Journal of Trace Element Analysis

(2012) Vol. 1 No. 1 pp. 1-11

doi:10.7726/jtea.2012.1001
Research Article

Assessment of trace metal contents in chocolate samples

by Atomic Absorption Spectrometry

Sohaila Rehman* and Syed Muhammad Husnain

Received 16 September 2012; Published online 13 October 2012

© The author(s) 2012. Published with open access at uscip.org

Abstract
Trace metals (Pb, Cd, Ni, Fe, Cu, Zn and Mn) were assessed using Atomic Absorption Spectrophotometry
(AAS) in 32 commonly consumed cocoa products (chocolates) prepared by different national and
multinational companies. Microelement contents studied varied significantly in all type of chocolates (P <
0.01). The concentrations of Pb and Cd in cocoa powder were found to be highest, 1400 and 190 μg/L
respectively, whereas mean concentrations of these elements in cocoa-based chocolates were found to be 306
and 46.4 μg/L respectively. In sugar-based chocolates, the mean concentrations of Pb and Cd were 209.8 and
40.7 μg/L, respectively, whereas in samples of milk-based chocolates they were found to be 88 and 33 μg/L
respectively. Frequent consumption of chocolates enhances the intake of Pb and Cd metals in children.
Weekly intake of toxic metals Pb, Cd and Ni were also calculated. Mean concentrations of Pb and Cd were
found below the provisional tolerable weekly intake as defined by FAO/WHO. All essential elements were
assessed for their weekly intake with the dietary reference intakes (DRI), whereas the percentage
contributions of Pb and Cd were calculated from provisional tolerable daily intake (PTDI). Results were
validated through the analysis of certified reference materials and determined metals concentrations were in
good agreement with certified levels. Data was interpreted through cluster analysis and pattern recognition.

Keywords: Trace metals; Chocolates; Cocoa Powder; Cluster analysis; DRIs; PTDIs.

1. Introduction
Chocolate products are among the most commonly purchased and globally inspired items. In
addition to being heavily advertised to children (most targeted population), they are also used in a
variety of foods recipes, especially cakes, tarts and cookies. Chocolate is composed of solid particles,
whose normal solid concentration is about 60 to 70% sugar, cocoa and milk. Beside these main
solid constituents, other ingredients like hydrogenated vegetable oil, salts, buffering agents,
permitted emulsifier and cocoa butter could be source of metals, (Ciurea and Lipka, 1992). As
chocolates and cocoa powder are consumed all over the world, consumers are usually concern not
only about their nutritional status but for its health safety viewpoint as well.
______________________________________________________________________________________________________________________________
*Corresponding e-mail: sohaila@pinstech.org.pk; sohaila_arshad@hotmail.com 1
Chemistry Division, Directorate of Science, Pakistan Institute of Nuclear Science and Technology, P.O. Nilore,
Islamabad45650, Pakistan
 Sohaila Rehman and Syed Muhammad Husnain / Journal of Trace Element Analysis (2012) 1: 1-11

Essential trace metals are beneficial when present below the limit of tolerance, but can be toxic if
taken in excess. This transition between essentiality and toxicity varies from element to element.
Substantial evidence supports the importance of trace elements in human nutrition. These trace
elements play a crucial role in various biochemical functions of the body as some of these forms are
integral enzyme cofactors.

The possibility of the presence of lead (Pb) and cadmium (Cd) in chocolate is a matter of health
consideration because the chemical composition of cocoa allows strong binding of Pb and Cd, as
discussed by Valiente et al (1996). The presence of Pb and Cd in chocolate products could be
natural or due to processing (Dahiya et al., 2005; Lee and Low, 1985; Rankin et al., 2005). These can
be absorbed directly by the theobroma (cocoa tree), can be introduced during the preparation
process or result from contamination via utensils, environmental pollution or transportation and
storage, but, regardless, they may be found in the final product (Mesallam, 1987).

The American Environmental Safety Institute (AESI) claimed that the chocolate manufacturers
neither take appropriate measures to remove potentially dangerous levels of lead and cadmium
from their chocolate products nor notified consumers of their health risks (BBC NEWS, 2002). On
the contrary, world chocolate manufacturers have dismissed claims that their products pose any
health hazard due to Pb and Cd (BBC NEWS, 2002). Chocolates are also considered an important
source of nickel as discussed by Smart and Sherlock (1987), as this element is used as catalyst in
hydrogenation process. Moreover, cocoa butter that is used in chocolates may also contain nickel
(Dahiya et al, 2005). Ni contamination in chocolates could also be due to the type of containers used
for storage and transportation (Mesallam, 1987).

Analysis of copper contents in chocolate is also important since copper compounds are widely used
as fungicides in the farming of cocoa (Dos Santos et al, 2005). Copper in the human body plays a
role in the mobilization of tissue iron and the formation of mitochondrial heme (Lucia et al, 2005).
Iron, Mn and Zn are amongst the most important nutrient elements for humans and are also
important for a strong immune system (McCall et al., 2000), therefore it is important to determine
their concentrations in chocolate samples for nutritional adequacy.

As chocolate matrix is very complex and has high levels of organic compounds very specific
digestion method that restricts the losses of the analyte of interest but doesn’t introduce any
contamination is very important. In this study, the wet digestion method was used, followed by
analysis with Atomic Absorption Spectrophotometry of chocolate samples.
This study identified not only the levels of toxic and essential metals in chocolate samples but also
discusses the Dietary Reference Intake (DRI) and the percentage contribution of observed elements
in chocolate samples for nutritional adequacy. Provisional tolerable daily intake (PTDI) for Pb and
Cd has also been calculated.

2. Method and Materials

2.1 Instrumentation.
The Hitachi model Z-2000 polarized Zeeman Atomic Absorption Spectrophotometer was used in
this study. Two modes of atomization, graphite furnace AAS (GFAAS) and flame AAS (FAAS) were
used. The former was required for the determination of Pb, Cd and Ni while Fe, Mn, Cu and Zn were
measured by the latter. In AAS (GFAAS), argon was used as an inert purging gas up to 100 ml min -1
2
 Sohaila Rehman and Syed Muhammad Husnain / Journal of Trace Element Analysis (2012) 1: 1-11

during dry charring and cleaning stages while its flow was interrupted during the atomization
stage. The absorption signal during atomization step was recorded as a peak height. A water-cooled
premix, fishtail type burner having a 10 x 0.05 cm2 slot was used for the air-acetylene flame in
flame atomic absorption spectrometer (FAAS). The monochromator in the Z-2000
spectrophotometer is equipped with a diffraction grating having 1800 grooves/mm. The focal
length of the monochromator is 450mm and provides a dispersion of 1.2nm/mm. The spectral
range of the instrument is from 190 to 900 nm with a provision of four slit widths with spectral
band passes of 0.09, 0.2, 0.4 and 1.3 nm. Pb, Cd and Ni were determined using the operating
conditions and heating program employed by GFAAS whereas instrumental parameters of FAAS
were used for the rest of the elements.

2.2 Reagents and solutions

The concentrated acids HClO4 and HNO3 (Merck, Germany) used were of analytical grade. Stock
standard solutions (1000 ppm) of Cu, Cd, Mn, Fe, Pb, Ni and Zn were used in preparing subsequent
calibration curves after serial dilutions.

2.3 Sampling
Samples were randomly collected from the open market. After sample collection, information
relevant to nutritional composition, ingredients, weight, manufacturer and country of origin were
recorded. The chocolate samples were categorized first with respect to their composition as milk,
sugar and cocoa-based and with respect to the manufacturer’s origin as national or multinational.

2.4 Sample Preparation

Complete mineralization of the sample is the fundamental requirement for accurate spectrometric
analysis. Acids like HNO3 and HCLO4 were found to be the most suitable acids for the wet digestion
of chocolates. An appropriate amount of chocolate sample (500 mg approx) was weighed and
transferred to a 100 mL digestion flask fitted with a 30 cm long air condenser. 5 mL of HNO 3 (65%)
was added. The content was heated at 70°C for an hour. After cooling it at room temperature 1.5 mL
HClO4 (70%) was added and heating prolonged at 240°C until white fumes were produced (Fatima
and Rahman, 2009). Clear solution was transferred to 10 mL volumetric flask and made up to mark
with deionized water. Samples were analyzed in triplicate.

3. Statistical analysis of data

Experimental data was analyzed statistically using the multivariate cluster analysis (CA) technique
(Shittu and Badmus, 2009) using the Mintab 16 statistical package. Descriptive statistics were
performed with help of Excel 2007 (Microsoft) and Origin 6.1 (Origin Lab).

CA is a powerful method of data analysis that can accomplish the goal of pattern recognition
(Richard, 1993). It was introduced primarily by biologists to establish a relationship between
organisms. It is a well-established approach to visually identify samples of similar type, where
clustering is represented by dendrograms (Vandeginste et al, 1998).

The results of cluster analysis performed on the data using Euclidean distances as a measure of
similarity with ward linkage are presented in Fig. 1. It predicts six clusters:
Cluster 1: C1, C18, C24, C13, C9, C22
Cluster 2: C4, C10, C7, C25, C21
3
 Sohaila Rehman and Syed Muhammad Husnain / Journal of Trace Element Analysis (2012) 1: 1-11

Cluster 3: C2, C31, C17, C20, C32, C6, C15, C29

Cluster 4: C3, C12, C19, C8, C23
Cluster 5: C5, C16, C11, C28, C14, C26, C27
Cluster 6: C30

13.15
Euclidean distance

8.77

4.38

0.00
C-11
C- 8
C-14
3
C- 9
22
C-4
0
C- 7
C- 5
21
C- 2
C- 1
C-27
C-30
2
C-6

C-25
9
C- 3
C-12
9
C- 8
23
C-5

C-16
C- 1
C- 8
C-24
C- 6
C- 7
30
C-

C-

C-

C-

C-

C-
2

C-1

3
1

C-1

2
1

2
C-

Chocolate samples
Fig 1. Cluster analysis using Euclidean distance as a measure of similarity with ward linkage

It is observed that the cluster analysis cannot differentiate chocolate samples on the basis of their
types, e.g., milk, sugar and cocoa. But this elemental variation is supposed to be due to some
additional sources, like individual preparing process, composition of raw materials, packaging
materials and some other additional ingredients. The chocolate samples present in Cluster 1 and
Cluster 3 contain relatively high level of toxic metals (Pb, Cd and Ni), while the samples present in
Clusters 2, 4 and 5 have lower concentration. Cluster 6 contains only single sample (cocoa powder),
it indicates that its composition is different from the rest of the samples and also that it has the
highest concentration of Pb and Cd.

4. Results & Discussion

The limits of detection (LOD) and quantification (LOQ) for all the elements analyzed in the
chocolate samples were calculated as the blank signal plus three or ten times, respectively, its
standard deviation (Currie, 1999; Rehman et al., 2011), employing the optimized instrumental
conditions stated in Table 1 and Table 2.

4
 Sohaila Rehman and Syed Muhammad Husnain / Journal of Trace Element Analysis (2012) 1: 1-11

Table 1 Optimized instrumental parameters for Pb, Cd and Ni by GFAAS

Parameters Pb Cd Ni
Lamp Current (mA) 7.5 7.5 10
Wave length (nm) 283.3 228.8 232.0
Slit (nm) 1.3 1.3 0.2
Cuvette Pyro Tube HR
Carrier gas (Atomize) (mL/min) 30
Sample Volume (µL) 20
Temperature Start 80 80 80
(°C) End 140 140 140
Dry
Time Ramp 40 40 40
(Sec) Hold - - -
Temperature Start 600 300 1400
(°C) End 600 300 1400
Ash
Time Ramp 20 20 20
(Sec) Hold - - -
Temperature Start 2400 1500 2700
(°C) End 2400 1500 2700
Atomize
Time Ramp - - -
(Sec) Hold 5 5 5
Temperature Start 2700 1800 2800
(°C) End 2700 1800 2800
Cool
Time Ramp - - -
(Sec) Hold 4 4 4

Table 2 Optimized instrumental parameters for Fe, Mn, Cu and Zn by FAAS

Parameters Fe Mn Cu Zn
Lamp current (mA) 12.5 7.5 7.5 5.0
Wave length(nm) 248.3 279.6 324.8 213.9
Slit width(nm) 0.4 0.4 0.2 1.3
Burner height 7.5
Burner head Standard
Flame Air-acetylene
Oxidant gas pressure (kPa) 160
Fuel gas flow rate (L/min) 1.8 2.0 2.0 1.8

The measurements were validated by the analysis of standard reference materials (SRM) of the
National Institute of Standards & Technology (NIST), i.e., NIST-SRM-1567 wheat flour and certified
reference materials (CRMs) from IAEA, IAEA-A-11 Milk powder by the proposed method. The
results for Pb and Cd in reference materials are expressed in µg kg-1and for Ni, Fe, Mn, Cu, Zn in mg
5
 Sohaila Rehman and Syed Muhammad Husnain / Journal of Trace Element Analysis (2012) 1: 1-11

kg-1 are summarized in Table 3, along with respective standard deviations and percentage
recoveries. Determined values for metals in reference materials are in good agreement with the
certified values.

Table 3 Concentrations (mg kg-1) of Pb, Cd, Ni, Fe, Mn, Cu and Zn in Certified Reference Materials
(± standard deviations, N=3)
Certified
Reference Material Element Our values Recovery (%)
values
Pb 0.02 ± 0.01 0.03 ± 0.01 150
Cd 0.03 ± 0.01 0.03 ± 0.02 100
Ni 0.26 ± 0.02 0.31 ± 0.02 119
Wheat Flour NIST
Fe 14.10 ± 0.56 14.30 ±0.43 101
1567
Mn 9.40 ± 0.94 9.70 ± 0.32 103
Cu 2.10 ± 0.21 2.06 ± 0.17 98
Zn 11.60 ± 0.35 11.29 ± 0.22 97
Ni 0.93 NC 0.98 ± 0.02 105
Milk Powder IAEA Fe 3.65 ± 0.76 3.71 ± 0.30 101
(A-11) Mn 0.26 ± 0.01 0.39 ± 0.02 150
Zn 38.90 ± 2.30 40.10 ± 1.60 103
Note: NC = Not Certified

Table 4 lists the average concentrations of metals in the aforementioned categories of chocolates
with their respective ingredients. The highest concentration of Pb was detected in cocoa-based
chocolates. Cocoa beans contain plenty of carbohydrates in the form of soluble starch and insoluble
dietary fibers, so these could be the probable source of Pb (Valiente et al., 1996). The mean
concentration of Cd in all three varieties of chocolate samples was in the range of 33.1-46.4 µg kg-1,
which we attribute to Cd in raw cocoa beans possibly being high (Lee and Low, 1985). According to
the American Environmental Safety Institute (AESI),the concentrations of Pb in chocolate products
is between 0.002-0.105 mg kg-1, whereas Cd is between 0.002-0.136 mg kg-1, that is around two
times below the legally admissible values defined at the level of individual countries (American
Environmental Safety Institute-Fact Sheet, 2002).

Table 4: Concentrations of Pb, Cd (µg kg-1) Ni, Fe, Cu, Mn and Zn (mg kg-1) in chocolate samples of
different types
Elements Cocoa-based Sugar-based Milk-based Total
N=9 N=9 N=14 N=32
Mean 306 209 88 184
Pb Range 60-1400 98-540 29-250 29-1400
Mean 46.4 40.7 33.1 38.9
Cd Range 20-190 4.3-90 10-140 4.3-190
Mean 2.0 1.7 1.2 1.6
Ni Range 0.2-5 0.3-3 0.2-2 0.2-5
Mean 32 28 30 30
6
 Sohaila Rehman and Syed Muhammad Husnain / Journal of Trace Element Analysis (2012) 1: 1-11

Fe Range 15 -51 12 -59 3 - 60 3 - 60

Mean 3.6 4.3 4.4 4.1

Mn Range 1.4-7.1 1.6-8.1 1.6-8.1 1.4-8.1
Mean 2.5 3.2 3.1 2.9
Cu Range 1.4-4.4 1.9-4.5 1.7-4.2 1.4-4.5
Mean 10.3 15.3 12.7 12.7
Zn Range 3.2-19 8 - 29 4 - 27 3 - 29
Note: * = Concentration in µg kg-1 (ppb)

The quantified Pb, Cd, Ni and Fe concentrations are highest in cocoa-based chocolates compared to
milk and sugar-based chocolates. The nutritional status of chocolates with respect to Fe was found
to be excellent, as its mean concentration in all the distinct varieties of chocolates studied was high,
varying from 28 to 32 mg kg-1. The levels of Mn, Cu and Zn were found to be directly related to the
content of milk. Henceforth, they were higher in milk-based chocolates. Teams from the Federal
University of Santa Maria in Brazil deemed chocolate as an extremely rich source of many essential
minerals and it said that cocoa is the best natural food source for Fe and Zn and hence contributes
to a healthy diet (Ieggli et al., 2011).

All chocolates characterized in this work have also categorized according to their manufacturers,
i.e. National (N) or Multinational (MN). The levels of determined toxic and essential metals in these
two categories are presented in Fig. 2. Pb is significantly high in national brands of chocolates as
compared to multinational ones. It is also evident from Fig. 2 that Cd and Ni are also higher in
national brands.

Fig 2. Comparison of toxic and essential metals between national [N]and multinational [MN]
samples

7
 Sohaila Rehman and Syed Muhammad Husnain / Journal of Trace Element Analysis (2012) 1: 1-11

On the basis of these findings it is suggested that regulatory implications should be imposed on
local manufacturers to monitor Pb, Cd and Ni levels in their brands. Both N and MN chocolate show
compatible results for Fe, however Mn and Cu are high in MN, and Zn is high in N brands.

Cocoa powder was also analyzed for Pb, Cd, Ni, Fe, Mn, Cu and Zn as it’s frequently used in
confectionary items and in different types of chocolates or drinks as an ingredient. Except for Pb
and Cd, all other quantified elements are comparable in their contents with chocolate samples. The
concentration of Pb and Cd in cocoa powder samples was found to be 1400 and 190 µg kg-1,
respectively, which seemed very high, but as the quantity of cocoa powder used in confections is
smaller in comparison to routine consumption of chocolates, high levels of Pb and Cd might not
pose any deleterious health effects. In cocoa powder the maximum tolerable levels for Pb and Cd
have been set to 1 mg kg-1 and 0.3 mg kg-1 by Polish national standard proposed by Codex
Alimentarius (FAO/WHO, 2006) and by the legislation of some European countries respectively
Dahiya et al.(2005).The maximum tolerable levels for Cd has been set by different countries in their
reports (0.4 mg kg-1 in Germany, 0.5 mg kg-1 in Finland and Central European countries) Satarug et
al. (2000) and 1.0 mg kg-1 in Malaysia as reported by Lee and Low (1985). Cocoa powder, if taken
on routine basis, could be harmful for health. Based on the findings, it is suggested that control of
contamination during the preparation process and proper selection of raw materials by chocolate
processing companies can minimize the presence of toxic metals in their products.
A global comparison of elemental concentrations determined in chocolate samples in this study is
presented in Table 5 (Dahiya et al., 2005; da Silva et al., 2006; Dos Santos et al., 2005; Guldas et al.,
2008; Lee and Low 1985; Ieggli et al., 2011).

Table 5 Comparison of metal concentration (mg kg -1) in studied chocolate samples with the results
published in some other countries in the world
Countrie
s Pb* Cd* Ni Fe Mn Cu Zn
Turkey 30 - 40 20 - 30 1.3 - 4.8 2.5-3.7 - 5.7-10.6 12.1-16.7
0.04 -
India 49 - 8040 1- 2730 8.3 - - - -
USA 11.9 - 69.8 - - - - - -
Brazil - - - 1.2-140.8 43.1-52.2 26.6-31.2 7.5-23.3
Malaysia 1100-1940 280 - 420 - - - 2.9-6.3 -
Our 0.20 -
study 28.6-1400 4.3 -190 4.8 3.2-59.6 1.4-8.1 1.4-4.4 3.2-29.3
Note: * = Concentration in µg kg-1 (ppb)

Dietary Reference Intakes (DRIs) have also been calculated for this work to assess dietary safety
and adequacy. Calculations of DRIs were performed with the assumption of 10 g/person/day
chocolate intake. Fig. 3 presents percentage contribution of Fe, Cu, Zn and Mn in chocolate samples
with reference to DRIs values (Welz, 1999).

There are no established DRIs for Ni, however, tolerable upper intake level for this element is set at
1 mg per day (Food and Nutrition Board, National Academy Press, Washington 2001), therefore the
percentage contribution for Ni was calculated with the presumption of 300 µg per day intake.
Moreover percentage contribution for Pb and Cd was calculated using their PTDIs values (Dietary
8
 Sohaila Rehman and Syed Muhammad Husnain / Journal of Trace Element Analysis (2012) 1: 1-11

Supplement–Standard 173 Metal Contaminant Acceptance Level 2003).Our findings suggested that
the daily intake of Pb and Cd through chocolates do not pose any health risk to the general
population as intake levels are far below the maximum tolerable levels defined by WHO (FAO/WHO
2006).

Fig 2. Percentage contribution of Ni, Fe, Cu, Zn, Mn through chocolate samples calculated from DRIs
and Cd &Pb from PTDI

5. Conclusions
The AAS technique has been successfully employed here for the determination of essential and
toxic metals in a complex matrix like chocolate. In chocolates, the mean concentrations of Pb, Cd, Ni
and Fe were found to be higher in cocoa powder and cocoa-based products than in milk and sugar-
based chocolates. The frequent use of cocoa-based chocolates and confectionaries containing cocoa
powder should be reduced, otherwise intake of lead and cadmium will cross the limits prescribed
by the US. Raw materials should properly be handled and if necessary be assessed for metal
contents before use. These research findings will hopefully create consciousness towards frequent
utilization of chocolates.

Acknowledgement
Authors gratefully acknowledge and highly appreciate the experimental assistance of staff members
of the Atomic Absorption Spectrophotometry laboratory.

References
American Environmental Safety Institute, 2002. Fact Sheet–Lead in Chocolate: The Impact on Children's
Health.
BBC NEWS., 2002. Chocolate makers dismiss toxic claims. Available at:
http://news.bbc.co.uk/2/hi/business/1977780.stm

9
 Sohaila Rehman and Syed Muhammad Husnain / Journal of Trace Element Analysis (2012) 1: 1-11

Ciurea, I.C., Lipka, Y.F., 1992. Occurrence of cadmium in cocoa and coffee.Mitt.Geb.LebensmitteluntersHyg, 83,
197-199.
Currie, L. A., 1999.Nomenclature in evaluation of analytical methods including detection and quantification
capabilities (IUPAC Recommendations 1995).AnalyticaChimicaActa, 391:2, 105–126.
http://dx.doi.org/10.1016/S0003-2670(99)00104-X
Dahiya, S., Karpe, R., Hegde, A.G., Sharma, R.M., 2005. Lead, cadmium and nickel in chocolate and candies
from suburban areas of Mumbai, India. Journal of Food Composition and Analysis, 18:6, 517-522.
http://dx.doi.org/10.1016/j.jfca.2004.05.002
Da Silva, E.G.P., Santos, A.C.d.N., Costa, A.C.S., Fortunato, D.M.d.N.; Jose, N.M., Korn, M.G.A., dos Santos, W.N.L.,
Ferreira, S.L.C., 2006. Determination of manganese and zinc in powdered chocolate samples by slurry
sampling using sequential multi-element flame atomic absorption spectrometry. Microchemical Journal,
82, 159-162.
http://dx.doi.org/10.1016/j.microc.2006.01.008
Dietary Supplement–Standard 173 Metal Contaminant Acceptance Level. August 19, 2003.
Dos Santos, W.N., da Silva, E.G., Fernandes, M.S., Araujo, R.G., Costa, A.C., Vale, M.G. and Ferreira, S.L.C., 2005.
Determination of copper in powdered chocolate samples by slurry sampling flame atomic-absorption
spectrometry. Analytical and Bioanalytical Chemistry, 382, 1099-1102.
http://dx.doi.org/10.1007/s00216-005-3252-y
PMid:15891868
FAO/WHO 2006. Draft standards for chocolates and chocolate products. Joint FAO/WHO standards
programme. CODEX committee on cocoa products and cocoa chocolates, 19th session, 3–5 October 2001.
Fribourg, Switzerland, CX/CPC 01/3.
Fatima, I., and Rahman, S., 2009. Measurements of Toxic elements in Fish: An Indicator of Aquatic
Environment. Journal of the Chemical Society of Pakistan 31:2, 236-239.
Food and Nutrition Board 2001. Institute of Medicine: Dietary Reference Intakes: Vitamin A, Vitamin K,
Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium and
Zinc. National Academy Press, Washington.
Guldas, M., Adnan, F.D., and Biricik, F.B., 2008. Determination and comparison of some trace elements in
different chocolates types produced in Turkey. Journal of Food Agriculture & Environment, 6:3&4, 90–94.
Ieggli, C.V.S., Bohrer, D., do Nascimento, P.C., de Carvalho, L.M., 2011. Determination of sodium, potassium,
calcium, magnesium, zinc and iron in emulsified chocolate samples by flame atomic absorption
spectrometry. Food Chemistry 124:3, 1189-1193.
http://dx.doi.org/10.1016/j.foodchem.2010.07.043
Lee, C.K., Low, K.S., 1985. Determination of cadmium, lead, copper and arsenic in raw cocoa, semi-finished
and finished chocolate products.Pertanika 8, 243-248.
Lucia, R.C., Neuza, M.B.C., and Fernando, P.R., 2005. Copper-iron metabolism interaction in rats. Nutrition
Research, 25:1, 79-92.
http://dx.doi.org/10.1016/j.nutres.2004.07.003
McCall, K.A., Huang, C., and Fierke, C.A., 2000.Function and mechanism of zinc metalloenzymes.The Journal of
Nutrition 130, 1437S-1446S.
PMid:10801957
Mesallam, A.S., 1987. Heavy metal content of canned orange juice as determined by direct current plasma
atomic emission spectrophotometry (DCPAES). Food Chemistry, 26:1, 47-58.
http://dx.doi.org/10.1016/0308-8146(87)90166-X
Rankin, C.W., Nriagu, J.O., Aggarwal, J.K., Arowolo, T.A., Adebayo, K., Flegal, A.R., 2005. Lead Contamination in
Cocoa and Cocoa Products: Isotopic Evidence of Global Contamination. Environmental Health
Perspectives, 113:10, 1344-1348.
http://dx.doi.org/10.1289/ehp.8009
PMid:16203244 PMCid:1281277
Rehman, S., Adnan, M., Khalid, N., Shaheen, L., 2011. Calcium Supplements: An Additional Source of Lead
Contamination. Biological Trace Element Research 143:1, 178-187.
http://dx.doi.org/10.1007/s12011-010-8870-3
10
 Sohaila Rehman and Syed Muhammad Husnain / Journal of Trace Element Analysis (2012) 1: 1-11

PMid:20953844
Richard, C.G., 1993. Data Analysis for the Chemical Sciences: A guide to statistical techniques, VCH, New York.
Satarug, S., Haswell-Elkins, M.R., Moore, M.R., 2000.Safe levels of cadmium intake to prevent renal toxicity in
human subjects. British Journal of Nutrition, 84:6, 791-802.
PMid:11177195
Shittu, T.A., Badmus, B.A., 2009. Statistical correlations between mineral element composition, product
information and retail price of powdered cocoa beverages in Nigeria. Journal of Food Composition and
Analysis, 22:3, 212-217.
http://dx.doi.org/10.1016/j.jfca.2008.10.006
Smart, G.A., and Sherlock, J.C., 1987. Nickel in foods and the diet. Food Additives and Contaminants, 4:1, 61-
71.
http://dx.doi.org/10.1080/02652038709373616
PMid:3556677
Valiente, C., Molla, E., Martin-Cabrejas, M.M., Lopez-Andreu, F.J., Esteban, R.M., 1996.Cadmium Binding
Capacity of Cocoa and Isolated Total Dietary Fibre under Physiological pH Conditions.Journal of the
Science of Food and Agriculture, 72:4, 476-482.
http://dx.doi.org/10.1002/(SICI)1097-0010(199612)72:4<476::AID-JSFA682>3.0.CO;2-M
Vandeginste, B.G., Massart, D.L., Buydens, L.M.C., de Jong, S., Lewi, P.J., Smeyers-Verbeke, J., 1998. Handbook
of Chemometrics and Qualimetrics: Part B, Data Handling in Science and Technology Elsevier,
Amsterdam.
Welz, B., Sperling, M., 1999. Atomic Absorption Spectrometry. Wiley-VCH, Weinheim.
PMCid:24144

11