Food Chemistry 176 (2015) 367375

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Determination of methylmercury in marine biota samples

with advanced mercury analyzer: Method validation
Sabine Azemard, Emilia Vassileva
International Atomic Energy Agency, Department of Nuclear Sciences and Applications, Environment Laboratories, 4 Quai Antoine 1er, MC 98000, Monaco

a r t i c l e

i n f o

Article history:
Received 8 August 2014
Received in revised form 17 December 2014
Accepted 20 December 2014
Available online 29 December 2014
Keywords:
Methyl mercury
Advanced mercury analyzer
Marine biota
Sample preparation
Method validation
Traceability
Uncertainty

a b s t r a c t
In this paper, we present a simple, fast and cost-effective method for determination of methyl mercury
(MeHg) in marine samples. All important parameters inuencing the sample preparation process were
investigated and optimized. Full validation of the method was performed in accordance to the ISO17025 (ISO/IEC, 2005) and Eurachem guidelines. Blanks, selectivity, working range (0.093.0 ng),
recovery (92108%), intermediate precision (1.74.5%), traceability, limit of detection (0.009 ng), limit
of quantication (0.045 ng) and expanded uncertainty (15%, k = 2) were assessed. Estimation of the
uncertainty contribution of each parameter and the demonstration of traceability of measurement results
was provided as well. Furthermore, the selectivity of the method was studied by analyzing the same
sample extracts by advanced mercury analyzer (AMA) and gas chromatographyatomic uorescence
spectrometry (GCAFS).
Additional validation of the proposed procedure was effectuated by participation in the IAEA-461
worldwide inter-laboratory comparison exercises.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
Mercury (Hg) is a persistent global pollutant, particularly in the
form of methylmercury (MeHg), a potent neurotoxin produced in
the aquatic environment from inorganic mercury by sulfate and
iron-reducing bacteria, as well as methanogens (Siciliano,
ODriscoll, & Lean, 2002). Indeed, MeHg is recognized as a major
environmental pollution issue and health hazard for humans
(Qiu, 2013). Owing to its capability to permeate through biological
membranes, once MeHg enters the food chain, it is efciently accumulated and transferred to organisms at higher trophic levels
(Mason & Benoit, 2003). As a result, the fraction of MeHg from
the total Hg (THg) in muscle tissue of top predator sh can be up
to almost 100% (Senn et al., 2010). The majority of population is
exposed to Hg through consumption of marine and freshwater
biota, mainly sh and seafood products (Sunderland, 2007). Specifically, large predatory sh which are at the top of the foodchain,
such as swordsh and tuna, contain high levels of MeHg and are
signicant sources of human exposure to that contaminant. Public
health warnings and guidelines on consumption of sh containing
high levels of MeHg have been published by the U.S. Food and Drug
Administration (USFDA, 2004) and the European Union (European
Corresponding author.
E-mail address: e.vasileva-veleva@iaea.org (E. Vassileva).
http://dx.doi.org/10.1016/j.foodchem.2014.12.085
0308-8146/ 2014 Elsevier Ltd. All rights reserved.

Union, 2008). However, to this date, ofcial legislation establishing

the maximum level of MeHg threshold authorized in seafood for
human consumption has not been issued.
Typically, the determination of MeHg in biota samples involves
the following analytical steps: (i) extraction, (ii) separation and (iii)
detection. The most widely used procedures for extraction are
based on alkaline (Carrasco & Vassileva, 2014) and acidic leaching
(Hintelmann & Nguyen, 2005), both assisted by microwave
(Nevado, Martin-Doimeadios, Bernardo, & Moreno, 2005) and conventional heating (Carrasco & Vassileva, 2014; Clmens,
Monperrus, Donard, Amouroux, & Gurin, 2011; Hintelmann &
Nguyen, 2005). More recently, enzymatic digestion (Lopez,
Cuello, Camara, & Madrid, 2010; Reyes, Rahman, Fahrenholz, &
Kingston, 2008), has also been described to isolate MeHg from
biota matrices. As the extraction process must preserve the integrity of the original chemical forms, special attention should be paid
in order to prevent transformation of species (Reyes et al., 2008).
Final steps, i.e. separation and detection, are commonly tackled
using hyphenated techniques, which couple the high resolution
power of a separation method, namely gas chromatography (GC)
(Carrasco, Diez, & Bayona, 2009), high-performance liquid chromatography (HPLC) (Jagtap, Krikowa, Maher, Foster, & Ellwood, 2011),
and capillary zone electrophoresis (CZE) (Silva da Rocha, Soldado,
Blancoa, & Sanz-Medel, 2001), to a Hg-selective and sensitive
detector. The major drawback of the most commonly used

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S. Azemard, E. Vassileva / Food Chemistry 176 (2015) 367375

separation technique, GC, is the requirement for derivatization of

the ionic MeHg into volatile species, and subsequent solid phase
micro extraction (SPME) or purge and trap collection (Carrasco &
Vassileva, 2014; Yang, Truong, Chen, & Belzile, 2009). The most
widely used detectors are atomic uorescence spectrometry
(AFS) (Carrasco & Vassileva, 2014; Nevado et al., 2005) and inductively coupled plasma mass spectrometry (ICP-MS) (Hight & Cheng,
2006; Hintelmann & Nguyen, 2005; Vassileva, Wysocka, & Betti,
2014).
Consequently in interlaboratory comparison and prociency
test a common result is that less than 25% of participants reporting
THg can provide MeHg results (IAEA, 2012). Considering that more
restrictive regulations on MeHg levels in marine samples are
expected, the current analytical challenge faced is the development
and validation of ready-to-use analytical methods, covering the
broadest possible range of sample matrices. Those methods will
be ultimately implemented in laboratories devoted to routine analysis of MeHg.
Direct and automated mercury analyzers, such as advanced
mercury analyzer (AMA-254) and direct mercury analyzer (DMA80), have proven to be a valuable tool for the direct analysis of
THg in a variety of biota matrices (Carrasco, Benejam, Benito,
Bayona, & Diez, 2011; Gerstenberger, 2004; Gerstenberger &
Pearson, 2002) and are widely found in laboratories performing
THg measurement as a routine. In marine biota, organic mercury
can be considered as MeHg, with a negligible error. Therefore, by
selecting a proper organic mercury extraction, the direct mercury
analyzer can be potentially applied for mercury speciation. Such
application has already been reported (Carbonell, Bravo,
Fernandez, & Tarazona, 2009; Maggi, Berducci, Bianchi, Giani, &
Campanella, 2009; Scerbo & Barghigiani, 1998; Valega et al.,
2006) and the European Commission has recently published a
standard operating procedure based on AMA-254 or DMA-80
determination (Caldern, Gonalves, Cordeiro, & de la Calle,
2013; Cordeiro et al., 2013).
The aim of this study was to develop and validate a ready-touse analytical method, consisting of extraction of MeHg, and direct
mercury analyzer (AMA) determination, for routine analysis of
MeHg in marine biota samples, focusing on the reduction of waste
and extraction time. For evaluating the method over a broad spectrum of MeHg levels (0.0223.67 mg kg1), as well as MeHg/THg
ratios (1488%), the following certied reference materials (CRMs)
were used as common samples: IAEA-452 (scallop soft tissue),
IAEA-436 (tuna sh muscle tissue), DOLT-2 (dogsh liver) and
TORT-2 (lobster hepatopancreas). The full validation of the method
was performed in accordance to the ISO-17025 guideline (ISO/IEC,
2005) assessing the following parameters: linearity, working range
of the calibration curve, limits of detection and quantication,
repeatability, intermediate precision, recovery and trueness. Estimations of the individual uncertainty contributions of each parameter and the nal expanded uncertainties have also been
performed. Demonstration of traceability of measurement results
is provided as well.
The proposed method is very appropriate for application in
environmental monitoring studies, based on determination of
MeHg in marine biota samples and in food safety control, when
large number of samples needs to be analyzed.

liquid) is dried at 220 C and then thermally decomposed at

725 C. The gaseous decomposition products are carried in an oxygen stream through the catalytic section of the furnace, where
Mn3O4/CaO based catalyst allows complete oxidation, while
halogens and nitrogen/sulfur oxides are trapped. Subsequently,
the different mercury species are converted into elemental
mercury (Hg0) vapor and selectively trapped on a gold-based amalgamator. After ushing the system with oxygen the amalgamator
is rapidly heated, releasing the mercury vapor. The oxygen ow
carries the mercury vapor to the absorbance cell in the light path
of a single wavelength atomic absorption spectrophotometer. A
low pressure mercury vapor lamp is used at the working wavelength of 253.7 nm. The detector is connected to a computer for
data acquisition and analysis. Temperatures of both, drying and
decomposition stages, were set by default at 220 C and 725 C,
respectively. Drying time (s) was programed as 0.7 times the volume (lL) of the sample injected. Decomposition and waiting time
were 150 s and 45 s, respectively.

2.1.2. Hg speciation analysis by gas chromatography and pyrolysis and

AFS
Mercury speciation analysis was accomplished with a dual trap
desorption module TDM II interfaced to an Atomic Fluorescent
Spectrometer (AFS) model III detector via a Hg speciation GC and
pyrolysis module (Py). All the three modules were supplied by
Brooks Rand Labs (Seattle, WA, USA). Full details about the instrumentation can be found in Carrasco, & Vassileva, (Carrasco &
Vassileva, 2014).

2.2. Chemicals and reagents

Deionized water used for the preparation of all solutions in this
study was from Milli-Q Element system (Millipore, Bedford, MA,
USA). Certied reference materials (CRMs) applied as a test samples in method validation process were as follows: IAEA-452 (scallop soft tissue) and IAEA-436 (tuna sh muscle tissue) produced in
Environment Laboratories-IAEA, Monaco, DOLT-2 (dogsh liver)
and TORT-2 (lobster hepatopancreas) purchased from the National
Research Council of Canada (NRCC, Ottawa, Ontario, Canada).
Hydrochloric acid (HCl) (30%, Suprapur) or hydrobromic acid
(HBr) (47%, pro analysis), both from Merck, Darmstadt, Germany
were used for hydrolysis of investigated samples. 0.002 M sodium
thiosulfate solution (Suprapur, Merck) or a 1% (w/v) L-cysteine
(Sigma Aldrich, Steinheim, Germany) prepared in 12% (w/v) anhydrous sodium sulfate and 0.8% (w/v) sodium acetate (Suprapur,
Merck) were used for back-extraction.
Standard solution of inorganic mercury 1000 mg kg1 in 12% (v/
v) nitric acid was from TraceCert, Fluka, Steinheim, Germany.
Working solutions were prepared by dilution of the standard solutions in 1% (v/v) nitric acid (40%, Suprapur Merck), 0.1% (v/v) HCl
(Suprapur, Merck) and 0.2% potassium dichromate (analytical
grade, 10% (w/v), Merck). Calibration solutions in the range of
0.515 lg L1 were prepared daily by further dilution of working
solution in 0.002 M thiosulfate solution.
Detail about chemical used for GCPyAFS can be found in
Carrasco, & Vassileva, (Carrasco & Vassileva, 2014).

2. Experimental part

2.3. Sample preparation

2.1. Instrumentation

Two sample preparation procedures for extraction of MeHg

followed by AMA determination (herein denoted 12) were investigated. Three subsamples of the forth different CRMs were used in
each extraction procedure. Three procedural blanks were prepared
in parallel with each sample batch.

2.1.1. Advance mercury analyzer AMA-254

The analyses were carried out using an advance mercury analyzer (AMA-254, Altech Czech Republic). The sample (solid or

S. Azemard, E. Vassileva / Food Chemistry 176 (2015) 367375

2.3.1. Procedure 1
A 0.10.8 g portion of sample (or CRM) was weighted in a 50 mL
polypropylene centrifuge tube; 5 mL of 25% (v/v) HCl solution were
added and the mixture was vigorously shaken for 30 s. Then, 10 mL
of toluene were added in the tube and a vortex shaking method
was subsequently employed for 3 min to ensure the homogenization of the phases. The mixture was centrifuged at 5000 rpm for
520 min. A known volume (48 mL) of the upper organic phase
was removed and transferred to a second 50 mL polypropylene
centrifuge tube containing 510 mL of 0.002 M sodium thiosulfate
solution. This second tube was vigorously shaken (vortex, 3 min)
and centrifuged at 5000 rpm for 515 min. Two milliliters of the
lower aqueous phase, which contains the extracted organic mercury, was transferred to a 15 mL polypropylene container or glass
vial, using a Pasteur glass pipette. Then, an aliquot (50400 lL)
of the extract was directly analyzed with AMA. The thiosulfate
extract was found to be stable for 2 days at temperature of 4 C.
2.3.2. Procedure 2
0.10.5 g of sample was placed into a 50 mL polypropylene centrifuge tube and hydrolyzed with 10 mL of HBr. Twenty milliliter of
toluene was added and the mixture was homogenized for 2 min
and centrifuged for 10 min at 3000 rpm. The organic phase was
transferred to a tube containing 6.0 mL of 1% L-cysteine solution.
A second organic extraction was subsequently performed. A
0.5 mL aliquot of L-cysteine extract was immediately analyzed with
the AMA.
This procedure was proposed by the European Commission as a
standard operation procedure (SOP) for determination of MeHg by
direct mercury analyzer in sea food to all European Reference Laboratories for trace elements in food and feed (Caldern et al., 2013).
2.4. Determination of moisture content
Correction for dry-mass was obtained from 3 sub samples of
biota sample of minimum mass of 1.0 g. The material was dried
for 24 h in a ventilated oven at a temperature of 85 2 C. Then
weighing and repeated drying was performed until constant mass
was attained (0.0002 g difference between two successive weighs).
The loss of mass corresponds to the dry mass correction factor,
which was applied for the estimation of the combined uncertainty.
2.5. Experimental set up for method validation
Several parameters were evaluated for the validation of the proposed procedure, namely: selectivity; trueness by recovery, repeatability and within-laboratory reproducibility, instrumental/
method detection limits (LoDs) and quantication limits (LoQs),
range of linearity, measurement uncertainty, traceability of measurement results. Recovery, repeatability and intermediate precision were evaluated at 4 levels of concentrations. Furthermore,
stability studies of the solution were carried out.
The validation experiments were performed on six different
days. Independent samples were prepared on each single day.
Some of the experiments were used in the estimation of different
parameters.
During the validation study, an internal quality control (IQC)
procedure was adopted the blank level and the drift of the instrument readings were systematically checked analyzing one standard solution after every 10 samples.
Procedural blank was prepared together with unknown samples
in order to account for cross contaminations during the validation
study. It underwent the same analytical procedure as for biota
samples without adding biota matrix.
The calibration curve was prepared with several different mass
quantities, covering a range of mercury from 0.1 to 3 ng, in a x

369

volume (200 lL) of calibration standard prepared in thiosulfate

solution. In order to achieve such low mass levels, calibration standard were prepared from a working standard solution of
0.1 mg L1, obtained by sequential dilution of the stock standard
solution of 1 g L1. Calibration standards were measured at the
beginning of the sequence, followed by the procedural blanks,
and then the samples. Instrumental blank was obtained from the
measurement of the same volume of 0.002 M thiosulfate solution.
Calibration was performed at the beginning of the sequence, followed by the procedural blanks, and then unknown samples. To
monitor for instrumental drift the standard with the lowest Hg
content was randomly re-measured during the measurement
sequence.
In parallel, second calibration strategy based on bracketing
technique was also applied and obtained results compared. First,
a preliminary estimate of the analyte concentration in the test
sample is obtained. Second, two calibration standards at levels that
bracket the sample concentration as closely as possible are then
used, i.e. the two calibrant mass fraction value differs from the
sample mass fraction value in a factor no greater than 20%.
The absolute limit of detection (LoD) of the method was calculated as the mean of the blank plus three times the standard deviation of the blank in twelve replicates. The absolute limit of
quantication (LoQ) was calculated as the mean of blank plus ten
times the standard deviation of the blank in twelve replicates.
For method LoDs and LoQs, nal dilution and weight were taken
into account to calculate the nal values.
Repeatability (Sr) and intermediate precision (SR) were calculated by the application of one way of variance as prescribe in
ISO 5725 (ISO/IEC, 1994; Thompson, Ellison, & Wood, 2002).
For repeatability and intermediate precision, the experimental
plan was applied on four concentration levels (IAEA 452; IAEA
436, TORT-2 and DOLT-2), three repetitions per days and per levels
for a period of 6 days.
The same set of CRM samples were used for the trueness (recovery) studies at 4 different concentration levels.
2.6. Evaluation of the measurement uncertainty
All possible sources of uncertainty were carefully identied.
Afterwards, the uncertainty components were quantied and the
combined uncertainty is calculated. Combined standard uncertainties were obtained by propagating together individual uncertainty
components according to the ISO/GUM guide (JCGM, 2008). The
calculations were performed using Kragten spreadsheet approach
(Kragten, 1994). All uncertainties indicated in the nal results are
expanded uncertainties U = kuc where uc is the combined standard
uncertainty and k is a coverage factor equal to 2.
3. Results and discussion
3.1. Extraction procedure optimization
The effect of HCl concentration on the extraction recovery of
MeHg was investigated. Tuna sh CRM IAEA-436 was used in this
test. The use of 25% (v/v) HCl in the extraction step leaded to recovery rate of 99.9%. Recovery decrease was observed with further
increase of HCl concentration, probably due to the degradation of
the MeHg. Similar pattern was observed by Hintelmann and
Nguyen (2005) for nitric and sulfuric acid. Consequently, no higher
concentrations were further evaluated.
Additionally, the effects of solvent and back extraction duration
were also investigated. Maximum recoveries (i.e. 98%) were
obtained for 3 and 5 min of solvent and back extraction duration
respectively. Since no signicant differences were found between

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S. Azemard, E. Vassileva / Food Chemistry 176 (2015) 367375

3 and 5 min extraction time, the shortest time was selected. Overall, the extraction conditions were xed at 25% (v/v) HCl and 3 min
of shaking time for solvent and back extraction steps.
Ultraviolet (UV) and visible (Vis) radiations lead to degradation
of MeHg (Li et al., 2010). In this respect, parallel extractions, under
and without UV exposition, were conducted. No signicant differences in the recovery of MeHg from marine biota samples were
observed.
Although some published protocols (Caldern et al., 2013;
Maggi et al., 2009; Scerbo & Barghigiani, 1998; Valega et al.,
2006) recommend double solvent extraction, the quantitative
recoveries obtained for IAEA-436 using proposed methodology
demonstrates that single extraction is sufcient.
In order to avoid long centrifugation time, sample size and volume of back extracted toluene were kept as small as possible, but
sufcient to obtain a measurable signal in the measurement step.
In the case of formation of undesirable emulsion at the liquid
liquid interface, the nal volume of thiosulfate solution was
increased. The latter had to be optimized as a function of the volume of measured sample aliquot, because measurement time with
AMA strongly depends from the volume of the sample used in the
measurement step. Measurement time was from 4 to almost 8 min
for aliquot volumes from 50 to 400 lL, respectively.
3.2. Stability study
The variation of analyte level in the thiosulfate extract may give
signicant biases of measurement result. The stability was rst
checked by repeated measurements of one sample extract kept
at room temperature during 5 h, 2 days after their preparation
(extracts are stored at 4 C in between). No statistical differences
were observed in the different determination and it was concluded
that extracted solutions are stable at room temperature for at least
5 h. Five hours is the usual duration of the measurement sequence.
Additionally solutions can be kept at 4 C up to 2 days before the
measurement.
3.3. Comparison of both extraction procedures
The extraction procedure proposed in this study was then compared with the extraction procedure described in procedure 2,
where MeHg was extracted following the standard operation procedure distributed for the collaborative study in EU (Caldern et al.,
2013). For this comparison three CRMs namely IAEA-436, IAEA-452
and TORT-2 covering a wide range of MeHg concentration, (i.e.
0.023.67 mg kg1) and MeHg/THg ratio (from 14% to 88%) in different biota matrices were analyzed by applying both extraction
procedures. Obtained results are presented in Table 1. MeHg mass
fraction yielded by the methodology developed in this study is in
very good agreement with those obtained with procedure 2.
One of the advantages of the proposed extraction protocol is the
signicant reduction of the generated waste volume of organic
solvent. In addition damages of sample boat and auto sampler

holder were observed when applying procedure 2, mainly due to

the spread of cysteine solution. Conversely, after 200 injections
performed with the proposed extraction procedure, no visible
damages or depositions on the samples boats were observed.
As a result of the reduction of the number of extraction steps,
the extraction time was shortened and the production of waste signicantly reduced.
3.4. Validation
According to the ISO-17025 guidelines, validation is the conrmation by examination and the provision of objective evidence
that the particular requirements for a specic intended use are fullled (ISO/IEC, 2005). The factors inuencing the nal results were
systematically assessed, as follows:
3.4.1. Selectivity
The selectivity can be dened as the ability of a method to
determine accurately and specically the analyte of interest in
the presence of other components in the sample matrix
(Vessman et al., 2001).
In order to evaluate the selectivity of the proposed analytical
procedure (i.e. the absence of inorganic mercury in the nal
extract), samples of IAEA-436, IAEA-452 and DOLT-2 were treated
following the procedure 1 and the speciation of mercury in the
organic extracts determined after aqueous-phase ethylation,
purge-trap, gas chromatography separation, followed by pyrolysis
and atomic uorescence spectrometry detection (GCPyAFS) as
described in Carrasco and Vassileva (2014). Typical chromatograms for the IAEA 452 biota sample obtained with GCPyAFS
methodology are shown on Fig. 1; the two peaks (Tr: 2.3 and
4.9 min) correspond to MeHg and inorganic Hg. The absence of
inorganic mercury in the extract from IAEA 452 biota sample (a)
(same peak than in the procedural blank sample) shows the capacity of the proposed procedure to extract selectively the organic
mercury from biota samples. As comparison the chromatogram
(c) of the same sample (IAEA 452) solubilized in KOH/MeOH exhibit a high inorganic mercury peak, representing 86% from the total
mercury content.
The MeHg content in above mentioned CRM was measured in
parallel with GCPyAFS and AMA techniques and obtained results
are presented in Table 1. The agreement of obtained results within
their combined uncertainties conrms the selectivity of the proposed sample preparation procedure and the absence of inorganic
mercury in the nal extract.
3.4.2. Calibration curve, linearity and working range
For any quantitative method, it is necessary to determine the
range of analyte concentrations or property values over which
the method may be applied. This refers to the range of concentrations in the solutions actually measured rather than in the original
samples. In the present study this test was performed with 10 different concentrations. At the lower end of the concentration range,

Table 1
Comparison of results obtained for MeHga content (X U, mg kg1)b in marine biota CRM with the extraction procedure developed in this study and different detection methods
with the results obtained with EC recommended method.

IAEA 436
DOLT-2
TORT-2
IAEA 452
a

Certied values

Proposed procedure

GCPyAFSc Procedure

EC recommended procedure

3.67 0.42
0.693 0.053
0.152 0.013
0.022 0.004

3.39 0.51
0.743 0.111
0.149 0.022
0.021 0.003

3.80 0.45
0.737 0.088

0.017 0.002

3.75 0.45

0.147 0.022
0.019 0.005

Reported as mercury.
Uncertainties are reported as expanded uncertainties U = kuc (k = 2). Expanded uncertainties of the method proposed by EC were taken from the report of the collaborative study.
c
As described in Carrasco and Vassileva (2014).
b

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371

Fig. 1. Typical chromatograms (a) for the separation of mercury and MeHg from CRM IAEA-452 subjected to the optimized extraction procedure, (b) for the instrumental
blank and (c) for mercury and MeHg extracted by traditional alkaline digestion with 25% (w/v) KOH in methanol from CRM IAEA-452.

the limiting factors are the values of the limits of quantitation. At

the upper end of the concentration range, limitations are imposed
by various effects depending on the instrument response system.
The linear range of the calibration curve was from 0.09 to 3 ng as
absolute mass of mercury. The linear correlation coefcient (R2)
was found to be 0.9993.
The linearity was conrmed by visual inspection and checking
of the distribution of residuals. Residuals over the studied range
were all below 2% and were distributed randomly around zero.
Depending on the variation in the procedural conditions, i.e. sample weight, thiosulfate and injection volumes, this linear range
allowed the determination of methyl mercury for sample with concentrations in the range of 0.00220 mg kg1.
An alternative calibration approach, which has been tested, is
bracketing calibration. The results obtained with both calibration
strategies did not differ by more than 0.1%.
3.4.3. Estimation of limit of detection and limit of quantication
The absolute LoD and LoQ of the proposed methodology for
determination of MeHg in marine biota were found 0.045 and
0.09 ng, respectively. Procedural detection and quantication lim-

its depend on the mass of initial sample, injected volumes, volumes

of thiosulfate and toluene collected for back-extraction. Accordingly, under experimental conditions of 0.8 g of sample, injection
volume of 400 lL, 5 mL of thiosulfate and 8 mL of toluene collected
for back-extraction, detection and quantication limits were found
to be 0.0009 mg kg1 and 0.0017 mg kg1, expressed as Hg.
3.4.4. Repeatability and within-laboratory reproducibility
The repeatability and intermediate precision of the measurement procedure were evaluated following the steps described in
the Section 2.5. Repeatability varied from 1.3% to 3.9% and intermediate precision from 1.7% to 4.5% over the 4 CRMs used.
3.4.5. Recovery (trueness) study
The trueness of the method was assessed by using recovery
tests. For the 4 different matrices CRM, recoveries of MeHg with
proposed procedure were in the range of 92108%. Considering
the estimated uncertainty of the analytical recovery, no statistical
signicant difference could be identied between measured and
certied values in the tested matrices.

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S. Azemard, E. Vassileva / Food Chemistry 176 (2015) 367375

3.4.6. Accuracy prole

Another strategy recently used in validation practice is the construction of accuracy prole, called also total error approach
(Clmens et al., 2011). The accuracy prole is a decision-making
graphical tool aiming to help the analyst to decide whether an analytical procedure is t for purpose. The building of such prole
helps to determine the experimental tolerance intervals or limits,
which have to be compared with acceptable limits xed by the
analyst as a function of the analytical problem (Mermet &
Granier, 2012; Feinberg, 2007).
The accuracy prole was applied in the present study to demonstrate the ability of the proposed methodology to quantify MeHg in
biota samples using data obtained during repeatability and intermediate precision studies. The probability b and acceptance limits
were set to 90% and 20%, respectively; bracketing strategy was
used for calibration. Obtained prole presented in Fig. 2, shows
that all b-expectation tolerance intervals were comprised within
the acceptable limits.
3.4.7. Uncertainty
The estimation of combined uncertainty of measurement
results was done by applying two different approaches: the modeling approach recommended by ISO GUM (JCGM, 2008) and single
laboratory validation approach (Nordtest, 2012).
3.4.7.1. Modeling approach
The equations described in Table 2, represent our mathematical
model, used to calculate MeHg mass fractions in biota sample. The
value for each parameter in the described equations, obtained
either by an analytical measurement or a mathematical calculation, was with an associated standard uncertainty. The uncertainties of all input quantities are found and thereafter combined
into the combined standard uncertainty.
Bracketing calibration strategies was used in the modeling
approach. This type of calibration is in theory more advantageous
in term of uncertainty propagation as (i) the measurement cycle
is fast and the instrumental drift is minimized, (ii) the effect of
instrumental non-linearity between two calibration points is insignicant. Therefore, the uncertainty coming from the calibration
step of the measurement procedure was estimated according to
the Eq. (2), Table 2, which describes the application of bracketing
approach.
Beside the correction for recovery, the measured mass fractions
were also corrected for procedural blank and moisture content.
Typically, the relative expanded uncertainty on the MeHg content in biota sample was found to be 15% (k = 2). In this way, the
combined uncertainty statements that arise go far beyond the

simple repeatability calculations and reect our understanding of

the measurement process.
The contributions of the individual parameters are expressed in
terms of their relative contribution to the expanded uncertainty
and are as follows: the main uncertainty component (72%) was
originated from the uncertainty coming from recovery, followed
by the repeatability of the measurements (17.5%), the extraction
and injection volumes (5.9%). Finally, uncertainties coming from
preparation of standard solutions and other factors are found to
be 3.9% and 0.4%, respectively.
3.4.7.2. Single laboratory validation approach
The single-lab validation approach, contrary to the ISO GUM
modeling approach, does not go deeply into the measurement procedure and does not attempt to quantify all uncertainty sources
individually. Instead the uncertainty sources are quantied in large
batches via components that take a number of uncertainty
sources into account. Most of the data that are used come from validation of the analytical procedure. This is the reason for the word
validation in the name of the approach. This type of approach is
sometimes called the top-down approach and is also known as a
Nordtest approach (Nordtest, 2012). The equations for the singlelab validation approach are presented in Table 3.
The Eq. (1) includes the two main components of uncertainty
budget: u (Rw) which stands for the within laboratory reproducibility component of uncertainty and u (bias) accounting the uncertainty component taking into account possible bias. The within
laboratory reproducibility (intermediate precision) component
takes into account all uncertainty sources that are random in the
long term. The bias component takes into account the systematic
effects that cause long-term bias (but not those that just cause bias
within a given day). The long-term bias can be regarded as sum of
procedure bias (bias inherent in the nature of the procedure) and
laboratory bias (bias caused by the way how the procedure is
implemented in the laboratory).
The relative expanded uncertainty calculated with validation
data was found to be 20% (k = 2), which is consistent with the
one calculated by the modeling approach.
3.4.8. Traceability
A principal requirement exists in ISO 17025 (ISO/IEC, 2005) for
laboratories to produce measurements that are traceable to a common system of measurement, SI in this case, to ensure comparability of measurement results. A typical chemical measurement
involves a number of steps as illustrated in Table 2. The way to
demonstrate traceability is to use an uncertainty budget, where
all the parameters inuencing the nal result are systematically

130%
120%

Recovery

110%
100%
90%
80%
70%
0.0

0.5

1.0

1.5

2.0

2.5

W(MeHg) in mg kg-1 as Hg
Fig. 2. Accuracy prole.

3.0

3.5

4.0

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S. Azemard, E. Vassileva / Food Chemistry 176 (2015) 367375

Table 2
Modeling approach for the calculation of combined uncertainty of MeHg mass
fraction in biota sample.
Preparation of calibration standards

CD i CM 

mi1
mM
m1


   
mM md 1 1 m1 md 2 2
mi1 md i i

Table 3
Single laboratory validation approach for the calculation of combined uncertainty of
MeHg mass fraction in biota sample.
Combined standard uncertainty (as relative)

uc

q
u2Rw u2bias

Sample bracketing calibration and other corrections

C meas

CD

i1



 AS  Aicorr  ABlk C D i  Ai1corr  AS ABlk


Ai1corr  Aicorr

Random effect (as relative)

2
uRw SRw

Absorbance correction

Acorr Astd  AI

Blk

Recovery calculation

ubias

n
1 X
C CRM n
R 
C CRM cert
n
1

Final calculation with corrections for recovery and moisture content

p
1 X C meas  V t1  V thio
1
CS 

f
p
1W
ms  V t2  R
1

Parameter

s
Pn
2
i1 biasi

clab i  cref i
cref i
s
Pp
2
i1 uc ref i

uc

5
ref

Index

Mass fraction (mg kg1)

Working calibration standard

Parameter

Index

Mass fraction average

(mg MeHg kg1 as Hg)
Mass (kg)

Stock solution

u
S
RMS
C

lab
n
Rw
ref

Absorbance

Dilution step after dilution 1

(i P 2)
Sample

Recovery

CRM

Certied Reference Material

V
W

Volume (mL)
Moisture content of
biota sample (%)
Dilution factor

Std
meas

Calibration standard
Measured

cert
n, p
Blk
I_Blk
Corr

Certied
Number of repeats
Procedural blank
Instrumental background
Correction for instrumental
background
Added toluene
Collected toluene
Volume of sodium thiosulfate
Measured aliquot thiosulfate

q
RMS2bias u2ref

biasi

t1
t2
thio
aliquot

assessed. Key steps in the attainment of traceability were as

follows:
1. The analytical method used was properly selected and validated, both in terms of matrix composition and analyte
concentration
2. The unbroken chain linking the MeHg mass fraction to SI unit
was described with the mathematical model. Table 2 presents
the mathematical model of the analytical procedure, which is
completely understood. This model, together with sub-calculations or references to certied values, relates each of the input
quantity to SI units of the mol or the kilogram
3. The use of CRM for calibration and bias (recovery) estimation is
the way to link MeHg mass fraction to the common system of
reference SI. CRM with similar matrix was used also in the validation of sample preparation step

Standard uncertainty
Standard deviation
Root mean Square
Mass fraction (mg kg1)

A: candidate CRM IAEA 470 (Oysters)

6
5
4
3
2
ILC

90

ID-ICP-MS

GC-Pyr-AFS

Org Hg AMA

B: IAEA 461 (Clams)

80
70
60
50
40
30
ILC

3.4.9. Comparison with IAEA worldwide inter-laboratory comparison

assigned values and independent techniques
Alternative approach for the method validation indicated in the
ISO 17025 (ISO/IEC, 2005), is the participation in inter-laboratory

Laboratory
Number of CRM used
Within laboratory reproducibility
Certied reference material

7
W(MeHg) mg kg-1 as Hg

RMSbias

W(MeHg) mg kg-1 as Hg

Possible bias (as relative)

ID-ICP-MS

GC-Pyr-AFS

Org Hg AMA

Fig. 3. Comparison of the MeHg mass fractions in candidate CRM IAEA 470 (A) and
IAEA 461 (B) obtained from: the IAEA world-wide inter-laboratory comparison
(ILC), isotope-dilution inductively coupled plasma mass spectrometry (ID-ICP-MS);
gas chromatography coupled to atomic uorescence spectrometry (GCPyAFS)
and the method proposed in the present study (Org Hg AMA).

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S. Azemard, E. Vassileva / Food Chemistry 176 (2015) 367375

comparisons and comparison of obtained results with results from

independent method.
Accordingly, results obtained with developed in this study measurement procedure were compared with the assigned reference
value from the IAEA 461 worldwide inter-laboratory comparison
(ILC) on the determination of trace elements and methyl mercury
in biota sample (Clam) and IAEA 470 (candidate CRM) reference
value.
In addition they were compared with results from two independent methods namely; species specic isotope dilution ICP-MS
(Vassileva et al., 2014) and GCPyAFS (Carrasco & Vassileva,
2014). As can be seen from Fig. 3, an excellent agreement within
stated uncertainties was obtained. This agreement contributes for
further validation of the described analytical procedure for determination of MeHg in marine biota samples.
4. Conclusion
A method for the routine determination of MeHg in marine
biota samples, based on solvent extraction of organic mercury species, back-extraction into aqueous phase and direct mercury analyzer determination has been validated. Important parameters
inuencing the extraction procedure efciency, such as acid concentration, solvent and back-extraction time were optimized. The
validation of the methodology was performed according to the
ISO-17025 guideline, using four different certied reference materials. Selectivity was evaluated by the analysis of the same extracts
by GCAFS hyphenated method. An uncertainty budget was built
for the analytical procedure, allowing for the quantication of
the relative uncertainty contributions for each parameter in the
measurement procedure and the determination of their relative
contributions to the nal expanded uncertainty. Contributions
arising from recovery and repeatability of the measurements dominated the uncertainty budget. Overall, the proposed method is
simple, cost-effective and allows the analysis of 20 samples per
working day. As a consequence from the single solvent extraction
step the volume of generated waste is considerably decreased. In
light of the results presented in this study, the methodology proposed could ultimately be a ready-to-use analytical method for
the determination of MeHg in marine biota samples.
Acknowledgements
The Agency is grateful for the support provided to its Environment Laboratories by the Government of the Principality of Monaco. The IAEA-NAEL in Monaco operates under an agreement
between the IAEA and the Government of the Principality of Monaco. Dr. Luis Carrasco is acknowledged for the helpful comments
and remarks during this study.
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