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Determination of Mineral Elements in Milk Products by

Inductively Coupled Plasma - Optical Emission Spectrometry

Article in Analytical Letters · May 2014

DOI: 10.1080/00032719.2013.878842

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Determination of Mineral Elements in

Milk Products by Inductively Coupled
Plasma-Optical Emission Spectrometry
a a a b
Naeem Khan , Ji Yeon Choi , Eun Yeong Nho , In Min Hwang ,
a c d a
Girum Habte , Murad Ali Khan , Kyung Su Park & Kyong Su Kim
a
Department of Food and Nutrition , Chosun University , Gwangju ,
Republic of Korea
b
World Institute of Kimchi , Gwangju , Republic of Korea
c
Department of Chemistry , Kohat University of Science &
Technology , Kohat , Pakistan
d
Korea Institute of Science and Technology , Seoul , Republic of
Korea
Accepted author version posted online: 24 Feb 2014.Published
online: 02 Jun 2014.

To cite this article: Naeem Khan , Ji Yeon Choi , Eun Yeong Nho , In Min Hwang , Girum Habte , Murad
Ali Khan , Kyung Su Park & Kyong Su Kim (2014) Determination of Mineral Elements in Milk Products by
Inductively Coupled Plasma-Optical Emission Spectrometry, Analytical Letters, 47:9, 1606-1613, DOI:
10.1080/00032719.2013.878842

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 Analytical Letters, 47: 1606–1613, 2014
Copyright # Taylor & Francis Group, LLC
ISSN: 0003-2719 print=1532-236X online
DOI: 10.1080/00032719.2013.878842

Atomic Spectroscopy

DETERMINATION OF MINERAL ELEMENTS IN MILK

PRODUCTS BY INDUCTIVELY COUPLED
PLASMA-OPTICAL EMISSION SPECTROMETRY
Downloaded by [Cho Sun University], [Naeem Khan] at 16:55 04 June 2014

Naeem Khan,1 Ji Yeon Choi,1 Eun Yeong Nho,1

In Min Hwang,2 Girum Habte,1 Murad Ali Khan,3
Kyung Su Park,4 and Kyong Su Kim1
1
Department of Food and Nutrition, Chosun University, Gwangju,
Republic of Korea
2
World Institute of Kimchi, Gwangju, Republic of Korea
3
Department of Chemistry, Kohat University of Science & Technology,
Kohat, Pakistan
4
Korea Institute of Science and Technology, Seoul, Republic of Korea

The determination of mineral elements in milk products is of great importance since their
excess or deficiency may affect human health. In this study, calcium, magnesium, potass-
ium, sodium, phosphorus, and iron were determined in milk products from South Korea.
The samples were wet-digested by microwave-assisted combustion using nitric acid and
hydrogen peroxide, and analyzed by inductively coupled plasma-optical emission spec-
trometry. The method was validated by sensitivity, linearity, precision, recovery checks,
and the analysis of a milk powder certified reference material. The elemental concentrations
were similar in the milk products. Calcium and potassium were the most abundant, followed
by phosphorus, sodium, magnesium, and iron. All concentrations were comparable to
previous values and were within the limits of recommended dietary allowances and tolerable
upper levels.

Keywords: ICP-OES; Milk; Mineral elements; Yogurt

INTRODUCTION
Milk products are diverse food products that contain essential elements
required for normal human metabolic processes and development (Stawarz,
Formicki, and Massányi 2007). In the recent past, manufacturers around the world
have developed a large variety of milk products. Regular monitoring is important to
evaluate and monitor the manufacturing processes (Lukáčová et al. 2012).

Received 28 October 2013; accepted 14 December 2013.

Address correspondence to Prof. Kyong Su Kim, PhD., Department of Food and Nutrition,
Chosun University, Gwangju 501-759, Republic of Korea. E-mail: kskim@chosun.ac.kr

1606
 MINERAL ELEMENTS IN MILK PRODUCTS BY ICP-OES 1607

Approximately 4% to 6% of human body weight is composed of mineral

elements which are essential in the diet. They have important roles in living systems,
regarding regulation of enzyme activities, control of normal osmotic pressure and
acid–base balance, facilitation of membrane transfer of essential nutrients, and
maintenance of nerve and muscular irritability. Any increase or deficiency of these
essential mineral elements, beyond their critical limits, may cause disturbances in
the whole physiological system (Freeland-Graves and Trotter 2003; Anderson
2004). Milk and yogurt are a good source of essential elements in human nutrition
(De la Fuente et al. 2003).
Researchers have developed technologies for determination of elements in foods
that include inductively coupled plasma optical emission spectroscopy (ICP-OES)
(Özcan and Akbulut 2007; Sola-Larrañaga and Navarro-Blasco 2009; Kira and
Downloaded by [Cho Sun University], [Naeem Khan] at 16:55 04 June 2014

Maihara, 2007; Guler 2007; Khan et al., 2013), flame atomic absorption spectrometry
(Sanchez-Sagarra et al. 2000; De la Fuente et al. 2003; Kondyli, Katsiari, and Voutsinas
2007), instrumental neutron activation analysis (Kira and Maihara, 2007; Avegliano,
Maihara, and Fernando da Silva 2011), and inductively coupled plasma-mass spec-
trometry (Khan et al. 2014). The accurate determination of elements in milk products
is largely dependent upon the critical step of sample digestion, due to the possible risks
of cross-contamination and loss of analyte (Fuente and Juarez, 1995). Traditional
digestion methods include wet digestion and dry ashing. These methods are time-
consuming and provide low efficiency. The most useful digestion method involves
minimal sample treatment to reduce the time and cross-contamination or losses (Kira
et al., 2007). Khan et al. (2013) evaluated dry ashing, wet digestion, and microwave
digestion for the determination of selenium, chromium, and molybdenum in infant
formula. The results showed that microwave digestion provided satisfactory results.
Inductively coupled plasma – optical emission spectrometry (ICP-OES) with
conventional digestion methods has been used for the determination of essential ele-
ments in food. Previous studies have determined several elements in milk from Brazil
(Avegliano et al. 2011) and yogurt from Spain (Sanchez-Sagarra et al. 2000) and
Turkey (Guler 2007), but a comprehensive study on milk products has not yet been
performed. Yogurt with added fruit is complex and requires a defined methodology
for elemental analysis. Therefore, in continuation of our previous work involving
minor and trace elements (Khan et al. 2014), this study was designed to determine
sodium, potassium, calcium, phosphorus, magnesium, and iron in milk and yogurt
by ICP-OES. Microwave digestion was employed to eliminate the inaccuracy of
conventional sample preparation methods and decrease the analysis time. This
method was evaluated for the determination of essential elements in milk and yogurt.
The results were compared to critical levels specified by Food and Nutrition Board
for recommended dietary allowances and tolerable upper levels to evaluate the
nutritional contribution of the milk products to consumers.

EXPERIMENTAL
Instrumentation
A Multi-wave 3000 microwave combustion system (Anton Paar GmbH, Graz,
Austria) was used to digest the samples. This instrument was programmable for
 1608 N. KHAN ET AL.

time and power between 600 and 1500 W and equipped with 16 high pressure poly-
tetrafluoroethylene vessels (MF 100) as digestion tubes. Each vessel had an internal vol-
ume of 100 mL, capable of a maximum pressure of 115 bar and a temperature of 310
C.
The inductively coupled plasma-optical emission spectrometer (ICP-OES)
was a Varian 730-ES simultaneous CCD (Wyndmoor, PA, USA) with a SeaSpray
concentric nebulizer (Glass Expansion, Pocasset, MA) and a cyclonic spray
chamber. After scanning a blank, a standard solution, and a sample solution, in the
programmed wavelength range, background correction wavelengths were selected
manually at appropriate positions for each analyte. Instrument configuration and
general experimental conditions are summarized in Table 1.
Downloaded by [Cho Sun University], [Naeem Khan] at 16:55 04 June 2014

Reagents
All reagents used were of analytical grade (Merck, Darmstadt, Germany) unless
otherwise stated. A multi-element (1000 mg=L) and phosphorus stock solutions
(1000 mg=L) (AnApure Kriat Co, Ltd. Daejeon, Korea) were used for calibration
curves and recovery experiments. Concentrated nitric acid and hydrogen peroxide
(Dong Woo Fine-Chem, Iksan, Korea) were used for digestion. The non-fat milk pow-
der certified reference material (NIST-1549) was purchased from the National Institute
of Standards and Technology (Gaithersburg MD, USA). Ultrapure deionized water
(18.2 MX cm) was obtained from a Milli-Q Plus water purification system (Millipore,
Bedford, MA, USA). All containers were soaked in 10% v=v HNO3 overnight, and
then thoroughly rinsed with deionized water and dried before use. The reagents,
digestion vessels, glassware, and plasticware were carefully checked to avoid cross-
contamination.

Sample Preparation and Calibration

Two hundred and seven samples of milk products (milk and yogurt) commer-
cially available to consumers were collected from South Korean supermarkets. These
included 73 milk samples (37 plain milk, 36 skim milk), 63 plain yogurt (31 drinking,
32 paste), and 71 yogurt with added fruit (36 paste, 35 drinking). The samples were
purchased in triplicate from June to September, 2013. The samples were stored in
a refrigerator until analysis, which was performed before the expiration date.
For microwave digestion, 0.5 to 1.0 g were accurately weighed directly into the
digestion vessels. 7 mL of concentrated nitric acid and 1.0 mL of hydrogen peroxide

Table 1. ICP-OES conditions for determination of elements in milk products

Radiofrequency generator frequency, MHz 27.12

Radiofrequency power, kW 1.3
Spray chamber Cyclonic
Plasma viewing mode Axial
Processing mode Area
Argon gas flow, L=min Plasma (16), Auxiliary (1.5), Nebulizer (0.94)
Read delay, s 30
Rinse, s 30
Metal (wavelength, nm) Ca (315.880), Mg (279.071), Na (588.983), K (766.455),
P (213.617), Fe (259.933)
 MINERAL ELEMENTS IN MILK PRODUCTS BY ICP-OES 1609

were added. The digestion was performed by 1000 W at 80
C for 5 min, 1000 W at
50
C for 5 min, and 1000 W at 190
C for 20 min. After cooling for 30 minutes, the
contents were transferred to 50 mL volumetric tubes (Corning NY, Mexico) and
diluted to 25.0 g for analysis. Blank solutions were prepared by the procedure above
without sample (Khan et al. 2013).
Quantitative analyses were performed by an external calibration technique.
Standard calibration solutions of all analytes were prepared from stock solutions
in 19.6% (w=w) nitric acid. Calibration curves were prepared from six standards so
that concentrations in the samples were within the linear range. To monitor instru-
mental drift, standards were analyzed as samples on regular basis. Blank solutions
were also analyzed to monitor cross-contamination and losses (Khan et al. 2013).
Downloaded by [Cho Sun University], [Naeem Khan] at 16:55 04 June 2014

Quality Assurance
Quality parameters were measured for the determination of calcium,
magnesium, potassium, sodium, phosphorus, and iron in milk products. Correlation
coefficients (R2) were determined from the calibration curves to evaluate linearity
(Pacquette et al. 2012; Khan et al. 2013). To evaluate the sensitivity of the analytical
method, limits of detection (LODs) and limits of quantification (LOQs) were
determined as three and ten times of the average standard deviation of ten determi-
nations of blank divided by the slope of the standard curves, respectively (Thompson
et al., 2002). Precision was evaluated through determination of the coefficient of
variation (CV) by measuring the relative standard deviation of 10 replicates of one
sample (Chudzinska and Baralkiewicz 2011). For accuracy, the analytes were
determined in the CRM. Quality control was verified by the recovery experiments
for the six selected elements. Spiking was performed 500 and 1000 mg=kg and
the recoveries were measured (Khan et al. 2014).

Statistical Analysis
The concentrations of the elements are reported as the mean  standard
deviation of triplicate measurements. One way analysis of variance (ANOVA) was
used to determine significant differences (P < 0.05) within means following Tukey’s
honestly significant difference (HSD) test and Duncan test, in the Statistical Package
for Social Sciences (SPSS) Software, Version 20 (IBM, New York, USA). Analytical
measurements that differed significantly (P < 0.05) among the samples are designa-
ted by different letters as superscripts.

RESULTS AND DISCUSSION

Table 2 lists analytical method validation parameters including linearity,
sensitivity, precision, and recoveries. Table 3 reports the accuracy of the method
for the non-fat milk powder certified reference material. Table 4 lists the concen-
trations of the elements in milk and yogurt. Table 5 is a comparison of the results
of this study with literature values and standards recommended by the Food and
Nutrition Board.
 1610 N. KHAN ET AL.

Table 2. Analytical method validation

Correlation Limit of detection Limit of quantification Relative precision Recovery

Element coefficient (R2) (mg=kg) (mg=kg) (%) (%)

Ca 0.9996 0.009 0.029 1.95 104.5a

Mg 0.9997 0.003 0.010 1.64 98.5
Na 0.9994 0.004 0.012 2.75 97.4
K 0.9995 0.002 0.005 1.77 98.3a
P 0.9998 0.021 0.066 2.98 96.8a
Fe 0.9996 0.022 0.075 3.10 96.5

a
Elements spiked at 1000 mg=kg, others were spiked at 500 mg=kg.
Downloaded by [Cho Sun University], [Naeem Khan] at 16:55 04 June 2014

Validation of the Analytical Method

Quality parameters, including detection limits, linearity, precision, accuracy,
and recovery, demonstrated that this method fulfilled the criteria of the Association
of Official Analytical Chemists (AOAC) (AOAC 2012; Khan et al. 2013). The LOD
values were between 0.002 and 0.022 (mg=kg) and the LOQ values were 0.005–0.075
(mg=kg) (Table 2), confirming the sensitivity. Correlation coefficients (R2) were
at least 0.9994 (Table 2), showing good linearity for the metals. The coefficient of
variation (CV) values showed the precision was between 1.64% and 3.1%. These
values are in accordance with the criteria specified by the AOAC (AOAC 2012).
The recovery values were between 97.4% (sodium) and 104.5% (calcium). The analy-
sis of the CRM gave recoveries in the range of 92.3% (sodium) to 108.4% (iron).
These results were within the confidence interval (P < 0.05) for the certified values.
Based on these results for the quality parameters, the analytical method was in
accordance to the specified criteria of AOAC (AOAC, 2012; Khan et al., 2014), and
provided rapid determination of six elements in the dairy products.

Analysis of Milk and Yogurt

The concentrations of the elements were similar in the milk and yogurt. The
minor and trace elements have been reported by Khan et al. (2014). The concentra-
tions of calcium (957.0–1304 mg=kg) and potassium (1044–1288 mg=kg) were the
highest, followed by phosphorus (448.4–824.4 mg=kg), sodium (219.1–345.1 mg=
kg), magnesium (90.90–136.9 mg=kg), and iron (10.88–40.18 mg=kg) (Table 4).
A similar trend has been reported in previous studies (Sánchez-Segarra et al. 2000;
Sola-Larrañaga and Navarro-Blasco 2009; Avegliano et al. 2011).

Table 3. Analysis of non-fat milk powder certified reference material (NIST-1549)

Element Certified value (mg=kg) Observed value (mg=kg) Recovery (%)

Ca 13000  500 12608  416 97.0

Mg 1200  30 1151  31 95.9
Na 4970  100 4956  274 92.3
K 16900  300 16344  256 96.7
P 10600  200 9820  132 92.6
Fe 1.78  0.1 1.93  0.001 108.4
 MINERAL ELEMENTS IN MILK PRODUCTS BY ICP-OES 1611

Table 4. Concentrations (mg=kg) of mineral elements in milk products by ICP-OES

Milk Plain yogurt Yogurt with added fruit

Plain Skim Drinking Paste yogurt Paste yogurt Drinking

Element (n ¼ 37) (n ¼ 36) yogurt (n ¼ 31) (n ¼ 32) (n ¼ 36) yogurt (n ¼ 35)

Ca 1085ab  0.29 1184bc  0.26 957.0a  0.30 1259a  0.27 1304c  0.26 971.4a  0.31
Mg 104.8abc  0.03 115.2bcd  0.04 90.90a  0.03 128.7cd  0.04 136.9d  0.03 92.66ab  0.03
Na 256.7ab  0.07 219.1a  0.05 273.9ab  0.05 322.6bc  0.05 345.1c  0.03 295.7bc  0.08
K 1195bc  0.30 1115ab  0.25 1094ab  0.26 1202bc  0.23 1288c  0.23 1044a  0.26
P 824.4d  0.56 674.4c  0.56 448.4a  0.24 555.6b  0.47 568.2b  0.31 508.2ab  0.32
Fe 26.99b  0.08 12.12a  0.01 10.88a  0.01 12.83a  0.01 14.36ab  0.01 40.18c  0.02
Downloaded by [Cho Sun University], [Naeem Khan] at 16:55 04 June 2014

Note: Values with different letters within a row differ significantly (P < 0.05). Each sample was analyzed
in triplicate.

By comparison with the published literature, the concentrations of calcium,

magnesium, potassium, and sodium in the present study were approximately the
same as reported for yogurt with added fruit and milk from Spain (Sachez-Sagarra
et al. 2000; De la Fuente et al. 2003; Sola-Larrañaga 2009), and goat milk in Turkey
(Garcı́a et.al. 2006). However, calcium, phosphorus, magnesium, and sodium were
lower and potassium was higher compared to dairy products from Turkey (Guler
2007). Compared to pasteurized milk from Brazil, the calcium concentrations were
the same, potassium was lower, while sodium and iron were higher (Avegliano
et al. 2011). On the other hand, iron was higher in the present study than reported
for yogurt with added fruit and milk from Spain (Sanchez-Sagarra et al. 2000), goat
milk from Turkey (Guler 2007), and milk and dairy products from Turkey (Garcı́a
et al. 2006) (Table 5). Thus, the results of the present study were comparable to
literature values.

Table 5. Comparison of mineral elements concentrations (mg=kg) in present study with previous studies
and recommended levels by the Food and Nutrition Board

Reported studies Recommended levels

a b c d e f
Element Present study 1 2 3 4 5 6 RDIg TUIh

Ca 957.0–1304 980 1200 970 1340 1342–1455 1166 1000–1200 2500

Mg 90.90–136.9 98 140 91.8 120 510–587 – 330–350 350
Na 219.1–345.1 370 – 372 510 433–520 445 1300–1500 2300
K 1044–1288 1190 – 1344 1240 409–511 1465 2000–2300 4700
P 448.4–824.4 – 1150 785 – 823–1052 – 580–700 4000
Fe 10.8840.18 1.2 – 0.29 0.70 3.88–7.62 0.4 6-8 45

a
Sachez-Sagarra et al. 2000.
b
De la Fuente et al. 2003.
c
Sola-Larrañaga and I. Navarro-Blasco 2009.
d
Garcı́a et al. 2006.
e
Guler 2007.
f
Avegliano et al. 2011.
g
Recommended dietary. allowance.
h
Tolerable upper level.
 1612 N. KHAN ET AL.

Calcium is known to be anti-carcinogenic (Shin et al. 2002), and magnesium is

required for insulin activity and is therefore important for prevention of type 2 dia-
betes (Huerta et al. 2005). Sodium is needed for acid-base balance (Anderson 2004).
Iron is an essential component of hemoglobin and is involved in several oxidation
reduction reactions (Achanta et al. 2007), while phosphorus is essential for bone
development (Heaney 2009). The Food and Nutrition Board, Institute of Medicine,
National Academies, Washington, DC, USA, has recommended critical values for
these elements called recommended dietary allowances (RDA) and tolerable upper
levels (TUL) (Table 5) (FNB 2005). Looking to these critical levels, the results
obtained in the present study were within the specified limits, demonstrating that
a positive contribution of the mineral elements to the diet of consumers is provided
by milk and yogurt. In addition, all the concentrations were below the tolerable
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upper levels. In summary, the reported method provides a basis for confirmation
that the elemental concentrations of milk and yogurt are suitable for human
consumption.

CONCLUSIONS
Microwave assisted digestion with ICP-OES determination of sodium, potass-
ium, calcium, magnesium, phosphorus, and iron was shown to be suitable for the
analysis of milk products. This method may be applied to ensure that the mineral
concentrations are within recommended values. The concentrations in the milk pro-
ducts were Ca ’ K>Na>P>Mg>Fe. These results are comparable to previous
reports and within critical limits. These results confirmed the nutritional contri-
bution of milk products to the human diet and demonstrated that all values were
below tolerable upper limits.

FUNDING
The authors greatly acknowledge the financial support through research funds
for this study from Chosun University, Gwangju, Republic of Korea.

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