Article

Cite This: J. Agric. Food Chem. 2019, 67, 13694−13705 pubs.acs.org/JAFC

Development of a Solid-Phase Microextraction-Gas

Chromatography/Mass Spectrometry Method for Quantifying
Nitrogen-Heterocyclic Volatile Aroma Compounds: Application to
Spirit and Wood Matrices
Magali Picard,*,† Carla Garrouste,† Christelle Absalon,‡ Marie-Françoise Nonier,† Nathalie Vivas,†
and Nicolas Vivas†
†
Demptos Research Center, Centre d’Etude Structurale et d’Analyse des Molécules Organiques (CESAMO) and ‡CESAMO,
Institut des Sciences Moléculaires, Univ. Bordeaux, 351 Cours de la Libération, 33405 Talence, France
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*
S Supporting Information

ABSTRACT: Over wood aging, matured spirits developed a complex aromatic bouquet where roasted-like notes were often
perceived. Since many nitrogen heterocycles were related to these olfactory nuances, a headspace solid-phase microextraction
(HS-SPME) coupled to gas chromatography−mass spectrometry was developed and validated to quantify them in both spirit
and wood matrices. The various parameters affecting the extraction of the analytes from both spirit and wood samples were first
investigated (i.e., fiber coating phase, dilution, pH and volume sample, adding salt, extraction time and temperature, and
incubation time) to determine the best compromise for a single-run analysis of the whole set of studied compounds. Good
linearity (R2 > 0.99), repeatability, reproducibility, accuracy and low detection, and quantification limits were obtained, making
this analytical method a suitable tool for routine analysis of the selected nitrogen compounds. Fifteen pyrazines, three pyrroles,
and three quinolines were quantified in a series of oak wood and commercial spirit samples where some of them were identified
for the first time. The significant impact of some barrel features and the spirit in-wood maturation step on the N-heterocycle
profile in both matrices were finally discussed.
KEYWORDS: wood aging bouquet, spirit and oakwood matrices, roasted-like aromas, nitrogen-heterocyclic compounds,
headspace solid-phase microextraction (HS-SPME)

■ INTRODUCTION
Nowadays, a wide range of spirits are matured in oak casks,
especially whiskies, brandies, Cognacs, Armagnacs, rums, and
liquors. In addition to the raw material and the typical
expression of the terroir, it is widely accepted that wood aging
accounts among the most important contributors of the quality
of distilled beverages, by increasing complexity and removing
the immature character of the raw distillate.1,2 Indeed, over the
maturation time upon wood, major changes occur within the
volatile fraction of spirits, mainly driven by complex
interactions with wood-derived components.2 The develop-
ment of this aromatic complexity and subtlety, also known as
spirit aging bouquet, integrates a large palette of nuances where
roasted- and nutty-derived aromas are often cited in matured
spirit description.3,4 Pyrazines and pyrroles are potent and
characteristic volatile aroma compounds found in a wide range
of raw and processed foods. They were identified in the
volatiles of roasted peanuts,5 cocoa,6 or coffee.7 Their sensory
properties are generally associated with pleasant roasted, nutty,
cereal, licorice, coffee, and woody nuances8,9 (Table 1). Wang
et al. (1969)10 also postulated that pyrazines played an
important role in the roasted barley flavor since their removal
resulted in a loss of the typical desirable roasted aroma. The
relevance of pyrazines to the nutty character of a new-make
malt spirit was then confirmed by Boothroyd et al. (2014).11
Quinoline derivatives constitute another type of nitrogen-
containing heterocycles. Detected in coffee, black tea,
tobacco,12,13 as well as in whiskies,14 they are characterized
by medicinal and phenolic aromatic nuances, which are
particularly appreciated in peated whiskies.4 Indeed, peat can
be used as an external source of flavor, producing a smoke
“peak reek” aroma into the final spirit.15 Interestingly,
Nishimura and Masuda (1971)14 suggested that the presence
of quinoline derivatives in whiskies can be attributed to peat
smoke since these compounds were only found in malt barley
kilned with peat fire.
All of these N-heterocycles belong to the Maillard reaction-
type volatile aroma compounds, the most important and
complex reaction in flavor chemistry occurring during the
thermal food process. To date, some N-heterocyclic com-
pounds of interest have been detected in spirit beverages. Kahn
(1969)16 and Worben et al. (1971)17 identified several alkyl-
pyrazines in Scotch whiskies, which was confirmed by
Nishimura and Masuda (1971)14 in Japanese whiskies and
later by Boothroyd et al. (2014)11 in a larger set of Scotch
whiskies. Moreover, pyrazines were also found in other
distillates, including Cognac,18 tequila,19 rum,20 and other
Received: September 10, 2019
Revised: November 8, 2019
Accepted: November 11, 2019
Published: November 22, 2019

© 2019 American Chemical Society 13694 DOI: 10.1021/acs.jafc.9b05716

J. Agric. Food Chem. 2019, 67, 13694−13705
Journal of Agricultural and Food Chemistry Article

Table 1. Odor Characteristic, Boiling Point, Decimal Logarithm of Octanol−Water Partition Coefficient (Log P) and Ions
Monitored in SIM Detection for each Nitrogen-Heterocyclic Compound Studied
compound aroma description molecular mass (g/moL) boiling point (°C)a log P specific ions (m/z)b
2-methylpyrazine nutty 94.1 137 0.2 67/94
2,3-dimethylpyrazine nutty, meaty 108.1 154 0.6 42/81/108
2,5-dimethylpyrazine nutty, meaty roast 108.1 154 0.6 42/81/108
2,6-dimethylpyrazine nutty, sweet, chocolate 108.1 154 0.6 42/81/108
2,3,5-trimethylpyrazine nutty, caramel, roasted peanut, burnt 122.2 171 1.9 42/81/122
2,3,5,6-tetramethylpyrazine walnut, green, cocoa 136.2 190 1.3 42/54/136
2-ethyl-3-methylpyrazine toasted nutty, peanut, cereal 122.2 170 1.1 67/94//121/122
2-ethyl-3,5(6)-dimethylpyrazine roasted, cocoa, peanut, coffee, woody 136.2 181 1.5 108/135/136
5-ethyl-2,3-dimethylpyrazine burnt, roasted cocoa 136.2 191 1.5 54/135/136
2,3-diethyl-5-methylpyrazine hazelnut, roasted, meaty 150.2 192 1.9 56/135/150
2-acetylpyrazine popcorn, roasted, hazelnut 122.1 189 0.2 80/94/122
2-acetyl-3-methylpyrazine nutty, hazelnut, roasted 136.2 165 0.2 43/93/136
2-acetyl-3,5(6)-dimethylpyrazine nutty, hazelnut, peanut 150.2 70 (7 mmHg) 1.1 107/108/150
pyrrole nutty, sweet 67.1 130 0.8 41/67
2-acetylpyrrole licorice, walnut 109.1 220 0.9 66/94/109
2-acetyl-1-methylpyrrole earthy, nutty, smoky 123.1 201 0.7 80/108/123
quinoline medicinal, tobacco, leather 129.1 237 2.0 102/129
2-methylquinoline medicinal 143.2 247 2.6 115/128/143
6-methylquinoline animal, leather, phenolic 143.2 260 2.6 115/143
a
Except for 2-acetyl-3,5(6)-dimethylpyrazine, all boiling points were obtained at 760 mm Hg. bQuantifier ions are in bold.

Figure 1. General chemical structures of the nitrogen-heterocyclic compounds studied. R = H, CH3, or CH2CH3.

types of liquors.21 In addition, several researches also noted the
presence of pyrazines and pyrroles in wood,22 wood smoke.23
Kim et al. (1974)24 and Maga and Chen (1985)25 identified
eight alkyl- and acetyl-pyrazines present at various concen-
trations depending on the wood source. However, despite
there is a wealth of information on the occurrence of nitrogen-
heterocyclic aroma compounds in food, they have received
little attention in the spirit aging context, regarding the wide
range of data collected on other wood-derived volatile aroma
compounds such as whisky lactones, volatile phenols, phenolic,
and furanic aldehydes.26−28
Besides, the complexity of spirit and wood-derived volatile
aroma compound profiles represents a current challenge from
an analytical standpoint and, more particularly, when
compounds at low concentration have to be not only identified
but also quantified. Headspace solid-phase microextraction
(HS-SPME) is considered one of the most effective sample-
enrichment technique as it is solvent-free, accurate, sensitive,
and easy-to-automate.29 The present study was undertaken to
gain knowledge on the composition of spirit and wood samples
in terms of nitrogen-heterocyclic compounds. Indeed, although
some quantitative analyses of N-heterocycles in whiskies and
Chinese liquors were reported,30,31 no previous study
investigated in-depth how maturation in wood barrels impacts
on the spirit-related roasted and peated volatile aroma
compounds. A HS-SPME extraction method, coupled to
GC/MS analysis, was therefore developed and validated by
assaying twenty-one N-heterocyclic metabolites, to address in a
single-analytical run their spirit and wood full profiling. As
spirit and wood matrices had different volatile and nonvolatile
profiles, the various parameters were optimized in each matrix
independently. To our knowledge, it is the first time that a
study was designed to develop such an analytical approach,
well-suited for each matrix. It offered the opportunity to
simultaneously examine, from both qualitative and quantitative
standpoints, a large set of the targeted nitrogen-heterocyclic
compounds covering a wide range of physico-chemical
properties and characterized by roasted- and nutty-derived
notes (Table 1).
■ MATERIALS AND METHODS
Chemicals and Reference Compounds. Absolute ethanol
(purity >99.8%) was obtained from Merck (Darmstadt, Germany)
and used as a solvent for preparing the stock solutions of analytes as
well as the wood solutions. Ultrapure water (18.2 M Ω.cm) was
obtained from a Milli-Q purification system (Millipore, Saint-
Quentin-en-Yvelines, France), and sodium chloride (99%) was
supplied by Sigma-Aldrich (Fontenay-sous-bois, France).
The general chemical formulas of the twenty-one studied N-
heterocyclic compounds are presented in Figure 1. They are derived
from either pyrazine, pyrrole, or quinoline chemical backbones. All
standard compounds were obtained from Sigma-Aldrich (Fontenay-
sous-bois, France): 2-methylpyrazine (CAS registry no. 109-08-0,
purity 99%), 2,3-dimethylpyrazine (CAS registry no. 5910-89-4,
purity 95%), 2,5-dimethylpyrazine (CAS registry no. 123-32-0, purity
98%), 2,6-dimethylpyrazine (CAS registry no. 108-50-9, purity 98%),
2,3,5-trimethylpyrazine (CAS registry no. 14667-55-1, purity 99%),
2,3,5,6-tetramethylpyrazine (CAS registry no. 1124-11-4, purity 98%)
2-ethyl-3-methylpyrazine (CAS registry no. 15707-23-0, purity 98%),
2-ethyl-3,5(6)-dimethylpyrazine (mixture of isomers, CAS registry no.
27043-05-6, purity 95%), 5-ethyl-2,3-dimethylpyrazine (CAS registry
no. 15707-34-3, purity 96%), 2,3-diethyl-5-methylpyrazine (CAS

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J. Agric. Food Chem. 2019, 67, 13694−13705
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registry no. 18138-04-0, purity 99%), 2-acetylpyrazine (CAS registry
no. 22047-25-2, purity 99%), 2-acetyl-3-methylpyrazine (CAS registry
no. 23787-80-6, purity 98%), 2-acetyl-3,5(6)-dimethylpyrazine
(mixture of isomers, CAS registry no. 54300-08-2, purity 98%),
pyrrole (CAS registry no. 109-97-7, purity 98%), 2-acetylpyrrole
(CAS registry no. 1072-83-9, purity 98%), 2-acetyl-1-methylpyrrole
(CAS registry no. 932-16-1, purity 98%), quinoline (CAS registry no.
91-22-5, purity 98%), 2-methylquinoline (CAS registry no. 91-63-4,
purity 95%), 6-methylquinoline (CAS registry no. 91-62-3, purity
98%), and 3,4-dimethylphenol (CAS registry no. 95-65-8, purity
98%).
Spirit and Wood Samples. Method Optimization and
Validation. For spirit analysis, the method was optimized and
validated using a commercial whisky (40% of ethanol) spiked with the
studied nitrogen heterocycles at 500 μg/L. Concerning the wood
analysis, a nontoasted oak chip sample (25 g) was extracted in a 500
mL of hydroalcoholic solution (50% of ethanol) during 12 h at room
temperature under magnetic stirring. The oakwood mixture was then
filtered using a filter paper, and the filtrate was spiked with 500 μg/L
of the studied volatile aroma compounds.
Method Application. Once the analytical parameters were
optimized, a series of six different commercial oak chips (Oak In
Wine, Arobois, Gangnac-Sur-Cere, France) were analyzed. They were
issued from French (Quercus petraea and Quercus robur) and
American (Quercus alba) oak wood species and were toasted at
three temperature levels in an industrial kiln, based on the cooper’s
skills (light: 120−130 °C; medium: 160−170 °C, and heavy: 210−
220 °C). Oak chips solutions were prepared in the same way that of
during the method optimization step (25 g in a 500 mL water/ethanol
solution, 50/50; v/v) and were analyzed using the validated
methodology. The analytical method was also applied to 41
commercial spirit samples (alcohol between 40 and 70%), issued
from various brands, and including 6 Armagnacs, 3 Cognacs, 3
brandies, 10 rums, and 19 whiskies. Six of them were non matured
upon wood, while the others covered a wide range of maturation
periods in wood barrels (from 1 to 48 years, Table 5). Each sample
was analyzed in duplicate for both spirit and wood matrices.
Preliminary Optimization of Several Analytical Parameters
during the Extraction Step. In food analysis, various factors are
reported to influence the effectiveness of SPME in concentrating
analytes.29,32 In this study, two kinds of parameters were more
particularly studied in relation to either the sample (pH, volume,
dilution factor, and sodium chloride content) or the fiber (coating,
adsorption temperature and time, and incubation time) (Table S1,
Supporting Information). The optimized SPME parameters were
selected to achieve the highest extraction efficiency, by comparing the
peak area of analytes obtained under each test condition. All
experiments were performed in duplicate.
SPME Fiber Coatings. Several fibers (Supelco, Bellefonte, PA,
USA) coated with various stationary phases and film thicknesses were
compared: polydimethylsiloxane 100 μm (PDMS), carboxen-poly-
dimethylsiloxane 85 μm (CAR-PDMS), and divinylbenzene-carbox-
en-polydimethylsiloxane 50/30 μm (DVB/CAR/PDMS). They were
conditioned prior to use by insertion into the GC injector, as
recommended by the manufacturer.
Sample Preparation. The impact of the dilution was first
investigated by comparing results obtained from the solution sample
diluted by factors 2, 4, and 10. The pH was then modified by adding
directly in 25 mL samples, a few drops of 1 M HCl and 1 and 10 M
NaOH solutions, to compare the relative efficiency of extractions at
pH 2.5, 4, 7, and 9, respectively. The optimization of sample
preparation also focused on the volume phase for which three
volumes (3, 5, and 10 mL) were tested. The effect of salt addition on
the extraction was finally studied, by increasing the sodium chloride
content in the sample (0, 1.5, and 3 g).
Fiber Adsorption Parameters. Both the temperature and time of
HS-SPME significantly affect the equilibrium during extraction and
thus influence the compound extraction. Operating conditions were
optimized while setting SPME extractions of samples at different
adsorption temperatures (40, 50, 60, and 70 °C) and times (20, 30,
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40, 50, and 60 min). The incubation time was also optimized by
evaluating extraction efficiency after 5, 10, and 15 min.
HS-SPME Optimized Procedure. The 2 mL initial samples were
diluted 10-fold in MQ water to reach a final volume of 20 mL, and pH
was adjusted to 4 and 7 for wood and spirit solutions, respectively,
using 1 M HCl and 1 M and 10 M NaOH solutions. To a 20 mL
headspace vial containing 3 g of sodium chloride was added either a
10 mL spirit or a 5 mL wood-diluted sample spiked with 10 or 5 μL of
internal standard 3,4-dimethylphenol solution (100 mg/L in alcoholic
solution), respectively. The vial was then tightly sealed with a PTFE-
lined cap, and the solution was homogenized with a vortex shaker
before loading onto an auto-sampling device. In the optimized
conditions, extractions were performed using the CAR-PDMS-coated
fiber and the extraction program consisted in swirling the vial for 5
min at 60 °C under regular agitation (200 rpm, 10 s intervals), then
inserting the fiber into the headspace for 30 min at 60 °C as the
solution was swirled again, and finally transferring the fiber to the
injector for desorption at 250 °C for 5 min.
GC−MS Instrumentation and Analytical Conditions. Chro-
matographic analyses were carried out using a Trace GC Ultra system
(Thermo Fisher Scientific, CA, USA) coupled to an ISQ quadrupole
mass spectrometer, equipped with a Triplus autosampler (Thermo
Fisher Scientific) and an Optima Waxplus capillary column (30 m ×
0.25 mm ID, 0.25 μm film thickness, Macherey-Nagel, Düren,
Germany). The carrier gas was Helium N55 at a constant flow of 1.2
mL min−1. The oven temperature was raised from 40 °C (1 min) to
160 °C at a rate of 5 °C/min and then ramped at a rate of 15 °C/min
to 260 °C (final isotherm for 5 min). The ion source was set at 200
°C and the transfer line between GC and MS at 250 °C. Detection
and quantitation were carried out in electron ionization (70 eV) and
the selected-ion monitoring (SIM) mode, based both on the retention
time of each compound and the selection of specific ions (two or
three ions for each analyte: one or two for confirmation and one for
quantification, on the basis of the best measured signal-to-noise ratio;
Table 1). The 3,4-dimethylphenol internal standard was quantified
with an ion mass-to-charge (m/z) of 122, and the qualifier ion was an
m/z of 107. Before the first daily analysis, a blank test was performed
to check possible carryover between samples.
Method Validation. The method linearity was evaluated both in
whisky and wood matrices used in the method optimization through
10 calibration levels covering a 0.5−1000 μg/L range of
concentrations for the various analytes. The performance of the
proposed optimized method was evaluated in terms of limits of
detection (LOD) and quantification (LOQ), defined as the
concentrations giving signal-to-noise ratios of 3 and 10, respectively.
Based on linear regression analysis, LOD and LOQ were calculated
from the calibration line, at low concentrations and using the
following formulas: LOD = 3Sa/b and LOQ = 10Sa/b, where Sa is the
standard deviation of the response, and b the slope of the calibration
curve.33 To evaluate repeatability (intraday precision), 10 replicates of
(i) the same spirit and (ii) wood solutions were spiked at 50 μg/L
with reference standards. The reproducibility of the method (interday
precision) was determined by analyzing 12 replicates of the same
whisky and wood solution spiked at 50 μg/L over a period of 1
month. The accuracy of the analytical method was evaluated by
calculating the recoveries on a non matured spirit and a light-toasted
oakwood extract solution using a standard addition technique.
Accuracy is reported for each assay as the percentage recovery of a
known amount of analyte added to the spirit and wood samples. Prior
to sample preparation, each matrix was spiked with three different
concentration levels: 10 μg/L (low level), 100 μg/L (medium level),
and 500 μg/L (high level).
Statistical Analysis. For each optimization step, data were
statistically analyzed with one-way analysis of variance (ANOVA) to
determine any significant differences. The F-ratio served as a marker
of the discrimination ability, and the statistically significant level was
set at 5% (p value <0.05).
All quantitative data obtained on the set of wood samples were
analyzed using a two-way ANOVA (factor 1: wood species, factor 2:
toasting degree) to correlate N-heterocycles concentrations to wood
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Figure 2. Impact of (a) fiber coating phase, (b) sample dilution, (c) sample pH, and (d) sample volume on SPME extraction efficiency in spirit and
wood matrices. Letters indicate significant differences at p value <0.05.
specificities and identify interactions between the considered factors.
A one-way ANOVA, at a significance level of 95%, was applied to the
two global clusters of spirit samples (matured and non matured) for
all the volatile aroma compound concentrations. A one-way ANOVA
test was also specifically performed on the matured and non matured
samples within each kind of spirit clusters (i.e., Armagnac, Cognacs,
and brandies/rums/whiskies). All statistical analyses were conducted
using XLSTAT Sensory software (Addinsoft, Paris, France, version
2019.2.2).
■
RESULTS AND DISCUSSION
Development of the HS-SPME Analytical Method:
Preliminary Optimizations. Although pyrazines, pyrroles,
and quinolines belong to the N-heterocyclic chemical family,
they present a wide range of chemical properties such as
boiling points and polarities (log P), which may directly affect
their extraction (Table 1). Therefore, it was necessary to
determine the best compromise within the optimized
conditions for getting in a single-run analysis the whole set
of studied heterocyclic compounds. In this optimization stage,
the impact of various operating conditions on HS-SPME
performance was evaluated, including fiber coating phase,
sample preparation (pH, volume, dilution, salt addition), and
fiber adsorption parameters (extraction temperature and time,
incubation time).
For better clarity, N-heterocycles were classified in three
groups according to similarities into their chemical structures:
alkypyrazines and pyrrole (group 1), acetylpyrazines and
acetylpyrroles (group 2), and quinolines (group 3). The effect
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of each parameter was studied individually, in the two distinct
matrices (Figures 2 and 3).
During the first steps of the development process (SPME
fiber selection and optimization of the sample preparation), the
program of the headspace SPME was set on the following
parameters: incubation/extraction temperature: 40 °C; in-
cubation time: 15 min; extraction time: 30 min.
Selection of the Fiber Coating. Since SPME is by nature an
equilibrium technique, the aim is to reach the equilibrium
between the sample matrix and the coating of the SPME device
as quickly as possible. Because both matrix and coating are
competing for analytes, the affinity of coating for targeted
analytes is crucial in SPME sampling. The extraction of solutes
from the aqueous phase into the SPME fiber is controlled
among others by the partition coefficients (usually known as
log Ko/w or log P). To investigate the extraction yields of the
studied analytes, three fiber coatings were tested: PDMS,
CAR/PDMS, and DVB/CAR/PDMS. PDMS fiber is the most
useful coating for nonpolar analytes, whereas CAR/PDMS and
DVB/CAR/PDMS fibers provide the best extraction efficien-
cies for a wide range of analytes with different polarities and
molecular weights.32 For this first-optimization step, the
parameters related to the sample preparation were first fixed
as follows for both matrices: pH 4; 10 mL volume; 10-fold
dilution; 3 g of sodium chloride addition. The comparison of
absolute peak areas between the experiments revealed that
CAR/PDMS was the most efficient coating phase for pyrazines
and pyrroles as it presented the significant greatest specificity
in both matrices (p value <0.05; Figure 2a). Better results were
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Figure 3. Effect of (a) adsorption temperature and (b) fiber exposure time on SPME extraction efficiency in spirit and wood matrices.
obtained for quinolines when polyphasic coating fibers were
used, but no significant differences were observed between
CAR/PDMS and DVB/CAR/PDMS phases. Interestingly, it
was reported that combining polar and nonpolar coatings on
the same fiber (typically a mixed CAR/PDMS coating phase)
improves the extraction efficiency of polar organic analytes
within a polar matrix, while the simultaneous extraction of
interferents is not possible.34 These results were also confirmed
by previous studies where the CAR/PDMS coating fiber was
chosen in heterocyclic compounds quantitation for wine35 or
meat36 applications. Altogether, the CAR/PDMS fiber was
selected for subsequent studies.
Dilution Effect. The percentage of the protic solvent is
usually the most influencing variable in SPME-based analysis
since a high content may dramatically reduce fiber extraction
efficiency and also damage the polymer-coating phase of the
SPME fiber. As ethanol is one of the main components in
spirit, the effect of the ethanol content on the aroma
compound extraction must be taken into account. Thus, the
high ethanol concentration (40−50%, v/v) of both spirit and
wood extract solutions required sample dilution. Three ethanol
level contents were compared, and the initial spirit and wood
samples were diluted in water by 2, 4, and 10 times. The other
parameters related to the sample preparation were fixed as
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follows for both matrices: pH 4; 10 mL volume; 3 g of sodium
chloride. From our results and also on the basis of previously
published data,37 decreasing the ethanol content had a
beneficial effect on extraction, with significant higher area
responses found in samples diluted 10 times (p value < 0.01)
in spirit and wood matrices, respectively (Figure 2b). Thus, a
10-fold dilution was adopted for the next experiments.
Influence of Sample pH. Once the fiber coating and the
dilution factor had been selected, the influence of matrix pH
on extraction was studied. In this experiment and for both
matrices, the sample volume and the salt content were always
kept at 10 mL and 3 g, respectively. Since the N-heterocyclic
compounds are weak basic compounds, the pH of the sample
potentially impacts their extraction. Indeed, some previous
studies adjusted the pH solution in developing SPME
analytical method.35,38 For both matrices, the least efficient
conditions were found at pH 2.5 where the response areas for
the whole set of analytes were significantly lower than in other
pH conditions. It was especially verified for pyrazine and
pyrrole derivatives for which the response factor was divided
by a factor 2 to 7 (p value <0.01; Figure 2c). A slight but not
significant increase in peak areas was obtained at pH higher
than 4 in spirit and wood matrices, meaning that pH had less
effect on extraction than other parameters. However, it was
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Table 2. Validation Parameters (Linearity, LOD and LOQ, Repeatability, and Reproducibility) of the Optimized SPME-GC/
MS Method Applied to Wood Matrix
intraday precision interday precision
linearity sensitivity (repeatability) at 50 μg/L (reproducibility) at 50 μg/L
concentration LOD LOQ
compound range (μg/L) R2 slope intercept (μg/L) (μg/L) RSD (%) RSD (%)
2-methylpyrazine 4.9−994 0.991 0.1698 +0.0005 0.069 0.233 4.5 14.4
2,3-dimethylpyrazine 5.2−1038 0.991 0.2647 +0.0892 0.117 0.391 4.5 15.0
2,5-dimethylpyrazine 5.1−1029 0.991 0.3608 −0.0373 0.153 0.511 7.9 13.9
2,6-dimethylpyrazine 4.6−923 0.993 0.5399 +0.1082 0.185 0.617 6.1 14.7
2,3,5- 4.7−951 0.997 0.2662 +0.0477 0.066 0.221 7.4 12.1
trimethylpyrazine
2,3,5,6- 4.7−940 0.992 0.9977 +0.1137 0.373 1.242 8.5 18.9
tetramethylpyrazine
2-ethyl-3- 5.5−1095 0.991 0.6354 +0.2159 0.303 1.011 3.8 16.6
methylpyrazine
2-ethyl-3,5- 6.2−1230 0.991 0.9808 +0.2461 0.517 1.722 6.2 16.4
dimethylpyrazine
2-ethyl-3,6- 6.2−1230 0.991 1.9527 +0.7053 1.055 3.518 6.7 14.5
dimethylpyrazine
5-ethyl-2,3- 4.9−994 0.990 0.0509 +0.0193 0.022 0.076 5.0 13.8
dimethylpyrazine
2,3-diethyl-5- 5.9−1182 0.992 2.8401 +1.0429 1.411 4.705 4.9 12.8
methylpyrazine
2-acetylpyrazine 6.4−1276 0.995 0.648 −0.0057 0.025 0.083 9.4 13.7
2-acetyl-3- 5.7−1132 0.993 0.3378 +0.050 0.140 0.468 7.6 11.1
methylpyrazine
2-acetyl-3,5- 5.1−1025 0.994 0.4795 +0.0338 0.167 0.557 9.4 9.1
dimethylpyrazine
2-acetyl-3,6- 5.1−1025 0.992 0.1379 −0.0095 0.055 0.184 8.9 11.1
dimethylpyrazine
pyrrole 4.9−982 0.991 0.0762 −0.002 0.031 0.101 9.0 17.0
2-acetylpyrrole 4.8−958 0.997 0.078 −0.0008 0.018 0.060 5.4 17.1
2-acetyl-1- 6.2−1238 0.991 0.8506 +0.2395 0.445 1.092 8.0 12.9
methylpyrrole
quinoline 4.6−918 0.992 0.5974 −0.3586 0.879 2.929 9.0 18.2
2-methylquinoline 6.9−1393 0.991 0.2228 −0.3005 0.540 1.802 9.2 16.3
6-methylquinoline 4.9−990 0.993 0.4795 −0.3923 0.722 2.408 9.6 16.6

noticed a significant increase in absolute areas for quinolines at
pH 4 in the wood matrix as well as a better resolution of
chromatographic peaks in the spirit matrix at pH 7.
Accordingly, we chose to buffer the sample pH to 4 and 7
for wood and spirit matrices, respectively.
Sample Volume. As previously described, the sample
volume is an important SPME parameter to be optimized
since it is directly related to the method sensitivity.39 To
address the impact of this parameter on compounds extraction
performance, three volumes (3, 5, and 10 mL) were compared
in both matrices. This step was performed in the optimized
conditions previously determined, and the added content of
sodium chloride was fixed at 3 g. Results obtained in the spirit
matrix highlighted that a 3 mL sample volume led to
contrasting results between analytes. Indeed, the best
extraction efficiency was observed for alkylpyrazines and
pyrrole, whereas lowest responses were obtained for the
other analytes. Moreover, a 10 mL sample provided the best
sensitivity for quinoline compounds where the abundances
increased by a factor of 6 compared to the other conditions (p
value <0.001). Consequently, considering the extraction
efficiency for all the set of compounds and as alkylpyrazines
and pyrrole were more easily detectable, a volume of 10 mL
was set for the spirit samples. On the other hand, a 5 mL
volume appeared to be the best choice for wood extracts since
a significant gain in efficiency was observed for alkylpyrazines
13699
and pyrrole (p value <0.05) as well as, even not significant, a
trend to a better extraction for acetylpyrazines, acetylpyrroles,
and quinolines (Figure 2d).
Salt Addition. Most studies have shown that the addition of
a salt (usually sodium chloride) enhances the ionic strength
and thus reduces the solubility of analytes, which are more
easily retained.29 To check this “salting out” effect, 10 and 5
mL samples for the spirit and wood matrix, respectively,
without or with sodium chloride (1.5 and 3 g) were compared.
As expected, results highlighted that, for all the analytes studied
in both matrices, responses clearly increased with the sodium
chloride content (p value <0.001), with the highest peak areas
obtained in the most saturated sample (data not shown). A 3 g
addition of sodium chloride was thus adopted for the study.
Adsorption Temperature and Time. Once the best
conditions for the fiber coating phase and the sample
preparation were identified and adopted, the next steps of
the SPME optimization method were focused on the
adsorption time and temperature. Time and temperature are
parameters closely related to each other.40 Indeed, higher
temperatures increase the partial vapor pressure of analytes and
enable shorter exposure time mainly for highly volatile
components. However, an excessive increase in temperature
can cause premature desorption of analytes with the coating
phase beginning to lose its ability to absorb them. Practically,
the effect of temperature was investigated by analyzing spiked
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Journal of Agricultural and Food Chemistry Article

Table 3. Validation Parameters (Linearity, LOD and LOQ, Repeatability, and Reproducibility) of the Optimized SPME-GC/
MS Method Applied to Spirit Matrix
intraday precision interday precision
linearity sensitivity (repeatability) at 50 μg/L (reproductibility) at 50 μg/L
concentration LOD LOQ
compound range (μg/L) R2 slope intercept (μg/L) (μg/L) RSD (%) RSD (%)
2-methylpyrazine 0.49−497 0.998 0.3108 −0.0009 0.25 0.84 6.6 15.7
2,3-dimethylpyrazine 0.52−519 0.990 0.4026 +0.0652 0.117 0.391 6.6 15.1
2,5-dimethylpyrazine 0.51−514 0.995 0.7312 −0.0164 0.093 0.309 6.9 18.4
2,6-dimethylpyrazine 0.46−461 0.994 0.9474 +0.1203 0.123 0.411 7.0 18.3
2,3,5- 0.47−475 0.992 0.4589 +0.0317 0.067 0.225 8.4 18.2
trimethylpyrazine
2,3,5,6- 0.47−470 0.994 0.6773 +0.070 0.266 0.888 9.0 13.8
tetramethylpyrazine
2-ethyl-3- 0.55−548 0.992 0.9239 +0.0754 0.163 0.546 7.5 17.4
methylpyrazine
2-ethyl-3,5- 0.62−615 0.993 1.2768 +0.1144 0.236 0.788 9.7 15.8
dimethylpyrazine
2-ethyl-3,6- 0.62−615 0.993 3.0134 +0.2965 0.560 1.866 9.6 14.8
dimethylpyrazine
5-ethyl-2,3- 0.49−497 0.990 0.0928 +0.0109 0.016 0.053 9.2 16.7
dimethylpyrazine
2,3-diethyl-5- 0.59−591 0.994 4.1585 +0.6307 0.845 2.818 9.8 15.7
methylpyrazine
2-acetylpyrazine 0.64−638 0.993 0,0286 +0.0016 0.022 0.087 8.3 16.9
2-acetyl-3- 0.57−566 0.998 0.5623 +0.0172 0.054 0.180 8.4 16.5
methylpyrazine
2-acetyl-3,5- 0.51−512 0.995 0.7744 +0.0028 0.092 0.306 7.5 15.8
dimethylpyrazine
2-acetyl-3,6- 0.51−512 0.997 0.2159 −0.0034 0.021 0.069 7.2 12.2
dimethylpyrazine
pyrrole 0.49−491 0.995 0.1452 +0.0131 0.017 0.058 5.7 18.7
2-acetylpyrrole 0.48−479 0.990 0.0146 +0.0084 0.03 0.09 6.7 16.7
2-acetyl-1- 0.62−619 0.997 1.534 +0.0778 0.188 0.626 8.0 16.4
methylpyrrole
quinoline 0.46−459 0.995 2.235 +0.0186 0.216 0.722 5.9 12.5
2-methylquinoline 0.69−696 0.993 2.252 −0.3119 0.559 1.865 9.2 16.1
6-methylquinoline 0.49−495 0.994 2.3402 −0.2367 0.381 1.271 8.5 17.2

spirit and wood matrices at 40, 50, 60, and 70 °C, with a
constant incubation time set at 15 min and extraction time at
30 min (Figure 3a,b). As can be observed in both matrices, the
extraction performance for alkylpyrazines and pyrrole,
acetylpyrazines and acetylpyrroles, increased from 40 to 60
°C before decreasing at 70 °C. Concerning quinoline
compounds, a global increase in the response peak areas was
observed over the extraction temperature range. Thus, an
adsorption temperature of 60 °C seemed like a relevant
compromise for the whole set of studied N-heterocyclic
compounds. The exposure time is also an important parameter,
which influences the partition of solutes between sample
solution and fiber coating, inducing a direct influence on the
analysis time and performance. Although a longer time favors
the analyte occupation of more sites on the fiber, an excessive
time may sometimes cause desorption.29
To optimize the SPME extraction period, a range of
extraction times (20−60 min) were tested at 60 °C, with a
previous incubation time set at 15 min. Figure 3c,d presents
the different extraction profiles of N-heterocyclic compounds
in spirit and wood matrices, respectively. For alkylpyrazines
and pyrrole in the spirit matrix, a slight increase in peak areas
was observed between 20 and 30 min, followed by a decrease
until 60 min (Figure 3c), whereas a constant decrease was
observed over the studied times in wood samples (Figure 3d).
A similar trend was found for acetylpyrazines and acetylpyr-
13700
roles in both matrices, with an equilibrium reached after 30
min of exposure. The same response profile was obtained in
both matrices for quinoline derivatives over the time range,
with the highest peak areas obtained at 30 min followed by a
decrease until 60 min. Consequently, a 30 min fiber exposure
time was adopted to achieve the best performance analysis of
the twenty-one compounds studied.
Incubation Time. The time period before inserting the fiber
into the sample was finally optimized. Several incubation times
(5, 10, and 15 min) were thus examined. Although no analyte
loss was observed, increasing the incubation time to 10 or 15
min did not give any significant efficiency gain (data not
shown). Therefore, as a compromise between quick analysis
run and good method sensitivity, a 5 min incubation time was
selected for subsequent experiments.
Validation of the Analytical Method. All validation
parameters, such as linearity, detection and quantification
limits, precision, and accuracy, were evaluated under optimum
selected conditions.
Calibration. Both spirit and wood matrices were spiked with
the studied analytes at various concentrations to obtain 10
calibration levels (from 0.5 to 500 μg/L for the spirit matrix
and from 5 to 1000 μg/L for the wood matrix). The calibration
curves were plotted as the relative peak area ratio (analyte vs
internal standard) and as a function of the added compound
concentration. From the data obtained, the optimized method
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Journal of Agricultural and Food Chemistry Article

showed linear functions throughout the concentration range in Table 4. F-Ratio Values Obtained in a Two-Way ANOVA
both matrices, with coefficients of determination ranging Performed on the N-Heterocycles Mean Concentrations
between 0.990 and 0.998 (Tables 2 and 3). Obtained in the Wood Samples. Factor 1 Referred to the
Sensitivity. Limits of detection (LOD) and quantification Oak Wood Species (American and French Oak Woods) and
(LOQ) varied according to the analyte. In the wood matrix, Factor 2 to the Toasting Levels (Light, Medium and
they were found between 0.03 and 1.41 μg/L and from 0.06 to Heavy)a
4.71 μg/L, respectively (Table 2). In the spirit matrix, LODs
interaction of
and LOQs were between 0.02 and 0.85 μg/L and from 0.05 to factors factors
2.82 μg/L, respectively (Table 3). In both mixtures, 2,3-
compound oak toasting oak x toasting
diethyl-5-methylpyrazine presented the highest detection and
quantification limits. Moreover, LODs and LOQs determined 2,6-dimethylpyrazine 0.43 56.06*** 2.67
in wood were slightly higher than those found in spirit 5-ethyl-2,3- 56.31*** 47.31*** 11.30**
dimethylpyrazine
probably due to differences in the matrix composition. In the 2-acetylpyrazine 11.47 81.76*** 3.46
study of complex mixtures, characterization and quantitation of 2-acetyl-3-methylpyrazine 3.12 3.49 11.81
analytes are often impaired by matrix effects, which directly 2-acetyl-3,5- 14.52** 0.14 5.92
affect the release and thus the extraction efficiency of volatile dimethylpyrazine
aroma compounds. Headspace partitioning differences are 2-acetyl-3,6- 8.60* 16.09** 8.69**
observed according to the hydrophilic−hydrophobic ratio dimethylpyrazine
within the sample. Other major sources of matrix effects were pyrrole 212.0*** 121.29*** 82.26***
also previously reported, such as the retentive effect of the 2-acetylpyrrole 8.53* 7.37* 5.38*
nonvolatile fraction and the volatile fraction composition.41,42 2-acetyl-1-methylpyrrole 0.004 6.05 2.74
Precision and Accuracy. Repeatability and reproducibility quinoline 2.74 4.29 18.12
were evaluated by relative standard deviations. To evaluate the 2-methylquinoline 0.002 2.07 0.40
repeatability, 10 identical spiked samples (wood and spirit, at 6-methylquinoline 40.07*** 1.88 5.12
50 μg/L) were analyzed. The coefficients obtained were below a
Asteriks *, **, and *** indicate significant differences at p values
10% for all the compounds. Concerning reproducibility for <0.05, <0.0,1 and <0.001, respectively.
each matrix, the relative standard deviations were calculated for
12 independent assays, run under the same analytical tryptophan under roasting conditions, their formation is
conditions and over a 1 month period. Results deviations assumed to proceed via alkylated indoles by ring enlargement
were below 20%, ranging from 9.1% (2-acetyl-3,5-dimethylpyr- reactions or intramolecular cyclizations. To our knowledge, no
azine) to 18.9% (2,3,5,6-tetramethylpyrazine) in the wood quantitative data for the current set of N-heterocyclic markers
sample (Table 2) and from 12.2% (2-acetyl-3,6-dimethylpyr- had been published in wood. Their average concentrations
azine) to 18.7% (pyrrole) in the spirit sample (Table 3). The ranged from 2.04 to 14.51 μg/L (corresponding to 0.04 to 0.29
accuracy of the analytical method was evaluated by calculating μg/g of dry wood) for alkylpyrazines (2,6-dimethylpyrazine
the individual analyte recoveries at three different spiking levels and 5-ethyl-2,3-dimethylpyrazine), 0.79 to 4.69 μg/L (i.e.,
in each matrix. The recovery rate of the method was 0.016 to 0.094 μg/g of dry wood) for acetylpyrazines, 0.62 to
satisfactory since it ranged from 70.5 to 120.5% for all the 9.27 μg/L (i.e., 0.012 to 0.19 μg/g of dry wood) for pyrrole,
studied compounds in wood (Table S2) and from 78.4 to 9.09 to 41.77 μg/L (i.e., 0.18 to 0.84 μg/g of dry wood) to
121.6% in spirit (Table S3). acetylpyrroles derivatives and from 10.85 to 18.52 μg/L (i.e.,
Proof Applicability: Quantitation of Nitrogen-Heter- 0.22 to 0.37 μg/g of dry wood) for quinolines (Figure 4). The
ocyclic Compounds in Wood and Spirit Matrices. analysis of the variance pointed out the influence of the wood
Influence of Oak Species and Toasting Conditions on N- species and its toasting level on many N-heterocyclic markers.
Heterocycles Profiling in Wood. Thirteen N-heterocyclic Concerning the wood effect, significantly higher contents of 5-
compounds were detected in oakwood samples. 2,3,5,6- ethyl-2,3-dimethylpyrazine, 2-acetyl-3,5-dimethylpyrazine, 2-
Tetramethylpyrazine was found at lower concentrations than acetyl-3,6-dimethylpyrazine, pyrrole, 2-acetylpyrrole, and 6-
its LOQ, and thus, 12 compounds were quantified (Table 4). methylquinoline were found in French oak compared to
Albeit previous studies had reported the presence of some American oak. Moreover, these results highlighted that the
alkylpyrazines in oak and wood smoke,9,25 neither 2,3,5,6- toasting level also significantly impacted on total N-
tetramethylpyrazine and 5-ethyl-2,3-dimethylpyrazine nor 2- heterocyclic aroma compounds concentrations (Figure 4).
acetyl-1-methylpyrrole and quinolines derivatives were identi- More specifically, alkylpyrazines, acetylpyrazines, pyrrole, and
fied so far in the wood matrix. All the studied N-heterocycles acetylpyrrole contents increased with the toasting level,
originated from the Maillard reaction, which is one of the most whereas no particular trend was observed for quinolines in
important routes to aromas in heated foods. Typically, in the our conditions. The components mainly affected by the
Maillard reaction, the carbonyl group of a reducing sugar reacts toasting degree were 2,6-dimethylpyrazine, 5-ethyl-2,3-dime-
with the amino group of an amino acid, leading to a complex thylpyrazine, 2-acetylpyrazine, and pyrrole (p value <0.001),
network of further chemical reactions and the release of followed by 2-acetyl-3,6-dimethylpyrazine (p value <0.01) and
multiple volatile aroma compounds. More particularly, after 2-acetylpyrrole (p value <0.05) (Table 4). These results
the initial Maillard reaction step, pyrazines and pyrroles were suggested that the toasting intensity had a clear effect on the
issued from reductones via Strecker degradation and Amadori level of nitrogen-heterocyclic compounds in wood, with
rearrangement, followed by further various condensation and medium and heavy toasting contributing to increase the
cyclisation mechanisms.43,44 Baltes and Knoch (1993)45 occurrence of the assayed volatile aroma compounds. These
proposed, meanwhile, a synthesis pathway for alkylquinolines findings were in agreement with other results applied to food
formation. After an initial Maillard reaction between sugars and analysis and covering the same range of temperature. It was
13701 DOI: 10.1021/acs.jafc.9b05716
J. Agric. Food Chem. 2019, 67, 13694−13705
Journal of Agricultural and Food Chemistry Article

Figure 4. Distribution of nitrogen-heterocyclic compound concentrations in wood samples according to their origin species (French vs American)
and toasting level (light, medium, and heavy). Alkylpyrazines group includes the two pyrazines quantified in the wood matrix (i.e. 2,6-
dimethylpyrazine and 5-ethyl-2,3-dimethylpyrazine). Letters indicate significant differences at p value <0.05.

Table 5. Mean Concentrations (μg/L, ± SD) of Targeted Nitrogen-Heterocyclic Compounds in the Series of 41 Commercial
Spirits Analyzeda
kind of spirit maturity alkylpyrazines acetylpyrazines pyrrole acetylpyrroles quinolines
Armagnac (n = 1) non matured 11.7 ± 1.6 12.4 ± 2.1 n.d. 23.7 ± 5.6 3.4 ± 0.7
brandy (n = 2) non matured 12.7 ± 2.6 0.5 ± 0.2 0.7 ± 0.3 5.9 ± 1.3 4.3 ± 1.6
rum (n = 2) non matured 27.8 ± 2.6 7.6 ± 2.6 n.d. 4.5 ± 0.3 2.1 ± 0.6
whisky (new-make malt; n = 1) non matured 16.8 ± 0.1 5.1 ± 0.4 7.8 ± 0.3 22.7 ± 2.2 5.1 ± 0.8
mean concentrations in non matured spirits (n = 17.3 ± 2.7 7.2 ± 2.1 b 4.3 ± 0.3 b 13.3 ± 2.3 b 5.1 ± 0.8
6)
Armagnac (n = 5) matured (15−48 11.9 ± 3.1 27.1 ± 4.2 32.6 ± 8.6 35.9 ± 9.9 7.2 ± 3.7
years)
brandy (n = 1) matured (7 years) 6.1 ± 0.4 1.6 ± 2.6 13.7 ± 4.3 9.3 ± 2.5 10.5 ± 4.1
Cognac (n = 3) matured (3−10 years) 11.3 ± 2.0 29.8 ± 4.3 8.0 ± 3.1 17.4 ± 5.6 1.6 ± 0.5
rum (n = 8) matured (1−12 years) 24.6 ± 3.3 35.1 ± 9.5 39.2 ± 9.7 20.5 ± 7.5 7.9 ± 3.6
whisky (n = 18) matured (3−21 years) 12.7 ± 2.4 39.3 ± 10.7 40.7 ± 12.1 45.2 ± 12.7 16.9 ± 5.8
mean concentrations in matured spirits (n = 35) 13.3 ± 2.3 26.3 ± 6.6 a 26.8 ± 7.5 a 25.7 ± 7.9 a 8.6 ± 3.6
a
n.d.: not detected. Letters indicate significant differences between the two clusters (non matured and matured spirits) at p value <0.05.

assumed that temperature accounts among the key factors formation rate of the resulted aroma compounds. Accordingly,
conditioning the kinetics of the Maillard reaction and thus the previous studies related to roasted coffee or sesame seeds46,47
13702 DOI: 10.1021/acs.jafc.9b05716
J. Agric. Food Chem. 2019, 67, 13694−13705
Journal of Agricultural and Food Chemistry
reported, within a constant roasting time, a continuous
increase in pyrazines and pyrroles derivatives when the process
temperature was increased up to 250 °C. Strong interactions
between the wood species and the toasting degree were also
observed for pyrrole (p value <0.001), 5-ethyl-2,3-dimethyl-
pyrazine and 2-acetyl-3,6-dimethylpyrazine (p value <0.01),
and 2-acetylpyrrole (p value <0.05) (Table 4). This suggests
that the relationship between increasing the toasting level and
the concentration of N-heterocyclic volatile aroma compounds
also depends on the type of wood since medium or heavy
toasted-French oak shows a higher release of these molecular
markers compared to American oak. In the basic fraction of a
charred American oak wood (Quercus alba), Maga (1987)23
identified several pyrazines, which are considered as important
sensory contributors. He also postulated that these compounds
were generated from nitrogen-containing macromolecules by
the charring process and could be transferred into alcoholic
beverages over the maturation period in wood barrels. Taking
into account the recent identification of melanoidins in oak
wood,48 the difference currently observed between American
and French oakwood species, on the release of nitrogen
heterocycles after the toasting step, might be thus related to
their initial content in available nitrogen precursors.
N-Heterocycle Profiling in Commercial Spirits and
Correlation with Wood Maturation. The set of 21 nitrogen-
heterocyclic compounds was quantified in a series of 41 non
matured or matured spirits. The average concentrations of the
volatile aroma compounds are presented in Table 5. Several
alkylpyrazines (such as 2-methylpyrazine, 2,3-dimethylpyra-
zine, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, 2,3,5-trime-
thylpyrazine, 2-ethyl-3-methylpyrazine, 2-ethyl-3,6-dimethyl-
pyrazine, and 2,3,5,6-tetramethylpyrazine) were previously
detected in whisky and rum and quantified from few to
several hundred micrograms per liter (μg/L).17,30 Moreover,
quinoline 2- and 6-methylquinolines were previously identified
in whisky.49 However, to our knowledge, it was the first time
that 2-ethyl-3,5-dimethylpyrazine, 5-ethyl-2,3-dimethylpyra-
zine, 2,3-diethyl-5-methylpyrazine, acetylpyrazine, and acetyl-
pyrrole derivatives were reported in spirit beverages.
Alkylpyrazines, acetylpyrazines, pyrrole, acetylpyrroles, and
quinolines were detected in non matured spirits with global
concentrations ranging from 7.3 μg/L for pyrrole to 17.3 μg/L
for alkylpyrazines. Within the non matured sample cluster,
alkylpyrazines and pyrrole were found in higher concentrations
in rums and new-make whiskies. As a consequence, the impact
of the raw material could not be excluded to explain their
occurrence in the final spirit. For the other assayed
compounds, no significant differences were observed according
to the kind of spirit. This above finding suggested that some of
them could either be initially present in the raw material or
formed at high temperature from precursors during the spirit
production process and prior to barrel aging (that is, during
the distillation step in the spirit production process, malting
and/or peating step(s) in the whisky elaboration).10,50
Interestingly, the ANOVA test applied to mean N-heterocycle
concentrations in the two global spirit clusters (matured vs non
matured in wood barrels) revealed that matured spirits had
significantly higher concentrations of acetylpyrazines, pyrrole,
and acetylpyrroles than non matured ones (p value <0.05;
Table 5). The same significant trend (p value <0.05) between
non matured and matured samples was observed when spirits
were clustered according to the nature of the raw material (that
is, cluster 1: Armagnacs, brandies, and Cognacs made from
13703
Article
grapes; cluster 2: rums made from molasses, and cluster 3:
whiskies made from barley). Because the former results of the
current study had shown that these N-heterocycles were
aromatic chemical markers also well present in oak wood, these
latter findings highlighted the essential role played by the
maturation step in modifying their content in spirits by
transferring them from the oak wood barrel to the raw distillate
over the aging period.
The current-optimized SPME-GC/MS methodology was
able to quantify in both wood and spirit matrices and in a
single chromatographic run and at low level, twenty-one
nitrogen-heterocyclic compounds characterized by roasted-
and nutty-derived notes. As a result, it was shown that the
barrel aging process played an important role in the
modulation of the N-heterocycle profiling in both wood and
spirit extracts since the geographical origin and the toasting
level of wood as well as the spirit maturation in woods barrels
significantly affected the contents of alkylpyrazines, acetylpyr-
azines, pyrrole, and acetylpyrroles in spirit samples. Never-
theless, further research work is still required to better clarify
the impact of the barrel toasting process on nitrogen-
heterocyclic compound profiling at different depths in wood,
as well as the evolution of these volatile roasted-aroma
compounds in a spirit matured upon various wood barrel
conditions. With regard to their common chemical structure,
the sensory interactions between the nitrogen heterocylic
compounds and their role in enhancing nutty and roasted
notes in the spirit aging bouquet represent another area for
future investigations.
■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jafc.9b05716.
Sample and SPME parameters studied during the
optimization stage of the analytical method are
presented in Table S1, N-heterocyclic volatile aroma
compounds recoveries obtained in a light-toasted
oakwood and non matured spirit using the optimized
SPME-GC/MS is presented in Tables S2 and S3,
respectively (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: picard@demptos.fr. Phone: +33 (0) 5 40 00 38 01.
ORCID
Magali Picard: 0000-0001-9083-8250
Notes
The authors declare no competing financial interest.
■
ABBREVIATION USED
HS-SPME, headspace solid-phase microextraction; GC/MS,
gas chromatography−mass spectrometry; LOD, limit of
detection; LOQ, limit of quantification
■
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13705 DOI: 10.1021/acs.jafc.9b05716

J. Agric. Food Chem. 2019, 67, 13694−13705