Simultaneous RP-HPLC Method for Estimation of Ezetimibe and Simvastatin in Bulk and Dosage Forms

R Siva Kumar*, MR Santhanakrishnan, P Kumar Nallasivan and R Venkatanarayanan

Dept. Pharma. Analysis, RVS College of Pharmaceutical Sciences, Sulur, Coimbatore- 641 402. Tamilnadu, India.

 

* Corresponding Author E-mail: andrilan@rediffmail.com

 

 

ABSTRACT

A simple, specific, accurate, precise, rapid and reproducible reverse HPLC method has been developed for the simultaneous estimation of ezetimibe and simvastatin in bulk and tablet dosage forms using C18 column (symmetry, 4.6 mm x 25 cm) in isocratic mode with methanol: water (95:05 v/v) as a mobile phase with the detection wavelength of 248 nm and the flow rate was 0.8 ml/ min. The retention time of ezetimibe and simvastatin was found to 3.2 min and be 4.9 min, respectively. Linearity of the ezetimibe and simvastatin was found in the range of 5-70 µ g/ml and 5 - 50 µ g/ml, respectively. The recoveries of ezetimibe and simvastatin were found to be in the range of 99.27-101.10% and 99.63-100.50%, respectively. The proposed method was validated suitably and can be used for routine analysis.

 

 KEY WORDS              Simultaneous estimation, RP-HPLC method, Simvastatin, Ezetimibe.

 

INTRODUCTION:

Ezetimibe (EZT) and Simvastatin (SMT) combination tablets  are  newly  marketed  and  both  are  used  as hypolipidemic   agents.   EZT   chemically   is,   1-(-4- Fluorophenyl)-(3R)-[3-(4-fluorophenyl)-3(S)-hydroxy propyl]– (4S)-(4-hydroxyphenyl)-2-azetidinone. It is a selective   cholesterol   absorption   inhibitor,   which potentially inhibits the absorption of biliary and dietary cholesterol. Additional studies in human indicated that EZT does not affect serum fat-soluble vitamins1. SMT is chemically  butanoic acid, 2,2-dimethyl -1,2,3,7,8,8a hexahydro-3,7-dimethyl-8-{2-[tetrahydro-4-hydroxy-6- oxo-2H-pyran-2-yl]-ethyl}-1-naphthalenyl ester. It is a HMG  CoA  reductase  inhibitor,  which  blocks  the synthesis  of  cholesterol  in  liver  by  competitively inhibiting  HMG  CoA  reductase  activity2.  SMT  is official in British Pharmacopoeia and also in United States  Pharmacopoeia.  EZT  is  not  official  in  any Pharmacopoeias.       Few       methods       such       as spectrophotometric method 3-7, HPLC method 8-13  and HPTLC  method14  have  been  reported  for  individual drugs   in   formulation.   One   UV-spectrophotometric method15  and one HPTLC16   method is available for simultaneous  estimation  of  these  combinations.  The reported procedures are time consuming, expensive and relatively complicated. The aim of this study was to develop a precise, specific, fast, accurate and sensitive method  for  simultaneous determination of  EZT  and SMT combination by RP-HPLC method.

 

MATERIALS AND METHODS:

EZT  and  SMT  were  obtained  from  Micro  Labs,  Hosur, India. Methanol HPLC grade of Rankem, water HPLC grade of Qualigens were used for the study. The liquid chromatographic system consisted of the following components: Waters  HPLC  system containing 515  pump system, with dual wavelength absorbance detector 2487 and Rheodyne injector with 20 µ l fixed loop. Chromatographic analysis was performed using Empower software on a symmetry  C18 column  (250x4.5  mm)  at  ambient temperature.

 

Preparation of mobile phase:

Mobile Phase was prepared by mixing 950 ml of methanol, with 50 ml of water to get the proportion of 95:05 v/v. The mobile phase was sonicated for 15  min and then it  was filtered through a 0.45 µ membrane filter paper.

 

Preparation of stock solution of simvastatin and ezetimibe:

The standard stock solutions of 1000 µ g/ml of EZT and SMT were prepared by dissolving 100 mg of EZT and SMT in mobile phase, in a 100 ml volumetric flask and made up to the volume. EZT and SMT solution were further diluted with mobile phase to obtain final concentration of 100 µ g/ml and stored under refrigeration.

 

Chromatographic Conditions:

A reverse phase C18 column equilibrated with mobile phase methanol: water (95: 05 v/v) was used. Mobile phase flow rate was maintained at 0.8 ml/min and effluents were monitored at 248 nm. The sample was injected using a 20 µ l fixed  loop.  All  determinations  were  performed  at ambient column temperature.

 

Fig. 1 A Typical Chromatogram of Ezetimibe (EZT) and Simvastatin (SMT). (AU indicates Absorbance Unit)

 

Calibration curve:

Aliquots of standard stock solution of EZT and SMT stock solution were taken in 10 ml volumetric flasks and diluted up to the mark with mobile phase in such a way that final concentrations of  EZT and SMT were in the range of 5-50 µ g/ml and 5-70 µ g/ml, respectively. Triplicate injections of 20 µ l were made two times for each concentration of each drug separately and chromatographed under the conditions as described above. Evaluation of  two  drugs  was performed and peak areas were recorded. The plots of peak area verses respective concentration of EZT and SMT were found to be linear in the range of 5-50 µ g/ml and 5-70 µ g/ml with co-efficient of correlation (r2) 0.9995 and 0.9996 for EZT and SMT, respectively.

 

Determination of EZT and SMT in their combined dosage forms:

Twenty  tablets  were  taken  and  weighed.  Powder equivalent to 10 mg of EZT and 10 mg of SMT were accurately   weighed   and   transferred   to   100   ml volumetric flask and 50 ml of mobile phase was added to the same and flask was sonicated for 5 min. The flask was shaken, and the volume was diluted to the mark with the same mixture. The above solution was filtered using Whatman filter paper No.1. Appropriate volume of the aliquot was transferred and diluted with mobile phase to obtain 50 µ g/ml of EZT and SMT. The solution was injected at above chromatographic condition and peak areas were measured. The quantification was carried out by keeping these values to the straight line equation of calibration curve. The method was validated for precision, specificity, detection of limit, quantification limit and robustness. The validation parameters are summarized in Table 1.

 

Accuracy:

The accuracy of the method was determined by calculating recoveries of EZT and SMT by method of standard addition. Known amount of standard EZT and SMT corresponding to 80%, 100%, and 120% of the label claim was added to a pre quantified sample solution, and the amount of EZT and SMT were estimated by measuring the peak areas and by fitting these values to the straight line equation of calibration curve. The results of studies along with its evaluation are given in Table 2.

 

Parameter	EZETIMIBE	SIMVASTATIN
Linearity Range (µ g/ml)	5-50	5-70
Correlation    Coefficient (r2) ± S.D*	0.9995±0.1162	0.9996±0.2864
* Retentiontime(min.)±S.D	3.2±0.017	4.9± 0.025
Resolution	9.46
Tailing factor	1.03	1.01
Theoretical plates	6655	9333
Limitofdetection(µg/ml)	0.01	0.01
Limitofquantification(µg/ml)	0.04	0.04
Precision    (RSD*,    %)   Intraday (n=5)	0.20-0.95	0.23-1.64
* Repeatability(RSD ,%)n=5	0.254	0.171

 

Mean of five determinations (n=5). SMT and EZT denotes simvastatin and ezetimibe, respectively.

 

Precision:

The intra day precision study of EZT and SMT was carried out by estimating the corresponding responses five times on the same day and the results are reported in terms of relative standard  deviation (RSD,  Table  1).  EZT  and  SMT  were stable up to 24 h only.

 

Specificity:

The specificity of the RP-HPLC method was determined by complete separation of EZT and SMT as shown in Fig No. 1 with parameters like retention time (tR), resolution (Rs) and tailing factor (T). Here tailing factor for peaks of SMT and EZT was less than 2% and resolution was satisfactory. The peaks obtained for EZT and SMT were sharp and have clear base line separation.

 

Detection limit and quantification limit:

A calibration curve was prepared by using concentration in the expected detection limit range of 0.1-4 µ g/ml for EZT and SMT. The standard deviation of y-intercepts of regression lines were determined and kept in the following equation for the determination of detection limit and quantification limit. Detection limit = 3.3 σ/s; quantification limit =  10  σ/s;  Where σ  is  the  standard deviation of  y- intercepts of regression lines and s is the slope of the calibration curve.

 

Robustness:

Robustness  of   the   method   was   studied   by   deliberate variations of the analytical parameters such as flow rate (0.8 ± 0.2 ml/min), concentration of methanol (85 ± 2 %) and also by observing the stability of the drugs for 24 h at 350 temperature in the mobile phase.

 

Table 2: Recovery Studies Of Ezt And Smt In Combined Dosage Form

 

Formulations	Ezetimibe			Simvastatin
	% Added	% Recovered  ± % RSD*	% Recovery	% Added	% Recovered  ± % RSD*	% Recovery
Brand A	80	79.89±0.015	99.86	80	80.29±0.308	100.36
	100	99.98±0.010	99.98	100	99.95±0.010	99.95
	120	120.07±0.085	100.05	120	119.87±0.085	99.89
Brand B	80	79.98±0.315	99.96	80	79.29±0.230	99.11
	100	101.28±0.250	101.28	100	100.05±0.030	100.05
	120	121.97±0.356	101.65	120	120.11±0.071	100.09

*Mean of five determinations (n=5). SMT and EZT denotes simvastatin and ezetimibe, respectively.

 

 

TABLE 3: ASSAY RESULTS OF COMBINED DOSAGE FORM

 

Formulations	Labeled amount (mg/tablet)		Amount obtained  (mg/tablet ± RSD*)		% Assay*
	EZT	SMT	EZT	SMT	EZT	SMT
Brand A	10	10	9.96±0.044	9.92±0.103	99.60	99.20
Brand B	10	10	10.05±0.021	10.11±0.066	100.50	101.10

* denotes average of five determinations. SMT and EZT denotes simvastatin and ezetimibe, respectively.

 

RESULT AND DISCUSSION:

Optimization of the mobile phase was performed based on   resolution,   asymmetric   factor   and   peak   area obtained for both EZT and SMT. The mobile phase methanol:   water   (95:05   v/v)   was   found   to   be satisfactory and gave two symmetric and well resolved peaks for EZT and SMT. The resolution between EZT and SMT was found to be 9.46 which indicates good separation of both the compounds. The retention time for EZT and SMT were 3.2 min and 4.9 min, respectively (Fig. No 1). The asymmetric factors for EZT and SMT were 1.03 and 1.01, respectively.

 

The calibration curve for EZT was obtained by plotting the peak area of EZT versus the concentrations of EZT over the range of 5-50 µ g/ml, and it was found to be linear with r2 = 0.9995. Similarly, the calibration curve for SMT was obtained over the range of 5-70 µ g/ml and was found to be linear with r2 = 0.9996. The data of regression analysis of the calibration curves and the validation parameters are summarized in Table 1. The quantitation limit for EZT and SMT were 0.04 µ g/ml, which suggest that a nanogram quantity of both the compounds can be estimated accurately.

 

The recoveries of EZT and SMT were found to be in the range of 99.63-100.50% and 99.27-101.10%, respectively. The system suitability test parameters are shown in Table 1. The chromatographic method was applied to the determination of EZT and SMT in their combined dosage forms (Tablet formulation A and B).

 

The  result for  EZT  and  SMT  were  comparable with the corresponding labeled amounts (Table 3).

 

Proposed study describes a new RP-HPLC method for estimation of EZT and SMT combination in mixture using simple mobile phase. The method gives good resolution between both the compounds with a short analysis time. The method  was  validated and  found to  be  simple, sensitive, accurate and precise. Percentage recovery shows that the method is free from interference of the excipients used in the formulation. Therefore, the proposed method can be used for routine analysis of EZT and SMT in their combined dosage form.

 

ACKNOWLEDGEMENTS:

The authors are grateful to the Management, RVS College of Pharmaceutical Sciences, Sulur, Coimbatore, and Pharmafabrikon, Otthapatti, Madurai, for providing the required facilities and also to M/S Micro labs, Hosur for providing the gift sample of Simvastatin and Ezetimibe.

 

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Accepted on 28.07.2008   © RJPT All right reserved