DEVELOPMENT AND VALIDATION OF NOVEL RP-HPLC METHOD FOR THESIMULTANEOUS ESTIMATION OF EZETIMIBE AND BEMPEDOIC ACID IN A TABLETDOSAGE FORM

DEVELOPMENT AND VALIDATION OF NOVEL RP-HPLC METHOD FOR THESIMULTANEOUS ESTIMATION OF EZETIMIBE AND BEMPEDOIC ACID IN A TABLETDOSAGE FORM

Vandana Jain *, Daksha Rahamatkar and Shubham Nikam

Department of Quality Assurance, Oriental College of Pharmacy, Sanpada, Navi Mumbai, Maharashtra, India.

ABSTRACT: A simple, novel, precise, and cost-effective reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated to simultaneously estimate ezetimibe and bempedoic acid in the marketed tablet dosage form. The chromatographic separation was carried out on a Prontosil C18 (250 x 4.6 mm, 5 µm) column using a mobile phase of Acetonitrile: water (60:40 v/v). The flow rate was 1.0 mL/min with detection at 225 nm using UV detector. The retention time for bempedoic acid was 4.7 min, and for ezetimibe 5.7 min. Ezetimibe showed a linear response in the concentration range of 20-60 µg/mL. Bempedoic acid showed a linear response in 180-540 µg/mL concentration range. The correlation coefficient ('r²' value) for ezetimibe and bempedoic acid was 0.9982 and 0.9998, respectively. The results of analysis have been validated as per different validation parameters. The percentage recoveries obtained for ezetimibe and bempedoic acid range from 98%-102%.

Keywords: RP-HPLC, Ezetimibe, Bempedoic acid, Method Development, Validation

INTRODUCTION: Ezetimibe is an azetidine derivative, it prevents absorption of cholesterol by blocking the Niemann Pick C1 like 1(NPC1L1) protein on epithelial cell of gastrointestinal tract, and in hepatocytes ¹. Ezetimibe is chemically (3R, 4S)-1-(4-fluorophenyl)-3-[(3S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl) azetidin-2-one and it belongs to the class cholesterol-lowering medications. Bempedoic acid is chemically 8-hydroxy-2, 2, 14, 14-tetramethyl pentadecane dioic acid and belongs to the adenosine triphosphate-citrate lyase (ACL) inhibitors ⁴.

Bempedoic acid and Ezetimibe are used in combination for the treatment hypercholesterolemia and ASCVD by reducing lipid parameters and Attenuating hsCRP levels ^3-4. Several spectroscopic RP-HPLC and UPLC-MS have been reported to estimate ezetimibe and bempedoic acid individually and in combination with other drugs. Therefore, it was thought worthwhile to develop an accurate, precise, and Cost-effective rapid RP-HPLC method for simultaneous estimation of ezetimibe and bempedoic acid in the tablet dosage form.

MATERIALS AND METHOD:

Instrumentation: Chromatography was performed on Shimadzu prominence – i 2030 system with lab solution software for data processing. Separation and quantitation were made on Prontosil C 18 column (250×4.6 nm, 5µm).

Chemicals and Reagents: Ezetimibe (99.10) and bempedoic acid (99.8) reference standard was a gift sample from Alkem laboratory Mumbai. Acetonitrile (Merck) and Milli-Q water (HPLC Grade) were used for preparing the mobile phase.

Selection of Wavelength: Each solution was scanned using a double beam UV visible spectrophotometer between the range 200nm to 400nm, and overlain spectra were obtained.

The wavelength selected was 225, which is an isosbestic point. The overlaid spectra of bempedoic acid and ezetimibe are shown in Fig. 1.

FIG. 1: UV SPECTRUM OF EZETIMIBE AND BEMPEDOIC ACID

Chromatographic Condition: Method was developed using a Prontosil C18 (250 x 4.6 mm, 5 µm) column. Mobile phase Acetonitrile: water (60:40) was used. Detection wavelength 225nm was selected by scanning standard drug solution over a wide range of wavelengths 200-400 nm using a spectrometer. The pump's flow rate was set 1.0 mL/min and the volume 10µl. The column temperature was set as 30°C.

Preparation of Mobile Phase: 60 volumes of HPLC grade acetonitrile and 40 volumes of water were used as the mobile phase.

Preparation of Diluent: Based on the Solubility of drug, diluent was selected as Water: Acetonitrile: Methanol (20:40:40).

Preparation of Standard Stock Solution: About 10 mg of each of reference standard of ezetimibe and bempedoic acid was weighed accurately and transferred to two separate 10 mL volumetric flask. Both drugs were dissolved in 60 % of diluent with shaking and volume was made up to the mark with diluent to get 1000 µg/mL of standard stock solution of each drug.

Standard Final Solution: Take 3.6 mL of standard stock solution of bempedoic acid and 0.2 mL of standard stock solution of ezetimibe transferred to 10 mL volumetric flask. Volume was made up to with diluent to get 360 µg/mL of bempedoic acid and 20µg/mL of ezetimibe standard final solutions.

Preparation of Sample Solution: Ten tablets were weighed and crushed to get a fine powder. The tablet powder equivalent to 360 mg of bempedoic acid and 20 mg of ezetimibe was transferred to a 100 mL volumetric flask and dissolved in diluent and the flask was kept in ultrasonication for 30 min. Finally, the volume was made up to the mark with the help of diluent. This solution was further diluted by taking 1ml from above solution and making up the volume up to 10 mL with diluent.

Method Development: Chromatographic separation was achieved by using Prontosil C18 (250 x 4.6 mm, 5 µm) column with acetonitrile and water in the ratio of (60:40) as the mobile phase at the flow rate of 1.0 mL/min and column temperature 30°C the detection was carried out at 225 nm. The developed, optimized method resulted in the elution of bempedoic acid at 4.7 min and ezetimibe at 5.7 min. The total run time was 10 min.

TABLE 1: OPTIMIZED CHROMATOGRAPHIC CONDITIONS FOR EZETIMIBE AND BEMPEDOIC ACID

Chromatograms of standard and sample of ezetimibe and bempedoic acid are shown in Fig. 2 and 3, respectively. The optimized chromatographic conditions are tabulated in Table 1.

RESULT AND DISCUSSION: The developed RP-HPLC method for ezetimibe and bempedoic acid was validated as per ICH guidelines.

Specificity: Specificity is the ability to assess the analyte unequivocally in the presence of components that may be expected to be present⁶.

Method specificity was determined by observing and comparing the test result obtained for the sample solution with the standard result obtained for a pure drug. The blank, standard drug and sample chromatogram is shown in Fig. 2, 3, and 4.

FIG. 2: CHROMATOGRAM OF BLANK SOLUTION

FIG. 3: CHROMATOGRAM OF STANDARD SOLUTION OF BEMPEDOIC ACID AND EZETIMIBE

FIG. 4: CHROMATOGRAM OF SAMPLE SOLUTION OF BEMPEDOIC ACID AND EZETIMIBE

System Suitability: system suitability parameter was studied to verify the optimum conditions. System suitability test was performed as per USP guidelines on the chromatograms. The different parameter was evaluated, such as retention time, tailing factor, theoretical plate, and resolution. The obtained results are summarized in Table 2.

TABLE 2: SYSTEM SUITABILITY PARAMETER RESULTS

Linearity: The standard curve was obtained within the 10 -30 µg/mL concentration range for ezetimibe and 180 -540 µg/mL for Bempedoic acid. The linearity of this method was evaluated by linear regression analysis. The linearity graph was plotted

by taking the concentration of the drug on the X-axis and the corresponding peak area on the Y-axis, as shown in Fig. 5 and Fig. 6. The linearity data is summarized in Table 3.

TABLE 3: LINEARITY DATA OF EZETIMIBE AND BEMPEDOIC ACID

FIG. 5: CALIBRATION CURVE OF EZETIMIBE              

FIG. 6: CALIBRATION CURVE OF BEMPEDOIC ACID

Precision: Precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling if same sample under the prescribed conditions ⁶.

System precision was performed by injecting six replicate injection of the standard solution of ezetimibe (20µg/ml) and bempedoic acid (360µg/mL). The average, standard deviation (SD) and % RSD of the area in six replicate injection was calculated and reported. And method precision was performed by injecting replicate injection of sample solution of ezetimibe (20μg/mL) and bempedoic acid (360μg/mL).

Its % assay, average, standard deviation (SD), and %RSD were calculated and reported. The result of system precision and method precision are summarized in Table 4.

TABLE 4: SYSTEM PRECISION & METHOD PRECISION RESULTS

Accuracy / Recovery Studies: To study the accuracy and reproducibility of the proposed method recovery experiment were carried out ⁶. A fixed amount of preanalyzed sample was taken and the standard drug was added at 50%, 100% and 150% levels. Each level was repeated three times. The content s of ezetimibe and bempedoic acid found by the proposed method is shown in Table 5. The mean of recoveries of bempedoic acid and ezetimibe was 98.88% and 100.02%, respectively, which shows no interference from the excipient.

TABLE 5: ACCURACY DATA OF EZETIMIBE AND BEMPEDOIC ACID

Assay of Marketed Formulation: Ten tablets were weighed and powdered finely. tablet powder equivalent to 360 mg of bempedoic acid and 20 mg of ezetimibe was transferred into 100 mL volumetric flask add 60 ml diluent sonicate for 30 min make up the volume up to mark. This solution was further diluted by taking 1ml from the above solution in 10 mL volumetric flak and making up the volume up to 10 mL with diluent to obtain 360 µg/mL of bempedoic acid and 20µg/mL of ezetimibe.

TABLE 6: ANALYSIS OF THE FORMULATION

Robustness: Robustness is a measure of its capacity to remain unaffected by small deliberate in the chromatographic method parameters and provides an indication of its reliability ⁶.

This was done by small, deliberate changes in chromatographic conditions at 3 different levels and retention time of ezetimibe and bempedoic acid. The factors selected were flow rate, column temperature, and wavelength.

It was observed that there were no deliberate changes in the chromatogram, which demonstrate that the RP-HPLC method developed is robust. The result is described in Table 7.

TABLE 7: RESULT OF ROBUSTNESS STUDY

CONCLUSION: Based on the results, it is concluded RP-HPLC method was successfully developed for simultaneous estimations of bempedoic acid and ezetimibe pharmaceutical formulation. Both drugs have good resolution with short analysis time 10 min. The developed HPLC method was found to be simple, accurate, linear, precise and robust.

ACKNOWLEDGEMENT: Authors are thankful to the Oriental college of pharmacy for providing the necessary facilities.

CONFLICTS OF INTEREST: Nil

REFERENCES:

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   How to cite this article:

   Jain V, Rahamatkar D and Nikam S: Devlopment and validation of novel RP-HPLC method for the simultaneous estimation of ezetimibe and bempedoic acid in a tablet dosage form. Int J Pharm Sci & Res 2022; 13(10): 4680-85. doi: 10.13040/IJPSR.0975-8232.13(11).4680-85.

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