Method Development and Validation for Simultaneous Estimation of Bempedoic Acid and Ezetimibe in Pharmaceutical Dosage Form by
RP- HPLC
Supriya Vanga1, Aneesa1*, C. Parthiban1, M. Sudhakar2
1Department of Pharmaceutical Analysis, Malla Reddy College of Pharmacy,
Maisammaguda, Secunderabad 500010, Telangana, India.
2Department of Pharmaceutical Biotechnology, Malla Reddy College of Pharmacy,
Maisammaguda, Secunderabad 500010, Telangana, India.
*Corresponding Author E-mail: draneesa.16@gmail.com
ABSTRACT:
A straightforward, accurate, and precise method has been successfully developed for the simultaneous estimation of Bempedoic acid and Ezetimibe in both bulk and tablet dosage forms. This achievement marks a significant advancement in pharmaceutical analysis. The method employed a chromatographic approach using a standard BDS C18 column measuring 150 x 4.6 mm with a particle size of 5μ. The mobile phase, comprising awell-balanced blend of 0.01N Ammonium acetate Buffer and Methanol in a 55:45 ratio, was efficiently pumped through the column at a constant flow rate of 1 ml/min. The critical buffer used in this methodology was 0.01N Ammonium acetate buffer, maintaining a stable temperature at 30°C. The optimized wavelength of 230 nm was selected for analysis. The retention times for Bempedoic acid and Ezetimibe were precisely determined as 2.219 minutes and 2.794 minutes, respectively, demonstrating the method's reproducibility with %RSD values of 0.3 and 0.4. Impressive recoveries of 100.25% for Bempedoic acid and 99.72% for Ezetimibe were achieved. Furthermore, the method exhibited impressive sensitivity with LOQ and LOD values of 0.07, 0.24, and 0.01, 0.04, respectively, derived from the regression equations. This method's exceptional efficiency not only reduced retention times but also minimized overall run time, offering a practical, cost-effective solution suitable for routine quality control testing within the pharmaceutical industry. Its simplicity and accuracy make it a valuable addition to pharmaceutical analysistechniques
KEYWORDS: Bempedoic acid, Ezetimibe, Methanol and Retention time.
Bempedoic Acid is a first-in-class adenosine triphosphate citrate lyase (ACL) inhibitor that is taken once daily to lower LDL cholesterol levels in statin-resistant patients1,2, Espersion therapeutics Inc.3 developed bempedoic acid, which was approved by the FDA on February 21, 20204. On February 26, 2020, NEXLIZET, a combination product of bempedoic acid and ezetimibe, was approved. The combination medication used to treat hypercholesterolemia. Structurally Bempedoic acid is also known as 8-hydroxy- 2,2,14,14- tetramethylpentadecanedioic acid. It is a prodrug that needs to be activated in the liver5. The very-long-chain acyl-CoA synthetase-1 (ACSVL1) enzyme is responsible for its conversion to the pharmacologically active metabolite ETC-1002-CoA6,7. The enzyme ATP lyase (also known as ATP synthase) is essential for cholesterol synthesis. After the parent drug is activated in the liver by coenzyme A (CoA), ETC- 1002-CoA directly inhibits this enzyme 8,9.
Ezetimibe is an azetidine derivative and a lipid lowering compound that inhibits intestinal cholesterol and phytosterol absorption10,11. The discovery and research of this drug began early 1990s, Ezetimibe structure consists of (3R,4S)-1-(4-fluorophenyl)-3-[(3S)-3-(4-fluorophenyl)-3- hydroxypropyl]-4-(4-hydroxyphenyl) azetidin-2-one12, Ezetimibe is used as an adjunctive therapy to a healthy diet to lower cholesterol levels in primary Hyperlipidemia, mixed Hyperlipidemia, Homozygous familial hypercholesterolemia and phytosterolemia. Ezetimibe mediates its blood cholesterol-lowering effect via selectively inhibiting the absorption of cholesterol and phytosterol by the small intestine without altering the absorption of fat- soluble vitamins and nutrients13,14,15.
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.
Structure of Ezetimibe
• WATERS HPLC 2695 SYSTEM equipped with quaternary pumps, Photo Diode Array detector and Auto sampler integrated with Empower 2Software.
• UV-VIS spectrophotometer PG Instruments T60 with special bandwidth of 2mm and 10mm and matched quartz cells integrated with UV win 6 Software was used for measuring absorbance of Ezetimibe and Bempedoic acid solutions.
Ezetimibe and Bempedoic acid pure drugs (API), Combination Ezetimibe and Bempedoic acid tablets (Bempetol-EZ, Ezetimibe 180mg, Bempedoic Acid 10mg), Distilled water, Acetonitrile, Phosphate buffer, Methanol, Potassium dehydrogenate ortho phosphate buffer, Ortho-phosphoric acid. All the above chemicals and solvents are from Rankem.
|
Mobile Phase |
: |
Methanol: 0.01N Ammonium acetate (45:55) |
|
Flow rate |
: |
1 mL/min |
|
Column |
: |
BDS C18 (4.6 x 150mm,5µm) |
Detector wavelength
|
:
|
228nm
|
Column temperature
|
:
|
25°C
|
Injection volume
|
:
|
10L
|
|
Run time |
: |
5min |
|
Diluent |
: |
Water and Acetonitrile in the ratio 50:50%V/V |
Preparation of buffer:
Accurately weighed 0.77g of Ammonium acetate was taken in a 1000mL of Volumetric flask add about 900 mL of milli-Q water added and degas to sonicate and finally make up the volume with water to get 0.01N Ammonium acetate buffer.
0.1% OPA Buffer: 1ml of Conc. Ortho Phosphoric acid was diluted to 1000mL with water.
Buffer: 0.01N Ammonium acetate
Accurately weighed 5mg of Ezetimibe, 90mg of Bempedoic acid and transferred to 50mL volumetric flask. 3/4th of diluents was added to both of these flasks and sonicated for 10min. Flasks were made up with diluents and labeled as Standard stock solution 1 and 2. (50µg/mL of Ezetimibe and 900µg/mL of Bempedoic acid)
(100% solution): 1ml from stock solution was pipetted out and taken into a 10mL volumetric flask and made up with diluent. (5µg/mL Ezetimibe of and 90µg/mL of Bempedoic acid)
5 tablets were weighed and the average weight of each tablet was calculated, then the weight equivalent to 1 tablet was transferred into a 10mL volumetric flask, 5mL of diluents was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC filters (100µg/mL of Ezetimibe and 1800µg/mL of Bempedoic acid)
(100% solution): 0.5mL of filtered sample stock solution was transferred to 10mL volumetric flask and made up with diluent.(5µg/mL of Ezetimibe and 100µg/mL of Bempedoic acid).
The system suitability parameters were determined by preparing standard solutions of Ezetimibe (5ppm) and Bempedoic acid (90ppm) and the solutions were injected six times and the parameters like peak tailing, resolution and USP plate count were determined.
The % RSD for the area of six standard injections results should not be more than 2%.
|
S no |
Bempedoic acid |
Ezetimibe |
|
||||
|
Inj |
RT (min) |
USP Plate Count |
Tailing |
RT (min) |
USP Plate Count |
Tailing |
Resolution |
|
1 |
2.215 |
6895 |
1.21 |
2.788 |
5146 |
1.35 |
4.5 |
|
2 |
2.217 |
6785 |
1.20 |
2.789 |
5136 |
1.34 |
4.6 |
|
3 |
2.217 |
6826 |
1.21 |
2.789 |
5175 |
1.34 |
4.5 |
|
4 |
2.218 |
6859 |
1.20 |
2.791 |
5163 |
1.35 |
4.4 |
|
5 |
2.218 |
6835 |
1.21 |
2.794 |
5178 |
1.34 |
4.5 |
|
6 |
2.220 |
6848 |
1.20 |
2.799 |
5169 |
1.35 |
4.5 |
Checking of the interference in the optimized method. We should not find interfering peaks in blank and placebo at retention times of these drugs in this method. So this method was said to be specific.
|
Bempedoic acid |
Ezetimibe |
||
|
Conc (μg/mL) |
Peak area |
Conc (μg/mL) |
Peak area |
|
0 |
0 |
0 |
0 |
|
22.5 |
1177262 |
1.25 |
62001 |
|
45 |
2393207 |
2.5 |
124761 |
|
67.5 |
3623397 |
3.75 |
183899 |
|
90 |
4745941 |
5 |
247225 |
|
112.5 |
5908806 |
6.25 |
307584 |
|
135 |
7148259 |
7.5 |
365385 |
Six linear concentrations of Bempedoic acid (22.5-135µg/mL) and Ezetimibe (1.25-7.5µg/mL) were injected in a duplicate manner. Average areas were mentioned above and linearity equations obtained for Bempedoic acid was y = 52795x+7346 and of Ezetimibe was y = 11385x + 2353.2 Correlation coefficient obtained was 0.9999 for the two drugs.
Calibration curve of Ezetimibe
Three levels of Accuracy samples were prepared by standard addition method. Triplicate injections were given for each level of accuracy and mean. %Recovery was obtained as 100.85% and 99.96% for Bempedoic acid and Ezetimibe respectively.
|
% Level |
Amount Spiked (μg/mL) |
Amount recovered (μg/mL) |
% Recovery |
Mean % Recovery |
|
50% |
45 |
45.42 |
100.94 |
100.85% |
|
|
45 |
45.98 |
102.18 |
|
|
45 |
45.66 |
101.47 |
||
|
100% |
90 |
90.34 |
100.38 |
|
|
90 |
91.25 |
101.39 |
||
|
90 |
89.73 |
99.70 |
||
|
150% |
135 |
135.74 |
100.55 |
|
|
135 |
135.78 |
100.58 |
||
|
135 |
135.61 |
100.45 |
Accuracy table of Ezetimibe
|
% Level |
Amount Spiked (μg/mL) |
Amount recovered (μg/mL) |
% Recovery |
Mean %Recovery |
|
50% |
2.5 |
2.51 |
100.75 |
99.96% |
|
2.5 |
2.50 |
99.68 |
||
|
2.5 |
2.49 |
99.51 |
||
|
100% |
5 |
4.99 |
101.36 |
|
|
5 |
5.04 |
99.57 |
||
|
5 |
5.01 |
100.30 |
||
|
150% |
7.5 |
7.43 |
99.02 |
|
|
7.5 |
7.46 |
99.45 |
||
|
7.5 |
7.55 |
100.67 |
e) Precision:
From a single volumetric flask of working standard solution six injections were given and the obtained areas were mentioned above. Average area, standard deviation and % RSD were calculated for two drugs. % RSD obtained as 0.5% and 1.0% respectively for Bempedoic acid and Ezetimibe. As the limit of Precision was less than “2” the system precision was passed in this method
|
S. No |
Area of Bempedoic acid |
Area of Ezetimibe |
|
1. |
4775481 |
244586 |
|
2. |
4746259 |
246985 |
|
3. |
4756932 |
247625 |
|
4. |
4775892 |
241586 |
|
5. |
4728632 |
246985 |
|
6. |
4793258 |
243287 |
|
Mean |
4762742 |
245176 |
|
S.D |
23389.6 |
2422.7 |
|
%RSD |
0.5 |
1.0 |
LOD sample Preparation: 0.25mL each from two standard stock solutions was pipetted out and transferred to two separate 10mL volumetric flasks and made up with diluents. From the above solutions 0.1ml each of Ezetimibe, Bempedoic acid, solutions respectively were transferred to 10mL volumetric flasks and made up with the same diluents.
LOQ sample Preparation: 0.25mL each from two standard stock solutions was pipetted out and transferred to two separate 10mL volumetric flask and made up with diluent. From the above solutions 0.3mL each of Ezetimibe, Bempedoic acid, and solutions respectively were transferred to 10mL volumetric flasks and made up with the same diluent.
|
Molecule |
LOD |
LOQ |
|
Bempedoic acid |
0.07 |
0.24 |
|
Ezetimibe |
0.01 |
0.04 |
Robustness conditions like Flow minus (0.9mL/min), Flow plus (1.1mL/min), mobile phase minus (65B:35A), mobile phase plus (55B:45A), temperature minus (25°C) and temperature plus (35°C) was maintained and samples were injected in duplicate manner. System suitability parameters were not much affected and all the parameters were passed. %RSD was within the limit.
|
S. No |
Condition |
%RSD of Bempedoic acid |
%RSD of Ezetimibe |
|
1 |
Flow rate 0.9mL/min |
0.3 |
0.6 |
|
2 |
Flow rate 1.1mL/min |
0.9 |
0.9 |
|
3 |
Mobile phase (-) 65B:35A |
0.6 |
0.3 |
|
4 |
Mobile phase (+) 55B:45A |
0.4 |
0.8 |
|
5 |
Temperature (-) 25°C |
0.7 |
1.3 |
|
6 |
Temperature (+) 35°C |
0.6 |
1.1 |
h) Degradation Studies:
Degradation studies were performed with the formulation and the degraded samples were injected. Assay of the injected samples was calculated and all the samples passed the limits of degradation
|
S. No. |
Degradation Condition |
% Drug Degraded |
% drug undegraded |
|
1 |
Acid |
92.15 |
7.85 |
|
2 |
Alkali |
96.03 |
3.97 |
|
3 |
Oxidation |
97.08 |
2.92 |
|
4 |
Thermal |
98.19 |
1.81 |
|
5 |
UV |
99.03 |
0.97 |
|
6 |
Water |
99.74 |
0.26 |
Degradation Data of Ezetimibe
|
S. No. |
Degradation Condition |
% Drug Degraded |
% drug undegraded |
|
1 |
Acid |
92.82 |
7.18 |
|
2 |
Alkali |
95.74 |
4.26 |
|
3 |
Oxidation |
96.63 |
3.37 |
|
4 |
Thermal |
98.14 |
1.86 |
|
5 |
UV |
98.14 |
1.86 |
|
6 |
Water |
99.45 |
0.55 |
A simple, accurate, precise method was developed for the simultaneous estimation of the Bempedoic acid and Ezetimibe in Bulk and Pharmaceutical dosage form. Retention time of Bempedoic acid and Ezetimibe were found to be 2.219min and 2.794min. %RSD of the Bempedoic acid and Ezetimibe were and found to be 0.3 and 0.4 respectively. %Recovery was obtained as 100.25% and 99.72% for Bempedoic acid and Ezetimibe respectively. LOQ, LOD values obtained from regression equations of Bempedoic acid and Ezetimibe were 0.07, 0.24 and 0.01, 0.04 respectively. Regression equation of Bempedoic acid is y =52795x + 7346, and y =11385x+2353 of Ezetimibe. Retention times were decreased and that run time was decreased, so the method developed was simple and economical that can be adopted in regular Quality control test in industries.
The authors have no conflicts of interest regarding this investigation.
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Received on 31.10.2023 Revised on 17.05.2024 Accepted on 24.10.2024 Published on 18.12.2024 Available online on December 21, 2024 Asian J. Pharm. Tech. 2024; 14(4):325-329. DOI: 10.52711/2231-5713.2024.00053 ©Asian Pharma Press All Right Reserved
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