Journal of Research Analytica

Ology Science
Research Article
Journal of Research Analytica
A Simple Reversed Phase HPLC Method for
Quantitative Estimation of Calcium Orotate and
Its Degradation Products in Solid Dosage Form
Abstract
A rapid, simple, sensitive and stability indicating reversed phase high performance liquid chromatographic
method was developed for estimation of calcium orotate in pharmaceutical preparations and in dissolution
medium. The new RP-HPLC method was validated according to ICH, FDA and USP guidelines with respect
to accuracy, precision, specificity, linearity, solution stability, robustness, sensitivity and system suitability. The
method was developed by using an isocratic condition of mobile phase comprising ammonium acetate buffer
15 mM (pH 5.5) and acetonitrile in a ratio of 60:40 v/v at a flow rate of 0.8 mL/min over C-18 (ODS, 250 × 4.6
mm) column at ambient temperature. Recovery was found between 98.0%~102.0%. Intraday and inter-day
precision studies of the new method were found less than the maximum allowable limit (% RSD ≤ 2.0 according
to FDA). The method showed linear response over the entire working range with a correlation coefficient (r)
value of 0.999. LOD and LOQ were found 0.002 µg/mL and 0.008 µg/mL respectively. Forced degradation
study was performed to establish the specificity and stability indicating property of current method. Practical
applicability was checked by analyzing market sample. So this method can be efficiently applied for quantitative
estimation of calcium orotate salt in pharmaceutical formulation.
Keywords: Calcium orotate; RP-HPLC; Stability-indicating; Forced degradation
Md. Sarowar Jahan 1 , Md. Hadiul Islam 2 , Maksudur Rahman Khan 1 , Abul Hossain 1 , Ruhul Kayesh 2 and
Asma Rahman 3 *
1
Department of Biochemistry and Molecular Biology, University of Dhaka, Bangladesh
2
Department of Pharmaceutical Chemistry, University of Dhaka, Bangladesh
3
Centre for Advanced Research in Sciences, University of Dhaka, Bangladesh
*
Corresponding author: Asma Rahman
Received : October 03, 2016; Accepted: November 07, 2016; Published: November 11, 2016
E-mail: pharma@du.ac.bd (A.R)
Copyright: ©2016 OLOGY Group.
J Res Anal. (2016) Volume 2 • Issue 4 ISSN : 2473-2230
1

Citation: Jahan SM, Islam HM, Khan MR, Hossain A, Kayesh R, Asma Rahman. A simple reversed phase HPLC method for quantitative estimation of
calcium orotate and its degradation products in solid dosage form. J Res Anal. 2016; 2(4): 108-112.
Introduction
Calcium orotate is the calcium salt of orotic acid (Figure 1).
Chemically, orotic acid is 1,2,3,6-tetrahydro-2,6-dioxo-4-
pyrimidinecarboxylic acid. It is white to almost white crystalline
powder with poor water solubility [1].This salt is mainly given as
calcium supplement in people who do not get enough calcium
from daily food intake [2,3]. It is also used as medication to
treat conditions caused by low calcium levels such as bone loss
(osteoporosis), weak bones (osteomalacia/rickets), decreased
activity of the parathyroid gland (hypoparathyroidism), and a
certain muscle disease (latent tetany) [3,4]. It may also be used in
certain patients to make sure they are getting enough calcium, e.g.,
women who are pregnant, nursing, or postmenopausal, people
taking certain medications such as phenytoin, phenobarbital, or
prednisone [3].
As a source of calcium supplement calcium supplement it is
far superior to all other conventional calcium salts now being used
[5]. For example, calcium orotate is absolutely free of any side
effects. Conventional calcium salts have certain problems when
applied in osteoporosis with concomitant arteriosclerosis of the
abdominal aorta. Calcium orotate, on the other hand, protects
the body from arteriosclerosis. Calcium orotate can penetrate the
complex cell membranes and therefore, it can compensate for a
disturbed calcium transport through these cell membranes. In
addition, calcium orotate has a special affinity for bradytrophic
tissue (e.g. Cartilage) where it is metabolized [6]. Parallel studies
have shown that a defective calcium transport through the
cell membrane is of great pathogenetical significance. So, this
substance is a very satisfactory agent in the re-calcification of
metastatic defects in the skeletal system and in other calcium
deficiency syndrome [4].
Calcium orotate can be assayed by complexometric titration,
polarographic method, UV or chromatographic method [1]. An
UV method has been stated for estimation of calcium orotate
in bulk and in tablets [7]. To the best of our knowledge,
there is no HPLC analytical method for its estimation in bulk
or in pharmaceutical formulations. Here, we developed a
rapid, sensitive and stability indicating RP-HPLC method for
quantification of calcium orotate and this was validated following
the of FDA, USP and ICH guidelines.
Materials and methods
Working standard of calcium orotate (98.5% potency) was
a kind gift of ACI Pharmaceuticals Ltd., Bangladesh. HPLC
grade acetonitrile and methanol were obtained from Active Fine
Chemicals Ltd., Bangladesh. For the estimation of calcium orotate
formulated as tablets, samples produced by three renowned
pharmaceutical industries in Bangladesh were collected from the
market. These samples were coded as S1, S2 and S3.
O
HN
O
N
H
O
O
Ca
N
H
NH
Figure 1: Chemical structure of calcium Orotate.
O
O
O
O
HPLC system
High Performance Liquid Chromatographic system
(Shimadzu-UFLC Prominence, Japan), equipped with an auto
sampler (Model- SIL 20AC HT) and UV-Visible detector
(Model-SPD 20A) was used for the analysis. The data was
recorded using LCsolutions software. Analytical reversed phase
C-18 column [ProntoSIL C18, 4.6 x 250 mm, India.] was used
to analyze the standards and samples.
Preparation of mobile phase: 1.15 g of ammonium acetate
was dissolved in 900 mL of HPLC grade water. The pH was
adjusted to 5.5 ± 0.1 with dilute acetic acid or ammonia solution
and then volume was made up to 1000 mL with water of same
quality. Then this buffer and HPLC grade acetonitrile were mixed
together at a ratio of 60:40 v/v. Finally it was filtered through a
0.22 µm millipore filter and sonicated.
Preparation of standard solutions: 25 mg of calcium
orotate was taken in 100 mL distilled water and sonicated for
10 minutes to dissolve completely. Thus stock solution of 250
µg/mL was found. 5 mL of this solution was taken into another
50 mL volumetric flask and diluted with distilled water to get
nominal concentration of 25 µg/mL. The stock solution was
further diluted with distilled water.
Sample preparation: Twenty tablets of each sample were
grinded to make powder and then powdered tablet equivalent
to 50 mg of calcium orotate was dissolved in 100 mL distilled
water by proper sonication. Then 5 mL of this solution was taken
in another 100 mL volumetric flask and volume was adjusted by
distilled water.
Chromatographic condition: An isocratic mobile phase was
consisted of acetate buffer and acetonitrile at a ratio of 60:40.
Flow rate of mobile phase was 0.8 mL/min through C18 column
for 5 minutes. The injection volume was 20 µL. Before analysis,
all standard and sample solutions were filtered through 0.2 µm
filter tips. The column eluent was monitored with UV detection
at 275 nm.
Parameters of method validation
Specificity: The specificity of the LC method was evaluated
to ensure that there was no interference from the degradation
products, excipients or other impurities in the pharmaceutical
formulation [8-10].
Solution stability: The solution stability of standards and
samples was established under normal bench top conditions,
normal storage conditions, and sometimes in the instrument
to determine where special storage conditions are necessary
or not, for instance, refrigeration or protection from light.
For this method, stability of standard solution was studied by
keeping a single concentration by diluting standard solution in
tightly capped volumetric flask at room temperature (25°C) on
laboratory bench and at 5°C in refrigerator for 72 hours. The
solutions were analyzed by HPLC at 0 hr, 24 hr, 48 hr and 72 hr.
Forced degradation: In this stage, forced degradation studies
were undertaken to degrade the sample (e.g., drug product
or API) deliberately. These studies were used to evaluate an
analytical method’s ability to measure an active ingredient and
its degradation products without interference. Samples of drug
2 ISSN : 2473-2230
J Res Anal. (2016) Volume 2 • Issue 4
Ology Science

Citation: Jahan SM, Islam HM, Khan MR, Hossain A, Kayesh R, Asma Rahman. A simple reversed phase HPLC method for quantitative estimation of
calcium orotate and its degradation products in solid dosage form. J Res Anal. 2016; 2(4): 108-112.
product (spiked placebo) and drug substance were exposed to
acid (0.1N~1.0N HCl), base (0.1N~1N NaOH), oxidizing
agent (10% ~30% H 2
O 2
solution )
, reducing agent (10% ~30%
Na bisulphate solution) and water for 24 hours to produce at
least 10% degradation. The degraded samples were then analyzed
using the method to determine if there are interferences with the
active or related compound(s).
Linearity and range: Five different concentration levels of
calcium orotate (20 µg/mL, 22 5µg/mL, 25 µg/mL, 27.5 µg/mL,
and 30 µg/mL) were prepared from stock solution. The average
peak areas were plotted against concentrations. The linearity of
the proposed method was evaluated by using calibration curve.
Accuracy: The accuracy of an analytical method expresses
the nearness between the expected value and the value found.
In present study, successive analysis (n=3) for three different
concentrations (20 µg/mL, 25 µg/mL, 30 µg/mL) of standard
solution of active drug were carried out to determined the
accuracy of proposed method.
Precision: The precision of the proposed method was
checked by intra- and inter-day repeatability of responses after
replicate injections and expressed as %RSD amongst responses
using the following formula
% RSD = (Standard deviation/Mean) x 100%
Robustness: Robustness measures the capacity of an
analytical method to remain unaffected by small but deliberate
variations in the parameters of the method. Robustness provides
some indication of reliability of the analytical method during
normal usage.
System suitability: System suitability is commonly used
ensure a method’s adequacy for a particular analysis. The following
parameters are verified in system suitability tests: number of
theoretical plate, tailing factor, resolution and repeatability at
100% test concentration.
Limit of detection (LOD) and limit of quantification
(LOQ)
LOD is the lowest amount of analyte in a sample that can
be detected but not necessarily quantified under the stated
experimental conditions. On the other hand LOQ is the lowest
amount of analyte in a sample that may be determined with
acceptable accuracy and precision.
Results and Discussion
HPLC method development
Calcium orotate is slightly soluble in water and practically
insoluble in methanol and ethanol [1]. It is also slightly soluble
in acetonitrile. The pKa value of orotic acid is 2.83 [11,12] which
suggests a pH of buffer solution should be 4.83 or above. So
we tried different ratio of ammonium acetate buffered solution
and acetonitrile to find optimum condition. Finally, 10 mM
buffer (pH 5.5) solution and acetonitrile in a ratio of 60:40
v/v produced well shaped and sharp chromatographic peak of
calcium orotate at 2.9 minute. The method was summarized in
Table 1. Typical chromatogram of standard clcium orotate was
shown in Figure 2.
Validation result
Specificity: The specificity of the LC method was evaluated
to ensure that there was no interference from the degradation
products, excipients, or other impurities in the region of actives.
The specificity was studied by injecting the unstressed and
stressed standard solutions, samples, and blanks.
Solution stability: Two vials of standard solution were
kept for solution stability study. One vial was kept in ambient
condition and another in refrigerator. AUC (area under curve)
changes were recorded by this developed method using the same
machine and column. Day to day area changes were observed
with respect to initial area (0 th hour). Low % RSD indicates
no major variation among the AUC recorded on different day,
which inturn is an indicator of solution stability of calcium
orotate. Results were shown in Table 2.
Forced degradation: Stressed samples were analyzed
against freshly prepared standard. Degradation was measured in
terms of AUC change between the stressed sample and freshly
prepared standard. The drug found fairly stable in acidic, basic
and aqueous environment, but it was degraded significantly by
oxidation and reduction. This study substantiates the use of
water as diluting solution. These data can be proved useful in
formulation development of this drug. Results were summarized
in Table 3.The chromatograms of degradation products were
shown in Figure 3.
Linearity, range and accuracy: Linearity of calcium orotate
was examined over 50% ~ 200% of nominal concentration
(i.e. 12.5 µg/mL to 50 µg/mL). Linearity curve was created by
plotting AUC of chromatograms against the corresponding
concentration. The method showed good linear response of
concentration with correlation coefficient (r) value of 0.999.
Linear equation was found as below:
y=67622x + 27665; y=AUC of chromatographic peak,
x=Concentration
This method followed Beer- Lambert law over a wide range
of concentration between 12.5 µg/mL to 50 µg/mL. Accuracy of
this method was calculated from the percent recovery of known
concentration calculated by linear equation. The % recoveries
were found above 99% and % RSD among the recoveries was
found very low which indicated accuracy of this method. Results
were shown in Table 4.
Precision: Precision (both intraday and inter-day) was
checked in terms of %RSD of recovered concentrations. The
%RSD values depicted in Table 5 showed that the proposed
method provides acceptable intra-day and inter-day variation of
the reproducibility of current method.
System suitability: All the system suitability parameters met
the acceptance value. Table 6 summarized the results.
Table 1: HPLC chromatographic conditions in developed method.
HPLC Method
Column
Flow rate
Monitoring wave length
Tempearture
Retention time
15mM Ammonium Acetate Buffer (pH 5.5) :
Acetonitrile = 60:40 (%v/v)
C18, 5µm , 250 x 4.6 mm
0.8 ml/min
275 ƞm
Ambient
2.90±0.1 minute
Ology Science J Res Anal. (2016) Volume 2 • Issue 4 ISSN : 2473-2230
3

2.90/ Calcium Orotate
Citation: Jahan SM, Islam HM, Khan MR, Hossain A, Kayesh R, Asma Rahman. A simple reversed phase HPLC method for quantitative estimation of
calcium orotate and its degradation products in solid dosage form. J Res Anal. 2016; 2(4): 108-112.
uV
150000
Detector A - Ch1
100000
50000
0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
min
Table 2: Solution stability of calcium orotate with respect to AUC changes.
Figure 2: Chromatogram of Calcium Orotate.
Testing Time (hour) AUC of ambient sample (n=3) AUC of refrigerator sample (n=3)
0 th 1754900 1754900
24 th 1725575 1728495
48 th 1721739 1725106
72 th 1719795 1718489
% RSD 0.950 0.923
Table 3: Result of forced degradation study.
AUC of standard solution Type of Degradation AUC of degraded sample (n=3) % Loss Remark
1095920
(n=6)
Acid Hydrolysis 1083416 1.1 % Less sensitive
Basic hydrolysis 1067725 2.5% Less sensitive
Water hydrolysis 1072920 2.0% Less sensitive
Oxidation 356503 67% Highly sensitive
Reduction 673860 38% Highly sensitive
uV
5000
2500
3.10 / Calcium Orotate
3.73 / Degradant -A
4.63 / Degradant -B
uV
100000
50000
Calcium Orotate / 3.1
uV
750
500
250
1 Det
0
0.0 2.5 5.0 7.5 10.0
min
0
a
Summary (Compound)
Red 1. lcD
Calcium Orotate / 3.0
Unknown Degradant -B / 4.4
0.0 2.5 5.0 7.5 10.0
min
c
Unknown Degradant - C / 6.1
1 Det A
uV
0
50000
25000
0 5 10
min
0
b
Summary (Compound)
Ox 1.lcd
Calcium Orotate / 2.9
2 3 4 5
min
Figure 3: Chromatograms of forced degradation: (a) Acid hydrolysis, (b) Basic hydrolysis, (c) Reduction, (d) Oxidation.
d
Oxidized Product / 3.6
1 Det A Ch1
1 Det A Ch1
4 ISSN : 2473-2230
J Res Anal. (2016) Volume 2 • Issue 4
Ology Science

Citation: Jahan SM, Islam HM, Khan MR, Hossain A, Kayesh R, Asma Rahman. A simple reversed phase HPLC method for quantitative estimation of
calcium orotate and its degradation products in solid dosage form. J Res Anal. 2016; 2(4): 108-112.
Robustness: This method also showed acceptable changes
in robustness study and established its stability and reliability
thereby. Result was shown in Table 7.
Market product analysis: To evaluate the practical
applicability of the developed method, three different brands
of calcium orotate formulated as tablets by three renowned
pharmaceutical industries in Bangladesh were collected to
determine the assay and dissolution by this method. All
three brands claim 400 mg calcium orotate per tablet in their
formulation. Results were presented in Table 8.
Table 4: Result of linearity, range and accuracy.
Injected Conc.
(µg/mL)
% of
nominal
Average AUC
(n=6)
Recovered
Concentration
%
Recovery
12.5 50% 862820 12.35 98.8
20 80% 1378895 19.9 99.9
22.5 90% 1542656 22.4 99.5
25 100% 1721739 25.05 100.2
27.5 110% 1896123 27.63 100.5
30 120% 2071599 30.22 100.7
50 200% 3398864 49.85 99.7
% RSD of recoveries 0.646%
Table 5: Result of intraday and inter-day precision.
Run Order
% RSD intraday of
variation (n=6)
Day-1,Analyst-1 0.2433
Day-2,Analyst-2 0.5802
Day-3,Analyst-3 1.1214
Table 6: System suitability parameters.
% RSD of inter-day
variation
0.2490
Parameter Average (n=6) % RSD
Acceptance
Limit [8-10]
Peak Area 1721739 0.276 %RSD