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Performance improvement and single laboratory

validation of classical qualitative methods for the
Cite this: DOI: 10.1039/c5ay01822f
detection of adulterants in milk: starch, chlorides
and sucrose
Carina de Souza Gondim, Roberto Cesar Santos de Souza, Marina de Paula Penna e
Palhares, Roberto Gonçalves Junqueira and Scheilla Vitorino Carvalho de Souza*

Received 13th July 2015
Accepted 5th October 2015
DOI: 10.1039/c5ay01822f
www.rsc.org/methods
Milk stands out among the foods that are subject to adulteration. Several studies have shown that milk
adulteration is a reality in many countries, despite the existing inspection routines. Furthermore, there is
an aggravating factor in that there are no reports in the literature concerning the validation of the
classical qualitative methods that are commonly used to detect fraud in milk and that are described in
the legislation. In the present study, a novel validation approach was applied on classical qualitative
methods related to the detection of the density restoratives starch, chlorides and sucrose in raw milk,
considering the official and modified versions. Rates, unreliability regions, detection limits, accordance,
concordance, selectivity and robustness were estimated. Although simple, the proposed modifications
significantly increased the efficiency of the methods, generating a lower frequency of errors or false
results and a reduction in the detection limits and uncertainty ranges, consequently enhancing the
capability of detecting adulterations.

Although they are published in the legislation of several

1. Introduction
The foods or ingredients that are most likely to be targets for
adulteration include those that are of a high value and undergo
a number of processing steps before reaching the consumer
market.1 This context highlights why milk and milk products
are so vulnerable to several types of adulterations.2 Milk can be
adulterated with water and then mixed with starch, salt or
sugar.3 These density restoratives are one class of substances
that are more frequently used in milk fraud.3,4
Recent studies reported adulterations in milk despite exist-
ing legislation and inspections by federal and state agencies,
and they still represent a challenge to the productive sector and
regulatory bodies.2,5–7
Most of the quantitative methods related to the quality
control of milk are normalized. However, the qualitative
methods for the detection of adulterations in milk are not
normalized.8–10 Chemometric strategies and infrared spectros-
copy have been jointly used to detect different adulterants in
milk.4,11–13 However, the classical qualitative tests are still widely
used to detect milk adulteration by official laboratories and
dairy industries.
Federal University of Minas Gerais (UFMG), Faculty of Pharmacy (FAFAR), Department
of Food Science, Av. Antônio Carlos, 6627, Campus da UFMG, Pampulha, 31270-010,
Belo Horizonte, MG, Brazil. E-mail: scheilla@bromatologiaufmg.com.br; Fax: +55-31-
3409-6908; Tel: +55-31-3409-6918
countries,8,9,14,15 validation studies of the qualitative methods
for detecting adulterants in milk are not reported in the litera-
ture. Some works involve a limited evaluation of classical
methods for the detection of density restoratives in milk.16
In several sectors of the food chain, the importance of
qualitative methods has increased. These methods have oen
been selected for screening and can be implemented in labo-
ratories of different sizes and personnel qualications because
they provide objective and rapid results, with low costs and
simplicity of use.17,18 Despite recent publications focusing on
the proposal for harmonized and detailed protocols, validation
of qualitative methods is still a critical issue for food analysis
laboratories.18–21
All the work required to maintain a framework for moni-
toring food quality should be based on the degree of reliability
of the analytical methods.22 Thus, in the dairy industry,
adequate control methods are required to evaluate the
authenticity of milk and milk products.11 The complexity, vari-
ability and instability of the milk matrix in association with
a lack of expertise in analyzing milk products are important
aspects that can highly affect the reliability of the analytical
control, even if consolidated methods are adopted.23
This paper presents the optimization and application of
a novel validation approach in the tness for purpose assess-
ment of classical qualitative methods related to the detection of
starch, chlorides and sucrose in raw milk by considering both

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Analytical Methods
versions as described in the legislation and with modications
for performance improvement.
2. Experimental
2.1 Samples
Raw cow milk samples (3 L) were obtained from the experimental
farm of the Ministry of Agriculture Livestock and Supply/Veterinary
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School at the Federal University of Minas Gerais State by milking
20 different cows that were managed under strictly controlled
livestock conditions. These samples were homogenized, packaged
in polypropylene gallons and kept under refrigeration (2 to 7
C)
until the beginning of the experiments, which were performed
within a maximum period of 48 hours. Each sample was analyzed
for its density, freezing point and titratable acidity, with the aim of
checking its consistency with regulated ranges.24
Sample collection was performed for the eight analytical
batches involved in the optimization and validation of each
method (starch, chlorides and sucrose) by considering both
versions (official and modied) (Fig. 1).
2.2 Chemicals and equipment
All reagents used were of analytical grade. Carboxymethylcel-
lulose, sodium chloride and starch were purchased from Vetec
Quı́mica Fina Ltd. (Rio de Janeiro, RJ, Brazil). Sucrose and
pectin were supplied by Synth (Diadema, SP, Brazil) and xan-
than gum and maltodextrin by Sigma-Aldrich Co. Ltd. (Gil-
lingham, Dorset, UK). Iodine, potassium iodide, silver nitrate,
potassium chromate, resorcinol and hydrochloric acid were
purchased from FMaia (São Paulo, SP, Brazil), Vetec Quı́mica
Fina Ltd. (Rio de Janeiro, RJ, Brazil) and Synth (Diadema, SP,
Brazil). Puried water was obtained using a Milli-Q Direct
system (Billerica, MA, USA).
The equipment was calibrated, including water bath (314-
8DN, Nova Ética), micropipets (LM 10000, HTL Lab Solutions;
Finnpipette F3, Thermo Scientic; and Transferpette S, Brand),
weighing machines (AUX 220, Shimadzu; and BK 300, Gehaka),
freezing point equipment (PZL 900, PZL), milk densimeter (5784,
Incotherm) and thermometers (7665.02.0.00, Incotherm).
2.3 Methods
The presence of starch in milk was determined aer heating the
milk and adding an iodine/potassium iodide solution (Lugol's
Fig. 1 Representation of the raw milk sampling procedure used for the optimization and validation of the methods for detecting starch, chlorides
and sucrose. Method 1.1: official method for starch detection; method 1.2: modified method for starch detection; method 2.1: official method for
chloride detection; method 2.2: modified method for chloride detection; method 3.1: official method for sucrose detection; and method 3.2:
modified method for sucrose detection.
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solution). The heating causes the opening of the helical struc-
ture of starch, allowing for the adsorption of iodine and the
development of a characteristic blue color aer cooling.9 The
identication of chlorides was based on a reaction with silver
nitrate in the presence of a potassium chromate indicator. The
development of the yellow color indicated a positive result or
the presence of chloride in amounts greater than the normal
range in milk.9 Sucrose was detected in milk through the
development of a deep-red color that resulted from the
condensation of ketoses with resorcinol in an acid medium.25
2.4 Experimental design
The optimization and validation approach, performed as
described by Gondim et al.,18 included eight analytical batches
for each method and their respective versions (official and
modied) (Fig. 1):
(i) Optimization;
(ii) Validation – preliminary tests;
(iii) Validation – study of rates, uncertainty or unreliability
regions (UR), detection limits (DL), accordance (ACO) and
concordance (CON) – run 1;
(iv) Validation – study of rates, UR, DL, ACO and CON – run 2;
(v) Validation – study of rates, UR, DL, ACO and CON – run 3;
(vi) Validation – study of selectivity in the presence of known
interferences;
(vii) Validation – study of robustness; and
(viii) Validation – tness for purpose assessment.
2.5 Optimization
The optimization steps were conducted to test changes in the
analytical procedures of the methods described in legislation
aiming to reduce false results and increase the sensitivity and
selectivity of the methods.
Raw milk samples were spiked with starch levels between
0.01 and 1.2 g L1, between 0.92 and 2.27 g L1 for chlorides
(considering the native concentrations determined9); and
between 0.3 and 3.0 g L1 for sucrose, plus the blanks (unspiked
samples).
2.6 Validation
2.6.1 Preliminary tests. Spiked and unspiked samples were
prepared and analyzed randomly in a single analytical batch,
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with 10 replicates per level, blind to the analysts. These exper-
iments were started with the same concentration ranges
described in Section 2.5. When necessary, new ranges were
tested to obtain positive results between 0 and 100%.18
2.6.2 Rates, unreliability regions, detection limits, accor-
dance and concordance. Raw milk samples were spiked with
the analytes at different concentrations, with 30 independent
replicates per level, plus the unspiked samples. The samples
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were prepared and analyzed randomly in three different
analytical batches, which involved three different times and
analysts, with 10 replicates per level plus the unspiked samples,
which was blind to the analysts. This experiment covered the
conditions for repeatability and intermediate precision.18
Starch was added at 21 levels between 0.01 and 1.20 g L1,
sodium chloride was added to produce 15 levels between 0.92
and 2.24 g L1 chlorides, and sucrose was added at 12
concentrations between 0.3 and 3.6 g L1.
The rates were calculated by using contingency tables. The
UR limits were estimated by non-linear regression, based on the
probabilities of 0.05 and 0.95 of having positive results. The DL
was stated as the upper limit of the UR. For the ACC and CON
values, calculated by equations based in combinatorial
concepts, the acceptability criterion was stated as a minimum of
0.8, which was outside the UR, given that this value corre-
sponded to the possibility of obtaining a single false negative
result in each analytical batch.18,26
2.6.3 Selectivity – interferents. Unspiked and spiked
samples at levels corresponding to the lower concentrations at
which 100% reliability rates (RLRs) were obtained in the
previous step of the validation process were used for the selec-
tivity evaluation, with 10 independent replicates per level. These
concentrations were set as 0.3 and 1.0 g L1 of starch; 1.51 and
1.87 g L1 of chlorides; and 2.4 and 3.6 g L1 sucrose, respec-
tively, for the modied and official methods. Samples were
analyzed randomly in a single analytical batch, which was blind
to the analyst.18
Carboxymethylcellulose, xanthan gum, maltodextrin and
pectin were investigated as potential interferents. These
carbohydrates were spiked at a 0.12 g L1 level, which was the
highest concentration at which the raw milk sample showed no
visually noticeable change. Starch, chlorides and sucrose were
also tested as potential interferents in other qualitative
methods, and set at 1.0, 1.87 and 3.6 g L1, respectively, to
correspond to the lowest level with a 100% RLR for the official
methods.
The studied compound was designated as an interferent if
the RLR was less than 90%, corresponding to the possibility of
obtaining a single false negative result.18
2.6.4 Robustness. Full factorial experiments were per-
formed. For each experiment, the analyte in question was added
to raw milk samples at concentrations corresponding to the
lowest levels in which RLRs of 100% were obtained for each
method, with 10 independent replicates per treatment. The
samples were analyzed randomly in a single analytical batch,
and they were blind to the analyst.18
For the starch detection method, the experiment involved
three factors and two levels as follows: the iodine (A and B) and
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potassium iodide (C and D) brands used in the preparation of
Lugol's solution, and the heating time of the sample (8 and 12
min), for a total of eight treatments.
Two factors and two levels were considered for the chloride
and sucrose methods, for a total of four treatments. For the
chloride method, potassium chromate (A and B) and silver
nitrate (A and B) brands were used, and for the sucrose method,
resorcinol brands (A and B) and the heating times for the
samples (3 and 7 min) were used.
The factors and levels were considered to be critical for
method performance when the RLR was less than 90%, corre-
sponding to the possibility of obtaining a single false negative
result.18
2.6.5 Assessment of tness for purpose. Milk samples were
adulterated with 150 g L1 water.7 These samples were spiked
with each analyte at 3 concentrations, in 3 replicates, including
the following: unspiked samples, samples spiked at the DL of
the modied methods, and samples spiked at the DL of the
official methods. The density was measured for each replicate
and compared with the permitted range as described in the
legislation, or 1028 to 1034 g L1.24
The tness for purpose of the method was considered
satisfactory if the starch, sodium chloride and sucrose
concentrations necessary to restore the density of the samples
that had been adulterated with water were equal to or greater
than the DL of the methods.
3. Results and discussion
3.1 Starch
3.1.1 Optimization. Considering the occurrence of false
negative results, a new criterion for positive results was
proposed (Fig. 2a.1). A greyish-green color was observed for
positive samples spiked at lower levels. This color was different
from the blue color observed for samples spiked at the upper
levels and from the yellow color formed for unspiked samples
(Fig. 3a). Then, the criterion suggested was the observation of
a color different from that obtained for a non-blind unspiked
sample, as shown in Fig. 2a.2.
3.1.2 Validation
3.1.2.1 Rates, unreliability regions, detection limits, accor-
dance and concordance. The preliminary tests indicated the
range of starch concentrations between 0.01 and 1.2 g L1 for
the subsequent steps of the validation study, i.e., provided 0%
and 100% of the positive results for both methods.
For both studied criteria, the analysis of unspiked samples
resulted in a 100% selectivity rate (SLR) and a 0% false positive
rate (FPR), indicating that the matrix components did not
interfere in the SLR. Considering the official method as well as
the modied method, a trend of larger percentages of false
results was evidenced by the lower concentrations. For the
official method, 100% false negative rates (FNRs) were ob-
tained at levels from 0.01 to 0.2 g L1, and no false negative
results were observed for levels corresponding to starch at 1.0,
1.1 and 1.2 g L1. For the modied method, 100% FNRs were
achieved only at the 0.01 g L1 starch level, and false negative
results were not observed from the 0.3 g L1 level (Table 1).
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Fig. 2 Flowchart of the qualitative analytical methods for starch (a), chlorides (b) and sucrose (c) detection with 1 being the official method and 2
being the modified method (modified step marked in gray).

These ndings demonstrated that the proposed modication The performance curves are shown in Fig. 4a.1 and a.2. In
reduced the false negative errors, enhancing the sensitivity of considering the blue color observation criterion, the UR was
the method. estimated to be between 0.2 and 0.8 g L1 starch. A lower UR

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Fig. 3 Colors observed in the optimization of the method for detecting starch, chlorides and sucrose in milk. (a) 1 – milk sample without addition
of Lugol's solution: observation of a white color (color index: 255r 255g 255b); 2 – the unspiked sample: observation of a yellow color (color
index: 255r 246g 133b) with the Lugol's solution; 3 – a spiked sample at 0.3 g L1: observations of a greyish-green color (color index: 208r 197g
151b or 230r 217g 165b) different from the blue color and from that obtained for the unspiked samples; and 4 – a spiked sample at 1.2 g L1:
observation of a blue color (color index: 107r 120g 164b) as established in the official method. (b) 1 – unspiked sample with a native chloride
concentration of 0.9 g L1: observation of brick-red color (color index: 200r 80g 60b); 2 and 3 – spiked samples at 1.0 and 1.1 g L1 chlorides:
observation of intermediate colors (color indexes: 250r 168g 95b and 254r 207g 140b); 4 and 5 – spiked samples at 1.2 and 1.5 g L1 of chlorides:
observation of yellow color (color index: 255r 246g 133b). (c.1) 1 – unspiked sample: observation of white color (color index: 255r 255g 255b); 2, 3
and 4 – spiked samples at 0.3 1.2 and 2.1 g L1 sucrose: observation of intermediate colors (color indexes: 225r 163g 128b, 201r 116g 105b and
245r 132g 102b); and 5 – spiked samples at 3.0 g L1 of sucrose: observation of pinkish color (color index: and 220r 86g 66b). (c.2) 1 – unspiked
sample: observation of a colorless liquid; 2 and 3 – spiked samples at 0.3 and 1.2 g L1 sucrose: observation of intermediate colors (color indexes:
142r 107g 75b and 117r 73g 48b); and 4 and 5 – spiked samples at 2.1 and 3.0 g L1 sucrose: observation of deep-red color (color index: 95r 46g
32b and 100r 35g 17b).

was achieved for the comparison criterion with the unspiked
sample, ranging from 0.02 to 0.2 g L1. In addition to having
presented a signicant reduction in the DL value (approxi-
mately four times), the narrower UR demonstrated that the
proposed modication, although simple, also contributed to an
improvement in the uncertainty.
Generally, adequate precision was observed for both
methods because the ACC and CON values outside the UR were
higher than 0.8, except for one value of ACC at the 0.9 g L1
level, when using the official method. For the unspiked
samples, these parameters were estimated as 1 (Table 1).
Silva16 studied the starch detection method in pasteurized
milk with 27 assays. This author observed 100% negative results
for unspiked samples analyzed in three replicates. Positive
results of 100% were achieved when this sample was split, and
the samples were spiked at eight levels between 0.1 and 25 g L1
and analyzed in three replicates. In the present study, no posi-
tive results were obtained for the 0.1 g L1 level when the official
method was applied. When the modied method was consid-
ered, a 53.3% rate of positive results was achieved. However, it is
important to highlight the importance of the representative-
ness, number of samples and replicates in studies involving
qualitative methods; and considered in the present study.18,27,28
The qualitative method for starch detection in milk
proposed by India-Ministry of Health and Family Welfare of
India,14 similar to the Brazilian official method, is described at
a DL of 1 g L1 that is consistent with the 0.8 g L1 estimated in
the present study, considering the official procedure. It is
important to note that the 2005 valid version of the Indian
manual has no data about the DL.29
Infrared spectroscopy techniques associated with chemo-
metric strategies were used to discriminate between milk
samples that were adulterated with starch when the concen-
tration of the adulterating solutions equalled or exceeded 1.5
and 5 g L1.12,13 These new methodologies could detect adul-
terated samples at levels signicantly higher than the DL esti-
mated in this study for both classical methods.
3.1.2.2 Selectivity – interferents. The methods were regarded
as selective for the potential interferents carboxymethylcellu-
lose, xanthan gum, pectin, sodium chloride and sucrose, having
obtained 100% of negative and positive results for samples
without and those spiked with starch, respectively. A lack of
selectivity was observed for maltodextrin. This could be because
the starch had not been fully hydrolyzed during maltodextrin
production30–32 or because of the capability of these methods to
detect the products of incomplete starch hydrolysis. This
second alternative could be investigated because it would allow
an expansion of the scope of these methods for the detection of
starch and its derivatives.
3.1.2.3 Robustness. Robustness was demonstrated for the
reagent brands and heating time for both methods because
100% positive results were obtained for each treatment, i.e.,
there was no change in the RLRs.
3.2 Chloride
3.2.1 Optimization. When the chloride content in a milk
sample is considered normal, part of the silver nitrate reacts
with chloride to form silver chloride, and the excess silver
nitrate reacts with potassium chromate, forming silver chro-
mate with a brick-red color. When the chloride content is high,
the consumption of silver nitrate will be greater. The quantity of
silver chromate and the intensity of the brick-red color will
consequently be low, and the yellow color of potassium chro-
mate will predominate.10
In the rst analyses employing the official method, a brick-
red color was observed for the blank samples with the addition
of silver nitrate. However, this color faded depending on the
agitation to which the sample was subjected, which led to false-
positive results.
Silver chromate has a solubility product constant (Ksp) lower
than that of silver chloride (1.2  1012 and 1.8  1010 mol
L1, respectively), but its solubility is approximately ve times
greater than that of silver chloride (6.5  105 and 1.3  105

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Table 1 False negative rate (FNR), sensitivity rate (SNR), false positive rate (FPR), selectivity rate (SLR), accordance (ACC) and concordance (CON)
obtained for unspiked and spiked samples with starch, sodium chloride and sucrose, for both official and modified methods

Official method Modied method

Density ACC (analytical batch) ACC (analytical batch)

restorative FNR or SNR or FNR or SNR or
(g L1) FPRa SLRa 1 2 3 CON FPRa SLRa 1 2 3 CON
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Starch
0.0a 0.0 100.0 1.0 1.0 1.0 1.0 0.0 100.0 1.0 1.0 1.0 1.0
0.01 100.0 0.0 1.0 1.0 1.0 1.0 100.0 0.0 1.0 1.0 1.0 1.0
0.02 100.0 0.0 1.0 1.0 1.0 1.0 93.3 6.7 0.6 1.0 1.0 0.9
0.03 100.0 0.0 1.0 1.0 1.0 1.0 93.3 6.7 0.6 1.0 1.0 0.9
0.04 100.0 0.0 1.0 1.0 1.0 1.0 96.7 3.3 0.8 1.0 1.0 0.9
0.05 100.0 0.0 1.0 1.0 1.0 1.0 83.3 16.7 0.5 1.0 0.8 0.7
0.06 100.0 0.0 1.0 1.0 1.0 1.0 90.0 10.0 0.6 0.8 1.0 0.8
0.07 100.0 0.0 1.0 1.0 1.0 1.0 73.3 26.7 0.4 0.6 0.8 0.6
0.08 100.0 0.0 1.0 1.0 1.0 1.0 60.0 40.0 0.5 0.5 1.0 0.4
0.09 100.0 0.0 1.0 1.0 1.0 1.0 53.3 46.7 0.6 0.5 1.0 0.4
0.1 100.0 0.0 1.0 1.0 1.0 1.0 33.3 66.7 1.0 1.0 1.0 0.3
0.2 100.0 0.0 1.0 1.0 1.0 1.0 20.0 80.0 1.0 1.0 0.5 0.6
0.3 86.7 13.3 1.0 0.6 0.6 0.8 0.0 100.0 1.0 1.0 1.0 1.0
0.4 60.0 40.0 1.0 0.5 0.8 0.4 0.0 100.0 1.0 1.0 1.0 1.0
0.5 36.7 63.3 1.0 0.8 1.0 0.3 0.0 100.0 1.0 1.0 1.0 1.0
0.6 33.3 66.7 1.0 1.0 1.0 0.3 0.0 100.0 1.0 1.0 1.0 1.0
0.7 23.3 76.7 0.5 1.0 1.0 0.5 0.0 100.0 1.0 1.0 1.0 1.0
0.8 6.7 93.3 0.6 1.0 1.0 0.9 0.0 100.0 1.0 1.0 1.0 1.0
0.9 6.7 93.3 0.6 1.0 1.0 0.9 0.0 100.0 1.0 1.0 1.0 1.0
1.0 0.0 100.0 1.0 1.0 1.0 1.0 0.0 100.0 1.0 1.0 1.0 1.0
1.1 0.0 100.0 1.0 1.0 1.0 1.0 0.0 100.0 1.0 1.0 1.0 1.0
1.2 0.0 100.0 1.0 1.0 1.0 1.0 0.0 100.0 1.0 1.0 1.0 1.0

Chloride
0.90 0.0 100.0 1.0 1.0 1.0 1.0 0.0 100.0 1.0 1.0 1.0 1.0
0.92 100.0 0.0 1.0 1.0 1.0 1.0 100.0 0.0 1.0 1.0 1.0 1.0
0.93 100.0 0.0 1.0 1.0 1.0 1.0 80.0 20.0 0.8 1.0 0.8 0.9
0.95 100.0 0.0 1.0 1.0 1.0 1.0 100.0 0.0 1.0 1.0 1.0 1.0
0.96 100.0 0.0 1.0 1.0 1.0 1.0 60.0 40.0 0.6 0.8 0.8 0.8
1.02 100.0 0.0 1.0 1.0 1.0 1.0 50.0 50.0 0.4 0.5 0.4 0.5
1.14 90.0 10.0 0.5 1.0 1.0 0.8 50.0 50.0 0.5 0.4 0.5 0.5
1.26 70.0 30.0 0.4 0.6 0.6 0.6 20.0 80.0 0.8 0.6 0.5 0.7
1.39 60.0 40.0 0.5 0.5 0.6 0.5 10.0 90.0 0.8 1.0 0.5 0.5
1.51 10.0 90.0 1.0 0.6 0.8 0.8 0.0 100.0 1.0 1.0 1.0 1.0
1.63 0.0 100.0 1.0 1.0 1.0 1.0 0.0 100.0 1.0 1.0 1.0 1.0
1.75 10.0 90.0 0.8 1.0 0.6 0.8 0.0 100.0 1.0 1.0 1.0 1.0
1.87 0.0 100.0 1.0 1.0 1.0 1.0 0.0 100.0 1.0 1.0 1.0 1.0
1.99 0.0 100.0 1.0 1.0 1.0 1.0 0.0 100.0 1.0 1.0 1.0 1.0
2.11 0.0 100.0 1.0 1.0 1.0 1.0 0.0 100.0 1.0 1.0 1.0 1.0
2.24 0.0 100.0 1.0 1.0 1.0 1.0 0.0 100.0 1.0 1.0 1.0 1.0

Sucrose
0.0a 6.7 93.3 1.0 1.0 0.6 0.9 0.0 100.0 1.0 1.0 1.0 1.0
0.3 93.3 6.7 1.0 0.8 0.8 0.9 100.0 0.0 1.0 1.0 1.0 1.0
0.6 80.0 20 1.0 0.5 0.6 0.6 90.0 10.0 1.0 1.0 1.0 1.0
0.9 53.3 46.7 0.5 0.5 0.5 0.5 66.7 33.3 1.0 0.6 0.8 0.8
1.2 50.0 50.0 0.5 0.5 0.4 0.5 30.0 70.0 0.5 0.5 0.5 0.6
1.5 23.3 76.7 0.6 1.0 0.4 0.6 6.7 93.3 0.4 0.8 0.5 0.6
1.8 36.7 63.3 0.5 0.5 0.4 0.5 10.0 90.0 0.8 1.0 0.8 0.9
2.1 30.0 70.0 0.5 0.8 0.5 0.6 6.7 96.7 0.8 0.8 0.8 0.8
2.4 23.3 76.7 0.8 0.6 0.5 0.6 0.0 100.0 1.0 0.8 1.0 0.9
2.7 16.7 83.3 1.0 0.8 0.5 0.7 0.0 100.0 1.0 1.0 1.0 1.0
3.0 10.0 90.0 1.0 1.0 0.5 0.8 3.3 96.7 1.0 1.0 1.0 1.0
3.3 3.3 96.7 1.0 1.0 0.8 0.9 0.0 100.0 0.8 1.0 1.0 0.9
3.6 0.0 100.0 1.0 1.0 1.0 1.0 0.0 100.0 1.0 1.0 1.0 1.0
a
FPR and SLR for unspiked samples, i.e., 0 g L1 density restorative; number of replicates for each level: 30; criterion for ACC and CON evaluation:
minimum of 0.8 outside the unreliability region (marked in bold).

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Fig. 4 Performance curves with their respective experimental data (A), unreliability regions and detection limits for starch (a), chlorides (b) and
sucrose (c) for 1 – the official method and 2 – the modified method.

mol L1, respectively33). Thus, by adding silver nitrate to an which were above the normal range. However, intermediate
aqueous medium with a low chloride content, the silver chro- colors were observed even for 1.0 g L1 to 1.1 g L1, which
mate can be solubilized aer manually shaking the test tube, were concentrations within and above the range stated as
making it difficult to visualize the characteristic color of this normal for milk, respectively. Considering these ndings, it
salt, and then giving false positive results. was not possible to suggest a new criterion for positive
A full factorial experiment was designed with two factors and samples (Fig. 3b.1 and b.2).
three levels for each one, including the following: the type of
movement (pendular, circular and inversion) and the number
of movements (10, 20 and 30), with triplicates for each treat- Table 2 Evaluation of different types and numbers of movements for
ment. The experimental design was set up with an analysis of the manual homogenization of test tubes, when considering unspiked
and spiked (2.27 g L1 of chlorides) samplesa
the blank samples and samples with 2.24 g L1 of chlorides and
the higher concentration of this analyte was chosen for the Unspiked Sample spiked at 2.27
optimization study. Homogenization with 30 pendular move- sample g L1 of chlorides
ments was considered the best condition, i.e., it did not lead to Type of Number of
movement movements R1 R2 R3 R1 R2 R3
false or inconclusive results (Table 2).
False negative results were also obtained for some spiked Circular 10 I I I P P P
samples, when an intermediate color was observed, different 20 I I I P P P
from both characteristic colors yellow and brick-red. Subse- 30 I P I P P P
quent studies were performed to dene the expected color for Pendular 10 N N N I I I
20 N N N P P I
samples containing chloride within and above the range 30 N N N P P P
considered normal for milk (0.8 to 1.0 g L1), in triplicate. The Inversion 10 P P P P P P
unspiked sample, with a native chloride concentration of 0.9 g 20 P P P P P P
L1, was spiked with sodium chloride solutions to obtain six 30 P P P P P P
nal concentrations between 1.0 and 1.5 g L1. a
R: replicate; N: negative result (brick red color characteristic of silver
The characteristic brick-red color appeared in the chromate – color index: 200r 80g 60b); I: inconclusive result (color
unspiked sample. The yellow color was observed for all index: 250r 168g 95b or 254r 207g 140b); P: positive result (yellow
color – color index: 255r 246g 133b).
samples containing chlorides between 1.2 and 1.5 g L1,

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Analytical Methods
In the official method, potassium chromate solution is
added, followed by silver nitrate solution and homogenization.
Thus, silver chromate precipitation can occur even for milk
samples containing high levels of chlorides, generating a brick-
red color, which can lead to false-negative results.
With the aim of improving the sensitivity of the method and
ensuring that all the chlorides present in the sample will
precipitate as silver chloride before the reaction between the
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chromate and the silver nitrate, an inversion in the solution
addition sequence was proposed, as described by Tronco.10
Furthermore, a manual homogenization step aer the addition
of the rst solution was suggested, considering the standard-
ized conditions (Fig. 2b.1 and b.2).
3.2.2 Validation
3.2.2.1 Rates, unreliability regions, detection limits, accor-
dance and concordance. The range of chlorides from 0.92 to 2.24
g L1 tested in the preliminary tests was used in the validation
process.
In the analysis of unspiked samples, SLR and RLR were null
for both methods. With respect to spiked samples, the FNR
ranged from 100 to 0%, resulting in sensitivity rates (SNRs)
between 0 and 100%, which showed higher FNRs for the lowest
concentrations, and also for both methods. However, FNRs of
0% were observed for the official method at levels between 1.87
and 2.24 g L1 chlorides, and for the modied method, the
same FNR value was found over a wider range (from 1.51 g L1).
These results indicated that the proposed modications
contributed to an increase in the sensitivity of the method by
the reduction of false negative errors (Table 1).
The prole of the performance curves showed that the
changes suggested have also led to an improvement in the
parameter uncertainty and DL (Fig. 4b.1 and b.2). Considering
the official method, the UR was estimated to be between 1.08
and 1.65 g L1, and for the modied method, the UR was
between 0.8 and 1.42 g L1 chlorides. The DLs were estimated to
be 1.65 and 1.42 g L1 for the official and modied methods,
respectively. These limits were near the 1.6 g L1 chlorides that
represented a theoretical limit based on the stoichiometry of
the reaction, considering the volume (4.5 mL) and the concen-
tration (0.1 mol L1) of the silver nitrate solution.
The standardization of both procedures for chloride detec-
tion was evidenced by the satisfactory values (greater than or
equal to 0.8) for ACC and CON at levels outside the UR, except at
1.75 g L1 for the official method.
Silva16 evaluated the sensitivity of the chloride detection
method in pasteurized milk samples spiked with commercial
common salt at nine concentrations between 0.1 and 1 g L1. For
each level, an analysis was performed in three replicates. The
results demonstrated that this test was capable of detecting
concentrations as low as 0.27 g L1 salt (0.16 g L1 chlorides).
Positive results were observed for 1.14 g L1 chlorides, when
considering the official method. In the Silva16 study, the native
chloride concentration was not considered. As mentioned previ-
ously, this work did not account for the importance of the repre-
sentativeness and number of replicates in qualitative methods.
The chloride detection method described by India-Ministry
of Health and Family Welfare of India14 was similar to that of
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Paper
the official Brazilian method. A DL of 0.2 g L1 sodium chloride
(0.12 g L1 chlorides), which was lower than the amount esti-
mated in the present study, was reported. However, the exper-
iments or criteria adopted for the estimation of the limit were
not described.
3.2.2.2 Selectivity – interferents. The selectivity was observed
for carboxymethylcellulose, xanthan gum, maltodextrin, pectin,
starch and sucrose for both chloride detection methods with
RLRs of 100%, considering the unspiked and spiked samples
with sodium chloride in the presence of these compounds.
3.2.2.3 Robustness. In a robustness assessment of the offi-
cial method, brand B of silver nitrate was indicated as a factor
that affected the performance of the method because RLRs of
80% were obtained with the experiments involving this brand.
When considering the experiments performed with brand A,
RLRs $ 90% were observed. For the modied method, the
robustness was demonstrated for all factors and levels studied
when RLRs $ 90% were obtained in all treatments.
3.3 Sucrose
3.3.1 Optimization. The results obtained for the unspiked
samples indicated a lack of selectivity for this method, with an
FPR of 10%. FNRs between 100 and 50% were also observed in
all studied levels.
When heated with acid, the poly- and oligosaccharides are
hydrolyzed to simpler sugars and dehydrated to furfural and
derivatives. Ketoses are more rapidly dehydrated. Dehydrated
ketose reacts with resorcinol producing a deep red color.
Aldoses may react and produce a faint pink color. The method
described in the Brazilian legislation (Fig. 2c.1) therefore
indicated the wrong principle and criterion for positive
results, i.e., the reaction of the resorcinol with aldoses and the
formation of a pinkish color, respectively. These misconcep-
tions could explain the occurrence of false positive results
when the described criteria were applied. The observed
pinkish color was likely formed from the reaction of aldoses
with resorcinol, which was derived from the hydrolysis of
lactose.
The procedure described by India-Ministry of Health and
Family Welfare of India29 was examined and modications
described in Fig. 2c.2 were proposed based on this method. It is
important to consider that the nal resorcinol concentration
was increased in the modied method, with the aim of
improving the sensitivity.
In Fig. 3c.1 and c.2, the colors observed for milk samples
containing sucrose concentrations from 0.0 to 3.6 g L1 for both
methods are presented. In the official method, there is a slight
increase in the intensity of the pink color with the increasing
concentration of sucrose. This nding occurs because the color
visualization is hampered by milk coagulation as a result of the
addition of concentrated hydrochloric acid to the samples. In
the modied method, the results can vary for unspiked samples
between a colorless liquid and an intermediate slightly
yellowish color, which does not characterize a positive result.
The orange to deep-red color results could be observed in
samples containing approximately 1.2 to 3.6 g L1 sucrose.
This journal is © The Royal Society of Chemistry 2015

Paper
Then, the observation of a color from orange to deep-red was
suggested for reporting positive results, as shown in Fig. 2c.2.
3.3.2 Validation
3.3.2.1 Rates, unreliability regions, detection limits, accor-
dance and concordance. In the preliminary tests, the sucrose
concentrations between 0.3 and 3.6 g L1 provided 0% and
100% positive results for both methodologies and were selected
for the next step of the validation process.
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In the analysis of blank samples, SLRs of 93.3 and 100% and
FPRs of 6.7 and 0% were obtained for the official and modied
methods, respectively. For the official method, these results
could be attributed to the reaction of aldoses, resulting in
lactose hydrolysis, with resorcinol discussed above. In the
spiked samples, the percentage of positive results was enhanced
with the increasing concentration, for both methods. The FNR
and SNR ranged from 93.3 to 0% and 6.7 to 100% for the official
method, and from 100 to 0% and 0 to 100% for the modied
method (Table 1). The frequency of positive results was higher
for the modied method, indicating an improvement in the
sensitivity.
The URs were estimated between 0 and 3.2 g L1 and from
0.3 to 1.9 g L1 sucrose for the official and modied methods,
respectively (Fig. 4c.1 and c.2). The range of uncertainty was
reduced with the proposed modications because this param-
eter was estimated at a lower and narrower concentration range
for the modied method.
The DLs were established as 3.2 g L1 for the official
method and 1.9 g L1 for the modied method. The dra of
the Indian manual14 describes a method with a DL of 1 g L1.
Liu, Ren, Liu & Guo4 suggested a method that could accu-
rately detect sucrose in milk with a concentration as low as
0.5% (w/w), using a Fourier transform infrared spectroscopic
instrument.
With the exception of an ACC value obtained for unspiked
samples by the official method and despite the difficulty of
interpreting the results inherent in this method because of the
milk coagulation, the values presented in Table 1 for ACC and
CON indicated a satisfactory standardization of both methods,
in terms of repeatability and intermediate precision conditions,
respectively.
3.3.2.2 Selectivity – interferents. In both the official and
modied methods, the selectivity in the presence of the density
restoratives (carboxymethylcellulose, xanthan gum, maltodex-
trin, pectin, starch and sodium chloride) studied was observed,
with RLRs $ 90%.
3.3.2.3 Robustness. Robustness was demonstrated for the
official method for all the studied factors and levels (100% of
RLR). For the modied method, RLRs of 60% were obtained in
all treatments with 3 minutes of heating and the robustness of
this method was then assured when the heating time varied
from 5 to 8 minutes.
3.4 Complementary evaluation of tness for purpose
In considering the modied method, it was observed that aer
the fraud with 150 g L1 water, the addition of starch, sodium
chloride and sucrose at the respective DLs was not sufficient to
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Analytical Methods
restore the density. These results indicated that greater levels of
these adulterants would be necessary and detectable.
For the official method, the density was restored for sodium
chloride and sucrose when the samples contained density
restoratives at the DL, indicating no need for greater amounts of
these restoratives. However, the analytes were detected at these
levels.
These results indicated that the modied methods for
detecting starch, chlorides and sucrose and the official method
for starch detection were able to detect milk fraud at concen-
trations lower than the levels required for masking fraud by
adding 150 g L1 water.
For all adulterated samples no false results were obtained,
considering the official and modied versions, conrming their
applicability.
4. Conclusions
For the starch detection method, a new criterion for positive
results was suggested. The standardization of the manual
homogenization and an inversion in the sequence of reagent
addition were the proposed modications for the chloride
method. Considering the method used for sucrose detection,
a ltration step and the use of a resorcinol solution were
incorporated; moreover, a change was made in the criterion for
positive results.
A performance improvement was observed for the modied
methods proposed in the present study, specically with respect
to the reduction of false responses, uncertainty and detection
limit.
The main weak point of these methods is that they are based
on sensorial detection, depending on human senses to record
and interpret the response; so that the experience and training
of analysts may affect the results.
Despite the current trend of replacing the classical qualita-
tive methods with instrumental techniques, high performance
in detection of adulterants in milk was demonstrated, which
can signicantly impact on protecting the health and rights of
consumers.
Acknowledgements
The authors would like to acknowledge Otávio Augusto Mazzoni
Coelho, Ronália Leite Alvarenga and Linda Lys Calderaro
Lafayette for their assistance in the rst stages of the execution
of this project; the experimental farm of the Ministry of Agri-
culture Livestock and Supply (MAPA)/Veterinary School at the
Federal University of Minas Gerais State (EV/UFMG) for allow-
ing the use of their facilities and providing the raw cow milk
samples; the Ezequiel Dias Foundation (Funed) for their
support of the density and freezing point analyses; and the
Brazilian agency CAPES for nancial support.
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This journal is © The Royal Society of Chemistry 2015