A new stability indicating RP-UFLC method for the simultaneous estimation of Ezetimibe and Rosuvastatin combined dosage forms (Tablets)

 

Dipak Chandrakant Kulkarni¹, Anima Sunil Dadhich¹*,

Mukthinuthalapati Mathrusri Annapurna²

¹Department of Chemistry, GITAM School of Science, Visakhapatnam, India.

²GITAM School of Pharmacy, GITAM (Deemed to be University), Visakhapatnam, India.

 

ABSTRACT:

The combination of Ezetimibe and Rosuvastatin is used for lowering the cholesterol along with proper diet. A new stability indicating RP-UFLC method has been developed and validated for the estimation of Ezetimibe and Rosuvastatin in tablet dosage forms. Shimadzu Model CBM-20A/20 Alite UFLC system with Phenomenex C₁₈ column (PDA detector) was used for the present chromatographic study. Both the drugs obeyed Beer-Lambert’s law over the concentration range 0.1-150 mg/ml and the linear regression equations are found to be y = 43364x + 8195.5 and y = 49468x – 7194 (R² = 0.9999) (R² = 0.9999) Ezetimibe and Rosuvastatin respectively. The LOD and LOQ values were found to be 0.0287 mg/ml and 0.0957 mg/ml for Ezetimibe and for that of Rosuvastatin the LOD and LOQ values were found to be 0.0291 mg/ml and 0.0892 mg/ml for respectively. Stress degradation studies were performed and the method was validated as per ICH guidelines.

 

 

 

INTRODUCTION: 

Ezetimibe (EZ) (CAS No. 163222-33-1) is chemically designated as (3R, 4S) - 1 - (4 - fluorophenyl) - 3 - [(3S) - 3 - (4 - fluorophenyl) - 3 - hydroxypropyl] - 4 - (4 - hydroxyphenyl) azetidin - 2 - one¹ (C₂₄H₂₁F₂NO₃·H₂O) with molecular formula 427.43grams/mole (Figure 1A). Rosuvastatin (CAS No. 287714-41-4) is chemically designated as (3R, 5S, 6E) - 7 - [4 - (4 - fluorophenyl) - 2 - (N – methyl methane sulfonamido) - 6 - (propan - 2 - yl) pyrimidin - 5 - yl] - 3, 5 – dihydroxy hept - 6 - enoic acid¹ (C₂₂H₂₈FN₃O₆S) with molecular formula 481.5 grams/mole (Figure 1B). The combination of these two drugs is used for the treatment of Hyperlipidemia.

 

 

Mohammed et al., developed RP-HPLC method² for the simultaneous determination of Rosuvastatin and Ezetimibe using Waters C18 column and mobile phase consisting of a mixture of acetonitrile: water: 0.02 M phosphate buffer pH 8 (40: 10: 50, v/v) with flow rate 1.0 ml/min (PDA detection at 230 nm) and the linearity was observed over the concentration range 30-90 μg/ ml for both the drugs. Mathrusri Annapurna et al., developed a stability indicating RP-HPLC method³ for the simultaneous determination of Rosuvastatin and Ezetimibe using C18 column and mobile phase consisting of a mixture of sodium acetate buffer (pH 4.0) and acetonitrile (30:70, %v/v) with a flow rate of 1.2 mL/min. (UV detection at 254) and the linearity was observed over the concentration range 0.5-250 μg/ml for both the drugs. Varma et al.,developed RP-HPLC method⁴ for the simultaneous determination of Rosuvastatin and Ezetimibe using Sunfire BDS C18 column and mobile phase consisting of a mixture of Ammonium acetate buffer (pH adjusted to 6.50 ± 0.05 with dilute formic acid solution) and acetonitrile (55:45, v/v) with UV detection at 230 nm. Linearity was observed over the concentration range 98.19-294.56 µg/ml and 99.12- 297.36 µg/ml for Rosuvastatin (Rt 2.74) and Ezetimibe (Rt 4.80) respectively. Anuradha et al., developed RP-HPLC method⁵ for the simultaneous determination of Rosuvastatin and Ezetimibe using Hypersil BDS C18 column and mobile phase consisting of a mixture of 0.05 M phosphate buffer (pH 2.5) and Methanol with UV detection at 242 nm. Linearity was observed over the concentration range 5-80 µg/ml for Rosuvastatin and Ezetimibe respectively. Swathi Sri et al., developed RP-HPLC method⁶ for the simultaneous determination of Rosuvastatin and Ezetimibe using Enable C18G column and mobile phase consisting of a mixture of acetonitrile and water (75:25) with UV detection at 252 nm. Linearity was observed over the concentration range 5-40 µg/ml for Rosuvastatin and Ezetimibe respectively. Joshi et al., developed RP-HPLC method⁷ for the simultaneous determination of Rosuvastatin and Ezetimibe using Phenomenex Gemini C18 column and mobile phase consisting of a mixture of acetonitrile, methanol and water (50: 10: 35) (pH adjusted to 3.0 using 0.5% ortho phosphoric acid). Smita et al., developed RP-HPLC method⁸ for the simultaneous determination of Rosuvastatin and Ezetimibe using Licrosphere C18 column and mobile phase consisting of a mixture of methanol: acetonitrile: phosphate buffer (pH 3.5) (60:20:20) with UV detection at 279 nm. Linearity was observed over the concentration range 5-10 mg/ml for Rosuvastatin and Ezetimibe respectively. Mathrusri Annapurna et al., developed a stability-indicating RP-HPLC method⁹ was developed and validated for the simultaneous determination of Rosuvastatin and Ezetimibe in tablet dosage forms using C 18 column with mobile phase consisting of tetra butyl ammonium hydrogen sulphate-acetonitrile (32:68, v/v)  with a  flow rate  of 1.0  ml/min (UV  detection 254  nm). Linearity was observed over the concentration range 0.1-200 μg/ml for both Rosuvastatin 0.9998) and Ezetimibe.

 

Smita et al., developed a micellar liquid chromatographic method¹⁰ for the simultaneous determination of Rosuvastatin and Ezetimibe.  Chromatographic study was performed using 5% n-Butanol in 0.05 mole/litre Tween-20 (pH adjusted to 5.5 + 0.01 with o-phosphoric acid) as mobile phase and Licrosphere C18 column (UV detection 314 nm) and the linearity was observed over the concentration range 5-25 μg/ ml for both the drugs. At present the authors have proposed a new stability indicating validated RP-UFLC method for the determination of Rosuvastatin and Ezetimibe in tablets.

 

 

MATERIALS AND METHODS

Rosuvastatin and Ezetimibe API (> 99.9 purity) were obtained from Glenmark Pharmaceuticals Ltd., India. HPLC grade acetonitrile, acetic acid, hydrochloric acid, sodium hydroxide and hydrogen peroxide (30% w/v) were (AR grade) procured from Merck, India and Milli-Q ultrapure water used for the study. and other chemicals such as and purchased from Merck (India). A mixture of 0.1% Acetic acid: Acetonitrile (35: 65) was chosen for the chromatographic study (Flow rate: 1.0 mL/min; UV detection: 240 nm; Injection volume: 20 µl). A UFLC system, Shimadzu Model CBM-20A/20 Alite with Phenomenex C₁₈ column (PDA detector) was used for the chromatographic study and the run time was 10 min.

 

Preparation of Rosuvastatin and Ezetimibe stock solutions (1000 µg/mL)

25 mg of each of Rosuvastatin and Ezetimibe were separately weighed accurately and transferred in to a 25 mL volumetric flask and dissolved in HPLC grade acetonitrile (1000 µg/mL) and these stock solutions were diluted with the mobile phase as per the requirement and used for the study.

 

Method validation¹¹

Linearity

Drug solutions containing both Rosuvastatin and Ezetimibe (0.1-150 µg/mL) were prepared n from their stock solutions on dilution and each of these solutions were injected into the UFLC system (n=3) and the respective chromatograms were recorded. The peak area of Rosuvastatin and Ezetimibe were noted and finally the mean peak area was calculated. A calibration curve was plotted by plotting the concentration of Rosuvastatin and Ezetimibe solutions on the x-axis and the corresponding mean peak area on the y-axis.

 

Precision, Accuracy and Robustness studies

Intra-day and inter-day precision studies of Rosuvastatin and Ezetimibe were performed (10, 20 and 40 µg/mL) within the linearity range on the same day and on three different days i.e. Day 1, Day 2 and Day 3 respectively and the % RSD was calculated. The accuracy study was performed by spiking the Rosuvastatin and Ezetimibe tablet formulation (each 10 µg/mL) solutions (80, 100, 150%) with a known concentration of Rosuvastatin (API) and Ezetimibe (API). 20 µL of these resulting solutions containing both Rosuvastatin and Ezetimibe were injected thrice in to the UFLC system and mean peak area was calculated and thereby the % RSD. Robustness study of Rosuvastatin and Ezetimibe (10 µg/mL) was performed by incorporating small changes in the optimized chromatographic conditions such as flow rate (± 0.1 mL/min), detection wavelength (± 2 nm) and mobile phase composition (± 5%). 20 µL of the solution containing both Rosuvastatin and Ezetimibe were injected (n=3) in to the UFLC system with the above mentioned conditions for the robustness study and the mean peak area from the respective chromatograms and thereby the mean peak area as well as the % RSD were calculated.

 

Assay of Rosuvastatin and Ezetimibe tablets

Tablets containing Rosuvastatin and Ezetimibe are available with brand names Rozavel-EZ, Roseday-EZ, Roszet, Drose-E, Rosuzet-E etc with different label claims consisting of Rosuvastatin and Ezetimibe 10/10, 10/5, 40/10 mg. 20 tablets of a combined dosage form of Rosuvastatin and Ezetimibe were accurately weighed, powdered and powder equivalent to 25 mg of each of Rosuvastatin and Ezetimibe of two different brands was carefully transferred to two separate 25 ml volumetric flasks and the combined solution of Rosuvastatin and Ezetimibe was extracted with HPLC grade acetonitrile, sonicated and filtered. These solutions were diluted further with the mobile phase and 20 µL of each combined formulation solution was injected in to the system (n=3) and the average peak area was calculated from the respective chromatograms and thereby the amount of Rosuvastatin and Ezetimibe was calculated from the calibration curve.   

 

Stress degradation studies¹²

The stability studies of Rosuvastatin and Ezetimibe were performed to determine the specificity of the method. Stress degradation studies such as acidic hydrolysis, basic hydrolysis, oxidation and thermal degradations were performed and 20 µl of these solutions were injected in to the UFLC system. Acidic degradation was performed by heating Rosuvastatin and Ezetimibe combined solution with 1 mL of 0.1 N HCl solution was heated to 80⁰C in a thermostat for 30 mins and then the stressed sample was cooled, neutralized with 1.0 mL 0.1N sodium hydroxide solution, diluted as per requirement with the mobile phase and then 20 µl of the resulting solution was injected in to the UFLC system. Alkaline degradation was performed by heating Rosuvastatin and Ezetimibe combined solution with 1 mL of 0.1 N sodium hydroxide solution was heated to 80⁰C in a thermostat for 30 mins and then the stressed sample was cooled, neutralized with 1.0 mL 0.1N HCl solution, diluted as per requirement with the mobile phase and then 20 µl of the resulting solution was injected in to the UFLC system. Thermal degradation was performed by heating Rosuvastatin and Ezetimibe combined solution to 80⁰C in a thermostat for 30 mins and then the stressed sample was cooled and diluted as per requirement with the mobile phase and then 20 µl of the resulting solution was injected in to the UFLC system. Oxidative degradation was performed by heating Rosuvastatin and Ezetimibe combined solution with 1.0 mL 30% hydrogen peroxide solution to 80⁰C in a thermostat for 30 mins and then the stressed sample was cooled and diluted as per requirement with the mobile phase and then 20 µl of the resulting solution was injected in to the UFLC system.

 

RESULTS AND DISCUSSION:

The authors have developed a new validated stability indicating RP-UFLC method for the quantification of Rosuvastatin and Ezetimibe in API and tablets. The chromatographic study was performed using Shimadzu Model CBM-20A/20 Alite UFLC system with Phenomenex C₁₈ column and a mixture of 0.1% acetic acid: acetonitrile (35: 65) with flow rate 1.0 mL/min (UV detection 240 nm on isocratic mode for the present study. The injection volume was 20 µl and the run time was 10 min. The analytical techniques so far developed were compared with the present proposed method and the details were given in Table 1.

 

Table 1: Review of literature

 

Method validation

Both Ezetimibe and Rosuvastatin obeyed Beer-Lambert’s law over the concentration range 0.1-150 mg/ml with % RSD 0.21-0.62 for Ezetimibe and 0.11-0.76 for Rosuvastatin (Table 2) and the linear regression equations are found to be y = 43364x + 8195.5 and y = 49468x – 7194 (R² = 0.9999) (R² = 0.9999) Ezetimibe and Rosuvastatin  respectively. The LOD and LOQ values were found to be 0.0287 mg/ml and 0.0957 mg/ml for Ezetimibe and for that of Rosuvastatin the LOD and LOQ values were found to be 0.0291 mg/ml and 0.0892 mg/ml for respectively and the representative chromatograms were shown in Figure 2A-Figure 2C.

 

In precision study the % RSD was found to be 0.22-0.48 and 0.54-0.71 (Intraday) and 0.34-0.81 and 0.55-0.92 (Inter-day) for Ezetimibe and Rosuvastatin respectively (Table 3) which is less than 2.0 indicating that the proposed method is precise. In accuracy study the % RSD was found to be 0.91-1.22 and 0.79-0.87 for Ezetimibe and Rosuvastatin respectively (Table 4) which is less than 2.0 indicating that the proposed method is accurate. The percentage recovery in accuracy studies was found to be 99.00-99.94 and 99.73-99.94% for Ezetimibe and Rosuvastatin respectively. The % RSD in robustness study was found to be 0.41-1.03 for Ezetimibe and 0.23-0.92 for Rosuvastatin which was less than 2% indicating that the method is robust (Table 5).

 

Table 2: Linearity Study

Conc. (µg/ml)	*Mean peak area		% RSD*
	Ezetimibe	Rosuvastatin	EZ	RS
0	0	0	0	0
0.1	4968	4384	0.21	0.38
0.2	9927	8702	0.35	0.61
0.5	24985	23491	0.28	0.55
1	48365	44913	0.47	0.27
2	98754	88945	0.54	0.45
5	249587	225468	0.29	0.67
10	488003	438667	0.62	0.59
20	982541	870219	0.57	0.62
50	2401466	2245973	0.37	0.76
100	4913249	4356837	0.24	0.34
150	7451584	6482571	0.38	0.11

*Mean of three replicates

 

Figure 2A: Blank

Figure 2B: Typical chromatogram of Rosuvastatin API (100 µg/mL) (Rt 2.576 min) Theoretical plates: 6091.566; Tailing factor: 1.467

Figure 2C: Typical chromatogram of Rosuvastatin (10 µg/mL) (Rt 2.563 min) Theoretical plates: 6111.428; Tailing factor: 1.517 Ezetimibe (10 µg/mL) (Rt 3.631 min) Theoretical plates: 8914.256; Tailing factor: 1.424; Resolution: 7.497

Figure 2D: Typical chromatogram of tablet formulation Rosuvastatin (10 µg/mL) (Rt 2.560 min) Theoretical plates: 5734.786; Tailing factor: 1.502 Ezetimibe (10 µg/mL) (Rt 3.625 min) Theoretical plates: 8602.834; Tailing factor: 1.422; Resolution: 7.302

 

Table 3: Precision Study

*Mean of three replicates

Table 4: Accuracy Study

*Mean of three replicates

 

Table 5: Robustness Study (20 µg/ml)

*Mean of three replicates

 

 

Assay of Rosuvastatin and Ezetimibe tablets

The assay of combined dosage forms of Rosuvastatin and Ezetimibe (Tablets) was performed for two different brands available in India.  The resulting extracted solutions from the tablet dosage forms were diluted as per the requirement and 20 µl of these two branded products were injected (n=3) in to the UFLC system under the optimized chromatographic conditions and the peak area of Rosuvastatin and Ezetimibe was noted and thereby the mean peak area was calculated and then the percentage of purity of Rosuvastatin and Ezetimibe was calculated from the linear regression equation. The percentage of purity of Rosuvastatin and Ezetimibe was found to be 99.52-99.75 for Ezetimibe and 99.68-99.87 for Rosuvastatin (Table 6). The typical chromatogram of Rosuvastatin and Ezetimibe tablet dosage form was shown in Figure 2D and no interference of excipients was observed.

 

Stress degradation studies

Ezetimibe and Rosuvastatin were eluted at 3.635 and 2.561 mins respectively with theoretical plates greater than 2000 and tailing factor less than 1.5 which are within the acceptable criteria. During the acidic degradation study Ezetimibe and Rosuvastatin were eluted at 3.635 and 2.550 mins respectively. 3.03% Ezetimibe and 14.38% Rosuvastatin had undergone degradation. The theoretical plates were greater than 2000, the tailing factor were less than 1.5 and that of resolution was greater than 2.0 (7.485) which are within the acceptable criteria. During the thermal degradation study Ezetimibe and Rosuvastatin were eluted at 3.632 and 2.554 mins respectively. 0.21% Ezetimibe and 20.84% Rosuvastatin had undergone degradation. The theoretical plates were greater than 2000, the tailing factor were less than 1.5 and that of resolution was greater than 2.0 (7.285) which are within the acceptable criteria. During the alkaline degradation study Ezetimibe and Rosuvastatin were eluted at 3.625 and 2.544 mins respectively. 0.21% Ezetimibe and 20.84% Rosuvastatin had undergone degradation. The theoretical plates were greater than 2000, the tailing factor were less than 1.5 and that of resolution was greater than 2.0 (7.285) which are within the acceptable criteria. The proposed method is highly specific as Ezetimibe and Rosuvastatin peaks did not interfere with any other degradant peaks. The % recovery in all the degradation studies was shown in Table 7 and the chromatograms obtained during the degradation studies were shown in Figure 3.

 

 

Table 6:  Assay of Ezetimibe and Rosuvastatin tablets

*Mean of three replicates

 

Table 7: Stress degradation study

*Mean of three replicates

 

 

CONCLUSIONS:

The authors have proposed a new robust stability indicating RP-UFLC for the simultaneous determination of Ezetimibe and Rosuvastatin and the method was validated as per ICH guidelines. The method is specific as no interference of degradants with the drug peaks was observed and also no interference of excipients was observed during the assay of tablet formulations. The proposed method is quite simple, precise and accurate and is suitable for the estimation of Ezetimibe and Rosuvastatin even in biological fluids as well as for the pharmacokinetic studies.

 

ACKNOWLEDGEMENT:

The authors are very much grateful to Glenmark Pharmaceuticals Ltd. (India) for providing the gift samples of Ezetimibe and Rosuvastatin. The authors declare no conflict of interest.

 

REFERENCES:

1.      Neil, M. J. O; The Merck Index, Merck Research Laboratories, 14^th edition, Merck and Co., Inc., Whitehouse Station, New Jersey, (2006).

2.      Mohammed, I. B., Vanitha Prakash, K. and Krishna Mohan, G. (2013). RP-HPLC method for the simultaneous estimation of Rosuvastatin and Ezetimibe from their combination tablet dosage form, International Journal of Chemical and Analysis Science, 4, 205-209.

3.      Mathrusri Annapurna M, Bukkapatnam V and Bandaru SP. Stability indicating liquid chromatographic method for the simultaneous determination of Rosuvastatin and Ezetimibe in pharmaceutical formulations. Adv Pharm Bull. 2014; 4(4): 405-411.

4.      Varma PSRChNPD, Lakshmana Rao A and Dinda SC. Development and validation of stability indicating RP-HPLC method for simultaneous estimation of Rosuvastatin and Ezetimibe in combined tablet dosage form. Rasayan Journal of Chemistry. 2012; 5(3):269-279.

5.      Anuradha KG and Vishal DS. Development and validation of a stability-indicating reversed-phase HPLC method for simultaneous estimation of Rosuvastatin and Ezetimibe from their combination dosage forms, Eurasian Journal of Analysis Chemistry. 2010; 5(3): 280-298.

6.      Swathi Sri D, Hemant Kumar T, Vara Prasada Rao K and Srinivasa Rao Y. Validated RP-HPLC method for simultaneous determination of Rosuvastatin calcium and Ezetimibe in pharmaceutical dosage form. International Journal of Pharmacy and Pharmaceutical Sciences. 2015; 7(4): 209-213.

7.      Joshi H, Yadav SK, Seth AK. New analytical method development and validation of rosuvastatin and ezetimibe in tablet dosage form. International Journal of Drug Discovery and Medical Research. 2012; 1(2): 22-30.

8.      Smita S., Sharma MC, Kohli D and Chaturvedi SC. Simultaneous estimation of Rosuvastatin calcium and Ezetimibe in tablet dosage form by reverse phase high performance liquid chromatography, Optoelectronics and Advanced Materials: Rapid Communications. 2010; 4(2): 238-241.

9.      9) Mathrusri Annapurna M, Bukkapatnam V, Bandaru SPK and Grandhi NS. Simultaneous determination of Rosuvastatin and Ezetimibe in pharmaceutical formulations by stability indicating liquid chromatographic method. Journal of Bioequivalence and Bioavailability. 2014; 6(5): 174-180.

10.   Smita S, Sharma MC, Kohli DV and Chaturvedi SC. Micellar liquid chromatographic method development for determination of Rosuvastatin calcium and Ezetimibe in pharmaceutical combination dosage form. Der Pharma Chemica 2010; 2: 371-377.

11.   ICH Q2 (R1) Validation of analytical procedures: Text and methodology (2005).

12.   ICH Q1A (R2) Stability testing of new drug substances and products (2003).

 

 

 

 

Accepted on 18.10.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(10):4917-4923.