Abstract A simple, rapid reverse phase high-performance liquid chromatographic method was developed and validated for the simultaneous estimation of Ezetimibe and Simvastatin in bulk and pharmaceutical dosage forms. Chromatography was carried out by using Chromosil C-18,column having 250 x 4.6mm internal diameter with a mixture of Methanol:Acetonitrile:0.1%Orthophosphoric Aid in the ratio of 75:20:05 (v/v/v) as mobile phase. Determination of the different analytical parameters such as linearity, precision, accuracy, and specificity, limit of detection (LOD) and limit of quantification (LOQ) was done. The calibration curve was found to be linear for each analyte in the desired concentration range. The average recovery was found to be 99.88 and 100.12 for Ezetimibe and Simvastatin respectively. The proposed method is highly sensitive, precise and accurate, which was evident from the LOD value of 1.2ppm and 0.25ppm for Ezetimibe and Simvastatin respectively and hence the present method can be applied successfully for the quantification of active pharmaceutical ingredient (API) content in the combined formulations of Ezetimibe and Simvastatin

G Krishnaveni

P V V Sathyannarayana

CiteSeerX

International Journal of Pharmaceutical and Life Sciences 
ISSN 2305-0330 
Volume 2, Issue 2: March 2013 
Page 60 
 
METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS DETERMINATION 
OF EZETIMIBE AND SIMVASTATIN IN COMBINED PHARMACEUTICAL DOSAGE 
FORM BY RP-HPLC METHOD 
G.Krishnaveni*1, P.V.V. Sathyannarayana.2  
 
1Department of Chemistry, K.B.N.College, Vijayawada, India.  
2Professor of Chemistry (Retired), ANU, Nagarjuna Nagar, India. 
 
Abstract 
A simple, rapid reverse phase high-performance liquid chromatographic method was 
developed and validated for the simultaneous estimation of Ezetimibe and Simvastatin in 
bulk and pharmaceutical dosage forms. Chromatography was carried out by using Chromosil 
C-18,column having 250 x 4.6mm internal diameter with a mixture of 
Methanol:Acetonitrile:0.1%Orthophosphoric Aid in the ratio of 75:20:05 (v/v/v) as mobile 
phase. Determination of the different analytical parameters such as linearity, precision, 
accuracy, and specificity, limit of detection (LOD) and limit of quantification (LOQ) was done. 
The calibration curve was found to be linear for each analyte in the desired concentration 
range. The average recovery was found to be 99.88 and 100.12 for Ezetimibe and 
Simvastatin  respectively. The proposed method is highly sensitive, precise and accurate, 
which was evident from the LOD value of 1.2ppm and 0.25ppm for Ezetimibe and 
Simvastatin respectively and hence the present method can be applied successfully for the 
quantification of active pharmaceutical ingredient (API) content in the combined formulations 
of Ezetimibe and Simvastatin. 
 
Key Words: Ezetimibe, Simvastatin, HPLC, Development, Validation. 
 
 
 
 
 
 
 
 
 
 
 
*Corresponding Author  
G. Krishnaveni  
e-Mail id: chemperuri@yahoo.co.in 
 
International Journal of Pharmaceutical and Life Sciences 
ISSN 2305-0330 
Volume 2, Issue 2: March 2013 
Page 61 
 
Introduction 
Ezetimibe is a drug that lowers cholesterol. It acts by decreasing cholesterol absorption in 
the intestine. It may be used alone (marketed as Zetia or Ezetrol), when other cholesterol-
lowering medications are not tolerated, or together with statins (e.g.,ezetimibe/simvastatin, 
marketed as Vytorin and Inegy) when statins alone do not control cholesterol. 
Even though ezetimibe decreases cholesterol levels, the results of two major, high-quality 
clinical trials (in 2008 and 2009) showed that it did not improve clinically significant 
outcomes, such as major coronary events, and actually made some outcomes, such as 
artery wall thickness, worse. Indeed, a panel of experts concluded in 2008 that it should 
"only be used as a last resort".[1] In one of those studies, a head-to-head trial in 2009, a 
much less expensive medication (extended-release niacin) was found to be superior. 
Ezetimibe actually increased the thickness of artery walls (a measurement of 
atherosclerosis) and caused more major cardiovascular events.[2] A more positive view of the 
benefits of Ezetimibe is offered by Britain's NICE statement which however was published in 
2007 and may not have been updated to reflect the results of the above mentioned trials.[3] 
 
Figure 1: Stricture of Ezetimibe 
Ezetimibe localises at the brush border of the small intestine, where it inhibits the absorption 
of cholesterol from the intestine. 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[4] as well as in hepatocytes.[5] In addition to this direct effect, decreased 
cholesterol absorption leads to an upregulation of LDL-receptors on the surface of cells and 
an increased LDL-cholesterol uptake into cells, thus decreasing levels of LDL in the blood 
plasmawhich contribute to atherosclerosis and cardiovascular events.[6] 
Common adverse drug reactions (≥1% of patients) associated with ezetimibe therapy are 
headache and/or diarrhea (steathorrea). Infrequent adverse effects (0.1–1% of patients) 
include: myalgia and/or raised liver function test (ALT/AST) results. Rarely (<0.1% of 
patients), hypersensitivity reactions (rash, angioedema) or myopathy may occur.[7] Side-
effects include gastro-intestinal disturbances; headache, fatigue; myalgia; rarely arthralgia, 
hypersensitivity reactions (including rash, angioedema, and anaphylaxis, hepatitis; very 
rarely pancreatitis, cholelithiasis, cholecystitis, thrombocytopenia, raised creatine 
kinase, myopathy, and rhabdomyolysis.[8] 
Simvastatin is a hypolipidemic drug used to control elevated cholesterol, 
or hypercholesterolemia. It is a member of the statin class of pharmaceuticals. 
Simvastatin is a synthetic derivate of a fermentation product of Aspergillus terreus. The drug 
is marketed under the trade name Zocor, as well as generically. 
International Journal of Pharmaceutical and Life Sciences 
ISSN 2305-0330 
Volume 2, Issue 2: March 2013 
Page 62 
 
 
 
Figure 2: Stricture of Simvastatin 
The primary uses of simvastatin is for the treatment of dyslipidemia and the prevention 
of cardiovascular disease.[1] It is recommended to be used only after other measures such 
as diet, exercise, and weight reduction have not improved cholesterol levels.[9] 
Common side effects (>1% incidence) may include abdominal pain, diarrhea, indigestion, 
and a general feeling of weakness. Rare side effects include joint pain, memory loss, and 
muscle cramps.[10] Cholestatic hepatitis, hepatic cirrhosis, rhabdomyolysis and myositis have 
been reported in patients receiving the drug chronically.[11] 
 
Method and Material 
Chemicals and Reagents  
Ezetimibe   and Simvastatin as pure standard reference drugs were purchased from Reddy’s 
Laboratory, Hyderabad and pharmaceutical formulation from local market were used for this 
present study. Acetonitrile, Methanol and Orthophosphoric acid (all HPLC grade) were 
purchased from Merck Specialties Private Limited, Mumbai, India. 
 
Instrumentation  
To develop a High Pressure Liquid Chromatographic method for quantitative estimation of 
Simvastatin  and Ezetimibe  , an isocratic PEAK HPLC instrument with Hypersil C18 column 
(250 mm x 4.6 mm, 5μ) was used. The instrument is equipped with a LC 20AT pump for 
solvent delivery and variable wavelength programmable LC – 7000 UV-detector. A 20μL 
Rheodyne inject port was used for injecting the samples. Data was analyzed by using PEAK 
software. UV-2306 Spectrophotometer was used for wavelength checking. Denver analytical 
Balance was used to weigh the drug. 
 
Experimental Condition  
Flow rate of the mobile phase was changed from 0.5 – 1.5 ml/min for optimum separation. A 
minimum flow rate as well as minimum run time gives the maximum saving on the usage of 
solvents. It was found from the experiments that 1.0 ml/min flow rate was ideal for the 
successful elution of the analyte. The HPLC system was hence operated using an isocratic 
mode at a flow rate of 1.0 ml/min at 25 ± 2ºc. For analysis the most suitable mobile phase 
International Journal of Pharmaceutical and Life Sciences 
ISSN 2305-0330 
Volume 2, Issue 2: March 2013 
Page 63 
 
was found to be Methanol, Acetonitrile and 0.1% Orthophosphoric Acid 75:20:05 Detection 
was carried out at wavelength of 243 nm.  
 
Preparation of Mobile Phase  
For the preparation of mobile phase suitable for the present determination Methanol, 
Acetonitrile and 0.1% Orthophosphoric Acid of HPLC grade were mixed, filtered and 
degassed in such a way that the final volume consisted of these in the ratio 75:20:05 
respectively, whose pH was found to be to 5.6 
 
Preparation of mixed standard solution  
Ezetimibe   and Simvastatin (1mg/ml) standard stock solutions were prepared using mobile 
phase as a solvent. Aliquots of mixed standard solutions of Ezetimibe   and Simvastatin 
were diluted in mobile phase to get a final concentration of 50-100ppm. 
 
Preparation of sample solution of pharmaceutical formulation  
Pharmaceutical form containing 10 mg of Ezetimibe   and 10 mg of Simvastatin  was 
weighed and dissolved in 25 ml of mobile phase and sonicated for 15 min. Using methanol 
the volume was made up to 50 ml and filtered through 0.45μ membrane filter. The final 
mixed sample solution corresponding to 70 ppm of Ezetimibe and 70 ppm of Simvastatin 
was prepared. 
 
Recording of chromatograms  
After stabilization of the base line with the optimized chromatographic conditions standard 
solutions containing 50-100 ppm of Ezetimibe   and Simvastatin were injected and the 
corresponding chromatograms were recorded. Retention time of Ezetimibe   and Simvastatin 
were found to be 3.30 and 6.17 mins respectively. Likewise for sample solution 
chromatograms were recorded. Calibration curves were plotted using peak area retentions 
of standard drug peaks against concentration of corresponding standard solutions.  
 
Results and discussion  
Method validation  
The method was validated by determining linearity, precision, accuracy, specificity, 
ruggedness and robustness by analyzing 50-100 ppm of both Ezetimibe   and Simvastatin.  
 
 
S.NO TEST RESULT 
 
 
H.P.L.C  CONDITIONS  
1 ELUTION ISOCRATIC 
2 A.P.I       CONC 70ppm 
3 MOBILE PHASE Methanol:Acetonitrile:0.1%Orthophosphoric Aid 
International Journal of Pharmaceutical and Life Sciences 
ISSN 2305-0330 
Volume 2, Issue 2: March 2013 
Page 64 
 
S.NO TEST RESULT 
75:20:5 
4    PH 5.6 
5 COLUMN C18 
6 WAVE LENGTH 243nm 
7 FLOW 1ml\min 
8 RUNTIME 10min 
9 RETENSION TIME Ezetimibe  3.30 
Simvastatin  6.17 
10 AREA Ezetimibe  271253 
Simvastatin  366363 
11 TH.PLATES Ezetimibe  7684 
Simvastatin  24004 
12 TAILING FACTOR Ezetimibe  1.90 
Simvastatin  1.53 
13 PUMP PRESURE 9.8psi 
Table 1: Optimized chromatographic conditions for estimation of Ezetimibe   and 
Simvastatin  
 
Linearity 
The linearity of the response for Ezetimibe   and Simvastatin  assay method was determined 
by preparing and injecting standard solutions of Ezetimibe   and Simvastatin . The linear 
regression data for the calibration curves indicate that the response is linear over the 
concentration range studied with correlation coefficient (r2) value, slope and intercept as 
shown in table 3. 
 
Graph 1: Calibration Plot for Ezetimibe   and Simvastatin  
International Journal of Pharmaceutical and Life Sciences 
ISSN 2305-0330 
Volume 2, Issue 2: March 2013 
Page 65 
 
S.NO CONC IN 
PPM 
EZETIMIBE SIMVASTATIN 
1 50 199726 278226 
2 60 238655 326584 
3 70 271253 366363 
4 80 305211 427588 
5 90 342830 486767 
6 100 388644 543348 
Table.2: Linearity results 
 
Parameters Ezetimibe   Simvastatin  
Calibration range (ppm) 50-100 50-100 ppm 
Intercept 3285 1063 
Slope 3829 5380.96 
Correlation coefficient (r2) 0.999 0.999 
                                   Table 3: Regression analysis of the calibration curve 
Precision 
The precision of the assay was studied with respect to both repeatability and intermediate 
precision. Repeatability was calculated from six replicate injections of freshly prepared 
Ezetimibe   and Simvastatin  combined test solution in the same equipment at a 
concentration value of 70 ppm on the same day. The experiment was repeated by assaying 
freshly prepared solution at the same concentration additionally on two consecutive days to 
determine intermediate precision. Peak areas of the drugs were determined and precision as 
% RSD was reported.  
S.NO CONCENTRATION Ezetimibe   peak 
area 
Simvastatin  peak 
area 
1 70 PPM 271253 366363 
2 70 PPM 272219 365322 
3 70 PPM 272481 367819 
4 70 PPM 271685 365174 
5 70 PPM 272051 365315 
6 70 PPM 273272 367534 
  %R.S.D = 0.25 %R.S.D = 0.32 
Table.4 Intraday precision 
 
International Journal of Pharmaceutical and Life Sciences 
ISSN 2305-0330 
Volume 2, Issue 2: March 2013 
Page 66 
 
S.NO CONCENTRATION Ezetimibe   peak 
area 
Simvastatin  peak 
area 
1 70 PPM 274666 365521 
2 70 PPM 266547 365174 
3 70 PPM 268688 368714 
4 70 PPM 271303 365315 
5 70 PPM 272481 365174 
6 70 PPM 268039 366697 
  %RSD = 1.23 %RSD = 0.4 
Table.5 Inter day precision 
Parameters Ezetimibe   Simvastatin  
Theoretical plates 
(N) 
7684 24004 
Retention time 
(min) 
3.30 6.17 
Tailing factor 1.90 1.53 
LOD (ppm) 1.2ppm 0.25ppm 
LOQ (ppm) 4ppm 0.8ppm 
R.S.D. (%) 0.750.35 0.4 
                                Table 6.System suitability and validation parameters 
 
Figure.2 Typical chromatogram of standard Ezetimibe   and Simvastatin  
 
International Journal of Pharmaceutical and Life Sciences 
ISSN 2305-0330 
Volume 2, Issue 2: March 2013 
Page 67 
 
Recovery  
The recovery of the standard solutions was done by adding them to pre-analyzed sample 
solution at different levels i.e. 50%, 100%, and 150% separately to study the accuracy of the 
above method. The corresponding results were recorded. 
 
Recovery Conc. of sample EZETIMIBE  SIMVASTATIN EZETIMIBE % 
of recovery 
SIMVASTATIN 
% of recovery 
50% 50ppm 50.33 50.10 100.66 100.20 
75% 75ppm 74.92 74.85 99.89 99.8 
100 % 100ppm 99.1 100.35 99.1 100.35 
Table.7 Recovery of Olmesaartan, Simvastatin  
 
Specificity  
Specificity was performed to exclude the possibility of interference with excipients in the 
region of elution of Ezetimibe   and Simvastatin . The specificity and selectivity of the method 
was tested under normal conditions and the results of the tests proved that the components 
other than the drug did not produce a detectable signal at the retention place of Ezetimibe   
and Simvastatin. 
 
Limit of detection (LOD) and limit of quantification (LOQ)  
LOD and LOQ were determined from standard deviation of y-intercept of regression line and 
slope method as per ICH guidelines.  
 
Robustness 
Typical variations in liquid chromatography conditions were used to evaluate the robustness 
of the assay method. In this study, the chromatographic parameters monitored were 
retention time, area, capacity factor, tailing factor and theoretical plates. The robustness 
acceptance criteria set in the validation were the same established on system suitability test 
describe above. 
 
PARAMETER MODIFICATION EZETIMIBE 
AREA 
SIMVASTATIN 
AREA 
EZETIMIBE 
%OF 
CHANGE 
SIMVASTATIN 
% OF 
CHANGE 
Standard …………. 271253 366363 …….. …….. 
MP MeOH:CAN:0.1%OPA 
70:25:5 
273238 366694 0.73 0.09 
PH 5.1 270644 366121 0.225 0.07 
Wavelength 249nm 270583 365457 00.25 0.247 
Table.8 Robustness study 
 
International Journal of Pharmaceutical and Life Sciences 
ISSN 2305-0330 
Volume 2, Issue 2: March 2013 
Page 68 
 
Analysis of marketed formulations  
The validated HPLC method was adopted for the quantification of Ezetimibe   and 
Simvastatin  in their combined pharmaceutical dosage form and the typical chromatograms 
of the formulation are shown in fig. The results of analysis are given in Table 8. The contents 
of the pharmaceutical dosage form were found to be in the range of 100±2% with RSD less 
than 2% which indicate suitability for routine analysis of Ezetimibe   and Simvastatin  in 
pharmaceutical dosage forms. 
 
Drug FORMULATION DOSAGE SAMPLE 
CONC 
DRUG 
ESTIMATED 
% OF DRUG 
ESTIMATED 
EZETIMIBE 
 
VYTORIN 10 mg 70 ppm 69.87 99.81 
SIMVASTATIN VYTORIN 10 mg 70 ppm 69.71 99.586 
Table.9 Formulation 
 
Conclusion 
The proposed study describes a new RP-HPLC method using simple mobile phase for the 
estimation of Ezetimibe   and Simvastatin in combined pharmaceutical dosage formulations. 
The method was validated and found to be simple, sensitive, accurate and precise. It was 
also proved to be convenient and effective for the determination of Ezetimibe   and 
Simvastatin in the pharmaceutical dosage form. The percentage of recovery shows that the 
method is free from interference of the excipients used in formulation. Moreover, the lower 
solvent consumption along with the short analytical run time leads to cost effective 
chromatographic method. 
 
References 
1.  Mitka M (May 2008). "Cholesterol drug controversy continues". JAMA 299 (19): 
2266.  
2.  Taylor A.J., Villnes T.C., Stanek E.J., et al. (26 November 2009). "Extended-Release 
Niacin or Ezetimibe and Carotid Intima-Media Thickness". N Engl J Med 361 (22): 
2113–22 
3.  , http://guidance.nice.org.uk/TA132 
4.  Garcia-Calvo M, Lisnock J, Bull HG, Hawes BE, Burnett DA, Braun MP, et al. The 
target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1). Proc Natl Acad Sci U S 
A 2005;102(23):8132-7.  
5.  Temel, Ryan E., Tang, Weiqing, Ma, Yinyan, Rudel, Lawrence L., Willingham, Mark 
C., Ioannou, Yiannis A., Davies, Joanna P., Nilsson, Lisa-Mari, Yu, Liqing. Hepatic 
Niemann-Pick C1-like 1 regulates biliary cholesterol concentration and is a target of 
ezetimibe J. Clin. Invest. 2007 0: JCI30060 
International Journal of Pharmaceutical and Life Sciences 
ISSN 2305-0330 
Volume 2, Issue 2: March 2013 
Page 69 
 
6.  DiPiro JT, Talbert RL, Yee GC, Marzke GR, Wells BG, Posey LM, editors. 
Pharmacotherapy:a pathophysiologic approach. 7th ed. New York: The McGraw-Hill  
7. Companies, Inc.; 2008. 
8.  Rossi S, editor. Australian Medicines Handbook 2006. Adelaide: Australian 
Medicines Handbook; 2006.  
9.  http://www.hc-sc.gc.ca/dhp-mps/medeff/advisories-avis/public/_2005/ezetrol_pa-ap-
eng.php 
10. "Simvastatin". The American Society of Health-System Pharmacists. Retrieved 3 
April 2011. 
11. "Gen-Simvastatin - Drug Factsheets - C-Health". Retrieved 2007-08-15. 
12. R. Baselt, Disposition of Toxic Drugs and Chemicals in Man, 8th edition, Biomedical 
Publications, Foster City, CA, 2008, pp. 1431-1433. 


METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS DETERMINATION OF EZETIMIBE AND SIMVASTATIN IN COMBINED PHARMACEUTICAL DOSAGE FORM BY RP-HPLC METHOD

http://citeseerx.ist.psu.edu/viewdoc/download;jsessionid=3D290C0B854301F0934B74693E66F3D2?doi=10.1.1.1053.8712&rep=rep1&type=pdf

METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS DETERMINATION OF EZETIMIBE AND SIMVASTATIN IN COMBINED PHARMACEUTICAL DOSAGE FORM BY RP-HPLC METHOD

Abstract

Similar works

Full text

Available Versions

CiteSeerX