J. Dairy Sci.
98:5040–5051
http://dx.doi.org/10.3168/jds.2014-9055
© American Dairy Science Association®, 2015.
Evaluation of X-ray fluorescence spectroscopy as a method
for the rapid and direct determination of sodium in cheese
J. A. Stankey, C. Akbulut, J. E. Romero, and S. Govindasamy-Lucey1
Wisconsin Center for Dairy Research, University of Wisconsin, Madison 53706
ABSTRACT
Cheese manufacturers indirectly determine Na in
cheese by analysis of Cl using the Volhard method,
assuming that all Cl came from NaCl. This method
overestimates the actual Na content in cheeses when
Na replacers (e.g., KCl) are used. A direct and rapid
method for Na detection is needed. X-ray fluorescence
spectroscopy (XRF), a mineral analysis technique used
in the mining industry, was investigated as an alter-
native method of Na detection in cheese. An XRF
method for the detection of Na in cheese was devel-
oped and compared with inductively coupled plasma
optical emission spectroscopy (ICP-OES; the reference
method for Na in cheese) and Cl analyzer. Sodium
quantification was performed by multi-point calibra-
tion with cheese standards spiked with NaCl ranging
from 0 to 4% Na (wt/wt). The Na concentration of
each of the cheese standards (discs: 30 mm × 7 mm)
was quantified by the 3 methods. A single laboratory
method validation was performed; linearity, precision,
limit of detection, and limit of quantification were de-
termined. An additional calibration graph was created
using cheese standards made from natural or process
cheeses manufactured with different ratios of Na:K.
Both Na and K calibration curves were linear for the
cheese standards. Sodium was quantified in a variety of
commercial cheese samples. The Na data obtained by
XRF were in agreement with those from ICP-OES and
Cl analyzer for most commercial natural cheeses. The
XRF method did not accurately determine Na concen-
tration for several process cheese samples, compared
with ICP-OES, likely due to the use of unknown types
of Na-based emulsifying salts (ES). When a calibration
curve was created for process cheese with the specific
types of ES used for this cheese, Na content was suc-
cessfully predicted in the samples. For natural cheeses,
the limit of detection and limit of quantification for
Na that can be determined with an acceptable level of
repeatability, precision, and trueness was 82 and 246
Received November 2, 2014.
Accepted April 13, 2015.
1
Corresponding author: rani@cdr.wisc.edu
5040
mg/100 g of cheese, respectively. Calibration graphs
should be created with standards that reflect the con-
centration range, ratio, and salt type present in the
cheeses. This XRF method can be successfully used for
the rapid and direct measurement of Na content in a
wide variety of natural cheeses. Commercial process
cheese manufacturers use proprietary blends of ES. We
did find that the XRF technique worked for process
cheese when the calibration graphs were created with
the specific types of ES actually used.
Key words: sodium, X-ray fluorescence spectroscopy,
cheese
INTRODUCTION
The Na content of cheese is typically determined cou-
lometrically using a Cl analyzer (Johnson and Olson,
1985), which is rapid and easy to operate. However,
this method indirectly determines Na by measuring the
amount of Cl ions present in the cheese. This is a major
problem when determining the Na content of cheeses
containing potassium chloride (KCl), which is often
used as a Na replacer. Additionally, the Cl analyzer is
not used for process cheeses, which are manufactured
with various Na-based emulsifying salts (ES), such as
sodium citrate or sodium phosphate. Thus, when Na
replacers are used in cheese, Na must be determined via
an alternative method. In these cases, it is preferable to
directly measure the Na content.
Direct measurement of Na content in foods is com-
monly done using atomic absorption spectroscopy or
inductively coupled plasma optical emission spectros-
copy (ICP-OES). The ICP-OES and atomic absorp-
tion spectroscopy techniques require costly ultra-pure
reagents, are laborious, and require complex sample
preparation (e.g., ashing, digestion; Dolan and Ca-
par, 2002; Ehling et al., 2010). Thus, a more practi-
cal chemical method is needed that is accurate (direct
measurement of Na), quicker, and cheaper for use
in routine quality control, which would allow cheese
plants to easily monitor changes in salt content during
the manufacturing process.
The X-ray fluorescence analysis (XRF) method has
previously been used to determine the concentration
 DETERMINATION OF SODIUM IN CHEESE
of elements in a diverse number of applications includ-
ing metal, cement, oil, polymer, plastics, mining, and
minerals (Herbert and Street, 1974; Potts et al., 1984;
Brouwer, 2003; West et al., 2010). X-ray fluorescence is
also a rapid, precise, nondestructive, and a potential al-
ternative method for direct Na determination in foods
(Jastrzebska et al., 2003; Pashkova, 2009; Rinaldoni
et al., 2009; Smagunova and Pashkova, 2013). Several
studies have used XRF for the measurement of individ-
ual elements in milk and dairy products such as infant
formula powders (Chan and Palmer, 2013; Fernandes
et al., 2014). These XRF spectrometers have 2 basic
components: the excitation source and the detection
system. First, the sample is irradiated with X-rays and
fluorescent X-rays are excited in the sample (Brouwer,
2003). These secondary X-rays are then measured for
quantitative analysis of its elemental composition in
the detection system (Brouwer, 2003). When an atom
is hit with the incoming photon, an electron from the
innermost orbital is expelled, which causes the atom
to be in an excited, but unstable, state. To regain its
stability, the atom must fill this gap by transferring
an electron from the higher-energy outer orbital to the
lower-energy inner orbital, which emits excess energy
as an X-ray photon (Brouwer, 2003). Each element has
discrete energy levels (e.g., dependent on the energy of
the electron) so that the emitted radiation is charac-
teristic for the particular element (e.g., “fingerprint”).
A spectrum is created with distinct peaks for each ele-
ment present in the sample, and the peak areas deter-
mine intensity; calibration is needed to relate intensity
to actual concentrations. Sodium is the lightest element
capable of being detected using energy dispersive XRF
(Brouwer, 2003).
X-ray fluorescence analysis was used for analysis of
various elements in dairy foods; determination of P in
melted and cottage cheeses (Jastrzebska et al., 2003),
trace elements (i.e., Ba, Cu, Cr, Ni, and V) in Bryndza
sheep cheese (Suhaj and Korenovska, 2008), and Na
concentrations in several types of milks, dairy products,
and infant formulas (Pashkova, 2009). These studies
prepared cheese samples either by oven-drying, grind-
ing, and pressing into pellets (Suhaj and Korenovska,
2008; Pashkova, 2009) or by microwave digestion of
cheese (Jastrzebska et al., 2003) before XRF analysis.
Some studies have successfully used XRF for the mea-
surement of the mineral content of some dairy samples
without any pretreatment; for example, Rinaldoni et
al. (2009) successfully used XRF for the determina-
tion of Ca, K, P, Fe, and Zn in undiluted skim milks,
ultrafiltered milks, and yogurts diluted with distilled
water. This suggests that the XRF technique might be
capable of Na analysis in cheeses without any pretreat-
ment (e.g., no drying, ashing, or wet digestion).
5041
The objective of this study was to develop, and
validate, a method for the rapid, precise, and direct
determination of Na in natural and process cheeses us-
ing XRF technology. Calibrations were created using
a variety of cheese matrices containing different Na
levels. These calibration cheese matrices were created
by experimentally manufacturing a variety of unsalted
cheeses (i.e., Cheddar, Mozzarella, Gouda, pizza, and
nonfat) and spiking them with increasing levels of Na.
The XRF equipment parameters, current, voltage, and
runtime, were studied to improve the intensity of the Na
measurements. Different sample preparation methods
(shredded versus sliced, as well as the dimensions of the
slices) were also evaluated. The method was validated
in terms of linearity, repeatability, reproducibility, and
trueness. The reference method, ICP-OES (Poitevin et
al., 2009), was used to validate the XRF technology; the
Cl analyzer method was used for comparison purposes.
MATERIALS AND METHODS
XRF Equipment
The Na contents of experimental cheese standards
and commercial cheese samples were measured on an
XRF instrument (X-Supreme8000, Oxford Instruments,
Oxford, UK) and operating software (X-Supreme,
version 1.99 build 45). The detector was purged with
high-purity He gas (Airgas, Madison, WI). Preliminary
experiments were carried out to determine XRF oper-
ating parameters, such as, voltage, current, and run-
time (which influence peak intensity). The equipment
was run at the following voltage and current levels: 4
kV at 750 μA for 180 s. Background corrections were
applied for Na, K, and Cl. The software calculates the
background corrections using the Lucas-Tooth and
Price intensity-based X-ray correction model. The
Lucas-Tooth and Price algorithm assumes that chemi-
cal variations within the sample (i.e., matrix effects)
proportionally influence the fluorescent intensity of the
element of interest (e.g., Na; Potts et al., 1984). The
correction model uses terms that correct for absorp-
tion and enhancement effects from other elements (e.g.,
elements of interest are excited by incoming radiation
but can also be excited by the fluorescence of other
elements; Brouwer, 2003).
The analysis volume of the X-Supreme8000 instru-
ment was ~174 mm3, which was approximately an area
of 62 mm2 by height of 0.7 mm. Thus, samples needed
to be ≥0.7 mm thick and have a diameter of ≥8.8
mm. The samples were introduced into plastic cups
(dimensions: 41 mm diameter × 28 mm height; Plastic
Secondary Safety Window, Oxford Instruments). The
sample cup consisted of 3 parts: film (4-μm-thick Film
Journal of Dairy Science Vol. 98 No. 8, 2015
5042 STANKEY ET AL.
Poly-4, Oxford Instruments), inner cup, and outer cup.
The plastic cups were reusable and the film was dis-
posable. The outer cup was placed in a platform ring
and film was laid across the platform. The inner cup
was pushed down into position so that the film was a
taut surface for the sample. The sample cup containing
the prepared sample was placed in the XRF chamber,
which has a 10-sample autosampler. After the chamber
door was closed, the autosampler moved the sample to
the analysis position where the sample was continually
rotated while being irradiated. The continuous rotation
of the sample helps to eliminate any nonhomogeneity
effects from the surface of the sample (Brouwer, 2003).
The accuracy of the XRF technique greatly depends
on the uniformity and amount of the material the X-
rays have to pass through. In this study, several sample
properties, such as the format, thickness, and cheese
composition, were evaluated to determine their influ-
ence on Na measurements. This particular piece of
equipment is simple to operate, provides rapid (live)
results, and is robust for potential quality analysis in a
manufacturing plant type of setting.
Preparation of Cheese Samples and Standards
for XRF Calibration
Cheese standards, containing a wide range of Na
concentrations, were prepared using natural cheeses
(Cheddar, Gouda, pizza, Mozzarella, and nonfat), which
were made using standard manufacturing protocols by
licensed Wisconsin cheesemakers at the University of
Wisconsin–Madison dairy processing plant. Because Na
naturally occurs in milk, it was not possible to create a
blank Na cheese matrix. Therefore, the performance of
the method was evaluated using the standard addition
method. The Na quantification was performed utilizing
a multi-point calibration with unsalted cheeses. Freshly
pressed blocks of cheese (≤1 d old) were obtained after
milling, before dry salting. The cheese was finely shred-
ded in a food processor (Cuisinart Inc., Greenwich,
CT). Different amounts of NaCl (analytical grade,
Fisher Scientific, Fair Lawn, NJ) were stirred into the
shredded cheese with a spoon for 1 min (e.g., direct
salting) to prepare cheese standards with different salt
contents (i.e., 0 to 4.0 g of NaCl/100 g of cheese). The
salted cheeses were then pressed in tri-cornered poly-
propylene beakers (250 mL, Fisher Scientific) using a
hydraulic press (670 kPa for 1 h at 22°C). The pressed
cheese blocks were sealed in resealable plastic bags and
stored for 1 wk at 4°C to allow for Na equilibration.
Using this dry-salting method, a maximum amount
of salt (i.e., 4.0 g/100 g of cheese) could be added.
Above this level, cheese texture became crumbly and
difficult to work with. The XRF readings for these
Journal of Dairy Science Vol. 98 No. 8, 2015
high-salt (i.e., ≥4.0 g/100 g of cheese) standards had
much larger standard deviations due to the crumbly
nature of the cheese sample. To address this issue (i.e.,
to have a calibration with >6% Na), several process
cheese standards were made. Laboratory-scale process
cheeses were prepared using the method described by
Brickley et al. (2008). These high-salt cheese standards
were made by adding different amounts of a single type
of Na-based ES, trisodium citrate (analytical grade,
Fisher Scientific), to unsalted pizza cheese base (i.e.,
1.0, 2.0, 3.0, 5.0, and 7.0 g of trisodium citrate/100 g of
cheese). The molten cheeses were then poured into tri-
cornered polypropylene beakers, covered with plastic
wrap, and sealed in plastic bags that were stored for 1
wk at 4°C to allow for Na equilibration.
Cheese Standards with Varying Na and K
Concentrations. A separate calibration plot for vari-
ous concentrations of K was created by replacing some
NaCl with KCl (i.e., 460, 510, 560, 620, 660, 680, and
750 mg of Na/100 g of cheese and 340, 310, 250, 230,
200, 140, and 105 mg of K/100 g of cheese, respectively)
in an unsalted Cheddar cheese sample. This calibration
plot was validated with a Cheddar cheese that was
manufactured in the University of Wisconsin-Madison
dairy plant, containing K-based salt replacers.
Process Cheese Standards with K-Based
Emulsifying Salts. A regular-salt Cheddar cheese
(1.8% salt) was manufactured at the University of
Wisconsin-Madison dairy plant and then used as the
cheese base for the creation of various process cheese
standards. Two ES, dipotassium phosphate (DPP;
BK Giulini, Ludwigshafen, Germany) and disodium
phosphate (DSP; BK Giulini), were blended in vary-
ing concentrations up to 2.0% (wt/wt) DSP and up to
1.75% (wt/wt) DPP. The process cheese standards (n
= 8) were prepared using the small-scale process cheese
method described by Brickley et al. (2008). The cheeses
were sealed in plastic bags and stored at 4°C for 1 wk.
These cheeses were sampled and analyzed by ICP-OES
to quantify the actual amount of Na and K in order to
create a calibration for measuring Na in the presence of
K for commercial process cheeses (n = 8).
Samples and Sample Preparation. To optimize
the intensity of the Na peak in the samples, cheeses
were introduced into the XRF equipment in different
formats, as follows:
(a) Discs (30 mm): cheese discs (30 mm diameter
× 7 mm height) were prepared from the pressed
block or commercial samples using a Hobart deli
slicer (model 410, Hobart Manufacturing Co.,
Troy, OH) and a cork borer. The XRF equip-
ment requires that samples have a diameter
between 18.5 and 40 mm (the diameter of the
 DETERMINATION OF SODIUM IN CHEESE
sample cup). We decided to cut the cheeses to a
diameter of 30 mm. For process cheese singles,
individual cheese slices were stacked so that
they had a height of approximately 7 mm. Five
30-mm-diameter samples were taken from each
stack. Process cheese food was cooled in a freezer
at −20°C for 10 min before sampling to firm up
the cheese and allow for easier and more uniform
slicing. The cheese discs were stored in resealable
plastic bags at 4°C until analysis.
(b) Shredded cheese: cheese samples were shredded
using a food processor (model DLC-2011CHB,
Cuisinart Inc.) for 10 to 15 s to produce uniform
cheese particles. The shredded cheese (5 or 10
g) samples (n = 5) were added to the prepared
sample cups and measured by the XRF. This ex-
periment was repeated but the shredded cheese
(5 or 10 g) samples were pressed to remove any
air gaps using a nylon plug (54-ZX1223, Oxford
Instruments), which fit tightly inside the inner
cup.
In addition to the cheese standards, various com-
mercial samples of natural (n = 7) and process (n = 3)
cheese were purchased locally and sampled in disc form
(30 mm diameter × 7 mm height).
XRF Method Validation
Single laboratory validation (SLV) was carried out
because our laboratory was the only laboratory with
access to this XRF instrument; the SLV procedure was
performed according to AOAC guidelines (AOAC In-
ternational, 2012). Measurements were carried out us-
ing blind duplicates of commercially purchased natural
cheeses (n = 13) with varying chemical composition
and Na contents. Blocks of cheese were divided in half,
repackaged, labeled with random 2- or 3-digit numbers,
and used to prepare the samples (slicing and sampling)
for XRF analysis. At least 12 samples were taken from
each cheese, sealed in a resealable plastic bag, and
stored at 4°C until analysis. Duplicate samples from
each bag were measured on the XRF equipment using
the same calibration method (calibration range was
from 250 to 1,600 mg of Na/100 g of cheese). Measure-
ments taken with the XRF equipment were carried out
4 times (instrument sessions) spanning 2 wk. Inter- and
intraday parameters were referred to as repeatability (r)
and within-laboratory reproducibility (R), respectively
(Horwitz and Albert, 2006). Parameters including r, R,
relative standard deviation (RSDr and RSDR), rela-
tive repeatability (rrel), and relative within-laboratory
reproducibility (Rrel) values were calculated according
to standard protocols (IDF, 1991). The Horitz ratio
5043
(HorRatR), which indicates reliability of these values
(Horwitz and Albert, 2006), was determined using the
equation
HorRatR = RSDR ÷ PRSDR = RSDR ÷ 2C−0.15, [1]
where PRSDR is the predicted relative within-labo-
ratory standard deviation (Masotti et al., 2012). The
PRSDR is equivalent to 2C−0.15, where C is the mass
fraction of the Na concentration. The HorRatR is a
unitless value used to express the acceptability of a
chemical method of analysis with respect to precision
(Horwitz and Albert, 2006). The expected range for a
HorRatR value in a SLV study is between 0.3 and 1.5,
ideally <1.0 (Poitevin et al., 2009).
The XRF software reports raw data as intensity
counts per second (cps; the area under the curve) ver-
sus energy (keV) and converts intensity to Na concen-
tration using the Lucas–Tooth and Price matrix correc-
tion algorithm. The limit of detection (LOD) and the
limit of quantification (LOQ), expressed as milligrams
per 100 g of cheese, were estimated using the standard
deviation (SD) from the analysis of at least 4 reagent
blanks (unsalted or unbrined cheese standards made
without any additional salt), run on a single day, and
measured over 3 separate days (instrument sessions).
The SD for intensity of the background noise for these
blanks was used to calculate the LOD and LOQ. Spe-
cifically, LOD was calculated as 3 × SD and the LOQ
was calculated as 3 × LOD.
ICP-OES Analysis
Analysis of Na and K was done using the standard
AOAC method 984.27 (Poitevin et al., 2009) for ICP-
OES (Vista-MPX Simultaneous ICP-OES, Varian Inc.,
Palo Alto, CA). Readings were taken at 589.6 and 769.9
nm for Na and K, respectively (Mozafar et al., 1990;
Govindasamy-Lucey et al., 2007). The same samples
used for XRF analysis were saved for ICP-OES test-
ing. The values obtained from ICP-OES analysis for
Na and K were used to create the calibration curves in
the XRF software.
Chloride Analyzer
Sodium contents of all cheese standards and commer-
cial samples were also measured indirectly by potentio-
metric Cl titration method (M926 Chloride Analyzer,
Nelson & Jameson Inc., Marshfield, WI; ISO, 2006;
Johnson and Olson, 1985). These values were used for
comparison with the results from the XRF analysis
method.
Journal of Dairy Science Vol. 98 No. 8, 2015
5044 STANKEY ET AL.

Statistical Analysis

Unless otherwise specified, 3 replicates were per-

formed for each sample. An ANOVA was carried out
with PROC GLM in SAS (version 9.3; SAS Institute
Inc., Cary, NC). Scheffé’s multiple-comparison test was
carried out to evaluate differences in the treatment
means at a significance level of P < 0.05.

RESULTS AND DISCUSSION

XRF Operating Parameters: Effect of Current,

Voltage, and Runtime on Na Peak Intensity

Preliminary work was carried out to enhance the Na

peak intensity by changing the current, voltage, and
runtime. Figure 1a shows an XRF spectrum scan ob-
tained for a commercial sample of low-moisture, part-
skim (LMPS) Mozzarella. Sodium, being one of the
lightest elements, was detected first (at photon energy
~1.6 × 10−19 kJ) and the intensity was also low (~5
cps), compared with the other elements. A portion of
Figure 1a was enhanced to show the region of interest
(ROI), where the Na peak was detected (Figure 1b).
The ROI for the Na peak was between 1.53 and 1.76 ×
10−19 kJ. Further experiments were carried out in which
the current, voltage, and runtime were changed to try
to increase the peak area and overall Na intensity at
the ROI (Figure 2). The area under the curve increased
with increasing voltage (Figure 2a) and decreased with
increasing current (Figure 2b). The runtime was in-
creased from 90 to 180 s, which increased the overall
number of readings (cps) taken, which improved the
signal to noise ratio (data not shown). Subsequent mea- Figure 1. An example of (a) a typical X-ray fluorescence spec-
troscopy (XRF) spectrum scan showing all the different elements for
surements for the calibrations of Na in cheeses, and for a commercial low-moisture, part-skim Mozzarella cheese (2% Na, wt/
the SLV procedure, were carried out using the following wt) sample, and (b) an enlarged portion of the spectrum scan to show
conditions: current of 750 μA and a voltage of 4 kV for the Na peak. Test was run at 5 kV at 600 μA for 90 s on a cheese
disc (30 mm diameter × 7 mm height) at ambient temperature. cps =
a 180-s runtime. counts per second.
The XRF equipment used for these experiments
had an autosampler feature (but without an internal
temperature controller), which allowed 10 samples to Smagunova and Pashkova (2013) found discoloration
be loaded and then analyzed consecutively. Cheese in both skim and whole milk powder-pellets and oil-
samples exhibited oiling off and possible moisture ing off in whole milk powder-pellets after successive
loss after 10 consecutive runs, which suggests that measurements and long measurement times (>20 min
the samples were being heated within the instrument per sample; sample chamber temperature was ~38°C).
chamber (initial cheese temperature: 5°C; cheese tem- Smagunova and Pashkova (2013) reported that the
perature after 10th measurement of the same sample: melting of fat contributes to scattering (enhancement
~35°C). A single full-fat Cheddar cheese sample was of the XRF intensity) effects when measuring elements
measured 10 consecutive times (run time: 90 s; total like Na due to primary and secondary radiation. Oiling
residence time: 15 min) to determine SD between the off was not observed when the experiment was repeated
repeated intensity readings. A layer of oil (oiling off) with a reduced-fat Cheddar cheese sample, and for this
was observed on the sample film after the final reading cheese type Na intensity was not affected by 10 con-
and Na intensity decreased with increasing residence secutive runs (P > 0.05). To prevent excessive oiling
time in the autosampler (results not shown). Similarly, off and to ensure consistency between samples, use of

Journal of Dairy Science Vol. 98 No. 8, 2015

 DETERMINATION OF SODIUM IN CHEESE
Figure 2. Changes in the intensities and peak areas of the Na peak
(region of interest: 1.53 to 1.76 10−19 kJ) with (a) changing voltage lev-
els (4 to 9 kV) at constant current (300 μA) and (b) changing current
(400 to 600 μA) and voltage levels (5 to 7 kV). Both experiments ran
for 90 s using a commercial low-moisture, part-skim Mozzarella cheese
(2% Na, wt/wt) disc (30 mm diameter × 7 mm height) at ambient
temperature. cps = counts per second.
the autosampler was discontinued and only one sample
was analyzed at a time (run time: 180 s; total residence
time: 4 min; cheese temperature after analysis: ~10°C).
Effect of Cheese Form, Thickness, and Composition
on Na Peak Intensity
The fluorescence emitted by a material and its sub-
sequent detection is greatly dependent on the sample
density, thickness, and composition (Szczerbowska-
Boruchowska, 2012). We explored the effect of different
formats and dimensions of samples.
Cheese Forms. A low-fat (6% fat) Cheddar cheese
was prepared in 2 different forms: sliced into discs or
shredded using a food processor. Sodium intensity was
5045
higher when samples were in the sliced disc form (66
cps) compared with when the same samples were shred-
ded and introduced in the XRF cup (34 cps). When
the cheese was shredded and placed in the sample cup,
air gaps remained between the individual shredded
particles, which influenced the intensity. Na intensity
was improved when a nylon plug (54-ZX1223, Oxford
Instruments) was used to manually compress the shred-
ded cheese. Despite varying the mass of shredded cheese
used, the intensity remained significantly lower (5 g =
46.9 cps, 10 g = 46.6 cps) than when the samples were
in a disc form (P < 0.05). Thus, the mass of shredded
cheese sample used did not have a significant effect on
the intensity of the Na peak.
Cheese Thickness. More X-ray radiation will gen-
erally be absorbed as the sample thickness increases
because more radiation is unable to leave the sample
(Brouwer, 2003). A commercial Colby cheese sample
was sliced to 3 different thickness using a deli slicer
(3, 7, or 17.5 mm) and cut into 30-mm-diameter discs.
Samples (n = 5) were run using the predetermined
operating conditions (4 kV at 750 μA for 180 s) and
replicated 3 times. The area under the Na peak varied
with sample thickness. When compared with the actual
Na concentration (744 mg of Na/100 g of cheese, deter-
mined by ICP-OES), the XRF concentrations using the
thicker slices (7 and 17.5 mm) were closer to the actual
concentration (697 and 657 mg of Na/100 g of cheese,
respectively). Thinner (3 mm) slices overestimated the
Na concentration (825 mg of Na/100 g of cheese) com-
pared with the actual Na concentration (P < 0.05).
Thicker samples (i.e., >5 mm) are usually preferred for
analysis because more radiation will be reflected back
and reach the detector than thinner samples (Brouwer,
2003).
Cheese Composition. It is desirable to create a
single method using the XRF equipment that can mea-
sure the Na concentration in a wide range of cheeses,
regardless of manufacturer, cheese type, or age. To de-
termine whether compositional differences (e.g., mois-
ture, fat, or protein contents; Table 1) in the cheese
matrix affected the accuracy of the XRF equipment in
determining changes in Na concentration, we created
6 unique calibration curves using a variety of cheese
types (i.e., Cheddar, Gouda, pizza, Mozzarella, nonfat,
and process cheese), which varied in moisture, fat, and
protein contents. The 6 calibration curves ranged from
0 to 4% NaCl (wt/wt); other mineral components were
not modified. The Na concentrations for the 6 calibra-
tion curves were combined into a master linear best-fit
calibration curve, shown in Figure 3, along with their
corresponding regression equations and coefficient of
determination values. The master curve was linear (R2
≥ 0.99), which suggests that the compositional differ-
Journal of Dairy Science Vol. 98 No. 8, 2015
5046 STANKEY ET AL.

Table 1. Composition of the experimental cheeses (before salt addition)

Cheese Moisture1 Protein2 Fat3 Na4

(no salt added) (%) (%) (%) (mg/100 g)
Cheddar 40.9c 23.5b 31.1a 30.1a
Gouda 44.2b 23.5b 29.4b 25.7a
Mozzarella 44.7b 30.3a 20.9c 38.5a
Nonfat 58.9a 34.6a 1.21d 38.6a
Pizza5 43.3bc 31.0a 21.7e 30.6a
a-e
Means within a column with different superscripts differ (P < 0.05).
1
Determined as 100 – total solids.
2
Total percentage N × 6.35 by Kjeldahl.
3
Determined by Mojonnier.
4
Determined by Cl analyzer.
5
Used as a base to make the laboratory-scale process cheese.

ences between cheese types did not affect the ability of
the XRF to successfully determine Na. These findings
are similar to other studies looking at the effect of fat
content in milk powders on the determination of sev-
eral minerals using XRF (Gunicheva, 2010; Smagunova
and Pashkova, 2013). Based on this result, any natural
cheese type (regardless of gross composition) can be
successfully analyzed using any of the 6 calibration
methods created with an individual cheese type.
XRF Method Validation
Linearity Range and Calibration Curve. Lin-
earity (R2 ≥ 0.99) was observed in all the calibration
Figure 3. Calibration curve for Na concentration established using
a variety of cheeses: Gouda (), Mozzarella (‫)چ‬, Cheddar (), process
(‫)ڐ‬, pizza (), and nonfat () spiked with NaCl and ranging in total
Na content from 30 to 1,800 mg of Na/100 g of cheese. The Na content
was calculated from X-ray fluorescence spectroscopy (XRF) intensity
versus Na content determined by inductively coupled plasma-optical
emission spectroscopy (ICP-OES). Solid line (and regression equation)
represents the linear regression curve for all types of cheese tested (i.e.,
master calibration curve). Values are means of 3 replicates; error bars
indicate SD.
curves when background corrections were applied (Fig-
ure 3). The range of Na in the calibration curves (100
to 1,500 mg of Na/100 g of cheese) encompassed the
typical Na contents found in most commercial natural
cheeses (300 to 700 mg of Na/100 g of cheese) as well
as commercial process cheeses (930 to 1,600 mg/100 g;
Agarwal et al., 2011).
One calibration curve was selected that had the larg-
est range of Na concentrations to verify the trueness of
the method. The regression equation for the calibra-
tion curve was y = 0.9856x + 21.569. The coefficient
of determination calculated from the weighted linear
regression analysis was 0.99 (linear over the range).
Sensitivity (LOD and LOQ). Dairy products con-
tain at least a trace amount of Na; thus, the creation
of a true blank was not possible. Using the conditions
described in the established method, the LOD, calcu-
lated as 3 × SD of an unsalted cheese sample, was
equal to 82 mg of Na/100 g of cheese. The LOQ for
an established method (i.e., the smallest amount of Na
that can be quantitatively determined in cheese with
suitable precision and accuracy; AOAC International,
2012) was calculated as 3 × LOD, which was equal to
246 mg of Na/100 g of cheese. The procedure for XRF
as described in this study was therefore suitable for
the analysis of low-Na cheeses (i.e., containing ≤280
mg/100 g of cheese).
Trueness. The trueness (that is, how close a result
is to the true value) was determined on a wide range of
commercial cheese (both natural and process) samples
(n = 10) for comparison with the ICP-OES and Cl
analyzer methods (Table 2). The Na data obtained by
XRF analysis were in close agreement with ICP-OES
for most of the commercial cheeses except for Muenster
(brand B), low-Na Cheddar cheese (brand C), and the
process cheeses: deli deluxe reduced fat pasteurized
process American cheese slices (brand A), deluxe pas-
teurized process American cheese product slices (brand
B), and pasteurized prepared American singles cheese

Journal of Dairy Science Vol. 98 No. 8, 2015

 DETERMINATION OF SODIUM IN CHEESE 5047
Table 2. Comparison of Na (mg/100 g) in commercial cheeses measured by inductively coupled plasma-optical emission spectroscopy (ICP-
OES), X-ray fluorescence spectroscopy (XRF), and Cl analyzer

Na2 (mg/100 g of cheese)

Cl
Cheese type Brand1 ICP-OES XRF analyzer SEM P-value Ca3 K3
Brick B 568a 620a 630a 23.44 NS 561 56
Colby B 569a 652a 657a 23.94 NS 545 48
Deli deluxe reduced fat pasteurized A 1,448a 1,366b 544c 10.25 <0.0001 1741 123
process American cheese slices
Deluxe pasteurized process American B 1,392a 1,509a 684b 46.13 <0.01 1192 72
cheese product slices
Low-moisture, part-skim Mozzarella B 563a 733a 633a 43.24 NS 635 47
Medium aged Cheddar B 540a 633a 642a 27.18 NS 652 42
Monterey Jack B 638a 682a 679a 7.93 NS 582 39
Muenster B 581b 679a 643a 7.94 <0.01 591 68
Pasteurized prepared American singles A 1,018a 604b 527b 24.50 <0.02 1202 310
cheese product
Low-Na Cheddar cheese C 262a 304b 306b 3.28 <0.001 771 97
a-c
Means within a row with different superscripts differ in Na concentration (P < 0.05).
1
Letters A, B, and C refer to the different manufacturers/distributors of the cheeses.
2
Mean values of 3 determinations.
3
Measured by ICP-OES method.

product (brand A). The discrepancy between ICP-OES
and XRF data for the Muenster cheese may have been
due to a sampling issue, as Muenster cheese is typically
brine-salted (our calibration method was created by
direct-salting of curd). The commercial cheese samples
were prepared for XRF analysis by slicing blocks pur-
chased from the grocery store into discs. Samples for
ICP-OES analyses were shredded first and then ashed,
which eliminates any possible variation in Na due to
the sample location from within a block of brine-salted
cheese.
The Cl analyzer accurately predicted Na content of
most of the natural cheeses (made without any K-salts)
when values were compared with ICP-OES with the
exception of the Muenster (brand B) and low-Na Ched-
dar cheese (brand C).
To verify the need for the standards to be an accu-
rate representation of our samples, an additional set of
XRF calibration standards were formulated by varying
both K and Na concentrations. Initially, aqueous solu-
tions were prepared that contained NaCl and KCl at
different levels (up to 50% substitution) to determine
whether the XRF method was capable of accurately
measuring the changes in Na intensity when K was
present at much higher quantities. The intensities for
each of the Na-K blend standards were plotted against
the known concentrations. Linearity (R2 > 0.98) was
observed for both Na and K in the blend standard solu-
tions, which demonstrated that XRF could accurately
predict Na when K was present in increasing quantities
in solutions (results not shown). Cheese standards were
then prepared from an unsalted Cheddar with different
ratios of Na:K as described in the methods section. Due
to the different composition of the cheese matrix (i.e.,
increased K concentration), an additional correction
factor (i.e., Ca) was applied to the regression model for
these standards. Linearity for Na (R2 = 0.98) and K
(R2 = 0.99) were both observed in the Cheddar cheese
calibrations made with varying Na to K ratios (Figure
4a).
Trueness was assessed on experimental Cheddar
cheeses manufactured with different K-salt based
replacers. Data obtained from XRF was compared
against ICP-OES and Cl analyzer methods (Table 3).
This XRF calibration method allowed for closer (bet-
ter) prediction (compared with ICP-OES) of the Na
content (P > 0.05) in the presence of high levels of K.
The XRF method was more accurate when compared
with the Cl analyzer, which was unable to predict (P <
0.05) Na levels accurately in cheeses manufactured with
K-salt replacers (Table 3).
Precision Parameters (Repeatability and Re-
producibility). The amount of Na determined in du-
plicate analyses of each of the 13 commercial natural
cheeses on 4 different days is presented in Table 4. The
RSDr varied between 0.9 and 13.3%, and the RSDR
ranged from 0.9 to 13.3%. For the 15 natural cheese
samples, the calculated HorRatR value ranged from 0.2
to 2.9 (mean = 0.74). The HorRatR value is a perfor-
mance parameter, which indicates the acceptability of
a chemical method of analysis with respect to precision.
Generally, a HorRatR <1 is considered acceptable for
SLV studies (Masotti et al., 2012). Some other stud-
ies have suggested that the extrapolations of HorRatR

Journal of Dairy Science Vol. 98 No. 8, 2015

5048 STANKEY ET AL.
Table 3. Comparison of Na (mg/100 g) of experimental Cheddar cheeses made with varying concentrations
of K (mg/100 g) measured by inductively coupled plasma-optical emission spectroscopy (ICP-OES), X-ray
fluorescence spectroscopy (XRF), and Cl analyzer
Na2 (mg/100 g of cheese)
Sample K1 ICP-OES
b b
High-K Cheddar 357 433
Low-K Cheddar 70 643a
a,b
Means within a row with different superscripts differ in Na concentration (P < 0.05).
1
Measured by ICP-OES method.
2
Mean values of 3 determinations.
values to a SLV study are expected to be from 0.3 to
1.5 (Poitevin et al., 2009). Consistent deviations from
the ratio on the low side (values <0.3) may indicate
unreported averaging or excellent training and operator
Figure 4. Calibration curves for Na () and K () established
using (a) natural cheeses spiked with NaCl and KCl or (b) process
cheeses made with disodium phosphate and dipotassium phosphate
emulsifying salts. The Na or K content was calculated from X-ray
fluorescence spectroscopy (XRF) intensity versus the Na or K content
determined by inductively coupled plasma optical emission spectros-
copy (ICP-OES). Solid lines represent the linear regression curves.
Values are means of 4 replicates; error bars indicate SD.
Journal of Dairy Science Vol. 98 No. 8, 2015
XRF Cl analyzer SEM P-value
a
468 597 5.10 <0.01
697a 631a 15.8 NS
experience, whereas consistent deviations on the high
side (values >1.5) may indicate inhomogeneity of the
test samples, need for further method optimization,
more training, operating below the limit of determina-
tion, or an unsatisfactory method.
The HorRatR values were ≥1.0 for the following
cheeses: Asiago (brand G), LMPS Mozzarella (brand
F), Swiss (brand H), and Parmesan (brand B) cheeses
(as denoted by the italicized font in Table 4). The salt
contents of the 2 LMPS Mozzarella cheeses tested were
very different (417 and 828 mg/100 g, Table 4). The
LMPS Mozzarella cheeses samples originated from 2
different manufacturers. Commercial cheeses of the
same variety have a wide range of salt levels (Agar-
wal et al., 2011). Additionally, the LMPS Mozzarella,
Asiago, Swiss, and Parmesan cheeses were all brine
salted. Brine salting results in a Na gradient in cheeses
(Guinee, 2004). Because the sample preparation for
the XRF method involved using a disc of the following
dimensions being cut (30 mm diameter × 7 mm height)
from somewhere in the block, the within-sample varia-
tion could have been due to an existing salt gradient
within the cheese block. This within-sample (block)
variation can significantly affect the repeatability, re-
producibility, and subsequently the calculated HorRatR
parameters. When the brine-salted cheeses were exclud-
ed from Table 4, the HorRatR range decreased to 0.2
and 0.8 (average = 0.46). Brine-salted cheeses for XRF
analysis should therefore be ground (like the prepara-
tion step for ICP-OES digestion) to have homogeneous
samples and eliminate salt-gradient effects. Calibration
curves should be made specifically for brine-salted
cheeses thus ensuring that the sample taken for analysis
is homogeneous and fully representative of the cheese
block. A suggestion is grinding a portion of the cheese
block (as is done in Cl analysis sample preparation),
weighing a predetermined amount of the ground cheese
into a sample cup, and removing most of the surface
irregularities (air gaps) with a nylon plunger. It should
be noted that the calibration method must be created
using the same sample preparation procedure, as will
be used for cheese analysis.
 DETERMINATION OF SODIUM IN CHEESE 5049
Table 4. Statistical analysis of data corrected for outliers for determining precision parameters1 for the within-laboratory study on 13 commercial
natural cheese samples for the determination of Na in cheese by X-ray fluorescence spectroscopy (XRF)

Na content r RSDr rrel R RSDR Rrel PRSDR

Cheese type Brand2 (mg/100 g) (mg/100 g) (%) (%) (mg/100 g) (%) (%) (%) HorRatR
3
Asiago G 1,323 131 3.5 9.9 150 4.0 11 3.8 1.0
Brick B 788 28.7 1.3 3.7 28.7 1.3 3.7 4.1 0.3
Colby H 782 20.8 0.9 2.7 22.2 1.0 2.8 4.1 0.2
LMPS4 Mozzarella3 F 417 155 13.3 37 155 13.3 37 4.5 2.9
LMPS4 Mozzarella H 828 70.6 3.0 8.5 74.7 3.2 9.0 4.1 0.8
Mild aged Cheddar H 648 21.6 1.2 3.3 21.6 1.2 3.3 4.3 0.3
Monterey Jack H 692 30.8 1.6 4.4 30.8 1.6 4.4 4.2 0.4
Muenster B 800 20.1 0.9 2.5 20.1 0.9 2.5 4.1 0.2
Parmesan I 653 36.5 2.0 5.6 55.3 3.0 8.5 4.2 0.7
Parmesan3 B 698 97.3 5.0 14 106 5.4 15 4.2 1.3
Provolone J 662 37.1 2.0 5.6 37.1 2.0 5.6 4.2 0.5
Sharp Cheddar B 701 22.8 1.2 3.3 22.8 1.1 3.3 4.2 0.3
Swiss3 H 325 68.0 7.5 21 80.5 8.9 25 4.7 1.9
1
r = repeatability; RSDr = relative standard deviation of repeatability; rrel = relative repeatability; R = within-laboratory reproducibility; RSDR
= relative standard deviation of within-laboratory reproducibility; Rrel = relative within-laboratory reproducibility; PRSDR = predicted RSDR;
HorRatR = Horwitz ratio (RSDR/PRSDR).
2
Letters B, F, G, H, I, and J refer to the different manufacturers/distributors of the cheeses.
3
Italicized rows indicate data sets with HorRatR ≥ 1.0.
4
Low-moisture, part-skim.

The rrel and Rrel values ranged from 2.5 to 37% and
2.5 to 37%, respectively, which represent expected vari-
ability between results when a sample was analyzed in
duplicate; these values were higher for the brine-salted
cheeses (as denoted by the italicized font in Table 4).
When the values from brine-salted cheeses are excluded,
then rrel and Rrel values ranged from 2.5 to 8.5% and 2.5
to 9.0%, respectively.
XRF Analysis of Process Cheeses
The XRF method was unable to accurately predict
the Na content of 2 of the commercial process cheese
samples: deli deluxe reduced fat pasteurized process
American cheese slices (brand A) and pasteurized
prepared American singles cheese product (brand A;
Table 2). The incorrect estimation of Na concentration
by XRF is due to an enhancement effect because the
intensity of the X-rays of the element of interest (i.e.,
Na) depends not only on the concentration of Na in
the sample, but also on the overall mineral composition
(Brouwer, 2003). Variations in the matrix mineral com-
position (e.g., addition of different ES in process cheese
manufacture) result in changes in the mean absorption
coefficients of both the primary radiation source and
the fluorescence radiation of the element of interest.
We did not observe these problems in estimating Na
concentration in natural cheeses because it is a simpler
mineral system; in our standards the only elements with
varying concentrations were Na and Cl. The absorption
effects from the additional elements (e.g., found in ES)
may result in either a decrease or an increase in the
measured Na intensity, depending on whether the el-
emental composition changes diminish or augment the
mass absorption coefficient (Brouwer, 2003).
We hypothesized that the XRF underestimated Na
concentration because the XRF mineral composition
of these standards did not accurately reflect those of
the samples being analyzed. The deli deluxe reduced
fat pasteurized process American cheese slices (brand
A) and pasteurized prepared American singles cheese
product (brand A) both contained higher K concentra-
tions (123 and 310 mg/100 g of cheese, respectively as
determined by ICP-OES; Table 2) than the calibration
standards we used for this XRF method (range: 30 to
60 mg/100 g of cheese). Despite using K or Ca (which
were also present in higher concentrations in these
process cheeses) as background correction factors, the
calculated Na values were still less (P < 0.05) than the
reference ICP-OES values.
As expected, the Cl analyzer underestimated the Na
content of the process cheeses (Table 2), which was due
to the indirect method for Na calculation used by the
Cl analyzer instrument. Because process cheeses are
manufactured with ES that contain Na but do not con-
tain Cl (e.g., Na-phosphate or Na-citrate), Na cannot
be accurately calculated using the Cl analyzer method
(Table 2).
Commercial process cheeses are manufactured with a
wide range (blends) of ES; they sometimes also contain
K-based salt replacers. A calibration plot was created
using process cheese standards made with varying con-
centrations of Na- and K-based ES. A linear relation-
ship (R2 ≥ 0.99) was obtained between known Na or K

Journal of Dairy Science Vol. 98 No. 8, 2015

5050 STANKEY ET AL.

Table 5. Comparison of Na content (mg/100 g) of commercial process cheeses measured by inductively coupled plasma optical emission
spectroscopy (ICP-OES), X-ray fluorescence spectroscopy (XRF), and Cl analyzer

Na2 (mg/100 g of cheese)

Cl
Cheese type Brand1 ICP-OES XRF analyzer SEM P-value K3
American pasteurized process cheese product singles D 1,326a 965b 825c 43.4 <0.05 342
Deli deluxe sharp Cheddar pasteurized process cheese slices A 1,527a 1,185b 920c 64.1 <0.05 124
Pasteurized prepared American cheese product A 1,331a 1,034b 692c 59.1 <0.05 340
Pasteurized prepared American singles cheese product A 1,033a 1,134b 635c 19.0 <0.01 320
Pasteurized prepared American singles cheese product A 1,036a 1,280b 595c 39.9 <0.01 300
Pasteurized process American cheese food singles E 1,267a 1,024b 808c 38.8 <0.05 235
Pasteurized process American cheese product slices D 1,162a 1,296b 694c 18.7 <0.01 156
Swiss style pasteurized process cheese product singles D 1,249a 1,241a 761b 40.1 <0.05 206
a–c
Means within a row with different superscripts differ in Na concentration (P < 0.05).
1
Letters A, E, and D refer to the different manufacturers/distributors of the cheeses.
2
Mean values of 3 determinations.
3
Measured by ICP-OES method.

concentration (obtained by ICP-OES) and calculated
concentrations from XRF technique when the back-
ground corrections were applied for these experimental
process cheese samples (Figure 4b).
Trueness was assessed on a wide range of com-
mercial process cheese samples by comparison of the
XRF method against the ICP-OES and Cl analyzer
methods (Table 5). Our experimentally created process
cheese calibration was only able to accurately (P > 0.05
compared with Na result from ICP-OES) calculate Na
in 1 out of the 8 commercial process cheese samples,
that is, Swiss-style pasteurized process cheese product
singles (brand D; Table 5). Two explanations for this
are possible. First, the specific types of ES that were
used to make these commercial process cheese samples
likely affected the XRF results. Second, the process
cheese standards for our calibration curves were made
using only 2 specific types of ES (i.e., DSP and DPP).
During the manufacture of commercial process cheeses,
blends of often 3 or more different types of ES are used.
Our sample calibration standards did not accurately
reflect the matrix chemical composition of all of the
different commercial process cheese samples. When ES
are added during the manufacture, a range of different
type of salt complexes can be formed, involving Ca-
casein crosslinking and new forms of phosphate salts
[e.g., Ca-pyrophosphate complex (Lucey et al., 2011)].
In addition, some ES salts remain undissolved in pro-
cess cheese and some other soluble forms of calcium
phosphate may be present. Thus, all of these complex
salt interactions in the process cheese would likely cre-
ate interferences for the XRF technique unless custom
calibration curves can be created. It is likely that the
accuracy of the XRF would improve if calibration stan-
dards were a true representation of the samples being
measured, as occurred in our experimental process
cheese work (Table 5). This method shows promise for
the direct and rapid measurement of process cheeses
at a manufacturing plant where standards can be used
for calibration that are representative of the process
cheese samples being manufactured. A process cheese
manufacturer would be unlikely to encounter the prob-
lems we faced because they would create calibration
standards using their manufactured cheeses as the base
material.
As expected, the Cl analyzer significantly (P < 0.05)
underestimated Na concentration in process cheeses
when compared with the ICP-OES reference values.
The XRF method was compared with the Cl analyzer
method (Table 5). As expected, the Cl analyzer was
unable to accurately predict Na levels in any process
cheeses containing higher K levels or when non-Cl-based
Na-ES (e.g., disodium phosphate, trisodium phosphate,
sodium metaphosphates, or trisodium citrate) are used
in process cheese manufacture.
CONCLUSIONS
The XRF technique was demonstrated to be a rapid
and reliable method for the routine determination of
Na in natural cheeses. No complex sample preparation
was required; a slice of cheese could easily be used to
accurately measure Na content. However, for brined-
salted cheeses, we suggest that the cheeses be ground
so as to present homogeneous samples and eliminate
salt-gradient effects with discs. Although the XRF
method worked for our experimental process cheeses,
cheese plants would need to create unique calibration
curves specific to the types (blends) of ES used in pro-
cess cheese. When we made process cheeses with known

Journal of Dairy Science Vol. 98 No. 8, 2015

 DETERMINATION OF SODIUM IN CHEESE
types of ES, we were able to accurately determine the
Na in the cheeses because we created calibration stan-
dards with these ES. The sensitivity of the technique
allowed for the rapid (<5 min) determination of Na in
natural cheeses with an LOQ of 245 mg of Na/100 g of
cheese, which would make it suitable for the analyses
of even low Na cheeses (e.g., ≤280 mg of Na/100 g
of cheese). The technique was relatively easy to use
once a standard method and calibration graph were
developed. The good linearity and satisfactory per-
formance in terms of recovery, trueness, and precision
factor support the adoption of this method for in-plant
monitoring of Na in natural cheeses where Na replacers
are being used.
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Journal of Dairy Science Vol. 98 No. 8, 2015