Food Anal.

Methods
DOI 10.1007/s12161-012-9498-z

Development and Analytical Validation of Robust

Near-Infrared Multivariate Calibration Models
for the Quality Inspection Control of Mozzarella Cheese
Bruno G. Botelho & Bruna A. P. Mendes & Marcelo M. Sena

Received: 3 July 2012 / Accepted: 24 August 2012

# Springer Science+Business Media, LLC 2012

Abstract This paper proposed and validated robust diffuse

reflectance near-infrared methods for the direct determination of fat and moisture in cow mozzarella cheeses using
partial least squares regression. They were developed under
the realistic conditions of routine analysis in a state laboratory of quality inspection control and were used for analyzing a great variety of mozzarella samples manufactured by
different manufacturing procedures and originating from the
whole state of Minas Gerais, Brazil (more than 100 different
producers). A robust methodology was implemented, including the detection of outliers and the harmonization of
the multivariate concepts with the traditional univariate
guidelines. The models were constructed in the ranges from
38.7 to 58.0 % w/w on dry basis for fat and from 41.5 to
55.1 % w/w for moisture, providing root mean square errors
of prediction of 2.1 and 0.9 %, respectively. Both methods
were validated through the estimation of figures of merit,
such as linearity, trueness, precision, analytical sensitivity,
ruggedness, bias, and residual prediction deviation. Once
the methods were adopted, their performances were monitored for approximately 1 year through control charts and
were considered satisfactorily stable with prediction errors
within the established limits. Beyond these specific
B. G. Botelho : M. M. Sena (*)
Departamento de Qumica, ICEx, Universidade Federal de Minas
Gerais,
31270-901, Belo Horizonte, MG, Brazil
e-mail: marcsen@ufmg.br
B. A. P. Mendes
Instituto Mineiro de Agropecuria (IMA),
BR 040, Km 527,
32145-900, Contagem, MG, Brazil
M. M. Sena
Instituto Nacional de Cincia e Tecnologia em Bioanaltica (INCT
Bio),
13083-970, Campinas, SP, Brazil

methods, it was also pursued to present a complete methodology for multivariate analytical validation, an important
aspect for the implementation of near-infrared spectroscopy
methods in the routine of food quality inspection.
Keywords Quality control . NIR . PLS . Multivariate
analytical validation . Dairy analysis

Abbreviations and Symbols

BF
BrownForsythe test
CLS
Classical least squares
DW
DurbinWatson test
FOM
Figures of merit
IMA
Instituto Mineiro de Agropecuria
LV
Latent variables
MLR
Multiple linear regression
NAS
Net analyte signal
NIRS
Near-infrared spectroscopy
PCR
Principal components regression
PLS
Partial least squares
RJ
RyanJoiner test
RMSEC
Root mean square error of calibration
RMSECV Root mean square error of cross-validation
RMSEP
Root mean square error of prediction
RPD
Relative prediction deviation
RSD
Relative standard deviation
SD
Standard deviation
SDsd
Standard deviation of the successive
differences
SDV
Standard deviation of validation errors
SEL
Selectivity
SEN
Sensitivity

Analytical sensitivity

Instrumental noise

Food Anal. Methods

Introduction
Mozzarella is a cheese made from pasteurized and partly
skimmed cow milk, widely found around the world, and
originated from Campania region in Southern Italy. Also
called pizza cheese, it is the most consumed cheese in the
USA, Brazil, and other countries, mainly due to its use in
pizzas, sandwiches, and snacks (Harbutt 2009). The State of
Minas Gerais, the second most populous in Brazil, with
about 20,000,000 inhabitants and an area of 586,528 km2,
is the largest national cheese producer, with about half of the
production (215,000 tons/year). From this total, 24 % corresponds to mozzarella cheese (Teixeira et al. 2007). The
mozzarella production in Brazil is regulated by the guidelines of the Ministry of Agriculture, Livestock and Food
Supply (MAPA for its initials in Portuguese), which establishes limits for fat, a minimum of 35 % w/w on dry weight
basis, and moisture, a maximum of 60 % w/w (MAPA
1997). For this quality inspection, nitrate content and phosphatase assay are also required, unlike other parameters,
such as protein and pH. According to this regulation, the
fat determination is carried out by acid hydrolysis (Gerbers
method). This method is based on the use of a butyrometer,
which presents several disadvantages. The determination
cannot be automated and involves a certain risk in handling
highly concentrated sulfuric acid. In addition, handling the
butyrometer requires practical skills, which has a negative
effect on the robustness and precision of the method
(Badertscher et al. 2007). The moisture determination is
carried out by the oven-drying gravimetric method, a simple
but slow method, since each analysis spends from 3 to 6 h
(Bradley and Vanderwarn 2001). In general, the traditional
analytical methods of major food components, such as fat
and moisture, are slow, relatively expensive, and demand
highly qualified staff. These classical methods are not effective enough to cover the growing demand and low costs
required in the quality control of industries or quality inspection of public agencies (Rodriguez-Otero et al. 1997).
A promising alternative to overcome the drawbacks of
the classical methods of food analysis is the joint use of
near-infrared spectroscopy (NIRS) and multivariate calibration. This combination provides methods that are rapid,
nondestructive, environmentally friendly, and solvent-free,
with no or low residue generation. Besides, these methods
require the use of minimum of sample pretreatment and
have sufficient accuracy and sensitivity with less human
intervention. The first paper that suggested the NIRS method for quantification in dairy products was published by Ben
Gera and Norris (1968), who have studied the influence of
fat content in milk using multiple linear regression (MLR).
But, in general, the widespread use of the NIRS method has
only emerged in the late 1980s, when the more accurate and
robust multivariate calibration models partial least squares

(PLS) and principal component regression (PCR) replaced

MLR in most of practical applications. Pierce and Wehling
(1994) have published the first method using NIRS and PLS
to determine components of cheddar cheese. Since then,
several articles have been published using NIRS and multivariate calibration for predicting quality parameters, such as
fat, protein, moisture, and others, of different types of
cheese (fresh, Danbo, Emmental, and cheddar) and derivatives (Wittrup and Norgaard 1998; Blazquez et al. 2004;
Karoui et al. 2006; Lucas et al. 2008; Pi et al. 2009; Ntsame
Affane et al. 2011). Specifically, no paper has been published using NIRS and multivariate calibration for determing parameters of mozzarella cheese.
Some calibration models based on NIRS are not reliable
and stable enough when used practically for routine analysis. It is necessary to improve and assure the reliability of
these models (Huang et al. 2008). Most of the papers cited
in the last paragraph have not incorporated sufficient variance in their models for a wider and robust use in quality
control inspection, analyzing cheese samples obtained from
a small number of producers. None of these models have
been applied in real routine analysis or have their reliability
assessed in this context. Moreover, none of these papers
have performed an analytical validation. A problem related
to this validation is the traditional guidelines in food analysis that have been conceived in a univariate way and need to
be harmonized in order to encompass the specific aspects of
NIRS technology related to multivariate methods. This is
the case of the Brazilian regulations (MAPA 2009) and most
of the international guidelines at which it is based on (ICH
1995, 1997; FAO 1998; Thompson et al. 2002; EC 2002).
None of these guidelines have at least mentioned the word
multivariate. Examples of conceptions that need to change
may be cited. The requirement of total selectivity of the
analytic signal does not make sense for multivariate methods, since they are precisely useful when a selective wavelength does not exist. It is also not possible to obtain the
traditional calibration curves (signal as a function of concentration) in multivariate calibration. In the past few years,
the advances in multivariate theory have also brought the
concept of net analyte signal (NAS), which allows extracting the specific information of the analyte from the whole
signal and can be used for estimating some figures of merit
(FOM). Discussions about how to harmonize univariate
guidelines with multivariate methods have been the subject
of recent papers, in general (Olivieri et al. 2006; Valderrama
et al. 2009) or in specific areas, such as in the analysis of
pharmaceuticals (Silva et al. 2012; Ferreira et al. 2012) and
agricultural products (Valderrama et al. 2007).
The aim of this paper was to develop two simpler, robust,
and rapid methods for directly determining fat and moisture
in cow mozzarella cheeses, which originated from different
origins and manufactured by different manufacturing

Food Anal. Methods

procedures (more than 100 producers), by using NIRS and

PLS. These methods should be used for the quality inspection control of these cheeses in Instituto Mineiro de
Agropecuria (IMA), the official laboratory of the State of
Minas Gerais, in the city of Contagem, Brazil. Models for
nitrate and phosphatase were not possible to build because
almost no sample present results above the limits of quantitation of the respective reference methods. This work also
searched for performing the analyses under the realistic
environment of the quality inspection control, carrying out
a full analytical validation, and assessing permanently the
performance of the methods through a post-model stability
monitoring.

Materials and Methods

Instruments and Software
The spectra were recorded on a Perkin Elmer Spectrum 400
FTIR spectrophotometer (Waltham, MA, USA), equipped
with a diffuse reflectance accessory. Data were handled using
the MATLAB software, version 7.13 (The MathWorks,
Natick, MA, USA). The PLS routine came from the PLS
Toolbox, version 6.5 (Eigenvector Technologies, Manson,
WA, USA), and a homemade routine was also employed for
the detection of outliers.

resolution of 8 cm1. Ten replicate spectra of the empty

plate were also recorded in the same conditions in order to
estimate the instrumental noise. During all these measurements, the temperature of the room was controlled at 221
C. The analyses by the reference methods were performed
according to the standard literature (Pomeranz and Meloan
1994) without replicates. Fat determination was carried out
with Gerbers method. One gram of a cheese sample was
accurately weighed in the butyrometer, in which 10 ml of a
sulfuric acid solution (d01.605 gcm3) and 1 ml of isoamyl
alcohol were added; then, the filler opening was sealed and
the butyrometer was shaken and tempered for 15 min in a
65 C water bath; the shaking and heating were repeated
until total dissolution of the sample; finally, the fat column
was adjusted to the zero point and the absolute fat content
was read off from the lower end of the meniscus. Moisture
determination was carried out by the oven-drying gravimetric method. Three grams of a cheese sample was placed
in a plate and spread with the same amount of predried
fine sand. The sand aids in more rapid and efficient
drying of the sample. This mixture was homogenized
and oven-dried at 105 C until it reached a constant weight,
taking 36 h.

Multivariate Analytical Validation

Detection of Outliers

Reagents and Samples

All the reagents and materials, sulfuric acid, isoamyl alcohol, and purified sand, were of analytical grade, purchased
from certified suppliers, and used without further purification. One hundred twenty-three mozzarella cheese samples
manufactured by different manufacturing procedures and
originating from more than 100 different producers from
all over the state of Minas Gerais, Brazil were collected
and analyzed in 2009 and 2010. The cheese samples were
transported to the laboratory under refrigeration at 4 C, left
for 30 minutes until thermal equilibrium, and analyzed
immediately after opening the sample. The slices were
obtained from the internal part of the cheese samples, avoiding their external surface, which can be subjected to
degradation.
Procedures
The cheese samples were sliced, milled with an industrial
mixer, and transferred to a Perkin Elmer plate in a sufficient amount to cover the bottom of the plate (about 40 g).
Then, the reflectance module was pressed against the sample surface. The spectra were collected as an average of 32
scans, from 1,000 to 2,500 nm with 2 nm steps, with a

Outliers are samples that are very different from the rest of
the data set and their detection is crucial when developing
multivariate NIRS models. This detection can be performed
in several ways, and this paper adopted a robust methodology for detecting samples with extreme leverages, large
residuals in the X block (spectral data) or large residuals in
the Y block (property values) (ASTM 2012; Valderrama et
al. 2007). If a sample presents a leverage, it means, a
measure of the influence of each sample on the PLS model,
larger than a limit value, it is considered an outlier. Outliers
with large residuals in X are detected by comparison of the
total standard deviation, s(e), of the unmodeled spectral data
with the respective standard deviation of each sample, s(ei).
If a sample presents s(ei)>2.5s(e), it is deleted at the 99 %
confidence level. Outliers with large residuals in Y can be
detected by comparing the root mean square error of calibration (RMSEC) with the prediction errors. If a sample has
an error of prediction larger than three times the RMSEC
value, it is deleted. A full description of this methodology
including the equations used can be found elsewhere
(Valderrama et al. 2007; Silva et al. 2012). Outliers in the
validation set were also detected by the jackknife (externally
studentized) residuals test, according to the appropriate reference (Souza and Junqueira 2005).

Food Anal. Methods

Net Analyte Signal

The NAS can be defined as the part of the analytic signal
that is uniquely related to the analyte/property of interest
and orthogonal to the space spanned by the interferences
(Lorber et al. 1997). The NAS vector, ^
xNAS
A;i , can be defined
for each sample i by the following equation:

 T  
T
NAS
b
b
b
b
1
xA;i
xA;i PNAS;k b
xA;i I  XA;k X
A;k
b A;k is the matrix containing
where I is the identity matrix, X
exclusively the information from the interferences (except the
analyte k), the superscripted plus sign indicates the Moore
Penrose pseudoinverse matrix, and PNAS,k is a matrix which
projects a given vector onto the NAS space. In some situa^ A;k , since the space
tions, it is not possible to estimate X
spanned by the interferences cannot be modeled (e.g., it is
not practical to obtain a cheese without fat). In these cases, an
alternative estimation of NAS is more useful, from the regression vector of a PLS model with A latent variables (LV), b:
 T 1 T
b
xNAS
b xi
2
A;i b b b
The norm of each vector provides a scalar NSi for each
sample, which is intuitively analogous to a selective univariate analyte signal. The NAS can be employed in multivariate calibration for estimating some FOM, such as selectivity
and limits of detection and quantitation.
Figures of Merit
The analytical validation was performed in order to harmonize univariate and multivariate concepts, searching for
adapting the requirements of the Brazilian (MAPA 2009)
and international guidelines (ICH 1995, 1997; FAO 1998;
Thompson et al. 2002; EC 2002) to the state of the art of
how to estimate FOM in multivariate calibration (Olivieri et
al. 2006; Valderrama et al. 2009). Thus, the following FOM
were estimated: trueness, precision, linearity, working
range, selectivity, sensitivity, analytical sensitivity, ruggedness, bias, and residual prediction deviation (RPD). The
limits of detection and quantitation could be calculated
based on NAS, but they were not estimated in this work
because they are not needed in calibration methods for
major food components at high concentrations, such as fat
and moisture in cheese.
Trueness and precision can be estimated in a similar way
for both univariate and multivariate methods. Trueness was
evaluated through the relative errors of prediction for each
sample. Nevertheless, the trueness of multivariate methods
is rather established through more specific parameters not
mentioned in the traditional guidelines of food analysis,

such as RMSEC and root mean square error of prediction

(RMSEP). RMSEP is more robust than RMSEC, since it is
estimated from external validation samples. Precision was
assessed at two levels, repeatability and intermediary precision.
Repeatability was obtained by the same analyst determining in
the same day three samples at different content levels, in six
replicates for each one. Intermediary precision was estimated
by three different analysts determining the same three samples
six times, in different days. Since there is no calibration curves
for multivariate models, the linearity used to be estimated
through the correlation coefficient (r) of a plot of reference
versus predicted values. But an r value close to 1 cannot be
solely considered appropriate to assure the linearity of the
method (Thompson et al. 2002). It is essential to verify previously the random behavior of the residuals of this fit. This is
often performed by visual inspection of a plot of the residuals,
which may sometimes be considered quite subjective. In order
to adopt more objective criteria to assess linearity, this work
adapted the methodology used by Souza and Junqueira (2005)
for the univariate ordinary least squares method. Thus, the
random behavior was verified by applying the tests of Ryan
Joiner (RJ), BrownForsythe (BF), and DurbinWatson (DW)
to the model residuals, in order to evaluate their normality,
homoscedasticity, and independency, respectively. The main
test is DW (Durbin and Watson 1951), used to check the
absence of autocorrelation, in which the standard deviation of
the ordered residual data (SD) is compared with the standard
deviation of the successive differences (SDsd) of the same data.
If the data are randomly and normally distributed and homoscedastic, there should be no statistical difference between SD
and SDsd. On the other hand, the parameters of the previously
mentioned pseudounivariate calibration curve could also be
used to express linearity. The working range of the method
can be established by confirming that the method provides
acceptable degrees of linearity, trueness, and precision.
For multivariate calibration methods, the selectivity (SEL)
is calculated as the ratio between the norm of each NAS
vector, jjNASi jj, and the norm of the respective spectrum,
jjxi jj. It should be stressed that this definition is only useful
within a certain group of samples of similar qualitative composition. As each sample has a different SEL, an average
value is used to characterize the method. Since multivariate
methods do not require signal resolution, it is also important to
stress that SEL is not useful for quality control purposes. It
only shows how much of the analytic signal is used in the
multivariate model. The sensitivity (SEN) of inverse methods,
such as PLS and PCR, can be calculated from:
SEN 1=kbk

Since SEN is dependent on the analytical technique

employed, a more comparable FOM is the analytical sensitivity
(), which is calculated as the ratio between SEN and the

Food Anal. Methods

instrumental noise (). In the case of diffuse reflectance measurements, can be estimated through the pooled standard deviation
of a certain number of replicate spectra of the empty cell.
g SEN="

The inverse of (1) provides an estimation of the minimum concentration difference that is distinguishable by the
method considering the random experimental noise as the only
source of error, regardless of the specific technique employed.
Ruggedness evaluates the susceptibility of an analytical
method to deliberate minor changes in experimental conditions.
In this work, it was assessed by varying two factors at two
levels, according to a 22 experimental design: the cheese sample pretreatment (only sliced or sliced, milled, and pressed) and
the type of cell used to obtain the reflectance measurements
(the plate from the spectrophotometer manufacturer or a simple
Petri dish). The bias is an FOM that evaluates the presence of
systematic errors in the model and is calculated only for the
validation set, as follows (ASTM 2012):
 ref

nv
X
yi  byi
bias
5
nv
i1
where yref
yiare the reference and predicted property values,
i andb
respectively, and nv is the number of samples in the validation
set. The standard deviation of validation errors (SDV) is also
estimated and used in a Students t test to determine if the
validation estimates present a statistically significant bias. The
calculated t value is then compared to the critical t value with
the appropriate number of degrees of freedom, which is equal
to nv.
s
P ref
2
yi  b
yi  bias
SDV
6
nv  1
Another FOM appropriate for assessing the predictive
ability of the calibration models is the RPD, also known as
relative predictive determinant (Williams 2001). The RPD is
the ratio of natural variation in the calibration or validation
samples to the size of probable errors occurring during the
prediction, and it is more useful for comparing models on
different data sets or in absolute terms. It is calculated for the
calibration and validation sets according to the Eqs. 7 and 8,
where RMSECV is the root mean square error of cross
validation and SDcal and SDval are the standard deviations
of the reference values for the calibration and validation
sets, respectively.
RPDcal SDcal =RMSECV

RPDval SDval =RMSEP

Post-Model Monitoring
Once the NIRS methods were established and validated,
they were implemented as a replacement for the traditional
ones in IMA for the routine quality control inspection of
mozzarella cheeses. During the year 2011, 53 cheese samples (about 10 % of the total number of mozzarella samples
analyzed) were randomly and equally temporarily spaced
analyzed for both the reference and NIRS methods, in order
to monitor the performance of the developed models. The
trueness of these determinations was evaluated according to
the Brazilian regulations (MAPA 2009). These results were
also plotted in control charts and their normal and independent distribution assessed through the RJ, BF, and DW tests.

Results and Discussion

PLS Model Development
The NIR spectra of the 123 analyzed samples of mozzarella
cheeses are shown in Fig. 1. By observing these spectra, it
was possible to attribute the most intense and broad bands
centered at 1,454 and 1,936 nm to vibrations of water,
corresponding to the first overtone of the OH stretch and
to the combination of the stretching and bending modes of
OH, respectively. Other observed peaks can be related to
the fat content of the cheeses and to the vibrations of the
lipid molecules, such as the band centered at 1,210 nm that
is attributed to the second overtone of CH stretch; the
peaks at 1,730 and 1,763 nm, related to the first overtone
of CH stretch; and the peaks at 2,308 and 2,348 nm,
attributed to the combination bands arising from the CH
stretch and deformation in a CH2 group (Lucas et al. 2008;
Pi et al. 2009).
The samples were split into 82 for the calibration set and
41 for the validation set by using the KennardStone algorithm, which assured the presence of the most representative
samples in the calibration set through a uniform scanning of
the spectral data (Kennard and Stone 1969). In Fig. 1, the
presence of nonlinear baseline deviations, typical of diffuse
reflectance measurements and caused by the multiplicative
light scattering, can also be noted. Thus, the data were
preprocessed by multiplicative scatter correction in order
to overcome this problem (Isaksson and Naes 1988), previously to the mean centering.
The number of LVs in each model was chosen by contiguous block (with nine splits) cross-validation, based on
the lower values of RMSECV. The best models were initially selected with four and seven LVs for moisture and fat,
respectively. Interval PLS models (with 10 intervals) were
also tried, but all the results were worse than the PLS
models using the whole spectra. The regression coefficients

Food Anal. Methods

Fig. 1 NIR spectra of 123
samples of mozzarella cheeses

of the PLS models for fat and moisture determinations are

shown in Fig. 2. By observing Fig. 2a, it was possible to
identify the spectral regions that most contributed to the fat
prediction. As it was expected, the highest coefficients were
related to second and first overtones of lipid CH stretch
(around 1,210, 1,730, and 1,763 nm). Nevertheless, it is
interesting to note that the most intense contribution came
from the region between 1,870 and 1,890 nm, which can be
attributed to the combination band of CO and OH
Fig. 2 Regression coefficients
of the PLS models for a fat and
b moisture determination

vibrations of carboxylic acids (Workman and Weyer

2008), which are present in mozzarella cheeses at lower
contents. In Fig. 2b, the regions that most contribute to the
moisture predictions can be indicated; the most positive
coefficients are between 1,850 and 1,910 nm, corresponding
to the combination band of OH of water. On the other
hand, the most negative coefficients were related to lipid
CH vibrations, since there is an inverse relation between
water and fat contents in cheeses. The peaks were observed

Food Anal. Methods

around 1,440 nm and between 1,690 and 1,780 nm, attributed to the first overtone combination of CH and to the first
overtone of CH stretch, respectively.

samples, used 6 LVs, and accounted for 98.2 % of the

variance in X and 94.1 % in Y.
Analytical Validation

Optimization of the Models Through the Detection

of Outliers
After the PLS models were constructed, they were optimized by using the procedure for the detection of outliers.
The outliers were detected at 99 % confidence level, and the
results were summarized in Table 1. The outliers can be
removed within a limit of 2 out of 9 (22.2 %) of the total
number of samples, as established by Brazilian (MAPA
2009) and international guidelines (Horwitz 1995; Souza
and Junqueira 2005). The optimization of the validation
set was only performed after finishing the optimization of
the calibration set. Besides, no more than three rounds of
outlier detection (four models) should be performed, in
order to avoid the snowballing effect, when repetitive
rounds continue to identify outliers (Valderrama et al.
2007). As can be seen from Table 1, for the fat model, eight
outliers were detected in the calibration set (corresponding
to 9.8 % of the samples) and four in the validation set (also
9.8 %). From these 12 outliers, 3 and 9 were detected based
on high leverage and high Y residuals, respectively. For the
moisture model, 9 outliers were identified in the calibration
set (11.0 %) and 6 in the validation set (14.6 %); from these
15 outliers, 3, 1, and 11 were detected based on high
leverage, high X residuals, and high Y residuals, respectively. Therefore, the optimized PLS model for fat determination was built with 74 calibration and 37 validation samples,
used 8 LVs, and accounted for 99.1 % of the variance in X
and 89.8 % in Y. The optimized model for moisture determination was built with 73 calibration and 35 validation

Table 1 Results for the optimization of the PLS models (in italics for
the final ones) through the detection of outliers
Model
Fat
Number of calibration samples
Number of validation samples
Number of LV
RMSEC (%)
RMSEP (%)
Moisture
Number of calibration samples
Number of validation samples
Number of LV
RMSEC (%)
RMSEP (%)

1st

2nd

3rd

4th

82
41
7
3.1
3.2

77
41
8
2.1
3.0

74
37
8
1.8
2.1

82
41
4
1.8
2.2

77
41
8
1.0
1.8

74
41
8
0.8
1.7

73
35
6
0.6
0.9

Table 2 summarizes the parameters estimated for evaluating

the FOM for the proposed methods. In the following subsections, they are specifically discussed.
Linearity
As previously discussed, the linearity was estimated through
the fit of the reference versus predicted values. The residuals
for the fat and moisture models are shown in Fig. 3. In most
chemometrics literature, these residuals used to be plotted as a
function of the reference values, which is not strictly corrected. The residuals should have been plotted as a function
of the predicted values, since they were not correlated with
them. If the data is ordered using the reference values, they
will always present some correlation, with r equals 1 minus
the r of the calibration curve (Draper and Smith 1998). As can
Table 2 Parameters for evaluating the main FOM of the developed
NIRS methods for the determination of fat and moisture in cheese
FOM

Parameter

Fat

Moisture

Value
Trueness
Precision

Relative errors
(max/min)
RSD
repeatabilitya
RSD intermediate
precisiona

Linearity

DW test
Slopeb
Interceptb
Correlation
coefficientb
Working range
Selectivity
Sensitivity
Analytical
sensitivity ()
1
Bias
RPD
RPD calibration
RPD validation
a

8.7/8.1 %

5.3/4.0 %

0.7 %
0.2 %
0.3 %
3.2 %
8.4 %
4.0 %
1.80
1.000.02
0.012.70
0.9442

0.5 %
0.4 %
0.4 %
1.4 %
2.6 %
1.2 %
1.91
1.000.03
0.001.40
0.9702

38.7 to 58.0 %
0.07
0.009c
1.8 %1

41.5 to 55.1 %
0.11
0.012c
2.4 %1

0.6 %
0.7751.942 %
2.1
2.0

0.4 %
0.0010.933 %
2.9
2.4

Results for three samples at three different content levels

Values for the line fitted to the calibration samples

Values expressed as the ratio between log (1/R) and percent

Food Anal. Methods

Fig. 3 Residuals for the
calibration (triangles) and
validation (circles) samples.
PLS models for a fat and b
moisture determination

be observed in Fig. 3, no systematic trend was apparent in the

residual distributions. Nevertheless, the random behavior of these
residuals was confirmed by appropriate statistical tests (Souza
and Junqueira 2005). At 95 % confidence level, the residuals of
the two models were considered normally distributed and homoscedastic according to the RJ and BF tests, respectively. In
addition, their independency was certified by the values of the
DW test (Table 2), which were within the limits of the acceptance
range, between 1.50 and 2.50. Once the linear models were
considered valid assumptions, the parameters of the fit of the
reference versus predicted values can be seen in Table 2.
Trueness, Precision, and Working Range
The trueness of the methods can be evaluated through the
RMSEP (absolute) values of 2.1 and 0.9 % for fat and
moisture, respectively (Table 1). It can also be evaluated
based on the observed relative errors, between 8.7 and
8.1 % for fat determination and between 5.5 and 4.0 %
for moisture determination. All of these values were in
accordance with the Brazilian guidelines (ICH 1997), which
established acceptance limits for relative errors between 20
and +10 %. The precision was evaluated through the relative
standard deviation (RSD). For both methods, these values

varied between 0.2 and 0.7 % at the level of repeatability and

between 1.2 and 8.4 % at the level of intermediary precision
(Table 2). All of these results were also in accordance with the
Brazilian guidelines (MAPA 2009), which prescribes a maximum RSD of 4 % for repeatability and 10 % for intermediate
precision, considering the level of the concentration of the
analytes (above 100 gkg1). The results of trueness and precision allow assuring that both methods were considered
accurate (EC 2002). Considering the linearity and accuracy
studies, the working ranges of the methods were established
from 38.7 to 58.0 % w/w for fat and from 41.5 to 55.1 % w/
w for moisture. It is important to realize that these ranges are
not necessarily identical to the useful ranges of the calibration.
While the calibration may cover a wide concentration range,
the remainder of the validation will cover a more restricted
range (Thompson et al. 2002).
Selectivity and Analytical Sensitivity
As previously discussed, the SEL values have no practical
interest for quality control. The obtained results just indicated
that 7 and 11 % of the analytic signal were used for predicting
fat and moisture, respectively. Since the SEN values are not
appropriate for comparison with other methods, the of the

Food Anal. Methods

Table 3 Results for the ruggedness evaluation, according to a 22 full
factorial design
Effect

Fat (%)

Moisture (%)

Plate
Pretreatment
Plate pretreatment

0.17
1.49
0.39

0.23
0.32
0.18

effect on the results at 95 % confidence level. As can

be seen in Table 3, all the estimated effects were within
the confidence intervals of 1.79 and 0.92 % for fat
and moisture, respectively, both calculated (Bruns et al.
2006) as the product of the error of the effect estimation
and the appropriate t value (2.31, with 8 degrees of
freedom and at 95 % confidence level).
Bias and RPD

methods (Table 2) was also calculated based on the estimated

of 0.005. The inverse of indicated that the methods were
able to discriminate minimum concentration differences of
0.6 % for fat and 0.4 % for moisture, considering the random
instrumental noise as the only source of errors.
Ruggedness
Two factors of practical interest were chosen for the
ruggedness evaluation. The objective was to know if it
would be possible to replace the plates from the instrumental manufacturer by less expensive Petri dishes for
the reflectance measurements. And also, if a simpler
sample pretreatment, without milling, would provide
the same results. A 22 full factorial design in triplicate
indicated that the two studied factors had no significant
Fig. 4 Control charts for
monitoring the stability of the
NIRS models: a fat and b
moisture. The sample number
on the x axis corresponds to a
homogeneous sampling from
January to December 2011

The bias was calculated based only on the validation

samples and its estimation for both models, as shown in
Table 2. From the values of bias and SDV, t00.005 was
estimated for the moisture model, which was below the
critical t of 2.026 with 37 degrees of freedom and at
95 % confidence level. For the fat model, t02.426 was
estimated, which can indicate the absence of a statistically significant bias only at 98 % confidence level
(tcrit 02.431). RPD values higher than 2.4 are considered
desirable for good calibration equations, whereas equations with RPD less than 1.5 are considered unusable
(Williams 2001; Patil et al. 2010). Thus, the RPD values for the moisture model were considered good,
whereas the RPD values for the fat model were considered satisfactory (Table 2).

Food Anal. Methods

Post-Model Monitoring
Once the methods were validated and adopted for the
quality control inspection, they were monitored in order
to assess their stabilities. The results are shown as
control charts in Fig. 4. The limits marked in these
control charts are based on the Brazilian guidelines
(MAPA 2009), which established for monitoring the
stability of quantitative methods that two thirds of the
samples should have relative errors within 15 % (alert
limits), while one third can present maximum allowable
errors of 33 % (action limits). Since only three samples (5.6 % of the total) for the fat model and one
sample (1.9 %) for the moisture model present relative
errors above 15 % and below 33 %, both models were
considered satisfactorily stable. In addition, the random
behavior of these errors was also analyzed by statistical
tests. The fat model (Fig. 4a) presented a DW value of
1.60, confirming the random distribution of their errors.
On the other hand, the moisture model (Fig. 4b) presented a DW value of 0.99, indicating the presence of
systematic trend. In spite of this, this model is still
being used since it is in accordance with the adopted
guidelines (MAPA 2009).

Conclusions
This paper developed and validated two robust multivariate diffuse reflectance NIRS methods for determining fat and moisture in cow mozzarella cheese. These
methods have been developed under real conditions of
routine analysis in a state laboratory of food quality
inspection control. They present several advantages over
the current methods, such as low cost, simplified procedure, no need for reagents, and no chemical waste
generation. Their main advantage is the rapidity of the
analyses, since only about 3 min is spent per determination. Thus, the NIRS method is about 30 times faster
than the Gerbers method for fat determination and 80
times faster than the gravimetric method for moisture
determination. Both methods have been validated in
accordance with Brazilian and international guidelines,
being considered linear, accurate, sensitive, and rugged.
In addition, once the methods were adopted, their performances were monitored for approximately 1 year
through control charts, demonstrating that they continue
to provide acceptable predictions. Finally, more than
these specific methods, we have aimed to present a
complete and robust multivariate methodology. This
methodology harmonizes the traditional univariate validation with the requirements of multivariate calibration,

which is essential for the implementation of new NIRS

methods in the routine analysis of food quality control.
Acknowledgments B.G.B thanks CAPES and CNPq for fellowships.

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