Food Chemistry 200 (2016) 245–248

Contents lists available at ScienceDirect

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

Analytical Methods

Method development for the determination of calcium, copper,

magnesium, manganese, iron, potassium, phosphorus and zinc in
different types of breads by microwave induced plasma-atomic emission
spectrometry
Nil Ozbek, Suleyman Akman ⇑
Istanbul Technical University, Faculty of Arts and Sciences, Department of Chemistry, 34469 Maslak, Istanbul, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: A novel method was developed for the determination of calcium, magnesium, potassium, iron, copper,
Received 14 May 2015 zinc, and manganese and phosphorous in various kinds of breads samples sold in Turkey by microwave
Received in revised form 15 September plasma-atomic emission spectrometry (MIP-AES). Breads were dried at 100 °C for one day, ground thor-
2015
oughly and then digested using nitric acid/hydrogen per oxide (3:1). The analytes in certified reference
Accepted 10 January 2016
Available online 11 January 2016
wheat flour and maize flour samples were determined in the uncertainty limits of the certified values
as well as the analytes added to the mixture of ground bread and acid mixture prior to digestion were
recovered quantitatively (>90%). Therefore, all determinations were made by linear calibration technique
Keywords:
Microwave induced plasma-atomic
using aqueous standards. The LOD values for Ca, Cu, Fe, K, Mg, Mn, P and Zn were 13.1, 0.28, 4.47, 118,
emission spectrometry 1.10, 0.41, 7550 and 3.00 ng mL 1, respectively. No spectral interference was detected at the working
Bread wavelengths of the analytes.
Elemental determination Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction gas-consumption because nitrogen is maintained mostly extracted

Bakery products, especially bread, is the most popular and
stable food in the world since prehistoric ages. It is consumed com-
monly and excessively in all meals, especially in Middle East coun-
tries, including Turkey as an indispensable food. Although it is
simply prepared from dough and water, many ingredients such
as preserving agents, leavening agents, milk, egg, sugar, spice,
dried-fruit, nuts, seeds, corn flour, oaten, whole wheat flour, etc.
are added to improve its ease of manufacturing, shelf life, taste
and nutrition value. Therefore, its mineral content is expected to
be influenced from those of its ingredients as well as baking
conditions.
Microwave induced plasma-atomic emission spectrometry
(MIP-AES) is a novel promising technique which uses a 2.45 GHz
microwave magnetic field and nitrogen gas to sustain a high tem-
perature plasma (approximately 5000 K). The most attractive
advantage of MIP-AES over atomic emission spectrometry
(ICP-AES) and inductively coupled plasma-mass spectrometry
(ICP-MS) techniques is the low running costs due to
⇑ Corresponding author.
E-mail address: akmans@itu.edu.tr (S. Akman).
from air by a nitrogen generator. In other words, it runs on air. The
advantage of MIP-AES over flame atomic absorption spectrometry
(FAAS) is that there is no flammable and toxic gases (e.g. acetylene,
nitrogen peroxide) as well as no manual handling of gas-cylinders
in the laboratory. Another advantage of MIP-AES compared to AAS
is that refractive and/or carbide forming elements can be more
easily determined because temperature of plasma is much higher
than those of flames and graphite furnace. Therefore, MIP-AES is
a competitive technique with many challenges and advantages.
Since MIP-AES is commercially available since 2012, the number
of papers was very limited. MIP-AES has been used for the determi-
nation of Si in diesel and biodiesel (Amais, Donati, Schiavo, &
Nobrega, 2013), Cu, Fe, Mn and Zn in animal feed and fertilizer
(Li, Simmons, Shrader, Herrman, & Dai, 2013) and boron determi-
nation in steel (Hettipathirana, 2013) and wine (Ozbek & Akman,
2015) and recently Ca, K and Mg in cheese samples (Ozbek &
Akman, 2016).
The aim of this study was to develop a practical and cheap
method for the determination of calcium, magnesium, potassium,
iron, copper, manganese, zinc and phosphorous in various types
of breads by MIP-AES which came on the market a few years

http://dx.doi.org/10.1016/j.foodchem.2016.01.043
0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
246 N. Ozbek, S. Akman / Food Chemistry 200 (2016) 245–248
ago. The experimental conditions for this new generation plasma
technique were optimized and the figures of merit were evaluated.
2. Experimental
2.1. Instrumental
An Agilent 4200 Microwave Plasma Atomic Emission Spectrom-
eter (Agilent Technologies, Melbourne, Australia) equipped with an
Inert One Neb nebulizer and a double-pass glass cyclonic spray
chamber (Agilent Technologies, Melbourne, Australia) was used
throughout this study. Nitrogen was extracted from air using a
F-DGSi, Thyster 8/1 LV, (Innovative Gas System Co., Evry, France),
dual flow nitrogen generator. Sample and waste tubings were
orange/green and blue/blue solvent resistant, respectively (Agilent
Technologies, Melbourne, Australia). Before every sample reading,
10 s of uptake time and 20 s of torch stabilization time were set,
whereas for emission measurement of each sample, 5 s read time
with 3 replicates were applied. Torch alignment and wavelength
calibration were carried out using a wavelength calibration solu-
tion (Agilent Technologies, Melbourne, Australia) and automati-
cally optimized according to MP Expert Software program
(version 1.1.1, Agilent Technologies, Melbourne, Australia).
Instrumental parameters for MIP-AES are given in Table 1.
A ContrAA-700 high resolution continuum source atomic
absorption spectrometer (Analytik Jena, Germany) equipped with
flame (HR-CS FAAS) and graphite furnace (HR-CS GFAAS) atomizers
were used for the verification of determinations. All instrumental
parameters were adjusted according to the recommendations of
the manufacturer.
2.2. Reagents and solutions
For all dilutions, water with 18.2 MO cm resistivity obtained
from a TKA reverse osmosis and a deionizer system (TKA Wasser-
aufbereitungsstandards, system GmbH, Niederelbert Germany)
was used. Calibration standards were prepared from 1000 mg L 1
of calcium, magnesium, potassium, iron, copper, manganese, zinc
and phosphorous solutions (Carlo Erbaa, Radona, Italy) daily. For
validation purposes, wheat flour-NCS ZC 7303 (LGC Ltd., UK),
maize flour-NCS ZC 73010 (LGC Ltd., UK) certified reference mate-
rials (CRMs) were used.
2.3. Procedure
One slice (approximately 25 g) from different types of bread
samples obtained from markets were dried at 100 °C in an oven
for 2 days. Samples were ground using agate mortar and blended
thoroughly. 1.0 g of portions were weighed precisely and dissolved
in a mixture of 3 mL of HNO3 (65% w/v) and 1 mL of H2O2 (35% w/v)
at 100 °C for 2 h. Digests were then diluted appropriately and aspi-
rated to the plasma. Blanks were subjected to the same procedure.
Results were given as the average of at least 3 replicates.
Table 1
Operating conditions for MIP-AES.
Parameter Ca Cu Fe
Wavelength, nm 393.366 324.754 371.993
Viewing position 0 0 20
Nebulizer Flow, L min 1 0.65 0.7 0.85
Background correction Auto Auto Auto
Stabilization time (s) 20 20 20
Sample uptake delay (s) 30 30 30
3. Results and discussion
3.1. Optimization of MIP-AES
The Achilles heel of this technique is the limitation of total dis-
solved salt (TDS) concentration of samples. The accumulation of
salts may cause the blockage in the torch and nebulizer and may
break the torch. The limiting TDS concentration for the system
used in this study is 4%. Therefore, the dilution of the sample as
much as possible reduces the risk of the accumulation of salt in
the torch and prevents its damaging. It should be noted that too
much dilution may cause the concentration of the analyte fall
under LOD. Therefore, the wavelengths and dilution were chosen
considering all those criteria and the preliminary emission values
for the digests of samples. In order to prevent the blockage of torch
and nebulizer and the damage of torch due to carbon accumulation
from sample matrix, samples were diluted as much as possible (to
get lower TDS).
To dilute the samples as much as possible as well as to stay
above LOD for all analytes, a systematic study was performed to
select the most suitable wavelengths and to make a single dilution
of the sample digests. After a series of optimization, 393.366 nm
285.213 nm, 766.491 nm, 371.993 nm, 324.54 nm, 403.076 nm,
213.857 nm and 214.915 nm were selected as working wave-
lengths for the determination of calcium, magnesium, potassium,
iron, copper, manganese, zinc and phosphorous, respectively and
sample digests were diluted to 10 mL. No spectral interference
from sample concomitants was observed for the wavelengths stud-
ied. For the highest sensitivity, nebulizer pressure and viewing
position were optimized automatically by the instrument. The
concentrations of analytes in digests were in the linear range of
calibration curves and generally above LOD. The last but not least,
no accumulation from sample matrix and damage of torch
occurred during the whole study.
3.2. Figures of merit
The method detection limit (MDL) or in other words limit of
detection (LOD) was calculated as 3 times the standard deviation
(r) for 10 repetitive aspiration of a blank subjected to the same
procedure as that applied for the digestion of samples. (3r/slope
of calibration graph). The limit of quantification (LOQ) was calcu-
lated similar to LOD with only difference that 10 times of the stan-
dard deviation was taken. Analytical parameters were summarized
in Table 2.
LOD values for Cu, Fe, Mn and Zn in this study (0.28, 4.47, 0.41
and 3.00 ng mL 1, respectively) were better or very close than/to
those found by Li et al. (2013) who found 2.2, 4.1, 1.5 and
2.3 ng mL 1, respectively using standard deviations of 50 ng mL 1
of matrix-free aqueous solutions of analytes for LOD in an old ver-
sion of this instrument. The analytes in two certified reference
flour (wheat and maize), which are the most similar CRMs to the
bread matrix, were found in the uncertainty limits of the certified
values (Table 3). Moreover, as shown from Table 4, the analytes
K Mg Mn P Zn
766.491 285.213 403.076 214.915 213.857
0 0 0 0 0
1 0.7 0.85 0.55 0.4
Auto Auto Auto Auto Auto
20 20 20 20 20
30 30 30 30 30
 N. Ozbek, S. Akman / Food Chemistry 200 (2016) 245–248 247

Table 2
Figures of merit of the method.

Ca Cu Fe K Mg Mn P Zn
Wavelength, nm 393.366 324.754 371.993 766.491 285.213 403.076 214.915 213.857
Linearity, lg mL 1
0–20 0–20 0–20 0–20 0–20 0–20 0–20 0–20
R2 0.9999 0.9999 1.000 0.9996 0.9997 0.9994 0.996 1.000
LOD, ng mL 1 13.1 0.28 4.47 118 1.10 0.41 7550 3.00
LOQ, ng mL 1 43.8 0.93 14.9 393 3.66 1.38 25.100 10.1

Table 3
Accuracy test for CRMs (N:3).

Maize flour NCS ZC73010 Wheat NCS ZC73030

Certified value Found valuea Certified value Found valuea
Ca, % 0.0055 ± 0.0008 0.0062 ± 0.0005 0.033 ± 0.002 0.029 ± 0.006
Cu, mg kg 1 0.66 ± 0.08 0.61 ± 0.11 2.4 ± 0.1 2.4 ± 0.2
Fe, mg kg 1 13.3 ± 1.5 14.4 ± 1.5 20 ± 3 18.9 ± 2.1
K, % 0.129 ± 0.007 0.137 ± 0.008 0.21 ± 0.01 0.24 ± 0.02
Mg, % 0.018 ± 0.002 0.019 ± 0.003 0.048 ± 0.002 0.045 ± 0.007
Mn, mg kg 1 1.55 ± 0.08 1.53 ± 0.05 10.8 ± 0.4 11.3 ± 0.9
P, % 0.061 ± 0.003 – 0.15 ± 0.01 0.17 ± 0.02
Zn, mg kg 1 2.9 ± 0.3 2.5 ± 0.5 12.4 ± 0.6 13.7 ± 1.5
a
Mean ± tr/(N)1/2 (95% confidence interval).

Table 4
Recoveries of analytes added to a bread sample prior to digestion.

Analytes Added (concentration in the Recovery added to a bread sample prior to digestion were recovered quanti-
sample solution) (lg mL 1) (%) tatively using aqueous standards for calibration which showed that
Ca 10 96.7 no analyte loss was occurred during digestion as well as the emis-
Cu 10 97.1 sion sensitivities of the analytes were not significantly influenced
Fe 10 102.5 from sample matrix. Therefore, linear calibration using aqueous
K 10 101.3
standards (r2 > 0.999) were reliably applied for all quantifications
Mg 10 104.6
Mn 10 99.1 without needing analyte (or standard) addition technique. The
P 10 90.1 RSD values of repetitive aspirations of the sample digests were
Zn 10 102.8 around 5–10% (N:3) depending on the element.

Table 5
Concentrations of calcium, magnesium, potassium, iron, copper, manganese, zinc, and phosphor in various kinds of bread samples, N:3a.
1 1 1 1 1 1 1 1
Bread Type Ca, mg g Cu, mg kg Fe, mg kg K, mg g Mg, mg g Mn, mg kg P, mg g Zn, mg kg
White flour bread 0.53 3.01 17.9 2.29 0.38 9.85 1.61 10.2
0.79 2.31 16.7 2.77 0.35 6.97 5.95 8.77
0.38 3.64 17.9 2.22 0.49 15.6 2.69 15.1
0.41 2.82 52.8 1.76 0.32 8.10 2.11 26.8
0.40 3.46 18.0 2.00 0.41 8.53 2.13 14.0
Whole meal brown bread 0.51 4.92 40.4 3.82 0.84 31.5 3.06 25.6
0.55 4.76 28.6 3.71 0.87 32.8 2.97 38.5
1.01 4.28 22.9 3.09 0.76 26.1 3.10 38.4
Rye bread 1.16 3.81 20.9 2.56 0.48 12.2 1.93 27.2
0.41 2.68 19.0 2.37 0.51 10.8 2.53 14.2
0.47 4.39 21.2 2.48 0.61 20.3 3.08 18.4
Whole wheat bread with multi grains 1.25 4.38 68.7 2.63 0.64 21.9 1.93 48.6
0.48 5.14 20.6 2.86 0.71 20.2 3.03 19.6
1.34 4.71 51.9 2.33 0.64 21.6 2.66 38.3
Corn flour bread 1.39 2.97 17.8 3.53 0.66 7.64 3.54 19.0
0.53 2.34 13.3 2.99 0.73 4.93 2.80 22.1
Whole wheat flour bread 1.03 4.29 26.6 3.18 0.72 26.7 2.61 19.7
0.61 3.86 27.7 2.88 0.61 16.9 2.24 17.1
1.10 3.65 17.3 2.94 0.62 19.2 2.27 19.3
1.03 3.60 29.3 2.99 0.62 19.8 2.11 16.4
White and whole flour mixed 1.01 3.15 18.6 2.54 0.44 17.9 1.61 13.4
0.70 3.77 15.0 2.03 0.42 13.6 2.06 14.3
1.05 3.21 24.8 2.76 0.51 13.2 1.56 14.2
1.07 4.26 31.0 2.93 0.70 22.8 2.95 34.1
0.72 2.92 15.2 1.97 0.41 10.6 2.40 11.5
a
The precisions were not shown not to make the table complicated. The instrumental RSD values were 5–15% depending on the element.
248 N. Ozbek, S. Akman / Food Chemistry 200 (2016) 245–248
Finally, the concentrations of calcium, magnesium, potassium,
iron, copper, manganese, zinc, and phosphorous in different types
of non-branded and branded bread samples produced in Turkey
were determined and the results were given in Table 5. The solid
residue of the sample digests were determined gravimetrically
(heated to dryness and the residue was weighed) and found to
be below 3%. Therefore, as expected, no problem occurred with
respect to accumulation of deposits in the torch during the whole
analysis. The RSDs of the results (repeatability) using the same
instrument on the same day were below 10% for all the analytes.
Moreover, the reproducibility was tested by HR CS FAAS or HR CS
GFAAS depending on element using the same bread digests. The
mean results of analytes obtained by HR CS AAS were 85–115%
of those found by MIP-AES.
The analyte concentrations in bread samples were not too much
different from those found in different countries (Hussein &
Bruggeman, 1997; Khaniki, 2005; Onianwa, Adeyemo, Idowu, &
Ogabiela, 2001; Pennington et al., 1995a, 1995b, 1995c). Federal
Department of Agriculture (FDA) recommended the daily intakes
of calcium, magnesium, potassium, iron, copper, manganese, zinc,
and phosphorous as 1000 mg, 400 mg, 3500 mg, 18 mg, 2 mg,
2 mg, 15 mg and 1000 mg, respectively. The analytes daily taken
from 100 g (4 slices approximately) of bread are as follows: Ca:
40–100 mg, Mg: 40–70 mg, K: 200–300 mg, Fe: 2–5 mg, Cu:
0.3–0.5 mg, Mn: 0.7–3 mg, Zn: 1.5–8 mg, P: 200–300 mg.
Obviously, bread, even if consumed at high amounts, provides only
a small fraction of the recommended daily intakes of analytes
(1/10–1/2) except Mn by adults and children depending on the
bread type (U.S. Food and Drug Administration, 2014).
4. Conclusion
It was proved that MIP- AES is an appropriate technique for the
determination of calcium, magnesium, potassium, iron, copper,
manganese, zinc and phosphor in bread. MIP-AES is a novel instru-
ment produced for multi-element analysis. It uses only nitrogen
extracted from air by nitrogen generator. The remarkable advan-
tages of MIP-AES compared to AAS and ICP are its low running
costs and laboratory safety since expensive, toxic or flammable
gases are not required. Therefore, this technique is fast, practical,
cheap and environment friendly. Upon development of instrumen-
tal and experimental conditions, MIP-AES would be one of the
basic techniques due to its simplicity and low running costs.
Acknowledgements
We are grateful to Agilent Technologies and SEM, Agilent
Authorized Distributor in Turkey for providing MP-AES 4200 spec-
trometer. Also thanks to Nur Sebahi for her helpful work during
this study.
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