Ch. Narasimharaju BH¹, G. DevalaRao² and Rajendran Rukmani³

¹A.S.N.Pharmacy College, Burripalem Road, Tenali - 522 201 (Inda).

²KVSR Siddhartha College of Pharmaceutical Sciences, Vijayawada - 520 010 (India).

³Jaya College of Pharmacy, Thiruninravur - 602 024 (India).

Corresponding Author E-mail: bhchnraju@yahoo.com

Abstract

This paper describes about validated Reverse phase High Performance Liquid Chromatographic method for determination of Ezetimibe in bulk and pharmaceutical formulations has been developed. Chromatographic separation was carried out using a mobile phase of Potassium dihydrogen phosphate buffer (containing triethylamine) and acetonitrile (55:45v/v) on a C18 –ODS –Hypersil (5 µ, 250×4.6 mm) column in isocratic mode at a flow rate of 1.5ml/minute with UV detection at 234nm. Atorvastatin calcium was used as internal standard. The chromatographic method was linear over the concentration range of 50-250ìg/ml. The Limit of Detection and Limit of Quantitation were 0.828 and 2.5 µg/ml respectively. The mean percentage purity and recovery were found to be 101.22% and 100.36% respectively. Suitable method was developed to determine Ezetimibe in bulk and pharmaceutical formulations.

Keywords

RP -HPLC; Ezetimibe; Tablets

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Copy the following to cite this article: Narasimharaju B. C, DevalaRao G, Rukmani R.Reverse Phase High Performance Liquid Chromatographic Determination of Ezetimibe in Bulk and Pharmaceutical Formulations. Biomed. Pharmacol. J.2008;1(2)

Copy the following to cite this URL: Narasimharaju B. C, DevalaRao G, Rukmani R.Reverse Phase High Performance Liquid Chromatographic Determination of Ezetimibe in Bulk and Pharmaceutical Formulations. Biomed. Pharmacol. J.2008;1(2). Available from: http://biomedpharmajournal.org/?p=489

Introduction

Ezetimibe is a new class of lipid lowering drug, which differs from other classes of cholesterol reducing compounds. It is chemically   as (3R, 4S) – 1 – (4-Flourophenyl –3 Hydroxy propyl]-4-(Hydroxy phenyl-2-azetidinone.

James E.Patrick et al studied the Ezetimibe pharmacokinetics². Literature survey reveals the availability of few analytical methods such as HPCL^3-5 ,LC-MS⁶,derivative spectrophotometry⁷ and voltammetry⁸ for  determination   of Ezetimibe in pharmaceutical formulations .In the present investigation the authors  propose a simple, sensitive and  reproducible RP-HPLC   method for the determination of Ezetimibe.

Expermental

Instrument

Shimadzu HPLC SPD-10ATVP, Japan equipped with UV visible detector Was used for HPLC analysis.

Chemicals and materials

Potassium dihydrogen phosphate, triethylamine and acetonitrile (HPLC grade) were procured from E.merck India, Limited. Water was purified by double distillation and filtered through 0.2μ pore sized membrane filter. Ezetimibe was obtained as a gift sample from Hetero Drug House Private Limited, Hyderabad, India. Two marketed brands of Ezetimibe were used for estimation.

The chromatographic separation was carried out in an isocratic mode utilizing Hypersil C₁₈ column with dimensions (5μm, 250mmx4.6mm) as stationary phase and the mobile phase composed of buffer and acetonitrile in 55:45 ratios at a flow rate of 1.5ml/minute. The buffer was prepared using 1.3609g of potassium dihydrogen phosphate and 1.0ml of triethylamine Acetonitrile and water in 3:1 ratio was used as diluent.

Preparation of standard solutions

In Chromatographic determination, separate stock solutions of reference compound and internal standard were prepared by weighing the appropriate amount into volumetric flasks and made up to volume using the diluent. Specific volume of each solution relating to the required concentration were pipetted into volumetric flask and made to volume to obtain serial dilutions using the diluent.

Sample preparation

Twenty tablets of both brands were weighed and powdered in a mortar. In Chromatographic method, a quantity equivalent to 50 mg was weighed accurately and transferred into 100ml volumetric flask along with the internal standard. About 75 ml of diluent was added and sonicated for 30 minutes and made to volume. This solution was then filtered through membrane filter. First 10ml of solution was discarded and then diluted suitably to get a concentration of 100 μg/ml.

Method validation

Linearity

Calibration curve for chromatographic method was constructed by plotting ratio of peak areas (standard/internal standard) against concentration of solution. Figure 1 shows HPLC-UV chromatogram of Ezetimibe. Linear ranges and correlation coefficient⁹ Obtained for chromatographic method were depicted in table 1.

Figure 1: CHROMATOGRAM of  EZETIMIBE (100 mcg/ml).                                                   Click here to View figure

 

Table 1: Analytical Performance Parameters of Ezetimibe

PARAMETERS	EZETIMIBE
	RP-HPLC METHOD
Linearity dynamic range (LDR;mg/ml) Correlation coefficient (r) Slope (m) Intercept (c)	50 – 250   0.9993   0.00862   0.05576

 

System suitability testing

System suitability for chromatographic method was determined by parameters like plate number; tailing factor, resolution factor and relative standard deviation. The data’s comply with ICH Standards¹⁰ and were presented in table 2.

Table 2: System Suitability Parameters (RP-HPLC).

Parameters	Ezetimibe	Internal standard
Retention Time (minutes)   Tailing Factor   Resolution Factor   Number Of Theoretical Plates	3.91   1.04   4.932   8298	5.64   1.01   4.932   7817

 

Tablet assay

Quantitative analysis was carried out in marketed brands of Ezetimibe, which are depicted in table 3.

Table 3: Quantification Parameters of Ezetimibe.

Sample	Label claim		RP-HPLC method
			Assay	%R.S.D
Tablet A Tablet B	10 10	101.22 103.30		0.1281       0.1307

 

Precision and accuracy

Precision was assessed by determining the relative standard deviation, Accuracy was determined by recovery studies, which was performed by spiking sample with known concentrations of standard. The datas were depicted in table 4.

Table 4: Recovery studies of Ezetimibe.

Sample concentration         μg/ml	Fortified concentration                       μg/ml	Percentage Recovery
		Tablet A	Tablet B
100	150 200 250	100.50 100.61 100.71	100.81 100.93 100.71

 

Results

The linearity was obeyed in the concentration range of 50-250 μg/ml in Chromatographic method. From the chromatogram, the retention times were found to be 3.91 minutes and 5.64 minutes for Ezetimibe and Atorvastatin respectively. The Limit of Detection and Limit of Quantitation were 0.828 and 2.5 μg/ml respectively. Quantitative estimation of the tablet showed average percentage purity of 101.22% with mean recovery of 100.36%. The system suitability parameters indicate that the developed method has acceptable accuracy and precision. In the above determined method, the relative standard deviation was below 1, which is a good index of accuracy and precision.

Conclusion

The developed method was simple, rapid, accurate, and reproducible was more suitable to determine Ezetimibe in bulk and pharmaceutical formulations.

Acknoledgement

The authors are grateful to m/s Hetero Drug House Private Limited, Hyderabad, for providing the authentic of Ezetimibe sample and Dr. Ceeal Analytical Lab, Chennai, for providing the facilities to carry out the present investigation.

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