{"id":489,"date":"2015-01-22T07:15:59","date_gmt":"2015-01-22T07:15:59","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=489"},"modified":"2020-04-25T06:22:52","modified_gmt":"2020-04-25T06:22:52","slug":"reverse-phase-high-performance-liquid-chromatographic-determination-of-ezetimibe-in-bulk-and-pharmaceutical-formulations","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol1no2\/reverse-phase-high-performance-liquid-chromatographic-determination-of-ezetimibe-in-bulk-and-pharmaceutical-formulations\/","title":{"rendered":"Reverse Phase High Performance Liquid Chromatographic Determination of Ezetimibe in Bulk and Pharmaceutical Formulations"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Ezetimibe is a new class of lipid lowering drug, which differs from other classes of cholesterol reducing compounds. It is chemically\u00a0\u00a0 as (3R, 4S) \u2013 1 \u2013 (4-Flourophenyl \u20133 Hydroxy propyl]-4-(Hydroxy phenyl-2-azetidinone.<\/p>\n

\"Vol_1_No_2_Reve_ChNa_sch1\"<\/a><\/p>\n

James E.Patrick et al studied the Ezetimibe pharmacokinetics2<\/sup>. Literature survey reveals the availability of few analytical methods such as HPCL3-5<\/sup> ,LC-MS6<\/sup>,derivative spectrophotometry7<\/sup> and voltammetry8<\/sup> for\u00a0 determination\u00a0\u00a0 of Ezetimibe in pharmaceutical formulations .In the present investigation the authors \u00a0propose a simple, sensitive and\u00a0 reproducible RP-HPLC\u00a0 \u00a0method for the determination of Ezetimibe.<\/p>\n

Expermental<\/strong><\/p>\n

Instrument<\/strong><\/p>\n

Shimadzu HPLC SPD-10ATVP, Japan equipped with UV visible detector Was used for HPLC analysis.<\/p>\n

Chemicals and materials<\/strong><\/p>\n

Potassium dihydrogen phosphate, triethylamine and acetonitrile (HPLC grade) were procured from E.merck India, Limited. Water was purified by double distillation and filtered through 0.2\u03bc 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.<\/p>\n

The chromatographic separation was carried out in an isocratic mode utilizing Hypersil C18<\/sub> column with dimensions (5\u03bcm, 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.<\/p>\n

Preparation of standard solutions<\/strong><\/p>\n

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.<\/p>\n

Sample preparation<\/strong><\/p>\n

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 \u03bcg\/ml.<\/p>\n

Method validation<\/strong><\/p>\n

Linearity<\/h3>\n

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\u00a0<\/sup>Obtained for chromatographic method were depicted in table 1.<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 1:\u00a0<\/strong>CHROMATOGRAM of \u00a0EZETIMIBE (100 mcg\/ml).<\/strong><\/p>\n

\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0<\/strong><\/p>\n

Click here to View figure<\/a>
\n<\/strong><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Table 1: Analytical Performance Parameters of Ezetimibe <\/strong><\/p>\n\n\n\n\n\n
\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 PARAMETERS<\/strong><\/td>\n\u00a0\u00a0 \u00a0EZETIMIBE<\/strong><\/td>\n<\/tr>\n
\n
\u00a0\u00a0 RP-HPLC METHOD<\/h6>\n<\/td>\n<\/tr>\n
\u00a0<\/strong><\/p>\n

Linearity dynamic range (LDR;<\/strong>mg\/ml)<\/strong><\/p>\n

Correlation coefficient (r) <\/strong><\/p>\n

Slope (m)<\/strong><\/p>\n

Intercept (c)<\/strong><\/p>\n

 <\/p>\n

 <\/td>\n

 <\/p>\n

 <\/p>\n

50 \u2013 250<\/p>\n

 <\/p>\n

0.9993<\/p>\n

 <\/p>\n

0.00862<\/p>\n

 <\/p>\n

0.05576<\/p>\n

 <\/p>\n

 <\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

System suitability testing<\/strong><\/p>\n

System suitability for chromatographic method was determined by parameters like plate number; tailing factor, resolution factor and relative standard deviation. The data\u2019s comply with ICH Standards10<\/sup> and were presented in table 2.<\/p>\n

Table 2: System Suitability Parameters (RP-HPLC).<\/strong><\/p>\n\n\n\n\n
\u00a0<\/strong><\/p>\n

Parameters<\/strong><\/p>\n

\u00a0<\/strong><\/td>\n

\u00a0<\/strong><\/p>\n

Ezetimibe<\/strong><\/td>\n

\u00a0<\/strong><\/p>\n

Internal standard <\/strong><\/td>\n<\/tr>\n

\u00a0<\/strong><\/p>\n

Retention Time (minutes)<\/strong><\/p>\n

\u00a0<\/strong><\/p>\n

Tailing Factor<\/strong><\/p>\n

\u00a0<\/strong><\/p>\n

Resolution Factor<\/strong><\/p>\n

\u00a0<\/strong><\/p>\n

Number Of Theoretical Plates<\/strong><\/p>\n

\u00a0<\/strong><\/td>\n

 <\/p>\n

3.91<\/p>\n

 <\/p>\n

1.04<\/p>\n

 <\/p>\n

4.932<\/p>\n

 <\/p>\n

8298<\/td>\n

 <\/p>\n

5.64<\/p>\n

 <\/p>\n

1.01<\/p>\n

 <\/p>\n

4.932<\/p>\n

 <\/p>\n

7817<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Tablet assay<\/strong><\/p>\n

Quantitative analysis was carried out in marketed brands of Ezetimibe, which are depicted in table 3.<\/p>\n

Table 3: Quantification Parameters of Ezetimibe.<\/span><\/strong><\/p>\n\n\n\n\n\n
Sample<\/strong><\/td>\nLabel claim<\/strong><\/td>\nRP-HPLC method<\/strong><\/td>\n<\/tr>\n
Assay<\/strong><\/td>\n%R.S.D<\/strong><\/td>\n<\/tr>\n
Tablet A<\/strong><\/p>\n

Tablet B<\/strong><\/td>\n

\u00a0\u00a0\u00a0\u00a0\u00a0 10<\/p>\n

10<\/td>\n

101.22<\/p>\n

103.30<\/p>\n

 <\/td>\n

\n

\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 0.1281<\/p>\n

\u00a0\u00a0\u00a0\u00a0\u00a0 0.1307<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Precision and accuracy<\/strong><\/p>\n

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.<\/p>\n

Table 4: Recovery studies of Ezetimibe.<\/strong><\/p>\n\n\n\n\n\n
\u00a0<\/strong><\/p>\n

Sample concentration <\/strong><\/p>\n

\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 \u03bcg\/ml<\/strong><\/td>\n

\u00a0<\/strong><\/p>\n

Fortified concentration<\/strong><\/p>\n

 <\/p>\n

\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 \u03bcg\/ml<\/strong><\/td>\n

\u00a0<\/strong><\/p>\n

Percentage Recovery<\/strong><\/td>\n<\/tr>\n

Tablet A<\/strong><\/td>\nTablet B<\/strong><\/td>\n<\/tr>\n
 <\/p>\n

 <\/p>\n

 <\/p>\n

 <\/p>\n

 <\/p>\n

 <\/p>\n

 <\/p>\n

100<\/p>\n

 <\/p>\n

 <\/p>\n

 <\/p>\n

 <\/p>\n

 <\/td>\n

 <\/p>\n

 <\/p>\n

 <\/p>\n

 <\/p>\n

 <\/p>\n

150<\/p>\n

200<\/p>\n

250<\/td>\n

 <\/p>\n

 <\/p>\n

 <\/p>\n

 <\/p>\n

100.50<\/p>\n

100.61<\/p>\n

100.71<\/p>\n

 <\/p>\n

 <\/p>\n

 <\/td>\n

 <\/p>\n

 <\/p>\n

 <\/p>\n

 <\/p>\n

100.81<\/p>\n

100.93<\/p>\n

100.71<\/p>\n

 <\/p>\n

 <\/p>\n

 <\/p>\n

 <\/p>\n

 <\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Results<\/strong><\/p>\n

The linearity was obeyed in the concentration range of 50-250 \u03bcg\/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 \u03bcg\/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.<\/p>\n

Conclusion<\/strong><\/p>\n

The developed method was simple, rapid, accurate, and reproducible was more suitable to determine Ezetimibe in bulk and pharmaceutical formulations.<\/p>\n

Acknoledgement<\/strong><\/p>\n

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.<\/p>\n

References<\/strong><\/p>\n

    \n
  1. The Merck index, Thirteenth edition, Merck and co., Inc.U.S.A, 2002: pp 3949.<\/li>\n
  2. James E. Patrick, Teddy Kosoglou, Kathe L. Stabber, Kevin B. Alton, Stephen E.Maxwell, Yali Zhu, Paul Statkevich, Robert Iannucci, Swapan Chowdhury, Melton Affrime, and Mitchell N. Cayen, Disposition of the Selective Cholesterol Absorption Inhibitor Ezetimibe in Healthy Male Subjects, Journal of Drug metabolism and Disposition,2002:30(4); 430-437.<\/li>\n
  3. MuhammadAshfaq, IslamUllahkhan, SyedShanazQutab, SyedNaeemrazzaq, HPLC Determination of Ezetimibe and simvastatin in pharmaceutical Formulations,Chil .chem.soc, 52 no 3(2007).1220-1223.<\/li>\n
  4. Carlucci, P.Mazzeco,L.Biordi, M.Bologna, J.Pharm.Biomed.Anal, 10 (9), 693 (1992).<\/li>\n
  5. Ochiai, N.Uchiyama, K.Imagaki, S.Hata,T.Kamei.,J.Chromatogr.B.Biomed<\/li>\n
  6. .Sci.Appl,.694(1),211(1997).<\/li>\n
  7. Barrett,JHuclova.V.BorekDohalsky,B.Nemec,I.Jelinek,J.Pharm.Biomed.Anal,41(2), 517(2006).<\/li>\n
  8. Wang,M.Asgharnejad ,J.Pharm.Biomed,Anal,21(6),1243(2000).<\/li>\n
  9. Corah.S.AOzkan,Pharmazie,61(4),285(2006)..<\/li>\n
  10. Frederick J Gravette\u2019s, Lancy B and Wallnau, Essential of Statistics for the behavioral Sciences, 2nd<\/sup> Edition, West Publishing Company, 1995, PP2-4, 173-175.<\/li>\n
  11. ICH Steering Committee, Validation of analytical procedures\/methodology, ICH Harmonized Tripartite Guidelines, 1996.<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

    Introduction Ezetimibe is a new class of lipid lowering drug, […]<\/p>\n","protected":false},"author":2,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[5],"tags":[],"class_list":["post-489","post","type-post","status-publish","format-standard","hentry","category-vol1no2"],"_links":{"self":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/489","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/comments?post=489"}],"version-history":[{"count":5,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/489\/revisions"}],"predecessor-version":[{"id":32912,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/489\/revisions\/32912"}],"wp:attachment":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/media?parent=489"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/categories?post=489"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/tags?post=489"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}