A.V. Satish Siva Kumar et al.

/ Journal of Pharmacy Research 2012,5(11),5018-5021

Research Article Available online through
ISSN: 0974-6943 http://jprsolutions.info
Simultaneous Determination of Ezetimibe and Simvastatin in Pharmaceutical
Dosage form by Validated RP-HPLC and UV- Spectrophotometric Methods
A.V. Satish Siva Kumar*, C.V. Nagendra Kumar Gupta, M. Lohita, V. Amrutha, P. Ramalingam.
*Centre for Pharmaceutical Research, Division of Pharmaceutical Analysis and Quality assurance,
Raghavendra Institute of Pharmaceutical Education and Research (RIPER), Anantapur Dist, India – 515 721
Received on:17-06-2012; Revised on: 19-07-2012; Accepted on:24-08-2012

ABSTRACT
A simple, rapid and precise method is developed for the quantitative simultaneous determination of Ezetimibe and Simvastatin in combined pharmaceu-
tical-dosage forms. Two methods are described for the simultaneous determination of Ezetimibe and Simvastatin. The first method was based on UV-
Spectrophotometric determination of two drugs, using simultaneous equation method. It involves absorbance measurement at 228.8 nm (λmax of Ezetimibe)
and 238.4 nm (λmax of Simvastatin) Methanol: Water (90:10); For UV Spectrophotometric method, linearity was obtained in concentration range of 2 – 18
µg/ml, for both the drugs; with regression 0.999 and 0.999, intercepts 0.004 and 0.005 and slope 0.040 and 0.062 for Ezetimibe and Simvastatin
respectively. Recovery was in the range of 99 –107%; the value of standard deviation and % R.S.D. were found to be < 2 %; shows the high precision of
the method.. The second method was based on HPLC separation of the two drugs in reverse phase mode using these C18, 4.5µ (250 X 4.6 mm). The
accuracy and reliability of the method was assessed by evaluation of linearity (1-18 µg/spot for both Ezetimibe and Simvastatin), precision (intra-day and
inter-day % RSD >2 for Ezetimibe and Simvastatin), accuracy (98-102% for Ezetimibe and Simvastatin) and specificity, in accordance with ICH
guidelines. Both these methods have been successively applied to pharmaceutical formulation and were validated according to ICH guidelines.

Key words: RP-HPLC, UV-spectrophotometer, fixed dose combinations, Ezetimibe, Simvastatin.

INTRODUCTION
Ezetimibe (Fig 1), (1-(4-flurophenyl)-3(R)-[3(S)-(4-flurophenyl) –3- dimethylbutanoate. This drug , which acts by inhibiting 3-hydroxy-3 methyl
hydroxypropyl] –4(S) (4 –ydroxyphenyl) azetidin- 2-one), which belongs lutarylcoA reductase is used in the treatment of hypercholesterolemia.
to a group of selective and very effective 2-azetidione cholesterol absorption
inhibitors acts at the level of cholesterol entry into enterocytes [3]. It prevents
transport of cholesterol through the intestinal wall by selectively blocking
the absorption of cholesterol from dietary and billiary sources. This reduces
the overall delivery of cholesterol to the liver, thereby promoting the
synthesis of LDL receptors and a subsequent reduction in serum LDL-C [4-
5]
. Clinical studies have shown that coadministration of ezetimibe with
statins could provide an additional reduction in LDL cholesterol as well as
total cholesterol [6].

Fig 2- Structure of Simvastatin.

A few methods based on HPLC [7-9], UV [10], LC-MS [11, 12] and GC-MS [13]
were reported earlier for the determination of Simvastatin individually and
in combination with other drugs. A few analytical procedures were also
proposed for the determination of ezetimibe in dosage forms [14] in human
Fig 1- Structure of Ezetimibe.
serum, urine and feces [15]. Although the combinational use of Simvastatin
and ezetimibe is continuously increasing, simultaneous analysis of these
Simvastatin (Fig 2) is a methylated analog of lovastatin, is the lactone form two components in their pharmaceutical preparation is not official in Indian
of (1S, 3R, 7S, 8S, 8aR)-8-{2-[(2R, 4R)-4-hydroxy-6-oxo oxotetraHydro- Pharmacopoeia, British Pharmacopoeia, United states and European
2-yl]ethyl}-3, 7-dimethyl-1, 2, 3, 7,8,8a-hexahydroNapthalen-1-yl2,2 Pharmacopoeia. There is an urgent need to develop and validated analytical
methods for the simultaneous analysis of Simvastatin and Ezetimibe in
*Corresponding author. pharmaceutical dosage forms. We describe herein a simple, sensitive and
A.V. Satish Siva Kumar, validated stability indicating HPLC method utilizing isocratic mobile phase
Pharmaceutical Analysis and Quality Assurance, with short retention time for the simultaneous determination of these two
Raghavendra Institute of Pharmaceutical Education components in pharmaceutical formulations like tablets. The developed
and Research (RIPER), method can be successfully applied to quality control and other analytical
KR palli cross, Chiyyedu, purposes.
Anantapur Dt, India – 515 721.

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 A.V. Satish Siva Kumar et al. / Journal of Pharmacy Research 2012,5(11),5018-5021
EXPERIMENTAL /min and UV detection was carried out at 232 nm. Stock solution was
prepared by dissolving 10 mg of Ezetimibe and Simvastatin in 100 mL
Materials and Reagents volumetric flask with Mobile phase (MeOH:H2O – 90:10). From the above
Working standards of pharmaceutical grade Simvastatin and Ezetimibe stock solution, dilutions were made in the conc. range of 1-18µg/mL of
obtained as generous gifts from Hetero Drugs Erragadda (Hyderabad, India), Ezetimibe and Simvastatin respectively. A volume of 20µL is injected into
respectively. Fixed-dose combination tablets (Simvas–EZ), containing 10 the column. All measurements were repeated three times for each
mg Simvastatin and 10 mg Ezetimibe were procured from Hetero Drugs. concentration and calibration curve was constructed by plotting the peak
Chemicals and reagents of analytical-grade were purchased from Merck area ratios of analyte to the corresponding drug concentration.
grade and were purchased from Qualigens fine Chemicals, Mumbai, India.
Analysis of Pharmaceutical Dosage Forms
Preparation of Mobile Phase and Stock Solution To determine the content of Ezetimibe and Simvastatin simultaneously in
tablets (label claim: 10 mg Simvastatin and 10 mg Ezetimibe, film coated);
UV- Spectrophotometer twenty tablets were weighed; their average weight determined and were
UV-Vis Double beam spectrophotometer 2200 (Systronics) with spectral finely powdered. The correct amount of powder was dissolved in mobile
bandwidth of 2 nm and 10mm matched quartz cells was used. Standard phase by stirring for 30 min. The excipients were separated by filtration.
stock 10 mg of each in 10 mL of (MeOH: H2O-9:1). From these stock Appropriate aliquots were subjected to above methods and the amount of
solutions, working standard solutions having concentration 10 µg / mL each Simvastatin and Ezetimibe were determined and was reported in Table 1.
were prepared by appropriate dilutions. They were scanned in the
Table 1: Analysis Data of Tablet Formulations.
wavelength range of 200- 400 nm and the overlain spectrum was obtained
(Fig 3). Two wavelengths 228.8 nm (λmax of Ezetimibe) and 238.4 nm (λmax S. No. Parameters UV-Spectrophotometer RP-HPLC
of Simvastatin) were selected for the formation of simultaneous equation. Ezetimibe Simvastatin Ezetimibe Simvastatin
The calibration curves were found to be linear in the concentration range of
2- 18 µg/mLG1, for each drug. The absorptivity coefficients of each drug at 1 Label Claim (mg) 10 10 10 10
both wavelengths were determined. The concentration of two drugs in the 2 *Drug Content 10.03 10.5 10.009 10.234
mixture were calculated using equations, 3 ± S.D 0.014 0.212 0.062 0.713
4 % R.S.D 0.282 0.140 0.619 0.791
CEze = A2 ay1 – A1 ay2/ ax2 ay1 – ax1ay2 (1)
* Value for drug content (mg) is the mean of 3 estimations; S.D is Standard
CSim = A1 ax2 – A2 ax2/ ax2 ay1 – ax1ay2 (2) Deviation and R.S.D. is Relative Standard Deviation.

Where A1 and A2 are absorbance of mixture at 228.8 nm and 238.4 nm; ax1
Recovery Studies
and ax2, absorptivities of Ezetimibe at 228.8 nm and 238.4 nm, respectively;
To check the accuracy of the developed methods and to study the interference
ay1 and ay2, absorptivities of Simvastatin at 228.8 nm and 238.4 nm,
of formulation additives, analytical recovery experiments were carried out
respectively. C Eze and CSim are concentrations of Ezetimibe and Simvastatin by standard addition method, at 80, 100 and 120 % level. From the total
in mixture. amount of drug found, the percentage recovery was calculated. The results
are reported in Table 2.
Table 2: Recovery Studies.
UV-Spectrophotometer RP-HPLC

Excess Drug *Recovery %R.S.D Excess Drug *Recovery %R.S.D

Ezetimibe Ezetimibe

80 9.04±0.014 0.155 80 9.07±0.01 0.11

100 9.93±0.021 0.213 100 9.86±0.04 0.04
120 10.88±0.014 0.129 120 11.07±0.04 0.36
Simvastatin Simvastatin
80 8.83±0.049 0.554 80 9.04±0.009 0.09
100 10.04±0.028 0.028 100 9.83±0.01 0.01
120 11.1±0.028 0.254 120 11.05±0.05 0.452

*Recovery is the mean of Three Estimations.

Fig 3- Wavelength selection of Ezetimibe and Simvastatin.
HPLC Method
LC system used consisted of pump (model Agilent; LC – 1120) with universal
loop injector (Rheodyne 7725 i) of injection capacity 20 µL. Detector
consists of UV Detector; the column used Phenomenox Luna C18, 4.5µ
(250 X 4.6 mm) at ambient temperature. Different mobile phases were
tested in order to find the best conditions, for separating both the drugs
simultaneously. The optimal composition of mobile phase was determined
to be Methanol and Water in the ratio 90:10. The flow rate was set to 1 mL
RESULTS AND DISCUSSION
Both, UV Spectrophotometric, RP-HPLC methods were found to be simple,
accurate, economic and rapid for routine simultaneous estimation of
Ezetimibe and Simvastatin, in tablet dosage forms. For UV
Spectrophotometric method, linearity was obtained in concentration range
of 2 – 18 µg / mL, for both the drugs; with regression 0.9998 and 0.9999,
intercept – 0.004 and – 0.005 and slope 0.04 and 0.062 for Ezetimibe and
Simvastatin, respectively. Recovery was in the range of 100 – 105 %; the
value of standard deviation and % R.S.D. were found to be < 2 %; shows
the high precision of the method. The all parameters are shown in Table 3.

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Table 3: Parameters of UV – Spectrophotometer.
Parameter Ezetimibe Simvastatin

Absorption maximum (nm) 228.8 238.4

Beer’s limit (µg/ml) 2-18 2-18
Regression equation
a. Slope 0.04 0.062
b. Intercept 0.004 0.005
c. Correlation coefficient 0.999 0.999
Molar Absorptivity (M-1cm-1) 16376 26156
Sandell’s Sensitivity (ng) 25 16
% RSD >2 >2
LOD & LOQ (µg/ml) 0.33 % 1 0.26 & 0.8

In HPLC method, HPLC conditions were optimized to obtain, an adequate

separation of eluted compounds. Initially, various mobile phase compositions
were tried, to separate drugs. Mobile phase and flow rate selection was
based on peak parameters (height, tailing, theoretical plates, capacity factor),
run time etc. The system with Methanol: Water (90: 10 v/v) with 1 mL /
min flow rate is quite robust. A typical UV spectrum and HPLC
chromatogram for Ezetimibe, Simvastatin is shown in Fig 4 and 5. The
optimum wavelength for detection was 232 nm at which better detector
response for drugs was obtained. The average retention times for Ezetimibe
and Simvastatin was found to be 3.573 and 5.687 min, respectively.
According to USP XXIV (621), system suitability tests are an integral part
of chromatographic method. To ascertain its effectiveness, system suitability
tests were carried out on freshly prepared stock solutions. The parameters
obtained are shown in Table 4. The calibration was linear in concentration
range of 1 – 18 µg / mL, with regression 0.999 and 0.999, intercept - 14296
and – 555.9 and slope 12200 and 14446 for Ezetimibe and Simvastatin,
respectively. The low values of % R.S.D. indicate the method is precise and
accurate. The mean recoveries were found in the range of 98 – 102 %.
Fig 5- Chromatogram of Ezetimibe and Simvastatin.
Sample to sample precision and accuracy were evaluated using, three samples
of three different concentrations, which were prepared and analyzed on
same day. Day to day variability was assessed using three concentrations
analyzed on three different days, over a period of one week. These results
show the accuracy and reproducibility of the assay. Thus, it was concluded
that there was no significant difference on the assay, which was tested on an
intra – day and inter – day basis. The % R.S.D. values reported in Table 5,
shows that proposed methods provides acceptable intra – day and inter –
day variation of Ezetimibe and Simvastatin.
The proposed methods are accurate, simple, rapid and selective for the
simultaneous estimation of Ezetimibe and Simvastatin in tablet dosage
forms. Hence, it can be conveniently adopted for the routine quality control
analysis in the combination formulations. As the drug combination is available
in market, hence, work is toward development of an analysis.

ACKNOWLEDGEMENT
The authors are thankful to Hetero Drugs (HYD), for providing drug samples
and Raghavendra Institute of Pharmaceutical Education and Research
(RIPER), Anantapur, Andhra Pradesh, India, for providing facilities to
carry out this work.

REFERENCES
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Source of support: Nil, Conflict of interest: None Declared

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