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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Department of Physical and Analytical Chemistry, Faculty of Sciences, University of Jan, E-23071 Jan, Spain
Dpartment de Chimie, Facult des Sciences, Universit Abdelmalek Essaadi, Ttouan, Maroc
c
Department of Physical and Analytical Chemistry, E.P.S. of Linares, University of Jan, E-23700 Linares, Jan, Spain
b
a r t i c l e
i n f o
Article history:
Received 6 October 2008
Received in revised form 14 January 2009
Accepted 1 March 2009
Keywords:
Metals
Vegetables
Certied reference material
Microwave digestion
Graphite furnace atomic absorption
spectrometry
a b s t r a c t
A simple and expeditious method for the determination of trace metals (cadmium, chromium, copper,
manganese and lead) is proposed. The metals are extracted from their matrix by using nitric acid and
hydrogen peroxide in a closed-vessel microwave digestion system for their subsequent detection by
graphite furnace atomic absorption spectrometry (GFAAS). The sample preparation procedure facilitates
the overall analytical process and enables the construction of calibration curves from inorganic standards.
The ensuing method provides good linearity and sensitivity for the ve metals, with limits of detection
and quantization spanning the ranges 0.052.20 and 0.157.34 lg/kg, respectively. This sensitivity level
is quite appropriate for the intended application. Accuracy was assessed by using a certied reference
material (NCS ZC85006 Tomato), for which the proposed method provided amounts of metals consistent
with their certied values. The proposed method was applied to tomato, pepper and onion, which are
widely consumed in Mediterranean countries.
2009 Elsevier Ltd. All rights reserved.
1. Introduction
Humans have traditionally been exposed to low concentrations
of metals mainly via water and foods; some, however, are exposed
to toxic concentrations via professional activities such as mining
or, anecdotally, through suicidal attempts. With time, the range
of potentially toxic metal sources has expanded substantially by
effect of the increasing agricultural and industrial activities (Fraga,
2005). Heavy metal contamination in agricultural environments
can come from atmospheric fall-out, pesticide formulations, contamination by chemical fertilizers and irrigation with water of poor
quality (Marcovecchio, Bott, & Freije, 2007). A risk of contamination of the food chain may arise when heavy metals accumulate in
plant tissues at concentrations above the threshold levels believed
to threaten the health of humans or animals feeding on the same
crops (Mantovi, Bonazzi, Maestri, & Marmiroli, 2003).
Lead, cadmium, arsenic and mercury are the metals and metalloids with the highest toxic potential among those present in foods.
As such, they have elicited a response in most countries in the form
of legal restrictions on their presence in foods (EEC, 2006; Jrup,
2003; Reilly, 2002). Other metals including copper, iron, tin, zinc,
antimony, aluminium, magnesium, manganese, molybdenum and
chromium, which are present in trace amounts in the human diet,
* Corresponding author. Tel./fax: +34 953 648560.
E-mail address: eballes@ujaen.es (E. Ballesteros).
0308-8146/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.03.010
591
2. Experimental
2.1. Instrumentation
A PerkinElmer Analyst 800 atomic absorption spectrometer
(AAS) equipped with two fully integrated atomizers (viz. a burner
system for ame atomization and a graphite furnace for electrothermal atomization), a model AS-800 autosampler and a cooling
system were used. The assembly was operated from an interfaced
computer running AA WinLab software. A transversely-heated
graphite furnace featuring full stabilized temperature platform
was used in all tests. The AAS instrument was equipped with a
deuterium and longitudinal Zeeman background corrector for
ame atomization and electrothermal atomization, respectively.
PerkinElmer hollow cathode lamps were used as line sources
for all analytes.
A high performance O-I-Analytical model 7295 microwave
digestion apparatus was used under the operating conditions to digest samples, and a Mettler AJ100 microbalance (0.1 lg sensitivity)
was employed to weigh samples.
2.2. Reagents and chemical standard
Experimental work was done with nitric acid (65 wt%), hydrogen peroxide (30 wt%, Merck, Darmstadt, Germany) and multi-element calibration standard-2A (10 lg/ml, Agilent, Palo Alto, CA).
Aqueous standards were prepared by appropriate dilution of a
Table 1
Selected lines, pretreatment and atomization temperatures for GFAAS.
Element
Wavelength
(nm)
Temperature of ashing
(C)
Atomization temperature
(C)
Pb
Cr
Mn
Cu
Cd
283.3
357.9
279.5
324.8
228.8
400
1500
1300
1200
500
1600
2300
1900
2000
1600
592
similar. Fig. 1B shows the inuence of the volume of hydrogen peroxide at a constant volume of 6 ml of nitric acid on the digestion of
a tomato sample the results for pepper and onion were similar.
Based on the results, the volumes of nitric acid and hydrogen peroxide should be at least 6 and 2 ml, respectively. This led us to use
a mixture consisting of 6 ml HNO3 and 2 ml H2O2 to digest the
three types of samples.
The pyrolysis and atomization temperatures were further optimised by using curves obtained from samples that were processed
as described above. Table 1 summarises the optimised temperature
programs, which were also used for calibration.
The amount of sample was optimised by using volumes of 5, 10,
20, 30 and 40 ll of a 10 lg/l multi-element standard solution. The
optimum volume was found to be 20 ll for Cr, Cu and Pb, 15 ll for
Mn and 30 ll for Cd.
modier in order to diminish the matrix effect of the types of vegetables; 1 ll of 10 g/l magnesium nitrate was thus selected as
modier.
We also studied the effect of various species commonly accompanying the target analytes in the samples on the determination of
5 lg/kg Cd and Mn, and 10 lg/kg Cr, Cu and Pb, by adding a known
amount of each potential interferent to the analyte solution. The
tolerated level was taken to be the foreign species concentration
causing an error not exceeding 5% the analyte concentration. As
can be seen from Table 2, the proposed method is quite tolerant
of other species present in the samples. The least tolerated species
were phosphate and sulphate in the determinations of Cd and Mn,
and potassium in that of Mn. On the other hand, the determinations of Pb and Cr tolerated the presence of all studied interferents
at concentrations more than one hundred times those of the
analytes.
We used the previously optimised operating conditions to assess the performance of the proposed method (see Table 3). The
method was calibrated with seven aqueous standards of the metals
including a blank. The regression coefcients obtained were all
greater than 0.992. In order to evaluate the precision of the method, ten independent analyses of a solution containing a 2.0 lg/l
concentration for Cd and Mn, and one of 5 lg/l for Cu, Cr, and Pb,
were performed. As can be seen in Table 3, the reproducibility, expressed as relative standard deviation, ranged from 2.6% to 4.2%.
The limits of detection (LOD) and quantication (LOQ) were calculated as the blank signal plus three or ten times, respectively, its
standard deviation (Currie, 1999). All LOQs were below the maximum residue limits (MRLs) for metals (Pb and Cd) in vegetables
set by the European Community (EEC, 2006).
Accuracy of the developed method for determination of metals
was veried by analysis of the certied reference material (NCS
ZC85006 Tomato). This material was previously used to validate
a method for the determination of heavy metals by inductively
coupled plasma optical emission spectrometry and ame atomic
absorption spectrometry following co-preconcentration of the
metals (Feist, Mikula, Pytlakowska, Puzio, & Buhl, 2008). As can
be seen in Table 4, the amounts of metals found in the reference
material were consistent with the certied values.
Due to the unavailability of reference materials on the market
for onion and pepper, the robustness of the method was also
checked by performing a recovery test on these types of vegetables.
For this purpose, each type of sample was spiked at two different
concentrations (viz. 7 and 10 lg/kg for Cu, Cr and Pb, and 3 and
7 lg/kg for Mn and Cd) prior to processing as described above.
All samples were analysed in triplicate (n = 3) and their standard
deviations calculated. The recoveries ranged from 96% to 105%,
which testies to the applicability of the proposed method to vegetable samples.
The proposed method was applied to the determination of metals in vegetable samples (tomato, pepper and onion). Each type of
Fig. 1. Inuence of the volume of nitric acid (A) and hydrogen peroxide (B) on the
microwave digestion of pepper and tomato samples, respectively.
Table 2
Tolerance of interferents.
Species
Ca
K
Na
PO34
SO24
Fe
Cr
Cu
Mn
Pb
1000
2000
10
5
5
2000
2000
2000
1000
2000
2000
100
500
2000
1000
100
10
500
500
10
500
5
5
100
2000
1000
1000
2000
2000
2000
593
r*
LR* (lg/kg)
LOD* (lg/kg)
LOQ* (lg/kg)
RSD* (%)
MRL* (mg/kg)
Cu
Cr
Pb
Mn
Cd
0.923
0.958
0.995
0.989
0.952
7.5100
5.080
3.080
0.540
0.225
2.20
1.33
0.81
0.13
0.05
7.34
4.43
2.69
0.44
0.15
3.7
4.2
3.4
3.4
2.6
0.10
0.050
r, regression coefcient; LR, linear range; LOD, limit of detection; LOQ, limit of quantitation; RSD, relative standard deviation (n = 10); MRL, maximum residue limit (6).
Table 4
Determination of Cu, Cr, Pb, Mn and Cd (lg/g) dry mass in certied reference material
NCS ZC85006 Tomato.
Metal
Certied value
Found*
Cu
Pb
Mn
Cd
Cr
21.1 2.5
4.97 0.54
87.1 7.3
0.82 0.09
Not certied
20.57 1.8
4.78 0.35
87.8 6.6
0.83 0.07
(SD, n = 3).
Table 5
Analytical application of proposed method to vegetable samples.
Metal
Cu
Cr
Pb
Mn
Cd
Tomato
Found* (lg/kg)
Pepper
Found* (lg/kg)
Onion
Found* (lg/kg)
78.8 5.1
33.3 1.4
59.5 3.2
52.9 3.0
19.4 0.8
71.6 2.1
30.3 1.4
34.5 0.5
67.7 3.6
31.9 2.3
22.3 1.1
35.8 4.8
14.2 0.8
13.4 0.4
19.9 0.6
75.9 6.9
40.9 3.5
81.7 2.5
35.2 0.9
36.9 2.4
69.1 4.1
29.1 0.8
18.1 1.0
70.6 4.1
34.7 0.8
37.8 1.4
34.5 0.9
11.8 0.7
12.7 0.8
15.0 1.1
75.1 2.7
75.7 3.0
80.9 4.0
23.9 0.8
25.1 1.0
27.7 2.1
59.6 0.6
12.2 0.9
40.6 1.5
27.3 0.4
26.8 1.5
22.9 0.7
12.1 0.7
13.2 0.6
10.7 0.4
(n = 3, SD).
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