Food Anal.
Methods
DOI 10.1007/s12161-016-0432-7
Interlaboratory Validation of Modified Classical Qualitative
Methods for Detection of Adulterants in Milk: Starch,
Chloride, and Sucrose
Carina de Souza Gondim 1 & Roberto Gonçalves Junqueira 1 &
Scheilla Vitorino Carvalho de Souza 1
Received: 2 November 2015 / Accepted: 1 February 2016
# Springer Science+Business Media New York 2016
Abstract Interlaboratory validation procedures were pro-
posed and performed to confirm the effectiveness of mod-
ified classical qualitative methods for the detection of adul-
terants in milk, including starch, chloride, and sucrose,
which were previously validated by a single laboratory
approach. Raw milk samples that were adulterated with
150 g L−1 of water and 0.0, 0.3, 0.8, and 1.2 g L−1 of
starch, 0, 1.5, 2.0, and 2.5 g L−1 of chlorides, and 0.0,
2.4, 3.0, and 3.6 g L−1 of sucrose were sent to 10 collabo-
rators in Brazil that represent the government, food con-
trol, food industry, and university affiliations. Reliability
rates of 93 to 100, 98 to 100, and 99 to 100 % were ob-
tained for the starch, chlorides, and sucrose methods, re-
spectively. The prediction intervals for the probability of
detection proved the sensitivity and selectivity of the
methods. Concordance values were greater than 0.85 to
starch, 0.98 to chlorides, and 0.99 to sucrose, indicating
precision and that the procedures were properly standard-
ized between the collaborators. The estimated detection
limits and unreliability regions confirmed the fitness of
the modified methods for their respective purposes.
Keywords Collaborative studies . Classical methods . Milk
adulteration . Thickeners . Qualitative methods
* Scheilla Vitorino Carvalho de Souza
scheilla@bromatologiaufmg.com.br
1
Faculty of Pharmacy (FAFAR), Department of Food Science, Federal
University of Minas Gerais (UFMG), Av. Antônio Carlos, 6627,
Campus da UFMG, Pampulha, 31270-010 Belo Horizonte,
MG, Brazil
Introduction
Due to the growing demand of milk and the price changes that
occur because of seasonal fluctuations, milk is often adulter-
ated to produce it in bulk quantities to meet the consumer
demand, to reduce the cost of the final product, and to increase
profit (Almeida et al. 2012; Rani et al. 2012). Starch, sodium
chloride, and sucrose have been illegally used as intentional
adulterants in milk to restore the density to established limits
and to mask another type of fraud—the addition of water
(Tronco 2010; Kartheek et al. 2011; Souza et al. 2011).
Different techniques and analytical procedures have been
reported for the detection of adulterants in milk (Kartheek
et al. 2011; Ellis et al. 2012). Sensorial techniques, in which
human senses are used to record and interpret the response
(Trullols et al. 2004), are represented by methods that involve
immunological responses (Renny et al. 2005) and chemical
reactions (India 2005; Brasil 2006; Tronco 2010; Souza et al.
2011). The instrumental techniques have included conven-
tional and alternative methods, such as potentiometric
(Veloso et al. 2002), chromatographic (Rani et al. 2012), spec-
troscopic (Almeida et al. 2012; Jawaid et al. 2014; Botelho
et al. 2015; Liu et al. 2015), amperometric (Silva et al. 2012),
conductometric, and spectrophotometric (Lima et al. 2004)
impedance sensors (Das et al. 2011) and digital image analysis
(Santos et al. 2012; Santos and Pereira-Filho 2013).
In this context, the advanced association of chemometric
methods with instrumental techniques can be highlighted for
food fraud detection (Almeida et al. 2012; Ellis et al. 2012;
Santos et al. 2012; Santos and Pereira-Filho 2013; Jawaid
et al. 2014; Botelho et al. 2015; Scampicchio et al. 2015).
These methodologies have advantages as the ability to analyze
samples with little or no preparation, ease of use, fast data
collection, and the capability to be used as a fingerprint tech-
nique (Karoui and De Baerdemaeker 2007; Santos and

Pereira-Filho 2013; López et al. 2014). However, performance
studies have shown low sensitivity and detection limits upper
to the classical methods (Botelho et al. 2015; Gondim et al.
2015; Liu et al. 2015).
To monitor the authenticity of milk, laboratories re-
quire economical, reliable, and reproducible techniques
(Rani et al. 2012). Qualitative methods have been ap-
plied for the analysis of milk and their products, and
these methods have been used by dairy industries to ver-
ify conformity to quality requirements, by authorities for
sanitary purposes and by researchers (Brasil 2011; Souza
et al. 2011).
Although recent reports have presented procedures for the
validation of these qualitative methods, a harmonized valida-
tion protocol is still required (Langton et al. 2002; Ellison and
Fearn 2005; Macarthur and Holst 2012; Gondim et al. 2014).
This is evidenced in studies involving qualitative methods for
detection of adulterants in milk. These studies are rare, are
limited regarding the parameters established in the procedures
mentioned above, and are restricted to in-house processes
(Silva 2013; Botelho et al. 2015).
Regardless of the method and the analytical technique, a
fully validated process, which includes in-house and collabo-
rative studies, is necessary both to assess or verify the quality
of the work done by a laboratory and to provide reliable data
which can support the industry and regulation agencies (Ginn
et al. 2006; López et al. 2015) and generate improved infor-
mation on milk fraud.
The methods reported here have been previously vali-
dated using a single laboratory process (Gondim et al.
2015), indicating their suitability for interlaboratory study.
In this study, these modified classical qualitative methods
for the detection of starch, sodium chloride, and sucrose
were validated using collaborative trials. Also, all aspects
of the organization of a collaborative study for the vali-
dation of qualitative methods are presented in detail, in-
cluding experimental design, statistics, and criteria for ho-
mogeneity and stability tests and method performance
assessment.
Material and Methods
An interlaboratory validation process consists in many
steps beyond the analysis of the test materials by collab-
orating laboratories. It covers the preparation of test ma-
terials, the evaluation of homogeneity and stability, the
distribution of test materials, besides the data analysis,
and, finally, the report of results (AOAC 2002; Langton
et al. 2002; Ellison and Fearn 2005; Thompson et al.
2006; Macarthur and Holst 2012). In Fig. 1, a scheme of
a minimum design for the validation of qualitative
methods by collaborative study is presented.
Food Anal. Methods
Fig. 1 Proposed scheme for the validation of qualitative methods by„
collaborative study: main steps, minimal design, acceptability criteria,
and estimated performance parameters
Test Materials
Samples
Three raw cow milk samples produced by a herd of 50
animals were taken from a refrigerated (4 to 7 °C) stor-
age tank on the experimental farm of Professor Hélio
Barbosa of the Veterinary School/Federal University of
Minas Gerais State (EV/UFMG). These samples of 15,
15, and 22 L were collected for the interlaboratory vali-
dation of the starch, chlorides, and sucrose methods, re-
spectively. The samples were kept refrigerated (4 to 7 °C)
in polypropylene gallons from transportation until the
time of preparation of the test materials, which occurred
immediately after the arrival of the samples to the
laboratory.
Preparation
Reliability is a property of qualitative methods related to
trueness and precision properties in quantitative analysis
and can be defined as the percentage of right results
obtained in independent tests performed to sample clas-
sification (Cardenas and Valcarcel 2005). Reliability rates
(RLRs) obtained for the single laboratory validation pro-
cess of the methods (Gondim et al. 2015) were used to
establish the concentration of the adulterants in the
samples.
Each sample was split into four batches that were spiked
with ultrapure water and a thickener aqueous solution to pro-
duce a target concentration of 150 g L−1 for the water and four
target concentrations for the restoratives which corresponded
to the upper and lower concentrations at which 100 % RLRs
were obtained in the previous validation process in addition to
the intermediate concentration and zero, as shown in Table 1.
For the starch method, an aliquot of 280 mL of the raw milk
sample was packaged into 50-mL conical polypropylene tubes
that were each filled with 20 mL using a macropipette. These
unadulterated samples were reserved as non-blind unspiked
samples for distribution to collaborators.
The spiking solutions were prepared with analytical grade
starch, sodium chloride (Vetec Química Fina Ltd., Rio de
Janeiro, RJ, Brazil) and sucrose (Synth, Diadema, SP,
Brazil) to final concentrations of 15, 100, and 140 g L−1, re-
spectively. Each batch was continuously mixed in a mechan-
ical stirrer (Fisatom 713 T) at 22.6×g during the test material
preparation. The milk was transferred using measuring cylin-
ders (1000 and 2000 mL) to a 10-L bowl and homogenized for
10 min. Ultrapure water was added to the milk using a
Food Anal. Methods
Step 1
Sampling and test materials
preparation
Step 2
Homogeneity tests
Step 3
Stability tests – isochronus desing
Step 4
Stability tests – classical desing
Step 5
Distribution of test materials
Step 6
Data analysis and reporting results
Fitness for purpose
Minimum design parameters:
Laboratory collaborators = 10;
Representative samples for the test materials formulation:
composed samples (for example for processed foods, samples
obtained from different brands and batches);
Test materials = 4, covering 4 concentration levels,
corresponding to the lower, an intermediate and the upper
levels at which 100 % of reliability rate were obtained in
previous single laboratory validation, plus the blank;
Replicates of blank - negative materials = 180 (10 for each
laboratory collaborator, 10 for homogeneity test, 30 for stability
tests and 40 for leftovers);
Replicates of positive materials = 110 to 250 (5 to 15 for each
laboratory collaborator, 10 for homogeneity test, 30 for stability
tests and 20 to 60 for leftovers).
Evaluation of the variation between-bottle of test materials:
Analysis in duplicate of 10 test materials replicates of each
concentration, randomly taken, by qualitative method previously
validated, under repeatability conditions.
Acceptability criterion = reliability rate ≥ 90 %.
Note: if a quantitative method is available, proceed as Thompson,
Ellison and Wood (2006). The within-bottle homogeneity will be
assured for the minimal intake of the analytical aliquot.
Stability of test materials stored at critical transportation
conditions:
Analysis in duplicate, by qualitative method previously validated,
under repeatability conditions, of 30 test materials replicates of
each concentration, randomly taken and kept at 3 conditions (10
at critical temperature during the minimal time for transportation ,
10 at critical temperature during the maximum time for
transportation and 10 under the ideal condition),
Acceptability criterion = reliability rate ≥ 90 %.
Note: if a quantitative method is available, proceed as
Thompson, Ellison and Wood (2006).
Stability of test materials during the time of the collaborative
trial:
Analysis in duplicate, by qualitative method previously validated,
under repeatability conditions, of 10 test materials replicates of
each concentration, randomly taken, on deadline for analysis,
Acceptability criterion = reliability rate ≥ 90 %.
Note: if a quantitative method is available, proceed as Thompson,
Ellison and Wood (2006).
For each laboratory:
Number of test materials = 10 negative test materials replicates
and from 5 to 15 positive test materials replicates of each
concentration;
Documents = analytical procedure, materials receipt and report
forms;
General instructions.
Data analysis:
Outliers analysis, assessment of false negative rates, false
positive rates, RLR, accordance, concordance, probabilities of
detection and detection limits.
Publication and distribution of the report results with a certificate
of participation, preserving the identity of the participants
concerning the results.
 Food Anal. Methods

Table 1 Preparation of the test

materials Thickener final Volume (mL) Number of Volume per
concentration tubes filled tube (mL)
(g L−1) Raw milk Water Thickener Total
solution

Starch
0.0 3570 630 0 4200 190 20
0.3 3570 546 84 4200 190 20
0.8 3570 406 224 4200 190 20
1.2 3570 294 336 4200 190 20
Chlorides
0.0 4675 825 0 5500 260 20
1.5 3570 567 63 4200 190 20
2.0 3570 546 84 4200 190 20
2.5 2550 375 75 3000 120 20
Sucrose
0.0 5355 945 0 6300 190 30
2.4 5355 837 108 6300 190 30
3.0 5355 810 135 6300 190 30
3.6 5355 783 162 6300 190 30

Thickener aqueous solution, starch 15 g L−1 , sodium chloride 100 g L−1 , and sucrose 140 g L−1 . Final concen-
tration of water 150 g L−1 . The chloride concentrations were estimated based on the native content of the raw milk
sample, which was determined in triplicate using the quantitative titrimetric method (Brasil 2011). The quantities
of milk, water, and the aqueous solutions of the restoratives were adjusted to obtain exact amounts for the
preparation of the test materials; however, this adjustment generated additional material that was not bottled

measuring cylinder (1000 mL), followed by homogenization Stability Tests

for 10 min. The aqueous spiking solutions of the thickeners
were incorporated and homogenized for 10 min.
Each adulterated batch was packaged into 50-mL conical
polypropylene tubes that were filled with 20 mL for starch and
chlorides and with 30 mL for sucrose, which were measured
using a micropipette. The tubes were closed with screw caps,
sealed with Parafilm® and labelled with code numbers that
were randomly generated. All of the test materials were refrig-
erated at 4 to 7 °C. The number of filled tubes was sufficient to
carry out the homogeneity and stability tests and to conduct
the collaborative trials, as well as leftovers, in case of need for
replacement materials.
Homogeneity Tests
The homogeneity was assessed for each batch of materials
using both qualitative and quantitative approaches under re-
peatability conditions. For each interlaboratory trial, 20 test
material replicates of each adulteration concentration were
randomly taken (Fig. 2). Ten of them were analyzed in dupli-
cate using the modified qualitative method that was previously
validated. An RLR greater or equal to 90 % confirmed the
homogeneity of each batch of materials. This criterion was
based on the possibility of obtaining a false result for each
adulteration level (Gondim et al. 2014).
The short-term stability was verified for each batch of mate-
rials using a qualitative approach and considering two designs.
First, we used an isochronous design in which failures in
maintaining the temperature conditions during the transport of
the test materials were simulated. Thirty replicates of the test
material were taken randomly from each adulteration level.
Ten were refrigerated at 4 to 7 °C, and 20 were incubated at
30 to 40 °C. After 5 h, 10 replicates were returned to the
refrigeration temperature. After 10 h, another 10 materials
were removed from the 30 to 40 °C temperature condition
(Fig. 2). All of the 30 materials were analyzed in duplicate
under repeatability conditions. The RLR values were estimat-
ed for the three treatments and compared, considering the
refrigeration condition as a reference. Second, we used a clas-
sical design in which the stability was investigated during the
time between the preparation and the deadline for the analysis
of the test materials in the collaborative trial. Ten replicates of
the test materials of each adulteration concentration that
were refrigerated at 4 to 7 °C were taken randomly on
the deadline (48 h after milking) for analysis by the collab-
orators (Fig. 2). These materials were analyzed in duplicate
under repeatability conditions. The RLR was estimated and
compared with that obtained in the homogeneity assess-
ment (zero time). The stability was considered when the
RLR was greater or equal to 90 %.
Food Anal. Methods
Fig. 2 Organization of interlaboratory trials, including homogeneity and stability tests, for validation of the modified classical qualitative methods for
the detection of adulterants in milk: starch, chlorides, and sucrose
Organization of Interlaboratory Studies
Ten laboratories that represented government (3), food control
(3), food industry (3), and university affiliations (1) took part
in the interlaboratory studies.
Test materials were taken randomly from each batch of ma-
terials and kept refrigerated during transportation to the collab-
orators. The materials were delivered within a period that en-
abled the analysis to be conducted within 48 h of milking.
For each trial, the collaborators received 40 blind and coded
test materials for a total of 400 results. For the starch method, 10
test material replicates of each adulteration concentration, 0.0,
0.3, 0.8, and 1.2 g L−1, were provided. For the chloride method,
we provided 10 replicates for the concentrations of 1.5 and
2.0 g L−1, 15 replicates of 2.5 g L−1, and 5 replicates of
0.0 g L−1. For the sucrose method, 10 replicates were provided
for each adulteration concentration, 0.0, 2.4, 3.0, and 3.6 g L−1
(Fig. 2).
In addition to the test materials, each laboratory received the
following: (i) a non-blind unspiked sample—one replicate of
an unadulterated sample as a reference for reporting the results
(only for the starch method); (ii) a copy of the method (analyt-
ical procedure); (iii) a report form for the analytical data (anal-
ysis date, laboratory identification, brand and lot of reagents,
manufacturer and model of equipment, calibration certificates
of the measuring instruments such as glassware used in the
preparation of solutions), results (positive or negative), obser-
vations (observed color or reference to index color code), com-
ments, and suggestions; and (iv) a material receipt form.
At the time of receiving, the following parameters were
evaluated: (i) temperature of the test materials; (ii) integrity,
sealing and labeling (including the code) of the tubes; (iii) the
presence of the unspiked sample (only for the starch method);
and (iv) the documents (report form and analytical procedure).
The collaborators were instructed to: (i) keep samples re-
frigerated until the time of analysis; (ii) conduct the analysis of
all replicates within the deadline, under repeatability condi-
tions (analyze the samples in a single analytical batch); (iii)
strictly follow the given analytical procedure; (iv) report to the
organizer any abnormality or loss of the test materials so that a
new shipment of materials could be sent to replace them to
adhere to the deadline; and (v) report the results to the orga-
nizer within 2 days after the analysis.
Statistical Analysis
The raw data received from the collaborators were evaluated
for gross errors of transcription or typing (Ellison and Fearn
2005). Once the principal aim of the study was to validate the
qualitative methods, the raw data were used to analyze possi-
ble discrepancies between the classes of laboratories repre-
sented in this study by a hierarchical cluster analysis (HCA)
with the criterion of Ward’s method (Ward 1963).
In qualitative method validation, some parameters, such as
trueness, precision, and measurement uncertainty, cannot be
applied as currently defined for quantitative measurement
methods (Ellison and Fearn 2005). Trueness can be defined
as a measure of how well a binary classification test correctly
identifies or excludes a condition represented by the propor-
tions of true and false positive results, i.e., the false positive
rate (FPR) and the false negative rate (FNR), and by RLR, as
previously defined (Ellison and Fearn 2005; NATA 2013;
Gondim et al. 2014).
The interlaboratory validation is considered as a complete
validation procedure because the two extreme conditions of
precision can be evaluated—repeatability and reproducibility
(Van der Voet et al. 1999). In the context of qualitative anal-
ysis, Langton et al. (2002) proposed the statistics accordance
(ACC) and concordance (CON) to assume the role of repeat-
ability and reproducibility, respectively, for qualitative analy-
sis. These two statistics indicate whether the particular

procedure used is sufficiently standardized (Langton et al.
2002; Ellison and Fearn 2005; Gondim et al. 2014).
Thus, the results reported by each laboratory were assessed
based on the characteristic parameters of the qualitative
methods: FPR, FNR, RLR, ACC, and CON. The rates that
were estimated were designated as satisfactory if the RLR was
greater than or equal to 90 %. The ACC and CON were de-
termined to be acceptable when the estimated values were
greater than or equal to 0.80 (for the materials that were dis-
tributed in at least 10 replicates) or 0.60 (for materials that
were distributed in 5 replicates). These criteria were based
on the possibility of obtaining a false result for each adultera-
tion concentration (Gondim et al. 2014).
Additionally, the results were used to estimate the detection
limits (DLs) and the prediction intervals for the probability of
detection when the methods were applied in a new laboratory
at each concentration, according to Gondim et al. (2014) and
Macarthur and Holst (2012), respectively.
Methods
Starch
The milk sample heating results in the opening of the starch
helical structure. Then, the iodine/potassium iodide solution
(Lugol’s solution) is adsorbed, producing a blue characteristic
color upon cooling (Brasil 2006).
Analytical Procedure Allow the test materials to warm to
room temperature. Measure 10 mL of test material and trans-
fer it to a test tube using a volumetric pipette. Heat in a boiling
water bath for 5 min. Cool under running water. Add two
drops of Lugol’s solution with a dropper and shake manually.
Follow the same procedure for the non-blind unspiked sam-
ple. Immediately, observe the formed color. Report a positive
result when the color of the test material is different from the
one obtained for the non-blind unspiked sample (grayish
green to blue) and a negative result when it is similar (yellow
color).
Note 1: If the readings of the test materials take more than
10 min after the addition of Lugol’s solution to the non-blind
unspiked sample, repeat the addition to the control sample.
Note 2: Index color references for reporting the following
results: (i) negative result, yellow color (color index 255r 246g
133b); or (ii) positive result, grayish green color (color index
208r 197g 151b or 230r 217g 165b) and blue color (color
index 107r 120g 164b) (Gondim et al. 2015).
Sodium Chloride
Principle The detection of sodium chloride was based on its
reaction with silver nitrate in the presence of potassium
Food Anal. Methods
chromate with a yellow color, indicating a positive result
(Brasil 2006).
Analytical Procedure Allow the test material to warm to
room temperature. Measure 10 mL and transfer it to a test tube
with a volumetric pipette. Add 4.5 mL of silver nitrate solution
(0.1 mol L−1) with a graduated pipette. Shake manually with
30 pendular moves. Add, with a graduated pipette, 0.5 mL of
potassium chromate solution (50 g L−1). Shake manually
again with 30 pendular moves. Report a positive result when
the color of the test material is yellow and a negative result
when the color is brick-red or intermediate.
Note 1: Index color references for reporting the results: (i)
negative result, brick-red color (color index 200r 80g 60b) and
intermediate colors (color indexes 250r 168g 95b and 254r
207g 140b); or (ii) positive result, yellow color (color index
255r 246g 133b) (Brasil 2006; Tronco 2010; Gondim et al.
2015).
Sucrose
Principle The detection of sucrose was indicated by the de-
velopment of an orange-to-deep-red color due to the conden-
sation of ketoses with resorcinol in an acid medium (India
2005; Nigam and Ayyagari 2007).
Analytical Procedure Allow the test material to warm to
room temperature. Measure 15 mL with a graduated pipette
and transfer it to a test tube. With a volumetric pipette, add
1 mL of concentrated hydrochloric acid. Allow to stand for
10 min. Filter through qualitative filter paper. Take 1 mL of the
filtrate and transfer to another test tube with a volumetric
pipette. Add 5 mL of resorcinol solution (10 g L−1 in hydro-
chloric acid solution—1:1.5, v/v) with a volumetric pipette.
Heat in a boiling water bath for 5 min. Report a positive result
when the color of the test material changes from orange to
deep-red and a negative result when an intermediate color or
a colorless solution is observed.
Note 1: Index color references for reporting the results:
(i) negative result, colorless liquid and intermediate color
(color index 142r 107g 75b); or (ii) positive result, inter-
mediate color (color index 117r 73g 48b) and deep-red
colors (color index 95r 46g 32b and 100r 35g 17b)
(Gondim et al. 2015).
Results and Discussion
Homogeneity and Stability Tests
The homogeneity was observed for all test materials that were
prepared for the validation of the starch, chlorides, and sucrose
methods. For the adulterated materials without the thickeners,
Food Anal. Methods
negative results of 100 % (or 100 % of RLR and 0 % of FPR)
were obtained. For the materials containing the thickeners,
positive results of 100 % (or 100 % of RLR and 0 % of
FNR) were achieved. According to Ellison and Fearn
(2005), the inhomogeneity is difficult to detect when a quali-
tative method is used. However, for positive samples, if the
lack of homogeneity does not significantly influence the re-
sults, the homogeneity can be assumed.
The short-term stability of the test materials was demon-
strated. For the isochronous design, the results indicated that
failures in maintaining the temperature conditions during the
transport did not affect the results. RLRs of 100 % were ob-
served for all test materials submitted to the three treatments
with refrigeration and incubation at 30 to 40 °C and for 5 and
10 h.
The same results were achieved for the classical design
because a 100 % RLR was estimated for all materials that
were analyzed within the deadline established for the analysis
by the collaborators (48 h after milking), as observed at the
moment of preparation of the materials (zero time).
Hierarchical Cluster Analysis
The results of HCA hierarchical clustering are presented in the
dendrogram (Fig. 3). In the first group observed, the four
segments included in the study (government, food control,
food industry, and university) were represented by seven lab-
oratories (3, 4, 5, 6, 8, 9, and 10). The second group has two
laboratories (2 and 7) representing the government and food
industry. Laboratory 1, industry representative, presented the
unequal performance in relation to the others. In general, HCA
showed that the affiliation of the laboratory did not influence
the results obtained by each laboratory and, consequently, the
process of validation of the qualitative methods.
4 the single laboratory (Gondim et al. 2015) and interlaboratory
8
6 the DL of 0.2 g L−1, which was calculated in the previous
10
9
5 As mentioned, interlaboratory validation studies were not
3
7 largely, the use of instrumental techniques as infrared spec-
2 troscopy (IR) methodologies. For these methods, the reported
1
0 10 20 30 40 50 60 70 80
Variance Weighted Distance Between Cluster Centers
Fig. 3 Hierarchical clustering analysis (HCA) dendrogram with the
distances between the laboratories
Interlaboratory Study Results
Starch
Considering each collaborator separately, for the adulterated
materials without the thickeners, the FPR ranged from 0 to
10 %, resulting in a RLR from 100 to 90 %. Three false
positive results were observed by laboratories 1, 2, and 7.
Similar results of the RLR were obtained for materials with
0.3 g L−1 of starch for laboratories 2 and 7, in which each
reported one negative result. These results implicated in
FNR and RLR of 10 and 90 %, respectively. However, for
laboratory 1, the estimated RLR was 40 %. A FNR and a RLR
of 0 and 100 %, respectively, were observed for the materials
containing 0.8 and 1.2 g L−1 of starch by all laboratories.
Considering all of the data, the RLR ranged from 93 to
100 % for the adulterated materials that were also spiked with
thickeners, and the RLR was 97 % for the adulterated material
without thickeners, indicating that the modified method ex-
hibits both sensitivity and selectivity, respectively (Table 2).
Satisfactory values of the ACC and CON were estimated
for all laboratories and concentrations of starch, indicating an
adequate standardization of the analytical procedure under the
repeatability and reproducibility conditions, respectively, ex-
cept for the ACC obtained by laboratory 1 for the 0.3 g L−1 of
starch (Table 3).
The estimates of the statistical parameters that were used to
describe the prediction intervals for the probability of detec-
tion are presented in Table 4 (Macarthur and Holst 2012). The
probability to detect starch in materials containing from 0.3 to
1.2 g L−1 ranged from 0.92 to 1.00, while the probability of
reporting false positive results was 0.03. These findings con-
firmed the sensitivity and selectivity of the method.
The limits of unreliability regions (URs) and the DLs esti-
mated via non-linear regression (Gondim et al. 2014) for both
processes are shown in Table 5. The DL estimated for starch in
the collaborative trial was 0.32 g L−1. This value was close to
single laboratory process. The prediction interval estimated
for the DL according to the Macarthur and Holst (2012) pro-
cedure included these limits (Fig. 4).
reported in the literature for these scopes. In-house validation
processes of qualitative methods for starch detection include,
DLs ranged between 1.5 and 5 g L−1 (Zang et al. 2014;
Botelho et al. 2015), lower than that obtained in both single
and interlaboratory validation processes of the modified meth-
od. Milk powder adulterated with starch was also analyzed by
IR, and a complete discrimination from samples adulterated
with 2 % of starch was reported (Capuano et al. 2015). In a
Table 2 False positive (FPR), false negative (FNR), and reliability (RLR) rates that were obtained in the interlaboratory validation of the modified classical qualitative methods for detection of starch,
chlorides, and sucrose in milk

Laboratory Starch (g L−1) Chlorides (g L−1) Sucrose (g L−1)

number
0.0 0.3 0.8 1.2 0.9 (native) 1.5 2.0 2.5 0.0 2.4 3.0 3.6

FPR RLR FNR RLR FNR RLR FNR RLR FPR RLR FNR RLR FNR RLR FNR RLR FPR RLR FNR RLR FNR RLR FNR RLR

1 10 90 60 40 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100
2 10 90 10 90 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100
3 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100
4 0 100 0 100 0 100 0 100 13 87 0 100 0 100 0 100 0 100 0 100 0 100 0 100
5 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100
6 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100
7 10 90 10 90 0 100 0 100 7 93 0 100 0 100 0 100 0 100 0 100 0 100 0 100
8 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 10 90 0 100 0 100
9 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100
10 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 100
Total 3 97 7 93 0 100 0 100 2 98 0 100 0 100 0 100 0 100 1 99 0 100 0 100
n 100 100 100 100 150 100 100 50 100 100 100 100

The values of the RLR highlighted in bold italics indicated results that were lower than 90 %, which were unsatisfactory
n total of test materials distributed among the collaborating laboratories
Food Anal. Methods
Food Anal. Methods

Table 3 Accordance (ACC) and

concordance (CON) values that Laboratory ACC
were obtained in the number
interlaboratory validation of the Starch (g L−1) Chlorides (g L−1) Sucrose (g L−1)
modified classical qualitative tests
for detection of starch, chlorides, 0.0 0.3 0.8 1.2 0.9 1.5 2.0 2.5 0.0 2.4 3.0 3.6
and sucrose in milk
1 0.80 0.47 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
2 0.80 0.80 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
3 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
4 1.00 1.00 1.00 1.00 0.75 1.00 1.00 1.00 1.00 1.00 1.00 1.00
5 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
6 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
7 0.80 0.80 1.00 1.00 0.87 1.00 1.00 1.00 1.00 1.00 1.00 1.00
8 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.80 1.00 1.00
9 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
10 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
CON 0.94 0.85 1.00 1.00 0.96 1.00 1.00 1.00 1.00 0.98 1.00 1.00

Values of ACC and CON highlighted in bold italics indicated results that were lower than 0.80 (for the materials
that were distributed in at least 10 replicates) or 0.60 (for the materials that were distributed in 5 replicates), which
were unsatisfactory

performance study of the official method for starch detection
in pasteurized milk carried out by Silva (2013), the detection
of starch was observed in 100 % of the samples adulterated
with concentrations between 0.1 and 25 g L−1 of starch, in
accordance with this interlaboratory study.
Table 4 Statistical parameters and lower (5 %) and upper (95 %)
estimated limits for the detection probability of the thickeners for the
modified classical qualitative methods for the detection of starch,
chlorides, and sucrose in milk
Thickener N X P sR Lower
(g L−1) limit
Generally, the results obtained in the interlaboratory vali-
dation process for the modified qualitative method for the
detection of starch in milk were influenced by the false results
reported by laboratory 1 for the concentration of 0.3 g L−1.
The intermediate color formed at this concentration was dif-
ferent from the yellow color observed for the non-blind
unspiked sample, and it was also different from the blue color
established in classical tests as the criterion for positive results,
which was only observed for samples adulterated with large
concentrations of starch. Thus, these results could have oc-
curred because the modified procedure (criterion) was not
Upper
limit understood by this specific collaborator.

Starch
Chlorides
0.0 100 3 0.03 0.005 0.022 0.038
0.3 100 92 0.92 0.019 0.887 0.950
The FPR ranged from 0 to 13 %, resulting in a RLR from 100
0.8 100 100 1.00 0.00 0.971 1.00
to 87 % for the data from each collaborator for the adulterated
1.2 100 100 1.00 0.00 0.971 1.00
Chlorides
0.0 150 3 0.03 0.007 0.007 0.046 Table 5 Limits of unreliability regions and detection limits (DL) that
were estimated for the modified classical qualitative methods for the
1.5 100 99 0.99 0.003 0.984 0.998 detection of starch, chlorides, and sucrose in milk via the single
2.0 100 100 1.00 0.00 0.971 1.00 laboratory and interlaboratory processes
2.5 50 50 1.00 0.00 0.942 1.00
Method Limits of unreliability regions (g L−1)
Sucrose
0.0 100 1 0.01 0.003 0.002 0.038 Single laboratory validationa Interlaboratorial validation
2.4 100 99 0.99 0.003 0.962 0.998
3.0 100 99 0.99 0.003 0.971 0.998 Lower Upper (or DL) Lower limit Upper (or DL)
3.6 100 100 1.00 0.00 0.971 1.00
Starch 0.02 0.20 0.03 0.32
N total number of analyses, X total number of positive results, p estimated Chlorides 0.80 1.42 1.00 1.62
mean probability of detection of the thickener, sR standard deviation of Sucrose 0.30 1.90 0.43 1.99
the estimates of the probability of detection from the individual laborato-
a
ries (Macarthur and Holst 2012) Gondim et al. 2015.
 Food Anal. Methods

1
with chlorides and it was 98 % for the material adulterated
0.9 with water that was not spiked with the thickeners (Table 2).
0.8 An ACC value of 0.75 was observed for laboratory 4, 0.87
0.7 for laboratory 7, and 1.00 for all of the other laboratories for
Probability

0.6 the adulterated test materials without the addition of sodium

0.5 chloride. Only the first disagreed with the established criteria.
0.4 However, this did not result in a CON value lower than 0.80.
0.3 For the other adulteration chloride concentrations, the estimat-
0.2 ed ACC and CON values were satisfactory. These results in-
0.1 dicated precision, i.e., that the validated procedure was suffi-
0 ciently standardized (Table 3).
0 0.2 0.4 0.6 0.8 1 1.2 The DL estimated by non-linear regression for this
Starch (g.L-1) interlaboratory trial was 1.38 g L−1 (Gondim et al. 2015),
1 similar to the calculated DL from the single laboratory process
0.9 (1.42 g L−1, Table 5). Considering the probability of detection
0.8 of 0.95 and the respective prediction interval (Macarthur and
0.7 Holst 2012), the DL was close to these values (Fig. 4).
Probability

0.6 The probability of reporting false positive results for the

0.5 modified method for the detection of chlorides was 0.03,
0.4 and the probability of detection was 0.99 to 1.00 for the test
0.3 materials containing 1.5 to 2.5 g L−1 of chlorides (Table 4),
0.2 proving the selectivity and sensitivity of the method.
0.1 Silva (2013) assessed the official method for chloride de-
0 tection in pasteurized milk and obtained 100 % of positive
0.9 1.4 1.9 2.4 results in samples added with commercial common salt at
Chlorides (g.L-1) concentrations from 0.27 to 1 g L−1. In this collaborative
1 study, upper concentrations were evaluated, although 100 %
0.9 of positive results were also observed.
0.8
0.7 Sucrose
Probability

0.6
0.5
0.4
0.3
0.2
0.1
0 from 2.4 to 3.6 g L−1 ranged from 0.99 to 1.00, while the
0 1.2 2.4 3.6
Sucrose (g.L-1)
Fig. 4 Estimates of lower and upper limits for the detection probability
and prediction interval for the limit of detection for the modified classical
qualitative methods for the detection of starch, chlorides, and sucrose.
Dashed lines, projection from the probability of detection 0.95 to give
lower (5 %) and upper (95 %) limits of a prediction interval for the limit of
detection (Macarthur and Holst 2012)
materials that did not contain sodium chloride, and these re-
sults indicated the selectivity of the method. Laboratories 4
and 7 reported 13 and 7 % false positive results, respectively.
The sensitivity was demonstrated by 0 % of FNR, i.e., an RLR
of 100 %, for all of the adulterated test materials that were
spiked with sodium chloride. Based on all of the data, the RLR
was 100 % for the adulterated materials that were also spiked
All of the estimated parameters for the sucrose method were
satisfactory. For laboratory 8, for the test materials that
contained 2.4 g L−1 of sucrose, the estimated FPR, RLR, and
ACC parameters were not the maximum values due to one false
result. The probability to detect sucrose in materials containing
probability of reporting false positive results was 0.01.
The DL calculated by the non-linear model in this
interlaboratory trial was similar to that reported in the previous
single laboratory study (1.90 and 1.99 g L−1, respectively),
indicating the agreement in the results (Table 5). A little higher
DL value and respective interval was achieved by the
Macarthur and Holst (2012) procedure (Fig. 4).
The method of detection of sucrose was evaluated during
the final round of the interlaboratory trial. Thus, the best re-
sults can be justified by the understanding of the modified
procedure that was adopted in this study and by an improved
familiarization by the collaborators of the process of the
interlaboratory validation.
All samples of pasteurized milk adulterated with sucrose in
concentrations from 0.45 to 9 g L−1 were detected as positive
in an evaluation of the official method of sucrose detection
Food Anal. Methods
performed by Silva (2013), which corroborates the results
observed in this interlaboratory validation. Studies of IR and
high-performance thin layer chromatography (HPTLC)
methods for, respectively, detection and quantification of su-
crose in milk were reported. For the qualitative method (IR),
samples were detected as positive in concentrations as low as
5 g L−1. The DL and quantification limit of the HPTLC meth-
od were calculated as 3.58 and 10 ng L−1 (Rani et al. 2012; Liu
et al. 2015), respectively. The performance of these method-
ologies was lower and upper than that observed for the mod-
ified method validated here. These results highlight that in-
strumental techniques do not always have better performance
than simple and low-cost qualitative techniques as used in this
work.
Collaborator’s Comments
Starch
Laboratory 9—The reading was performed immediately after
the addition of Lugol’s solution, and the total reading time of
the 20 samples was an average of 6 min. After reading the first
20 samples, we added Lugol’s solution again to the non-blind
unspiked sample to monitor the remaining test materials.
Conducting the tests in this way facilitated the analysis be-
cause the color that formed disappeared after some time.
Later, all samples that tested positive for starch showed a blue
precipitate, and some of them presented a bluish ring on the
surface.
Conclusions
This interlaboratory study provided a complementary and fi-
nal assessment of the fitness for purpose of the modified clas-
sical methods for detection of thickeners in milk. These
methods may be an important and reliable tool for researchers,
industries, and regulation agencies for the monitoring and
control of milk adulteration. The detailed and systematized
scheme for the organization of a collaborative study for vali-
dating qualitative methods was successfully applied.
Acknowledgments The authors acknowledge the experimental farm of
BProfessor Hélio Barbosa^ of the Veterinary School/Federal University of
Minas Gerais State (EV/UFMG) for allowing the use of their facilities and
animals during this project and for providing the raw cow milk samples.
We acknowledge Pedro Paulo Borges Santos for his analytical assistance
and the Brazilian agency BCoordenação de Aperfeiçoamento de Pessoal
de Nível Superior (CAPES)^ for financial support. The authors also ex-
press their appreciation to the collaborators for their participation in the
study: GMO Centro de Pesquisas e Controle de Qualidade Ltda.;
Hidrocepe Servicos de Qualidade Ltda. Epp; Ita Alimentos—Laticínios
Ita Indústria e Comércio de Alimentos Ltda.; Itambé Alimentos S.A.;
Laboratório de Análise Físico-Química em Alimentos do Laboratório
de Química Agropecuária do Instituto Mineiro de Agropecuária
(LAFQ/LQA/IMA); Laboratório de Química Bromatológica da
Fundação Ezequiel Dias (FUNED); Laboratório Nacional Agropecuário
de Minas Gerais do Ministério da Agricultura, Pecuária e Abastecimento
(LANAGRO-MG/MAPA); Laboratório de Bromatologia—Unidade de
Pesquisa Análise de Alimentos da Faculdade de Farmácia da
Universidade Federal de Minas Gerais (BRO-UPAA/FAFAR/UFMG);
NUGAP—Núcleo Global de Análise e Pesquisa Ltda.; and Trevo
Alimentos—Nogueira e Rezende Indústria de Laticínio Ltda.
Compliance with Ethical Standards
Conflict of Interest Carina de Souza Gondim declares that she has no
conflict of interest. Roberto Gonçalves Junqueira declares that he has no
conflict of interest. Scheilla Vitorino Carvalho de Souza declares that she
has no conflict of interest.
Ethical Approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Informed Consent Not applicable.
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