for the
determination of volatile phenols in beer. Journal of Chromatography A, 1217,
6013–6021.
Poole, C. F. (2013). Derivatization reactions for use with the electron-capture
detector. Journal of Chromatography A, 1296, 15–24.
Rudnitskaya, A., Polshin, E., Kirsanov, D., Lammertyn, J., Nicolai, B., Saison, D., et al.
(2009). Instrumental measurement of beer taste attributes using an electronic
tongue. Analytica Chimica Acta, 646(1–2), 111–118.
Saison, D., De Schutter, D. P., Delvaux, F., & Delvaux, F. R. (2008). Optimisation of a
complete method for the analysis of volatiles involved in the flavour stability of
beer by solid-phase microextraction in combination with gas chromatography
and mass spectrometry. Journal of Chromatography A, 1190, 342–349.
Scherer, R., Roger, W., Kowalski, C. H., & Teixeira, G. H. (2010). Determinação de (E)-
2-nonenal em cervejas brasileiras utilizando microextração em fase sólida do
headspace e cromatografia gasosa acoplada a espectrometria de massas. Revista
Ciência Tecnologia de Alimentos, 30(1), 161–165.
Silva, G. A., Maretto, D. A., Bolini, H. M. A., Teófilo, R. F., Augusto, F., & Poppi, R. J.
(2012). Correlation of qualitative sensorial descriptors and chromatographic
signals of beer using multivariate calibration strategies. Food Chemistry, 134,
1673–1681.
Siqueira, P. H., Bolini, H. M. A., & Macedo, G. A. (2008). Beer production and its
effects on the presence of polyphenols. Brazilian Journal of Food and Nutrition,
19(4), 491–498.
Snedecor, S. W., & Cochran, W. G. (1989). Statistical methods (6th ed.). Ames: Iowa
State University. 503.
Sterckx, F. L., Saidson, D., & Delvaux, F. R. (2010). Determination of volatile
monophenols in beer using acetylation and headspace solid-phase
microextraction in combination with gas chromatography and mass. Analytica
Chimica Acta, 676, 53–59.
 G.C. da Silva et al. / Food Chemistry 167 (2015) 71–77 77

Thompson, M., Ellison, S. L. R., & Wood, R. (2002). Harmonized guidelines for single
laboratory validation of methods of analysis (IUPAC Technical Report). Pure and
Applied Chemistry, 74, 835–855.
Tian, J. (2010). Application of static headspace gas chromatography for
determination of acetaldehyde in beer. Journal of Food Composition and
Analysis, 23, 475–479.
Valente, A. L. P., & Augusto, F. (2000). Microextração por fase sólida. Química Nova,
23(4), 523–529.
Zapata, J., Mateo-Vivaracho, L., Lopez, R., & Ferreira, V. (2012). Automated and
quantitative headspace in-tube extraction for the accurate determination of
highly volatile compounds from wines and beers. Journal of Chromatography A,
1230, 1–7.