Indo American Journal of Pharmaceutical Research, 2014

ISSN NO: 2231-6876

METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS

ESTIMATION
OF
ROSUVASTATIN
CALCIUM
AND
EZETIMIBE
IN
A
PHARMACEUTICAL FORMULATION BY RP-HPLC METHOD
Putchakayala Purnachandra Rao, Dondeti Mogili Reddy and D.Ramachandran*
Acharya Nagarjuna University, Nagarjuna Nagar, Guntur, Andhra Pradesh, India-522510.
ARTICLE INFO
Article history
Received 18/12/2014
Available online
28/12/2014
Keywords
ROSUVASTATIN
CALCIUM, EZETIMIBE,
RP-HPLC Method,
Phenomenax C18 Column,
Acetonitrile,
Ammonium Acetate,
Glacial Acetic Acid And
Validation.

ABSTRACT
An isocratic Simultaneous estimation by RP-HPLC Method was developed and validated for
the quantification of ROSUVASTATIN CALCIUM and EZETIMIBE in tablet dosage form.
Quantification was achieved by using a reversed-phase C18 column (Phenomenax C18
Column , 5, 250 mm 4.6 mm) at ambient temperature with mobile phase consisting of
30mM Ammonium acetate buffer(pH:3.6) : Acetonitrile (40:60). The flow rate was 1.0
ml/min. Measurements were made at a wavelength of 227nm. The average retention time for
ROSUVASTATIN CALCIUM and EZETIMIBE were found to be 3.09 min and 5.13. The
proposed method was validated for selectivity, precision, linearity and accuracy. The assay
methods were found to be linear from 60-140g/ml for ROSUVASTATIN CALCIUM and
60-140g/ml for EZETIMIBE. All validation parameters were within the acceptable range.
The developed method was successfully applied to estimate the amount of ROSUVASTATIN
CALCIUM and EZETIMIBE in tablet dosage form.

*Corresponding author

Copy right 2014 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

www.iajpr.com

Page

Please cite this article in press as Putchakayala Purnachandra Rao et al. Method Development and Validation For The
Simultaneous Estimation of Rosuvastatin Calcium and Ezetimibe In A Pharmaceutical Formulation By RP-HPLC Method. Indo
American Journal of Pharm Research.2014:4(12).

5828

Department of Chemistry,
Acharya Nagarjuna University, Nagarjuna Nagar,
Guntur, Andhra Pradesh, India-522510
purna74061@yahoo.co.in, dittakavirc@gmail.com.
+918897419125, +919866965335

Vol 4, Issue 12, 2014.

Putchakayala Purnachandra Rao et al.

ISSN NO: 2231-6876

INTRODUCTION
Rosuvastatin is a member of the drug class of statins, used in combination with exercise, diet, and weight-loss to treat high
cholesterol and related conditions, and to prevent cardiovascular disease. The primary use of rosuvastatin is for the treatment of
dyslipidemia. It is recommended to be used only after other measures such as diet, exercise, and weight reduction have not improved
cholesterol levels. Rosuvastatin is a synthetic, enantiomerically pure antilipemic agent. It is used to lower total cholesterol, low density
lipoprotein-cholesterol (LDL-C), apolipoprotein B (apoB), non-high density lipoprotein-cholesterol (non-HDL-C), and trigleride (TG)
plasma concentrations while increasing HDL-C concentrations. High LDL-C, low HDL-C and high TG concentrations in the plasma
are associated with increased risk of atherosclerosis and cardiovascular disease. The total cholesterol to HDL-C ratio is a strong
predictor of coronary artery disease and high ratios are associated with higher risk of disease. Increased levels of HDL-C are
associated with lower cardiovascular risk. By decreasing LDL-C and TG and increasing HDL-C, rosuvastatin reduces the risk of
cardiovascular morbidity and mortality.
Rosuvastatin is a competitive inhibitor of HMG-CoA reductase. HMG-CoA reductase catalyzes the conversion of HMG-CoA
to mevalonate, an early rate-limiting step in cholesterol biosynthesis. Rosuvastatin acts primarily in the liver. Decreased hepatic
cholesterol concentrations stimulate the upregulation of hepatic low density lipoprotein (LDL) receptors which increases hepatic
uptake of LDL. Rosuvastatin also inhibits hepatic synthesis of very low density lipoprotein (VLDL). The overall effect is a decrease in
plasma LDL and VLDL. In vitro and in vivo animal studies also demonstrate that rosuvastatin exerts vasculoprotective effects
independent of its lipid-lowering properties. Rosuvastatin exerts an anti-inflammatory effect on rat mesenteric microvascular
endothelium by attenuating leukocyte rolling, adherence and transmigration.The drug also modulates nitric oxide synthase (NOS)
expression and reduces ischemic-reperfusion injuries in rat hearts.Rosuvastatin increases the bioavailability of nitric oxideby
upregulating NOS and by increasing the stability of NOS through post-transcriptional polyadenylation. It is unclear as to how
rosuvastatin brings about these effects though they may be due to decreased concentrations of mevalonic acid.
Structure of ROSUVASTATIN CALCIUM:

Page

5829

EZETIMIBE
Ezetimibe is an anti-hyperlipidemic medication which is used to lower cholesterol levels. Specifically, it appears to bind to a
critical mediator of cholesterol absorption, the Niemann-Pick C1-Like 1 (NPC1L1) protein on the gastrointestinal tract epithelial cells
as well as in hepatocytes.
Ezetimibe localizes and appears to act at the brush border of the small intestine and inhibits the absorption of cholesterol.
This leads to a decrease in the delivery of intestinal cholesterol to the liver.

www.iajpr.com

Vol 4, Issue 12, 2014.

Putchakayala Purnachandra Rao et al.

ISSN NO: 2231-6876

Structure of EZETIMIBE:

Ezetimibe is in a class of lipid-lowering compounds that selectively inhibits the intestinal absorption of cholesterol and
related phytosterols. Ezetimibe, administered alone is indicated as adjunctive therapy to diet for the reduction of elevated total-C,
LDL-C, and Apo B in patients with primary (heterozygous familial and non-familial) hypercholesterolemia. It is also used in
combination therapy with HMG-CoA reductase inhibitors. Ezetimibe has a mechanism of action that differs from those of other
classes of cholesterol-reducing compounds (HMG-CoA reductase inhibitors, bile acid sequestrants, fibric acid derivatives, and plant
stanols). Ezetimibe does not inhibit cholesterol synthesis in the liver, or increase bile acid excretion but instead localizes and appears
to act at the brush border of the small intestine and inhibits the absorption of cholesterol, leading to a decrease in the delivery of
intestinal cholesterol to the liver. This causes a reduction of hepatic cholesterol stores and an increase in clearance of cholesterol from
the blood; this distinct mechanism is complementary to that of HMG-CoA reductase inhibitors.
EXPERIMENTAL
Equipments:
The chromatographic technique performed on a Shimadzu LC20-AT Liquid chromatography with SPD-20A prominence
UV-visible detector and Spinchrom software, reversed phase C18 column (Phenomenax 5, 250 mm 4.6 mm) as stationary phase.
Thermo Electron Corporation double beam UV-visible spectrophotometer (vision pro-software), Ultrasonic cleaner, Shimadzu
analytical balance AY-220, Vaccum micro filtration unit with 0.45 membrane filter was used in the study.
Materials:
Pharmaceutically pure sample of ROSUVASTATIN CALCIUM and EZETIMIBE were obtained as gift samples from
Chandra Labs, Prashanthinagar, Kukatpally, and Hyderabad, India. The purity of the drug was evaluated by obtaining its melting point
and ultraviolet (UV) and infrared (IR) spectra. No impurities were found. The drug was used without further purification.
HPLC-grade Acetonitrile ware from standard reagents pvt ltd. Ammonium acetate (AR grade) was from Merck.
A tablet formulation of ROSUVASTATIN CALCIUM and EZETIMIBE (10 mg and 10mg label claims)
Chromatographic conditions:
The sample separation was achieved on a C18 (5 , 250 4.6 mm) PHENOMENAX column, aided by mobile phase mixture
of Ammonium acetate Buffer(pH:3.6) : Acetonitrile (40:60), that was filtered and degassed prior to use, at a flow rate of 1ml/min.
Injection volume is 20 l and detected at 227 nm at ambient temperatures.

Analysis of formulation:
Preparation of standard solution:
50mg of standard ROSUVASTATIN CALCIUM and 50 mg EZETIMIBE was weighed and transferred into 50 ml of
volumetric flask and dissolved in mobile phase. The flask was shaken and volume was made up to mark with mobile phase to give a

www.iajpr.com

Page

Mobile phase:
Added 40 volumes of buffer, 60volumes of Acetonitrile sonicated for 10 min.

5830

Preparation of mobile phase:

Buffer Preparation:
Weighed accurately about 2.31 gms of Ammonium acetate and dissolved with 200ml of HPLC Grade water than make up to
1000 ml with HPLC grade water then adjusted the pH:3.6 with glacial Acetic acid and filtered through a 0.45 membrane filter.

Vol 4, Issue 12, 2014.

Putchakayala Purnachandra Rao et al.

ISSN NO: 2231-6876

primary stock solution containing 1000g/ml ROSUVASTATIN CALCIUM and 1000g/ml of EZETIMIBE. From the above
solution 5ml of solution is pipette out into a 50 ml volumetric flask and volume was made up to mark with mobile phase to give a
solution containing 100g/ml ROSUVASTATIN CALCIUM and 100g/ml of EZETIMIBE.
Preparation of sample solution (Tablet Formulation):
For the estimation of the drug in tablet formulation twenty tablets were weighed and their average weight was determined.
The tablets were then finely powdered. Appropriate quantity equivalent to 50mg ROSUVASTATIN CALCIUM and 50 mg
EZETIMIBE ware accurately weighed and The powder was transferred to 100 ml volumetric flask and shaken vigorously with
mobile phase and sonicated for 15 min and volume made up to the mark with mobile phase. The solution was shaken vigorously and
filtered by using whatmann filter no.41. from the above filtered clear solution 5ml of sample pipetted out into a 25 ml volumetric flask
volume made up to the mark with mobile phase to give a solution containing 100g/ml ROSUVASTATIN CALCIUM and 100g/ml
of EZETIMIBE.
RESULTS AND DISCUSSIONS
Determination Of Working Wavelength(max):
5 mg of the ROSUVASTATIN CALCIUM standard drug was taken in a 10 ml volumetric flask and dissolved in methanol
and volume made up to the mark, from this solution 0.2ml is pipetted into 10 ml volumetric flask and made upto the mark with the
methanol to give a concentration of 10 g/ml . The above prepared solution is scanned in uv between 200-400 nm using methanol as
blank. The max was found to be 246nm
5 mg of the EZETIMIBE standard drug was taken in a 10 ml volumetric flask and dissolved in methanol and volume made
up to the mark, from this solution 0.2ml is pipette into 10 ml volumetric flask and made upto the mark with the methanol to give a
concentration of 10 g/ml. The above prepared solution is scanned in uv between 200-400 nm using methanol as blank. The max was
found to be 231nm.
The Isosbestic Point of ROSUVASTATIN CALCIUM and EZETIMIBE were found to be 227nm. The U.V Graph shown in
Figure No. 1.

Figure No. 1: U.V Graph of ROSUVASTATIN CALCIUM and EZETIMIBE.

Page

5831

After several initial trails with mixtures of methanol, water, ACN and buffer in various combinations and proportions, a trail
with a mobile phase mixture of 30mM Ammonium acetate Buffer (pH:3.6) : Acetonitrile (40:60) brought sharp and well resolved
peaks. The chromatogram was shown in Figure No. 2.

www.iajpr.com

Vol 4, Issue 12, 2014.

Putchakayala Purnachandra Rao et al.

ISSN NO: 2231-6876

Figure No. 2: Chromatogram of ROSUVASTATIN CALCIUM and EZETIMIBE.

METHOD VALIDATION:
Linearity:
Linearity was studied by analyzing five standard solutions covering the range of 60-140 g/ml for ROSUVASTATIN
CALCIUM and 60 to 140g/ml for EZETIMIBE of the drug. From the primary stock solution 0.6ml,0.8ml,1.0ml,1.2ml,1.4 ml of
aliquots are pipette into 10 ml volumetric flasks and made up to the mark with the mobile phase to give a concentrations of 60g/mL ,
80g/mL ,100g/mL ,120g/mL and 140 g/mL of ROSUVASTATIN CALCIUM and 60g/mL , 80g/mL ,100g/mL ,120g/mL
and 140 g/mL of EZETIMIBE .
Calibration curve with concentration verses peak areas was plotted by injecting the above prepared solutions and the obtained data
were subjected to regression analysis using the least squares method.
Method precision (repeatability):
The precision of the instrument was checked by repeated injections and measurement of peak areas and retention times of
solutions (n = 6) for,100 g/ml of ROSUVASTATIN CALCIUM and 100 g/ml of EZETIMIBE without changing the parameter of
the proposed chromatographic method.
Limit of detection and limit of quantification:
The limit of detection (LOD) and limit of quantification (LOQ) were separately determined based on standard deviation of
the y-intercept and the slope of the calibration curve by using the below equations.

Page

Accuracy (recovery study):

The accuracy of the method was determined by calculating the recoveries of ROSUVASTATIN CALCIUM and
EZETIMIBE by the standard addition method. Known amounts of standard solutions of ROSUVASTATIN CALCIUM and
EZETIMIBE were added at 10% concentration to pre quantified sample solutions of ROSUVASTATIN CALCIUM (100, 120,
140g/ml) and EZETIMIBE (100, 120, 140g/ml) (Figure No.5.1 and 5.2). The amount of ROSUVASTATIN CALCIUM and
EZETIMIBE recovered was estimated by using the following formulas.

5832

LOD = 3.3 /S
LOQ =10 /S
Where,
= the standard deviation of the response
S = the slope of the calibration curve

www.iajpr.com

Vol 4, Issue 12, 2014.

Putchakayala Purnachandra Rao et al.

ISSN NO: 2231-6876

Specificity:
In an assay, demonstration of specificity requires that it can be shown that the procedure is unaffected by the presence of
impurities or excipients. In practice, this can be done by spiking the drug substance or product with appropriate levels of impurities or
excipients and demonstrating that the assay results are unaffected by the presence of these extraneous materials. There should be no
interference of the diluents, placebo at retention time of drug substances.
Robustness:
Robustness is the measure of a method remain unaffected by small, deliberate changes in method parameters like flow rate
and detection wavelength on assay of the analyte of interest. Here the detection wavelength varied 2nm and flow rate was varied 0.2
ml/min. The results were shown in (Table no.4)
Ruggedness:
The ruggedness of the method was studied by analyzing the sample and standard preparations by two analysts. The %RSD
assay values between two analysts was calculated i.e., (limit <2%).
This indicates the method was rugged. The results were shown in Table no.5.
DISCUSSION
In RP HPLC method, the primary requirement for developing a method for analysis is that the using different solvents and
buffers and columns to get better retention time and theoretical plates, and better cost effective and time saving method than the
previously developed methods. The Isosbestic Point of ROSUVASTATIN CALCIUM and EZETIMIBE were found to be 227nm
(Figure No: 1) by scanning in UV region. The chromatographic method was optimized with mobile phase consisting of 30mM
Ammonium acetate Buffer: Acetonitrile (40: 60) and C18 Phenomenax column. All the validation parameters were studied at a
the wavelength 227nm. Accuracy was determined by calculating the recovery (Table No.3) and the results were in acceptable range
(limit 98-102%). The method was successfully used to determine the amount of ROSUVASTATIN CALCIUM and EZETIMIBE
present in the Tablet. The results obtained were in good agreement with the corresponding labeled amount (Table No.3). The method
was linear in the concentration range of 60 to 140 g/ml for ROSUVASTATIN CALCIUM and 60 to 140g/ml for EZETIMIBE
(Figure no.3 ,Table No.1 and 1.1). Precision was calculated as repeatability (% RSD) for the drug (Table No.6). Robustness and
ruggedness results were in acceptable range (Table No.4 and Table No.5).Summary of all validation parameters for method is given in
Table No.8. By observing the validation parameters, the method was found to be simple, sensitive, accurate and precise. Hence the
method can be employed for the routine analysis ROSUVASTATIN CALCIUM and EZETIMIBE in tablet dosage form.
Table No: 1
Concentration (g/ml)
60
80
100
120
140

Peak Area
1489.674
2287.146
2883.389
3757.693
4368.058

Peak Area
1495.165
2017.299
2555.586
3174.141
3774.290

www.iajpr.com

Page

Concentration (g/ml )
60
80
100
120
140

5833

Table No: 1.1

Vol 4, Issue 12, 2014.

Putchakayala Purnachandra Rao et al.

ISSN NO: 2231-6876

Figure No. 3: Linearity (calibration) curve of ROSUVASTATIN CALCIUM and EZETIMIBE.

Table No.2: LOD and LOQ values Calculated from calibration curve.

LOD
LOQ

g/mL
2.89
8.75

ROSUVASTATIN CALCIUM
SD
Slope
31.6

g/mL
3.65
11.07

36.14

EZETIMIBE
SD

Slope

31.6

28.57

Table No.3: Recovery data.

LEVEL

III

Amount of
Standard Spiked (%)
10%
10%
10%
10%
10%
10%
10%
10%
10%

%Recovery of
ROSUVASTATIN CALCIUM

%Recovery of
EZETIMIBE

101.78%

101.58%

99.81%

98.10%

99.67%

101.89%

Table No.4: Results of Robustness study.

Parameter
Flow Rate(0.8ml)
1.2ml
1.0ml
Wave Length 227nm
225nm
229nm

Rt of ROSUVASTATIN CALCIUM
3.770
2.543
3.033
3.047
3.040
3.041

Parameter
Flow Rate(0.8ml)
1.2ml
1.0ml
Wave Length 227nm
225nm
229nm

Rt of EZETIMIBE
6.170
4.160
5.100
5.103
5.107
5.101

Tailing factor
1.790
1.585
1.823
1.820
1.833
1.807

Tailing factor
1.851
1.676
1.769
1.742
1.709
1.706

www.iajpr.com

Theoretical Plates
2533
2460
2930
2462
2472
2441

Theoretical Plates
3765
3451
3529
3521
3584
3596

5834

II.

1
2
3
1
2
3
1
2
3

Amount of
Sample taken (%)
80
80
80
100
100
100
120
120
120

Page

I.

S.No

Vol 4, Issue 12, 2014.

Putchakayala Purnachandra Rao et al.

ISSN NO: 2231-6876

Table No.5: Results of Ruggedness.

Analyst-1
Analyst-2
Analyst-1
Analyst-2

%Assay
99.04
100.31
99.89
99.47

ROSUVASTATIN CALCIUM
EZETIMIBE

%RSD
0.52%
0.21%

Table No.6: Method Precision (Repeatability).

S.No.
1
2
3
4
5
6
Mean
SD
%RSD

ROSUVASTATIN CALCIUM
Rt
Area
3.097
2883.380
3.080
2866.524
3.080
2993.631
3.090
2913.628
3.073
2909.442
3.083
2920.572
3.0838
2914.530
0.0085
43.807
0.27
1.50

EZETIMIBE
Rt
Area
5.137 2555.586
5.113 2505.453
5.117 2549.701
5.130 2547.409
5.110 2567.784
5.127 2561.177
5.122 2547.852
0.011 22.072
0.21
0.87

Table No.7: Assay Results.

2907.773
2926.735
2893.900
2900.841
2905.211
2906.892
2887.707
2877.263
2900.841
2906.452
2887.707
2891.994
233.9
100.01
1165.6
10
99.65
9.93
99.3

EZETIMIBE
2554.668
2562.605
2549.999
2537.676
2538.187
Average 2548.627
2535.153
2516.744
2537.676
2547.593
2535.153
Average 2534.464
mg
233.9
mg
100.04
mg
1165.6
mg
10
99.11
mg
9.89
%
98.9

mg
mg
mg
mg
%
mg
%

Page

5835

ROSUVASTATIN CALCIUM
Standard Area
1
2
3
4
5
Average
Sample area
1
2
3
4
5
Average
Tablet average weight
Standard weight
Sample weight
Label amount
std.purity
Cal.:
%Assay

www.iajpr.com

Vol 4, Issue 12, 2014.

Putchakayala Purnachandra Rao et al.

ISSN NO: 2231-6876

Table No.8: Validation parameters of evaluated method.

S.
No

Parameter

Limit

1.

ACCURACY(%Recovery)

98-102%

2.

Linearity concentrations Range(

g/mL)
Regression coefficient (R2 value)

NLT 0.99

3.

Precision (% RSD)
Method precision
(Repeatability)
(%RSD, n = 6)

NMT 1% (For Rt)

NMT 2%
(For Area)

4.

Robustness

It should meet System

suitability criteria

Complies

5.

Ruggedness
(Intermediate Precision)
(%RSD analyst to analyst
variation)

NMT2%

0.52% For Rosuvastatin Calcium and 0.21% for

EZETIMIBE

Value Obtained
99.67 to 101.783% (ROSUVASTATIN CALCIUM)
98.10 to101.58% (EZETIMIBE)
60 to 140 g/ml (ROSUVASTATIN CALCIUM)
R2=0.997
and 60 to 140g/ml(EZETIMIBE)
R2=0.9986
%RSD of Rt=0.27% and %RSD of Area 1. 26%
(ROSUVASTATIN CALCIUM)
%RSD of Rt=0.21% and %RSD of Area 0.87%
(EZETIMIBE)

CONCLUSION
From the above experimental results and parameters it was concluded that, this newly developed method for the simultaneous
estimation of ROSUVASTATIN CALCIUM and EZETIMIBE was found to be simple, precise, accurate and high resolution and
shorter retention time makes this method more acceptable and cost effective and it can be effectively applied for routine analysis in
research institutions, quality control department in meant in industries, approved testing laboratories.
ACKNOWLEDGMENTS
We are grateful to Chandra Labs, Prashanthinagar, Kukatpally, Hyderabad, India, for providing gift sample of drugs and
Laboratory for research work. We are thankful to Dr. D Ramachandrarn, Acharya nagarjuna university, Department of chemistry for
constant encouragement.

Page

5836

ABRAVATIONS
HPLC: High Pressure Liquid Chromatography
UV : Ultra violet
IR: Infrared
LOD: Limit of Detection
LOQ: Limit of Quantification
ICH: International Conference on Harmonisation
r2 : Correlation coefficient
Gms : Grams
g/mL : Microgram / Millilitre
ml : Millilitre
mg : Milligram
% : Percentage
% RSD : Percent relative standard deviation
SD: Standard Deviation
min : Minutes
hr : Hour
L : Microlitre
m : Micrometre

www.iajpr.com

Vol 4, Issue 12, 2014.

Putchakayala Purnachandra Rao et al.

ISSN NO: 2231-6876

REFERENCES
1. ICH, Q2A validation of analytical procedure: Methodology International Conference on Harmonization, Geneva, October 1994.
2. ICH, Q2B Validation of analytical procedure: Methodology International Conference on Harmonization, Geneva, March 1996.
3. http://www.drugbank.ca/drugs/DB01098
4. http://www.drugbank.ca/drugs/DB00973
5. http://linkinghub.elsevier.com/retrieve
6. http://www.ncbi.nlm.nih.gov/pubmed/20922955
7. http://www.drugbank.ca/drugs/DB00458
8. http://www.researchgate.net/publication/259139852 RP HPLC method for simultaneous estimation of Rosuvastatin and
Ezetimibe from their combination tablet dosage form
9. http://www.medlineindia.com.

Page

5837

54878478451141248

www.iajpr.com