View of RP-HPLC METHOD DEVELOPMENT FOR THE SIMULTANEOUS DETERMINATION OF SIMVASTATIN AND EZETIMIBE

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Vol. 05,Special Issue 01

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(ICOSD-2020) January 2020, Available Online:

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RP-HPLC METHOD DEVELOPMENT FOR THE SIMULTANEOUS DETERMINATION OF SIMVASTATIN AND EZETIMIBE

Aarti Nadwana^a, R.k. Nema^b, Achla Vyas^c

aLakshami Narain Institute of Pharmacy (RCP), Revati, sawer Road, Indore, M.P., India.

Abstract- A simple, sensitive and specific RP-HPLC method was developed and validated for the simvastatin and ezetimibe tablet. The mobile phase for these method, composed of ethanol: acetonitrile: orthophosphoric acid (65:30:5) was used at 0.1ml/mint. Flow rate, C18

hypersil column was used and isocratic elution best at 25◦c-5◦c.UV detector was used, detect at 245 for starting 10 min and 253 for 15-20 min. The all validation parameters were examined in acceptable range. This work is accepted with application of precise, accurate and simple. Determination of the different analytical parameters such as linearity, precision, accuracy, specificity, LOD and LOQ was done. The proposed method was applied successfully for simultaneously determination of Simvastatin and Ezetimibe.

Keywords: Ezetimibe, Simvastatin, reverse phase high performance liquid chromatography, tablets.

1. INTRODUCTION 1.1 Ezetimibe

It’s a therapeutically important drug that inhibit the protein transporter on small intestine brush border(which active transport of cholesterol) and also inhibits phytosterol absorption .It’s used for to treat high blood cholesterol and other lipid abnormalities. Ezetimibe has no effect on the triglycerides (lipid soluble vitamins).Their effective effect was increased with statins drugs.

Trade name-ZETIA

Figure 1.Chemical Structure of Ezetemibe 1.2. Simvastatin

Simvastatin used for hypercholesterolemia that the primary risk for cardiovascular disease, the drug leading lowering the high blood cholesterol levels. It acts as HMG-CoA inhibitor.

The statins drugs reduce total cholesterol level in serum, specially LDL (low density lipoprotein) level. Also prevent and reduce the development of peripheral vescular disease, protect against orthosclerotic plaque growth via their anti-inflammatory and anti- thrombotic effect. Its synthesis from the fungus Aspergillus terrus. It target a specific enzyme HMG-CoA, this enzyme responsible for the cholesterol synthesis. Used along with exercise, diet and weight loss to decrease the risk of heart problems.

Trade name:-ZOCOR

Figure 2: Chemical Structure of Simvastatin

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1.3 Mechanism of action

2. MATERIAL AND METHOD 2.1 Chemical and reagents

Ezetimibe and simvastatin were purchased from local market of pharmaceutical formulation, Indore (M.P.).Acetonitrile, methanol and orthophosphoric acid was purched from drugs market Indore (M.P.).

2.2 Experimental condition

Flow rate of mobile phase was changed from 0.5-1.5ml/min. for optimum separation. A minimum flow rate as well as minimum run time gives the maximum saving on the usage of solvents. 0.1ml/min. flow rate at 25±2◦c was operating at isocratic mode. UV wavelength for that 253nm.

2.3 Instrumentation

An isocratic PEAK HPLC Instrument was used with C18hypersile column (250mm×4.6mm, 5µ) was used. UV detector LC-7000 was used and instrument is equipped with a LC 20AT pump for solvents delivery. For injecting the sample 20µl Rheodyne inject part was used.

Uv spectrophotometer was used for wavelength checking.

2.4 Preparation of mobile phase

For the preparation of mobile phase the methanol, acetonitrile and 0.1%orthophosphoric acid was used in ratio (65:30:5v/v) respectively has HPLC grade were mixed ,filtered and degassed , the PH was found to be 5.4.

2.5 Preparation of mixed standard solution

Both drug Ezetimibe and simvastatin (1mg/ml) standard stock solutions were prepared using mobile phase as a solvent. The final concentration were prepared 50-100ppm was mixed with standard solution were diluted in mobile phase.

2.6 Preparation of sample solution of pharmaceutical formulation

10mg Ezetimibe and 10mg Simvastatin was weigh and dissolved in 25ml of mobile phase and sonicated for 15min.sonicated, volume was made up by methanol up to 50ml and filter through membrane filter (0.45µ), finally the mixed sample was prepared corresponding to 70ppm of simvastatin and 70ppm ezetimibe.

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2.7 Recording of chromatogram

After stabilization the base line with the optimized chromatographic condition, the standard solution containing 50-100ppmof simvastatin and ezetimibe were injected and the chromatogram was recorded .3.40 and 6.20min.retention time for ezetimibe and simvastatin were found.

Calibration curve were plotted using peak area retention of standard drug peak against concentration of corresponding standard solution.

3. RESULT AND DISCUSSION 3.1 Estimation

An RP-HPLC method was developed for the simultaneous estimation of Ezetemibe and simvastatin in combined dosage forms, which can be conveniently employed for routine quality control in pharmaceutical dosage forms. The chromatographic conditions were optimized in order to provide a good performance of the assay. The standard and sample solution was prepared and chromatograms were recorded.

Graph 1-Typical chromatogram of standard Ezetemibe and Simvastatin

3.2 Method validation

The method was validated by determining linearity, precision, accuracy, specificity, ruggedness, and robustness by analyzing 50-100 ppm of both Ezetemibe and Simvastatin.

Table-1: Optimized Chromatographic Conditions for Estimation of Ezetemibe and Simvastatin

S.NO. Test

HPLC condition

Result

1 Mobile phase Methanol: acetonitrile:

orthophosphoric acid (65:30:5)

2 API concentration 70ppm

3 PH 5.4

4 Elution Isocratic

5 Runtime 12mint

6 Wavelength 253nm

7 Theoretical plate Ezetimibe-7684 Simvastatin-24004 8 Flow of mobile phase 1ml/mint

9 Column C18

10 Area Ezetimibe-271463

Simvastatin-365483 11 Retention time Ezetimibe-3.40

Simvastatin-6.20

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3.3 Linearity

The linearity for the simvastatin and Ezetimibe assay method was determined by preparing and injecting standard solution of simvastatin and ezetimibe. The linearity curve indicate that the response is linear over the concentration range, the correlation coefficient (r²) value, slop and intercept as show in table-

Table-2: Linearity result

S. NO. CONC. IN PPM EZETEMIBE SIMVASTATIN

1 50 199730 278230

2 60 238660 326590

3 70 271383 366340

4 80 305210 427599

5 90 342930 486777

6 100 388650 543358

Graph 2: Calibration plot for Ezetemibe and Simvastatin Table-3: Regression analysis of the calibration curve Parameters Ezetemibe Simvastatin Calibration range (ppm) 50-100 50-100 ppm

Intercept 3280 1060

Slop 3830 5380.76

Correlation coefficient 0.999 0.999 3.4 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 Ezetemibe and Simvastatin combined test solution in the same equipment at a concentration value of 70 ppm on the same day. Peak areas of the drugs were determined and precision as % RSD was reported.

Table-4: Intraday precision

S. No. Concentration Ezetemibe peak area Simvastatin peak area

1 70 PPM 271263 366373

2 70 PPM 272239 365320

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3 70 PPM 272490 367820

4 70 PPM 271695 365194

5 70 PPM 272065 365325

6 70 PPM 273382 367630

% R.S.D. = 0.23 %R.S.D. = 0.34 Table-5: - Inter-day precision

S.No. Concentration Ezetemibe peak area Simvastatin peak area

1 70 PPM 274676 365621

2 70 PPM 266650 365185

3 70 PPM 268690 368724

4 70 PPM 271405 365325

5 70 PPM 272491 365189

6 70 PPM 268539 366897

% R.S.D. = 1.54 %R.S.D. = 0.52 Table 6: System suitability of Ezetemibe and Simvastatin

PARAMETERS EZETEMIBE SIMVASTATIN Theoretical plates(N) 7684 24004

Retention time(min) 3.40 6.20 Tailing factor 1.96 1.83

LOD (ppm) 1.2ppm 0.25ppm

LOQ (ppm) 4ppm 0.8ppm

R.S.D. (%) 0.75 0.4

3.5 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

Table 7: Recovery of Ezetemibe and Simvastatin Specificity Recovery Conc. Of

Sample

Ezetemibe Simvastatin Ezetemibe % of recovery

Simvastatin

%of recovery

50% 50ppm 50.31 50.15 100.56 100.21

75% 75ppm 73.52 73.75 99.90 99.9

100% 100ppm 99.92 100.34 99.92 100.34

Specificity was performed to exclude the possibility of interference with excipients in the region of elution of Ezetemibe 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 Ezetemibe and Simvastatin.

3.6 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.

3.7 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:-

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Table 8: Robustness study Parameter Modification Ezetemibe area Simvast

at in area

Ezetemibe

% of

recovery

Simvastatin

% of recovery

Standard 271383 366340

MP MeOH;CAN;0.1%

OPA(65 :30:5)

273340 366690 0.73 0.09

pH 5.4 270654 366320 0.225 0.07

Wavelength 253nm 270693 365467 00.25 0.247

3.8 Analysis of marketed formulations

The validated HPLC method was adopted for the quantification of Ezetemibe and Simvastatin in their combined pharmaceutical dosage form and the typical chromatograms of the formulation are shown in fig. The results of the 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 Ezetemibe and Simvastatin in pharmaceutical dosage forms

Table-9: Formulation Drug Formulation Dosage Sample

Conc. Drug

estimated % of drug estimated

Ezetemibe Vytorin 10mg 70ppm 70.07 99.80 Simvastatin Vytorin 10mg 70ppm 69.65 99.61 4. CONCLUSION

The proposed study describes a new RP-HPLC method using simple mobile phase for the estimation of Ezetemibe 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 Ezetemibe and Simvastatin in the pharmaceutical dosage form. The percentage of recovery shows that the method is free from the interference of the excipients used in the formulation. Moreover, the lower solvent consumption along with the short analytical run time leads to the cost- effective chromatographic method.

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