Microchemical Journal 102 (2012) 2327

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Microchemical Journal
journal homepage: www.elsevier.com/locate/microc

Analysis of 20 trace and minor elements in soy and dairy yogurts by ICP-MS
E.J. Llorent-Martnez, M.L. Fernndez de Crdova, A. Ruiz-Medina, P. Ortega-Barrales
Department of Physical and Analytical Chemistry, Faculty of Experimental Sciences, University of Jan, Campus Las Lagunillas, E-23071 Jan, Spain

a r t i c l e

i n f o

Article history:
Received 5 October 2011
Received in revised form 9 November 2011
Accepted 9 November 2011
Available online 22 November 2011
Keywords:
Yogurt
Soy
Trace elements
ICP-MS
Microwave digestion
Nutritional value

a b s t r a c t
The content of 20 trace and minor elements in soy and dairy yogurts consumed in Spain has been determined
using inductively coupled plasma-mass spectrometry (ICP-MS) after microwave digestion. This work presents
two goals: a) to determine the nutritional value in terms of minor elements content, using recommended
daily allowance data; b) to determine the levels of trace toxic elements and compare them with the acceptable
daily intake values. The developed analytical method was validated by using both milk certied reference
materials and recovery experiments over different yogurt samples, obtaining satisfactory results in all cases. In
addition, a comparison between the levels of minor elements found in soy and dairy yogurts was performed.
Much higher concentrations of Cu and Mn were found in soybean products (up to 30-fold higher) and
minor differences were observed in the content of Fe and Zn. Other minor element found at higher concentrations in soy yogurts was Ni (effect usually observed in plant-origin foods), which was determined at
levels up to 450 ng g 1 in some samples. Finally, the levels of toxic elements were very low, even below
the detection limit in some cases.
2011 Elsevier B.V. All rights reserved.

1. Introduction
Milk and dairy products are a good source of many valuable nutrients and minerals in human diet. Among these products, yogurt is
gaining popularity due to its acceptability for consumers as well as
its nutritional properties and potentially benecial effects on human
health. One of the reasons for its rising popularity is the increasing
number of avours and yogurts enriched with fruit pieces, cereals
or other ingredients. Although dairy yogurts still represent the highest
percentage of consumed yogurts, soy-based ones are gaining ground
in the market. Soy yogurts are produced using soy milk (produced by
soaking dry soybeans and grinding them with water), yogurt bacteria
and sometimes additional sweeteners, in a similar way than dairy yogurts (we will refer only to yogurts obtained from cow's milk).
In addition to nutritional minerals, undesirable potentially toxic
elements can be present in these foods too. In fact, apart from those
communities exposed to high levels of pollution by industrial efuents or emissions rich in heavy metals, for most of individuals, food
is the most important source of potentially toxic elements, such as
Cd, Hg, Ni or Pb. The exposure of the organism to high levels of these
metals can cause different adverse effects, from contact dermatitis
(Ni) to neurological or carcinogenic diseases (Cd) [1]. As a result, routine control of their levels is required to ensure the safe consumption
of these products.
There are numerous bibliographic references on the mineral content and levels of trace elements in bovine milk [2-9]. However,
Corresponding author. Tel.: + 34 953 212757; fax: + 34 953 212940.
E-mail address: portega@ujaen.es (P. Ortega-Barrales).
0026-265X/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.microc.2011.11.004

fewer studies have been made on other dairy products, such as yogurt
[3,10,11] or other different types of milks [12,13]. Although the concentration of selected major and trace elements has been studied in
some soy-based formulations [14-16], to the best of our knowledge,
this study has not been performed in soy yogurt and milk. As a result,
this work presents two different goals for comparing soy and dairy
yogurts: a) to determine the levels of minor nutritional elements in
both types of yogurts and compare their nutritional value using the
recommended daily allowance; b) to determine the levels of potentially toxic trace elements and compare them with the acceptable
ADI data. In addition, samples of cow's milk and soymilk have also
been analysed and the obtained values compared, observing similar
patterns to those ones found in yogurts.
In this work, we made use of ICP-MS due to its well-known advantages of sensitivity, selectivity and multi-element analysis. ICP-MS
has been widely used for the analysis of trace elements in a wide variety of food samples [17-20] with satisfactory results. However, to
date the analysis of yogurts has been carried out only with atomic
absorption spectrometry (AAS) or ICP with atomic emission spectrometry (ICP-AES). Here, the preparation of samples was carried
out by microwave digestion, which has proven to be excellent in
the analysis of trace metals in different food [21-23]. With this technique, the samples are enclosed in the vessels, so cross contamination and loss of volatiles, critical aspects in trace and ultratrace
analysis, are eliminated.
The proposed method was applied to the quantitative analysis of
20 elements (Ag, Al, As, Ba, Be, Cd, Co, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb,
Sb, Sn, Tl, V, Zn) in yogurt and milk samples after microwave digestion with nitric acid. The validation of the method was performed

24

E.J. Llorent-Martnez et al. / Microchemical Journal 102 (2012) 2327

by determining these elements in two milk certied reference materials and carrying out additional recovery studies over different yogurts.
The method was applied to the analysis of bovine and soybean-based
milk and yogurt samples collected from Spanish supermarkets. Acceptable daily intake (ADI) and recommended daily allowance (RDA) data
were used to make a critical comparison between the food from each
origin.

2. Experimental
2.1. Instrumentation
The quadrupole inductively coupled plasma mass spectrometer
used in this work was an Agilent 7500a (Agilent Technologies, CA,
USA) equipped with a Babington nebuliser, a Peltier-cooled quartz
spray chamber and a standard torch (2.5 mm i.d.). Before each experiment, the instrument was tuned using an aqueous multi-element
standard solution (Agilent, Madrid, Spain) of 10 ng mL 1 each of Li,
Y, Co, Ce and Tl for consistent sensitivity ( 7Li, 89Y and 205Tl) and minimum doubly charged and oxide species levels ( 140Ce). Operating
conditions are shown in Table 1.
The samples were digested using the following system for the microwave digestion: Milestone Ethos MicroSYNTH oven with programmable power control (10 W increments, maximum power 1000 W)
with segmented rotor MPR-600 (operating pressure up to 35 bar maximum; operating temperature 260 C maximum) with 10 reaction
vessels.

2.2. Reagents
All calibration standard solutions were prepared from 100 g mL 1
multi-element standard solution (SCP Science, Paris, France) by dilution
with the suitable percentage of HNO3 in ultrapure water. In solution
(1000 g mL 1) was obtained from SCP Science. Analytical reagent
grade HNO3 65% (Sigma-Aldrich, Madrid, Spain) was additionally
cleaned by sub-boiling distillation. The sub-boiling still was built up
with components obtained from Savillex (www.savillex.com). The
certied reference materials, skim milk powder BCR-151 and skim
milk powder BCR-063R were obtained from Sigma-Aldrich (Madrid,
Spain).
Ultrapure deionised water (18.2 M cm) was obtained from a
Milli-Q system (Millipore, Bedford, MA, USA). All plastic containers
were soaked in 10% v/v sub-boiling HNO3 for at least 24 h, and then
rinsed extensively with Milli-Q water prior to use. All kinds of glassware were avoided to prevent metal releases. All plastic containers,
polyethylene asks, pipette tips, PFA Teon digestion vessels and reagents that came into contact with samples or standards were
checked for contamination.

Table 1
Operating conditions for ICP mass spectrometer.
Plasma conditions
RF power
Plasma Ar ow rate
Auxiliary Ar ow rate
Carrier Ar ow rate
Torch horizontal alignment
Torch vertical alignment
Sampling depth
Instrument
Sampler cone
Skimmer cone

1.2 kW
15 L min 1
0.89 L min 1
0.951.0 L min 1
(0.51.0) mm
0.20.5 mm
6.08.0 mm

Nickel, 1.0 mm orice diameter

Nickel, 0.4 mm orice diameter

2.3. Sample preparation and digestion

The yogurt and milk samples used for the experiments were
obtained from Spanish local supermarkets and consisted of 30 dairy
yogurts (14 regular, 8 skim, 8 with fruits and cereals), 18 soy yogurts
(10 regular, 8 with fruits and cereals), 10 cow's milks (7 whole, 3
skim) and 8 soy milks.
The digestion of the certied reference materials was carried out
by weighing 1 g of sample directly in the digestion vessel and adding
3 mL ultrapure water and 6 mL sub-boiled HNO3. For yogurt and milk
samples, 4 g were weighed in the corresponding vessel and 6 mL subboiled HNO3 were used for the digestion. Then, they were left open
for 15 min for reacting in order to reduce the built up of gases
which could increase the pressure inside the vessels. The power of
the microwave for each digestion step was optimised, from 500 up
to 1000 W. In all cases, the lowest power that provided the required
temperature was selected. The operating program for the microwave
digestion system for the analytical batch is shown in Table 2. The digestion was complete in all cases, as checked by the analysis of reference materials and recovery studies. As a result, the addition of
hydrogen peroxide was not required.
After cooling at room temperature, all the digestion liquors were
quantitatively transferred into plastic containers and diluted to
60 mL with ultrapure water and In was added as internal standard
to yield a concentration of 2 g L 1. Although Ga and Y were also
tested as internal standards, the best results were obtained using In
and hence it was the chosen one.
After each analytical batch, the vessels were cleaned using the
same microwave operating program used for samples, adding 10 mL
HNO3 to each digestion vessel. After cooling at room temperature,
all vessels were thoroughly rinsed with Milli-Q water.
2.4. Calibration procedure
For the quantitative analysis of the samples, calibration curves
were built on six different concentrations. Standard solutions were
prepared in 10% (v/v) sub-boiling HNO3 (the same percentage of
acid present in the samples) by diluting a multi-element standard solution containing all the elements.
The calibration curves were built from the quantication limit
(QL) of the corresponding element up to 200 ng mL 1 in all cases except Hg, which was prepared up to 5 ng mL 1 in order to avoid memory effects. The absence of polyatomic interferences was checked by
measuring several isotopes of the elements and checking the isotopic
ratio in the digested solution of the samples.
3. Results and discussion
The development of the method required the selection of the isotope masses for each element. When a given analyte had two or more
isotopes, at least two of its isotopes were monitored to check the absence of interferences. The isotopes nally selected were the most
abundant of each sought element showing less interference from
the digested matrix: 107, 109Ag, 27Al, 75As, 137Ba, 9Be, 111, 114Cd, 59Co,
53
Cr, 63, 65Cu, 57Fe, 199,202Hg, 55Mn, 95,98Mo, 60,62Ni, 206,208Pb, 121,123Sb,
116,118,120
Sn, 203,205Tl, 51V, 66,68Zn.
Table 2
Operating program for the microwave digestion.
Microwave digestion program
Step

Initial T (C)

Final T (C)

Time (min)

Power (W)

1
2
3
4

25
90
90
180

90
90
180
180

5
3
10
8

750
600
600
600

E.J. Llorent-Martnez et al. / Microchemical Journal 102 (2012) 2327

3.1. Validation of the method

Table 4
Analysis of the skim milk certied reference material BCR-063R.

In order to check the applicability of the proposed method to the

analysis of yogurt and milk samples, two certied reference materials
(BCR-151 and BCR-063R) available for skim milk (in powder form)
were analysed. In addition, taking into account that no certied material is available for yogurts, in this case recovery experiments were
performed.
On the one hand, the certied reference materials were analysed
by triplicate using the microwave digestion procedure previously described. The experimental results obtained were compared with
those ones provided by the manufacturer and they are depicted in
Tables 3 and 4. The Student's t statistical test (P = 0.005) was performed in order to check if there were any signicant differences between the certied and obtained values. For all the certied elements,
no signicant difference was observed. In addition, the indicative
values were close to the ones obtained by the proposed method, although the values for Co and Zn were slightly different.
On the other hand, recovery experiments were performed for the 20
selected elements (Ag, Al, As, Ba, Be, Cd, Co, Cr, Cu, Fe, Hg, Mn, Mo, Ni,
Pb, Sb, Sn, Tl, V, Zn) in soy and dairy yogurts. For this study, two concentration levels were selected, 100 and 2000 ng g 1. All elements were
determined at the lowest level, except Al and Fe, due to their high
QLs. In the case of Al, it is due to the high blank signal obtained and, in
the second case, the isotope 56Fe could not be measured using a simple
quadrupole and 57Fe had to be selected. In addition, the elements Cu,
Mn and Zn were also studied at the 2000 ng g 1 level, as they were
expected to be found in higher concentrations than the other trace
elements.
The recoveries, depicted in Table 5, were in the range 85110%
with relative standard deviations (n = 3) lower than 7% in all cases.
The obtained recoveries conrmed that no signicant metal losses
occurred during the digestion procedure.
3.2. Analytical parameters
All the measurements were carried out using the full quantitative
mode analysis. The correlation coefcients for all the calibration curves
were at least 0.998, showing good linear relationships throughout
the ranges of concentrations studied. The detection limits (DLs) for
all the selected elements, calculated as the concentration yielding
three times the blank signal, are shown in Table 6. As can be seen,
the DLs allowed the determination of both trace and minor elements
at the required levels.
Inter-day (n = 5) and intra-day repeatability data were obtained
for all the analysed elements. The inter-day data were generated on
different days using new instrument tuning and new calibrations
curves. The relative standard deviations were lower than 105 and
7% for all the elements for inter- and intra-day data, respectively.
Table 3
Analysis of the skim milk certied reference material BCR-151.
Element

Certied value (ng g 1)

Observed valuea (ng g 1)

Cd
Cu
Fe
Hg
Pb

101 8
5230 80
50100 1300
101 10
2002 26

98 5
5080 100
53500 3000
111 3
1940 50

Indicative value (ng g 1)

Observed valuea (ng g 1)

6
223
56
0.8
50000

18 1
244 20
54 5
n.d.
45500 200

Co
Mn
Ni
Tl
Zn
n.d. = not detected.
a
n = 3.

25

Element

Certied value (ng g 1)

Observed valuea (ng g 1)

Cu
Fe
Pb
Zn

602 19
2320 230
18.5 2.7
49000 600

585 30
2400 300
20 3
48000 3000

n = 3.

3.3. Results
The main goal of this study was to compare the levels of trace and
minor elements (including toxic heavy metals) in soy and dairy yogurts.
In addition, some milk samples (soy and cow) were analysed to try corroborating the differences found between soy and cow yogurts. Therefore, a higher number of yogurts were analysed in comparison to milk
samples. The number of soy products analysed was lower than cow
products due to their minor availability in the Spanish market. A total
of 66 samples were analysed, being the exact number of each type
indicated in Table 7. Each sample was independently digested and
analysed by triplicate. The range of concentrations found for each
type of sample is shown in Table 7.
The levels of some trace elements (Ag, Be, Sn) were below DL in all
the analysed samples. In other cases (Ba, Co, Cr, Mo, V) the observed
levels were similar in dairy or soybean-based products. The most significant results will be discussed in more detail in following sub-sections.
3.3.1. Toxic trace elements
The levels of potentially toxic trace elements (Al, As, Cd, Hg, Pb, Sb,
Sn, Tl) were very low or even below DL in all yogurt samples, except
Al, which was measured from 100 ng g 1 up to 1.2 g g 1. However,
it has been previously described that dairy and cereal products account for about 60% of the total dietary intake of Al and these levels
present no risk for healthy people [1]. In the case of Pb, the only regulated metal in milk and related products [24], the found levels were
below the maximum tolerated level, 20 ng g 1, in all cases.
3.3.2. Minor elements
As can be seen in Table 7, the main differences between dairy and
soy products were observed in the levels of Ni and minor nutritional
Table 5
Recoveries (%) for spiked yogurts.
Element

Spike recovery (%) RSDa (%)

Cow's yogurt

Ag
Al
As
Ba
Be
Cd
Co
Cr
Cu
Fe
Hg
Mn
Mo
Ni
Pb
Sb
Sn
Tl
V
Zn
a

n = 3.

Soy yogurt

100 ng g 1

2000 ng g 1

100 ng g 1

2000 ng g 1

88 5

101 4
107 3
99 4
91 5
99 2
106 5
93 6

108 6
101 4
90 5
99 3
101 3
92 4
98 5
90 6
105 5
103 4

109 7

106 5
105 7

96 3

104 5

92 4

99 3
98 4
106 5
89 4
101 3
103 4
98 7

110 5
100 3
93 5
105 4
100 3
94 5
105 3
95 6
106 4
106 4

106 6

109 6
104 6

102 2

99 5

26

E.J. Llorent-Martnez et al. / Microchemical Journal 102 (2012) 2327

Table 6
Detection limits for the analysed elements.
Element

DL (ng g 1)

Element

DL (ng g 1)

Ag
Al
As
Ba
Be
Cd
Co
Cr
Cu
Fe

0.7
80
3.0
1.0
1.5
1.5
1
5.0
1.5
35

Hg
Mn
Mo
Ni
Pb
Sb
Sn
Tl
V
Zn

12
1.2
1.0
5.0
0.7
1.2
1.5
1.0
0.5
20

elements (Cu, Fe, Mn and Zn). The highest differences in the levels of
nutritional elements were observed in Cu and Mn, which appeared at
much higher levels in soybean yogurts. The representation of the average levels of these elements is shown in Fig. 1.
Total dietary nickel intakes of humans vary with the amounts and
proportion of food of animal (low-nickel) or plant (high-nickel) origin consumed, and with the amount of rened or processed foods in
the diet. Some reports indicate intakes around 150 g daily [25-28].
Taking into account the recommendations from the World Health Organization (WHO), the threshold level for toxicity can be set at less
than 600 g/day [1]. The average nickel levels found in dairy and
soy yogurts were 14 and 195 ng g -1, respectively. Similar results
were also found in milk samples. These results are consistent with
the higher amount of Ni found in plant origin foods and other authors
have reported approximately the same levels in Spanish dairy yogurts
[29].
Considering the weight of one yogurt (approximately 125 g), the
intake of Ni would be 1.75 g and 24.3 g when consuming one
dairy or soy yogurt, respectively, which would not be considered
any problem for the health. However, Ni levels up to 450 ng g 1
were found in some soy yogurt (500 ng g 1 in milk samples), and
that would mean an intake of approximately 60 g Ni, which is 10%
of the toxicological threshold level. As a result, although no risk for
health arises from these data, it is clear that routine analyses of
trace elements are advisable for soy-related foods.
Table 8 shows the results obtained for minor nutritional elements
(Cu, Fe, Mn, Zn). In this table, the mean concentrations (g g 1 in fresh
weight) are indicated and the percentage of contribution to the RDA

Table 7
Results obtained in the analysis of yogurt and milk samples available in Spanish supermarkets, expressed in ng g 1.
Element

Cow yogurt
(n = 30)

Soy yogurt
(n = 18)

Cow milk
(n = 10)

Soybean milk
(n = 8)

Ag
Al
As
Ba
Be
Cd
Co
Cr
Cu
Fe
Hg
Mn
Mo
Ni
Pb
Sb
Sn
Tl
V
Zn

b DL
100800
b DL-12
40140
b DL
b DL
b DL-15
660
35180
15003600
b DL
20220
3575
630
b DL-7
b DL
b DL
b DL - 2
0.58
26004500

b DL
1001200
b DL-10
70260
b DL
b DL-6
132
475
3001350
30007100
b DL
1906000
50150
14450
b DL-6
b DL
b DL
b DL
0.511
15003500

b DL
60550
24
80160
b DL
b DL
124
750
40150
24003800
b DL-7
20130
4060
425
110
b DL
b DL
b DL
110
29003600

b DL
9003000
24
150500
b DL
1.55
540
1055
7001300
40009400
b DL-12
13002400
250440
100500
28
b DL-1.5
b DL
b DL
640
21003200

Fig. 1. Average concentration of Cu, Ni, Fe, Mn and Zn in the different yogurts analysed.

is calculated for each element, considering the intake of one yogurt.

The employed RDA data, which are the levels of intake of essential
nutrients considered to be adequate to meet the needs of practically
all healthy people, are those ones provided by the Commission of
the European Communities in 2008 [30]. The levels found in dairy
yogurt were similar to the ones reported in other works in which
different types of yogurts were analysed [10,31]. The only difference was observed in the higher levels of Fe here found, which
could be attributed not only to the addition of fruit pieces or cereals,
which causes an increase in the concentration of the mineral [10],
but also to the existence of Fe-enriched yogurts. In addition, the
levels of Fe can vary depending on different factors and are not always constant [11].
The comparison between the mean levels found in yogurts of cow
or soybean origin showed that soy-based ones presented higher
levels of Cu, Mn and Fe, providing increased percentages of contribution to the RDA values, especially in the cases of Cu and Mn, where
the increases were 8-fold and 30-fold, respectively. These results
Table 8
Mean daily intake and percentage of contribution to RDA for minor nutritional elements in yogurts.
Cu

-1 a

Level (g g )
Mean daily intake (g)b
RDA (mg)
% RDAb
a
b

Fe

Mn

Zn

Cow

Soy

Cow

Soy

Cow

Soy

Cow

Soy

0.1
12.5
1
1.25

0.8
100

2.9
362
14
2.6

4.6
575

0.06
6.25
2
0.3

1.8
150

3.5
437.5
10
4.4

2.5
312.5

10

4.1

7.5

Average level of the determined values (Table 7).

Calculated on the basis of the intake of one yogurt ( 125 g).

3.1

E.J. Llorent-Martnez et al. / Microchemical Journal 102 (2012) 2327

made sense taking into account that the levels of these elements are
higher in vegetables than in bovine milk and support the introduction
of soybean products in the diet.
Finally, the differences observed between dairy and soy yogurts
were similar to those ones found between cow's milk and soy milk
(Table 7), supporting the previously discussed results. These differences in different kinds of milk samples had already been reported
[32] and are analogous to the ones here mentioned.
4. Conclusions
In this paper, the determination of trace elements and minor nutrients in yogurt of bovine and soybean origin was carried out using
microwave digestion with nitric acid followed by ICP-MS analysis.
Forty-eight yogurt samples were analysed and the results obtained
were used to make a critical comparison between both types of yogurt in terms of RDA. To the best of our knowledge, this is the rst
time that this comparison has been reported in scientic literature.
In minor nutritional elements analysed, much higher concentrations
of Cu and Mn were found in soybean products and lower differences
were observed in the content of Fe and Zn. The RDA data support the
possibility of replacing dairy products with soybean-based ones for
individuals with intolerance to lactose. However, the content of Ni
was also higher in soybean yogurts and, therefore, they are not
recommended for patients with severe allergies to Ni. Finally, it is
worth mentioning that the levels of potentially toxic elements were
always below the toxicity threshold.
Acknowledgements
The authors are grateful to Centro de Instrumentacion CientcoTcnica (CICT) from the University of Jan for the availability of the
ICP-MS instrument.
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