Food Control 136 (2022) 108873

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Development, validation and application of a fast GC-FID method for the

analysis of volatile compounds in spirit drinks and wine
Mauro Paolini *, Loris Tonidandel , Roberto Larcher
Technology Transfer Centre, Fondazione Edmund Mach, via E. Mach 1, 38010, San Michele all’Adige, Italy

A R T I C L E I N F O A B S T R A C T

Keywords: In this work, a fast GC-FID method for the analysis of principal volatile compounds in spirit drinks and wines was
Fast GC-FID analysis presented. With a chromatographic run of 8 min were identified and quantified sixteen volatile compounds
Volatile compounds belonging to different chemical classes: acetals (1,1-diethoxyethane or acetal), aldehydes (acetaldehyde), alco­
Spirit drinks
hols (methanol, 1-hexanol, 2-methylbutan-1-ol, 2-methylpropan-1-ol, 2-phenylethanol, 3-methylbutan-1-ol,
Wine
butan-1-ol, butan-2-ol and propan-1-ol) and ethyl esters (ethyl acetate, ethyl formate and ethyl lactate). These
compounds are responsible for the quality of alcoholic beverages, affecting the aromatic composition and the
wholesomeness of the final products. For this reason, their quantification is essential to optimize the fermen­
tation conditions, storage techniques and distillation process.
Validation of the method was performed in terms of matrix effect, limit of detection, limit of quantification,
linearity, recovery, repeatability and reproducibility. Once validated, the developed method was tested suc­
cessfully on real enological samples, demonstrating its applicability in the daily practice of analytical
laboratories.

1. Introduction
By definition, any drinkable liquid containing ethanol of agricultural
origin between 0.5 and 70% (v/v) is considered as an alcoholic
beverage. The International Organization of Vine and Wine (OIV) has
established for wine a minimum alcoholic grade of 8.5% (v/v) (Inter­
national Organisation of Vine and Wine (OIV), 2015), while the Regu­
lation (EC) 787/2019 has defined for spirit drink an ethanol content
equal to or higher than 15% (v/v) (The European Parliament and of the
Council of the European Union, 2019).
Although ethanol and water are the most abundant components of
alcoholic beverages, there is a small fraction of volatile compounds,
called congeners, that are synthesized through a series of biological
mechanisms, such as yeast metabolism and enzymatic reactions
(Lachenmeier et al., 2008). 1-hexanol, methanol, acetaldehyde, acetal
(or 1,1-diethoxyethane), ethyl acetate, ethyl formate, ethyl lactate and
some higher alcohols (2-methylbutan-1-ol, 2-methylpropan-1-ol,
2-phenylethanol, 3-methylbutan-1-ol, butan-1-ol, butan-2-ol and
propan-1-ol) represent the main congener content in wines and spirit
drinks (Jung et al., 2010).
These compounds play an important role from the organoleptic point
of view (Ortega-Heras et al., 2002; Sáenz et al., 2010), and their quan­
tification demonstrates particularly interesting to understand the origin
of the raw material used, and to support the technological choices
during industrial transformation processes.
Methanol is an enzymatic product obtained from the hydrolysis of
the methoxyl groups of fruits pectins, with a characteristic alcoholic
smell (Berger, 2007; Hodson et al., 2017; Pang et al., 2017). Methanol
also has harmful effects on the human body (Vale, 2007; Feron et al.,
1991) and, for this reason, it was necessary to set concentration limits to
protect consumers’ health (The European Parliament and of the Council
of the European Union, 2019; International Organisation of Vine and
Wine (OIV), 2020).
1-hexanol is only partially an alcoholic fermentation product. It de­
rives from the enzymatic degradation of unsaturated C18-acids of the
marc, mostly obtaining C6-aldehydes which are then reduced by yeasts
to saturated alcohols (Joslin & Ough, 1978; López-Vázquez et al., 2010).
1-hexanol in alcoholic beverages impart green and floral notes, with a
sensory threshold of about 1 mg/L (Berger, 2007).
Higher alcohols, or “fusel alcohols”, are quantitatively the largest
group of volatile compounds in alcoholic beverages with flavour prop­
erties (Cortés et al., 2011). This class of compounds is characterized by

* Corresponding author. Technology Transfer Centre, Fondazione Edmund Mach, via E. Mach 1, 38098, San Michele all’Adige, Italy.
E-mail address: mauro.paolini@fmach.it (M. Paolini).

https://doi.org/10.1016/j.foodcont.2022.108873
Received 17 November 2021; Received in revised form 29 January 2022; Accepted 1 February 2022
Available online 2 February 2022
0956-7135/© 2022 Elsevier Ltd. All rights reserved.
M. Paolini et al.
molecules containing more than two carbon atoms and includes the
branched-chain alcohols and the aromatic ones. The most important
higher alcohols are 2-methylbutan-1-ol, 2-methylpropan-1-ol, 2-phenyl­
ethanol, 3-methylbutan-1-ol, butan-1-ol, butan-2-ol and propan-1-ol
(Victoria Moreno-Arribas & Polo, 2008). Butan-1-ol and butan-2-ol are
not compounds of alcoholic fermentation but substances coming from
the raw material; their threshold values are rather high and therefore
they do not contribute significantly to the flavour. The other higher
alcohols, instead, are produced from the degradation of amino acids by
yeasts during the fermentation process (Berger, 2007). 2-methylbuta­
n-1-ol and 3-methylbutan-1-ol, the mixture of which is also called iso­
amyl alcohols, are the most abundant and represent 40–70% of the total
higher alcohols fraction (Lambrechts & Pretorius, 2019). Higher alco­
hols are positively involved in the sensory quality of wines and distil­
lates, but in high concentration they impart a malty and burnt flavour.
Conversely, 2-phenylethanol is generally characterized by a pleasant
rose-like aroma (Swiegers et al., 2005).
Acetaldehyde is one of the most important saturated carbonyl com­
pounds produced by fermenting yeast as an intermediate in ethanol
production. Acetaldehyde is directly related to the quality of alcoholic
beverages, not only in terms of aromatic composition but also in terms of
wholesomeness (Feron et al., 1991). It is characterized by apple and nut
notes with sensory threshold of about 100 mg/L, and at high concen­
tration may cause off-flavours owing to the pungent odour (Ger­
oyiannaki et al., 2007; S.-Q. Liu & Pilone, 2000; Grumezescu & Holban,
2019). From the organoleptic point of view, the formation of acetal from
ethanol and acetaldehyde help to reduce the pungent odour and
contribute to giving soft notes (Berger, 2007).
Furfuraldehyde in marc distillates is formed by Maillard reactions of
unfermented sugars (pentoses) during the distillation process at high
temperature (Onishi et al., 1977; Ebeler et al., 2000). This compound is
characterized by a bitter almond smell with a perception threshold of
150 mg/L.
Fermentation-derived esters are a large group of volatile compounds
generally present in concentration higher than their odour threshold,
with mostly pleasant flavour properties (Berger, 2007; Arribas & Polo,
2009). Ethyl acetate is the most important ester in fermented products
and their distillates. This compound is largely responsible for the altered
sensory properties typical of acescency (Plata et al., 2003), but can have
favorable effects on aroma at concentrations below 80 mg/L
(Ribéreau-Gayon, 1978).
Ethyl formate is one of the less abundant ethyl esters in wine, usually
present in a concentration of a few mg/L and, in any case, under its
sensory threshold value (Margalit, 2014). In grape distillates, ethyl
formate is a product of head fraction so its concentration may be affected
by the intensity of the head cut during the distillation process
(López-Vázquez et al., 2010).
Ethyl lactate is a characteristic compound of malolactic fermentation
and it is produced by the esterification of lactic acid with ethanol
(Lasik-Kurdyś et al., 2018). This volatile compound has a sensory
threshold of about 100 mg/L and it is characterized by fruity, buttery,
and creamy aromas (Gámbaro et al., 2001; Berger, 2007).
The analysis of these volatile compounds has become of paramount
importance and, in order to achieve this scope, official analytical
methods were published. In 2000, the European Commission issued the
Regulation 2870 (TheCommission of the European Communities, 2000),
which was then supplemented by the Regulation 635/2016 (Commis­
sion Implementing Regulation (EU) 2016/635 of April 22, 2016
Amending the Annex to Regulation (EC) No 2870/2000 as Regards
Certain Reference Methods for the Analysis of Spirit Drinks, 2016),
laying down the reference methods to be used for analysing spirit drinks.
“The Compendium of International Methods of Analysis of Wine and
Musts” (Ed. 2018), issued by OIV, has instead defined the official pro­
cedure to quantify methanol in wine (OIV-MA-AS312-03A) (Interna­
tional Organisation of Vine and Wine, 2014).
Following the official methods reported above, several studies were
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Food Control 136 (2022) 108873
proposed regarding the determination of volatile compounds in different
spirit drinks and wine using gas chromatography separation and flame
ionization detector (GC-FID) (Rodríguez Madrera & Suárez Valles, 2007;
Yilmaztekin & Cabaroglu, 2011; Luís et al., 2011). As shown in pub­
lished works, traditional GC-FID technique requires long analytical run
time and long GC-columns to ensure good chromatographic separation
and to avoid co-elutions.
Agilent offers a new fast Intuvo GC-FID system that may cut down
total analytical time without compromising chromatographic separa­
tion. The new fast-cool technology reduces post-run cooling while steep
temperature gradients and high operating column pressure reduce
analytical run-time.
Moreover, the fast gas chromatography is characterized by narrow
and, hence, higher peaks with an improvement of analytical perfor­
mance in terms of limit of quantification (LOQ) and limit of detection
(LOD), as reported in “Practical fast gas chromatography: methods,
instrumentation and application” (Korytár et al., 2002).
The aim of this study was to develop and validate a fast, accurate and
precise method using the new Intuvo GC-FID system to speed up the
quantification of the principal volatile compounds in alcoholic bever­
ages, preserving or improving the efficiency of conventional approaches.
The developed method was then tested on samples of spirit drink and
wine provided by proficiency-testing programs and collected from
different market sites.
2. Material and methods
2.1. Chemicals
All standards at ≥99% purity (1-hexanol, 2-methylbutan-1-ol, 2-
methylpropan-1-ol, 2-phenylethanol, 3-methylbutan-1-ol, 4-methylpen­
tan-2-ol, acetal, acetaldehyde, benzyl alcohol, butan-1-ol, butan-2-ol,
ethyl acetate, ethyl formate, ethyl lactate, furfuraldehyde, methanol,
propan-1-ol) and ethanol (solvent) were purchased from Sigma-Aldrich
(Milan, Italy).
2.2. Sampling
In this study, eight different spirit drinks (Eau-de-vie de marc,
Armagnac, Rhum, Calvados, Grape marc distillate, Brandy, Whisky and,
Cognac) provided by BIPEA (Bureau Interprofessionnel d’Etudes Analy­
tiques) and four different wines (Fortified red wine, Sparkling red wine, Dry
white wine and Sweet Champagne) provided by UIV (Unione Italiana Vini)
were analysed.
BIPEA is an European scientific organization offering an interna­
tional interlaboratory ring test dedicated to the quantification of 1-hex­
anol, acetaldehyde, acetal, ethyl acetate, ethyl formate, ethyl lactate,
furfuraldehyde, methanol and higher alcohols (2-methylbutan-1-ol, 2-
methylpropan-1-ol, 2-phenyletanol, 3-mehylbutan-1-ol, butan-1-ol,
butan-2-ol, propan-1-ol) in spirit drinks.
UIV is the Italian Confederation of Vines and Wine that manages an
interlaboratory ring test related to the determination of acetaldehyde
and methanol in wines.
Moreover, 20 different samples of grape marc spirits and 20 different
samples of red wines were purchased from the market.
2.3. Standard solutions and samples preparation for spirit drinks
The preparation of samples and standard solutions was performed by
following the method reported in Regulation (EC) 2870/2000 (The
Commission of the European Communities, 2000). Six calibration levels,
ranging from 5 to 5000 μg/g, were obtained by dilution of the stock
solution with a hydroalcoholic mixture at 40% (v/v) ethanol. 4-methyl­
pentan-2-ol was chosen as internal standard (IS), due to the fact it is not
ordinarily present in the considered matrices and has shown good
chromatographic separation from the studied compounds
M. Paolini et al. Food Control 136 (2022) 108873

(Rodríguez-Bencomo et al., 2003; Gómez-Míguez et al., 2007). Simi­ Specifically, the temperature gradients (equation (1)) and the hold time
larly, the solution of quantification (named sol C) was prepared at a for the isothermal parts (equation (2)) of the original temperature pro­
concentration of 250 μg/g. gram were recalculated. equations (3) and (4) allow us to obtain the
For sample preparation, 9 ml of spirit drink were weighted in a phase ratio and the average linear velocity, respectively.
volumetric flask, 1 ml of the internal standard solution was added and
v2 β l1
the weight of the final mixture was recorded. tg2 = tg1 × × 2× equation 1
v1 β1 l2

2.4. Standard solution and samples preparation for wine v1 β l2

T2 = T1 × × 1× equation 2
v2 β2 l1
The calibration solutions containing all target compounds were
prepared at seven concentration levels between 5 and 500 mg/L, using a dc
hydroalcoholic solution at 10% (v/v) ethanol as solvent and 4-methyl­ β= equation 3
4df
pentan-2-ol as internal standard.
According to the OIV method OIV-MA-AS312-03A for methanol L
v= equation 4
determination (International Organisation of Vine and Wine, 2014), tm
before the GC analysis the wine sample was distilled. A volume of 9 mL
of the alcoholic distillate thus obtained were mixed with 1 mL of the where:
internal standard solution and analysed.
In order to obtain a correct quantification of acetaldehyde content, - tg1 and tg2 are the temperature gradient for original and new
the wine sample was previously neutralised to pH 7 with sodium hy­ conditions;
droxide to release the bound acetaldehyde fraction. Indeed, a portion of - v1 and v2 are the average linear velocity of gas (cm/sec) for original
SO2 free in wine binds to acetaldehyde and forms the non-volatile and new conditions;
adduct acetaldehyde hydroxysulfonate (Burroughs & Sparks, 1973). - T1 and T2 are hold time for isothermal part of separation for original
The alcoholic distillate was collected in an ice-cold calibrated flask. and new conditions;
- β1 and β2 are the phase ratio of the column for original and new
2.5. GC parameter optimization conditions;
- l1 and l2 are the length of column for original and new conditions;
The development of a fast GC method needs a careful setting of the - dc is the column diameter (μm);
experimental conditions with a tuned compromise between speed, - df is the film thickness (μm);
sample capacity, resolution and sensibility (Klee & Blumberg, 2002). - L is the column length (cm);
Therefore, the proposed method was obtained through a series of stages - tm is the retention time of unretained peak (sec).
in which the GC parameters were optimized in order to obtain the best
chromatographic separation by using the CP-Wax 57 CB column (25 m The standard mix (sol C) was analysed applying the new GC condi­
× 0.25 mm × 0.2 μm). tions (Table 1 – Step 1) and the chromatogram was reported in Fig. 1.
The first step was to determine the new oven temperature program, As shown, the 17 compounds are eluted in about 7 min with good
maintaining unchanged the carrier flow, injection volume, split ratio chromatographic separation for butan-2-ol (6), propan-1-ol (7), 2-meth­
and injector temperature. The following equations (equation 1, 2, 3 and ylpropan-1-ol (8), butan-1-ol (9), 4-methylpentan-2-ol (IS) (10), 2-meth­
4) were used to determine the system parameters required to optimize ylbutan-1-ol (11), 3-methylbutan-1-ol (12), ethyl lactate (13), 1-hexanol
performance using the short GC column (Grob & Barry, 2004). (14), furfuraldehyde (15), benzyl alcohol (16) and 2-phenylethanol

Table 1
Method development: GC parameter optimization.
Conventional method Fast method

Step 1 Step 2 Step 3 Step 4

GC column CP-Wax 57 CB CP-Wax 57 CB

50 m × 0.32 mm × 0.2 μm 25 m × 0.25 mm × 0.2 μm

Phase ratio 400 312

Carrier gas (ml/min) 1.8 1.8

Average linear velocity (cm/sec) 14 48

Volume (μL) and split ratio 1–40:1 1–40:1 0.5–70:1

Injector temp (◦ C) 240 240 300

Oven program
Initial temp (◦ C) 40 40 40
Initial hold time 10 min 2 min 2 min
ramp rate 1 5 ◦ C/min to 110 ◦ C 27 ◦ C/min to 110 ◦ C 50 ◦ C/min to 48 ◦ C
ramp rate 2 20 ◦ C/min to 190 ◦ C 106 ◦ C/min to 190 ◦ C 0.5 ◦ C/min to 49.5 ◦ C
ramp rate 3 80 ◦ C/min to 135 ◦ C
ramp rate 4 120 ◦ C/min to 220 ◦ C
final hold time 10 min 2 min 2 min

Detector temp (◦ C) 250 250

Total run time (min) 40 7 8

3
M. Paolini et al. Food Control 136 (2022) 108873

Fig. 1. The effect of the fast oven temperature ramp rate on analysis time and resolution.

Fig. 2. Resolving power, peak height and peak width under different split ratios.

(17). However, for acetaldehyde (1), ethyl formate (2), ethyl acetate (3),
acetal (4) and methanol (5) significant peak broadening with loss of
efficiency and resolution is observed.
In order to minimize the band-widths and improve the separation
efficiency, the injection volume was reduced (from 1 to 0.5 μL) while the
split ratio was increased (van Es et al., 1987). The effect of different split
ratios (40:1, 70:1 and 100:1) on resolving power, peak height and width
was reported in Fig. 2. The most narrow bands and the best resolution
between ethyl acetate and acetal was obtained by injecting 0.5 μL at
100:1 split ratio.
The drawback by using too high split ratios is the poor detection
limits (LODs), for this reason the split ratio 100:1 was discarded,
choosing 70:1 (Table 1 – Step 2).
The peak height and peak width were also investigated as a function
of the injector temperature. The temperature was increased from 240 to
300 ◦ C providing more narrow and higher peaks, particularly for acet­
aldehyde and methanol, as shown in Fig. 3. Higher injector temperature
benefits the quick vaporization of the sample liquid, and thus, helps in
the formation of a sample plug with more uniformity (G. Liu & Xin,
1989).

4
M. Paolini et al.
Fig. 3. Effect of injector temperature on peak height and peak width of acetaldehyde (1) and methanol (5).
Fig. 4. GC-FID chromatogram of the standard mixture (sol C).
The developed fast GC method was implemented by setting the
injector temperature at 300 ◦ C (Table 1 – Step 3).
The final step was to improve the resolution of the isoamyl alcohols
(2-methylbutan-1-ol (11) and 3-methylbutan-1-ol (12)) by modifying
the oven temperature program (Fig. 4). In order to achieve this scope,
the temperature ramp rate was reduced by increasing the analysis time
to 8 min (Table 1 – Step 4).
2.6. GC-FID analysis
The quantification of the sixteen target compounds (1-hexanol, 2-
methylbutan-1-ol, 2-methylpropan-1-ol, 2-phenylethanol, 3-methyl­
butan-1-ol, acetal, acetaldehyde, benzyl alcohol, butan-1-ol, butan-2-ol,
ethyl acetate, ethyl formate, ethyl lactate, furfuraldehyde, methanol and
propan-1-ol) was carried out using a GC-FID system. The official GC-FID
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Food Control 136 (2022) 108873
method (named A), reported by Regulation 2870 for spirit drinks (The
Commission of the European Communities, 2000) and by
OIV-MA-AS312-03A for wines (International Organisation of Vine and
Wine, 2014), a chromatographic run of 40 min, whereas the proposed
one (named B) takes only 8 min.
The short chromatographic separation was obtained using the new
Agilent Intuvo 9000 GC system equipped with an FID detector, an high-
speed injection system, a short capillary column and the rapid oven
heating/cooling. The optimized conditions were reported in Table 1 -
Step 4.
Data acquisition and analysis were performed using the Agilent
Technologies MassHunter Workstation software – Data Acquisition (ver.
B.07.05) and the Agilent MassHunter Workstation Software – Quanti­
tative Analysis (ver. B.08.00), respectively.
In order to compare the analytical results obtained with the proposed
M. Paolini et al. Food Control 136 (2022) 108873

Fig. 5. GC-FID chromatogram of a grape marc spirit sample (A) and a wine sample (B).

method (A) with those of the official\conventional one (B), commercial

AISC Ci
samples were analysed using both gas chromatographic methods. The RFi = × C equation 5
Ai C CISC
quantification of the target compounds using the official\conventional
method was carried out using a PerkinElmer® Clarus 500 GC System
where AISc and CISc are the area and concentration [μg/g] of internal
(Shelton CT, USA) equipped with an FID detector. The GC conditions
standard, whereas Aic and Cic are the area and concentration [μg/g] of
were reported in Table 1.
the compound of interest.
Data acquisition and analysis were performed using the TotalChrom
The final concentration of each compounds (Ci) in a spirit drink
Navigator - Clarus 500 software (ver. 6.3.2).
sample was determined using equation (6):
Ai WIS
2.7. Expression of the results for spirit drinks Ci = × × CIS × RFi equation 6
AIS Wsample

The concentration of each analyte was calculated in μg/g consis­ where AIS, Ai, WIS and Wsample are the area of internal standard, the area
tently with the Regulation (EC) 2870/2000 (The Commission of the of compound of interest, the weight of internal standard [g] and the
European Communities, 2000), using the response factors (RFi) obtained weight of sample [g], respectively. RFi is the response factor of the
from the solution of quantification (sol C) applying equation (5):

6
M. Paolini et al. Food Control 136 (2022) 108873

Table 2
Validation parameters at 40% (v/v) of ethanol: retention time (RT), linearity range, regression equation, correlation coefficient (R2), LOD and LOQ.
Compound RT (min) Linearity range (μg/g) Regression equation Correlation coefficient (R2) LOD (μg/g) LOQ (μg/g)

acetaldehyde 1.23 5–3000 y = 0.456x + 0.007 0.999 0.5 1.5

ethyl formate 1.45 5–3000 y = 0.452x + 0.002 0.999 0.3 1.0
ethyl acetate 1.67 5–3000 y = 0.534x + 0.001 0.999 0.3 1.0
acetal 1.71 5–3000 y = 0.712x + 0.006 0.999 0.3 1.0
methanol 1.80 5–3000 y = 0.499x + 0.001 0.999 0.5 1.5
butan-2-ol 2.86 5–5000 y = 0.854x + 0.002 0.999 0.3 1.0
propan-1-ol 3.00 5–3000 y = 0.831x + 0.002 0.999 0.3 1.0
2-methylpropan-1-ol 3.82 5–3000 y = 0.987x + 0.004 0.999 0.3 1.0
butan-1-ol 4.85 5–3000 y = 0.895x + 0.001 0.999 0.3 1.0
4-methylpentan-2-ol (IS) 5.41
2-methylbutan-1-ol 5.89 5–3000 y = 0.995x + 0.004 0.999 0.3 1.0
3-methylbutan-1-ol 5.91 5–3000 y = 1.387x + 0.001 0.999 0.3 1.0
ethyl lactate 6.61 5–3000 y = 0.456x - 0.001 0.999 0.3 1.0
1-hexanol 6.67 5–3000 y = 1.007x +0.003 0.999 0.3 1.0
furfuraldehyde 6.94 5–3000 y = 0.659x + 0.003 0.999 0.3 1.0
benzyl alcohol 7.62 5–3000 y = 1.308x + 0.008 0.999 0.3 1.0
2-phenylethanol 7.67 5–3000 y = 1.281x + 0.010 0.999 0.3 1.0

Table 3
Validation parameters at 10% (v/v) of ethanol: retention time (RT), linearity range, regression equation, correlation coefficient (R2), LOD and LOQ.
Compound RT (min) Linearity range (mg/L) Regression equation Correlation coefficient (R2) LOD (mg/L) LOQ (mg/L)

acetaldehyde 1.23 5–500 y= 0.477x + 0.006 0.999 0.5 1.5

ethyl formate 1.45 5–500 y= 0.442x + 0.002 0.998 0.3 1.0
ethyl acetate 1.67 5–500 y= 0.534x + 0.002 0.997 0.3 1.0
acetal 1.71 5–500 y= 0.706x + 0.004 0.999 0.3 1.0
methanol 1.79 5–500 y= 0.509x + 0.003 0.999 0.5 1.5
butan-2-ol 2.86 5–500 y= 0.833x + 0.001 0.996 0.3 1.0
propan-1-ol 3.00 5–500 y= 0.846x + 0.002 0.996 0.3 1.0
2-methylpropan-1-ol 3.84 5–500 y= 0.999x + 0.005 0.996 0.3 1.0
butan-1-ol 4.87 5–500 y= 0.913x + 0.002 0.995 0.3 1.0
4-methylpentan-2-ol (IS) 5.42
2-methylbutan-1-ol 5.89 5–500 y= 1.019x + 0.003 0.996 0.3 1.0
3-methylbutan-1-ol 5.91 5–500 y= 1.431x + 0.002 0.996 0.3 1.0
ethyl lactate 6.61 5–500 y= 0.462x - 0.001 0.998 0.3 1.0
1-hexanol 6.67 5–500 y= 0.989x +0.004 0.995 0.3 1.0
furfuraldehyde 6.94 5–500 y= 0.675x + 0.004 0.997 0.3 1.0
benzyl alcohol 7.62 5–500 y= 1.331x + 0.006 0.995 0.3 1.0
2-phenylethanol 7.67 5–500 y= 1.360x + 0.009 0.996 0.3 1.0

Table 4
Slope of the calibration curves, t-values and significance levels.
Compound Slope at 40% of ethanol Slope at 10% of ethanol t(10) calculated Significance level (α value)

acetaldehyde 0.456 0.477 1.77 0.10

ethyl formate 0.452 0.442 1.41 0.10
ethyl acetate 0.534 0.534 0.03 0.20
acetal 0.712 0.706 1.29 0.20
methanol 0.499 0.509 1.46 0.10
butan-2-ol 0.854 0.833 0.84 0.20
propan-1-ol 0.831 0.846 0.71 0.20
2-methylpropan-1-ol 0.987 0.999 0.60 0.20
butan-1-ol 0.895 0.913 1.28 0.20
2-methylbutan-1-ol 0.995 1.019 1.65 0.10
3-methylbutan-1-ol 1.387 1.431 1.59 0.10
ethyl lactate 0.456 0.462 0.64 0.20
1-hexanol 1.007 0.989 0.70 0.20
furfuraldehyde 0.659 0.675 1.37 0.10
benzyl alcohol 1.308 1.331 0.26 0.20
2-phenylethanol 1.281 1.360 1.55 0.10

analyte of interest, whereas CIS is the concentration [μg/g] of the in­ calculated in compliance with what reported by the method OIV-MA-
ternal standard solution which was added at the sample. The results can AS312-03A (International Organisation of Vine and Wine, 2014),
be expressed in mg/L passing through the density. using the following equation (7):
( )
CIS Ai
Ci = × − b equation 7
2.8. Expression of the results for wine samples m AIS

The concentration in mg/L of the interest compounds (Ci) was where AIS and CIS are the area and concentration [mg/L] of internal

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M. Paolini et al. Food Control 136 (2022) 108873

Table 5
Results of the Interlaboratory evaluation study for spirit drink samples, expressed in mg/L.
Eau-de-vie de marca Armagnaca Rhuma Calvadosa

Compound Our results Z-score Our results Z-score Our results Z-score Our results Z-score

acetaldehyde 100 0.00 54 0.75 74 0.29 32 0.80

ethyl formate 2 0.26 12 0.25 12 0.88 4 − 0.26
ethyl acetate 359 − 0.97 185 0.11 71 0.11 217 − 0.16
acetal 68 − 0.17 21 0.03 41 0.43 15 0.91
methanol 2931 − 0.39 155 0.26 27 − 0.95 303 0.60
butan-2-ol <1 – <1 – <1 – 91 0.19
propan-1-ol 283 − 0.53 109 − 0.07 153 − 0.57 135 0.47
2-methylpropan-1-ol 156 − 0.67 533 − 0.09 296 − 0.49 249 0.31
butan-1-ol 17 − 0.51 2 − 0.20 4 − 0.50 28 − 0.24
2-methylbutan-1-ol 69 − 0.75 273 − 0.94 160 − 0.38 175 − 0.07
3-mehylbutan-1-ol 287 − 0.93 1092 − 0.85 649 − 0.17 971 − 0.17
ethyl lactate 244 0.11 31 − 0.13 2 − 0.50 92 0.85
1-hexanol 5 − 0.65 5 − 0.96 1 − 0.89 32 0.60
furfuraldehyde 8 − 0.06 6 − 0.10 1 0.11 6 0.30
benzyl alcohol <1 – <1 – <1 – <1 –
2-phenylethanol 13 0.48 16 0.50 5 0.40 21 0.06
a
Declared by BIPEA.

Table 6
Results of the Interlaboratory evaluation study for wine samples, expressed in mg/L.
Fortified red winea Sparkling red winea Dry white winea Sweet champagnea

Compound Our results Z-score Our results Z-score Our results Z-score Our results Z-score

acetaldehyde 11 0.00 78 0.20 64 − 0.15 56 0.21

methanol 203 0.85 75 − 0.13 47 0.00 39 0.18
a
Declared by UIV.

standard, Ai is the area of the compound of interest, whereas m and b are
respectively the slope and intercept of the calibration curve.
2.9. Statistical analysis
The slope of the calibration curves obtained at the two different
concentration levels of ethanol (40 and 10% v/v) were compared by
applying a 2-tailed t-test for parallelism.
The non-parametric Wilcoxon test for paired samples was carried out
to compare the concentration values obtained using two gas chro­
matographic methods: the official method (A) and the fast one (B)
proposed in this work.
Parallelism test and Wilcoxon test were performed using the R sta­
tistical package (version 4.1.1).
3. Results and discussion
The Agilent Intuvo 9000 GC System turned out to be a very fast and
suitable tool for analysis of major volatile compounds in spirit drinks
and wine. The fast direct heating\cooling technology in combination
with the special Intuvo column (custom product characterized by a
planar design) made it possible to obtain a total analysis time of 8 min.
Compared to the official method, this is a 80% reduction in the total time
of analysis.
The fast-quantitative determination was achieved by applying the
optimized GC-FID conditions allowed to have a good chromatographic
separation of the sixteen target compounds (Fig. 5).
The method was validated in view of the Regulation (EC) 657/2002
(European Commission, 2002) concerning the performance of analytical
methods and the interpretation of results, by defining the limit of
detection (LOD), limit of quantification (LOQ), linearity (R2), recovery,
repeatability and reproducibility. LOD, LOQ and linearity were per­
formed at two different alcoholic strengths, 40 and 10% (v/v) of
ethanol, in order to simulate the spirit drink and wine conditions,
respectively.
The signal-response of the GC-FID system was evaluated at six con­
centration levels across a range of 5–5000 μg/g at 40% (v/v) of ethanol
(Table 2) and at seven concentration levels between 5 and 500 mg/L at
10% (v/v) of ethanol (Table 3), using the calibration standard mixtures.
Analysis was performed in triplicate for every concentration level, and
calibration curves were established for each of the considered com­
pounds. Calibration plots were made by plotting the relative analyte-to-
IS peak area ratio against the relative analyte-to-IS concentration ratio,
and the linearity was evaluated by the squared correlation coefficient
(R2).
The R2 values obtained for each compound were always equal to or
higher than 0.995, as shown in Table 2 and in Table 3; this indicated a
good fit and linearity for the calibration curves in relation to the scope of
the method.
The LOQ and LOD were estimated according to the “Harmonized
guidelines for single-laboratory validation of methods of analysis
(IUPAC Technical Report)” (Thompson et al., 2002). Specifically, the
LOD was calculated as 3*S0, where S0 was the precision estimate of 6
independent determinations of analyte concentration at the lowest
calibration point, while the LOQ was computed as 3*LOD.
The LOQ was found to be in the range from 1.0 to 1.5 μg/g and the
LOD between 0.3 and 0.5 μg/g at 40% (v/v) of ethanol (Table 2). In
Table 3 were reported the LOQ (1.0 ÷ 1.5 mg/L) and LOD (0.0 ÷ 0.5 mg/
L) values calculated at 10% (v/v) of ethanol.
In order to determine the recovery, eight different spirit drinks
(Grape marc distillate (47.6%) Armagnac (39.8%), Brandy (40.2%),
Whisky (40.0%), Calvados (39.9%), Eau-de-vie de marc (48.0%), Cognac
(40.1%) and Rum (44.7%)) and two different wines (red wine (12%) and
white wine (11%)) with known composition were spiked with the target
compounds increasing the concentration of 250 μg/g and 150 mg/L,
respectively.
The accuracy, expressed as recovery percentage, was valued in
triplicate and considered acceptable when falling within ±20% of the
theoretical concentration (European Commission, 2002). The recovery
values were found to be equal to or higher than 93% for all of the target

8
M. Paolini et al. Food Control 136 (2022) 108873

Fig. 6. Box plots of concentration of volatile compounds in grape distillate and wine samples with Wilcoxon test results. The two GC-FID methods were labelled with
“A” and “B” (official method, A; proposed method, B).

9
M. Paolini et al.
compounds in all the different alcoholic beverages considered.
The repeatability and reproducibility were studied analysing the
same ten commercial alcoholic beverages. The repeatability was eval­
uated as the relative standard deviation (RSD) of five injections and was
lower than 10% for all analytes, while the reproducibility (RSD) was
calculated analysing the samples over a period of 5 days. The results
obtained were in most cases better than 10%, going from 1.6 to 13%.
In conclusion, the new instrumental approach allowed us to obtain
the chromatographic separation of the interested compounds in only 8
min compared to the 40 min required by the official methods (The
Commission of the European Communities, 2000; International Orga­
nisation of Vine and Wine, 2014), while maintaining or even improving
the analytical performance. Specifically, the fast GC-FID system allowed
to reach lower LOQ and LOD values than those obtained by using the
conventional approach and reported in others studies (Rodríguez
Madrera & Suárez Valles, 2007; Yilmaztekin & Cabaroglu, 2011; Luís
et al., 2011).
Moreover, the slope of the calibration curves and validation pa­
rameters (LOD, LOQ and linearity) obtained at the two different con­
centration levels of ethanol are very similar to each other. The slopes,
reported in Table 4, were compared by applying a 2-tailed t-test for
parallelism of regression lines. On the basis of the results of the statis­
tical test (df = 10) it is concluded that the slopes obtained with ethanol
at 40 and 10% are not significantly different and that the regression lines
are parallel (Table 4). This means that the analytical performances of the
developed GC-FID method are not affected by the alcoholic strength of
the analysed beverage and that the matrix effect is negligible.
As reported in the Regulation (EC) 787/2019, the spirit drinks are
characterized by a sugar concentration (expressed as invert sugar)
ranging between 0 g/L like in "Whisky" and 400 g/L like in "Crème de
cassis" (The European Parliament and of the Council of the European
Union, 2019). In this regard, an experimental study was carried out in
order to investigate how the presence of sugar could influence the
quantification of the considered congeners.
Hydroalcoholic solutions, at 40% (v/v) ethanol, with different sugar
content were spiked with the target compounds at two concentration
levels, 20 and 100 μg/g, and then analysed. The quantification of the
volatile compounds fell within the acceptance range (±20% of the
theoretical value) up to a sugar concentration of 50 g/L. That means, the
spirit drinks with a sugar content higher than 50 g/L should be distilled
before the GC-FID analysis as for wine samples.
4. Application of the method on real enological samples
The reliability of the developed method was assessed by analyzing
the alcoholic beverage samples provided by BIPEA and UIV as part of
Interlaboratory Proficiency-testing programs.
Four different spirit drinks (Eau-de-vie de marc, Armagnac, Rhum and
Calvados) and four different wines (Fortified red wine, Sparkling red wine,
Dry white wine and Sweet Champagne) were analysed, and the results
were compared with those of the others laboratories using the Z-score
model.
The most common evaluation of Proficiency Testing results is based
on the Z-score introduced in the IUPAC Harmonized Protocol (Thomp­
son et al., 2006). Participant’s results are converted into a ‘Z-score’ by
the equation:
(X − μ)
Z= equation 8
σ
where X is the participant’s result, μ and σ are the assigned value and
the measure of its variability (assigned standard deviation), corre­
spondingly. In order to evaluate the performance of a laboratory with
respect to comparability, the pattern applied is: |Z-score| ≤ 2 the result
is satisfying, 2 < |Z-score| ≤ 3 the result is questionable/suspicious and |
Z-score| > 3 the result is dissatisfying.
10
Food Control 136 (2022) 108873
As shown in Tables 5 and 6, the Z-score values are lower than 2 for all
the analytical parameters, meaning that our results fall within the
variability range accepted by BIPEA and UIV for both spirit drink and
wine samples, respectively.
Furthermore, in wine samples it was also possible to quantify other
compounds beside methanol and acetaldehyde, such as ethyl acetate,
ethyl lactate and some higher alcohols having important flavour
properties.
In order to compare the official GC-FID method (A) with the devel­
oped one (B), 40 commercial samples of alcoholic beverages, including
20 grape marc spirits and 20 red wines, were analysed. The obtained
results were displayed in Fig. 6 using a box plot graphical representa­
tion. Higher alcohols (2-methylbutan-1-ol, 2-methylpropan-1-ol, 2-
phenylethanol, 3-methylbutan-1-ol, butan-1-ol, butan-2-ol and propan-
1-ol) were collectively considered and expressed as "total higher alco­
hols", while acetaldehyde and acetal, if both present, were jointly
referred to as "total acetaldehyde".
Acetaldehyde and acetal (1,1-diethoxyethane) are related to each
other through an acetylation reaction, influenced by ethanol content, pH
and temperature (Cole & Noble, 1997; Peynaud & Maurie, 1938). The
"total acetaldehyde" content (TA) was calculated applying the following
equation (9):
TA = A + (0.373 × L) equation 9
where A and L are the concentration of acetaldehyde and acetal,
respectively, whereas 0.373 is the decimal fraction of acetaldehyde
realized from acid hydrolysis of acetal (Morrison, 1962).
The non-parametric Wilcoxon test for paired samples was carried out
to compare the concentration values obtained using the two gas chro­
matographic methods. The level of significance was set at 0.05. The
results of the statistical test are reported in Fig. 6 and show that there are
no significant differences (p > 0.05) between the concentration distri­
butions determined with the official method (A) and those determined
with the newly developed one (B) for both grape distillate and wine
samples.
5. Conclusion
In the presented work, a simple and rapid GC–FID method for the
determination of principal volatile compounds in alcoholic beverages
has been proposed. The developed method was optimized and sixteen
volatile compounds characteristic of alcoholic beverages were quanti­
fied in only 8 min. The method was validated and successfully tested on
different commercial samples, demonstrating its applicability and
reliability.
On the basis of the obtained results, the developed GC-FID method
can be proposed as a fast quality control procedure available to the in­
dustry in order to support the technological choices and improve the
winemaking practices and the spirit drink production.
CRediT authorship contribution statement
Mauro Paolini: Conceptualization, Investigation, Formal analysis,
Validation, Visualization, Writing – original draft, preparation, Writing
– review & editing. Loris Tonidandel: Conceptualization, Writing –
review & editing. Roberto Larcher: Resources, Supervision, Concep­
tualization, Writing – review & editing.
Declaration of interests
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
M. Paolini et al. Food Control 136 (2022) 108873

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