Microchemical Journal 124 (2016) 1519

Contents lists available at ScienceDirect

Microchemical Journal
journal homepage: www.elsevier.com/locate/microc

Detection and quantication of milk adulteration using time domain

nuclear magnetic resonance (TD-NMR)
Poliana M. Santos a,, Edenir R. Pereira-Filho b, Luiz A. Colnago c
a
b
c

Department of Chemistry and Biology, Federal Technological University of Paran (UTFPR), Rua Deputado Heitor de Alencar Furtado, 5000, 81280-340 Curitiba, PR, Brazil
Department of Chemistry, Federal University of So Carlos (UFSCar), Rodovia SP 310, km 235, P. O. Box 676, 13565-905 So Carlos, SP, Brazil
Embrapa Instrumentation, Rua XV de Novembro, 1452, P. O. Box 741, 13560-970 So Carlos, SP, Brazil

a r t i c l e

i n f o

Article history:
Received 18 May 2015
Received in revised form 14 July 2015
Accepted 17 July 2015
Available online 26 July 2015
Keywords:
Milk adulteration
1
H TD-NMR
Multivariate analysis

a b s t r a c t
This study explores the possibilities for application of 1H Time Domain Nuclear Magnetic Resonance (1H
TD-NMR) as a rapid method for assessment of milk quality. Whey, urea, hydrogen peroxide, synthetic urine
and synthetic milk were added to the milk samples at concentrations of 5, 15, 25, 35 and 50% v/v. Discrete exponential analysis of the 1H TD-NMR relaxation decay revealed that the milk samples contained a single water component as well as that the T2 relaxation times differed signicantly with respect to the level of adulteration.
Regression models obtained with the full 1H TD-NMR (multivariate approach) and T2 value (univariate
approach) demonstrate a strong correlation to estimate the level of adulteration in milk samples, with standard
errors of prediction of 2.34 and 3.79% v/v, respectively. SIMCA and kNN classication models were developed to
classify control from adulterated milk samples and adulterated milk samples based on the level of adulteration.
The results obtained with both models showed a similar and quite satisfactorily predictability, with sensitivity
and specicity ranging from 0.66 to 1.00. This study clearly demonstrates that 1H TD-NMR could be applied as
an alternative rapid method for detecting and quantifying milk adulteration.
2015 Elsevier B.V. All rights reserved.

1. Introduction
The application of 1H time domain nuclear magnetic resonance
(1H TD-NMR) spectroscopy in food science has been widely investigated in the last few years [1]. Several factors have contributed to the
rapidly increasing use of this technique in the analysis and quality
control of foods, including: no sample preparation required, simple and
fast measurement procedures, instrumental stability, noninvasiveness,
and the potential for through-package analysis. Additionally, the low
cost of commercial benchtop NMR system, with permanent magnet
technology, has contributed to the growing popularity of TD-NMR.
In 1H TD-NMR spectroscopy, the proton relaxation (transversal
relaxation, T2) is monitored providing information about the mobility
of the nuclei. In the foodstuffs, 1H TD-NMR provide unique information
about the nuclei described in terms of bound water, free water and by
an exchange between these two states. One of the most reported applications of 1H TD-NMR in food science has focused in the meat analysis.
This technique was suggested as an alternative method for determination of water-holding capacity (WHC) [24] and the physical changes
during the conversion of muscle into meat [5], cooking [6] and freezing
storage [7]. 1H TD-NMR has been also demonstrated to be a powerful
technique for quality control of milk [8], cheese [9], honey [10], potato
Corresponding author. Tel.: +55 41 3279 4575.
E-mail address: polianasantos@utfpr.edu.br (P.M. Santos).

http://dx.doi.org/10.1016/j.microc.2015.07.013
0026-265X/ 2015 Elsevier B.V. All rights reserved.

[11], oil [12], intact fruits [13,14], and sauces [15], as it provides important information about sensory attributes, food texture and ripening
status, in a completely non-invasive way.
Recently, the feasibility to apply 1H TD-NMR to check the authenticity of food was investigated [16,17]. Ribeiro et al. [16] have used 1H TDNMR to detect honey adulteration. Pure blossom honey samples were
collected by beekeepers and adulterated by adding of high fructose
syrup at a ratio of 0, 10, 25, 50, 75 and 100% (w/w). The results showed
that the relaxation times were signicantly affected by adulterant concentration in pure honey, suggesting that 1H TD-NMR could be used to
discriminate pure blossom honey from honey adulterated with high
fructose corn syrup. In a different study, Zhang et al. [17] have applied
1
H TD-NMR to discriminate edible vegetable oil adulteration mixed
with used frying oil. Commercial corn, peanut, rapeseed and soybean
oil were purchased from a local supermarket and adulterated with frying oil collected from local restaurants. The T2 distribution curves
showed the presence of three peaks (three types of hydrogen protons),
and revealed that the major differences between the unused and used
oil appeared in the third peak. Using linear correlation against the
peak area and the percentage of adulteration, proved that 1H TD-NMR
could be used to detect adulteration of vegetable oils mixed with used
frying oil.
The goal of this study was to examine the feasibility of the application of 1H TD-NMR combined with multivariate analysis to identify
and quantify milk adulteration by the addition of tap water, whey,

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P.M. Santos et al. / Microchemical Journal 124 (2016) 1519

synthetic milk, synthetic urine, urea and hydrogen peroxide. According

to the database of food fraud developed by members of the US
Pharmacopeia Convention's Food Ingredients Intentional Adulterants,
milk is one of the most common targets for adulteration and the
major issue for the dairy industry [18]. The establishment of a general
rapid method can have practical use in the dairy industry for inspection
of milk authenticity.
2. Materials and methods
2.1. Samples
Bovine milk samples were furnished by Embrapa Southeast Livestock (Brazilian Agricultural Research Corporation) located at So Carlos
(So Paulo, Brazil). Milk samples were spiked with known levels of
whey, urea, hydrogen peroxide, synthetic urine [19] and synthetic
milk [20], following the same dilution process (5 to 50% v/v). A total of
78 milk samples were analyzed.
2.2. TD-NMR measurements
1

H TD-NMR experiments were evaluated using SLK-IF-1399 NMR

spectrometer (Spinlock Magnetic Resonance Solution, Cordoba, AG)
equipped with a Halbach permanent magnet of 0.23 T (9 MHz for 1H)
and 10 cm bore. 1H transverse relaxation time, T2, measurements
were performed using CPMG (CarrPursellMeiboonGill) pulse
sequence with a /2 pulse width of 16 s, time between echoes of
400 s and a recycle delay of 15 s.
2.3. Data analysis
The 1H TD-NMR relaxation curves were maximum normalized
(normalized against the rst echo amplitude) and analyzed by discrete
exponential tting in the Origin software version 8.1 (OriginLab,
Northampton, MA, USA). T2 values were obtained by mono-exponential
function according to the equation:


N
X
t
et
M0;n exp
Mt
T 2;n
n1
where M(t) is the residual magnetization at time t, M0,n is the concentration or magnitude parameter of the nth exponential,T2,n is the corresponding transverse relaxation time constant, and e(t) is the residual
error.
Multivariate analysis based on principal component analysis (PCA),
soft independent modeling of class analogy (SIMCA), k nearest neighbors (kNN), and partial least squares regression (PLSR) were applied
in the full 1H TD-NMR relaxation curves for exploratory analysis
(PCA), classication (SIMCA and kNN), and proposition of regression
models (PLSR). These analyses were developed in Pirouette software
version 4.5 (Infometrix Inc., Woodville, WA, USA). Prior to the
multivariate analysis, the 1H TD-NMR relaxation curves were maximum
normalized and mean centered.
PCA was applied to obtain a global view of the main variation in the
1
H TD-NMR relaxation data. In PCA, the original data matrix X is
decomposed into a score matrix T, loading matrix P and the residuals
are collected in a matrix E:

model was also evaluated using linear regression by correlating the T2

values against to the level of adulteration. Linear regression was
performed according to the following equation y = mx + b. The performance of multivariate and univariate models was evaluated in terms of
the gures of merit accuracy, precision and linearity.
SIMCA and kNN classication models were evaluated using the full
1
H TD-NMR relaxation decays. For each chemometric classication
technique two strategies were made: the rst model was developed
to classify the control milk samples from adulterated milk samples
(authentication model); the second one was evaluated to discriminate
the milk samples based on the level of adulteration. Both models were
evaluated using sensitivity and specicity parameters. To build the
classication models, the 1H TD-NMR decays were divided into two
thirds for the training set (calibration) and one third for the test set
(validation). Prior to the analysis, 1H TD-NMR decays were mean
centered.
3. Results and discussion
3.1. T2 relaxation measurements
Fig. 1 shows the maximum normalized 1H TD-NMR relaxation
curves of the control and milk samples adulterated with water (from
5 to 50% v/v). The effect of adulteration level in the T2 relaxation time
is visually apparent. Samples with high level of adulteration, those between 35 and 50% v/v, relaxed more slowly than the control samples.
In order to verify the number of components in the milk samples,
Laplace inversion was performed in the 1H TD-NMR decays. The results
showed that the T2 relaxation was characterized by one 1H component.
This result agrees with previously studies found in the literature, which
reported that the proton transverse relaxation in skin milk is mainly
mono-exponential due to the fast diffusive exchange of water between
the different compartments [2123].
Table 1 describes the T2 found by mono-exponential tting. The
average and standard deviation of T2 ranged from 0.178 0.002 s (for
control milk samples) to 0.358 0.011 s (for milk adulterated with
water in 50% v/v). This behavior was obtained for all adulterants, except
for hydrogen peroxide. Hydrogen peroxide rapidly decomposes to oxygen gas, which is paramagnetic, resulting in the decrease of T2 time. Results showed that samples adulterated with 5% and 15% v/v of hydrogen
peroxide relaxed faster than control milk samples (0.178 0.002 s). On
the other hand, samples with 35% and 50% v/v of hydrogen peroxide
showed similar T2 values as the milk samples adulterated at the same
level using other adulterants. This indicates that the dilution process is
driving the relaxation time.

X TPT E:
The scores contain information about the samples, and the loadings
information about the variables.
Partial least squares regression (PLSR) was used to predict the level
of adulteration (Y, dependent variables) from the instrumental data
(X, independent variables). The milk sample was randomly divided
into a calibration and validation sets for the PLSR modeling. Regression

Fig. 1. Maximum normalized 1H TD-NMR relaxation decays of control and milk samples
adulterated with water (550% v/v).

P.M. Santos et al. / Microchemical Journal 124 (2016) 1519

17

Table 1
Relaxation time constants (T2) obtained by discrete mono-exponential tting of the Carr-Purcell-Meiboom-Gill (CPMG) decays. The values are expressed as mean standard deviation.
Adulterant

Water
Whey
Synthetic milk
Synthetic urine
Hydrogen peroxide

T2 (s)
5%

15%

25%

35%

50%

0.188 0.002
0.188 0.002
0.188 0.002
0.188 0.004
0.135 0.006

0.213 0.003
0.209 0.004
0.212 0.002
0.215 0.003
0.161 0.006

0.241 0.003
0.233 0.002
0.239 0.002
0.243 0.004
0.205 0.005

0.284 0.014
0.279 0.014
0.281 0.013
0.295 0.013
0.283 0.014

0.358 0.011
0.335 0.011
0.360 0.012
0.369 0.011
0.403 0.017

This result clearly showed that 1H TD-NMR is a sensitive method for

monitoring the milk quality with signicant variation of T2 values
among control and adulterated milk samples.
3.2. PCA analysis
To illustrate the different behavior of the control and adulterated
milk samples, PCA was performed on the maximum normalized 1H
TD-NMR relaxation decays (Fig. 1). Due to the nonlinear variation of
the T2 values in the samples adulterated with hydrogen peroxide, the
1
H TD-NMR of this samples was not used in this analysis.
PC1, which accounted for 73.2% of the variation in the data, clearly
showed differentiation in the samples according to the dilution process.
Control milk samples (T2 ~ 0.178 s) were grouped at one end of PC1,
whereas milk samples adulterated in 50% (T2 ~ 0.362 s) were grouped
at the other end (Fig. 2). No signicant differences were observed
with relation to the type of adulterant. This agreed with the T2 values
shown in Table 1, as similar T2 values were obtained for all milk samples
diluted with targeted adulterant in the same level.
In summary, the PCA score plot indicated that the clustering of the
milk samples is presumably based on the variation of T2 values listed
in Table 1.
3.2. Regression models
Although the results obtained with PCA analysis suggested that only
the T2 variable is responsible for the main variation in the data, a multivariate regression model was developed using the full 1H TD-NMR
(PLSR model) and the performance was compared with the univariate
model. A summary of prediction performance of the models is shown
in Table 2. The best PLSR model was obtained with 2 latent variables
(LV), which accounts more than 90% of the total variance of the data.
The accuracy of the models was estimated mainly by RMSEP (root
mean square of prediction) [24,25]. PLSR model showed better accuracy
than the univariate model with RMSEP values of 2.35% (v/v), indicating

that the method provided results that agree with those in the reference
values (Table 2). Since these parameters are not recognized by ofcial
regulations, a non-paired t tests was applied to compare the reference
values with those predicted. No signicant differences were observed
at 95% condence level for all level of adulteration (estimated t values
below the critical t value), which conrms the accuracy of the proposed
method.
The precision of the models was assessed for levels of repeatability
and intermediary precision through the relative standard deviations
(RSD) [24,25]. The generated models properly showed similar precision,
with RSD b 1.95% for repeatability and 3.33% for intermediary precision
(Table 2). These values are within the limits dened by the Brazilian
guidelines [26], which establishes a maximum acceptable RSD of 4.9%
for repeatability and 10% for intermediate precision.
The linearity of the regression models was estimated by tting the
reference values versus predicted ones. Fig. 3 shows the regression
graphics obtained with PLSR (Fig. 3A) and univariate (Fig. 3B) models.
The slope, intercept and correlation coefcient for the models are also
shown in Table 2. PLSR model showed better correlation coefcient
(N 0.99) and slope (closer to 1), while univariate model showed better
intercept (closer to 0).
Overall, regression models developed using the full 1H TD-NMR
relaxation show better predictability than the model obtained by
using the T2 values (univariate model). The lower prediction
performance of the univariate model could be attributed to the error
generated during the application of the discrete exponential tting to
obtain the respective relaxation time.
A variety of methods has been used to quantify milk adulteration.
PLSR models developed using different NIR and MIR spectrometers
showed RMSEP values ranging from 0.83 to 4.74% [27]. Similarly, PLSR
models obtained with digital image showed a RMSEP of 5.85% [28].
3.3. SIMCA and kNN models
Based on the results obtained with the regression models, classication models were developed using the full 1H TD-NMR relaxation
curves. The performance of test set of SIMCA and kNN classication
models are presented in Table 3. In general, the models showed a similar
and quite satisfactorily predictability. The best SIMCA and kNN models
developed to discriminate control from adulterated samples (authentication models) were obtained using 34 principal components and 3
number of neighbors, respectively. The values of sensitivity, the ability
of a method to detect truly positive samples as positive, ranged from
Table 2
Regression models performance to quantify milk adulteration.
Figures of merit

Parameter

PLSR

Univariate

Accuracy
Precision

RMSEP
Repeatability
Intermediate precision
Slope
Intercept
Correlation coefcient

2.34
1.95b
3.33b
0.97a
0.64a
0.99a

3.79
1.93b
3.00b
0.95
0.25
0.94

Linearity

Fig.2. PCA score plot of the maximum normalized 1H TD-NMR relaxation decays of control
and adulterated milk samples.

a
b

Values for the line tted to the calibration samples.

Results for milk samples adulterated at 25% v/v.

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P.M. Santos et al. / Microchemical Journal 124 (2016) 1519

Fig. 3. Reference values versus estimated values by (a) PLSR and (b) univariate regression models.

0.66 (control samples) to 1.00 (adulterated samples). This means that

some false positives or unadulterated sample have been classied as if
they were adulterated. False positive results are a lesser concern, as all
positive samples may be conrmed by the reference methods. Specicity values, the ability of a method to detect truly negative samples as
negative ranged from 1.00 (control samples) to 0.66 (adulterated
samples). The greatest number of misclassications in these models
may be due to the close similarity between the control and samples
with lower concentration of adulterant, as 5% (v/v). Even though, none
of the adulterated samples were predicted as control using the model
developed (false negative). False negative results should be more
rigorously controlled due to the economic and safety issue.
SIMCA and kNN models based on the level of adulteration were also
obtained using 34 principal components and 3 neighbors, respectively.
These models demonstrated proper sensitivity and specicity, with
values higher than 0.66 and 0.80, respectively. Even though the
detection of control samples had a lower success rate than in the case
of adulterated milk samples, the model still gave similar or better
performance compared to literature data on milk classication from
infrared spectroscopy [27] and digital image [28].

4. Conclusion
This is the rst study that has demonstrated the feasibility of
TD-NMR to detect and quantify milk adulteration in a fast, nondestructive,
and noninvasive manner. This would mean that the possibility of using 1H
TD-NMR to measure milk adulteration without any sample preparation
and through milk package.
Regression models obtained with multivariate analysis (PLSR
method) showed better predictability when compared with univariate
model, with low RMSEP (b 2.35% v/v) and high correlation coefcient
(N0.99). SIMCA and kNN classication models discriminated control
Table 3
Sensitivity and specicity of SIMCA and kNN models. Results obtained with the training
set.
Authentication model

Level of adulteration model

Control

Adulterated

Control

5%

15%

25%

35%

50%

SIMCA
Sensitivity
Specicity

0.66
1.00

1.00
0.66

0.66
1.00

1.00
1.00

0.88
1.00

1.00
0.97

1.00
1.00

1.00
1.00

kNN
Sensitivity
Specicity

0.66
1.00

1.00
0.66

0.66
1.00

1.00
0.97

1.00
1.00

1.00
1.00

1.00
0.80

0.83
1.00

milk samples from several potential adulterants (whey, urea, hydrogen

peroxide, synthetic urine and synthetic milk) at level of
adulteration N 5% v/v.
Overall, 1H TD-NMR combined with chemometrics analysis may be
used for online automatization of milk analysis with a higher sampling
frequency, in portable equipments.
Acknowledgments
The authors would like to acknowledge Brazilian agencies FAPESP
(2009/01345-6) and CNPq (572859/2008-7) for their nancial support
of this research.
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