Food Anal.
Methods (2017) 10:1787–1799
DOI 10.1007/s12161-016-0718-9
Different Approaches for Digestion, Performance
Assessment and Measurement Uncertainty for the Analysis
of Cadmium and Lead in Feeds
Luciano Molognoni 1,2 & Jaqueline Zarpelon 3 & Leandro Antunes de Sá Ploêncio 1,3 &
Jacson Nascimento dos Santos 1,2 & Heitor Daguer 1
Received: 21 July 2016 / Accepted: 7 November 2016 / Published online: 6 December 2016
# Springer Science+Business Media New York 2016
Abstract Certain feedingstuffs are sources of contamination
for animals with cadmium (Cd) and lead (Pb). Different ap-
proaches for sample digestion, performance assessment, and
measurement uncertainty for the analysis of Pb and Cd by
flame atomic absorption spectrometry were compared. Full
method validation, including matrix effect and analytical
limits, were assessed. Despite having a lower performance
compared to the microwave digestion, dry ashing was a viable
alternative for sample digestion, leading to lower operating
costs. Interlaboratory validation proved to be a cheaper alter-
native, instead of the long traditional in-house approach. The
method was suitable for routine analysis of several feed sam-
ples. There was a greater amount of quantifiable results for Pb,
ranging from traces up to high concentrations (7430 ± 930 mg
kg-1). Cd was also quantified in some samples, ranging from
0.11 ± 0.01 to 8.71 ± 0.90 mg kg−1. The higher concentrations
of both metals were found in mineral mixtures and fish meal.
Keywords Dry digestion . Microwave digestion . Proficiency
testing . Top-down approach . Traditional approach
* Heitor Daguer
heitor.daguer@agricultura.gov.br
1
Ministério da Agricultura, Pecuária e Abastecimento, Laboratório
Nacional Agropecuário (SLAV/LANAGRO/RS), Rua João
Grumiché, 117, São José, SC 88102-600, Brazil
2
Instituto Catarinense de Sanidade Agropecuária (ICASA),
Florianópolis, SC, Brazil
3
Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil
Introduction
Toxicity and ecotoxicity assessment of metals such as cadmi-
um (Cd) and lead (Pb), mistakenly referred as Bheavy metals,^
shall be guided by their chemical properties and by the bio-
logical and environmental properties of the organisms at risk.
However, these relationships are still at an early stage of
knowledge (Duffus 2002). Furthermore, inorganic contami-
nants cannot be completely excluded of feed production and
thus of the food chain, since they are naturally present in
certain raw materials, such as the mineral supplements.
Thus, contamination with Cd and Pb should be minimized
and controlled, in order to reduce its hazardous effects on
animal health, as well as in humans (European Commission
2002a, b). Cd and Pb may present a cumulative nature and can
be transferred to the food chain without benefits for any bio-
logical function in both animals and humans (López-Alonso
and Fink-Gremmels 2012).
Cd and Pb are naturally found in phosphate rocks used in
the manufacture of fertilizers and formulations for feed sup-
plementation, constituting an important source of contamina-
tion for animals (EFSA 2004). Cd can accumulate in the kid-
neys, liver, and lungs, and competing with other minerals,
such as zinc, iron, copper, and selenium, due to their chemical
similarity. Exposure to Cd has been related to carcinogenic
effects in human prostate, lung, kidney, and liver (Waalkes
2000; Vassileva and Hoenig 2011). Pb accumulation occurs
mainly in the soft tissues and bones (EFSA 2009). Animal
studies have been providing evidence that Pb, in its inorganic
form, has carcinogenic effects on kidney and brain and there-
fore can trigger similar problems in humans (WHO Library
Cataloguing in Publication Data 2010).
To prevent food contamination by toxic metals, it is neces-
sary to identify and quantify these elements in the food chain.
With this purpose, several analytical techniques have been
1788
adopted. Flame atomic absorption spectrometry (FAAS), hy-
dride generation absorption spectrometry (HG AAS)
(Molognoni et al. 2016), graphite furnace absorption spec-
trometry (GF AAS) (Borges et al. 2015), inductively coupled
plasma–optical emission spectroscopy (ICP-OES) (Durduran
et al. 2015), and detection by inductively coupled plasma–
mass spectrometry (ICP-MS) (Vassileva and Hoenig 2011)
have been widely disseminated. However, the technique
choice by the control laboratory must take into account their
availability because of the cost, their sensitivity, and reproduc-
ibility. In addition, sample preparation process is another fun-
damental step and should be available within the same
perspective.
The various steps involved in an analytical process are
subject to errors that may alter the results considered as con-
ventionally true. Method validation is the confirmation,
through objective evidences, that the requirements for a spec-
ified analytical protocol are fulfilled (Associação Brasileira de
Normas Técnicas 2005). The Commission Decision 657/
2002/EC is traditionally considered an appropriate validation
guide, with great international acceptance (European
Commission 2002a, b). Currently, it has the highest amount
of publications involving its application (MacDonald et al.
2009; Lucatello et al. 2015).
There are different methods and calculation approaches
for estimating the measurement uncertainty (MU) (Medina-
Pastor et al. 2011; Rozet et al. 2011; Coelho et al. 2013).
The Guide to the Expression of Uncertainty in
Measurement (GUM) is an international consensus on
how to estimate the calculation due to their ability to cover
specific models with different approaches and types of
measurements (ISO/IEC Guide 98-3:2008 2008). In
Analytical Chemistry, a MU calculation approach does
not necessarily refer to the calculation method. In fact, it
is a strategy to quantify the relevant sources of uncertainty
of a measure. As proposed by GUM, in the traditional
bottom-up approach, each source of uncertainty is individ-
ually quantified, and their effects are combined as proposed
by using an expansion of Taylor series. The top-down (or
experimental) approach uses empirical data to represent the
entire measurement process, generally simplifying the esti-
mation of the overall uncertainty, without the need to study
and quantify each uncertainty component alone, as in the
GUM method (Medina-Pastor et al. 2011). These compo-
nents can be evaluated in combination through
interlaboratory studies (proficiency testing) or through in-
house validation (Magnusson et al. 2004; Eurolab 2007;
Ellison 2014). An interlaboratory validation approach must
be considered by control laboratories when a proficiency
test with a significant number of participants is available.
This is a cheaper alternative, since it uses empirical data
from collaborative studies, instead the long traditional pro-
cess of in-house validation (Thompson et al. 2006).
Food Anal. Methods (2017) 10:1787–1799
In this context, the main goal of this research was to com-
pare different approaches for the digestion of samples, the
analytical performance assessment, and the calculation of the
measurement uncertainty of a method for the analysis of Pb
and Cd in feedingstuffs by FAAS.
Materials and Methods
Reagents and Solutions
All chemicals were in analytical grade. Used water was ultra-
pure (18.3 MΩ cm resistivity). Cd and Pb reference solutions
(1000 mg L−1) were supplied by Fluka (Steinheim, Germany),
with minimum 99% purity. Hydrochloric (HCl), nitric
(HNO3), per chloric (HClO4), and sulfuric (H2SO4) acids
and ammonium hydroxide (NH 4OH) were supplied by
Sigma-Aldrich Co. (St. Louis, MO, USA). EDTA and
lantanium oxide (La2O3) were supplied by Vetec Química
Fina Ltda. (Duque de Caxias, Brazil). Cd standard solution
(100 mg L−1) was prepared by diluting the reference solution
in nitric acid solution 2%. All standard solutions were kept
under refrigeration until use. Buffer solution (pH 4.4) for Pb
analysis was prepared with EDTA (81.5 g), NH 4 OH
(0.01 mol L−1), HClO4 (30 mL), and La2O3 (25 g).
Instrument
The analytical and instrumental parameters were adjusted to
obtain the best sensitivity (Table 1). Sample digestion was
carried out in a Multiwave 3000 microwave system supplied
by Anton Paar GmbH, (Graz, Austria) or in a muffle furnace
with temperature ramp builder supplied by Fornos Jung Ltda.
(Blumenau, Brazil). Sample quantitation was performed in the
AAnalyst 200 FAAS spectrometer (PerkinElmer Inc.,
Waltham, MA, USA), equipped with deuterium arc back-
ground corrector and Echelle optical resolution system.
Acetylene (99.7% purity) and compressed air from a compres-
sor (with filter and dehumidifier) were used as fuel and carrier
gas, respectively. Hollow cathode lamps for the analyzed ele-
ments were from Lumina (PerkinElmer Inc., Waltham, MA,
USA). A 300D Nexion mass spectrometer with ICP-MS
(PerkinElmer Inc., Waltham, MA, USA) was also used to
assist method validation.
Sample Preparation
Before testing, all samples were grinded in a ZM 200 ultra-
centrifugal mill (Retsch GmbH, Haan, Germany), with sieves
of 1.0-mm trapezoid holes. Samples were stored in tightly
closed glass containers and kept at room temperature until
the digestion procedure.
Food Anal. Methods (2017) 10:1787–1799
Table 1 Instrumental settings
adopted for method development Instrumental settings
and validation for the samples
digestion and quantitation of lead Microwave
and cadmium in feedingstuffs Power ramp—10 min
Digestion length—35 min
Cooling time—15 min
Power—800 W
Maximum internal/external temperatures—240/210 °C
Maximum pressure—40.0 bar
ICP-MS
Power of radio frequency—1600 W
Main argon gas flow—18.0 L min−1
Auxiliary gas (argon) flow—1.2 L min−1
Nebulizer gas (argon) flow—1.13 L min−1
For the microwave digestion, portions of 0.50 ± 0.01 g of
sample were weighed directly into a microwave digestion
vessel. Then, HCl (2.0 mL) and HNO3 (6.0 mL) were added
to each vessel. A power ramp was adopted (Table 1). After
digestion, the samples were filtered and transferred to
25.0-mL volumetric flasks.
The dry digestion was carried out by firstly weighing
5.00 ± 0.10 g of sample in porcelain decomposition vessels.
Then, H2SO4 (1.0 mL) was added to each vessel. Vessels were
submitted to heating in a plate and then dry ashing was carried
out for 13 h, adopting a temperature ramp (Table 1). Ash were
diluted in HCl 8 mol L−1 in a water bath (100 °C), filtered, and
transferred to 25-mL volumetric flasks containing 1.0 mL of
buffer solution.
Traditional In-House Validation
In-house validation was conducted according to the
Commission Decision 657/2002/EC (European
Commission 2002a, b), in terms of precision (repeatability
and in-house reproducibility), recovery, analytical curves,
specificity/selectivity, decision limit (CCα), detection ca-
pability (CCβ), ruggedness, and stability. Additionally,
matrix effect, limit of detection (LOD), and limit of quan-
titation (LOQ) were also assessed, according to the
Brazilian Ministry of Agriculture, Livestock and Food
Supply (Brasil 2011).
Specificity/selectivity was checked by analyzing both
blank and incurred samples (n = 20). The results were assessed
by agreement between the measurements by FAAS and ICP-
MS performed for three samples. The t test (for two samples
assuming equal variances) was carried out to determine
1789
Muffle furnace
Time (h) Temperature (°C)
2 100 ± 10
4 200 ± 10
2 300 ± 10
5 450 ± 10
FAAS
Wavelength (λ)—228.80 nm (Cd), 283.31 nm (Pb)
Slit width—1.0 nm
Current—10.0 mA
Burner height—7.0 mm
Burner angle—0°
Fuel gas flow—2.5 L min−1
Type of oxidant—air with flow of 2.5 L min−1
significant differences between the values of the measure-
ments obtained by the two techniques.
Analytical curves were prepared with six concentration
levels (including zero), using unweighted linear functional
relationship of concentration (x) vs. absorbance (y). Solvent
analytical curves were prepared by using a 2% solution of HCl
and HNO3 1:3. The assessed linear ranges were 0–30 and 0–
200 mg L−1 for Cd and Pb for all the assessed dilutions and
wavelengths (228.80 and 283.31 nm), respectively. Linearity
was assessed with two repetitions per level, on three different
days. The acceptance criterion was the coefficient of determi-
nation (R2 > 0.99). The residual plots of each day of analysis
were examined for obvious patterns, and the Grubb’s test was
applied sequentially, until no further outliers were detected
(P < 0.05). The residues were also evaluated for violations
of the assumptions of normality employing the Anderson-
Darling test and homoscedasticity by applying the Bartlett
test. F tests were conducted to verify the regression and
lack-of-fit significance. Additionally, it was checked if signif-
icant difference between the derivatives of the analytical
curves could be noted when in-house reproducibility condi-
tions were applied. Statistical analyses were performed using
the Microsoft Excel and the Minitab 14 softwares.
The matrix effect was assessed to verify the increase or the
decrease in absorbance. The procedure was based on the anal-
ysis of the following three analytical curves: curve I was pre-
pared in solvent, by diluting the standard solution in acidic
solution; curve II was prepared by fortifying the blank sample
before digestion; and curve III was prepared by fortifying the
blank sample after digestion. The evaluation was performed
by evaluating the significant difference between the deriva-
tives obtained for each calibration curve. For this, the t test
(for paired samples) with 5% significance level was applied.
1790
Recovery and precision were assessed from the same exper-
iment. In total, aliquots of 21 blank samples were spiked into 3
groups of 7 aliquots each. The fortification levels were 7.5, 15.0,
and 22.5 mg kg−1 for Cd and 50.0, 100.0, and 150.0 mg kg−1 for
Pb. Analytical curves were prepared at each evaluation day.
Recovery was assessed in 3 days by considering the recovery
rate (−20 to +10%). Precision was evaluated for each analyte
and for each concentration level. Precision in terms of repeat-
ability was assessed by the analysis of seven aliquots on the
same day under the same analytical conditions, by the Horwitz
equation. Precision assessment in terms of in-house reproduc-
ibility was based on the coefficient of variation (CV < 10%) by
applying the variables day (n = 3) and analyst (n = 2).
Ruggedness was assessed by the Youden fractional facto-
rial design model. Seven factors that could influence the rou-
tine method were slightly varied. These factors were identified
with letters and consisted of volume of HCl (2.0 and 1.9 mL)
(A), volume of HNO3 (6.0 and 5.9 mL) (B), supplier of HCl
(two different suppliers) (C), size of the holes of the grinding
sieve (0.5 and 1.0 mm) (D), variation in the nebulization flow
(2% variation towards optimum condition) (E), variation in
burner height (7.0 and 6.9 mm) (F), and heating time of hol-
low cathode lamps (30 and 40 min) (G). Their results repre-
sented the average concentration of three points for each pa-
rameter. The evaluation was performed by comparing the de-
viation of ruggedness (standard deviation of the differences
(SDi)) with the deviation of the in-house reproductivity (Szi)
for each analyte. In addition, it evaluated if the effects were
significant by the principle of sparsity. For this, the Lenth’s
method was used, by estimating the standard error of con-
trasts. The margin of error (ME) was obtained with 95%
confidence.
The stability test was performed at room temperature by
applying the t test (for paired samples), with 5% significance
level. Differences among the averages for each day of storage
and the reference values were compared.
CCα was obtained by multiplying the standard deviation of
21 fortified blank samples by the average and by the 1.64
factor. CCβ was also obtained by multiplying the standard
deviation of 21 samples by 1.64 and adding this value to the
CCα. The determination of the analytical limits (LOQ and
LOD) was done from the analysis of seven replicates of blank
sample. LOD and LOQ were determined from three times and
from ten times the standard deviation of the average by the
slope of the calibration curve, respectively. These parameters
were determined by considering the same criteria established
for the analysis of mineral feed samples.
Interlaboratory Validation Approach
The accuracy of the alternative digestion by dry ashing (in
muffle furnace) was also checked by an interlaboratory vali-
dation approach. The method was evaluated by the
Food Anal. Methods (2017) 10:1787–1799
international proficiency testing using a poultry mixed
feed provided by FAO-IAG (International Group
Analytical–Section feedingstuff), with 119 laboratories from
48 countries (in total). To evaluate laboratory performance, the
z-score was calculated by the difference among the average
results of the laboratory and the target value (median), expressed
by the program, and divided by the overall variability. The result
was evaluated according to the following criteria: satisfactory
(z ≤ 2), questionable (2 < z < 3), and unsatisfactory (z ≥ 3).
Measurement Uncertainty
Measurement uncertainty was calculated by diffe-
rent approaches. All calculations were performed at
15.00 mg kg-1(Cd) and 100.0 mg kg−1 (Pb) concentration levels.
The traditional approach was performed according to the
Guide to the Expression of Uncertainty in Measurement (ISO/
IEC Guide 98-3:2008 2008). Sources of uncertainty were iden-
tified, evaluated, and then individually quantitated. These sources
were related to analytical weighing procedures (standards and
samples), dilutions, and absorbance interpolations in analytical
curves. The measurement model used in this approach was
L:V
C mg kg−1 ¼ ð1Þ
m
where Cmg kg−1 is the analyte concentration (mg kg−1), m is the
sample weight (g), V is the volume of stock solution after
digestion (mL), and L is the absorbance interpolations in an-
alytical curves (mg L−1).
The standard uncertainty due to weighing (m) took into
account the eccentricity error and the mass displayed by the
analytical balance. The latter was calculated by considering
the measurement uncertainty of the maximum error accepted
by the laboratory and the instrument resolution. This uncer-
tainty was calculated by the following equation:


∂m 2
u2 ðmðExt ; M t ; Ex; M ÞÞ ¼ :ðσExt Þ2
∂Ext


∂m 2
þ :ðσM t Þ2
∂M t


∂m 2
þ :ðσExÞ2
∂Ex


∂m 2
þ :ðσM Þ2 ð2Þ
∂M
where u(m) is the weighing standard uncertainty, Ex is the ec-
centricity error (g), M is the value displayed by the balance (g), t
is the alternative index for the tare of the balance, (σ xi)2 is the
2
output standard uncertainty, and ∂m
∂xi is the sensitivity coefficient.
Food Anal. Methods (2017) 10:1787–1799 1791

The standard uncertainty due to the diluting process (V) The top-down approach (in-house validation) was obtained
was calculated by considering the uncertainties, errors, and by combining the deviations related to repeated measurements
performance data of volumetric flasks. This uncertainty in repeatability and in-house reproducibility conditions. The
source was calculated by the following equation: contributions due to ruggedness deviation and bias were also

 considered. The model for the calculation of measurement
∂V 2 uncertainty was
u ðV ð V 1 ; V 2 ; …; V i ÞÞ ¼
2
:ðσV 1 Þ2
∂V 1

 u2 ðR&R&R&bÞ ¼ ðσS r Þ2 þ ðσS zi Þ2 þ ðσS Di Þ2
∂V 2
þ :ðσV 2 Þ2 þ …
∂V 2 þ ðσS bias Þ2 ð6Þ


∂V 2 where u(R & R & R & b) is the combined standard uncertainty
þ :ðσV i Þ2 ð3Þ
∂V i due to repeatability, in-house reproducibility, method rugged-
ness, and bias (dimensionless); (σSr)2 is the repeatability stan-
where u(V) is the volume standard uncertainty, V1 is the dard deviation (dimensionless); (σSzi)2 is the in-house repro-
uncertainty of the pipetted volume (mL), V2 is the un- ducibility standard deviation (dimensionless); (σSDi)2 is the
certainty of the final volume (mL), V1i is the infinite ruggedness deviation (dimensionless); (σSbias)2 is the bias de-
variable for volume (mL), and σVi is the sensitivity viation; and i is the number of applied variations.
coefficient. The top-down approach (proficiency testing (PT) scheme)
The standard uncertainty due to the sample concentration related to the interlaboratory validation was obtained from the
as a result of its interpolation in the calibration curve (L) was proficiency test’s participants standard deviation variability
calculated by adopting the least squares method by the follow- (SR), when a target value was assigned. The standard uncer-
ing equation: tainty was calculated by the following equation:
v6 ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ffi
u6
u6  2 7 7 σS R
7 uðPTÞ ¼ pffiffiffi ð7Þ
Sy uu6 1 1 C−x 7 n
uðLÞ ¼ *u6 4 þ þ   7
5 ð4Þ
b t N n N 2
∑ xi −x where u(PT) is the uncertainty calculated with data from pro-
i¼1
ficiency testing, σSR is the standard deviation of n results
where u(L) is the standard uncertainty due to the residue of the (mg kg−1), and n is the number of compliant results achieved
calibration curve, S Y is the residue standard deviation by the proficiency test’s participants.
(mg L−1), b is the angular coefficient (absorbance mg−1 L), n The measurement uncertainty due to the combination of
is the number of measurements in xi, C is the analyte concen- approaches was also estimated. Firstly, the traditional ap-
tration (mg L−1), N is the number of C measurements, x is the proach was combined with random effects obtained during
value of calibration standards, and i is the index for the num- in-house validation, represented by standard uncertainty
ber of measurements needed to the preparing of calibration (u(R & R & R & b)). Then, it was combined to the variability
curve. of the proficiency test’s participants, given to the standard
The combined standard uncertainty was calculated by com- uncertainty (u(PT)). The sources of uncertainty were individ-
bining the output standard uncertainties of the overall mea- ually assessed to check their contribution to the overall value,
surement model. The calculation was performed in accor- aiming to define the more appropriate approach to be applied
dance to the law of propagation of uncertainties by using a to the analytical method.
Taylor series expansion through the following equation: Finally, the expanded standard uncertainty was obtained by
multiplying the coverage factor (95.45%) by the combined


2 standard uncertainties, which have been obtained from the
∂C 2 2 ∂C
uc ðC ðm; V; LÞÞ ¼
2
:ðσmÞ þ :ðσV Þ2 different approaches by the following equation:
∂m ∂V

2 U ¼ k:uc ðyÞ ð8Þ
∂C
þ :ðσLÞ2 ð5Þ
∂L where U is the expanded standard uncertainty, uc(y) is the
combined standard uncertainty, and k is the coverage factor.
where uc is the combined standard uncertainty; C is the
2
measurand; ∂C∂xi are the sensitivity coefficients; m, V, and L Method Applicability
are the variables of the overall measurement model; and
(σxi)2 is the output standard uncertainty of each variable of After validation, method applicability was checked on several
the overall measurement model. samples of feedingstuffs collected by the Brazilian official
1792 Food Anal. Methods (2017) 10:1787–1799

feed inspection service, by adopting both digestion ap-
proaches. In total, 50 samples of feedingstuffs have been an-
alyzed, consisting of mineral feed with protein (n = 10), fish
meal (n = 3), pig feed (n = 2), cattle feed (n = 1), meat and
bone meal (n = 1), blood meal (n = 1), feather meal (n = 1),
and wheat flour (n = 1). All results were expressed as means of
triplicate analysis with the measurement uncertainty from the
more appropriate approach.
Results and Discussion
Due to the high chemical recalcitrance and long organic
chains, the elemental analysis of complex matrices, with var-
ied chemical composition, usually challenges digestion proce-
dures (Enders and Lehmann 2012). In this study, two distinct
digestion methods for the analysis of Cd and Pb in
feedingstuffs were developed. Several studies have been dem-
onstrating the advantages of microwave digestion, in compar-
ison to the classic dry digestion. Microwave has been related
to higher-efficiency digestion and lower contamination during
sample prepare. It is a faster process, which usually leads to
more precise results (MacCarthy and Ellis 1990; Aydin 2008;
Demirel et al. 2008; Yang et al. 2013). Moreover, with the
digestion of samples in a pressurized system, better recoveries
are achieved, because the loss of volatile analytes is mini-
mized (Aydin 2008). As it can be seen in Table 2, our results
demonstrate that the microwave digestion approach was effi-
cient, since adequate recoveries (among 80–110%) and low
coefficients of variance (CVs) were achieved. Mixing HNO3
and HCl was an adequate strategy to digest samples with high
amounts of organic compounds, as well as inorganic samples
such as mineral feeds. Passivation of the analytes has not been
observed by using this mixture. Moreover, there was no need
for using acids with high complexing properties (such as
fluoridric acid) for the digestion of mineral feeds, neither ad-
ditional reagents (such as hydrogen peroxide and HClO4) to
the decomposition of organic matter. This is advantageous for
the increased security of analysts in the handling of chemicals
and for the lower reagent consumption during routine
analysis.
In general, recovery rates were about 100% for both Pb and
Cd (Fig. 1). Recovery rates were 98 and 90% (Cd) and 106
and 91% (Pb) by microwave digestion and dry digestion, re-
spectively. Lower recoveries were already expected for Cd,
since it is a volatile species. On the other hand, the determi-
nation of Pb usually deals with many problems such as the
contamination of reagents and materials, which can lead to
expressive analytical blank solutions and higher analytical
values than the actual concentration. Since average recovery
rates were satisfactory for both digestion processes, such prob-
lems did not seem to influence the final results of this work.
Since the 80s, the calcination in muffle has been defended
as an open digestion system, mainly by its operational saving
(Adeloju 1989). This type of digestion is economically viable
and affordable, since it requires a smaller quantity of
chemicals and an easy to use equipment (muffle furnace), in
comparison to the high cost of a microwave system.
Furthermore, it results in fewer risks related to the use of
chemical reagents, lower background signals (due to the low
quantity of reagents), and the ability to handle considerably
larger amounts of sample, which avoids homogeneity issues
often found in these kinds of samples (Akinyele and Shokunbi
2015). In this research, other issues such as the volatility of the
analytes were round by transforming the matrix into sulfate
(CdSO4 and PbSO4) before calcination. This practice was also
successful when samples of fish were digested with H2SO4
before calcination, avoiding losses of Cd due to its volatility
(Molognoni et al. 2016). As it can be seen in the first
interlaboratory round, Cd results were far below the target

Table 2 Results of traditional in-

house validation for the analysis Conventional performance evaluation Analytes
of cadmium and lead in blank
samples of mineral feeds by Cd Pb
microwave digestion and flame
atomic absorption spectrometry Linearity (R2) >0.999 >0.999
Limit of detection (mg L−1) 0.01 0.16
Limit of quantitation (mg L−1) 0.04 0.52
Limit of decision (CCα) (mg kg−1) 17.05 114.00
Capacity of decision (CCβ) (mg kg−1) 19.09 127.99
Robustness deviation 0.04 0.02
In-house reproducibility deviation 0.10 0.06
Bias deviation 0.02 0.01
Fortified level (mg kg−1) 7.50 15.00 22.50 50.00 100.00 150.00
Recovery rate (%) 84.00 98.00 91.00 108.00 106.00 105.00
Repeatability (CV%) 2.64 3.60 0.03 5.55 1.04 0.01
In-house reproducibility (CV%) 9.89 0.38 8.22 5.70 0.40 1.13
Food Anal. Methods (2017) 10:1787–1799 1793

Fig. 1 Graphical plot of the

recovery of fortified blank sample
for the analysis of cadmium and
lead in feeds by microwave and
dry ashing

value (median), which demonstrated great losses of this ele- which was performed by previously transforming the analyte
ment due to its volatility, with recovery rate of only 26.9% and into sulfate, the volatility issue was solved. An acceptable
z-score around −5.0 (Fig. 2). In the second proficiency round, recovery of 82.2% has been then achieved; thus, a proficient

*This study: F-AAS; round 1: 36.7% used ICP-MS; 26.7% used FAAS; 20.0% used AAS-graphite furnace; and 16.6% used
ICP-AES; round 2: 31.5% used ICP-MS; 26.4% used AAS-graphite furnace; 23.7% used ICP-AES and 18.4% used FAAS;
Round 3: 36.4% used ICP-MS; 24.2% used FAAS; 21.2% used ICP-AES and 18.2% used AAS-graphite furnace.
Fig. 2 Proficiency testing rounds and respective z-scores considered for the interlaboratory validation approach for the analysis of cadmium in feeds*
1794 Food Anal. Methods (2017) 10:1787–1799

performance could be reported (z-score = −1.0). Finally, in the
third proficiency round, an even better accuracy was achieved
(z-score = −0.5; recovery = 95.6%) due to the optimization of
the acid addition before calcination. On the other hand, when
presented in the form of sulfate, the unsatisfactory solubility of
Pb and its contamination by the addition of acid lead to dis-
agree with the target value in the first proficiency round.
However, corrective actions, such as adding a buffer solution
and the use of analytical blank solutions, were enough to solve
these issues, as well as the chemical interference of double
sulfates in the flame. This was evidenced by the improvement
of laboratory’s accuracy along the three proficiency rounds,
leading to a perfect result (z-score = 0) (Fig. 3). As shown in a
study conducted by Enders and Lehmann (2012), dry-ashing
digestion methods can be modified to achieve a better recov-
ery, precision, and adsorption of analytes in the decomposition
vessels. With this aim, we modified a calcination method to
avoid losses due to Cd volatility and Pb solubility problems,
proving to be a viable and cheaper alternative. In general, the
d r y d i g e s t i on s ho w ed a w o r s e p e r f o r m a n c e a n d
reproducibility in relation to microwave digestion system, es-
pecially for Cd. However, all the results from both methods
were considered satisfactory, according to both validation
approaches.
Regardless of the analytical method and technique, the re-
liability of results must be checked by validation procedures
(Associação Brasileira de Normas Técnicas 2005). Method
validation by using an interlaboratory approach becomes a
possibility if a sufficient number of participants use similar
analytical methods and techniques. In addition, more than an
interlaboratory round is required, so that assessments must be
carried out and possible corrective actions can be taken. This
approach, if properly assessed, is an alternative to convention-
al collaborative and validation studies, which are known to be
more costly (Thompson et al. 2006). In this work, all the
results of conventional validation parameters were in accor-
dance with the criteria established by the Commission
Decision 657/2002/EC (Table 2) (European Commission
2002a, b). The method was linear in the adopted analytical
ranges, since the regression coefficients were greater than 0.99

*This study: F-AAS; round 1: 36.7% used ICP-MS; 23.3% used FAAS; 23.3% used AAS-graphite furnace and 16.7% used
ICP-AES; round 2: 36.4% used ICP-MS; 30.3% used AAS-graphite furnace; 21.2% used FAAS; and 12.1% used ICP-AES;
round 3: 38.8% used ICP-MS; 22.6% used AAS-graphite furnace; 19.3% used FAAS and 19.3% used ICP-AES.
Fig. 3 Proficiency testing rounds and respective z-scores considered for the interlaboratory validation approach for the analysis of lead in feeds*
Food Anal. Methods (2017) 10:1787–1799 1795

Table 3 Comparison of results

by flame atomic absorption Sample Digestion Determination Analytical technique
spectrometry (FAAS) and
inductively coupled plasma mass FAAS ICP-MS
spectrometry (ICP-MS) for the
specificity/selectivity evaluation Premix Microwave Cd NQ a <0.04 mg kg−1 a
−1
Pb 12.14 mg kg b 11.41 mg kg−1 b
−1
Mineral feed Microwave Cd 1.59 mg kg c 1.54 mg kg−1 c
Pb 2.36 mg kg−1 d 1.94 mg kg−1 d

Lines with results with the same lowercase letter do not differ significantly (P > 0.05). Limits of detection
(mg kg−1 ), 0.01 (Cd) and 0.16 (Pb)
NQ not quantifiable

in the 3 days of validation. On the other hand, neither extreme
values nor tendencies were identified in the analytical curves.
Statistical methods are often required to detect trends and
draw conclusions from experimental data (Granato et al.
2014). With this aim, the Anderson-Darling test was applied
to confirm the normal distribution of the residuals of the re-
gression, with P = 0.1108 > 0.05 values, P = 0.2111 > 0.05,
and P = 0.1374 > 0.05 for the first, second, and third days of
analytical curves, respectively. The distribution of the resid-
uals of the regression was homogeneous; thus, homoscedas-
ticity was demonstrated. Moreover, there was no significant
difference among the curve derivatives calculated for three
separate validation days (P > 0.05). By comparing the slopes
of the different analytical curves (solvent, matrix fortified be-
fore digestion, matrix fortified after digestion), no significant
effect matrix was evidenced (P > 0.05). Therefore, method
applicability was assessed using solvent analytical curves.
Accuracy and recovery were satisfactory, since recoveries
ranged from approximately −20 to +10%, with CV <10%
(Table 2). There was no significant difference among the re-
sults of the analysts when subjected to in-house reproducibil-
ity conditions (P > 0.05). Selectivity/specificity in feed matri-
ces was also considered satisfactory, since no significant dif-
ference could be reported among samples analyzed by ICP-
MS and FAAS (P > 0.05) (Table 3). Specificity is the unequiv-
ocal measurement capability of the analyte in the presence of
endogenous and exogenous matrix components. Mass spec-
trometry is an adequate tool to check the authenticity of the
analytes in both detection/quantitation methods due to its high
selectivity and specificity (European Commission 2002a, b).
According to the criteria of the Youden’s test, the FAAS
method has proved to be robust when subjected to small var-
iations. The deviation due to ruggedness was even lower than
the largest source of random error due to method validation,

Fig. 4 Lenth’s plot for the

ruggedness assessment of the
cadmium and lead analysis by
microwave digestion and flame-
atomic absorption spectrometry*

*ME: margin of error; volume of HCl (2.0 and 1.9 mL) (A); volume of HNO3 (6.0 and 5.9 mL) (B); supplier of HCl
(two different suppliers) (C); size of the holes of the grinding sieve (0.5 and 1.0 mm) (D); variation in the
nebulization flow (2% variation towards optimum condition) (E); variation in burner height (7.0 and 6.9 mm) (F) and
heating time of hollow cathode lamps (30 and 40 min) (G).
1796 Food Anal. Methods (2017) 10:1787–1799

* Expanded standard uncertainties calculated for the concentrations of 15 mg kg-1 (cadmium) and 100 mg kg-1 (lead).
PT scheme approach obtained from data of the third proficiency testing round for both analytes.
Fig. 5 Values of the expanded standard uncertainties obtained by different approaches*

the laboratory reproducibility (Table 2). Moreover, the Lenth’s
plot demonstrates that the assessed factors were much lower
than the calculated margin of error; thus, no influence on the
results could be noticed (Fig. 4). The stability study of the
digested stock solution demonstrated that up to the 40th day,
the solutions are still stable at room temperature (25 °C) for
both analytes, even by using a buffer solution during sample
preparation. The main goal of adding the buffer solution is the
formation of double sulfate with La, thus favoring the atomi-
zation of Pb in the flame (Gonçalves 1983). There was no
significant difference among the results of the solutions ana-
lyzed in each period and the reference value (obtained on day
1). This process simulated the laboratory routine, where solu-
tions are stored at room temperature in the waiting of larger-
volume samples to be injected onto the equipment. The meth-
od also had adequate sensitivity to its purpose, with LOD and

Table 4 Results of the overall uncertainties due to measurements of cadmium and lead in feeds by flame atomic absorption spectrometry, calculated by
the traditional approach

Type Measurement Quantity value Uncertainty value Divisor u(xi) Entrance unity Distribution Ci u(yi) Output unity Veff
variable

Quantitation of cadmium
B u(m) mass 0.5000 0.0005 2.02 0.0002 g t-Student −777.15 0.18 mg kg−1 ∞
B u(V) volume 25.00 0.01 2.02 0.01 mL t-Student 15.62 0.09 mg kg−1 ∞
A u(L) interpolation 0.30 0.03 2.23 0.02 mg L−1 t-Student 49.73 0.75 mg kg−1 12.09
in curve
Combined uncertainty (uc) = 0.77 Veff = ∞
Expanded uncertainty (U) = 1.56
Coverage factor (k) = 2.00
Quantitation of lead
B u(m) mass 0.5000 0.0005 2.02 0.0002 g t-Student −10,102.12 2.33 mg kg−1 ∞
B u(V) volume 25.00 0.01 2.02 0.01 mL t-Student 203.12 1.12 mg kg−1 ∞
A u(L) interpolation 2.00 0.004 2.23 0.002 mg L−1 t-Student 49.73 0.11 mg kg−1 12.09
in curve
Combined uncertainty (uc) = 2.59 Veff = ∞
Expanded uncertainty (U) = 5.18
Coverage factor (k) = 2.00

Type A method of evaluation of uncertainty by the statistical analysis of series of observations, type B method of evaluation of uncertainty by means other
than the statistical analysis of series of observation, u(xi) entrance standard uncertainty, Ci sensitivity coefficient, u(yi) output standard uncertainty, Veff
effective degrees of freedom
Food Anal. Methods (2017) 10:1787–1799 1797

Table 5 Analysis of cadmium

and lead in real feed samples by Total samples (n = 50) Contentsa (±MU mg kg−1) Digestion by
both microwaves and dry-ashing
digestions and quantitation by Cadmium Lead
flame atomic absorption
spectrometry Mineral feed (n = 30) Microwave
Minimum concentration 0.18 ± 0.02 9.03 ± 1.15
Maximum concentration 8.71 ± 0.90 7430 ± 930
Average 1.99 ± 0.22 403.58 ± 50.45
Unquantifiable samples n=5 n = 20
Mineral feed with protein (n = 10) Microwave
Unquantifiable samples n = 10 n = 10
Fish meal (n = 3) Dry ashing
Minimum concentration 0.11 ± 0.01 7.14 ± 0.90
Maximum concentration 0.15 ± 0.01 11.53 ± 1.44
Average 0.13 ± 0.01 9.35 ± 0.90
Pig feed (n = 2) Dry ashing
Minimum concentration <LOQ 0.55 ± 0.06
Maximum concentration <LOQ 2.74 ± 0.40
Average <LOQ 1.64 ± 0.11
Unquantifiable samples n=2 –
Cattle feed (n = 1) Dry ashing
Concentration <LOQ <LOQ
Meat and bone meal (n = 1)
Concentration <LOQ 4.95 ± 0.62
Blood meal (n = 1)
Concentration <LOQ <LOQ
Feather meal (n = 1)
Minimum concentration <LOQ 5.00 ± 0.62
Wheat flour (n = 1)
Minimum concentration <LOQ <LOQ
a
Measurement uncertainty (MU) was calculated by using combined approach (traditional + PT scheme) and an
effective degree of freedom that corresponds to a probability of coverage factor of approximately 95.45%; limits
of quantitation (LOQ)—cadmium 0.04 mg L−1 and lead 0.52 mg L−1

LOQ. In addition, settling other analytical limits such as CCα
and CCβ is important to estimate the results’ level of trust and
to prevent false-negative and false-positive results (Table 2).
Interlaboratory studies are a way to predict the performance
of analytical methods from external data, which are indepen-
dent from the accuracy obtained in-house. If laboratories use
in-house evaluations only, without an external reference, the
possibility of executing methods influenced by random vari-
ations or trends for long periods can raise, thus affecting the
final value of the measurand (Thompson et al. 2006). In this
context, the interlaboratory approach is effective for the per-
formance evaluation of the analytical methods. Based on the
proficiency rounds, it was possible to adjust the digestion
method initially developed, which resulted in consistent and
reliable measurements, compared to the conventional true val-
ue. On the other hand, in order to produce consistent data in its
routine, a laboratory should also implement an adequate mon-
itoring program of its performance. These procedures should
consider both in-house and interlaboratory studies, since they
complement each other and provide greater reliability to the
results.
Furthermore, regardless of the adopted validation ap-
proach, overall performance data are usually applied to the
calculation of measurement uncertainty (ISO/IEC Guide 98-
3:2008 2008). Figure 5 shows the values of the expanded
standard uncertainties calculated by in-house validation top-
down approaches, which were similar even when separately
evaluated. These were low values that underestimated the final
result. On the other hand, the value obtained by the traditional
GUM approach was higher than any source of variation rep-
resented by the in-house validation approach, such as preci-
sion, recovery, and ruggedness, because the major sources of
uncertainty were related to analytical processes represented by
the measurement model. In the determination of Cd, the larg-
est source of uncertainty was related to the interpolation of the
absorbance in the calibration curve. In the Pb analysis, the
weighing and diluting analytical procedures presented a wide
variation in comparison to other processes, such as random
1798
effects (Table 2). This can be understood because the method
was developed with constant masses and dilution processes.
Thus, maximum assigned errors, higher than the balance cal-
ibration errors, have been considered in addition to dilution
processes. Furthermore, these errors propagate several times,
such as in the preparing of analytical curves.
Combining different approaches to estimate the MU may
provide a better representation of the measurement process
due to the inclusion of random effects (type A uncertainties)
to the traditional modeling (Eurolab 2007). In this case, the
traditional approach combined to the top-down in-house val-
idation conferred no changes to the MU value, since adequate
precision, ruggedness, and recovery were achieved. This was
evident by finding the same measurement uncertainty values
for the traditional approaches, with and without combinations.
The top-down approach is often used to estimate the MU by
using proficiency testing data. Compared to the traditional
approach, its calculation is simple and practical (Ellison
2014). However, sometimes it can lead to values that do not
represent the method’s actual routine, for not containing all the
information that affect the measurement quality (Magnusson
et al. 2004). In this work, the use of the top-down PT approach
was considered adequate. This approach usually overesti-
mates or underestimates the result, when used for comparisons
to other values, specifications, and regulations. This was al-
ready discussed by Lecomte et al. (2012), who showed that
this approach has not been considered optimal for
underestimating results, in comparison to the traditional ap-
proach (Table 4). On the other hand, when combining the
traditional approach to the PT variations, the results of Pb
and Cd were better represented. This was due to a greater set
of hitting probability, within the acceptable range according to
the Guidelines on Measurement Uncertainty, which recom-
mends maximum MU values in specific concentrations of
the measurands (Codex Alimentarius Commission 2011). In
this research, maximum values of 16 and 22% were consid-
ered for Pb and Cd concentrations of 100 and 15 mg kg−1,
respectively (Fig. 5). The adopted MU approach was then
combined with PT scheme. This approach covered the proba-
bility of the results more appropriately and was considered
ideal for comparisons and decision making, increasing the
reliability of the results. The α and β errors were similar con-
tributions to the overall approach in the in-house validation.
Same sources, such as the precision deviations, are considered
for the calculation of both errors. The values were equal to or
greater than the total in-house approach, because these limits
are calculated with all fortification points applied to the meth-
od validation, and not only the levels of interest, such as the
other approach (Fig. 5).
The validated method proved to be suitable for the routine
analysis of a wide variety of samples (Table 5). There was a
greater amount of quantifiable results for the determination of
Pb, ranging from traces (0.55 ± 0.06 mg kg−1) up to extremely
Food Anal. Methods (2017) 10:1787–1799
high concentrations (7430 ± 930 mg kg−1). Cd was also quan-
tified in some samples, ranging from 0.11 ± 0.01 to
8.71 ± 0.90 mg kg−1. The higher concentrations of both metals
were found in mineral mixtures and fish meal.
Conclusion
Both digestion approaches developed in this work were effec-
tive, as demonstrated by their applicability. Dry ashing was a
viable alternative, leading to lower operating costs for the
digestion of samples, even after few adjustments and adapta-
tions. Despite having a lower performance compared to the
microwave digestion, the results were also satisfactory and
reproducible. The validation approaches used for both the
overall method and the alternative digestion method demon-
strate the importance of applying control tools for measuring
quality, whether being through in-house or interlaboratory ap-
proaches. However, linking approaches must be considered to
generate additional data. Regardless of the approach used for
estimating the uncertainty, its detailed study must always be
considered. An uncertainty estimation calculation cannot be
limited to validation studies only, as other sources can be
significant.
Acknowledgments We acknowledge Food and Agriculture
Organization of the United Nations and the Austrian Agency for Health
and Food Safety for authorizing the use of the FAO-IAG Ringtests data.
ICP-MS analyses were carried out at Universidade Federal de Santa
Catarina. We would like to acknowledge Vera Lucia Azzolin Frescura
Bascuñan for this facility. We are also grateful to Fundação de Amparo à
Pesquisa e Inovação de Santa Catarina (FAPESC) for funding this re-
search with fellowships (Grant No. 1683).
Compliance with Ethical Standards
Conflict of Interest Luciano Molognoni declares that he has no con-
flict of interest. Jaqueline Zarpelon declares that she has no conflict of
interest. Leandro Antunes de Sá Ploêncio declares that he has no conflict
of interest. Jacson Nascimento dos Santos declares that he has no conflict
of interest. Heitor Daguer declares that he 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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