Food Chemistry 229 (2017) 142–151

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

Analytical Methods

Discrimination of whisky brands and counterfeit identification

by UV–Vis spectroscopy and multivariate data analysis
Angélica Rocha Martins a, Márcio Talhavini b, Maurício Leite Vieira b, Jorge Jardim Zacca b,
Jez Willian Batista Braga a,⇑
a
Institute of Chemistry, Universidade de Brasília, Distrito Federal, Brazil
b
National Institute of Criminalistics, Brazilian Federal Police, Brazil

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

Article history: The discrimination of whisky brands and counterfeit identification were performed by UV–Vis spec-
Received 5 July 2016 troscopy combined with partial least squares for discriminant analysis (PLS-DA). In the proposed method
Received in revised form 2 February 2017 all spectra were obtained with no sample preparation. The discrimination models were built with the
Accepted 6 February 2017
employment of seven whisky brands: Red Label, Black Label, White Horse, Chivas Regal (12 years),
Available online 8 February 2017
Ballantine’s Finest, Old Parr and Natu Nobilis. The method was validated with an independent test set
of authentic samples belonging to the seven selected brands and another eleven brands not included
Keywords:
in the training samples. Furthermore, seventy-three counterfeit samples were also used to validate the
Whisky
UV–Vis
method. Results showed correct classification rates for genuine and false samples over 98.6% and
Chemometrics 93.1%, respectively, indicating that the method can be helpful for the forensic analysis of whisky samples.
Falsification Ó 2017 Elsevier Ltd. All rights reserved.
Forensic

1. Introduction alcohol content measurement and the electrical conductivity

Whisky is an alcoholic beverage with great worldwide accep-
tance and high commercial value. Due to these factors, this bever-
age has been a common target of various types of counterfeits,
such as: the presence of another type of colored beverage, dilution
with water and alcohol, and mixing or replacement with another
lower quality whisky using the original commercial bottle. There-
fore, methods that allow for beverage authentication are of great
interest to producers, regulatory agencies, consumer protection
and criminal investigative institutions (Mackenzie & Aylott, 2004).
According to Hoffman (2008) whisky can be defined as a dis-
tilled beverage made of cereals, yeast and water, which has been
matured in wood barrels for a certain period of time. The verifica-
tion that a beverage meets the requirements to be classified as
whisky can be accomplished by reference classical analysis of the
AOAC Official Methods of Analysis Handbook (AOAC, 2000) and
the Beverages and Vinegars Operating Manual of the Brazilian Min-
istry of Agriculture, Livestock and Supply (Ministry of Agriculture,
Livestock and Supply, 2010). Apart from these classical methods,
some forgeries can also be detected by the visual analysis of the
bottle and content characteristics, obtained evaporation residue,
⇑ Corresponding author at: Institute of Chemistry, Universidade de Brasília,
Campus Darcy Ribeiro, Asa Norte, PO Box 4478, CEP 70904-970 Brasília-DF, Brazil.
E-mail address: jez@unb.br (J.W.B. Braga).
(Talhavini & da Veiga, 2012). However, these analyses may not
be able to identify forgeries like the filling of a bottle of a high value
whisky with a lower value whisky. In these cases, the main analyt-
ical methods used to confirm authenticity are based on the deter-
mination of congeners present in whisky by gas chromatography
coupled with mass spectrometry detection with direct injection
or by the use of headspace solid phase microextraction (Câmara
et al., 2007; Demyttenaere, Sánchez Martínez, Verhé, Sandra, &
De Kimpe, 2003; Lee, Paterson, Birkmyre, & Piggott, 2001; Wang,
Wang, & Choong, 2004).
Currently, spectroscopy techniques and chemometric methods
have been employed in the analysis of beverages and food in order
to improve quality control, detection of forgery or adulteration,
and origin identification, among others (Alcázar, Jurado, Palacios-
Morillo, de Pablos, & Martín, 2012; Bertone, Venturello, Giraudo,
Pellegrino, & Geobaldo, 2016; Botelho, Reis, Oliveira, & Sena,
2015; Boyaci, Genis, Guven, Tamer, & Alper, 2012; Ferreira,
Galão, Pallone, & Poppi, 2014; Herranz, de la Serna, Barro, Martin,
& Cabezudo, 1989; Kaya-Celiker, Mallikarjunan, & Kaaya, 2015;
Lachenmeier, 2007; Picque et al., 2006). Among these papers the
work of Mignani et al. (2012) may be highlighted. They applied
UV–Vis, Near Infrared (NIR) and fluorescence spectroscopy to dis-
tinguish single-malt whiskies from commercial-grade blend and
to identify the geographic origin of production of the single-malt
types. In this work, 18 samples/brands were used: 15 of a

http://dx.doi.org/10.1016/j.foodchem.2017.02.024
0308-8146/Ó 2017 Elsevier Ltd. All rights reserved.
 A.R. Martins et al. / Food Chemistry 229 (2017) 142–151 143

single-malt produced in superior distilleries located in two differ-
ent macro-areas from Scotland and 3 samples from commercial-
grade blend of grain whisky. Initially Principal Component Analysis
(PCA) was applied to each spectroscopic data and the scores
obtained in each data were merged for Linear discriminant Analy-
sis (LDA). The method was able to identify the samples according
to the production area and to distinguish them from commercial-
grade blends. However, a single bottle of each sample was used,
which is a severe sampling limitation that does not include the
variability of each brand/sample for a proper validation of the
method.
Pontes et al. (2006) applied NIR, Soft Independent Modeling of
Class Analogy (SIMCA) and PCA to perform the classification and
verification of counterfeiting samples of four different beverages
types: whisky, brandy, rum and vodka. In this work, the training
sets were built with 10 samples from different batches of the same
manufacturer for each beverage type, totalizing 40 samples. The
validation was performed with 24 adulterated samples prepared
in the laboratory and 5 actual samples with suspicion of adulter-
ation provided by a regulatory agency. The PCA model was able
to group the original samples in 4 different classes, corresponding
to the 4 different types of beverage and the SIMCA model identified
the adulterated samples withy 100% correct classification, the
same results were observed in the actual samples provided by
the regulatory agency. However, the distinction between similar
brands of the same beverages type has been not evaluated.
McIntyre, Bilyk, Nordon, Colquhoun, and Littlejohn (2011) pro-
posed the use of Middle Infrared with an Attenuated Total Reflec-
tion (FTIR-ATR) immersion probe for in situ determination of the
ethanol concentration in whisky and the identification of counter-
feit samples. A total of 17 authentic and counterfeit samples were
used. The results of univariate and Partial Least Squares Regression
(PLSR) models were shown to be equivalent for ethanol analysis.
Counterfeit samples were analyzed by a PCA model built with
the dried residue and caramel solutions spectra. The PCA model
was able to distinguish between different caramel colorants and
ethanol concentrations. The obtained results were consistent with
those supplied by the whisky company. Nevertheless, the method
required some sample preparation for the identification of coun-
terfeits based on caramel addition, which increases significantly
the time required for the analysis.
Recently, Cantarelli, Azcarate, Savio, Marchevsky, and Camiña
(2015) proposed the discrimination of whiskies according to age
this work aims at proposing a simple method for direct analysis of
whisky by UV–Vis spectroscopy and PLS-DA in order to fulfill the
following objectives. First, to perform the discrimination between
authentic whisky brand samples with different ages, so to prevent
counterfeits such as filling of high value whisky bottle with a lower
value one. Second, to do the analysis of different common counter-
feits samples seized by the Brazilian Federal Police in order to
prove the efficiency of the method in real forensic cases. Discrim-
ination models for seven different brands were developed and their
efficiency evaluated with independent authentic samples coming
not only from the modeled brands but also from other eleven dif-
ferent brands not included in the training step. In addition, 73
counterfeited samples from the seven target brands were analyzed
and the occurrence of false positive and false negative errors were
assessed.
2. Materials and methods
2.1. Description of samples
2.1.1. Authentic samples
Authentic samples consisted of 164 bottles from eighteen dif-
ferent brands obtained through donations of the National Labora-
tory of Agriculture of Minas Gerais (LANAGRO – MG), DIAGEO
PLC, the Brazilian Federal Police and acquired in certified whisky
distributors in certified importation stores of Brasília city, Brazil.
All authentic samples acquired in Brasília presented importation
certificates, budget documents. Also, they were sealed and had
authentic commercial seals. In addition, all samples acquired in
Brasília were analyzed by the routine whisky reference methods
of the Brazilian Federal Police to ensure its authenticity. This rou-
tine is based on visual analysis of the bottle and content character-
istics, evaporation residue, alcohol content and electrical
conductivity measurements (Ministry of Agriculture, Livestock and
Supply, 2010. Table 1 shows the distribution of these samples
according their age and number of units in each brand. In addition,
these brands were selected to represent some of the most commer-
Table 1
List of authentic (A) and counterfeit (C) samples.
Brand Age Units

and brand by UV–Vis spectroscopy using PCA, LDA and Partial Johnnie Walker – Red Label (A) 8 years 32
Least Squares for Discriminant Analysis (PLS-DA). Six bottles of fif- Johnnie Walker – Black Label (A) 12 years 33
Johnnie Walker – Gold Label (A) 18 years 1
teen different brands were used and the measurements were
Johnnie Walker – Green Label (A) 15 years 1
acquired in each bottle. Five of this brands were of high commer- Logan (A) 12 years 1
cial value whiskies and for this reason were selected to be discrim- Grant’s Royal (A) 12 years 1
inated between each other and from the other ten brands. The Grant’s (A) 8 years 1
sample preparation involved the addition of a buffer solution for Grand Old Parr (A) 12 years 10
Dimple (A) 12 years 1
pH adjustment (pH = 12) and dilution. The PCA model was able Dimple (A) 15 years 1
to separate the samples according to the brands and the years of House of Lords (A) Unspecified 1
aging. The LDA model presented 99.15% of correct classification Jonnie Walker - Swing (A) 15 years 1
according to brands and 100% according to years of aging. The Glenfiddich (A) 12 years 2
Ballantine’s Finest (A) 8 years 19
PLS-DA models were built using the 5 samples of the high commer-
Chivas Regal (A) 12 years 11
cial value whiskies in the training set, and 100% correct classifica- Buchanan’s (A) Unspecified 1
tion according to years of aging was obtained. However, in this last White Horse (A) 8 years 32
work only a very small validation data set was used and the Natu Nobilis (A) 3 years 15
method was not tested with real false/adulterated samples in order Johnnie Walker Red Label (C) 8 years 23
Johnnie Walker Black Label (C) 12 years 15
to verify its efficiency. Ballantine’s (C) 8 years 8
Although different methods applying UV–Vis spectroscopy have White Horse (C) 8 years 9
been already published suggesting the possibility to discriminate Natu Nobilis (C) 3 years 9
whiskies according to brands and ages, there is still a lack of results Chivas Regal (C) 12 years 7
Grand Old Parr (C) 12 years 2
that prove the effectiveness of this approach by means of an appro-
priate validation utilizing both authentic and false samples and Total authentic (A) 164
Total counterfeit (C) 73
determining the proper figures of merit for the method. Therefore,
144 A.R. Martins et al. / Food Chemistry 229 (2017) 142–151
cialized whisky brands in Brazil, to enable the evaluation of the
discrimination between similar brands (i.e. Red label and Black
Label). This aimed at evaluating if the proposed method could solve
forensic cases such as a whisky bottle of a higher quality whisky
filled with a lower price/quality whisky (e.g. a Black Label bottle
containing Red label or Natu Nobilis). The other brands were used
to validate the method with authentic samples belonging to brands
not included in the training set.
2.1.2. Counterfeited samples
Table 1 also presents the description of the seventy-three coun-
terfeit samples belonging to the seven target brands selected in the
previous section. These samples were provided by the Brazilian
Federal Police and obtained from seizure operations. These sam-
ples were attested as counterfeit according to routine whisky anal-
ysis performed at the forensic laboratory of the National Institute
of Criminalistics of the Brazilian Federal Police in Brasília. Initially
all samples were submitted to inspection regarding the presence of
legal commercial seals, stamps and signals of seal’s violation. After
that, physical-chemical analyses included detection of particulates
on the liquid, ethanol concentration, determination of dry residue,
comparative profile GC/MS analysis, FTIR, pH and conductometric
analysis. The counterfeit samples were identified by the fulfillment
of at least one of the following requisites: presence of ions F
and
Cl
(indicating the presence of tap water), presence of caramel,
dyes and sugar cane spirit (Brazilian cachaça), mixtures of etha-
nol/water/dyes, but low concentration of ethyl esters.
2.2. UV–Vis analysis
All samples were analyzed in triplicate on UV–Vis spectropho-
tometer coupled to a peristaltic pump (Agilent Technologies,
model 8453, fitted with deuterium and tungsten lamps) using a
quartz flow cell with 1 mm of optical path and volume of 62 mL
(Hellma). Absorbance data were acquired at 1 nm resolution
between 190 and 1100 nm and exported into the spreadsheet
using the UV–Visible Chemstation software. Background measure-
ments were obtained from a 40:60 (v/v) ethanol:water solution
prepared from an absolute ethanol reagent (Dynamic, PA) in ultra-
pure water (Millipore Direct-Q 5). No sample preparation or dilu-
tion was carried out for the whisky analysis.
The flow cell was used to improve analytical throughput, by
minimizing the required sample volume in each analysis and opti-
mizing both the required time for filling and cleaning the quartz
cell. Initially, the quartz cell was cleaned by pumping a 40:60
(v/v) ethanol:water solution during 10 s. Then the whisky sample
was pumped to fill the flow cell and the flow was stopped for spec-
trum measurement. This procedure was repeated for the analysis
in each replicate of all samples. No normalization or correction
was performed in the spectra before chemometric analysis.
The analysis was conducted on different days, wherein the sam-
ples were randomly selected from all brands to prevent preferen-
tial selection of training samples that could introduce bias in the
results.
2.3. Chemometrics analysis
Data analysis was performed using the MATLAB software ver-
sion 7.12 (R2011b) and the PLS Toolbox version 6.5 applying the
discrimination modeling by PLS-DA. In order to appropriately
develop and validate a PLS-DA model a minimum number of sam-
ples of each brand is required. Therefore, according to the distribu-
tion of authentic samples presented in Table 1, only seven brands
were selected for the development of the PLS-DA models: Red
Label, Black Label, White Horse, Chivas Regal, Ballantine’s Finest,
Old Parr and Natu Nobilis. The samples of these brands were split-
ted systematically into two independent datasets, for use in the
training and validation steps. The samples were split so that at
least 2/3 of the samples of the seven target brands were present
in the training dataset (Table S1 at the Supplementary material).
The samples of all remaining authentic samples were used in the
validation dataset. It should be noticed that in the proposed
method a PLS-DA model is developed for each specific brand of
interest, so that, after this training, it can be applied to identify
genuine/authentic samples of these brands and different kinds of
adulterations of these brands (i.e. adding of other components in
the whisky, replace part or the entire volume of the bottle with a
lower quality/price whisky).
2.3.1. Partial least squares for discriminant analysis
PLS was originally developed as a multivariate calibration
method, but also found application in discrimination analysis
(Hobro, Kuligowski, Döll, & Lendl, 2010; Sun, 2009). Different from
PCA, which enables only an exploratory or unsupervised analysis of
the data, PLS-DA is a supervised model that requires known sam-
ples to be trained. Moreover, after the training and validation steps,
the PLS-DA model enables to conclude if an unknown sample
belongs or not to one of the classes/brands included in the training
step, or if the analyzed sample represent some type of counterfeit
or is an authentic sample belonging to a class/brand not included
in the developed model. Mathematically, PLS regression relates
the data matrix (X) and the dependent variable vector (y) contain-
ing the reference values of the interest property by means of a mul-
tivariate linear model in order to obtain a better estimation of y
from the instrumental data in X (Esbensen, 2009; Wold,
Sjöström, & Eriksson, 2001). In the case of PLS-DA the y vector con-
tains the identification of classes (in this case, whisky classes). The
response vector y is composed of 0’s and 1’s, where the value 1 is
assigned to samples belonging to the class being discriminated and
the value 0 to samples belonging to any other class/brand included
in the training set (de la Mata et al., 2012). This model decomposes
X and y and creates a new coordinate axes system called latent
variables, obtained in order to maximize the covariance between
X and y according Eqs. (1) and (2) (Naes, Isaksson, Feran, &
Davies, 2002).
X
A
X¼ ta pTa þ E ð1Þ
a¼1
X
A
y¼ ta qTa þ f ð2Þ
a¼1
where ta is the score vector, pa and qa are the loadings for the latent
variable a and E and f are the error matrices of X and y, respectively.
The estimation of the class value for an unknown sample presenting
a spectrum xi is obtained by:
^i ¼ xi WðPWÞ
1 q ¼ xi b
y ð3Þ
where W is the weight matrix of X, P is loading matrix of X and q
loafing vector of y, all obtained in the training step for A latent vari-
ables by the PLS-DA algorithm. According to Eq. (3), the training
parameters W, P and q can also be combined into a single vector
b, which can be multiplied by the spectrum of the unknown sample
and results in the estimated class value (y).
After model optimization, a discrimination threshold is calcu-
lated based on the dispersion of the estimated class values (y ^) for
the training samples minimizing the occurrence of false positive
and negative errors according a Bayesian approach. Details about
the mathematical background of PLS-DA and the threshold estima-
tion can be obtained in specific texts of the chemometrics (Ballabio
& Consonni, 2013; Barker & Rayens, 2003; Botelho et al., 2015;
 A.R. Martins et al. / Food Chemistry 229 (2017) 142–151
Brereton & Lloyd, 2014; Ferreira, 2015). An unknown generic sam-
ple i is classified as belonging to a given class/brand if its estimated
class value y ^i turns out to be above the corresponding discrimina-
tion threshold. Otherwise, this sample will be classified as belong-
ing to the class 0, which contain the samples of all other classes. It
should be noticed that the class value attributed to the samples
were arbitrarily defined as the dimensionless numbers 1 (one)
and 0 (zero) to identify the samples as belonging to the discrimi-
nated class or belonging to the other classes, respectively.
2.3.2. Optimization of the discrimination models and outlier
identification
The optimization of the discrimination models was performed
by the selection of the preprocessing method, the number of latent
variables and the elimination of outliers, which are defined as sam-
ples showing some type of departure from the bulk of the data.
The preprocessing method and the number of latent variables
(LV) were determined based on the root mean square error of cross
validation (RMSECV) values obtained by venetian blinds cross-
validation, which is estimated according to the following equation:
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Pn
i¼1 ðyi
yi Þ
^ 2
RMSECV ¼ ð4Þ
nc
where yi is the actual class value attributed to the sample i, value y ^i
is its estimate obtained by PLS-DA and nc the number of samples in
the training set.
The preprocessing method and the number of LV were chosen
as the condition that provided the lower RMSECV value or the
lower LV after the stabilization of the RMSECV. It is important to
note that the cross-validation classification error (CVCE), defined
as the correct classification rate obtained in the PLS-DA model in
the training using cross-validation, was not used in the paper for
the determination of the preprocessing method and LV in the
PLS-DA models. CVCE was not used because it does not penalize
samples presenting high errors in the estimated class value (y ^i ) if
the sample is correctly classified. However, for samples presenting
high errors in y^i the estimated class values might be an indication
of a sample with different characteristics of the training dataset,
which constitute an outlier. On the other hand, the RMSECV, ide-
ally provide the largest separation of the estimated values for the
discriminated class (y = 1) from the other classes (y = 0), minimiz-
ing the estimation errors y and, consequently, should provide
higher correct classification rates.
The optimizing step related to identification and exclusion of
outliers was based on the work of Borin and Poppi (2004), the
ASTM E1655-05 and adapted from da Silva, Talhavini, Zacca,
Maldaner, et al. (2014) and da Silva, Talhavini, Zacca, Trindade,
and Braga (2014). Three parameters were used for this purpose:
Q spectral residuals, Hotelling’s T2 and the errors in the estimated
class values, which are detailed below.
 Q spectral residuals: evaluate the lack-of-fit of each individual
spectrum in the PLS-DA model. It is determined by the sum of
the squares of the residual vector of each sample (ei ), expressed
for a sample i by Eq. (5):
Q i ¼ ei eTi ð5Þ
where ei is the decomposition residual obtained for sample i,
according Eq. (1). If a sample spectrum belongs to one of the
classes/brands included in the training set (predefined classes)
and does not contain any contamination or measurement error,
it should present a Q value compatible with the spectra used in
the training step. Otherwise, if the sample presents a Q value sig-
nificantly higher than the ones observed in the training set, this
will indicate the presence of some kind of adulteration com-
145
pound or by the sample belonging to a class/brand not included
in the training set.
 Hotelling’s T2: this parameter statistic measures how far a sam-
ple is from the center of the PLS-DA model. In other words,
while the Q statistic is based on the residual variance of each
sample, the T2 statistic refers to the modeled variance in each
sample within the model. For a sample i the Hotteling’s T2 is
determined by:
T 2i ¼ ti K
1 tTi ð6Þ
where K(A,A) is a diagonal matrix containing the eigenvalues
associated with the A latent variables of the PLS-DA model.
For objective decisions in outlier detection, threshold values for
Q residuals and T2 statistic can be estimated at a certain proba-
bility level. Details of the determination of these threshold val-
ues for T2 and Q residuals can be obtained at the work of Borin
and Poppi (2004).
 Errors in the estimated class values (y): samples of the training
set presenting high errors in the class values were identified by
the Student’s t-test for residuals described in the ASTM E1655-
05 [28], considering the correction for bias suggested by da
Silva, Talhavini, Zacca, Maldaner, et al. (2014), as follows:
ei
bias
t student;i ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffi ð7Þ
RMSEC bias 1
hi
^ i
yi )
where ei, and hi are the error in the estimated class value (y
for the sample i, RMSECbias is the root mean square error esti-
mated using the calibration/training samples after bias correc-
tion, bias is the observed bias for the class 1 (biasclass1) or 0
(biasclass 0), depending on the sample considered, and hi is the
sample leverage estimated as:
X
A
t2i;a
hi ¼ ð8Þ
a¼1 tTa ta
Based on these three criterions, the outliers in the training sam-
ples were identified in two steps:
1) If a sample spectrum presented the Hotelling’s T2 and a Q
residual value above the limits of 99.9%, the spectrum was
identified as an outlier and removed from the training
dataset;
2) Or if a sample spectrum presented a high error for the esti-
mated class value, according the Student residuals test with
bias correction defined by Eq. (7) above the critical value of
the t-Student distribution with 99.9% of confidence and nc-A-
2 degrees of freedom, it was also identified as an outlier.
The first criterion was applied in the same way for the valida-
tion samples. On the other hand, the t-test for Student residuals
was adapted to establish the upper limit for the estimates of the
class 1 and the lower limit for the estimates in class 0 according
Eqs. (9) and (10):
 qffiffiffiffiffiffiffiffiffiffiffiffiffiffi
yupperlimit;class1 ¼ 1 þ biasclass1 þ t 99;m RMSECbias 1
h c ð9Þ
 qffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ylowerlimit;class0 ¼ 0 þ biasclass0
t99;m RMSECbias 1
h c ð10Þ
where h  c is the mean leverage observed in the training samples,
biasclass0 and biasclass1 are the bias estimated for the classes 1 or 0
described by da Silva, Talhavini, Zacca, Trindade, et al. (2014),
respectively, t99.9, is the reference t-value of the t-Student distribu-
tion with 99.9% of confidence and nc-VL-2 degrees of freedom.
146 A.R. Martins et al. / Food Chemistry 229 (2017) 142–151
Samples in the validation set were considered outliers regard-
ing y if the estimated class value was larger than yupper,class 1 or
lower than ylower,class 0. According the second criterion, the valida-
tion samples identified as outliers will be the samples presenting
class values significantly higher than the estimated class values
observed in the training samples for the class 1 or significantly
lower than the estimated class values observed in the training
samples for the class 0, taking into account the 99.9% confidence
levels.
The identification and exclusion of outliers was performed in
two steps. Initially, a first model was built and outliers were
excluded from the training set. Then the model was calculated
with the remaining samples and a new identification of outliers
was performed. As the second model presented new outliers they
were excluded using the same criterions, and the model was recal-
culated again. The third model was considered optimized.
2.3.3. Validation
The validation of the method was performed with two inde-
pendent data sets that were not used in the training step, wherein
the first one was composed of authentic samples and the second
one of counterfeit samples. Initially, the Q residual, T2 value and
the estimated class value (y ^) of a sample i are compared with
the respective threshold values of each parameter, with 99.9% of
confidence. If the estimated value for Q residual, T2 were above
the threshold values or the class value (y ^i ) was out of the limits
defined by Eqs. (9) and (10), the sample i was either called not
compatible with the samples/brands included in the training step
or not belonging to the brands included in the discrimination
model (modeled classes or predefined classes). In a real forensic
analysis, it would be concluded that either this sample should
be analyzed again to confirm its condition or analyzed by the ref-
erence method in order to present the results of two independent
analysis and a solid result to present in court. Otherwise, if the
values of Q, T2 were below the thresholds and y ^i was within the
limits for the class values, the result of the discrimination model
indicated that the sample belonged to one of the predefined
classes and the result is recorded and included in the calculation
of the figures of merit to demonstrate the effectiveness of the pro-
posed method, based on the work of Trullols, Ruisánchez, and Rius
(2004) and Botelho et al. (2015). Fig. 1S at the Supplementary
material shows the analysis scheme adopted for the analysis of
the validation samples.
The figures of merit used for method validation were estimated
according the following equations (Botelho et al., 2015):
FN
FNR ¼ 100 ð11Þ
FN þ TP
FP
FPR ¼ 100 ð12Þ
FP þ TN
TP
STR ¼ 100 ð13Þ
FN þ TP
TN
SPR ¼ 100 ð14Þ
TN þ FP
EFR ¼ 100
ðFPR þ FNRÞ ð15Þ
where FP and FN are the number of samples classified respectively
as false positive and negative; TP and TN are the number of samples
classified respectively as true positive and true negative; FPR is the
false negative rate, FPR is the false positive rate, STR is the sensitiv-
ity rate, SPR is the specificity rate and EFR the efficiency rate.
It is important to note that the expected result for the analysis
of counterfeit whisky samples is to obtain Q residual, T2 and class
values (y^i ) out of their limits with 99.9% of confidence, because
they would not be compatible with the predefined brands used
in the training step. In addition, for the dataset composed by only
counterfeit samples, there are not true positives (samples belong-
ing to the discriminated class) or true negatives (samples belong-
ing to the other modeled classes). Therefore, in this case only
false positive errors can be estimated, which indicate that a coun-
terfeit sample was identified as belonging to the discriminated
class. As a consequence, the figures of merit described by Eqs.
(11)–(15) cannot be applied. Consequently, the efficiency of the
method was measured in terms of correct classification rate
(CCR), calculated as:
ðN
FP
FNÞ
CCR ¼ 100 ð16Þ
N
where N is the total number samples of the dataset composed of
only counterfeit samples.
Finally, all samples were analyzed in triplicate, so that a mis-
classification result was considered only if at least two replicates
of a same sample were classified incorrectly.
3. Results
Fig. 1 shows that there are differences in intensity between the
absorbance spectra of the whisky brands, where Black Label (iden-
tified in Black) shows the greatest intensity and the Natu Nobilis
(identified in cyan) shows the lower intensity. The absorbance in
the UV region can be explained by the presence of congeners
resulting from the maturation process in oak casks, the presence
of volatile phenolic compounds from peat burning in the drying
step of malted barley, as well as presence of caramel to adjust
the color of the drink (Cantarelli et al., 2015; Mackenzie & Aylott,
2004). Therefore, from Fig. 1 it is concluded that these factors lead
to a relationship between the aging time and congener content.
Boscolo, Andrade-Sobrinho, Lima-Neto, Franco, and Ferreira
(2002) associated UV–vis absorption in the region near 210 nm
and 282 nm with the presence of furfural and hydroxymethylfur-
fural in aged beverage in oak barrels. According to them, the fur-
fural is extracted from the oak barrel and hydroxymethylfurfural
is the main component of caramel, the latter being responsible
for about 60% of the absorbance at 282 nm.
Fig. 1 also shows that a significant difference in the absorption
spectra can be noticed at approximately 205 nm, where a clear
separation between the spectra of brands with 12 years aging
(Black Label, Old Parr and Chivas Regal) from the ones presenting
8 years or less (Red Label, Ballantine’s Finest, White Horse and
Natu Nobilis), wherein the brands 12 years aging presents higher
absorbance values. However, even with this qualitative informa-
tion from the UV spectra, it is still not possible to determine the
identity whiskies brands safely only by visual analysis of the spec-
trum, which justifies a multivariate data analysis. In fact, the appli-
cation of multivariate analysis (i.e. PLS-DA) is well known to be
able to detect patterns related both to the intensities and the shape
of spectroscopic data, even if these small changes are not visible to
human perception.
The pre-processing methods used for data analysis were mean
centering and first derivative, the latter based on the Savitsky-
Golay algorithm using a filter with 9 points and a second-order
polynomial window.
The parameters of the discrimination models are shown in
Table S2. It can be observed that the RMSEC and RMSECV present
similar values within all classes, indicating that there was no over-
fitting in the models. Furthermore, the threshold values were near
to the ideal value of 0.5, except for the Old Parr model, which indi-
cates that no significant systematic errors were observed in the
 A.R. Martins et al. / Food Chemistry 229 (2017) 142–151 147

Fig. 1. UV–Vis spectra of whiskies authentic samples of 12 and 8 years aging or less, used in the calibration set. ( ) Black Label, ( ) Old Parr, ( ) Chivas, ( ) Red Label,
( ) White Horse, ( ) Ballantine’s, ( ) Natu Nobilis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)

estimated class values for most models. However, due to the very
good discrimination observed between the brands (see below), the
deviation of the threshold value observed for the discrimination of
the brand Old Parr did not affect the results.
3.1. Method validation by the analysis of authentic samples
Fig. 2A shows the graph of Hotelling T2 versus the Q residuals
for the discrimination model of the brand Red Label. Since the ana-
lyzed data is in absorbance scale, which is a dimensionless param-
eter, the T2 and Q residuals also are dimensionless, so they are
presented in the figures in arbitrary units. It shows that most of
the authentic samples belonging to brands not included in the
training step present T2 and Q values above the confidence limit
of 99.9%. This result suggests that the model is able to identify
when an authentic whisky sample belonging to brands not
included in the model is not compatible with any of the brands
included in the training set (target brands). This makes it possible
to detect when a bottle of a high value whisky has been adulter-
ated by the total or partial replacement of its content with a lower
value whisky. This behavior was observed in all other brand dis-
crimination models.

Fig. 2. (A) Hotelling T2 versus Q residuals for the discrimination of Red Label samples from the other brands. (B) Zoom of the region within the 99.9% limit. (C) Estimated class
values for the discrimination of the authentic samples of Red Label ( ) from those of Black Label ( ), White Horse ( ), Chivas Regal ( ), Ballantine’s (e), Natu Nobilis ( ),
Grand Old Parr ( ), authentic samples from brands not included in the training set (d), ( ) discrimination threshold and ( ) limits for estimated class values.
148 A.R. Martins et al. / Food Chemistry 229 (2017) 142–151

The Fig. 2B shows a zoom of the graph presented in Fig. 2A. It
may be observed that only two replicates from the Black Label
brand exceeded the 99.9% confidence limits for both T2 and resid-
uals Q. However, as these replicates came from different samples,
this result does not characterize a classification error. Similarly, it
has also been observed that some replicates from different samples
of the brands not included in the training step also presented T2, Q
residuals or both below the 99.9% confidence limits. However, as
least two other replicates of these samples presented T2 and Q val-
ues above the limits, they were correctly identified as belonging to
unmodeled classes/brands. It is important to notice that the num-
ber of outliers identified in the training step was very low for all
models. The model presenting the larger number of excluded spec-
tra (outliers) was for the Old Parr brand (6 spectra out of 270 train-
ing spectra), as presented in Table S2. The occurrence of these
outliers may be explained by variations between the batches/lots
of these brands. Fig. 2C shows the estimated class values of the dis-
crimination between authentic samples of Red Label from the
authentic samples of the other brands. In this model, a class value
around 1 is expected for samples belonging to the Red Label brand,
around 0 for all the other brands included in the training set (mod-
eled brands) and outside the limits for the estimated class values
for the authentic samples belonging to the brands not included
in the training set. This graph shows a good separation between
the modeled classes (target brands). All Red Label samples have
been correctly classified (class values close to 1) and that the
remaining samples of the modeled brands were identified as other
brands (class value close to zero). Exception is made for one repli-
cate of Black Label that exceeded the discrimination threshold,
which does not configure an classification error as discussed previ-
ously. Also, it can be observed that some samples belonging to
unmodeled classes presented class values significantly higher than
1 or lower than zero. For this model the limits for the class esti-
mates were 1.35 and
0.55 for classes 1 and 0, respectively. There-
fore, 10 spectra can be excluded by the limits defined by Eqs. (9)
and (10). As a result of the combination of the estimates presented
in Fig. 2A–C, the model showed sensitivity, specificity and effi-
ciency rates of 100% as shown in Table 2. It is important to observe
that three training samples of Red Label exhibited class values rel-
atively higher than the behavior of the majority of the other train-
ing samples of this brand (replicate numbers between 37 and 47).
This behavior was attributed to differences in sample origin and
whisky production processes.
It should be pointed out that, normally, when a sample is iden-
tified as an outlier it means that, as a result of an experimental or
instrumental error, it should be removed from the dataset. In the
present work, this occurs only when a spectrum from the training
set or validation set composed by the authentic samples from the
modeled brands present T2 and Q residual or errors in the class val-
ues above the limits with 99.9% of confidence. As discussed before,
the discrimination model that presented the largest occurrence of
outliers was for the brand Old Parr, for which 6 out 270 (2.2%)
training spectra were considered as outliers. For the analysis of
the authentic samples from the eleven brands not included in
the training set (and the counterfeit samples discussed below) no
spectrum was removed from the analysis. In this case, the T2, Q
residuals or errors in the class values above the limits for these
samples indicate correctly that they do not belong to any of the
seven target brands (modeled classes).
The results of the estimated class values for all other modeled
brands are presented in Fig. S2, which indicate a very good discrim-
ination. The calculated figures of merit for these models are shown
in Table 2. The method presented excellent results for the discrim-
ination between authentic samples of whisky, with the lowest effi-
ciency rate of 98.6%.
3.2. Counterfeited samples analysis
The Fig. 3A shows the influence (T2) versus residuals Q graphs of
counterfeited samples predicted by Red Label discrimination
model. As expected, the majority of the counterfeit samples
showed T2 and residual Q values above the 99.9% confidence limits.
Fig. 3B shows a zoom of the graph below the confidence limit,
where can be seen that just 6 false samples (22 spectra) showed
T2 and residual Q values compatible with the authentic samples
values. However, among these 6 samples, only one sample pre-
sented estimated class values compatible with the ones observed
in the training samples (inside the limits defined by Eqs. (9) and
(10)). Therefore, just one false positive was observed and the cor-
rect classification rate was equal to 98.6% in the discrimination
model developed for identify the Red Label brand. This behavior
was observed in the models of all other brands. Fig. S3 (presented
in the Supplementary information) shows the expansion of T2 ver-
sus Q residuals graphs in the region of values below the 99.9% limit
for the other models.
Based on Fig. 3C it can be seen that most of the samples present
class values much greater than 1 or less than 0, been out of the lim-
its of Eqs. (9) and (10), indicating that these samples come from a
different population from those used in the training step. From the
219 spectra of the counterfeited samples 127 are out of the limits
established by the Eqs. (9) and (10). This behavior was also
observed in all other models (Fig. S4). Combining the results of
T2, residuals Q and the class values it can be concluded that the
Red Label model showed good discrimination between counterfeit

Table 2
Figures of merit of the models for the analysis of the authentic samples.

Red Label Black Label White Horse Chivas Regal Ballantine’s Finest Natu Nobilis Old Parr Other brands
NS 12 13 12 5 9 7 4 11
SNB 10 10 11 10 10 10 10 0
FP 0 0 0 0 1 0 1 0
FN 0 0 0 0 0 0 0 –
TP 12 13 12 5 9 7 4 –
TN 62 62 62 69 64 67 69 11
FPR* 0 0 0 0 1.5 0 1.4 –
FNR* 0 0 0 0 0 0 0 –
STR* 100 100 100 100 100 100 100 –
SPR* 100 100 100 100 98.5 100 98.6 –
EFR* 100 100 100 100 98.5 100 98.6 –
CCR* 100 100 100 100 98.6 100 98.6 100
*
Expressed as a percentage; NS = number of samples; SNB = number of samples that were classified as not belonging to the class modeled; FP = Number of false positives;
FN = number of false negative; TP = number of true positives; TN = number of true negatives; FPR = false positive rate; FNR = false negative rate; STR = sensitivity rate;
SPR = specific rate; EFR = efficiency rate; CCR = correct classification rate; Ball = Ballantine’s Finest.
 A.R. Martins et al. / Food Chemistry 229 (2017) 142–151 149

Fig. 3. (A) Hotelling T2 versus residual statistics (Q) for the analysis of the counterfeit samples through the discrimination model built with authentic training samples for the
brand Red Label. (B) Zoom of the region within the T2 and Q below the 99.9% confidence limit. (C) Estimated class values for the discrimination of Red Label ( ) from those of
Black Label ( ), White Horse ( ), Chivas Regal ( ), Ballantine’s (e), Natu Nobilis ( ), Grand Old Parr ( ), counterfeit authentic samples (d), discrimination threshold ( )
and limits for estimated class values ( ).

and authentic samples, since from a set containing 73 counter-
feited samples there was only one false positive. The calculated fig-
ures of merit are presented in Table 3. It can be seen that the
method was able to perform a good discrimination between
authentic and counterfeit whisky samples, with a correct classifica-
tion rate varying from 93.1 to 100%. It is important to note that the
results presented in Table 3 consider the analysis of the 73 coun-
terfeited samples in each one of the discrimination models devel-
oped for the seven target brands. Therefore, considering all these
models, 511 predictions (73 counterfeited samples  6 models)
were performed with only 8 false positive errors, given an average
correct discrimination rate of 98.4%.
The result of the analysis of the authentic samples belonging to
unmodeled classes and the counterfeit samples showed that the
PLS-DA models were efficient even with samples of brands not
belonging to any of the predefined classes (brands included in
the training set). Recently, Oliveri and Downey (2012) and
Rodionova, Titova, and Pomerantsev (2016) discussed that discrim-
inant models, as PLS-DA, can present serious problems in analysis
of future samples not included in the predefined classes and argue
that these models should be used only when all the classes belong-
ing to the class 0 are available to be included in the PLS-DA model.
However, in these papers (Oliveri & Downey, 2012; Rodionova
et al., 2016) the T2, Q residuals and the limits for the class values
were not considered for identification of samples incompatible
with the classes included training set. The results obtained in this
work showed that at least in some situations the use of these
parameters in the PLS-DA can reduce significantly the limitation

Table 3
Figures of merit of the models for the analysis of the 73 counterfeited samples.

Red Label Black Label White Horse Chivas Regal Ballantine’s Natu Nobilis Old Parr
N 23 15 9 7 8 9 2
NA 72 72 68 73 71 73 73
FP 1 1 5 0 1 0 0
CCR 98.6 98.6 93.1 100 98.6 100 100

N = number of counterfeit samples of each brand; NA = number of samples classified as not authentic by the criterions of T2, Q residuals or high errors in the class values;
FP = number of false positive errors; CCR = Correct Classification Rate, expressed in percentage.
150 A.R. Martins et al. / Food Chemistry 229 (2017) 142–151
described by Oliveri and Downey (2012) and Rodionova et al.
(2016).
4. Conclusion
The UV–Vis spectroscopy technique combined with PLS-DA
proved to be an efficient method for the discrimination between
seven brands of whisky. Spectra were acquired requiring only a
small volume and no sample preparation. The proposed discrimi-
nation models were promising, showing a small number of outliers
in the predefined/modeled brands and non-occurrence of overfit-
ting. Even though previous publications in the literature have
addressed the application of spectroscopy and chemometric meth-
ods for whisky analysis, the method presented in this work has
been properly validated. A representative dataset has been used.
It was composed of authentic and counterfeit samples from the
seven predefined brands and also of authentic samples from eleven
different brands, resulting in high overall efficiency (93.1%).
Therefore, the results suggest that the proposed method can be
used to certificate the contents of whisky bottles of the seven tar-
get brands. It is important to note that the range of application of
the method may be expanded by the inclusion of new brands
(other than the seven original ones) in the training step.
The classification of authentic samples with the modeled
brands showed very good results, wherein only 2 false positive
misclassification samples occurred. One of them for Ballantine’s
and the other for Old Parr (efficiency rate  98.6). Another impor-
tant aspect was the validation with authentic samples from eleven
brands not included in the predefined classes. In this case the mod-
els were able to identify all samples as not belonging to the brands
included in the training samples. The method also proved to be
effective for the analysis of different kinds of counterfeit samples
with high correct classification rates (93–100% depending on the
brand). Overall, only 8 false positive results were observed among
all of the models.
In order to obtain correct identification results with PLS-DA in
brands not included in the predefined classes and counterfeit sam-
ples it is necessary to observe not only the threshold value for class
delimitation, but all the results for the T2, Q residuals and limits for
estimates of the class numbers.
Acknowledgements
The authors wish to thank CAPES/PROFORENCE (process AUXPE
n° 3509/2014), CNPq (process n° 308748/2015-8) and FAPDF for
the financial aid, the University of Brasilia, the Brazilian Federal
Police, the Lanagro and Diageo by donating samples.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2017.
02.024.
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