Development of a RP-HPLC method for separation of ezetimibe in presence of atorvastatin caclium and simvastatin and its application for qunatitation of  tablet dosage forms

 

Kavita Wagh, Sandeep Sonawane*, Santosh Chhajad, Sanjay Kshirsagar

MET’S Institute of Pharmacy, Bhujbal Knowledge City, Adgaon, Nashik-422003, India

 

ABSTRACT:

A single, simple, accurate and precise RP-HPLC method has been developed for the estimation of ezetimibe in presence of atorvastatin calcium and simvastatin in bulk and marketed combined formulations. The chromatographic separation was achieved on C18 column (250 × 4.6 mm, 5 µ) using Acetonitrile: water 70:30 v/v as a mobile phase at flow rate of 1.2 mL/min. The separation was achieved in isocratic mode and the detection was performed at 242 nm. Further the developed method was validated as per the ICH Q2 (R1) and applied for quantitation of atorvastatin calcium-ezetimibe and ezetimibe – simvastatin in tablet formulations.

 

 

 

INTRODUCTION:

In hyperlipidemia decrease in low density lipoproteins (LDL) and high-density lipoprotein (HDL) is observed. Therefore, the use of both atorvastatin and ezetimibe in combination produces the additive effects in hyperlipidemia. When atorvastatin is used with ezetimibe in combination it causes reduction in LDL cholesterol levels as compared to double dose of individual drugs when it is used alone. The use of atrovastatin calcium, a HMG-CoA reductase inhibitor and ezetimibe, a selective cholesterol asorption inhibitor given in combination to enhance the effect of statin at a lower dose and reducion in side effects.[1]

 

When a patient on statins fails to control of LDL-cholesterol, a double dose of statin or the use of ezetimibe seems to be good choice. Compared to statins alone, the co-administration of ezetimibe with statin has previously been shown to produced significant reduction in the plasma levels of LDL-C.[2]

 

Chemically, atorvastatin calcium is[R-(R*, R*)]-2-(4-Flurophenyl)-β, -δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino) carbonyl]-1 H-pyrrole-1-heptanoic acidis HMG-CoA reductase inhibitor. Ezetimibe is (3R, 4S)-1-(4-fluorophenyl)-3-[(3S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl) azetidin-2-one selective inhibitor of intestinal cholesterol and related phytosterol absorbtion and simvastatin is [(1S, 3R, 7S, 8S, 8aR)-8-[2-[(2R, 4R)-4-hydroxy-6-oxooxan-2-yl]ethyl]-3, 7-dimethyl-1, 2, 3, 7, 8, 8a-hexahydronaphthalen-1-yl]2, 2-dimethylbutanoate HMG-reductase inhibitor.

 

There are few HPLC, UV spectroscopy, UPLC and HPTLC methods reported for the quantitation of atorvastatin calcium and ezetimibe.[3-8] and several methods for quantitation of ezetimibe and simvastatin in their combined dosage forms [9-18].

 

In present study, a single RP-HPLC method has been developed with the aim to separate atorvastatin calcium, ezetimibe and Simvastatin using single mobile phase. Further, the developed method was successfully applied for the quantitation of atorvastatin calcium and ezetimibe and for eztimibe and simvastatin in combined tablet formulations.

 

MATERIALS AND METHODS:

Chemicals and reagents:

The working standards of atorvastatin calcium, ezetimibe and simvastatin were received as gift samples from Blue Cross Pharmaceuticals Ltd., Nashik, respectively. HPLC grade acetonitrile, methanol was purchased from S D fine-chemicals ltd, Mumbai. Double distilled water used for the preparation of mobile phases was prepared freshly using double distillation assembly purchased from Borosil, Mumbai. Every time the prepared mobile phase was filtered through 0.45 µ × 47 mm membrane filter papers purchased from AxivaScichem Biotech, Delhi. The pH meter used for the adjustment of pH was purchased from Sytronics India Pvt. Ltd, Ahmedabad and sonicator from PCI Analytics Pvt. Ltd, Mumbai. Tablets were procured from local pharmacy containing 10 mg of simvastatin- 10 mg of ezetimibe and 10 mg of Atorvastatin calcium- 10 mg of ezetimibe.

 

Instrumentation and chromatographic conditions:

JASCO HPLC system equipped with dual PU-2080 plus pumps, multichannel UV-2075 UV detector and injection loop (20 µL capacity), Rheodyne manual loop injection system 7725i was used. Data were collected using Browin Chromatography software (version 1.5). The mobile phase was composed of Acetonitrile: water 70:30 v/v. Isocratic elution was carried out on a Phenomenex Hyperclone C18 column (250 × 4.6 mm, 5µ) at a flow rate of 1.2 mL/min. The detection was performed at 242 nm.

 

Preparation of standard solution:

Quantity equivalent to 10 mg of simvastatin, ezetimibe and atorvastatin calcium were weighed and transferred to separate 10 mL volumetric flasks and volume was made upto the mark with methanol. The resulting solutions were of 1000 µg/mL of simvastatin, ezetimibe and atorvastatin calcium, respectively.

 

Calibration curve standards for Simvastatin and Ezetimibe:

From the standard stock solution of simvastatin and ezetimibe six aliquots were prepare and diluted with mobile phase to get calibration curve standard with concentration of 2, 4, 6, 8, 10 and 12 µg/mL for simvastatin and 2, 4, 6, 8, 10 and 12 µg/mL for ezetimibe. These calibration curves were analyzed in triplicates and then mean peak area were plotted on y-axis against respective concentration on x-axis. The intercept, slope and co-efficient of regression were determined.

 

Calibration curve standards for Ezetimibe and Atorvastatin calcium:

From the standard stock solution of eztimibe and atorvastatin calcium six aliquots were prepare and diluted with mobile phase to get calibration curve standard with concentration of 2, 4, 8, 6, 10 and 12 µg/mL for ezetimibe and 2, 4, 6, 8, 10 and 12 µg/mL for atorvastatin calcium. These calibration curves were analyzed in triplicates and then mean peak area were plotted on y-axis against respective concentration on x-axis. The intercept, slope and co-efficient of regression were determined.

 

Estimation of simvastatin and ezetimibe in Tablets:

For analysis of tablet formulation, tablet containing 10 mg of simvastatin and 10 mg of ezetimibe were commercially available tablets are used. Tablets are weighed and finely powered. A tablet powered equivalent to 10 mg of simvastatin and 10 mg of ezetimibe to 100 mL volumetric flask and shaken with acetonitrile for 10 min. After filtration, the excipients were separated and the volume was made up to the 100 mL with the same solvent. From the stock solution, suitable aliquot was diluted with mobile phase to get concentration of 10 µg/mL of simvastatin and 10 µg/mL of ezetimibe and subjected to chromatographic analysis.

 

Estimation of Ezetimibe and Atorvastatin calcium in Tablets:

For analysis of tablet formulation, tablet containing 10 mg of ezetimibe and 10 mg of atorvastatin calcium were commercially available tablets are used. Tablets are weighed and finely powered. A tablet powered equivalent to 10 mg of ezetimibe and 10 mg of atorvastatin calcium to 100 ml volumetric flask and shaken with acetonitrile for 10 min. After filtration the excipients were separated and the volume was made up to the 100 mL with the same solvent. From the stock solution, suitable aliquot was diluted with mobile phase to get concentration of 10 µg/mL of ezetimibe and 10 µg/mL of atorvastatin calcium and subjected to chromatographic analysis.

 

Method validation:

Method was validated as per ICH Q2 (R1) for accuracy, precision, and specificity, limit of detection (DL) and limit of quantification (QL).

 

Limit of detection (DL) and limit of Quantification (QL)

Limit of detection and limit of quantification of simvastatin and ezetimibe and for atorvastatin calcium and ezetimibe were determined from standard deviation of y-intercept of regression line and slope method.

 

 

Accuracy and precision

Accuracy of method were determined by analyzing the standard samples at three concentration level of 80%, 100%, 120% by standard addition method across the range for Simvastatin and ezetimibe and for ezetimibe and atorvastatin calcium. The method precision was established by three replicates of three (80%, 100%, and 120%) for intraday precision and on three successive days for the intermediate precision. The percent recovery of added concentration and % RSD were taken as measures of accuracy and precision, respectively.

 

Specificity

To evaluate the specificity of the proposed method, blank tablets were chromatographed, absence of peaks in the chromatographic run at retention time of the Simvastatin and ezetimibe and ezetimibe and atorvastatin calcium was taken as indication of specificity.

 

Result and Discussion:

Various mobile phases were tried to get separation of ezetimibe, Simvastatin and atrovastatin calcium. It was observed that a mobile phase with composition of Acetonitrile: water 70:30 v/v was gave appropriate resolution of atrovastatin calcium, ezetimibe, simvastatin. The optimum wavelength for detection was 242 nm. The retention time of atrovastatin calcium, ezetimibe, simvastatin were observed at 1.56 min, 2.87 min, 8.60 min respectively (fig.1).

 

 

Fig. 1: Representative Chromatogram of atorvastatin calcium (1.56 min), ezetimibe (2.87 min) and simvastatin (8.60 min).

 

I] Simvastatin and Eztimibe:

In calibration studies, it was observed that Simvastatin was linear in the range of 2-12 µg/mL and ezetimibe in the range of 2-12 µg/mL. The calibration curves with their respective calibration curve equation and regression are depicted in figure 2 and figure 3, respectively.

 

Fig. 2: Calibration of Simvastatin

 

Fig.3: Calibration of Ezetimibe

 

Limit of detection (DL) and limit of quantification (QL):

The result obtained for DL and QL are summarized in Table 1, for simvastatin and ezetimibe respectively.

 

Table 1: DL and QL for simvastatin and ezetimibe

Accuracy and Precision Studies:

The results obtained for accuracy and precision are summarized in Table 2 and Table 3, for simvastain and ezetimibe, respectively. Mean values of concentration added were close to the concentration added and low values of %RSD indicates the acceptable accuracy and precision of the method.

 

Table 2: Accuracy and Precision study for Simvastatin

 

Table 3: Accuracy and Precision study for Ezetimibe

 

Fig. 4 - Representative Chromatogram of specificity

 

Specificity:

When blank tablets were analyzed as per the mentioned chromatographic conditions, no peak was obtained at the times of simvastatin and ezetimibe.

 

The results of specificity is depicted in Fig 4.

 

 

Analysis of formulation

The representative chromatogram  of simvastatin and ezetimibe in tablet formulation is presented in Fig 5. The chromatogram of the drug samples did not show a change in the retention time. There were no interferences from excipients, which are commonly present in the solution for drugs.

 

Fig.5- Representative Chromatogram of Formulation

 

II] Ezetimibe and Atrovastatin Calcium:

In calibration studies it was observed that ezetimibe was linear in the range of 2-12 µg/mL and atorvastatin calcium in the range of 2-12 µg/mL. The calibration curves with their respective calibration curve equation and regression are depicied in figure 6 and figure 7, respectively.

 

Fig. 6: calibration of ezetimibe

 

Fig. 7: Calibration of Atorvastatin calcium

Limit of detection (DL) and limit of quantification (QL):

The result obtained for DL and QL are summarized in Table 4, for ezetimibe and atrovastatin calcium respectively.

 

Table 4: DL and QL for ezetimibe and atrovastatin calcium

Parameter	Ezetimibe	Atorvastain calcium
DL	0.09	0.19
QL	0.27	0.60

 

Accuracy and Precision studies:

The results obtained for accuracy and precision are summarized in Table 5 and Table 6, for ezetimibe and atorvastatin calcium, respectively. Mean values of concentration added were close to the concentration added and low values of %RSD indicates the acceptable accuracy and precision of the method.

 

Specificity-

When blank tablets were analyzed as per the mentioned chromatographic conditions, no peak was obtained at the times of ezetimibe and atorvastatin calcium.

 

The results of specificity are presented in Fig 8.

 

Analysis of formulation:

Chromatogram for formulation of ezetimibe and atorvastatin calcium is presented in Fig. 9. The chromatogram of the drug samples did not show a change in the retention time. There were no interferences from excipients, which are commonly present in the solution for drugs.

 

Table 5: Accuracy and Precision study for ezetimibe

Concentration added(µg/mL)	AMOUNT FOUND				MEAN	SD	%RSD	% RECOVERY
4+3.2 7.2µg/mL 80%	DAY 1	7	7.01	7.01	7.006667	0.005774	0.0824	97.31481481
	DAY2	7.06	7.06	7.05	7.056667	0.005774	0.081816	98.00925926
	DAY3	7	7.04	7	7.013333	0.023094	0.329287	97.40740741
4+4 8µg/mL 100%	DAY 1	7.81	7.95	7.81	7.856667	0.080829	1.028796	98.20833333
	DAY 2	7.9	7.81	7.91	7.873333	0.055076	0.699522	98.41666667
	DAY 3	7.81	7.9	7.8	7.836667	0.055076	0.702795	97.95833333
4+4.4 8.8µg/mL 120%	DAY 1	8.6	8.6	8.8	8.666667	0.11547	1.332347	98.48484848
	DAY 2	8.7	8.7	8.6	8.666667	0.057735	0.666173	98.48484848
	DAY 3	8.5	8.5	8.7	8.566667	0.11547	1.347899	97.34848485

 

Table 6: Accuracy and Precision study for Atorvastatin calcium

Concentration added(µg/mL)	AMOUNT FOUND				MEAN	SD	%RSD	% RECOVERY
4+3.2 7.2µg/mL 80%	DAY 1	7.01	7.02	7.02	7.016667	0.005774	0.082283	97.4537037
	DAY2	7.04	7.04	7.09	7.056667	0.028868	0.409081	98.00925926
	DAY3	7	7.04	7.09	7.043333	0.045092	0.640215	97.82407407
4+4 8µg/mL 100%	DAY 1	7.81	7.79	7.81	7.803333	0.011547	0.147975	97.54166667
	DAY 2	7.94	7.87	7.76	7.856667	0.090738	1.154914	98.20833333
	DAY 3	7.8	7.9	7.8	7.833333	0.057735	0.737043	97.91666667
4+4.4 8.8µg/mL 120%	DAY 1	8.8	8.7	8.7	8.733333	0.057735	0.661088	99.24242424
	DAY 2	8.7	8.7	8.6	8.666667	0.057735	0.666173	98.48484848
	DAY 3	8.7	8.6	8.7	8.666667	0.057735	0.666173	98.48484848

 

Fig. 8 - Representative Chromatogram of specificity

 

Fig. 9 - Representative Chromatogram of formulation

 

 

CONCLUSION:

A single, simple, accurate and precise RP-HPLC method has been developed, optimized and validated for simultaneous estimation of ezetimibe, simvastatin and atorvastatin calcium in tablet dosage forms. This method reduces overall assay development time and solvents. All the parameters of validation are within the acceptable range. The developed method was specific as there was no any interfering peak at retention time of drugs. Hence the developed method was accurate and can be used for routine analysis of ezetimibe, Simvastatin and atorvastatin calcium in tablet dosage forms.

 

ACKNOWLEDGEMENT:

Authors are thankful to the trustees and management of MET’S Institute of Pharmacy, Bhujbal Knowledge city, Nasik for providing necessary analytical facilities and Blue cross Pharmaceutical Ltd., Nashik for providing gift samples of Ezetimibe, Atorvastatin calcium, Simvastatin.

 

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Received on 22.06.2017       Accepted on 19.08.2017     

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