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Evaluation of the uncertainty statement in the case of mercury speciation

analysis
Zsuzsa J
okai* and Peter Fodor

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Published on 26 June 2009 on http://pubs.rsc.org | doi:10.1039/B900414A

Received 13th January 2009, Accepted 17th June 2009

First published as an Advance Article on the web 26th June 2009
DOI: 10.1039/b900414a
Nowadays, the evaluation of the uncertainty statement is more and more incorporated into quality
assurance procedures. In this paper, the uncertainty statement of a mercury speciation analytical
method is presented using the relationships fixed by GUM (Guide to the Expression of Uncertainty
in Measurement). According to the developed analytical method, the sample is digested using an
alkaline phase, then phenylation is applied to derivatize the organomercury compounds found in the
digested solution. Finally, following solid phase microextraction (SPME) of the phenylated species,
detection with GC-AFS is carried out. The main purpose of this work is to identify the analytical steps
that have the strongest influence on the reproducibility of the measurements. The presented approach
is based on an investigation executed in more steps and so it provides an opportunity to determine the
uncertainty of each applied analytical step. Based on the completed experiments, the alkaline digestion
and the calibration were the most critical steps regarding reproducibility. Phenylation derivatization,
SPME and volume and weight measurements during sample preparation were less dominant processes
in this respect.

Introduction
Many important decisions are based on the results of chemical
quantitative analysis. The results are used, for example, to check
materials against specifications or statutory limits and to estimate yields and monetary value. Whenever decisions are based
on analytical results, it is important to give some indication of the
quality of the results.
In some sectors of analytical chemistry, introducing quality
assurance (QA) measures is required by law in order to prove
that laboratories are capable of and are providing data of the
required quality. Such measures include: the use of validated
methods of analysis, the use of defined internal quality control
procedures, participation in proficiency testing schemes,
accreditation based on ISO 170251 or establishing traceability
of the results of the measurements. Further information on
analytical QA procedures is provided by ref. 2.
As a consequence, chemists are, on their part, coming under
increasing pressure to demonstrate the quality of their results and
particularly to demonstrate their fitness for purpose by giving
a measurement of the confidence that can be placed on the result.
They have to include the degree to which a result would be expected
to agree with other results or with other own results, normally
irrespective of the analytical method used. One of the useful
methods for this purpose is the measurement of uncertainty.2
The definition of the term uncertainty (of measurement) used
in this work and adopted for the International Vocabulary of
Basic and General Terms in Metrology3 is:

Corvinus University of Budapest, Faculty of Food Science, Department of

Applied Chemistry, Hungary-1118, BudapestVill
anyi ut 29-31. E-mail:
zsuzsanna.szatura@uni-corvinus.hu; Fax: +36 1 466 4272; Tel: + 36 1
482 6164

This journal is The Royal Society of Chemistry 2009

A parameter associated with the result of a measurement,

that characterizes the dispersion of the values that could
reasonably be attributed to the measurand.
It is also important to distinguish between error and uncertainty. Error is defined as the difference between an individual
result and the true value of the measurand. Error is a single value,
as such, so the value of a known error can be applied as
a correction to the result. Uncertainty takes the form of a range
and if it is estimated for an analytical procedure and defined
sample type, it can be applied to all determinations so described.
In general, the value of the uncertainty can not be used to correct
a measurement result.4
The concept of measurement uncertainty has been recognized
by chemists for many years. A publication in 1993 on the Guide
to the Expression of Uncertainty in Measurement5 by ISO in
collaboration with BIPM, IEC, IFCC, IUPAC, IUPAC and
OIML, formally established general rules for evaluating and
expressing uncertainty in measurements across a broad spectrum
of measurements.
According to the so-called bottom-up approach, the process of
evaluating uncertainty involves mainly four stages: (1) creating
the equation for calculating the result, namely specification of the
measurand, (2) identifying all significant sources of uncertainty
in the method, (3) establishing of the magnitude of each potential
source of uncertainty identified from published or experimental
data, namely establishing of standard uncertainties and finally
(4) calculating of combined uncertainty.
In this work, an uncertainty statement connected with mercury
speciation analysis is presented. Nowadays, there is a growing
interest in mercury speciation in a variety of biological, industrial
and food samples for reasons of public health, since mercury is
a potential environmental toxicant. The main source of human
intake of mercury contaminants originates from methylmercury
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(MeHg) in fish and fishery products. Typical methylmercury

levels in fish and fishery products found in the literature are
between 0.050.3 mg g1.6,7 MeHg is particularly interesting due
to its high toxicity compared to inorganic mercury and its high
proportion among organomercury species in the environment.
Mercury species may induce alterations in the normal development of the brain of infants and may induce neurological changes
in adults. To protect public health, maximum levels of mercury
content in fishery products are laid down in relevant regulations.8
The 2003 recommendation from JECFA, the Joint FAO/WHO
(Food and Agriculture Organization of the United Nations and
World Health Organization) Expert Committee on Food Additives, is that the provisionally tolerable weekly intake (PTWI)
of MeHg is limited to 1.6 mg per kg of body mass.9 On the basis of
this recommendation and other studies, several national and
international regulatory bodies monitor fish samples to check the
levels of contamination and issue guidelines on the consumption
of fish and shellfish.
The measurement of mercury species requires hyphenated
techniques, which typically has an experimental process and
instrumentation with a greater level of complexity than those for
element measurement.
The mercury speciation techniques are based on successive
steps: (1) liberating mercury species from solid samples using
acidic leaching10 or alkaline digestion,11,12 (2) extraction of the
liberated analytes using organic solvents13,14 or solid phase
microextraction,15,16 (3) separation of species most often using
gas chromatography and (4) finally, detection of separated
components. Derivatization of the extracted monoalkylmercuric compounds into nonpolar molecules has been preferred
prior to analysis by gas chromatography, however, methods
without derivatization have also been developed.17 The most
common derivatization procedures used for mercury species are
highlighted by Dez and Bayona.16
The employed steps may vary from one procedure to another
but all the methods have their own particular sources of uncertainty and/or bias. For some techniques, for instance, mistakes
may occur due to sampling, storage, extraction, derivatization in
the case of gas chromatography, separation or calibration. These
effects cumulatively influence the uncertainty of final result and/
or can lead to bias.
The aim of this paper is to present a method for the evaluation
of uncertainty in the case of a mercury speciation analytical
procedure. As the first step of the procedure, the sample was
digested using an alkaline phase in order to liberate the required
compounds from the matrix. As organomercury compounds are
usually present as ionic species in diluted sample solutions, these
species were to be converted into volatile ones since gas chromatography was used for separation. Phenylation by sodium
tetraphenylborate was applied to derivatize organomercury
compounds. Finally, following solid phase microextraction of
the phenylated species, detection with gas chromatographyatomic fluorescence spectrometry (GC-AFS) was carried out.
The main purpose of this work is to try to find out which steps in
this multi-step procedure have the largest contribution to the
overall uncertainty. For this reason, an investigation executed in 4
steps was carried out. During experiments, the overall uncertainty
determined experimentally was applied as a combined standard
uncertainty and based on this knowledge the uncertainty of each
1230 | J. Anal. At. Spectrom., 2009, 24, 12291236

analytical step was calculated. Thus, it became known how the

experimentally determined overall uncertainty distributes among
the individual analytical processes.

Experimental
Instrumentation
A manual SPME device (Supelco, Bellefonte, PA, USA) equipped with a fused silica fiber coated with a 100 mm film of poly(dimethylsiloxane) (PDMS) was used for extraction in all
experiments. GC separation was carried out on a HP-5890 gas
chromatograph equipped with a 15 m long, 0.53 mm id, 1.5 mm
film thickness DB-1 capillary column. The end of the GC column
was connected to a 0.5 m long, 0.32 mm id deactivated fused
silica capillary. Both the DB-1 column and the fused silica
capillary are located in the GC oven and are subjected to the
same heating conditions. The fused silica capillary was driven
through a pyrolysis tube before being coupled to a PSA 10.750
(PS Analyticals, Orpington, U.K.) atomic fluorescence (AFS)
mercury detector. Argon was used as the carrier gas. The split/
splitless injector of the GC was fitted with a 0.75 mm id (SPME)
liner and was used in splitless mode.
The schematic diagram of the instrumental setup is presented
in Fig. 1. The operational parameters of the GC-pyrolysis-AFS
system are shown in Table 1.
For sample preparation, a Realsonic RS 16-F ultrasonic bath
(Realsonic, Budapest, Hungary) operated at 50 W (dm3)1
ultrasonic power was used. The prepared samples were centrifuged with a Hettich Mikro 22R centrifuge (Hettich, Tuttlingen,

Fig. 1 Schematic diagram of the GC-pyrolysis-AFS system.

Table 1 Operational parameters of the applied GC-pyrolysis-AFS

system
Parameter

Value

Column head pressure

Injector temperature
Initial oven temperature, hold time
Ramp
Final temperature, hold time
Pyrolysis temperature

100 kPa
190  C
100  C, 0.6 min
30  C min1
250  C, 1 min
800  C

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Germany). An electronically controlled magnetic stirring plate

was applied for the agitation of the sample solutions during
SPME sampling.

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Reagents and standards

Digestion of samples was carried out with 25% (w/v) potassium
hydroxide or 18% (w/v) sodium hydroxide in methanol, which
were prepared by dissolving appropriate amounts of KOH or
NaOH (Merck, Darmstadt, Germany) in methanol (Carlo Elba
Reagents, Milan, Italy).
Freshly prepared 1% (w/v) sodium tetraphenylborate
(NaBPh4) solution (Sigma-Aldrich, Budapest, Hungary) was
used as the derivatization reagent. The 1 M sodium acetate buffer
solution (NaAc) was prepared by dissolving appropriate
amounts of the salt (Reanal, Budapest, Hungary) in deionized
water (DIW), the pH was adjusted to 5.0 with acetic acid
(Reanal).
The methylmercury stock solution (ca. 1000 mg L1) was
prepared by dissolving appropriate amounts of crystalline
(98.4% purity) methylmercury chloride (MeHgCl, SigmaAldrich) in methanol. The solution was stored in the dark at 4  C
for maximum of 2 months. Working standards of MeHgCl were
prepared by serial dilution of the stock solution with DIW. The
10 mg mL1 MeHgCl solution was prepared weekly from the
stock solution while solutions of lower concentrations were
prepared daily.
Deionized water, R > 18.2 MU (Elgacan, Ultrapure, Vivendi
Water System Ltd., High Wycombe, Bucks, England), was used
in all experiments.
Two CRM samples, BCR 464 (IRMM) and TORT-2
(National Research Council of Canada, Ottawa, ON, Canada),
were employed for the presented experiments.
Analytical procedure
Sample preparation was based on the procedure reported
previously by J
okai and Abrank
o.18 Briefly, nominally 250 mg of
the samples was accurately weighted into a 30 mL glass vial and
46 mL of 25% (w/v) KOH in methanol or 18% (w/v) NaOH in
methanol (both were 4.5 M) were added. The vial was then sealed
with a PTFE-lined screw cap and was placed in an ultrasonic
bath for 3 h at 75  C. After the vial was allowed to cool to
ambient temperature, the procedure varied depending on the
MeHg content of the sample.
In the case of the BCR 464 sample, which is a high-mercury
content sample containing 5.12 mg g1 MeHg (as Hg) based on
dry weight, the entire digest was thoroughly rinsed into a 50 mL

volumetric flask. 1 mL of this solution was pipetted into a 10 mL

volumetric flask. Since the standard addition method was to be
used, 1 mL of aqueous MeHgCl standard was also added to the
solution before it was diluted to the nominal volume. Standard
addition levels corresponded to results 1, 2 or 4 the expected
analyte concentration of the sample solution.
In the case of the low-mercury content TORT-2 CRM
sample containing only 0.152 mg g1 MeHg (as Hg) based on dry
weight, after allowing the vial to cool to ambient temperature,
the digest was shaken for a few seconds and then 5 mL was
pipetted into a 6 mL glass centrifuge vial. The solution was
centrifuged for 15 min at 4100 g and 20  C. 1 mL of the supernatant was transferred to a 10 mL volumetric flask and the
standard addition method was carried out as described above.
In both cases, 1 mL of the 10 mL diluted (and spiked) solutions
was pipetted into 30 mL glass vials, each containing 10 mL of
1 M, pH 5 acetate buffer. At this point, a clean PTFE-coated
stirring bar was placed into the vial. Finally, 1 mL of freshly
prepared 1% NaBPh4 was added and the vial was immediately
sealed with a screw cap outfitted with a PTFE-coated rubber
septum. The vial was placed on a magnetic stirring plate and
during vigorous stirring (700 rpm), headspace SPME extraction
was carried out. After extraction, the fiber was introduced into
the heated inlet port of the GC-pyrolysis-AFS system for thermal
desorption and analysis.
Figures of merit
For MeHg, with the optimized analytical conditions, the detection limit of the method calculated as three times the standard
deviation (SD) of the blank value divided by the slope of the
calibration curve is 0.006 ng mL1. The quantification limit (QL),
calculated as the blank plus ten times the SD, is 0.018 ng mL1.
The range of linearity goes from 0.02 to 0.6 ng mL1. Reproducibility for methylmercury in the case of high-content
samples is 4%, in the case of low-content samples is 1015%,
determined experimentally.
Quality control of measurements
The mentioned two different CRMs were used for validation
purposes, namely BCR 464 tuna fish and TORT-2 lobster
hepatopancreas. The summary of the validation results is given
in Table 2.
To control the quality of this method, we have participated in
the CCQM P39 and CCQM P39.1 interlaboratory comparisons
as invited expert laboratory. These comparisons were an activity
of the Inorganic Analysis Working Group (IAWG) of CCQM

Table 2 Results of the validationa

BCR 464 (tuna fish)

TORT-2 (lobster hepatopancreas)

Measured MeHg (as Hg)/mg kg1 dry wt

Certified MeHg (as Hg)/mg kg1 dry wt

4.85  0.42
0.151  0.036

5.12  0.16
0.152  0.013

a
Each sample was digested in triplicate and standard addition was used for quantification. Sample preparation procedures differed depending on the
MeHg content (see text for details). The expanded uncertainties of the measured values are expressed as the 95% confidence interval (see Table 5, Table 6
and text for details).

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(Comite Consultatif pour la Quantite de Matiere) and were

organized by the Institute for Reference Materials and
Measurements (IRMM). CCQM P39 was the first and CCQM
P39.1 was the second CCQM comparison decided for the
quantification of MeHg. In the first case, a tuna fish test sample
with relatively high mercury content was analyzed, secondly
a real life salmon fish sample with significantly lower mercury
content was investigated. In both cases, our measurements were
in good agreement with those of other laboratories.19,20

Table 3 The sources of uncertainty and the standard uncertainties in the

case of the BCR 464 CRM sample
Type A estimation
Source of
uncertainty

Number of
measurements

Mean, xi

Standard
uncertainty, u(xi)

Results and discussion

c/pg mL1
V1/mL
V2/mL
V10/mL
L10/mL
L50/mL
V3/mL

6
6
6
6
6
6

202.75
0.9989
0.9989
9.9589
9.9839
49.9672
0.9989

Not quantified
0.0061
0.0061
0.0948
0.0332
0.1457
0.0061

The measurand model equation

Type B estimation

As it was mentioned above, the first step of the process of the

evaluation of uncertainty is to create a model equation for
calculating the result. In the experiments described, two CRMs
were analyzed. BCR 464 (tuna fish) is considered a matrixmatching CRM in the case of modelling MeHg determination
in seafood samples. However, the matching of a CRM with
a market sample in terms of the analyte concentration is
also a basic requirement. In this aspect, TORT-2 CRM sample,
a low-mercury content sample, was suitable for modelling the
analysis of real-life samples.
On the basis of the described analytical procedures, the
following two model equations can be created for the measurand.
Eqn (1) relates to the measurements of the high-content BCR
464 sample, while eqn (2) describes the process of MeHg determination in the low-content TORT-2 sample:
,
cV1 V2 V10
,
V1
y
L10 L50 m  106
(1)
V3

Source of
uncertainty

xi  a

Type of
distribution

Standard
uncertainty

m/g

0.2500  0.0001

Rectangular

5.77  105

Table 4 The sources of uncertainty and the standard uncertainties in the

case of the TORT-2 CRM sample
Type A estimation
Source of
uncertainty

Number of
measurements

Mean, xi

Standard
uncertainty, u(xi)

c/pg mL1
V1/mL
V2/mL
V10/mL
L10/mL
V5/mL
V3/mL

6
6
6
6
6
6

63.56
0.9989
0.9989
9.9589
9.9839
4.9712
0.9989

Not quantified
0.0061
0.0061
0.0948
0.0332
0.0487
0.0055

Type B estimation

,
cV1 V2 V10
,
V1
y
L10 V5 m  106
V3

(2)

where y is the measurand (MeHg (as Hg) content, mg g1 dry

weight); c is the concentration (pg mL1) of the final solution
used for SPME; V1, V2, V3, V5, V10, L10 and L50 indicate the
volumes during the sample preparation procedure (V1: 1 mL of
the 10 mL diluted and spiked solutions, V2: 1 mL of the 1%
NaBPh4 reagent, V3: 1 mL of the 50 mL digested and diluted
sample solutions in the case of the BCR 464 sample and 1 mL of
the 5 mL digested samples in the case of the TORT-2 CRM
sample, V10: 10 mL of the acetate buffer, V5: 5 mL of the alkaline
reagent in the case of the TORT-2 sample, L50: 50 mL of the
stock solutions from the digested samples in the case of the BCR
464 sample, L10: 10 mL of the spiked solutions) and m is the mass
of the CRMs weighed. V1, V2, V3, V5 and V10 represent pipetting
processes, L10 and L50 represent the adjusting of solutions to the
nominal volume in a volumetric flask.
Identifying and quantifying the sources of uncertainty
Since this method has a great level of complexity, numerous
sources of uncertainty have to be taken into account.
1232 | J. Anal. At. Spectrom., 2009, 24, 12291236

Source of
uncertainty

xi  a

Type of distribution

Standard
uncertainty

m/g

0.2500  0.0001

Rectangular

5.77  105

Some of these (c, V, L, m) appear above in the equations,

while others (extraction, calibration, derivatization, SPME and
detection processes) are not included but their effects manifest in
the uncertainty of c. The sources of uncertainty in the case of the
BCR 464 and TORT-2 measurements are shown in Table 3 and
Table 4, respectively. Before combination, all uncertainty
contributions must be expressed as standard uncertainties (u(xi)),
that is, as standard deviations.
The V1, V2, V3, V5, V10, L10 and L50 uncertainty components
were evaluated experimentally from the statistical distribution of
the results of series of measurements and were expressed as
standard deviations. The ISO Guide refers to this case as Type A
estimation.
In the case of Type B estimation, the uncertainty is evaluated
from an assumed probability distribution based on experience or
other information. The uncertainty of the mass measurement (m)
derived from certificate, gives a limit (a) without specifying
a level of confidence. If there is a reason to expect that extreme
values are likely, it is normally appropriate to assume
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a rectangular distribution. In this case, on the basis of GUM, the

a/O3 figure gives the standard uncertainty (u(m)).
It can be seen, that the standard uncertainty of the concentration (u(c)) is not quantified. The reason for this is that the uncertainty of the analytical processes (extraction, calibration,
derivatization, SPME and detection) manifesting in the u(c) are
unknown. In order to determine the uncertainty of the mentioned
processes separately, an experiment of 4 steps was performed. It is
discussed in the section The uncertainty of extraction, calibration,
derivatization, SPME and detection processes.

ci



vy y xi uxi  yxi

vxi
uxi

(4)

Accordingly, the u(y,xi) is just a difference between the values of y calculated for [xi + u(xi)] and xi, respectively (eqn (5)),
and it denotes the uncertainty in y arising from the uncertainty
in xi.
u(y,xi) y(xi + u(xi))  y(xi)

(5)

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Calculating the combined uncertainty

The calculations presented in this work were made according to
the spreadsheet method given in the Eurochem guide.4
The general relationship between the combined standard
uncertainty (u(y)) of a value y and the uncertainty of the independent parameters (xi) on which it depends is
q q
(3)
u y Sci 2 uxi 2 Suy; xi 2

The uncertainty of extraction, calibration, derivatization, SPME

and detection processes
To establish the uncertainty of extraction, calibration, derivatization, SPME and detection processes separately, an investigation of 4 steps was performed. In these experiments, the overall
uncertainty determined experimentally (standard deviation of
peak areas or of measured concentration values) was considered
as the combined standard uncertainty and knowing this value,
the uncertainties of extraction, calibration, derivatization and
SPME processes were calculated using the adequate relationships
fixed by GUM. The results of these investigations and the
calculation can be seen in Tables 5 and 6.

where u(y) is the combined standard uncertainty; u(xi) is the

standard uncertainty of the several parameters; ci is a sensitivity
coefficient evaluated as the partial differential of y with respect
to xi. Provided, that y(xi) is linear in xi or u(xi) is small compared
to xi, the partial differentials can be approximated by

Table 5 The spreadsheet method and the experimental steps in order to determine the uncertainty of SPME, derivatization, digestion and calibration
processes in the case of the BCR 464 sample (see text for details)
c
xi
u(xi)

V1

202.75

c
V1
V2
V3
V10
L10
L50
m
y/mg g1
u(y.xi)
u(y.xi)2

4.84800

4.82115

V2

V3

V10

L10

0.9989
0.0061

0.9989
0.0061

0.9989
0.0061

9.9589
0.0948

9.9839
0.0332

49.9672
0.1457

0.2500
0.00006

202.75
1.005
0.9989
0.9989
9.9589
9.9839
49.9672
0.2500

202.75
0.9989
1.005
0.9989
9.9589
9.9839
49.9672
0.2500

202.75
0.9989
0.9989
1.005
9.9589
9.9839
49.9672
0.2500

202.75
0.9989
0.9989
0.9989
10.0537
9.9839
49.9672
0.2500

202.75
0.9989
0.9989
0.9989
9.9589
10.0171
49.9672
0.2500

202.75
0.9989
0.9989
0.9989
9.9589
9.9839
50.1129
0.2500

202.75
0.9989
0.9989
0.9989
9.9589
9.9839
49.9672
0.25006

4.85059
0.02685
7.21104

4.81869
0.00259
6.73  106

4.88656
0.02931
8.59  104

4.86424
0.03856
1.49  103

4.86226
0.01624
2.64  104

4.84696
0.01426
2.03  104

0.00104
1.09  106

The u(y) value depends on the actual investigation.

1 step. Investigation to determine the uncertainty of the SPME process
The RSD%
of the peak areas was 0.7027, consequently the u(y) 0.0341 mg g1 dry weight.
q

u y

L50

Su yXi 2 , where the xi is the SPME.

2 step. Investigation to determine the uncertainty of the derivatization process

The RSD% of the peak areas was 1.7182, consequently the u(y) 0.0833 mg g1 dry weight.
q
u y Su yXi 2 , where the xi are the parameters: SPME, derivatization, V1, V2,
.
and V10
3 step. Investigation to determine the uncertainty of the digestion process
The RSD%
of the peak areas was 3.5267, consequently the u(y) 0.1709 mg g1 dry weight.
q
u y Su yXi 2 , where the xi are the parameters: SPME, derivatization, digestion,
, , ,
,
,
V1 V2 V3 V10 L10 L50 and m.
4 step. Investigation to determine the uncertainty of the calibration
The RSD% of the peak areas was 4.2729, consequently the u(y) 0.2071 mg g1 dry weight.
q
u y Su yXi 2 , where the xi are the parameters: SPME, derivatization, digestion,

u(ySPME) 3.41  102 mg g1

u(yderivatization) 5.97  102 mg g1

u(ydigestion) 1.45  101 mg g1

u(ycalibration) 1.17  101 mg g1

V1, V2, V3, V10, L10, L50, m and calibration.

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Table 6 The spreadsheet method and the experimental steps in order to determine the uncertainty of SPME, derivatization, digestion and calibration
processes in case of TORT-2 sample (see text for details)
c
xi
u(xi)

V1

63.56

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c
V1
V2
V3
V10
L10
V5
m
y/mg g1
u(y.xi)
u(y.xi)2

0.15120

0.15037

V2

V3

V10

L10

V5

0.9989
0.0061

0.9989
0.0061

0.9989
0.0061

9.9589
0.0948

9.9839
0.0332

4.9712
0.0487

0.2500
0.00006

63.56
1.005
0.9989
0.9989
9.9589
9.9839
4.9712
0.2500

63.56
0.9989
1.005
0.9989
9.9589
9.9839
4.9712
0.2500

63.56
0.9989
0.9989
1.005
9.9589
9.9839
4.9712
0.2500

63.56
0.9989
0.9989
0.9989
10.0537
9.9839
4.9712
0.2500

63.56
0.9989
0.9989
0.9989
9.9589
10.0171
4.9712
0.2500

63.56
0.9989
0.9989
0.9989
9.9589
9.9839
5.0199
0.2500

63.56
0.9989
0.9989
0.9989
9.9589
9.9839
4.9712
0.25006

0.15128
8.34  104
6.95  107

0.15029
8.44  105
7.12  109

0.15241
9.11  104
8.29  107

0.15171
1.21  103
1.45  106

0.15269
5.11  104
2.60  107

0.15117
1.49  103
2.22  106

2.90  105
8.42  1010

The u(y) value depends on the actual investigation.

1 step. Investigation to determine the uncertainty of the SPME process
The RSD%
of the peak areas was 4.2576, consequently the u(y) 6.44  103 mg g1 dry weight.
q

u y

Su yXi 2 , where the xi is the SPME.

u (ySPME) 6.44  103 mg g1

2 step. Investigation to determine the uncertainty of the derivatization process

The RSD%
of the peak areas was 5.2525, consequently the u(y) 7.94  103 mg g1 dry weight.
q

b
u y Su yXi 2 , where the xi are the parameters: SPME,
u(yderivatization) 4.41  103 mg g1
.
derivatization, V1, V2, and V10
3 step. Investigation to determine the uncertainty of the digestion process
The RSD%
of the peak areas was 10.0993, consequently the u(y) 1.53  102 mg g1 dry weight.
q

u y

Su yXi 2 , where the xi are the parameters:

u(ydigestion) 1.29  102 mg g1

SPME,derivatization, digestion, V1, V2, V3, V10, L10, V5 and m.

4 step. Investigation to determine the uncertainty of the calibration
The RSD%
of the peak areas was 12.0431, consequently the u(y) 1.82  102 mg g1 dry weight.
q

u y Su yXi 2 , where the xi are the parameters: SPME,

derivatization, digestion, V1, V2, V3, V10, L10, V5, m and
calibration.

In the first step, the reproducibility of the SPME-desorptiondetection processes was investigated. For this purpose, in all
cases one previously derivatized solution (10 mL of acetate buffer
+ 1 mL of sample solution + 1 mL of NaBPh4 reagent solution,
see Analytical procedure section) was extracted and measured six
times. The standard deviation of the peak areas is derived just
from the uncertainty of SPME and desorption process in the
injector part of the GC and the GC-AFS system in this case.
Based on the RSD% of peak areas, the u(y) arising only from
the uncertainty of SPME-desorption-detection (u(ySPME)) was
determined. This value can be seen in Table 5 and Table 6
marked with an a.
In the second step, the uncertainty of the derivatization
process was studied. Six solutions were prepared (10 mL of
acetate buffer + 1 mL of sample solution + 1 mL of NaBPh4
reagent solution) from all samples and following derivatization,
these solutions were extracted and measured. The standard
deviation of the peak areas is derived now from the uncertainty
of the derivatization process, the above mentioned SPMEdesorption-detection processes and the V1, V2 and V10 sources.
Using eqn (5), the u(y,V1), u(y,V2) and u(y,V10) could be calculated, the u(ySPME) was already known and the u(y) was also
known (the relative standard deviation of peak areas was
1234 | J. Anal. At. Spectrom., 2009, 24, 12291236

u(ycalibration) 9.92  103 mg g1

referred to y), so the u(yderivatization) could be calculated by

applying eqn (3). This value can be seen in Table 5 and Table 6
marked with a b.
To determine the uncertainty of the alkaline digestion process,
six parallel digestions were carried out in the case of both CRM
samples. Calibration was not used, so its uncertainty does not
have to be taken into account. The effect of the above mentioned
uncertainty sources in addition to V3, V5, L10, L50 or V5, m and
digestion process manifested in the standard deviation of the peak
areas. Again using eqn (5), the individual u(y,xi) values could be
calculated in the cases of all mentioned sources, the u(ySPME) and
the u(yderivatization) were already known, so the u(ydigestion) could
be calculated, shown in Table 5 and Table 6 marked with a c.
Finally, u(ycalibration) was determined. These experiments were
similarly performed as before but in this case a standard addition
calibration was applied. The standard deviation of the measured
concentration values was derived from the uncertainty of all
mentioned sources plus that of the calibration process. With
knowledge of the previous results and using the adequate
relationships the u(ycalibration), could be determined, which is
presented in Table 5 and Table 6 marked with a d.
It can be seen that during these experiments, a reversed
calculation process was realized compared to the common
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protocol; namely with knowledge of the combined standard

uncertainty (the experimentally determined overall uncertainty
was considered as the combined standard uncertainty), the
uncertainty of the individual sources was calculated. Nevertheless, this method can not be considered as a new approach, it is
described in GUM, and its advantage lies in the fact that it
provides the opportunity to determine the uncertainty of the
above mentioned detection, SPME, derivatization, digestion and
calibration processes. In other words, by applying the described
method, it became known how the experimentally determined
overall uncertainty distributes among the individual analytical
processes.
Knowing each individual u(y,xi) value found in Table 5 and
Table 6 and using eqn (3), the combined standard uncertainty
referred to the whole analytical procedure can be determined. It
is 0.21 mg g1 in the case of BCR 464 and 0.018 mg g1 in the case
of TORT-2. The expanded uncertainty (U(y)) is obtained by
multiplying the combined standard uncertainty with a coverage
factor of 2, giving 0.42 mg g1 and 0.036 mg g1 in the case of BCR
464 and TORT-2, respectively.
The results of the experiments are presented in Fig. 2 and 3.
The values of u(y,xi) presented in Fig. 2 are taken from Table 5
and refer to the measurements made with the BCR 464 sample.

Fig. 2 Uncertainties in MeHg determination in the case of measurements made with the BCR 464 sample.

Fig. 3 Uncertainties in MeHg determination in the case of measurements made with the TORT-2 sample.

This journal is The Royal Society of Chemistry 2009

The u(y,xi) values shown in Fig. 3 are taken from Table 6 and
refer to the measurements made with the TORT-2 sample.
Based on our results, it can be concluded that irrespective of
the sample concentration, the alkaline digestion has the strongest
influence on the reproducibility of the measurement results while
the calibration has the second strongest influence on it.
As for digestion, there are a lot of important parameters in
connection with the use of an ultrasonic bath during the sample
preparation, for example the number of samples prepared
simultaneously, the volume of the samples, the volume of water
in the bath, the position of the samples in the bath, etc. The
effects of the treatment-time and the applied temperature were
investigated during method development, the influence of the
other factors were not studied. Consequently, the large uncertainty of the digestion process may come from this fact.
The uncertainty of the applied standard addition calibration
probably derives from the fact that separate standard addition
solutions were applied for each individual digested sample
although the matrix was in all cases the same. In this way, the
measurements were over-calibrated.
Since the uncertainty of the calibration process was established
on the basis of experimental results, all uncertainty sources
associated with the calibration process has been taken into
account by calculation. Apart from this fact, it may be interesting
to determine the uncertainty of the purity of the calibrant within
the uncertainty of the calibration process.
The purity of the MeHgCl standard is given on the
certificate as 0.9840  0.0050 with a level of confidence
95%, so the standard uncertainty of purity is given by
0:0050
uPurity
0:00255. Based on the model equations
1:96
(the purity is a multiplier factor in the equation) and using eqn
(5), the u(yPurity) is 1.24  102 mg g1 in the case of the BCR 464
sample, and 3.86  104 mg g1 in the case of the TORT-2 sample.
These values denote the uncertainty in y (MeHg as Hg content,
mg g1 dry weight) arising from the uncertainty in the purity of
the calibrant.
The u(ycalibration) was found to be 1.17  101 mg g1 in the case
of the BCR 464 and 9.92  103 mg g1 in the case of the TORT-2
(see values marked with a d in Tables 5 and 6), so it can be seen
how the u(yPurity) contributes to the u(ycalibration) in the two cases.
Moreover, it can be concluded that the SPME was a more
critical step regarding reproducibility in the case of the low
content TORT-2 sample compared to BCR 464, while the
uncertainty of the derivatization process proved larger in the case
of the high content BCR 464 sample. In the case of TORT-2,
the higher methanol and matrix concentration, while in the case
of BCR 464 the potential precipitation reaction between potassium and sodium tetraphenylborate can explain the above
mentioned symptom.18
According to our expectations, the volume and weight
measurements during sample preparation were less dominant
processes in this respect.
Finally, it should be emphasized that this investigation tended
to determine the uncertainty arising from the reproducibility, the
uncertainty associated with the determination of the bias was not
taken into account.
The recovery is always an important question, its study is
often a complicated problem.
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In any case, as a result of our experiments, it can be concluded

that the efficiency of the alkaline digestion is around 100% (see
Table 2) and neither the solid phase microextraction nor the
derivatization influences the accuracy because their efficiency is
maximum. The SPME is used in an equilibrium system and the
extraction of standard solutions is performed in the same
conditions as that of the sample solutions. Moreover, during
derivatization, the sodium tetraphenylborate reagent is many
orders of magnitude in excess compared to the analyte, and the
derivatization of standard solutions is performed in the same
conditions as that of the sample solutions.
It follows from the foregoing, that the calibration is the most
critical process regarding the accuracy of measurements. Since
organomercurials are very toxic, they must be handled rapidly
with the use of appropriate personal protection, so the preparation of standard solutions sometimes can not be precise
enough. The stability of the organomercury standard solution is
a much discussed question, so these facts can explain the
differences between the measured and the certified MeHg values
for the two CRM samples.

Conclusion
The uncertainty statement of the developed mercury speciation
analytical method was evaluated and is presented in this paper.
The presented study was based on an investigation executed in
4 steps. During the experiments, the overall uncertainty determined experimentally was considered as the combined standard
uncertainty and, with knowledge of this value, the uncertainty
of the individual analytical steps was calculated according to
GUM. By this means, it became clear how the uncertainty of the
individual analytical processes contributes to the experimentally
determined overall uncertainty. Consequently, this study
provides an opportunity to find the steps in a multi-step procedure that have the largest contribution to the overall uncertainty.
Measurements applied to one of the two CRM samples represented analysis of samples with a high MeHg content (a few mg
g1), measurements with the other CRM represented analysis of
samples with low (a few ng g1) MeHg content.
As a result of the completed experiments, it can be concluded
that the alkaline digestion and the calibration are the most critical steps regarding reproducibility irrespective of the measured
CRM sample. Phenylation derivatization, SPME and volume
and weight measurements during sample preparation are less
dominant processes in this respect.
Knowing the uncertainty of each step, critical analytical
processes can be planned more carefully in the future.

1236 | J. Anal. At. Spectrom., 2009, 24, 12291236

Safety
Organomercurials are very toxic, therefore, inhalation or skin
contact must be avoided. These compounds must be handled
with the use of appropriate personal protection equipment and in
an adequately ventilated environment.

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This journal is The Royal Society of Chemistry 2009