Accred Qual Assur (2016) 21:47–55
DOI 10.1007/s00769-015-1179-3
PRACTITIONER’S REPORT
Production and evaluation of a reference material for moisture,
ash, and total fat mass fractions, and titratable acidity in whole
milk powder
Renata Luana de Pádua Gandra1 • Ana Luiza Soares dos Santos1 • Pedro Paulo Borges dos
Santos1 • Ronália Leite Alvarenga1 • Roberto Gonçalves Junqueira1 •
Renata Adriana Labanca1 • Scheilla Vitorino Carvalho de Souza1
Received: 24 February 2015 / Accepted: 8 September 2015 / Published online: 29 September 2015
Ó Springer-Verlag Berlin Heidelberg 2015
Abstract Given the importance of reference materials
(RM) in the structuring and maintenance of quality man-
agement systems for food analysis laboratories, RM were
produced for the measurement of moisture, ash, total fat,
and titratable acidity in whole milk powder. Two types of
packaging and three storage temperatures were evaluated.
The RM were considered homogeneous. The materials that
were packaged in the pouches and those packaged in the
amber glass bottles with screw caps (stored at room tem-
perature) were not stable for moisture. Nevertheless,
considering the four measurands, the amber glass bottles
under refrigeration and freezing temperatures were the best
packaging and storage conditions, respectively. Homo-
geneity and stability studies were carried out according to
the ISO Guide 35, IUPAC harmonized protocol, and ISO
13528, considering the potential applicability of the pro-
duced materials as certified reference materials or even as
RM for use in proficiency testing schemes. For homo-
geneity, despite the different criteria, there was agreement
between the results. Considering the stability, the agree-
ment depended on the evaluation of the regression
assumptions, which is not required by the ISO Guide 35.
The contribution of uncertainty associated with homo-
geneity was greater than that related to stability. The results
indicated that the produced materials, i.e., the candidate
certified RMs, were suitable for assessing trueness and
& Scheilla Vitorino Carvalho de Souza
scheilla@bromatologiaufmg.com.br
1
Department of Food Science, Faculty of Pharmacy (FAFAR),
Federal University of Minas Gerais (UFMG), Av. Antônio
Carlos, 6627, Campus da UFMG, Pampulha, Belo Horizonte,
MG 31270-010, Brazil
could be submitted to further inter-laboratory
characterization.
Keywords Milk powder
Reference material

Quality control
Trueness
Homogeneity
Stability

Uncertainty
Introduction
Currently, quality becomes a key factor for the food
industry and leads to the introduction of legislation con-
cerning food safety and inspection. It is therefore necessary
to develop quality control programs and adopt surveillance
measures that are fast, precise, and objective [1, 2]. These
programs and measures depend significantly on the relia-
bility of the analytical results.
Calibration and the trueness assessment, which are used
in method validation and quality control processes, require
reference elements that can ensure the traceability to rel-
evant basic quantities, such as reference materials (RM) or
certified reference materials (CRM) [3–5].
According to the harmonized guidelines for in-house
method validation published by AOAC International, the
International Organization for Standardization (ISO), and
the International Union of Pure and Applied Chemistry
(IUPAC), the reference values needed for assessing true-
ness can be obtained using different types of materials,
including RM and CRM. As described herein, CRM can be
used in validation processes to simultaneously consider the
various aspects involved in the bias (method, laboratory,
and assay), assuming that there are no effects due to the
matrix variations. On the other hand, RM are used when
CRM are not available or to complement CRM studies
because RM include materials that are characterized by a
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RM producer but whose values are not accompanied by an
uncertainty statement or are otherwise qualified [6].
Internal quality control is one of the fundamental tools
used for quality assurance concerning the results obtained
in a laboratory quality management system [7, 8]. The
main objectives of this type of control are to monitor the
stability of the performance parameters of a previously
validated method during its routine use, to maintain the
statistical control of the measurement process [9], and to
indicate the acceptance or rejection of a set of results,
which may require test repetition [10]. Therefore, internal
quality control is required to provide evidence of how the
method performs during its application; thus, such an
activity should occur frequently to demonstrate the per-
formance of the methods that have been validated [6, 11].
Although RM and CRM play a key role in method
validation processes and in the development of internal
quality control programs for trueness assessments, there are
no available materials for all of the chemical analyses that
are currently performed in food laboratories. The few
available materials are expensive, primarily due to the
production and certification process costs. Hence, the
preparation and certification of a material that can be
applied in the quality assessment of milk and milk products
is fundamental for advancing chemical metrology and
analytical quality assurance in the dairy chain [12].
Because foods are complex mixtures, the properties of
measurands and matrix components must be considered
during the preparation, storage, transport, and analysis of
RM. Therefore, homogeneity and stability studies are
crucial steps in the evaluation and certification of these
materials [13–15].
Given the importance of and growing demand for
ensuring the reliability of tests that assess the safety and
quality of dairy products, this study aimed to produce and
evaluate the homogeneity and stability of different RM for
the measurands moisture, ash, total fat, and titratable acid-
ity in whole milk powder. The statistical criteria used to
assess homogeneity and stability, as established by differ-
ent guides and protocols related to the production of CRM
and of RM for use in proficiency testing (PT), were applied
and evaluated, considering the potential applications of the
produced materials.
Experimental
Laboratory performance assessment
The only CRM that was available on the market and has a
similar scope to that produced in the present study, CRM
BCR 380-R from the Institute for Reference Materials and
Measurements (IRMM, Joint Research Centre/European
123
Accred Qual Assur (2016) 21:47–55
Commission, Geel, Belgium), was acquired for the pre-
liminary evaluation of laboratory performance. The
material was kept under refrigeration from 2 °C to 8 °C and
analyzed at each analytical batch for homogeneity and
stability testing. The statistical analysis was conducted
using the z-score for the trueness assessment, as described
in the ISO Guide 33, with the assumption of normality
assessed by the Ryan–Joiner test and outliers evaluated
using the Grubbs test, at a 0.05 significance level [5]. The
Horwitz [16] and Thompson [17] models were adopted for
the estimation of the standard deviation for the proficiency
assessment (rp).
Production of materials
Preparation of materials
Immediately after processing, 50 kg of milk powder orig-
inating from the same batch was homogenized in the
industry and then placed in a polyethylene bag, wrapped in
a kraft paper bag, and transported to the laboratory. The
bag was stored at room temperature (19 °C to 25 °C) until
the materials’ preparation.
The milk powder was homogenized, and two batches of
RMs were prepared: The first was randomly packaged in
210 zippered metalized polyethylene terephthalate/low-
density polyethylene stand-up pouches (100 g) (metallized
PET/LDPE), and the second batch was randomly placed in
210 amber glass bottles (100 g) with screw cap. Both types
of packaging protected the contents from light due to the
metallized layer of the pouches and the amber pigment of
the bottles.
The pouches were sealed above the zipper using a
vacuum sealer (EZ HOME 201, Cotia, SP, Brazil), and the
bottles were closed with screw caps. The oxygen in head-
space was evacuated prior to sealing the packages. The
glass bottles were completely filled to avoid free space.
Extra care was taken to ensure an adequate closing when
sealing the pouches and capping the bottles to prevent
moisture from entering or escaping. The pouches and
bottles of each batch were divided into three sub-batches
and stored under different conditions, i.e., room (19 °C to
25 °C), refrigeration (2 °C to 8 °C), or freezing (–18 °C to
-24 °C) temperatures (Fig. 1), which were monitored
daily with calibrated thermometers. The pouches and bot-
tles were identified using self-adhesive plastic labels.
Homogeneity tests
Ten packaged units were randomly selected from each
batch and analyzed in duplicate for the measurement of
moisture, ash, total fat, and titratable acidity, totaling 160
determinations.
Accred Qual Assur (2016) 21:47–55 49

Fig. 1 Flowchart of the

preparation of the reference Whole milk powder
materials. RM1, RM2, and RM3
were packed in metallized
(50 kg)
polyethylene terephthalate/low-
density polyethylene pouches;
RM4, RM5, and RM6 in amber
glass bottles. They were stored
at either ambient temperature
(19 °C to 25 °C), refrigeration Batch 1
temperature (2 °C to 8 °C), or Batch 2
freezing temperature (–18 °C to Metallized
PET/LDPE stand-up Amber glass bottles
-24 °C) as indicated
pouches (25 kg)
(25 kg)

RM1 RM2 RM3 RM4 RM5 RM6

Ambient Freezing Refrigeration Ambient Freezing Refrigeration

temperature temperature temperature temperature temperature temperature

70 stand-up 70 stand-up 70 stand-up 70 amber glass 70 amber glass 70 amber glass

pouches pouches pouches bottles bottles bottles
(100 g) (100 g) (100 g) (100 g) (100 g) (100 g)

The uncertainty associated with homogeneity was esti-
mated as described in the ISO Guide 35 [18]. Because PT
could be an alternative application for the materials that
were produced, the data were also analyzed according to
the proficiency testing requirements of ISO 13528 [19] and
the IUPAC protocol [20]. The criteria adopted for evalu-
ating homogeneity were compared.
Stability tests
In a classical stability study, three units of each sub-batch
were selected randomly in 2-, 4-, and 6-month intervals and
analyzed in duplicate for the determination of moisture,
ash, total fat, and acidity, totaling 432 determinations.
The stability of the RM prepared was assessed by
regression analysis (the data obtained for each measurand
versus the time in months), as described in the ISO Guide 35
[18]. A significant slope indicated that the parameter chan-
ged with time, i.e., the material was unstable; the absence of a
significant slope signaled the stability of the material in the
time period evaluated. Regression analysis was processed
according to Souza and Junqueira: Outliers were treated by
the Jacknife standardized residuals test until the maximum
recommended limit of 22 %; the assumptions for linear
regression were evaluated (residual normality by the Ryan–
Joiner test, homoscedasticity by the Levene test modified by
Brown and Forsythe, and independence by the Durbin–
Watson test); and F tests were undertaken to check the
adjustment to the model [21]. The uncertainty associated
with stability was estimated as described in the ISO Guide 35
[18]. The data were also evaluated according to ISO 13528
[19] and the IUPAC protocol [20] due to the possible
application of the materials in PT schemes. In these cases, a
data analysis was conducted for each time period. The sta-
tistical criteria used to assess stability were also compared.
Analytical methods
The analytical methods used for the measurement of
moisture, ash, and total fat mass fractions, and titrat-
able acidity followed the procedures given in the ISO/IDF
or the official methods published by the Brazilian Ministry
of Agriculture, Livestock and Supply [22–24].
The moisture content was established by the reference oven
drying method, based on the removal of water from the milk
by heating 1 g of the sample between 100 °C and 104 °C
using a regular oven. The samples were immediately transferred
to desiccators, containing freshly dried silica gel with a hygro-
metric indicator, between the drying and weighing steps [22].
The method used to measure the ash content consisted of
heating 5 g of the milk sample using an open gas flame
with subsequent incineration at 550 °C in a muffle furnace
until complete mineralization of the organic matter. The
samples were kept in desiccators, containing freshly dried
silica gel with a hygrometric indicator, between the
incineration and weighing steps [22].
The mass fraction of fat was measured using the gravi-
metric reference Rose Gottlieb method, using approximately
1 g of milk sample; ammonium hydroxide to solubilize the
casein, neutralize acidity, and reduce the viscosity; ethyl
alcohol to break up the fat–casein emulsion; and the mixture
diethyl ether–petroleum ether to extract the fat [23].

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The routine method used to measure the acidity of the
milk consisted of titrating a portion of the milk powder
(corresponding to 5 g of non-fat solids, approximately 7 g
of milk sample) using an alkaline solution of known con-
centration, phenolphthalein as an indicator, and cobalt(II)
sulfate as a colorimetric standard. The result was expressed
as the volume of 0.1 mol/L NaOH solution required to
titrate 10 g of non-fat solids [24].
Results and discussion
Laboratory performance assessment
In the trueness assessment using the CRM BCR 380-R, the
assumption of normality was confirmed (p [ 0.10), and
there was no indication of outliers in any case (p [ 0.05).
Satisfactory z-scores (-2 B z B ?2) were obtained for
ashes and total fat. For the ashes, the z-scores ranged
between -0.2591 and 0.4924, and for the total fat, they
ranged between -1.5579 and 1.1786. Titratable acidity and
moisture were not compared in this trueness assessment
because these parameters were not evaluated in the CRM.
It is worth noting that the results obtained for both ash and
total fat were expressed on a dry weight basis for the
z-score estimation because the values determined by
the CRM manufacturer were expressed in this way.
Among the works available on the production of RM of
powder milk and similar matrixes, such as infant formula,
only one of them was conducted by an accredited labora-
tory [25]. However, the other studies contained no
information related to the laboratory’s performance, such
as the subcontracting of accredited laboratories, the use of
reference/normalized methods, or the use of CRM obtained
from other suppliers [26–29].
Production of materials
General characterization of the material
The means of the mass fractions found for moisture, and total
fat were 3.4 g/100 g and 26.0 g/100 g, respectively, and the
value for titratable acidity, 17.1 mL NaOH 0.1 mol/L, were
in agreement with the values established for the quality of
whole milk powder. The mean value obtained for ash
(5.9 g/100 g) was in agreement with the values reported
in the Brazilian food composition tables [30].
Homogeneity
The ISO 13528 [19] criteria are stricter than those estab-
lished by IUPAC [20], but in both cases, the standard
deviation between the samples was smaller than the critical
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Accred Qual Assur (2016) 21:47–55
value established for homogeneity. According to the
IUPAC [20], outliers were not detected by the Cochran test
(p [ 0.05) in any case. Adequate repeatability was
observed, which indicated the precision of the methods.
Thus, sufficient homogeneity between the bottles was
achieved for all measurands and materials. Considering the
variability within the bottles, homogeneity was assumed
for the analytical test portions that were used. Then, the
minimum sample intake of 1 g for moisture and fat, 5 g for
ash, and 7 g (corresponding to 5 g of non-fat solids) for
titratable acidity was defined (Table 1).
António [28] produced a similar CRM for the scope of
moisture, ash, protein, total fat, and titratable acidity in
infant formula. For the homogeneity studies, five materials
were analyzed in duplicate to evaluate the variability
between the samples, and one material was analyzed in six
replicates to study the variability within the sample. An
analysis of variance (ANOVA) was performed, but the
reference and criteria used were not reported. Hetero-
geneity was observed for the measurands moisture and
titratable acidity; hence, the material was considered
homogeneous only for ash, protein, and total fat. With
respect to repeatability, this author argued that the values
achieved were all satisfactory.
Stability
Stability testing aims to establish the degree of instability
of the candidate RM after preparation or to certify the
stability of the material. The long-term stability under
specified storage conditions is important to specify these
conditions and to study the stability of the material under
the same conditions. According to the ISO Guide 35
[18], in the absence of a well-defined kinetic mechanism,
a basic linear model can be expressed for a stability
study. In this case, for a stable RM, the slope is
expected to be zero.
In the ISO Guide 35 [18] is mentioned that regression
analysis is based on the assumption of normally distributed
data. Although it is not recommended by this Guide, ver-
ifying the compliance of all regression assumptions is of
fundamental importance.
Outliers were detected in most cases. When the
assumption tests were applied, the parameter moisture was
confirmed as critical, and it was not possible to draw any
conclusions concerning the stability of this measurand in
materials RM1, RM2, RM3, and RM4. Normality was not
confirmed in RM4 (p \ 0.01). The residuals were posi-
tively autocorrelated (p \ 0.10) in RM2, RM3, and RM4,
and heteroscedasticity (p \ 0.05) was observed in RM2
and RM4, which suggested that the simple linear regression
could not be applied to these data without weighting. Lack-
of-fit (to agree with Table 2) was detected for the materials
Accred Qual Assur (2016) 21:47–55 51

Table 1 Assessment of homogeneity of the produced reference materials according to the ISO 13528 and IUPAC harmonized protocol
Measurand/analyte Mean (n = 20) ISO 13528 Criteria IUPAC Criteria rp Conclusion
ss san ssam c

Materials packaged in metallized polyethylene terephthalate/low-density polyethylene stand-up pouches

Moisture (g/100 g) 3.4 0.022 0.046 0.000 0.0043 0.11 Homogeneous
Ash (g/100 g) 5.9 0.000 0.035 0.000 0.0068 0.18 Homogeneous
Total fat (g/100 g) 26 0.072 0.13 0.071 0.061 0.51 Homogeneous
Titratable Acidity (mL NaOH 0.1 mol/L) 17 0.10 0.081 0.10 0.036 0.41 Homogeneous
Materials packaged in amber glass bottles
Moisture (g/100 g) 3.4 0.032 0.049 0.032 0.0046 0.11 Homogeneous
Ash (g/100 g) 5.9 0.018 0.020 0.018 0.0060 0.18 Homogeneous
Total fat (g/100 g) 26 0.15 0.12 0.14 0.059 0.51 Homogeneous
Titratable Acidity (mL NaOH 0.1 mol/L) 17 0.077 0.074 0.077 0.034 0.41 Homogeneous
ss: standard deviation between samples; ISO 1358 homogeneity criterion: ss B 0.3rp, where rp is the target standard deviation
san: standard deviation of repeatability; ssam: sample standard deviation related to the between group variation; c: critical value for the test for
sufficient homogeneity; IUPAC repeatability criterion: san \ 0.5rp; IUPAC homogeneity criterion [20]: s2sam B c
No outliers were detected for any of the measurands (p [ 0.05)
Analytical sample intake of 1 g for moisture and fat, 5 g for ash, and 7 g for titratable acidity

RM1, RM2, RM3, and RM4, thus confirming that the
models did not accurately fit the data (Table 2).
In the assessment of significance of regression by
ANOVA, which indicates a lack of stability, as described
by ISO Guide 35 [18], RM1, RM2, and RM4 showed
significant regression (p \ 0.01), i.e., that the slope of the
regression line was significantly different from zero, for the
measurand moisture. All other materials and measurands
that were evaluated showed nonsignificant regressions
(p [ 0.05), which indicated stability, as illustrated in
Fig. 2.
Therefore, according to the ISO 35 [18] criteria, insta-
bility was observed in RM1, RM2, and RM4 only in terms
of moisture over the 6-month storage period of the milk
powder. Considering the linear regression analysis
according to Souza and Junqueira [21], RM1, RM2, RM3,
and RM4 could not be considered stable in their moisture
because some assumptions of regression were not met and
because deviation from the linear model was observed. It is
noteworthy that obtaining regression parameters estima-
tions and regression significance evaluations, as described
in ISO Guide 35 [18], without a previous check of the
regression assumptions could lead to erroneous and mis-
leading conclusions concerning the stability of the
materials.
Considering the stability test requirements of ISO 13528
[19], the critical values of 0.3rp, where rp is the standard
deviation for the proficiency assessment, were 0.034 g/100 g
for moisture, 0.055 g/100 g for ash, 0.153 g/100 g for total
fat, and 0.124 mL for titratable acidity. As recommended in
the IUPAC harmonized protocol [20], the 0.1rp criterion
was applied, and the most restrictive critical values were
obtained, including 0.011 g/100 g, 0.018 g/100 g, 0.051 g/100 g,
and 0.041 mL for moisture, ash, total fat, and titratable acidity,
respectively. Because a t test was conducted to test for
stability in the IUPAC protocol [20], this test was also
performed here.
With regard to RM1, RM2, RM3, and RM4, only the
measurand moisture was unstable, with differences
between the means of the homogeneity and stability tests of
0.30 g/100 g, 0.15 g/100 g, 0.08 g/100 g, and 0.85 mL. The
values obtained for ash, total fat, and titratable acidity
were 0.01 g/100 g, 0.00 g/100 g, and 0.01 mL for RM1;
0.015 g/100 g, 0.04 g/100 g, and 0.00 mL for RM2; and
0.01 g/100 g, 0.03 g/100 g, and 0.04 mL for RM3,
respectively, which were less than the critical values.
For RM4, the differences between the means were
0.019 g/100 g for ash, 0.03 g/100 g for total fat, and
0.00 mL for titratable acidity; therefore, this material was
considered stable for all measurands, except for the ash,
according to the IUPAC 0.1rp criterion [20]. Considering
the t test criterion, the instability was not observed for the
ash measurand in RM4.
RM5 and RM6 showed stability in terms of all mea-
surands. The differences between the means of the
homogeneity and stability tests were less than the critical
values in all cases. With regard to RM5, the value of the
difference between the means was 0.01 g/100 g for mois-
ture, 0.02 g/100 g for ash and total fat, and 0.03 mL for
titratable acidity, whereas for RM6, the differences
were 0.00 g/100 g for moisture, 0.01 g/100 g for ash,
0.05 g/100 g for total fat, and 0.03 mL for titratable acidity.
Therefore, according to the ISO 13528 [19] 0.3rp cri-
terion and the IUPAC harmonized protocol [20] t test

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Fig. 2 Typical linear regression y = (0.0070±0.0038)x + (3.4±0.0011) y = (-0.0035±0.0019)x + (5.9±0.0055)

plots obtained in the stability R² = 0.092, p = 0.072 R² = 0.088, p = 0.079
evaluation during 6 months of 4.0 7.0
storage for the reference

Moisture (g/100 g)
6.5

Ash (g/100 g)
material RM5 packaged in 3.5
amber glass bottles and stored at
refrigeration temperature. In the 3.0 6.0
equations above the diagrams,
y and x refer to the numerical
2.5 5.5
values on the respective axes. R2
is the coefficient of
determination, p significance 2.0 5.0
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Month Month

y = (0.0034±0.013)x + (26±0.039) y = (-0.0054±0.0072)x + (17±0.022)

R² = 0.0019, p = 0.80 R² = 0.016, p = 0.46
27.0 18.0

(mL NaOH 0.1 mol/L)

Total fat (g/100 g)

Titrable acidity
26.5 17.5

26.0 17.0

25.5 16.5

25.0 16.0
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Month Month

criterion, RM1, RM2, RM3, and RM4 showed instability
only in terms of moisture over the 6 months of storage of
the whole milk powder. RM5 and RM6 were considered
stable for all measurands. Considering the 0.1rp IUPAC
criterion, there was disagreement for the ash measurand in
RM4, which indicated that this is more restrictive than
even the t test criterion. The stability/instability results
found at 2 and 4 months were the same as those found at
6 months for both criteria.
Considering the acceptability criteria recommended by
ISO Guide 35 [18], ISO 13528 [19], and IUPAC protocol
[20], RM5 and RM6 showed stability for all measurands
(moisture, ash, and total fat mass fractions, and titrat-
able acidity) after 6 months of storage; however, instability
in terms of moisture was found for RM1, RM2, and RM4.
Therefore, these materials were considered stable only in
terms of ash, total fat, and titratable acidity, regardless of
the criteria and reference adopted, which indicated the
potential applicability of these materials as CRM or RM for
proficiency schemes.
There was disagreement between the stability results
found for the moisture in RM3 because, according to the
criteria of ISO Guide 35 [18], there was no indication of
instability, though instability was detected based on the
criteria used in the other references. This would not have
occurred if the significance of the slope of the regression
line had been evaluated after verifying the assumptions and
the linear model, according to Souza and Junqueira [21].
Additionally, despite the lower cost and convenience of
the metallized PET/LDPE stand-up pouch, this packaging
material was not adequate for the moisture measurand,
most likely due to failures in sealing. According to Alves
et al. [31], the increased moisture in the milk powder
results from the transfer of water vapor to and from the
package, which may occur due to the permeation of the
packing material or due to heat-sealing flaws.
Campos et al. [32] evaluated the influence of nitrogen
inertization on the shelf life of milk powder packaged in
metal packaging, and over the span of a year, they found
increased acidity and moisture (which was detected from
the first month) in the milk packaged under both normal
and modified atmospheres. According to these authors, the
changes were due to redox reactions in the product and the
packaging’s water vapor permeability, respectively.
Similar results were found by Krey and Souza [33], who
investigated the microbiological and physicochemical
quality of whole milk powder from 20 different batches
and, after 3 months of storage, observed increased levels of
acidity and moisture in some milk powder samples. In
terms of the fat content, no significant changes were
observed in 95 % of the batches analyzed.
In the present study, similar results were achieved at
freezing temperature compared with the data reported by
Antònio [28]. However, at the refrigeration temperature, at
which stability was observed for all measurands, and at
room temperature, at which instability was observed only

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Accred Qual Assur (2016) 21:47–55 53

Table 2 Evaluation of the regression assumptions and assessment of stability of the reference materials produced according to the ISO Guide
35. The reference materials differ in packaging and storage temperature as given in Fig. 1

ISO Guide 35
Assumptions
Criteria
Material, 2
Equation R n Normality Independency Homoscedasticity Lack-of-fit Regression
Measurand/analyte
RJ p d p tL p F p F p
RM1
Moisture y = (0.076 ± 0.0040)x + (3.4 ± 0.012) 0.91 35 0.99 p > 0.10 1.7 p > 0.10 2.7 0.062 8.5 1.1x10 -3
3.5x10 2
3.9 x10-19
Ash y = (-0.0027 ± 0.0021)x + (5.9 ± 0.0064) 0.046 36 0.97 p > 0.050 2.4 p > 0.10 0.11 0.95 0.46 0.64 1.6 0.21
Total fat y = (-0.0092 ± 0.0067)x + (26 ± 0.020) 0.062 30 0.99 p > 0.10 1.8 p > 0.10 0.095 0.96 0.81 0.46 1.9 0.18
Titratable acidity y = (-0.0047 ± 0.0082)x + (17 ± 0.025) 0.0092 37 0.98 p > 0.10 2.1 p > 0.10 1.4 0.27 0.076 0.93 0.33 0.57
RM2
Moisture y = (0.021± 0.0071)x + (3.4 ± 0.021) 0.19 38 0.99 p > 0.10 1.5 p < 0.10 3.9 0.017 31 1.7x10-8 8.3 6.7x10-3
Ash y = (-0.0022± 0.0015)x + (5.9 ± 0.0047) 0.071 31 0.99 p > 0.10 2.3 p > 0.10 2.2 0.12 0.28 0.76 2.2 0.15
Total fat y = (0.011± 0.011)x + (26 ± 0.033) 0.029 37 0.99 p > 0.10 1.9 p > 0.10 1.4 0.26 0.47 0.63 1.1 0.31
Titratable acidity y = (-0.0023± 0.0075)x + (17 ± 0.023) 0.0027 36 0.97 p > 0.050 2.0 p > 0.10 0.087 0.97 0.94 0.40 0.092 0.76
RM3
Moisture y = (0.0045± 0.0084)x + (3.4 ± 0.025) 0.0078 38 0.98 p > 0.10 0.97 p < 0.10 1.3 0.30 34 6.9x10-9 0.28 0.60
Ash y = (-0.0020± 0.0014)x + (5.9 ± 0.0044) 0.062 31 0.97 p > 0.10 2.2 p > 0.10 1.6 0.21 1.2 0.31 1.9 0.18
Total fat y = (-0.0045± 0.012)x + (25 ± 0.035) 0.0042 35 0.99 p > 0.10 2.4 p > 0.10 1.3 0.28 0.80 0.46 0.14 0.71
Titratable acidity y = (-0.014± 0.0072)x + (17 ± 0.022) 0.099 36 0.98 p > 0.10 2.3 p > 0.10 0.63 0.60 0.32 0.73 3.7 0.062
RM4
Moisture y = (0.22± 0.024)x + (3.3 ± 0.070) 0.72 38 0.92 p < 0.01 0.53 p < 0.10 10 4.6x10-5 13 6.1x10-5 94 1.4x10-11
Ash y = (-0.0036± 0.0020)x + (5.9 ± 0.0061) 0.083 37 0.97 p > 0.050 2.0 p > 0.10 0.15 0.93 0.089 0.92 3.2 0.084
Total fat y = (-0.0023± 0.011)x + (26 ± 0.033) 0.0014 35 0.99 p > 0.10 1.5 p > 0.10 0.57 0.64 1.0 0.38 0.048 0.83
Titratable acidity y = (0.0082± 0.0072)x + (17 ± 0.022) 0.036 37 0.97 p > 0.050 1.7 p > 0.10 0.29 0.83 1.3 0.28 1.3 0.26
RM5
Moisture y = (0.0070± 0.0038)x + (3.4 ± 0.0011) 0.092 36 0.99 p > 0.10 2.1 p > 0.10 0.83 0.49 0.22 0.81 3.4 0.072
Ash y = (-0.0035± 0.0019)x + (5.9 ± 0.0055) 0.088 36 0.99 p > 0.10 1.9 p > 0.10 1.5 0.23 0.34 0.71 3.3 0.079
Total fat y = (0.0034± 0.013)x + (26 ± 0.039) 0.0019 37 0.98 p > 0.10 1.5 p > 0.10 0.42 0.74 1.5 0.24 0.067 0.80
Titratable acidity y = (-0.0054± 0.0072)x + (17 ± 0.022) 0.016 37 0.98 p > 0.10 1.6 p > 0.10 0.33 0.80 0.55 0.58 0.56 0.46
RM6
Moisture y = (0.0032± 0.0039)x + (3.4 ± 0.011) 0.020 35 0.99 p > 0.10 2.2 p > 0.10 0.91 0.45 0.097 0.91 0.67 0.42
Ash y = (0.0018± 0.0016)x + (5.9 ± 0.0046) 0.039 35 0.98 p > 0.10 1.7 p > 0.10 0.11 0.96 0.99 0.38 1.3 0.26
Total fat y = (0.0094± 0.011)x + (26 ± 0.033) 0.021 36 0.99 p > 0.10 1.6 p > 0.10 0.96 0.42 3.2 0.052 0.74 0.40
Titratable acidity y = (-0.0062± 0.0083)x + (17 ± 0.025) 0.015 38 0.98 p > 0.10 1.8 p > 0.10 1.7 0.18 0.32 0.73 0.55 0.46

In the equations, y refers to the numerical values of the response when measured in g/100 g or mL (NaOH, 0.1 mol/L), depending on the
measurand, and x to the time measured in months; R2: coefficient of determination; n: number of observations; RJ: Ryan–Joiner correlation
coefficient; p: significance; d: Durbin–Watson statistic; tL: Levene t statistic; F: ratio of variances
Marked in gray is the evidence of a lack of compliance with the regression assumptions

Table 3 Relative standard uncertainty of homogeneity (bb) and long-term stability (lts) of the produced reference materials which differ in
packaging and storage temperature as given in Fig. 1
Measurand/ RM1 RM2 RM3 RM4 RM5 RM6
analyte
Relative standard uncertainty (%)
bb lts bb lts bb lts bb lts bb lts bb lts

Moisture – – – – – – – – 0.95 0.67 0.95 0.69

Ash 0.35 0.22 0.35 0.15 0.35 0.15 0.31 0.20 0.31 0.19 0.31 0.16
Total fat 0.28 0.16 0.28 0.25 0.28 0.28 0.56 0.25 0.56 0.30 0.56 0.25
Titratable acidity 0.61 0.29 0.61 0.26 0.61 0.25 0.45 0.25 0.45 0.25 0.45 0.29

for moisture, it can be said that better stability results were Milk contains an endogenous lipase, which could cause
obtained in this study. the hydrolysis of triglycerides resulting in increased free
Despite the increased moisture observed in RM1, fatty acids content and, hence, increased acidity; however,
RM2, RM3, and RM4, it is likely that there were no this enzyme is not free because it is associated with casein
lipolysis and oxidation reactions in the produced mate- micelles. Additionally, heating at 75 °C for 20 s (condi-
rials because the titratable acidity remained stable. tions similar to pasteurization) is sufficient to inactivate it.
According to Walstra et al. [34], microbial and enzy- Furthermore, the fat globule membrane protects the
matic deterioration are rare in milk powder. For triglycerides from an enzymatic attack, and the enzyme
microbial deterioration to occur, Aw should increase to cannot pass through it easily [34].
over 0.6, and the increase in moisture was not sufficient Lipolysis can also occur due to lipases produced by
to achieve high Aw values. Furthermore, the stability of microorganisms; however, because milk powder is a pro-
milk powder is due to the protection of its fat globules duct that has a water activity value (Aw) of 0.1, it is
by a layer of lactose, which prevents the contact of unlikely that it will undergo microbial and enzymatic
oxygen with fat. In addition, antioxidant compounds are changes and chemical interactions unless the Aw value
formed during high-temperature drying. reaches values greater than 0.6 [34].

123
54
Consequently, because only total fat was analyzed in the
present study, it was not possible to confirm the presence or
absence of lipid oxidation, which would be indicated by the
determinations of aldehydes, free fatty acids, and indices,
such as the peroxide index, which were not objectives of
this study.
Uncertainty of homogeneity and stability
The values of relative standard uncertainties obtained in the
homogeneity and stability studies according to ISO Guide
35 [18] for the six materials produced are given in Table 3.
Due to the lack of a specific criterion for the evaluation
of homogeneity in the ISO Guide 35 [18], it is up to the
RM producer to determine whether a specific value rep-
resenting bottle heterogeneity is acceptable. It was
determined that the uncertainty associated with homo-
geneity made a larger contribution than did the uncertainty
related to long-term stability for all measurands and
materials. The relative standard uncertainties due to the
homogeneity showed maximum values of 0.95 % for
moisture, 0.35 % for ash, 0.56 % for total fat, and 0.61 %
for titratable acidity; however, even considering both
contributions to the combined uncertainty, the values were
smaller than the expected measurement uncertainty. The
combined uncertainty estimated for ash and total fat were
also smaller than that declared for the IRMM CRM used
for the laboratory performance assessment, which corre-
sponded to 1.5 % and 0.45 % of the property values
certified for ash and total fat, respectively.
Therefore, further studies to characterize these materials
(using different methods or laboratories) are needed
because the uncertainty of this third component must be
combined to estimate the expanded uncertainty associated
with the property values of the RM produced [18].
Conclusions
The six materials produced were homogeneous in terms of
moisture, ash, total fat, and titratable acidity, according to
the requirements of ISO 13528 and the IUPAC harmonized
protocol, though the criteria of ISO 13528 are stricter. The
within-bottle homogeneity was assured for minimal intakes
of 1 g for moisture and total fat, 5 g for ash, and 7 g for
titratable acidity.
Stability was observed for all measurands (moisture,
ash, total fat, and titratable acidity) for the materials
packaged in amber glass bottles under refrigeration and
freezing temperatures for 6 months. Stability was not
guaranteed for moisture, considering the materials pack-
aged in metallized PET/LDPE stand-up pouches that were
stored at room, freezing, and refrigeration temperature and
123
Accred Qual Assur (2016) 21:47–55
those packaged in amber glass bottles stored at room
temperature. The stability tests’ results were in agreement
with the requirements of ISO 13528 and the IUPAC har-
monized protocol, though the criteria of the IUPAC are
more strict.
The materials stored in metallized PET/LDPE stand-up
pouches under refrigeration were considered stable in
terms of moisture only when the criteria of ISO Guide 35
were adopted; however, autocorrelation and deviation from
linearity were observed when verifying the assumptions of
regression, which confirmed the need for this approach
prior to testing the significance of the slope of the
regression.
The best packaging and storage conditions for the
materials produced, considering the four measurands, were
the amber glass bottles with screw caps under refrigeration
and freezing temperatures.
The contribution of uncertainty associated with homo-
geneity was greater than that associated with stability.
Standard uncertainties associated with homogeneity and
stability were smaller than the expected measurement
uncertainty, which indicated that the materials produced,
i.e., candidate CRM, may be submitted to further inter-
laboratory characterization using one or more methods of
demonstrable accuracy.
Acknowledgments The authors are grateful for the financial sup-
port provided by the Foundation for Research Support of the State of
Minas Gerais (FAPEMIG).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict
of interest.
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