Food Chemistry 181 (2015) 31–37

Contents lists available at ScienceDirect

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

Analytical Methods

Development and analytical validation of a screening method

for simultaneous detection of five adulterants in raw milk using
mid-infrared spectroscopy and PLS-DA
Bruno G. Botelho a, Nádia Reis b, Leandro S. Oliveira b, Marcelo M. Sena a,c,⇑
a
Departamento de Química, ICEx, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil
b
Faculdade de Farmácia, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil
c
Instituto Nacional de Ciência e Tecnologia em Bioanalítica (INCT Bio), 13083-970, Campinas, SP, Brazil

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

Article history: This paper proposed a new screening method for the simultaneous detection of five common adulterants
Received 30 August 2014 in raw cow milk by using attenuated total reflectance (ATR) mid infrared spectroscopy and multivariate
Received in revised form 11 January 2015 supervised classification (partial least squares discrimination analysis – PLSDA). The method was able to
Accepted 14 February 2015
detect the presence of the adulterants water, starch, sodium citrate, formaldehyde and sucrose in milk
Available online 20 February 2015
samples containing from one up to five of these analytes, in the range of 0.5–10% w/v. A multivariate
qualitative validation was performed, estimating specific figures of merit, such as false positive and false
Keywords:
negative rates, selectivity, specificity and efficiency rates, accordance and concordance. The proposed
Adulteration
Milk
method does not need any sample pretreatment, requires a small amount of sample (30 lL), is fast
Qualitative validation and simple, being suitable for the control of raw milk in a dairy industry or for the quality inspection
Partial least squares discriminant analysis of commercialized milk.
FT-IR Ó 2015 Elsevier Ltd. All rights reserved.
Screening analysis

1. Introduction citrate, sodium hydroxide, sodium chloride, sucrose, phosphates,

Milk can be defined as the fluid secreted by the female of all
mammalian species, primarily to meet the complete nutritional
requirement of the neonate, and it is the major component in
human diets in many parts of the world. In 2011, more than 735
million tons of milk were produced around the world (including
cow, buffalo, sheep, goat and camel milk). USA was the largest pro-
ducer, with 87.5 millions of tons, and Brazil was the fifth major
producer (31.7 millions of tons). However, as fast as the production
and demand for milk have grown, a larger frequency of sophisticat-
ed milk adulterations has been reported in different countries
(EMBRAPA, 2014).
The practice of unscrupulous producers that add adulterating
substances in milk in order to raise their profits is not a new
subject, but recently, it has attracted more attention worldwide.
In Brazil, two major milk adulteration scandals were recently
reported by the Federal Police. In 2007, the so-called ‘‘Operação
Ouro Branco’’ (White Gold Operation) reported the use of sodium
⇑ Corresponding author at: Departamento de Química, ICEx, Universidade Federal
de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil. Tel.: +55 31 34096389; fax:
+55 31 34095700.
E-mail address: marcsen@ufmg.br (M.M. Sena).
carbonates, bicarbonates and hydrogen peroxide to correct milk
defects, such as high acidity and microbial growth, and to increase
its volume (Hoorfar, 2012). In 2013, the ‘‘Operação Leite Com-
pen$ado’’ (Compensated Milk Operation) revealed the utilization
of fertilizers containing urea and formaldehyde to mask water
addition in milk (UOL, 2014). Souza et al. (2011) have analyzed
100 samples of Brazilian UHT milk and detected high rates of
non-conformities, such as 55% for urine, 44% for formaldehyde,
30% for hydrogen peroxide and 12% for chlorine.
Currently, the detection of adulterants in milk is performed by
using physicochemical methods based on specific reactions or dif-
ferences in the freezing point and specific gravity (IAL, 2005;
MAPA, 2006). These methods are time consuming, labor-intensive
and may not yield accurate results. In the last years, the use of
chemometric methods of supervised classification and/or multi-
variate calibration combined with near or mid infrared spec-
troscopy has allowed the development of rapid and non-
destructive methods to detect and/or quantify adulterants in milk,
such as urea, synthetic urine, synthetic milk, whey, hydrogen per-
oxide, water, melamine and ammonium nitrate (Kasemsumran,
Thanapase, & Kiatsoonthon, 2007; Nicolaou, Xu, & Goodcare,
2010; Santos, Pereira-Filho, & Rodriguez-Saona, 2013a, 2013b;
Zhang et al., 2014). An alternative method based on image analysis

http://dx.doi.org/10.1016/j.foodchem.2015.02.077
0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
32 B.G. Botelho et al. / Food Chemistry 181 (2015) 31–37
has also been applied for detecting and quantifying water and
sodium hydroxide as adulterants in milk (Santos, Wentzell, &
Pereira-Filho, 2012).
Once the addition of any kind of foreign substance in raw milk is
prohibited in Brazil, at any level, only qualitative methods are
described in Brazilian official guidelines for detection of adulter-
ation in milk (IAL, 2005; MAPA, 2006). It is more important to iden-
tify than to quantify possible adulterants. Qualitative analytical
methods provide a dichotomous variable as answer (1/0, yes/no,
presence/absence, pass/fail, accepted/not accepted). These meth-
ods have been considered a declining branch of analytical chem-
istry in recent decades, due to their association with the
identification of ionic species using chemical reactions. However,
with the advances in instrumental analysis and the increasing
demands for total indices and yes/no binary responses, qualitative
methods are now identified as promising tools to develop and
implement reliable vanguard (screening)-rearguard (confirmation,
quality control and information expansion) analytical strategies
(Valcárcel & Cárdenas, 2005). Rapid sample screening methods
can confirm the presence of the analytes of interest, simplifying
the analytical process, saving time and costs by reducing the
number of samples that demand the use of a more laborious
quantitative analysis (Magalhães et al., 2012; Pikeemaat, 2009).
Similarly to quantitative methods, qualitative methods present
in the official guidelines must be submitted to an analytical valida-
tion procedure. However, this subject has received less attention in
the literature. As the response provided by qualitative methods is
discrete, statistical tests and procedures used differ from the ones
used in quantitative validation. In 2002, European Community
released the first official guidelines for qualitative analytical
validation (EC, 2002). Since then, the interest on this subject has
continuously grown (Gondim, Junqueira, & Souza, 2011; NATA,
2013; Trullols, Ruisanchez, & Rius, 2004). With the increasing
number of developed chemometric methods of supervised classifi-
cation, the need to discuss multivariate qualitative validation has
increased, as it has been the case of multivariate quantitative
validation, which has gained more attention in the last years
(Botelho, Mendes, & Sena, 2013). Nevertheless, this subject is still
almost completely absent in the literature.
The main objective of this paper was to develop a rapid and
non-destructive qualitative method for the simultaneous detection
of five adulterants in raw cow milk, using attenuated total reflec-
tion (ATR) mid infrared spectroscopy and partial least squares dis-
criminant analysis (PLS-DA). There were chosen five adulterants
recently identified in Brazilian milk: water, starch, sodium citrate,
formaldehyde and sucrose. Water is the most common adulterant
used for increasing the milk volume by dilution, resulting in the
decrease of nutrition substances, such as protein and solid content.
Starch is used as thickener, increasing the solid content of adulter-
ated milk. Citrate is used as stabilizer and preservative, aiming to
avoid precipitation of nutrients. Formaldehyde is used for preserv-
ing adulterated milk from microbial contamination, since the
reduction of nutritional value due to water addition increases this
risk, and sucrose is used to restore the normal analytical values of
adulterated milk in physicochemical tests and to improve its sen-
sory properties. Considering the milk adulterations usually report-
ed, the simultaneous presence of different adulterants is common,
but most of the chemometric methods found in the literature have
determined only one adulterant at a time or at most binary mix-
tures (Kasemsumran et al., 2007; Santos et al., 2013b; Zhang
et al., 2014). In this paper, mixtures of higher order (up five adul-
terants) were also analyzed for specific detection of each adulter-
ant. The developed method was also validated, by the estimate of
specific figures of merit (FOM) used in qualitative analytical valida-
tion, such as sensitivity, specificity, efficiency rate, accordance and
concordance.
2. Materials and methods
2.1. Instruments and software
Mid infrared spectra were obtained using a Shimadzu IR Affi-
nity1 spectrophotometer (Shimadzu Corp., Kyoto, Japan) in
attenuated total reflectance (ATR) mode, equipped with a ZnSe cell.
Data were handled using MATLAB software, version 7.13 (The
MathWorks, Natick, MA, USA), the PLS-DA routine came from the
PLS Toolbox, version 6.5 (Eigenvector Technologies, Manson, WA,
USA), and Minitab 16 Demo Version (Minitab, Inc, State College,
PA, USA) was used for the mixture design.
2.2. Samples and reagents
Fifteen blank samples (300 mL) of whole cow’s milk were pro-
vided by the model experimental farm from the Veterinary School
of the Universidade Federal de Minas Gerais (UFMG, Belo Horizon-
te) and used as replicates in this investigation. Since its production
is controlled, it is guaranteed that no substances are added to this
milk. Thus, these blank samples did not contain any amount of
formaldehyde, sucrose or starch. Water is naturally present in milk,
being its major component (85–90% w/w), and sodium citrate is
commonly present in milk, but in lower concentrations, bellow
0.1% w/w (Fox & McSweeney, 1998). The milk was stored at
18 °C in 500 mL glass bottles, and heated up using a water bath
until it reached ambient temperature, daily. Sodium citrate,
formaldehyde, starch, sucrose (all of analytical grade) and distilled
water were used as adulterants. For the preparation of the samples,
all the adulterants were weighed in an analytical balance
(±0.0001 g). For the samples containing only one adulterant, water,
sucrose, formaldehyde, starch and sodium citrate were added in a
concentration range from 0.5% to 10.0% w/v, with 0.5% increments,
resulting in 20 adulterated samples for each adulterant. For the
samples with more than one adulterant, a simplex centroid mix-
ture design with five components, with the concentrations of each
adulterant ranging from 0.5% to 10.0%, and a maximum of 10.0% of
total adulteration. This mixture design resulted in 71 samples, con-
taining from two to five adulterants simultaneously (30 binary, 30
tertiary, 5 quaternary and 6 pentenary mixtures) and their compo-
sitions are presented in Table 1 of Supplemental materials.
2.3. Procedure
All the samples were analyzed directly, without any pre-treat-
ment, placing approximately 30 lL in the ATR accessory. The spec-
tra were collected as an average of 20 scans, from 600 to
4000 cm1. Before each measurement, a background correction
was performed to avoid atmospheric interference and reduce
instrumental noise. After each spectrum acquisition, the accessory
was cleaned with acetone P.A.
2.4. Qualitative analytical validation
The first step in qualitative multivariate validation is to deter-
mine the limit whether the sample is classified as 0 (negative/
not accepted/absence) or 1 (positive/accepted/presence). Modern
qualitative methods usually rely on instrumental reading for clas-
sifying a sample as positive or negative, and this limit value is
called threshold limit (TL), estimated using the Bayes Theory
(Gondim et al., 2011; Pulido, Ruisanchez, Boqué, & Rius, 2003).
The TL estimate has been extended from univariate to multivariate
methods, specifically for PLS-DA (Wise et al., 2006). Assuming that
all the samples in the training test belong to one of the proposed
 B.G. Botelho et al. / Food Chemistry 181 (2015) 31–37
classes, i.e., class A or class B (here, we assumed only two classes
for simplification), it can be stated:
PðAjyÞ þ PðBjyÞ ¼ 1
Using the Bayes’ Theory, it is possible to estimate the probabil-
ity that a sample is from class A given a particular value of y, P(A|y),
from the following equation:
PðyjAÞ  PðAÞ
PðAjyÞ ¼
½PðyjAÞ  PðAÞ þ PðyjBÞ  PðBÞ
where P(A) and P(B) are the probabilities for the observation of class
A or B in the future. If we assume that the probability of observing A
or B is similar to how many samples of A and B were in the original
training set, we can reduce this to:
PðyjAÞ
PðAjyÞ ¼
½PðyjAÞ þ PðyjBÞ
and
PðyjBÞ
PðBjyÞ ¼
½PðyjAÞ þ PðyjBÞ
These two distributions typically intersect in a unique value,
which leads to a single point where both P(B|y) and P(A|y) are
0.5. This point is selected as the threshold limit.
After the TL has been established, the next step is to determine
the numbers of false positive (FP) and false negative (FN) samples.
FP is the number of samples that do not belong to a class classified
as belonging to it, i.e., a sample that is not adulterated been classi-
fied as it is, and FN is the opposite parameter. Using FP and FN, the
following FOM are estimated, False Negative Rate (FNR), False Posi-
tive Rate (FPR), Selectivity Rate (STR), Specificity Rate (SPR) and
Efficiency Rate (EFR). FPR is defined as the ratio between FP and
the sum of FP and the total number of known negative samples
(TN).
FP
FPR ¼
FP þ TN
Analogously, the FNR is defined as the ratio between FN and the
sum of FN and the total number of known positive samples (TP).
FN
FNR ¼
FN þ TP
The STR is defined as the ratio between TP and the sum of TP
and FN, and the SPR in the ratio between TN and the sum of TN
and FP. In addition, EFR can be defined as the difference between
the total of results (100%) and the sum of FPR and FNR.
TP
STR ¼  100
TP þ FN
TN
SPR ¼  100
TN þ FP
EFR ¼ 100  ðFPR þ FNRÞ
Although some FOM used in qualitative and quantitative valida-
tions have the same name, the concepts attached to them and their
evaluation are different. While FPR, FNR and EFR express the true-
ness in qualitative analysis, STR and SPR are considered FOM relat-
ed to the selectivity of the method (Gondim et al., 2011; Trullols
et al., 2004). Another two FOM, accordance (ACO) and concordance
(CON), express the precision of qualitative methods. ACO is the
probability that two identical samples provide the same result
(both found positive or negative) when analyzed by the same
laboratory or batch, under repeatability conditions. For this
33
estimation, the probability that two samples provide the same
result is calculated, and then this probability is averaged over all
batches. The ACO can be estimated by the following equation:
ð1Þ
fkðk  1Þ þ ðn  kÞðn  k  1Þg
ACO ¼ ð10Þ
nðn  1Þ
where n is the total number of samples in each batch and k is the
number of positive results in each batch. CON is the qualitative
ð2Þ FOM equivalent to the quantitative estimation of intermediate pre-
cision or reproducibility. It estimates the probability of two testing
samples providing the same results in different laboratories or
batches in intermediary precision conditions. The CON can be esti-
mated using the following equation:
2½kðk  nbÞ þ nbðnb  1Þ  ACO½nbðn  1Þ
CON ¼ ð11Þ
n2 bðb  1Þ
ð3Þ
where b is the number of laboratories or analytical batches (Gondim
et al., 2011; Langton, Chevennement, Nagelkerke, & Lombard,
2002).
ð4Þ
3. Results and discussion
3.1. Mid infrared spectra
The spectra of all the samples are shown in Fig. 1. By observing
these spectra, it is possible to attribute the most intense absorption
peak around 3600–3000 cm1, which can be associated with
strong OAH stretching vibrations. The two small peaks near
2800 cm1 are related to milk’s fatty acid CH2 stretching. Another
peak, near 1700 cm1, is characteristic from amide I and II, which
can be related to milk protein content (Nicolaou et al., 2010;
Santos et al., 2013a). Initially, the region between 2430 and
2230 cm1 was removed due to the interference of atmospheric
CO2. Even using the instrumental atmospheric correction, some
samples presented a characteristic peak in this spectral region, so
it was removed from all samples.
ð5Þ
Fig. 2(a) shows a spectrum of a blank milk sample, without the
addition of any adulterant. As stated before, two distinguishable
peaks can be noticed, related to water and protein content.
Fig. 2(b) shows the spectrum of a milk sample adulterated with
10.0% v/v of water, and no significant changes can be visually not-
ð6Þ ed, compared with Fig. 2(a). Fig. 2(c) shows a spectrum for milk
with 10.0% w/v of starch. In comparison with Fig. 2(a), it is possible
to note slight changes in the fingerprint region, between 1200 and
900 cm1. The absorbance in this region can be associated with
skeletal mode vibrations of the a-1,4 glycosidic linkage, CAH bend-
ing, CAOAH bending and CAO and CAC stretching (Kizil,
Irudayaraj, & Seethraman, 2002). Fig. 2(d) shows the spectrum of
ð7Þ a sample adulterated with sodium citrate 10.0% w/v. Two charac-
teristics peaks around 1400 and 1600 cm1 can be related to sym-
metric and asymmetric stretching of CAO bonds from carboxylate
ð8Þ groups. Fig. 2(e) shows the spectrum of the adulteration of a blank
milk with 10.0% v/v of formaldehyde. A distinguishable peak
around 1000 cm1 can be noticed. This absorption peak can be
ð9Þ
related to formaldehyde CH2 rocking and wagging bending vibra-
tions (Pavia, Lampman, Kriz, & Vyvyan, 2008). Fig. 2(f) shows a
spectrum of a sample adulterated with 10.0% w/v of sucrose. The
presence of sucrose can be associated with the appearance of sev-
eral peaks in the fingerprint region (near 1200–1000 cm1), associ-
ated with CAH and CAO stretching, ketone C@O stretching and
bending, and CAO stretching and bending (Tewari & Malik,
2007). The region between 2000–900 cm1 presented the greatest
variations and is considered to be the most discriminative, contain-
ing characteristic bands for almost all the adulterants evaluated.
Thus, local models were tested and presented smaller classification
34 B.G. Botelho et al. / Food Chemistry 181 (2015) 31–37

Fig. 1. Mid infrared spectra of all the analyzed milk samples.

Fig. 2. Mid infrared spectra of (a) a blank milk sample; (b) a sample adulterated with water (10% w/v); (c) a sample adulterated with starch (10% w/v); (d) a sample
adulterated with sodium citrate (10% w/v); (e) a sample adulterated with formaldehyde (10% w/v); (f) a sample adulterated with sucrose (10% w/v). Spectral region used in
the models is marked by the dashed squares.

errors when compared to the full spectra models. All the models
developed and validated in this paper used the above cited region.
3.2. PLS-DA models
Five PLS1-DA models were built, one for each adulterant. Each
model was built using 155 samples (15 blank samples, 100 sam-
ples containing only one of the five adulterants, and 40 samples
containing the adulterant of choice for that model in mixtures with
others adulterants). Samples were split in training and test sets and
25% of the samples (38) of each model was selected for the test set,
chosen in equally spaced intervals of composition according to the
experimental design. To select the best number of latent variables
(LV), venetian blind cross validation was used, and the LV number
that presented the smallest cross validation classification error
(CVCE) was chosen (Table 1). Table 1 also presents the percentages
of variance accounted for each model, which were 99.5% in the X
block for all the models, and between 69.3% and 77.0% in the Y
block. The classification plots, with the y predicted values of all
samples, for all the models are shown in Fig. 3. In these plots, the
 B.G. Botelho et al. / Food Chemistry 181 (2015) 31–37 35

Table 1 coefficients. Nevertheless, as pointed out by Brown and Green

Number of latent variables (LV), variance accounted for in X and Y blocks, and CVCE (2009), this interpretation should not rely on the regression vec-
for the developed PLS-DA models.
tors, since they are dependent on the samples in the calibra-
Adulterant Number of Explained Explained Cross validation tion/training set, on the implicit covariance of the components,
latent variance – variance – classification and on the signal to noise ratio of the data. A better tool for the
variables X (%) Y (%) error (CVCE)
spectral interpretation of PLS models is the variable importance
Water 9 99.5 76.2 0.07 in projection (VIP) scores, which measure the importance of each
Starch 8 99.5 72.5 0.07
Sodium 9 99.5 77.0 0.02
variable in the projection used by a particular PLS model via their
citrate coefficients in every component, together with the significance of
Formaldehyde 9 99.5 69.3 0.04 each component in regression (Chong & Jun, 2005). Fig. 4 shows
Sucrose 9 99.5 73.1 0.07 the VIP scores for each model. There is a close agreement between
the spectral discussion presented in Section 3.1 and the variables of
greater importance observed through the highest VIP scores in
data were ordered always starting with the samples not containing Fig. 4.
the adulterant of the model. The threshold for each class/model As can be seen in Fig. 4, all the VIP scores presented distinguish-
was estimated using the Bayes’ theorem (Wise et al., 2006) in order able peaks in regions specifically characteristic of each adulterant,
to minimize classification errors. The Bayesian threshold estima- such as the two peaks between 1080–1040 and 1015–980 cm1 of
tion assumes that the predicted Y variance follows a distribution citrate (Fig. 4(c)), the broad band with two peaks between around
similar to what will be observed for future samples. Plots of the 1100 and 1000 cm1 of formaldehyde (Fig. 4(d)), and the two
PLS-DA predictions as a function of different concentrations are major peaks at 1080 and 980 cm1 for sucrose (Fig. 4(e)).
also shown in Fig. 1 of Supplemental materials for two adulterants,
sodium citrate and water. 3.3. Multivariate qualitative validation
For all the models, the data were preprocessed with first deriva-
tive and Savitzky–Golay smoothing, followed by mean centering. As stated previously, multivariate validation of analytical meth-
Other preprocessing were tested, such as orthogonal signal correc- ods is still an evolving subject, not well established, and the esti-
tion (OSC), multiplicative scatter correction (MSC) and standard mation of FOM for qualitative multivariate methods has received
normal variate (SNV), but all of them provided higher CVCE. even less attention. In this paper, some basic FOM were estimated.
The spectral interpretation of PLS or PLS-DA models is As can be seen in Table 2, seven different FOM were estimated for
sometimes carried out through the observation of the regression the training and test sets.

Fig. 3. Classification plots for (a) water; (b) starch; (c) sodium citrate; (d) formaldehyde; (e) sucrose. The following symbols apply for the samples of each class: 5 – blank
samples; / – water adulteration; } – starch adulteration; s – sodium citrate adulteration; 4 – formaldehyde adulteration; h – sucrose adulteration. The full and empty
symbols represent the samples allocated in the training and test sets, respectively. The solid lines indicate the threshold values for each model.
36 B.G. Botelho et al. / Food Chemistry 181 (2015) 31–37

Fig. 4. Variable importance in projection (VIP) scores for the models detecting adulterations with: (a) water; (b) starch; (c) sodium citrate; (d) formaldehyde; (e) sucrose.

Table 2 and citrate being responsible for this high FPR. Formaldehyde
Estimated FOM for evaluating the developed methods for simultaneous detection of and sucrose models presented similar FPR (0.0% and 1.0%, respec-
adulterants in milk.
tively) and FNR (3.2% and 4.8%, respectively) in the training set;
FPR (%) FNR (%) STR (%) SPR (%) EFR (%) and identical values for the test set, with a FPR of 4.2% and a FNR
Training set of 0% for both analytes. The citrate model presented the best
Water 4.0 3.2 96.8 96.0 92.8 results, with FPR and FNR of 0% for training and test sets. According
Starch 3.0 4.8 95.2 97.0 92.2 to the Directive 657/2002 of the European Community (EC, 2002),
Sodium citrate 0.0 0.0 100.0 100.0 100.0
the FNR should be smaller than 5%. False negative results should be
Formaldehyde 0.0 3.2 96.8 100 96.8
Sucrose 1.0 4.8 96.8 99.0 95.7 more rigorously controlled for consumer’s safety, avoiding that an
adulterated sample be classified as non-adulterated. False positive
Test set
Water 11.5 0.0 93.8 88.5 82.8 results represent a lower concern, because all positive samples in a
Starch 8.0 16.7 83.3 92.0 75.3 screening test may be confirmed by a reference method. Except for
Sodium citrate 0.0 0.0 100.0 100.0 100.0 the starch model, all of the proposed methods are in accordance
Formaldehyde 4.2 0.0 93.8 95.8 89.6
with the above cited validation guide.
Sucrose 4.2 0.0 93.8 95.8 89.6
The more representative FOM of trueness, EFR, was also shown
FPR – False Positive Rate; FNR – False Negative Rate; STR – Sensitivity Rate; SPR – in Table 2. EFR for the training set of each model ranged from 92.2%
Specificity Rate; EFR – Efficiency Rate.
to 100%, and for test sets ranged from 75.3% to 100%, with the two
worst results observed for the starch adulteration model.
The CON was estimated using three replicates of three different
Water adulteration model presented the highest FPR (4.0%) and levels (0.5%, 5.0% and 10% w/v) analyzed in repeatability conditions
the second highest FNR (3.2%) in the training set, and also the high- for each adulterant. The ACO was estimated using the same proto-
est FPR in the test set (11.5%). The greatest number of misclassifi- col employed for CON estimation, repeated in three different days
cations in this model may be due to the close similarity between by three different analysts. The water model presented the small-
the water–adulterated samples and the blank samples or samples est ACO and CON values (78% and 92%, respectively), with all other
of other adulterants in lower concentrations (Fig. 4(a)). Starch four models presenting 100% of both ACO and CON. These results
adulteration presented a low number of FPR and FNR (3.0% and suggest that the detection of adulteration with water was not so
4.8%, respectively) in the training set. However, this model pre- precise, maybe because of the large amount of water already pre-
sented relative high FPR and FNR for the test set (8.0% and 16.7%, sent in non-adulterated milk and the natural variation of its con-
respectively), with misclassification of samples containing water tent between 85% and 90% w/w.
 B.G. Botelho et al. / Food Chemistry 181 (2015) 31–37
4. Conclusion
A simple multivariate screening method for simultaneous
detection of five common adulterants in milk, based on ATR mid
infrared spectroscopy, was developed and validated. The method
was able to detect the specific presence of water, starch, sodium
citrate, formaldehyde and sucrose in raw cow milk samples in con-
centrations as low as 0.5% w/v. The analytical range chosen for all
the adulterants, between 0.5% and 10.0% w/v, was consistent with
the forensic interest, considering the reported cases of milk adul-
teration. All the models, except for the starch, provided results in
accordance with international validation guidelines (EC, 2002;
NATA, 2013). The proposed method requires a small amount of
sample (about 30 lL), is very rapid (up to 1 min for spectral acqui-
sition) and non-destructive, not demanding any sample prepara-
tion nor using reagents, and all the adulterants can be evaluated
simultaneously using the same spectra. The analytical validation
was carried out searching to extend the estimate of specific figures
of merit from univariate to multivariate qualitative methods. This
harmonization is important once it aims the acceptance of multi-
variate screening methods by the official guidelines. The simplicity
of the proposed method makes it suitable for quality control in dif-
ferent steps of the chain of production, such as the receipt of raw
milk in a dairy product factory or the detection of adulterants in
commercialized milk.
Acknowledgements
B. G. B. thanks CAPES and CNPq for fellowships and Denise
Aquino from LANAGRO (Pedro Leopoldo, MG) for providing the
milk 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.2015.
02.077.
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