Food Additives and Contaminants

Vol. 29, No. 4, April 2012, 679–693

Modelling uncertainty estimation for the determination of aflatoxin M1 in milk by visual and
densitometric thin-layer chromatography with immunoaffinity column clean-up
K.L. Carvalhoa, G.A.A. Gonçalvesa, A.L. Lopesa, E.A. Santosa, E.A. Vargasa and W.F. Magalhãesb*
a
Laboratory of Quality Control and Safety Food – LACQSA/LANAGRO-MG/MAPA, Av. Raja Gabaglia, 245, Cidade Jardim,
CEP 30380-090 – Belo Horizonte, MG, Brazil; bChemistry Department, Universidade Federal de Minas Gerais – UFMG, Av.
Pres. Antonio Carlos, 6627, Campus Pampulha, CEP 31270-901 – Belo Horizonte, MG, Brazil
(Received 26 November 2010; final version received 7 November 2011)

The uncertainty of aflatoxin M1 concentration in milk, determined by thin-layer chromatography (TLC) with
visual and densitometric quantification of the fluorescence intensities of the spots, was estimated using the cause-
and-effect approach proposed by ISO GUM (Guide to the expression of uncertainty in measurement) following
its main four steps. The sources of uncertainties due to volume measurements, visual and densitometric TLC
calibration curve, allowed range for recovery variation and intermediary precision to be taken into account in the
uncertainty budget. For volume measurements the sources of uncertainties due to calibration, resolution,
laboratory temperature variation and repeatability were considered. For the quantification by visual readings of
the intensity of the aflatoxin M1 in the TLC the uncertainty arising from resolution calibration curves was
modelled based on the intervals of concentrations between pairs of the calibration standard solutions. The
uncertainty of the densitometric TLC quantification arising from the calibration curve was obtained by weighted
least square (WLS) regression. Finally, the repeatability uncertainty of the densitometric peak areas or of the
visual readings for the test sample solutions was considered. For the test samples with aflatoxin M1 concentration
between 0.02 and 0.5 mg l1, the relative expanded uncertainties, with approximately 95% of coverage probability,
obtained for visual TLC readings were between 60% and 130% of the values predicted by the Horwitz model.
For the densitometric TLC determination they were about 20% lower. The main sources of uncertainties in both
visual and densitometric TLC quantification were the intermediary precision, calibration curve and recovery. The
main source of uncertainty in the calibration curve in the visual TLC analysis was due to the resolution of the
visual readings, whereas in the densitometric analysis it was due to the peak areas of test sample solutions
followed by the intercept and slope uncertainties of the calibration line.
Keywords: chromatographic analysis; clean-up – affinity columns; statistical analysis; regression; measurement
uncertainty; aflatoxins; mycotoxins – aflatoxins; mycotoxins; milk

Introduction determined by thin-layer chromatography (TLC) with

One of the most important metrological characteris-
tics of a measurement result is its uncertainty. It is a
worldwide consensus that a result is not complete
without an expression of its uncertainty (ABNT,
INMETRO 2003; BIPM et al. 1995) and its estima-
tion is a requirement for testing laboratories accred-
itation by the International Organization for
Standardization (ISO) (2005). The present paper
uses the methodology called the cause-and-effect
approach (Ellison and Barwick 1998a, 1998b;
Barwick and Ellison 1998), or GUM approach
(ABNT, INMETRO 2003; BIPM et al. 1995) or
bottom-up approach to estimate the uncertainty of
aflatoxin M1 concentration in bovine milk which was
visual and densitometric quantification. Within this
methodology, this work addresses especially to the
following issues:
. Evaluation of the uncertainty due to the
resolution of the intensity of aflatoxin
M1 in the TLC quantification by a visual
reading.
. Accounting for the heteroskedasticity of the
instrumental response (fluorescence intensity)
in the densitometric TLC calibration by using
weighted least squares (WLS).
. Inclusion of an uncertainty contribution due
to the uncorrected bias within an allowed
range for the recovery variation.

*Corresponding author. Email: welmag@ufmg.br

ISSN 1944–0049 print/ISSN 1944–0057 online

ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/19440049.2011.648959
http://www.tandfonline.com
680 K.L. Carvalho et al.
. Accounting for a within-laboratory reproduc-
ibility or intermediary precision through the
data of the analysis of control samples for
quality control.
Other approaches that estimate the uncertainty of
an analytical measurement make use of data obtained
from a method validation study (Ellison et al. 2000;
Barwick and Ellison 2000a, 2000b; Barwick et al. 2000)
or from interlaboratory studies through the results of
collaborative trials (Adriaan et al. 1998; Barwick and
Ellison 1998; Ellison 1998).
Recent publications have addressed considerations
to assess the uncertainty of aflatoxin M1 determination
(Calaresu et al. 2006; Populaire and Giménez 2006).
Calaresu et al. (2006) used data obtained from preci-
sion, trueness and ruggedness studies during method
validation to estimate uncertainty components.
However, the uncertainties due to the input quantities
appearing in their measurand equation and in their
cause-and-effect diagram were not included in their
uncertainty budget. For instance, glassware tolerances
were used to estimate uncertainties and not the data
from the calibration certificates and laboratory tem-
perature variation which appear in the cause-and-effect
diagram. A very simple measurand equation was
presented where only three input quantities appear:
the volume of the sample taken for analysis, the final
volume of the sample solution, and the concentration
of aflatoxin evaluated from the calibration curve.
However, nothing was presented about the least square
procedure used to fit the calibration curve data, nor
about both the calibration curve range or the values of
the intercept, the slope of the calibration curve, and
their uncertainties and covariance.
Populaire and Giménez (2006) compared the
uncertainty estimation obtained from the bottom-up
method with the top-down method. They concluded
that the main sources of uncertainties in different
analytical methods were mainly due to the intermedi-
ary precision, accuracy/recovery and, in some cases,
calibration. They also concluded that the uncertainties
due to weighting and volume measurement were in
general negligible. However, no details of their calcu-
lations were given, also they used a poor calibration
design with only one standard calibration solution.
The present paper presents a detailed uncertainty
budget to estimate the measurement uncertainty for
aflatoxin M1 determination in milk with immunoaffi-
nity column clean-up and thin-layer chromatographic
(TLC) determination with visual and densitometric
quantification.
Aflatoxin M1 (4-hydroxyaflatoxin B1, (6aR-cis)-
2,3,6a,9a-tetrahydro-9a-hydroxy-4-methoxycyclopenta
[c]furo[30 ,20 :4,5]furo[2,3-h][l]benzopyran-1,11-dione) is
a genotoxic carcinogenic hydroxylated metabolite of
aflatoxin B1 found in the milk of animals that have
consumed feedstuffs contaminated with aflatoxin B1.
Recent reports of the International Agency for
Research on Cancer (IARC) assessed the potential
carcinogenic risks to humans (World Health
Organization (WHO) and IARC 2002) of aflatoxin
M1, and concluded that even very low levels of
exposure to aflatoxins, i.e. 1 ng kg1 bw day1 or less
contribute to a risk of liver cancer (Byrne 2000). Due it
high human risks the European Union has established
a very restrict maximum residue limit (MRL) or
maximum permitted limit (MPL) for aflatoxin M1
content in milk at 0.050 mg kg1 (Commission of the
European Communities 1998, 2001, 2004, 2010).
European Union legislation (Commission of the
European Communities 2006) states that the uncer-
tainty of the analytical measurement for aflatoxin M1
contamination should be considered when reporting
and interpreting the analytical results (Commission of
the European Communities 2002, 2004, 2006). Besides,
European Union legislation (Commission of the
European Communities 2006) establishes the necessity
of declaring the correction of the aflatoxin M1
contamination by the recovery, or not, and what is
the recovery. It also establishes the minimum perfor-
mance criteria that an analysis procedure should meet
to be used in the analysis of aflatoxin M1 in milk that
was used when setting criteria for method perfor-
mance. MERCOSUR and Brazil (Agência Nacional de
Vigilância Sanitária (ANVISA) 2011; MERCOSUR/
GMC/RES 2002) have established limits of 0.5 and
5 mg kg1 for aflatoxin M1 in milk and powdered milk
along sampling plans (Mercosur), but no method
performance has been established, nor the necessity
of reporting the recovery or uncertainty of the analyt-
ical measurement. Recovery and measurement uncer-
tainty have been reported when demanded by
customers or according to institution policies as
defined by ISO/IEC 17025 (ISO 2005).
The LACQSA/LANAGRO-MG/MAPA labora-
tory is accredited according ISO/IEC 17025 by the
national metrology institute – the Instituto Nacional
de Metrologia Normalização e Qualidade Industrial
(INMETRO) – to realise the analytical procedure
for the determination of the herein reported ‘determi-
nation of aflatoxin M1 in milk by visual and densito-
metric thin-layer chromatography (TLC) with
immunoaffinity column clean-up’.
Materials and methods
Uncertainty estimation procedure
In the cause-and-effect approach for uncertainty esti-
mation, or bottom-up approach, the detailed knowl-
edge of the chemical analytical procedure is necessary
to enable the correct identification of the measurand
and of its sources of uncertainties. In this paper the

four steps presented elsewhere were strictly followed
(Carvalho, Santos, et al. Forthcoming).
Step 1: Measurand specification
Measurand specification is realised through the mea-
surand equation and the measurement procedure
(ABNT, INMETRO 2003; BIPM et al. 1995; Ellison
et al. 2000) or analytical procedure, which is normally
documented through the standard operation proce-
dures. The analytical procedure for aflatoxin M1
determination using TLC is summarised below.
Complete measurand equations, also presented
below, show in detail all the direct measurements
realised during the analytical procedure using TLC
with densitometric quantification (Equation 2) or with
visual quantification (Equation 3).
Analytical procedure
The analytical procedure for aflatoxin M1 determina-
tion was adapted from a published immunoaffinity
clean-up method (Dragacci et al. 2001). It can be
summarised in the following steps:
(1) A volume of 100 ml (Vs) of defatted milk was
taken after centrifugation and filtered by using
folded filter paper.
(2) Clean-up was done by using an immunoaffinity
column (VICAN or R-Biopharm), washing
with 5 ml of water, followed by the elution of
aflatoxin M1 with a solution of 2.5 ml of
acetonitrile and methanol (2:1, v/v) and 2.5 ml
of pure methanol. The sample eluate was
evaporated at 40 C under nitrogen bubbling
just before complete dryness (critical step).
(3) The dried extract was re-dissolved with the
addition of 100 ml (Vr) of toluene–acetonitrile
(9:1 v/v) and by homogenisation with sonica-
tion and vortexed mix.
(4) Application of 20 ml (Va) of the sample extract
obtained in step 3 and of 10 ml (Vp) of the
calibration aflatoxin M1 standard solutions (six
spots) was made on the TLC plate, followed by
elution with ether–methanol–water (96:3:1 v/v/
v).
(5) Detection and quantification step: the fluores-
cence intensities of the spots for the test
samples and the calibration solutions were
read under ultraviolet light ( ¼ 365 nm) by
visual comparison between sample spots and
calibration standard solutions spots or by using
a densitometer (dual wavelength flying spot
scanning densitometer; model CS-9301PC,
Shimadzu, São Paulo, Brasil) at a wavelength
of 365 nm.
Food Additives and Contaminants 681
Step 2: Identifying the sources of uncertainties
As proposed elsewhere (Carvalho, Santos, et al.
Forthcoming) the cause-and-effect, or Ishikawa, dia-
gram was constructed by considering the input quan-
tities that appear in the measurand equation as the
primary sources of uncertainties. Other sources of
uncertainties considered are the allowed range of
uncorrected bias due to the allowed variation of the
recovery ratio (RR), and the intermediary precision or
internal reproducibility or intra-laboratory reproduc-
ibility obtained from the analysis of control samples
for quality control.
Volumes
For the measurements of volume, four basic sources of
uncertainties were considered: instrumental uncer-
tainty (Joint Committee for Guides in Metrology
(JCGM/WG 2) et al. 2008; INMETRO 2009) due to
the formal calibration of the instrument of volume
measurement; uncertainty due to resolution for grad-
uated volume instruments; uncertainty due to the
variation of the laboratory temperature around the
mean laboratory temperature; and uncertainty due to
volume measurement repeatability.
Calibration
For TLC calibration, six standard calibration solutions
were used labelled P1, P2, . . . , P6 with concentrations
near to 1.0, 0.5, 0.3, 0.2, 0.1 and 0.05 mg ml1,
respectively. As presented by Carvalho, Santos, et al.
(Forthcoming), the resolution of the visual calibration
was modelled by considering the different intervals
(D’s) among the concentrations of the calibration
solutions. This model considers that when reading,
by visual comparison, the analyst can state that the
fluorescence of the sample spot is equal to the
fluorescence of one of the calibration standard solu-
tions, Pi, or that its fluorescence is found between two
successive solutions Pi–Piþ1. This model is represented
in Figure 1. From this model the uncertainty of the
visual quantification due to calibration resolution
••••• Δ(P4-P5) Δ(P3-P4) Δ(P2-P3) Δ(P1-P2)
P6 P5 P4 P3 P2 P1
••••• P3P4 P2P3 P1P2
••••• Δ(P2P3-P3P4) Δ(P1P2-P2P3)
Figure 1. Variations (D) between any pair Pi–Piþ1 of
standards and between half readings PiPiþ1–Piþ1Piþ2 used
to model the uncertainty due to the resolution of the visual
calibration of the fluorescence readings in the TLC.
682 K.L. Carvalho et al.
(uVCresol) was estimated for different fluorescence
readings as a function of the standard calibration
solution concentration, C(Pi) by using the rectangular
probability density function (PDF) of the following
equations (1a) and (1b):
   
CðPi Þ þ CðPiþ1 Þ CðPi Þ  CðPiþ1 Þ
uVCresol ¼ pffiffiffi ð1aÞ
2 2 3
The uncertainty obtained by Equation (1a) is
attributed to the averaged concentration of the stan-
dard solutions Pi and Pi þ 1:
½CðPi Þ þ CðPi þ1Þ=2
  
CðPi Þ þ CðPiþ1 Þ CðPiþ1 Þ þ CðPiþ2 Þ
uVCresol þ 2
2 2
 
CðPi ÞþCðPiþ1 Þ CðPiþ1 ÞþCðPiþ2 Þ
 2  2 
¼ pffiffiffi ð1bÞ
2 3
The uncertainty obtained by Equation (1b) is
attributed to the concentration:
f½CðPi Þ þ CðPiþ1 Þ=2 þ ½CðPiþ1 Þ þ CðPiþ2 Þ=2g=2
For each visual reading, the modelled resolution
uncertainty was combined with the reproducibility
standard deviation due to the two or three readings of
the TLC spots fluorescence intensities of different
analysts.
For the densitometric TLC calibration, the areas of
the calibration peaks, associated with the fluorescence
of each standard calibration solution spots on the TLC
plate, were fitted against the standard calibration
solution concentration using the weighted least
square (WLS). To estimate the uncertainties on the
densitometric TLC peak areas, the results of the
calibration curves obtained over 8 months were used.
The areas of the densitometric peaks of each of four
sets of calibration standard solutions were measured
on at least 3 days to obtain a model for the sample
standard deviations (square root of the variances) of
these areas as a function of the standard calibration
solution concentration. This model was used to weight
each densitometric peak area in the WLS fitting of the
calibration curves (see the last column of Table 2).
With this approach, the uncertainties of the intercept
and slope of the calibration curves take into account
the sources of uncertainties due to the intermediary
precision of the densitometric peak areas as well as,
and implicitly, that due to the preparation of the
standard calibration solutions, as required by
European Union norms (Commission of the
European Communities 2002, 2004). Although these
European laws do not specifically apply to mycotoxin
analysis, the use of the above approach to estimate
calibration uncertainty is consistent with statistical
concepts and is recommended in order to avoid the
underestimation of this source of uncertainty. This
model does not consider the intrinsic Poisson uncer-
tainty of the densitometric peak area of each individual
spot, which is negligible compared with the other two
sources of uncertainties considered.
Recovery
The analytical method presents a recovery ratio
variable from day to day, but no correction is applied
to the results. Therefore, the allowed recovery ratio
range was used to estimate a Type B uncertainty, as
described by Carvalho, Santos, et al. (Forthcoming).
Intermediary precision
Although the uncertainty of repeatability was included
in the volume and calibration uncertainty sources, it
also included an intermediary precision to account for
other sources of uncertainties from bath-to-bath, day-
to-day, sample preparation-to-sample preparation, etc.
variations.
Step 3: Estimating (quantifying) the standard
uncertainties of each source of uncertainty
The uncertainties due to volume measurements,
visual and densitometric TLC calibration and
quantification were estimated as described by
Carvalho, Santos, et al. (Forthcoming).
Recovery
When recovery is not used to correct the results, its
uncertainty is obtained from the allowed variation of
the recovery of the quality control samples analysed
with each bath of analysis. For the samples with
aflatoxin M1 contamination from 0.01 to 0.05 mg l1,
the allowed recovery ratio range is from 60% to 120%;
above this contamination this allowed range becomes
from 70% to 110% (Commission of the European
Communities 2006). Each of these ranges defines a
total amplitude 2a of the rectangular PDF for a Type B
estimation of the uncertainty.
Intermediary precision
The uncertainties of intermediary precision of the
densitometric and visual TLC methods were obtained
from the straight line model fitted to the data of the
standard deviation of the results of analysis of the
fortified blank samples with 0.02, 0.05 and 0.5 mg l1
for quality control, which was carried out over a period
of 11 months. This is a Type A estimation; and as only
three points were used to fit the straight line, the
degrees of freedom of this uncertainty component
was only 1.

Step 4: Calculating the combined and expanded
uncertainty of the measurand
To calculate the combined standard uncertainty of the
concentration of aflatoxin M1 in the test sample, the
classical uncertainties propagation law was used. To
obtain the expanded combined uncertainty, the stan-
dard combined uncertaity is multiplied by the coverage
factor to a coverage probability of 95%, as described
by Carvalho, Santos, et al. (Forthcoming). The cover-
age factor is obtained from the student PDF point of
probabilities, depending on the effective degree of
freedom calculated by the Welch-Satterthwaite equa-
tion (INMETRO 2009; Joint Committee for Guides in
Metrology (JCGM/WG 2) 2008; Carvalho, Santos,
et al. Forthcoming).
Results and discussion
Step 1: Measurand specification
After a detailed analysis of the standard operation
procedures the proposed measurand equations were:
For densitometric TLC quantification:
Vp  CSAA  Vr
CaflaM1 ¼  CFrecup þ Cprecint
Va  Vs
Vp  ðAPDA  aÞ  Vr 1
¼  þ Cprecint
b  Va  Vs RR
ð2Þ
For visual TLC quantification:
Vp  ðLVm þ CResol Þ  Vr
CaflaM1 ¼
Va  Vs
 CFrecup þ Cprecint ð3Þ
where CaflaM1 is the aflatoxin M1 concentration
(mg l1 or ppb) quantified in the milk sample; CSAA
is the concentration (mg ml1 or ppm) of the test sample
solution applied in the TLC plate; Vp is the volume (ml)
of the standard solutions of different concentrations
applied to the TLC plate to quantify the aflatoxin
contamination by using a densitometer calibration
curve or by visual comparison of the fluorescence
intensity related to aflatoxin M1 from the standards
and the test sample spots; Vr is the volume (ml) of the
solvent used to re-dissolve the sample after drying the
test sample extract obtained after the sample clean-up
through the elution of aflatoxin M1 from the
immunoaffinity column; Va is the volume (ml) of the
test sample solution applied to the TLC plate; Vs is the
test sample volume (ml) of the milk injected onto
the immunoaffinity column to be purified; APDA is the
area for the densitometric peak for the test sample
solution applied in the TLC plate; a and b are,
respectively, the intercept and slope of the densitom-
eter calibration curve; CFrecup ¼ 1/RR is the correction
factor of the unitary value due to the uncorrected
Food Additives and Contaminants 683
recovery; RR is the recovery ratio measured through
the analyses of a spike sample of quality control during
the bath of analysis; CResol is a null correction applied
to the average reading LVm due to the resolution of
the visual calibration (by introducing this null correc-
tion the not null uncertainty due to the visual reading
resolution was computed); Cprecint is the null correction
due to the intermediary precision; and LVm is the
mean visual reading for the test sample concentration
(mg ml1 or ppm) obtained by the average of three
individual readings of three different analysts for the
fluorescence intensity of the test sample spot compared
with the fluorescence of the six spots of the aflatoxin
M1 standard calibration solutions.
Note that CSAA is obtained from the intercept and
slope of the calibration curve by the equation:
APDA  a
CSAA ¼ ð4Þ
b
Therefore, the uncertainty of CSAA, u(CSAA), will
be dependent on the uncertainties and covariance of a
and b, but also on the repeatability uncertainty of the
instrumental response for the test sample solution
applied to the TLC plate, u(APDA).
Step 2: Identifying the sources of uncertainties
The cause-and-effect (Ishikawa) diagrams, obtained
for densitometric and visual methods, are showed in
Figures 2 and 3, respectively. All the input quantities
in Equations (2) and (3) are present in these
cause-and-effect diagrams. For Figures 2 and 3 some
symbols are defined as above; other symbols are as
follows: CP, concentration of the calibration standard
solutions; APDP, area of the densitometric peak for
the spots of the calibration standard solutions; Calibr,
calibration; Resol, resolution of the measurement
instrument; Repet, repeatability; and Lab Temp Var,
variation of the laboratory temperature around its
mean value.
Step 3: Estimating (quantifying) the standard
uncertainties of each source of uncertainty
Volume
The three Type B uncertainties of the volume
measurements due to the resolution, uResol(V), the
calibration, uCalib(V), and the total laboratory
temperature variation, DT, of 5 K around the mean
laboratory temperature, uVartemp(V), as well as the
Type A uncertainty due to the repeatability, uRep(V),
were obtained as shown elsewhere (Carvalho, Santos,
et al. Forthcoming).
The measurement function (INMETRO 2009; Joint
Committee for Guides in Metrology (JCGM/WG 2)
684 K.L. Carvalho et al.

Calibration
APDP
CSAA Allowed recovery variation
CP
a range

APDA uc(CaflaM1)
and
Purification U(CaflaM1)
Resol
Experimeters
Volumes
Lab Temp Var
Different days
Calibr

Repeat
Intermediary precision or internal
reproducibility or intralaboratorial Vp Vr Vs Va
precision

Figure 2. Cause-and-effect (Ishikawa) diagram to represent the sources of uncertainties to estimate the uncertainty of the
aflatoxin M1 content u(CaflaM1) for densitometric TLC quantification.

Allowed recovery variation

Resolution Repeatabilty LVm
range

Calibration

u(CaflaM1)
and
Purification
U(CaflaM1)
Experimeters Resol
Volumes
Lab Temp Var
Different days
Calibr

Intermediary precision or internal Repeat

reproducibility or intralaboratorial Vp Vr Vs Va
reproducibility

Figure 3. Cause-and-effect (Ishikawa) diagram to represent the sources of uncertainties to estimate the uncertainty of the
aflatoxin M1 content u(CaflaM1) for visual TLC quantification.

2008) or measurand equation
quantity is:
V ¼ Vnominal þCResol þ CCalib þ CVartemp þ CRep
By applying the law of uncertainty propagation on
Equation (5) the combined uncertainties of each of the
four measured volumes, V (Vs, Vr, Va or Vp), were
obtained by the equation:
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
uðVÞ ¼ u2Resol ðVÞ þ u2Calib ðVÞ þ u2Vartemp ðVÞ þ u2Rep ðVÞ
Table 1 shows the metrological characteristics of
the instruments used for volume measurement: their
standard uncertainties due to instrument resolution,
calibration, repeatability and laboratory temperature
variation, as well as the combined uncertainty of
volume measurement u(V) as calculated by
Equation (6).
of the volume came from the uncertainties u(a) ¼ sa and u(b) ¼ sb, and
from the covariance u(a,b) ¼ cov(a, b) of the intercept
and of the slope of the calibration straight line
ð5Þ
(Carvalho, Gonçalves, et al. Forthcoming, Carvalho,
Santos, et al. Forthcoming).
To quantify a test sample, a set of standard
calibration solutions with the concentrations presented
in Table 2 was used. The same set of calibration
standard solutions was used for visual and densito-
metric TLC quantification. The peak areas of these
ð6Þ calibration standard solutions for a typical calibration
are also shown in the Table 2.
The uncertainties (sample standard deviations) of
the APDP in the last column of Table 2 and the
uncertainties bars in Figure 4 clearly show the
heteroskedasticity of the instrumental responses, and
were experimentally obtained as presented above
through the linear model:

uðAPDPÞ ¼ 10:80037 þ 165:56443  CP ð7Þ

Calibration
The uncertainty of the concentration of the aflatoxin By fitting a straight line to the data of the Table 2
M1 test sample solution, u(CSAA), applied to the TLC using the WLS (the continuous line in Figure 4), the
plate determined by the densitometric quantification intercept a ¼ (7.83  17.00), the slope b ¼ (1767.18 
 Food Additives and Contaminants 685

Table 1. Metrological characteristics of the instruments used for volume measurements inp TLC
ffiffiffi quantification of aflatoxin M1:
scale division (d), scale space (SS), kd is the divisor in the equation: uResol(V) ¼ SS/[kd x 3], uncertainties due to resolution,
calibration laboratory temperature variation and repeatability, as well as the combined standard uncertainty for graduated
cylinder (Vs), automatic pipette (Vr) and microsyringe (Va and Vp).

Quantity Value d SS kd uResol (V) uCalib (V) uVartemp (V) uRep (V) u (V)

Vs 100 ml 1 ml 1.6 mm 4 0.14 ml 0.51 ml 0.030 ml 0.31 ml 0.61 ml

Vr 100 ml 1 ml –  0.29 ml 0.26 ml 0.030 ml 0.001 ml 0.39 ml
Va 20 ml 0.5 ml 1.5 mm 4 0.072 ml 0.085 ml 0.0061 ml 0.001 ml 0.11 ml
Vp 10 ml 0.5 ml 1.5 mm 4 0.072 ml 0.080 ml 0.0030 ml 0.001 ml 0.11 ml

Table 2. Calibration data of the TLC densitometric represent the uncertainty of any instrumental response
quantification.
within the calibration concentration range. The WLS
Standard CP (mg ml1) APDP u (APDP) and OLS lines cross each other at the crossing point
(0.268 mg ml1) marked in the Figure 4 with a signal
P6 0.036802 71.019 16.893 plus (þ). At concentrations lower than 0.268 mg ml1,
P5 0.101913 188.972 27.674 before the crossing point in Figure 4, the WLS line is
P4 0.184010 342.176 41.266
P3 0.331218 584.259 65.638 below the OLS line; after this concentration the WLS
P2 0.496827 920.293 93.057 line is above. This implies that the use of the OLS fit in
P1 0.993655 1696.175 175.314 the present case generates a proportional negative bias
for milk samples contaminated with aflatoxin M1 at
Note: CP, concentration of the calibration standard solution;
APDP, area of the densitometric peak for the spots of the levels of contamination lower than 0.268/
calibration standard solutions. 2 ¼ 0.134 mg ml1 and a positive bias above this con-
tamination. Figure 4 also shows the upper, UPL
(dashed line), and lower (dotted line), LPL, prediction
limits (lines) for 95% of confidence as obtained by the
WLS. Due the linear heteroskedasticity they are not
symmetrical around the centroid (median point, arith-
metic average), as occurs in the OLS, but are around
the barycentre (weighted centroid, weighted average),
where the prediction limit lines are the nearest to the
WLS fitted straight line. The region around the
barycentre of the calibration line is of better precision.
The area of the densitometric peak for a test sample
solution was 201.082, leading, according to Equation
(4) and the WLS calibration fitting parameters, to
CSAA ¼0.1094 mg ml1 and, according to Equation
(2), to an aflatoxin M1 content of 0.05468 mg l1. This
content on the basis of the OLS fit is 5.4% less than the
Figure 4. Calibration curve of the densitometric TLC content obtained from the WLS statistical method.
quantification as obtained by WLS (continuous line) and The barycentre and the centroid, which are marked
OLS fitting (dotted-dashed line). UPL and LPL are the upper
and the lower prediction limit curves for 95% of confidence. in Figure 4, are the points with coordinates
(0.0929 mg ml1, 172) and (0.3574 mg ml1, 634),
respectively.
115.91) ml mg1 and the covariance between the inter- Using the curves of the limit of prediction, as
cept and the slope cov(a, b) ¼ 1248.1 ml mg1 were recommended by the standard ISO 11843 (ISO 1997,
obtained. The intercept standard uncertainty of 2000) and described by Carvalho, Santos, et al.
17 ml mg1 shows that it is statistically equal to zero. (Forthcoming), the decision limit CC ¼
For comparison, the intercept and the slope of the 0.1171 mg ml1 (see CC in Figure 4) was obtained,
calibration line obtained from the ordinary least square corresponding, according to Equation (2), to aflatoxin
(OLS) fit (dash and dotted line in Figure 4) were M1 contamination CC ¼ 0.059 mg l1. Using the sim-
a ¼ 24.73 and b ¼ 1704.2 ml mg1. The uncertainties of plified methodology recommended in European
these OLS fitted parameters were not reported because Commission Decision 657 (Commission of the
they do not have any statistical meaning once the European Communities 2002), a decision limit of
residual standard deviation of the OLS fit does not CC ¼ 0.1132 mg ml1 corresponding to an aflatoxin
686 K.L. Carvalho et al.

M1 contamination of CC ¼ 0.055 mg l1 was found. Table 3. Modeling the typical uncertainty of resolution of
Note that these decision limits are nearly 10–20% the visual TLC calibration.
larger than the MPL of the European Union.
Standard CP
However, these calculated CC have only taken into solution (mg ml1) DCP Rr uResol (LVm)
account the calibration curve uncertainty. By including
the within-laboratory reproducibility they will become P1 1.000 – 0.787 0.227
higher. According to European legislation concerning Mean of P1P2 0.750 0.500 0.513 0.148
P2 0.500 0.350 0.294 0.0849
residues in animals and animal products for human
Mean of P2P3 0.400 0.200 0.222 0.0641
consumption, samples with residue levels above the P3 0.300 0.150 0.160 0.0462
limit of decision will be considered as being non- Mean of P3P4 0.250 0.100 0.132 0.0381
compliant (Commission of the European Communities P4 0.200 0.100 0.106 0.0306
2002, 2004; ISO 1997). Once, even taking into account Mean of P4P5 0.150 0.100 0.082 0.024
P5 0.100 0.075 0.061 0.018
the measurement uncertainty, such samples exceed the
Mean of P5P6 0.075 0.050 0.051 0.015
maximum residue limit (MRL) beyond reasonable P6 0.050 – 0.042 0.012
doubt (Commission of the European Communities
2002, 2004). Nowadays the decision limit is not applied Note: CP, typical standard concentration; DCP, concentra-
to decide about the compliance of the products tion variation between neighbouring calibration solutions;
Rr ¼ 2a is the range of the rectangular PDF. In the second
contaminated with aflatoxins. However, due to its column, values shown in bold are the concentrations of real
statistical consistency, it is expected that in the near standard calibration solutions; other values correspond to
future the decision limit will also apply to mycotoxins half readings.
as well as to all other products and contaminants.
In the visual quantification the readings of three
analysts for the fluorescence intensity of a sample spot Concentration variation between neighbours
standard solutions
were equivalent to those spots of the standard P5, 0.60
between the standards P5 and P6 (mean concentration
of P5 and P6), and the standard P5 again. These 0.50
y = 0.672x – 0.023
readings lead to a mean standard concentration R² = 0.951
LVm ¼ 0.0911 mg ml1 and standard deviation of 0.40
repeatability ¼ 0.0108 mg l1. This LVm corresponds,
ΔCP

0.30
according to Equation (3), to an aflatoxin M1 con-
tamination of 0.0455 mg l1. 0.20
This contamination, determined by the visual TLC
quantification, was 16.7% lower than that of the 0.10 y = 0.447x2 + 0.315x + 0.025
R² = 0.969
densitometric TLC quantification. For other contam-
0.00
ination levels the differences between these methods is
0.00 0.20 0.40 0.60 0.80
nearly between 15% and 20% and the signal of these
differences alternates randomly, and both analytical CP = Standard Concentration
procedures can be considered to be equally accurate Figure 5. Linear and the parabolic mathematical functions
concerning its trueness. modelling the aflatoxin M1 concentration variation, DCP,
The uncertainty of the mean read of the spot between successive calibration solution concentrations
fluorescence of the test sample in the visual TLC and between successive half calibration solution concentra-
tions (mean between two standards) used to estimate the
quantification has two components: repeatability and
uncertainty of resolution of the visual TLC calibration.
resolution. The first was obtained as the standard
deviation of three readings. To obtain a mathematical
model for the uncertainty due to the resolution of Commission 2010). The fourth column of Table 3
the visual TLC calibration according to Figure 1 and shows the calculated standard concentration variation
the strategy presented by Carvalho, Santos, et al. around each possible reading of the visual calibration
(Forthcoming), Table 3 was constructed. according to the parabolic model, which is used as the
Figure 5 shows the linear and parabolic (the range of the rectangular PDF (Rr) to estimate the
numerator in Equation 8) functions fitted to the data uncertainty of resolution of the mean visual reading,
of DCP against CP of Table 3. Owing to the coefficient uResol(LVm):
of determination, R2, the parabolic function is only
Rr 0:447CP2 þ 0:315CP þ 0:025
slightly better than the linear one. However, the uResol ðLVmÞ ¼ pffiffiffi ¼ pffiffiffi ð8Þ
parabolic function fits the data better at low concen- 2 3 2 3
trations, near the standard concentration of where CP is the concentration of a real or hypothetical
0.1 mg ml1, which correspond to the maximum per- standard calibration solution at the mean visual
mitted contamination level of 0.05 mg l1 (European reading.
 Food Additives and Contaminants 687

Recovery For the visual TLC quantification:

As mentioned above, the uncertainty due to the
recovery variation assumes two different values uðprecintÞ ¼ 0:0006 þ 0:2706  CaflaM1ðmg=kgÞ ð14Þ
depending on the contamination of the test sample.
The correction factor due to the recovery (CFrecup) is
equal to the inverse of the recovery ratio. For samples Step 4: Calculating the combined and expanded
with contamination in the range 0.01–0.05 mg l1, uncertainty of the measurand
for which the allowed range of the recovery ratio To combine the standard uncertainties of the input and
was 60–120%, the standard uncertainty of the recov- influence quantities, u(), the particular case of the
ery ratio u(RR) was estimated by (Carvalho, Santos, uncertainty propagation law by propagating the rela-
et al. Forthcoming, Carvalho, Gonçalves et al. tive uncertainties was not used, as often occurs (Ellison
Forthcoming): et al. 2000; Barwick and Ellison 2000a, 2000b;
1:20  0:60 Armishaw 2003; Calaresu et al. 2006), because the
uðRRÞ ¼ pffiffiffi ¼ 0:1732 ð9Þ present measurand equations do not only have multi-
2 3
plications and division operations. For densitometric
As CFrecup ¼ 1/RR, the uncertainty of the correc- TLC quantification the uncertainty propagation law
tion factor of recovery u(CFrecup) is (Carvalho, takes the form:
Gonçalves et al. Forthcoming, Carvalho, Santos,
et al. Forthcoming): uðcaflaM1Þ
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

uðRRÞ 1:20  0:60 uh i2 h i2
u @CaflaM1
u CFrecup ¼ ¼ pffiffiffi ð10Þ u @Vp uðVpÞ þ @CaflaM1 @CSAA uðCSAAÞ
RR2 2 3  RR2 u
u
u h i h i
u þ @CaflaM1 uðVrÞ 2 þ @CaflaM1 uðVaÞ 2
While for samples with contamination above u @Vr @Va
u
0.05 mg l1, for which the allowed range of recovery ¼u h
u i
was 70–110%, the standard uncertainty of the recovery u þ @CaflaM1 uðVsÞ 2
u @Vs
ratio u(RR) was estimated by (Carvalho, Gonçalves u
u
et al. Forthcoming, Carvalho, Santos, et al. t h
i2 h@CaflaM1
i2
Forthcoming) þ @CaflaM1
@CFrecup u CFrecup þ @Cprecint u Cprecint

1:10  0:70 vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

uh i2 h i2
uðRRÞ ¼ pffiffiffi ¼ 0:1155 ð11Þ u @CaflaM1 @CaflaM1
2 3 u u ð Vp Þ þ u ð APDA Þ
u @Vp @APDA
u
u h i2 h i2

uðRRÞ 1:10  0:70 u @CaflaM1 @CaflaM1
u CFrecup ¼ ¼ pffiffiffi ð12Þ uþ u ð a Þ þ u ð b Þ
RR2 2 3  RR2 u @a @b
u
u h i2 h i2
u @CaflaM1
u þ @Vr uðVrÞ þ @CaflaM1 u ð Va Þ
¼u
As the recovery ratio varies significantly even @Va
u
within a batch of analysis, the median value 0.90 of uh i2
u @CaflaM1
the allowed range of recovery ratio variation was used u @Vs uðVsÞ
u
for both the recovery ratio range in Equationspffiffi(10)
ffi u
u h
i2 h@CaflaM1
i2
and (12), leading to values ofpffiffi0.740741/(2
ffi 3) ¼ u @CaflaM1
u þ @CFrecup u CFrecup þ @Cprecint u Cprecint
0.21383 mg l1 and of 0.493827/(2 3) ¼ 0.14256 mg l1 u
t
as standard uncertainties for the correction factor of
þ2 @CaflaM1 @a
@CaflaM1
@b uða, bÞ
the recovery ratio.
ð15Þ

Intermediary precision The first and second forms of the above equation
The straight line fitted to three standard deviations of are based on the respective forms of the measurand
the spiked samples blanks with aflatoxin M1 standard equation presented in Equation (2). Here the partial
solutions for 0.02, 0.05 and 0.5 mg l1 aflatoxin M1 derivatives (differential quotient or differential coeffi-
contaminations, measured many times during the 11 cient) are the sensitivity coefficients given by the
months, leads to the following models for uncertainty following equations:
due to intermediary precision or intra-laboratory
@CaflaM1 ðAPDA  aÞ  Vr CaflaM1
reproducibility: ¼  CFrecup ¼
For the densitometric TLC quantification: @Vp b  Va  Vs Vp
ð16Þ
uðprecintÞ ¼ 0:2262  CaflaM1ðmg=kgÞ ð13Þ
688 K.L. Carvalho et al.

@CaflaM1 Vp  ðAPDA  aÞ  Vr where the partial derivatives (differential quotient or

¼  CFrecup
@Va b  Va2  Vs differential coefficient) are the sensitivity coefficients
CaflaM1 given by:
¼ ð17Þ
Va
@CaflaM1 ðLVm  CResol Þ  Vr
¼  CFrecup
@Vp Va  Vs
@CaflaM1 Vp  ðAPDA  aÞ
¼  CFrecup CaflaM1
@Vr b  Va  Vs ¼ ð26Þ
CaflaM1 Vp
¼ ð18Þ
Vr
@CaflaM1 Vp  ðLVm  CResol Þ  Vr
¼  CFrecup
@CaflaM1 Vp  ðAPDA  aÞ  Vr @Va Va2  Vs
¼  CFrecup CaflaM1
@Vs b  Va  Vs2 ¼ ð27Þ
CaflaM1 Va
¼ ð19Þ
Vs @CaflaM1 Vp  ðLVm  CResol Þ
¼  CFrecup
@Vr Va  Vs
@CaflaM1 Vp  ðAPDA  aÞ  Vr CaflaM1 CaflaM1
¼ ¼ ¼ ð28Þ
CFrecup b  Va  Vs CFrecup Va
ð20Þ
@CaflaM1 Vp  ðLVm  CResol Þ  Vr
¼  CFrecup
@CaflaM1 @Vs Va  Vs2
¼1 ð21Þ CaflaM1
@Cprecint ¼ ð29Þ
Vs
@CaflaM1 Vp  Vr CaflaM1 @CaflaM1 Vp  ðLVm  CResol Þ  Vr CaflaM1
¼  CFrecup ¼ ¼ ¼
DAPDA b  Va  Vs APDA  a @CFrecup Va  Vs CFrecup
ð22Þ
ð30Þ

@CaflaM1 Vp  Vr CaflaM1 @CaflaM1

¼  CFrecup ¼  ¼1 ð31Þ
@a b  Va  Vs APDA  a @Cprecint
ð23Þ
@CaflaM1 Vp  Vr CaflaM1
¼  CFrecup ¼ ð32Þ
@CaflaM1 Vp  ðAPDA  aÞ  Vr @LVm Va  Vs LVm  CResol
¼  CFrecup
@b b2  Va  Vs
CaflaM1 @CaflaM1 Vp  Vr CaflaM1
¼ ð24Þ ¼  CFrecup ¼
b @CResol Va  Vs LVm  CResol
The only covariance taken into account was that ð33Þ
between the intercept and the slope, u(a, b) ¼ cov(a, b), Tables 4 and 5 summarise the uncertainty
of the calibration curve of the densitometric TLC combination for a blank sample spiked with aflatoxin
quantification obtained from WLS fitting. M1 to a content of 0.05 mg l1, which is the European
For visual TLC quantification, the uncertainty Union maximum permitted value, and determined as
propagation law takes the form: 0.04553 mg l1 by visual TLC quantification and as
0.05468 mg l1 by densitometric TLC quantification.
uðcaflaM1Þ The standard uncertainties of the quantities in
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Tables 4 and 5, where an asterisk (*) is shown after the
uh i h i
u @CaflaM1 uðVpÞ 2 þ @CaflaM1 uðLVmÞ 2
u @Vp @LVm
quantity symbol, are obtained through the uncertainty
u
u combination of the uncertainties at the lines immedi-
u h i2 h i
u þ @CaflaM1 uðVrÞ þ @CaflaM1 uðVaÞ 2 ately before it. For the case of the volumes, this
u @Vr @Va
u combination is realised according to Equation (6). The
¼u h i2 h i2
u graduated cylinder used to measure Vs was the unique
u þ @CaflaM1 uðVsÞ þ @CaflaM1 uðC Þ
u @Vs @CResol Resol volume measurement instrument not formally cali-
u
u brated. However, the data for internal quality control
t h
2 h@CaflaM1
i
i2
þ @CaflaM1
@CFrecup u CF recup þ @Cprecint u C precint
of a set of such cylinders showed a maximum bias of
0.8834 ml. In a conservative way, this bias was used as
ð25Þ the half range of the rectangular PDF (see the
Table 4. Calculation of the combined uncertainty for densitometric TLC quantification of aflatoxin M1 in milk.

Input or influence quantities Probability density Uncertainty contribution

sources of uncertainties function (PDF) (distribution) ui(y) or u(y;xi)

Standard Sensitivity Degrees of

Symbol Value, xi Interval Units Type Name Divider, k uncertainty u(xi) coefficient, ci ui(y) or u(y;xi) freedom i or eff [ui(y)]4/i

CResol 0 0.125 ml B Rectangular 1.73205 0.072169 0.005468 0.000395 1.0E þ 99 2.4E–113

CVartemp 0 0.00525 ml B Rectangular 1.73205 0.003031 0.005468 0.000017 1.0E þ 99 7.5E–119
CCalib 0 0.16 ml B Normal 2 0.08 0.005468 0.000437 1.0E þ 99 3.6E–113
CRep 0 0.0003 ml A Normal 1 0.0003 0.005468 0.000002 9 8.04E–25
Vp* 10 ml 0.107785 0.005468 0.000589
CResol 0 0.5 ml B Rectangular 1.73205 0.288675 0.000547 0.000158 1.0E þ 99 6.2E–115
CVartemp 0 0.0525 ml B Rectangular 1.73205 0.030311 0.000547 1.66E–05 1.0E þ 99 7.5E–119
CCalib 0 0.51 ml B Normal 2 0.255 0.000547 0.000139 1.0E þ 99 3.7E–115
CRep 0 0.00055 ml A Normal 1 0.00055 0.000547 3.01E–07 9 9.08E–28
Vr* 100 ml 0.386364 0.000547 0.000211
CResol 0 0.125 ml B Rectangular 1.73205 0.072169 0.002734 0.000197 1.0E þ 99 1.5E–114
CVartemp 0 0.0105 ml B Rectangular 1.73205 0.006062 0.002734 1.66E–05 1.0E þ 99 7.5E–119
CCalib 0 0.17 ml B Normal 2 0.085 0.002734 0.000232 1.0E þ 99 2.9E–114
CRep 0 0.0006 ml A Normal 1 0.0006 0.002734 1.64E–06 9 8.04E–25
Va* 20 ml 0.111671 0.002734 0.0003053
CResol 0 0.25 ml B Rectangular 1.73205 0.144338 0.000547 7.89E–05 1.0E þ 99 3.8E–116
CVartemp 0 0.0525 ml B Rectangular 1.73205 0.030311 0.000547 1.66E–05 1.0E þ 99 7.5E–119
CCalib 0 0.8834 ml B Rectangular 1.73205 0.510031 0.000547 0.000279 1.0E þ 99 6.0E–114
CRep 0 0.3056 ml A Normal 1 0.3056 0.000547 0.000167 4 1.94E–16
Vs* 100 ml 0.612597 0.000547 0.000335
APDA 201.082 28.9060 1 A Normal 1 28.90599 0.000283 0.008179 16 2.79E–10
a 7.83 17.0011 1 A Normal 1 17.001138 0.000283 0.00481 4 1.33E–10
b 1767.18 115.911 ml mg1 A Normal 1 115.911 3.09E–05 0.003586 4 4.13E–11
Cov(a,b) –1248.1 –1248.1 ml mg1 A Normal 1 –1248.1 8.75E–09 –1.09E–05 – –
CSAA* 0.1094 mg ml1 0.018004 0.5 0.009002
CSAA* 0.1094 mg ml1 0.020287** 0.5 0.010143
CFrecup 1 0.493827 1 B Rectangular 3.46410 0.142556 0.045529 0.006491 1.0E þ 99 1.7E–108
1
Cprecint 0 0.01237 mg l A Normal 1 0.012370 1 0.01237 1 2.34E–08
Sum 2.387E–08
Summary
Relative Relative
Quantity Combined Effective Coverage Expanded standard expanded
nominal Estimated standard degrees of probability Coverage uncertainty uncertainty uncertainty
value Y value y uncertainty freedom eff P (%) factor k U(y) RSU REU
Food Additives and Contaminants

uc(y)

0.05 0.0547 0.016637 3.21 95 3.1824 0.052945 30.4 96.8

689

Notes: Only the correlation between the intercept and the slope of the calibration line was considered according to Equation (34).
**Value for the standard uncertainty of CSAA obtained without the covariance between the intercept and slope of the calibration straight line.
Table 5. Calculation of the combined uncertainty for visual TLC quantification of aflatoxin M1 in milk.

Input or influence quantities sources Probability density function Uncertainty contribution 690
of uncertainties (PDF) (distribution) ui(y) or u(y;xi)

Standard Sensitivity Degrees of

Symbol Value, xi Interval Units Type Name Divider, k uncertainty U(xi) coefficient, ci ui(y) or u(y;xi) freedom i or eff [ui(y)]4/i

CResol 0 0.125 ml B Rectangular 1.73205 0.072169 0.004553 0.000329 1.0E þ 99 1.2E–113

CVartemp 0 0.00525 ml B Rectangular 1.73205 0.003031 0.004553 1.380E–05 1.0E þ 99 3.6E–119
CCalib 0 0.16 ml B Normal 2 0.08 0.004553 0.000364 1.0E þ 99 1.8E–113
K.L. Carvalho et al.

CRep 0 0.0003 ml A Normal 1 0.0003 0.004553 1.366E–06 9 3.87E–25

Vp* 10 ml 0.107785 0.004553 0.000491
CResol 0 0.5 ml B Rectangular 1.73205 0.288675 0.000455 0.000131 1.0E þ 99 3.0E–115
CVartemp 0 0.0525 ml B Rectangular 1.73205 0.030311 0.000455 1.380E–05 1.0E þ 99 3.6E–119
CCalib 0 0.51 ml B Normal 2 0.255 0.000455 0.000116 1.0E þ 99 1.8E–115
CRep 0 0.00055 ml A Normal 1 0.00055 0.000455 2.504E–07 9 4.37E–28
Vr* 100 ml 0.386364 0.000455 0.000176
CResol 0 0.125 ml B Rectangular 1.73205 0.072169 0.002276 0.000164 1.0E þ 99 7.3E–115
CVartemp 0 0.0105 ml B Rectangular 1.73205 0.006062 0.002276 1.380E–05 1.0E þ 99 3.6E–119
CCalib 0 0.17 ml B Normal 2 0.085 0.002276 0.000194 1.0E þ 99 1.4E–114
CRep 0 0.0006 ml A Normal 1 0.0006 0.002276 1.366E–06 9 3.87E–25
Va* 20 ml 0.111671 0.002276 0.000254
CResol 0 0.25 ml B Rectangular 1.73205 0.144338 0.000455 6.571E–05 1.0E þ 99 1.9E–116
CVartemp 0 0.0525 ml B Rectangular 1.73205 0.030311 0.000455 1.380E–05 1.0E þ 99 3.6E–119
CCalib 0 0.8834 ml B Rectangular 1.73205 0.510031 0.000455 0.000232 1.0E þ 99 2.9E–114
CRep 0 0.3056 ml A Normal 1 0.3056 0.000455 0.000139 4 9.37E–17
Vs* 100 ml 0.612597 0.000455 0.000279

LVmRepet 0 0.010850779 mg ml1 A Normal 1 0.010851 0.5 0.005425 2 4.33E–10

LVmResol 0 0.035885331 mgm l1 B Rectangular 1.732051 0.020718 0.5 0.010359 1.0E þ 99 1.2E–107
LV mg ml1 0.023388 0.5 0.011694
CFrecup 1 0.740740741 1 B Rectangular 3.46410 0.213833 0.04553 0.009736 1.0E þ 99 9.0E–108
Cprecint 0 0.012920313 mg l1 A Normal 1 0.012920 1 0.012920 1 2.79E–08
Sum 2.83E–08
Summary
Combined Effective Relative Relative
Quantity standard degrees of Coverage Coverage Expanded standard expanded
nominal Estimated uncertainty freedom probability factor uncertainty uncertainty uncertainty
value Y value y uc(y) eff P (%) k U(y) RSU REU

0.05 0.045 0.01997 5.62 95 2.5706 0.051340 36.5263 93.8938

Note: No possible correlations were considered.


0.02
Uncertainty contributions
0.015
0.01
0.005
0 ibility, followed by the uncertainties due to the
Comb Unc CU-prec Int Prec
Figure 6. Contributions for the combined uncertainty of the
visual and densitometric TLC quantification of aflatoxin M1
in milk for a sample blank sample spiked to a content of
0.05 mg l1 of aflatoxin M1. For volume measurement, the
bars show the combined contribution of the four measured
volumes.
calibration component of Vs in Tables 4 and 5) to
estimate its calibration uncertainty.
The standard uncertainty of the concentration of
the sample solution applied in the TLC plate,
u(CSAA) ¼ 0.018004 mg ml1, was obtained from the
WLS parameters of the calibration straight line
through Equation (34) (see also Equation E.3.3 in
Ellison et al. 2000 and Equation 26 in Carvalho,
Santos, et al. Forthcoming):
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
u 2
u u ðAPDAÞ þ u2 ðaÞ þ CSAA2  u2 ðbÞ
u
t þ2  CSAA  covða, bÞ
uðCSAAÞ ¼
b2
As the co-variance (and correlation) between the
intercept and the slope of the calibration straight line is
always negative, u(CSAA) ¼ 0.020287 mg ml1 (see the
value with a double asterisk in the second line with the
label CSAA in Table 4) without this correlation is
higher than u(CSAA) ¼ 0.018004 mg ml1 (see the value
in the first line with the label CSAA in Table 4) when
this correlation is taken into account.
The combined standard uncertainties of aflatoxin
M1 content as determined by the densitometric and
visual TLC quantification were 0.017 and 0.020 mg l1,
respectively, for a blank sample of milk spiked with
0.05 mg l1 (Tables 4 and 5). These combined uncer-
tainties without the contribution of the intermediary
precision are reduced to 0.011 and 0.015 mg l1 (see the
bars labelled CU-prec in Figure 6), respectively, almost
equal to the uncertainties of the intermediary preci-
sions alone: 0.0124 and 0.0129 mg l1, respectively.
Figure 6 summarises the calculations in Tables 4
and 5, showing the combined uncertainties and their
contributions to the densitometric and visual TLC
quantifications of a 0.05 mg l1 aflatoxin M1 spiked
blank sample milk. For comparison, the values of the
combined uncertainties without the intermediary pre-
cision contribution (see CU-Prec in Figure 6) are
Food Additives and Contaminants 691
almost equal to the uncertainties of the intermediary
Densitometric
precisions alone. This result corroborates the proposed
Visual
use of the intermediary precision as a gross estimation
of the analytical method’s combined uncertainty
(Populaire and Giménez 2006). The higher contribu-
tions of the combined uncertainty come from the
intermediary precision or within-laboratory reproduc-
calib Recov Volumes calibration process and by the uncertainty due to the
allowed recovery variation range. The two last have
nearly the same contribution. The uncertainties due to
the volume measurements are almost negligible and
even the combination of their four contributions is
very low, 0.00144 and 0.00120 for densitometric and
visual quantification, respectively (see the last pair of
bars in Figure 6), corresponding to less than 10% of
the analytical methods’ combined uncertainties. Within
these uncertainties’ sources the most important is the
contribution due the volume measurement of 10 ml of
the calibration standard solution, Vp, which is respon-
sible for 41% of the total volume uncertainties in both
methods.
Conclusions
ffi
This paper showed in a detailed and conceptually
consistent manner the estimation of the combined
uncertainty of the aflatoxin M1 content in milk,
determined by visual and densitometric quantification
with immunoaffinity column clean-up, through the
ð34Þ
cause-and-effect methodology or bottom-up approach
recommended by the ISO through the ISO GUM
(ABNT, INMETRO 2003; BIPM et al. 1995). From
these estimations it seems that the uncertainty of the
densitometric TLC quantification is approximately
between 10% and 30% lower than that of the visual
TLC quantification, but their trueness is the same
(INMETRO 2009; Joint Committee for Guides in
Metrology (JCGM/WG 2) 2008).
The main source of uncertainty in both the visual
and the densitometric methods, as generally happens,
was the intermediary precision, which alone is a
reasonable estimation of the total (combined) analyt-
ical uncertainty (Populaire and Giménez 2006), which
justifies the use of the top-down approach to estimate
the analytical uncertainty. However, the systematic but
strenuous cause-and-effect (bottom-up) approach for
the estimation of analytical method uncertainty
enabled the authors to understand the most important
sources of uncertainties, which, if reduced, lead to an
efficient analytical method precision improvement.
Excepting the intermediary precision, the uncertainties
due to the calibration process and the allowed recovery
ratio range are equally the most important sources of
uncertainties of the TLC aflatoxin M1 analysis, while
the uncertainty contributions due to the volume
692 K.L. Carvalho et al.
measurements are practically negligible. The inclusion
of the analytical method uncertainty in the calculation
of the decision limit for product compliance, according
European legislations, increases the importance of a
realistic and conceptually consistent estimation of the
combined and expanded uncertainties of the analytical
procedures. By increasing the analytical precision,
reducing its uncertainty, the consumer and vendor
risks concerning the compliance decision are simulta-
neously decreased.
This work shows that the use of the OLS to fit the
data of measurement instrument calibration, when
they are the subject of heteroskedasticity, leads to
proportional bias and to incorrect estimation of the
measurement uncertainty. The OLS lower and upper
prediction limit lines for the heteroskedastic calibra-
tion data presented in this work are far from the fitted
calibration straight line at the barycentre and MPL
when compared with the WLS lower and upper
prediction limit lines. This implies that the uncertain-
ties of the analyte content at these levels, obtained
from the OLS fit, is much higher than the uncertainty
obtained from the WLS fit. As a consequence, the
decision limit (CC) of the OLS is also higher than the
CC of the WLS. This can be a great risk for consumer
health when using the decision limit as a compliance
criterion.
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