Bull Vet Inst Pulawy 50, 363-366, 2006
DETERMINATION OF TOTAL MERCURY
IN BIOLOGICAL MATERIAL
BY ATOMIC ABSORPTION SPECTROMETRY METHOD
JÓZEF SZKODA, JAN ŻMUDZKI AND AGNIESZKA GRZEBALSKA
Department of Pharmacology and Toxicology,
National Veterinary Research Institute, 24-100 Pulawy, Poland
e-mail: [email protected]
Received for publication May 05,2006.
Abstract
A very simple procedure is proposed for the
determination of total mercury in biological materials. The
atomic absorption spectrometry method (AAS) for mercury
determination was prepared. The Advanced Mercury Analyzer
AMA 254 spectrometer was tested in order to determine the
influence of the instrumentation on changes of the level of
mercury in different biological samples (animal tissues, milk,
feedstuffs). The method was validated in terms of basic
analytical parameters. The mean recoveries of mercury was
101.70% for muscle and 98.30% for liver, and analytical
detection limits was 0.001 µg/g. Certified reference materials
and participation in national and international proficiency
studies were used for analytical quality assurance programme.
Key words: mercury, atomic absorption
spectrometry, biological materials.
Mercury occurs naturally in the environment
and exists in several forms such as metallic mercury,
inorganic mercury and organic mercury. Metallic
mercury is still used in thermometers, barometers,
batteries, and electrical switches. Some inorganic
mercury compounds are used as fungicides and
medicinal products. Mercuric chloride is a topical
antiseptic or disinfectant agent. Other chemicals
containing mercury are still used as antibacterials. Most
of the mercury found in the environment is in the form
of metallic and inorganic mercury compounds (1, 15,
18). Metallic mercury is a liquid at room temperature; it
will evaporate into the air and can be carried for long
distances. Microorganisms convert inorganic mercury to
methylmercury. Inorganic or organic compounds of
mercury can be released to water or soil. Mercury can
enter and accumulate in the food chain. Some people
can be exposed to higher levels of mercury in the form
of methylmercury if they have a diet high in fish,
shellfish, or marine mammals that come from mercury
contaminated waters (7, 12, 34, 35). Inorganic mercury
accumulates mostly in the kidneys but methylmercury is
easily absorbed through the gastrointestinal tract and
penetrates to the brain (1, 11, 17, 36). Mercury has been
found in all foodstuffs (10, 12, 13, 21, 23, 28-32, 35,
37).
Commission Regulation (EC) No 466/2001 of 8
March 2001, and (EC) No 78/2005 of 19 January 2005
(4, 5), and Polish Regulation of Ministry of Health of 30
April, 2004 established the limits of Hg only for fish
(25). Maximum levels for Hg in other foodstuffs existed
in our country on legal basis until accession to the
European Union (Polish Regulation of Ministry of
Health of 13 January, 2003) (24). Actually, these values
are established as the levels of action in the National
Residue Control Plan in Poland which is realized by the
Ministry of Agriculture and Rural Development (38).
The limits for mercury are also established for feedstuffs
(range 0.1 – 0.40 mg/kg) by the Polish Regulation of the
Ministry of Agriculture and Rural Development of June
28, 2004 (26).
The exposure of mercury could be minimalized
by regular control of food and feed and setting mercury
maximum levels in these products. At the present time
the National Veterinary Residue Control Programme
which is organized in Poland according to Council
Directive 96/23/EC of 29 April 1966 and Polish
Regulation of the Ministry of Agriculture and Rural
Development of April 19, 2004 requires controlling the
levels of mercury in animal tissues, food of animal
origin, and feedstuffs. A fully validated analytical
method was developed to support introduction of
legislation to practice and necessity of mercury control
in monitoring programme.
Material and Methods
Reagents. Concentrated nitric acid was
analytical grade. Stock standard solution containing
1 000 µg of Hg /ml was reference solution from Beaker.
364
Working standard solutions were prepared by
dilution of stock and intermediate standards. The
working standards were as follows:
• for the first range calibration – 0.05, 0.10, 0.20,
0.30 and 0.50 µg of Hg/ml prepared from the
solution of 5.0 µg of Hg/ml concentration
• for the second range calibration – 1.0, 2.0, 3.0
and 5.0 µg of Hg/ml prepared from the solution
of 100.0 µg of Hg/ml concentration
In the first range calibration, to every 100 ml
flask 1, 2, 4, 6, and 10 ml of intermediate standard
solution, containing 5 µg of Hg/ml, were measured and
1 ml of concentrated nitric acid was added and made up
to 100 ml volume with deionized water. In the second
range calibration, to every 100 ml flask 1, 2, 3, and 5 ml
of intermediate standard solution, containing 100 µg of
Hg/ml, were measured and 1 ml of concentrated nitric
acid was added and made up to 100 ml volume with
deionized water. At the same time a zero calibration
solution was prepared in 100 ml flask, using 1 ml of
concentrated nitric acid made up to 100 ml with
deionized water. The stability of the standard solutions
in the darkness is one week for the first range calibration
and one month for the second range calibration.
Samples. Mercury was determined in animal
tissues, milk, eggs, and feedstuffs. Cod muscle CRM
142, Pig kidney BCR 186, Milk powder BCR 151 and
Milk powder BCR 150 were used as reference materials.
Apparatus. Advanced Mercury Analyzer
AMA 254 spectrometer (Altec Ltd., Czech Republic).
Oxygen of medical purity was used for sample
combustion.
Instrument calibration. The calibration curve
(first and second range) for the determination of
mercury was prepared using a blank and working
standard solutions.
Analytical
method
for
mercury
determination. Detailed instructions on the operation of
the AMA 254 spectrometer are described in the
operator’s manual.
The sample of known weight up to 300 mg or
known volume up to 500 µl was placed to a sampling
boat. The size of sample was adapted to expected
concentration of mercury. The sample was introduced
into a decomposition tube, dried for 70 s and thermally
decomposed for 100 s at 900°C. The decomposition
products of the sample are carried by oxygen flow to an
amalgamator. The amalgamator and block of measuring
cuvettes are thermostated to 120°C to prevent
condensation of water. After completing sample
decomposition and stabilization of temperature within
the amalgamator the content of mercury trapped in
amalgamator was measured in the optical cell system at
253.7 nm. After the atomization steps, concentrations of
the mercury was reported in the computer in µg of Hg/g
of wet weight of sample.
The calibration was periodically verified by
analysing the standard and certified reference materials
at the frequency of 20 readings. If the recovery was
outside the limits, the analysis was stopped. The
problem was corrected and the system was recalibrated.
Statistical analysis. The date obtained from the
analysis were evaluated in based statistical parameters
using computer program Excel.
Results
The Advanced Mercury Analyzer AMA 254
spectrometer was tested in order to determine the
influence of the instrumentation on changes of the level
of mercury in different biological samples (animal
tissues, milk, feedstuffs). A series of experiments were
conducted to establish optimum of analytical
parameters. Optimalization of analytical condition for
different biological matrices and selection of
instrumental programmes for this element were done.
The method was tested by studying of the
certified reference materials (Cod muscle CRM 142, Pig
kidney BCR 186 and Milk powder BCR 150 and 151)
with the certified values and was regularly evaluated by
participation in proficiency programmes organized by
Food Analysis Performance Assessment Scheme
(FAPAS) and European Community Reference
Laboratories (CRL ISS). The result of Z-score of
proficiency test organized by FAPAS in 2004 was -0.10,
however, the result of Z-score of the 9th proficiency test
in fish organized by CRL ISS in Rome in 2005 was 0.11. The results of the determination of mercury in
certified reference material and validation reports are
presented in Tables 1 and 2.
Table 1
Comparison of the found and certified values of mercury in the certified reference materials (accuracy), n = 10
Certified
materials
Cod muscle
CRM 142
Pig kidney
BCR 186
Milk powder
BCR 151
Milk powder
BCR 150
Certified
value µg/g
Mercury
Found µg/g
x ± SD
Recovery
(%)
0.559
0.494 ± 0.019
88.37
1.97
1.76 ± 0.023
89.42
0.101
0.092 ± 0.0007
90.59
0.009
0.010 ± 0.0006
108.89
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Table 2
Validation report – Hg determination by AAS (AMA-254)
Parameters
Results
Linearity (working range), mg/kg
0.002 – 0.500
Limit of detection (LOD), mg/kg
0.0004
Limit of quantification (LOQ),
mg/kg
Matrix
Levels of spiked samples, mg/kg
Repeatability
x, mg/kg
SD, mg/kg
RSD, %
Intralaboratory reproducibility
x, mg/kg
SD, mg/kg
RSD, %
Recovery, %
Uncertainty
combined (uc)
expanded (U)
coverage factor (k)
0.0005
0.010
Muscle
0.020
0.040
0.020
Liver
0.040
0.050
0.010
0.0004
7.51
0.020
0.0022
12.94
0.037
0.0023
6.94
0.021
0.004
1.79
0.039
0.0011
2.72
0.047
0.0011
3.25
0.011
0.0005
4.97
107.80
0.020
0.0013
6.59
102.00
0.038
0.0020
5.21
95.40
0.021
0.0005
2.22
103.00
0.039
0.0008
2.03
98.20
0.047
0.0011
2.34
93.70
0.0014
0.020 ± 0.003 mg/kg
2
Discussion
A very simple procedure is proposed for the
determination of total and inorganic mercury in
biological materials. Analytical and instrumental
conditions are the most important stages in mercury
analysis. Usually methods of atomic absorption
spectrometry (AAS), atomic fluorescence spectrometry
(AFS), or neutron activation analysis (NAA) are used in
the analysis (2, 3, 6, 12, 20, 22, 27, 30, 32). In addition,
methods based on mass spectrometry (MS),
spectrophotometry, and anodic stripping voltametry
(ASV) have also been used (14, 15). From the available
methods, cold vapour-AAS is the most widely used (2,
14-16, 19, 30, 33). The wide range of biological
materials (foods, feeds, plants, animal tissues and fluids)
is characterized by a wide variety of chemical analytical
measurements. In general, the classical method is
composed of 3 steps: 1) sample pre-treatment, 2) analyte
isolation/separation, and 3) quantitative measurement.
These various materials were determined without
preceding mineralization. Many of these materials
contained large quantity of fat, protein, and carbohydrate
which have influence on processing and determination
steps in the unvestigated method.
In this study, the procedure of the determination
of mercury in biological material by advanced mercury
analyser – AMA 254 was tested. Selection of the
operating parameters for AMA 254; oxygen flow, time
of drying and time of decomposition in tube at 900°C
were satisfactory to run all kinds of biological samples
and give the satisfactory analytical parameters (limit of
0.0013
0.050 ± 0.0027 mg/kg
2
detection – LOD, and limit of quantification – LOQ
below the levels of 0.001 mg/kg).
The method was validated in terms of the
linearity, LOD, LOQ, precision, recovery, and
uncertainty (8, 9). Results of the analysed certified
reference materials and inter-laboratory comparison, and
all validation data confirmed that the method could be
used as a routine procedure for the determination of
mercury levels in food and feed in official monitoring
control programme. Generally good results of precision
(RSD about 5% for muscles and 2% for liver),
recoveries (about 100% for all analysed samples) and
reasonable value of uncertainty additionally support the
described method as a routine procedure for mercury
determination in biological materials.
Value and simplicity of the developed method
was fully supported by validation results. The validation
procedure was in general agreement with the
Commission Decision of 12 August 2002
(2002/657/EC) implementing Council Directive
96/23/EC concerning the performance of analytical
methods and the interpretation of results. The proposed
analytical procedure is well suited for maximum level
control of mercury in food and feedstuffs as a screening
and confirmatory method in official food control
laboratory.
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