American Journal of Anal yt ical Chemistry, 2011, 2, 718-725
doi:10.4236/ajac.2011.26082 Published Online October 2011 (http://www.SciRP.org/journal/ajac)
A Validated Inductively Coupled Plasma-Optical Emission
Spectrometry (ICP-OES) Method to Estimate Free
Calcium and Phosphorus in In Vitro Phosphate Binding
Study of Eliphos Tablets
Venkata Vivekanand Vallapragada1,2*, Gopichand Inti1, J. Sri Ramulu2
1Invagen P h armaceuti c al Inc, Hauppauge, USA
2Department of Chemistry, Sri Krishna Devaraya University, Anantapur, India
Received June 24, 2011; revised July 25, 2011; accepted August 2, 2011
Abstract
An ICP-OES method has been developed to estimate Calcium and Phosphorous in In vitro phosphate binding
study of Eliphos Tablets. The method is selective and is capable of detecting calcium and phosphorous in the
presence of other trace elements. The method has been validated using RF power of 1500 watts, plasma flow
of 15L/min, Nebuliser flow of 0.8 L/min and plasma view at radial mode for calcium and axial mode for
phosphorus. The wavelength was monitored for calcium and phosphorous at 317.933 nm and 213.677 nm
respectively. The method has been validated in terms of specificity, precision, linearity, accuracy, limit of
quantification and ruggedness. The In vitro binding studies were performed for Eliphos Tablets at eight dif-
ferent phosphate concentrations by incubating at 37.0˚C and analysis was performed using the validated
method to estimate free calcium and phosphorus. The objective of the study is to provide an alternate In vitro
method to estimate the binding capacity of calcium acetate tablets to avoid the expensive in-vivo bio clinical
studies.
Keywords: ICP-OES, Phosphate Binding Study, Quantification, Eliphos Tablets, Free Calcium
1. Introduction
Calcium Acetate is the Active ing redient in Eliphos Tab-
letsTM and is indicated for haemodialysis and peritoneal
dialysis. In kidney disease, blood levels of phosphate
may raise leading to bone problems. Calcium Acetate
binds phosphate in the diet to lower blood phosphate
levels. This medication is used in kidney disease to con-
trol blood phosphate.
Phosphorus is an essential element necessary for the
normal function of human body, required for skeletal
construction and synthesis of DNA, proteins and adeno-
sine Triphosphate. In healthy individuals, serum phos-
phorus concentrations are maintained between 2.5 and
4.5 mg/dL through diet and renal excretion. In renal in-
sufficiency, phosphorus excretion declines and hyper-
phosphatemia develops. The body’s compensation me-
chanisms cause secondary hyperparathyroidism and renal
osteodystrophy.
Phosphate binders provide an effective means for ma-
naging serum phosphate. Commercially available phos-
phate binders include calcium carbonate, calcium acetate
and rarely aluminum hydroxide. Calcium carbonate’s
benefits are seen over a narrow gastric pH range, thereby
limiting the drug’s utility. Calcium acetate is effective
over a wide pH range [1,2]. Because of aluminum’s
known toxicities, aluminum based phosphate binders
have a limited place in therapy. Up to this time, no bio-
logical function has been attributed to this metal, and,
more importantly, aluminum accumulation in tissues and
organ results in their dysfunction and toxicity. Alumi-
num causes an oxidative stress within brain tissue. Since
the elimination half-life of aluminum from the human
brain is 7 year, this can result in cumulative damage via
the element’s interference with neurofilament axonal
transport and neurofilament assembly. Some experts be-
lieve it plays a role in leading to the formation of Alz-
heimer like neurofibrillary tangles [3-11].

V. V. VALLAPRAGADA ET AL.719
The general analytical techniques employed for the
inorganic metal impurities include titration, ion chroma-
tography, capillary electrophoresis and inductively cou-
pled plasma (ICP). Among the above-said techniques,
ICP is a versatile tool for detection and quantification of
elements in accurate manner. The ICP technique is based
on atomic spectrometry. Most specifically, the ICP-OES
is emission spectrometric technique that exploits the fact
that excited atoms emit energy at a given wavelength as
the electrons return to their ground state. A given ele-
ment emits energy at specific wavelengths peculiar to its
chemical character. The intensity of the energy emitted at
that wavelength is proportional to the amount of that
element in the analyzed sample.
ICP-OES has additional advantages over the other
techniques in terms of detection limits as well as speed
of analysis. In ICP-OES sample experiences tempera-
tures estimated to be in the vicinity of 10,000 K. This
results in atomization and excitation of even most re-
fractory elements with high efficiency so that detection
limits for these elements with ICP-OES can be well ov er
and order of magnitude better than the corresponding
values of other techniques. The limit of quantitation val-
ues of most of the elements in ICP-OES in parts per mil-
lion and even parts per billion. In number of analytical
applications speed can be an important factor. Those
advocating simultaneous ICP-OES regard it is the only
method worth considering for this task because it is so
much analyses sample in minutes is only fast enough if
the sample preparation time takes only a few minutes
[12,13]. In other technique like ion chromatography,
capillary electrophoresis stabilization is a time taking
process and sensitivities are low when compared with
ICP-OES. The titration methods are not accurate espe-
cially while estimating the elements at lower concentra-
tions and also errors could be expected.
We have come across few publications regarding in-
fluence of pH on In vitro disintegration of phosphate
binders. However we did not find any publication giving
details determining the calcium and phosphorus in In
vitro phosphate binding study of calcium acetate tablets
in the open literature. The phosphate binders disintegra-
tion is a pH dependant as well as formulation dependant.
It was found that disintegration in simulated gastric fluid
and distilled water yielded a h igher passing rate than that
of intestinal fluid. The percentage of products that passed
the study disintegration test in distilled water, gastric
fluid, and intestinal fluid were 80%, 80% and 73% re-
spectively [14-18]. This paper describes not only the
methodology and validation it also includes the study of
In vitro phosphate binding of Eliphos Tablets 667 mg by
Inductively Coupled Plasma. Free Calcium and Free
Phosphorus in the supernatant solution was determined
in eight different phosphate concentrations to estimate
the binding assay.
2. Experimental
2.1. Chemicals
Calcium Acetate was obtained from Kemira chemso-
lutions b.v (Netherlands). Sodium Phosphate tribasic
(Na3PO4) obtained from Spectrum Chemicals (USA).
Deionised water was obtained from an in-house TOC
water system (Sievers, USA). Eliphos Tablets are pur-
chased from Amsterdam pharmacy, New York and
manufactured by Hawthorn pharmaceuticals Inc (USA).
2.2. Equipment
A Perkin Elmer Inductively coupled Plasma system
equipped with Optical Emission Spectrophotometer and
system controlled through Win Lab32 software. The in-
cubator used for the experiment is Max Q 4000 of Thermo
Scientific.
2.3. Sample Preparation
A stock solution of Calcium Acetate was prepared 4.733
mg/ml (29.89 mM) by dissolving the appropriate amount
of the calcium acetate in deionised water. Sodium Pho-
sphate was not prepared as stock preparation and weighed
separately depending on validation experiment.
2.4. Method Development
The main objective of the study is to develop a suitable
ICP-OES method to quantitate calcium and phosphorus
in the presence of placebo and other matrix.
The placebo contains PEG 8000, Sodium Lauryl
Sulfate and Crospovidone. The other matrix means so-
dium phosophate buffer in which sodium is possible
interference in the development. During the method
development calcium standard was prepared at working
concentr ation 0.04733 mg/mL (0.2989 mM) in deionised
water. The calcium standard was monitored at different
possible emission lines of 317.933 nm, 315.887 nm,
393.366 nm, 396.847 nm, 422.673 nm and 227.546 nm
[19-22]. In among these emission lines at applied target
RF power of 1500 W the responses for calcium were
evaluated. The response of calcium is very high at emis-
sion line of 317.933 nm and moreover the baseline found
to be good, comparatively at other emission lines of cal-
cium. The calcium response was even good at radial view
mode. The possible interference with phosphorus, sodium
and placebo related interference were not observed at

V. V. VALLAPRAGADA ET AL. 720
this emission line.
The phosphorus standard was monitored at different
possible emission lines of 213.617 nm, 214.914 nm,
178.221 nm and 177.434 nm by aspirating the solution at
lowest buffer solution concentration (0.02817 mM). The
method was optimized at lowest phosphate concentration
during method development. The phosphate concentra-
tion is low and to get better sensitivity view mode was
selected as Axial. In axial, plasma views down the
central channel of the plasma and collects all the analyte
emission over the entire length of the remaining plasma
after the shear gas. This region is much larger than that
viewed by normal radial or no rmal side on ICP resulting
higher intensity for phosphorus.
2.5. ICP-OES Conditions
Calcium:
The RF power was used 1500 watts, Plasma flow was
used 15 L/min, Auxillary Flow was kept at 0.2 L/min,
Nebuliser Flow was kept at 0.8 L/min, Pump Rate was
kept at 1.5 ml/min, Calcium was monitored at wave-
length of 317.933 and plasma view was in Radial mode.
Phosphorus:
The RF power was used 1500 watts, Plasma flow was
used 15 L/min, Auxillary Flow was kept at 0.2 L/min,
Nebuliser Flow was kept at 0.8 L/min, Pump Rate was
kept at 1.5 ml/min, Phosphorus was monitored at wave-
length of 213.617 and plasma view was in axial mode.
2.6. Validation of the Method
2.6.1. Specificity
The International Conference on Harmonisation defines
specificity as the ability to assess unequivocally the
analyte in the presence of components that may be ex-
pected to be present, such as impurities, degradation
products and placebo matrix. Calcium Acetate solution at
0.04733 mg/mL solution (0.2989 mM) and placebo sam-
ple solution was separately aspirated at 317.933 nm into
ICP-OES to check the interference of any other elements
at that particular wavelength.
The 5.6334 mM Sodium phosphate solution (3.08
mg/ml) was prepared and aspirated into ICP-OES at
213.617 nm along with placebo solution to evaluate the
interference of any other elements or matrix.
2.6.2. Precision
Precision of the method is the degree of agreement
among the individual test results when the procedure is
applied repeatedly to multiple samplings of a homogenous
sample. The precision of the method was checked by
analyzing five replicates of Calcium Acetate and Sodium
Phosphate solutions at working concentrations and the
%RSD found to be with in the specification. Refer Table
1.
2.6.3. Linearity
The linearity of an analytical method is its ability to elicit
test results that are directly, or by a well defined mathe-
matical transformation, proportional to the concentration
of analyte in samples within a given range [23].
Linearity was evaluated by determining six working
solutions for Calcium ranging from 10%, 20%, 50%,
80%, 100%, and 150% w/v with respect to working con-
centration of 0.0473 mg/ml (0.2989 mM). The above
solutions were prepared from stock solution of Calcium
Acetate. Each solution was aspirated five times. The
mean responses recorded for calcium was plotted against
concentration. The correlation coefficient for calcium
was found to be 0.9995 which indicated good linearity.
Linearity was evaluated by determining nine working
solutions for sodium phosphate tribasic ranging from
0.014085 mM, 0.02817 mM, 0.14084 mM, 0.28167 mM,
1.12668 mM, 1.97169 mM, 2.8167 mM, 5.6334 mM and
8.450103 mM. Each solution was aspirated five times.
The mean responses recorded for phosphorus were plot-
ted against concentration. The correlation coefficient for
calcium was found to be 0.9999 which indicated good
linearity.
The Calibration equations for calcium and phospho-
rous were round to be y = 29317644x – 9591.581 and y =
265323.166 + 9502.654 respectively. Refer Figures 1
and 2 for the Linearity plots for calcium and phosphorus.
2.6.4. Accu racy
Calcium Acetate sample solutions were spiked with
placebo at different concentrations i.e. 60%, 80%, 100%,
120% and 140% with respect to working concentration
of 0.04733 mg/ml (0.2989 mM). Each spiked solution
Table 1. Precision results of calcium at emission line of
317.933 nm and phosphorus at emission line of 213.677 nm.
Aspirations Peak intensity
of calcium Peak intensity of phosphorus
1 125322.4 1502730.0
2 126382.7 1506216.4
3 128442.8 1508620.5
4 130034.5 1506005.3
5 131995.7 1498419.3
Average 128435.6 1504398.3
%RSD 2.10% 0.26%

V. V. VALLAPRAGADA ET AL. 721
LINEARIT Y STUDY
0
500000
1000000
1500000
2000000
2500000
00.01 0.02 0.030.04 0.05 0.060.07 0.0
8
Concentra tion (mg / ml)
Average Peak Intensity
rous accuracy has not been studied for this reason. Refer
Table 2 for recovery values.
2.6.5. Limit of Quantification
The limit of quantification of an individual analytical
procedure is the lowest amount of analyte in a sample,
which can be quantitatively determined with suitable pre-
cision and accuracy.
The precision of the method for calcium at limit of
quantification was checked by analyzing six replicates
prepared at concentration of 1.75 µg/ml (0.011 mM)
and calculating the percentage relative standard devia-
tion.
The precision of the method for phosphorus at limit of
quantification was checked by analyzing six replicates
prepared at concentration of 0.014085 mM phosphate
buffer solution and calculating the percentage relative
standard deviation.
Figure 1. Linearity graph of Calcium at emission line of
317.933 nm, plotted concentration on X-axis versus average
peak intensity on Y-axis. 2.6.6. Ruggedness
The ruggedness of a method was defined as degree of
reproducibility of results obtained by analysis of the
same sample under variety of normal test conditions such
as different labs, different analysts, different instruments,
and different lots of reagents. The recovery levels tested
in Section 2.6.4 (laboratory A) were again carried out in
laboratory B using a different instrument. The recovery
values were found to be comparable.
LINEARITY STUDY
0
500000
1000000
1500000
2000000
2500000
012345678
Concentration ( m M)
9
A
verage Peak Intensit y
3. In Vitro Phosphate Binding Study
The office of Generic drugs, a division of Food and Drug
Administration has recommended the In vitro phosphate
binding study of Calcium Acetate Tablets to determine
the binding capacity. Phosphorus binding is either a
chemical reaction between dietary phosphorous and
cation of the binder compound, resulting in the formation
of insoluble and hence unabsorbable phosphate com-
pounds or an adsorption of phosphorus ions on the sur-
face of binding particles or a combination of both pro-
cesses. The operational definition of phosphorus binding
varies in the three parts of this study. In theoretical
calculations, binding is defined as formation of insoluble
solid phosphate. For the in vitro experiments, the amount
of phosphorus that did no t pass through a millip ore filter
was regarded as bound by the binder. In this case
calcium is a positive ion and p hosphate binds to calcium
resulting insoluble calcium phosphate. The chemical
equation is shown below as reference.
Figure 2. Linearity graph of Phosphorus at emission line of
213.677 nm, plotted concentration on X-axis versus average
peak intensity on Y-axis.
was prepared in triplicate and aspirated. The recovery of
calcium in the presence of the placebo was calculated
against the calcium standard prepared at 0.04733 mg/ml
(0.2989 mM). Recovery of calcium ranged from 98.3% -
101.1%. The phosphorous recovery in the presence of
calcium acetate is not possible since phosphorous binds
to calcium and forms calcium phosphate. The phospho-



334343
2 2
3CaCH COO2NaPOCaPO6CH COONa
Insoluble form


V. V. VALLAPRAGADA ET AL. 722
Table 2. Percentage recovery of calcium at different con-
centration levels in the presence of placebo as a part of me-
thod validation under accuracy experiment.
Concentration level % Recovery Average recovery
60% Prep-1 98.3
60% Prep-2 98.3 98.5
60% Prep-3 98.9
80% Prep-1 100.1
80% Prep-2 99.5 100.1
80% Prep-3 100.5
100% Prep-1 100.3
100% Prep-2 100.4 100.5
100% Prep-3 100.7
120% Prep-1 101.1
120% Prep-2 100.8 100.9
120% Prep-3 100.7
140% Prep-1 100.8
140% Prep-2 99.4 100.3
140% Prep-3 100.8
3.1. Buffer and Sample Preparations
Phosphate Buffer Preparation
Sodium Phosphate buffer solutions were prepared at the
concentrations of 0.02817 mM, 0.14084 mM, 0.28167
mM, 1.12668 mM, 1.97169 mM, 2.8167 mM and 5.633 4
mM. The above phosphate buffer solutions pH were
observed 10.0, 10.5, 10.7, 11.0, 11.2, 11.3 & 11.5 respec-
tively. DI water was also placed in this experiment and
considered as a 0.0 mM phosphate buffer.
Transferred 150 ml of each buffer into each 12 incu-
bating flasks, weighed 12 Eliphos tablets and transferred
into individual incubating flasks shaked well to dissolve.
Twelve Calcium Acetate Tablets were tested at each buf-
fer stage and 96 tablets were used for this experiment.
All the samples were then placed on a BOD incubator
shaker at 37˚C for 2 hours to ensure the establishment of
phosphate binding equ ilibrium.
All the sample s were removed filtered throug h 0.45 µm
Nylon filter. The filtrates were studied to estimate the
concentrations of free calcium and phosphate. Some of
the filtrate portion used for the analysis of free calcium
and other portion used to estimate free phosphate. The
filtrates were diluted 1 ml to 100 mL with deionised
water in order to achieve within the range of ICP work-
ing concentration for Calcium where as filtrates were used
as is with out any further dilution s fo r phosphate.
3.2. Optimizing Incubating Time
To optimize the incubation time four tablets were sepa-
rately incubated in 5.6334 mM ph osphate buffer solution
of 150 ml at di fferent i nterval s li ke 60 mi nut es, 90 minutes,
120 minutes and 180 minutes. The binding assay was
calculated and found constant from 90 minutes to 180
minutes range. The incubation time has been elected as
120 minutes time for entire study for better precision and
accuracy.
3.3. Standards Preparation
Calcium Acetate standard was prepared at 0.04733 mg/
mL (0.2989 mM) in deionised water. Before adding the
tablets into phosphate buffers some of the portions were
separated and used as phosphate standard solutions. Refer
the typical ICP spectras for calcium and phosphorus in
Figures 3 and 4 respectively.
3.4. Calculations
The unbound phosphorus and calcium concentrations
remaining in each sample were calculated from indivi-
dual phosphate buffer standard preparations and calcium
standard solutions.
Free phosphor us or Cal ci um
Au C
As

where
Au is the mean corrected intensity of phosphorus/cal-
cium in sample preparation,
As is the mean corrected intensity of phosphorus/cal-
cium in standard preparation,
C is the molar concentration of phosphoru s/calcium in
standar d preparatio n.
Sodium phosphate added (mM)-free phosphorus found (mM)
%binding of phosphate 100
Sodium phosphate added (mM)

The phosphate binding was calculated by subtracting
free phosphorus found in samples from added sodium
phosphate in mM. Divided the result with sodium phos-
phate added and multiplied with hundred to convert into
percentage. The complete study data was tabulated in-
cluding the free calcium, free phosphate and binding

V. V. VALLAPRAGADA ET AL.723
assay of phosphate.
3.5. Discussion
The free calcium and free phosphate data clearly indi-
cates binding assay at different concentrations of phos-
phate buffer solutio ns. Refer Table 3. Calcium Acetate is
highly soluble compound. Calcium Acetate dissolves
readily thus making all the binder available for reaction.
In the case of poorly soluble compounds, however, only
Figure 3. Typical ICP spectra of Calcium standard at working concentration of 0.0473 mg/ml (0.2989 mM) at emission line of
317.933 nm.
Figure 4. Typical ICP spectra of Phosphorus at concentration of 5.633 mM at emission line of 213.677 nm.

V. V. VALLAPRAGADA ET AL. 724
Table 3. Average percentage binding data of Twelve tablets at each buffer stage, experimented with RF power of 1500 watts,
plasma flow of 15 L/min, Auxillary flow of 0.2 mL/min, Nebuliser flow of 0.8 L/min, pl asma view at radial mode for calcium,
axial view for phosphorus and wavelength of 317.933 for calcium and 213.617 for phosphorus.
Buffer Concentration Free calcium (mM) Free phosphate (mM) % Binding
0.000 mM buffer 27.241 0.000 0.0
0.028mM buffer 27.241 0.002 93.6
0.141mM buffer 27.473 0.009 93.3
0.282 mM buffer 25.012 0.008 97.0
1.127 mM buffer 22.092 0.018 98.4
1.972 mM buffer 20.494 0.014 99.3
2.817mM buffer 19.696 0.007 99.7
5.633 mM buffer 16.342 0.112 99.8
a small amount slowly gets dissolved. As this small
amount of dissolved binder reacts with phosphorus, the
concentration of dissolved binder falls, which in turn
allows further dissolution. This process continues until
equilibrium is reached. It follows that freely soluble com-
pounds would reach equilibrium quickly, whereas poorly
soluble compounds would take longer time. Calcium
Acetate dissolves readily, hence the equilibrium reaches
quickly. Calcium Acetate binds more than 90% at above
pH 6.0. Hence the phosphate buffer solutions were se-
lected at range of 0.02817 mM and 5.6334 mM. The
buffer solu tions pH ranges were found to be around 10.0
and 11.5 respectively. The pHs of the same solutions
were observed 7.0 and 8.5 after dissolving the Calcium
Acetate tablets in the mentioned phosphate buffers. Cal-
cium acetate binding drops to 0% at lower pH (pH 3.5),
this happens because at low pH, where hydrogen ion
concentration is high, H+ competes for phosphorus more
effectively than calcium [24].
In the experiment 0.0 mM phosphate buffer is nothing
but deionised water, basically no binding and complete
calcium to be found in the analysis and zero free phos-
phorus to be expected. The increase in the phosphate
concentration resulted binding and expected less free
phosphorus and the other hand free calcium was de-
creased since calcium was bounded to phosphate and
formed as calcium phosphate. At maximum precipitation
concentration of 5.6334 mM free calcium decreased sig-
nificantly since feasibility of maximum binding. The
phosphate binding assay versus phosphate concentration
was figured in Figure 5.
4. Conclusions
A validated and accurate ICP-OES method has been de-
veloped to estimate calcium and phosphorus in In vitro
phosphate binding study of Eliphos tablets. The method
is selective and is capable of quantitate calcium and
phosphorus in the presence of other trace elements. The
method has been validated in terms of specificity, preci-
sion, linearity, accuracy and limit of quantification. The
validated method can be used to estimate calcium and
phosphorus in In vitro studies of no t o nly Elip ho s tab lets,
any generic version of calcium acetate tablets. The In
vitro experiment was done using the validated method on
Eliphos tablets and results are in good agreement with
theoretical assumption. The benefit of the study is to use
an alternate In vitro method to estimate the binding ca-
pacity of calcium acetate tablets to avoid the expensive
in-vivo bio clinical studies.
5. Acknowledgements
The authors wish to thank Dr. Sudhakar Rao, president
of Invagen pharmaceutical Inc. for supporting this work.
We would also like to thank colleagues in analytical de-
partment of Invagen pharmaceutical Inc. for their
93.0
94.0
95.0
96.0
97.0
98.0
99.0
100.0
0.0 0.5 1.0 1.5 2.0 2.5 3.03.5 4.0 4.5 5.0 5.5 6.0
Concentration of Sodium Phosphate (mM)
% Phosphate binding
Figure 5. Graph plotted for percentage phosphate binding
of Eliphos tablets. Percentage phosphate binding on Y-axis
and different concentrations of sodium phosphate buffer on
X-axis.

V. V. VALLAPRAGADA ET AL.725
co-operation in carrying out this work.
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