To Develop and Validate the Stability Indicating Method for Ezetimibe and Rosuvastatin Using HPLC Method

 

Mohd Bilal Sufi; Sakshi Bobade *; Rajlaxmi Deolekar

     

Department of Pharmaceutics, New Montfort Institute of Pharmacy, Ashti, Dist. Wardha (M.S) 442202

 

*Correspondence: sakshibobade2000@gmail.com

DOI: https://doi.org/10.71431/IJRPAS.2026.5718            

Article Information		Abstract
Review Article Received: 20/07/2026 Accepted: 21/07/2026 Published:31/07/2026   Keywords Rosuvastatin, Ezetimibe, RP-HPLC, Method Validation, Stability-Indicating Method, Forced Degradation, ICH Q2(R2), Pharmaceutical Analysis.		A reverse-phase HPLC (RP-HPLC) method has been developed and validated which is simple, fast, accurate of Rosuvastatin and Ezetimibe in the pharmaceutical tablet dose. The separation was done on C18 column (250 × 4.6 mm, 5 µm) with Methanol acid (60:40, v/v) as mobile phase flow rate of 1.0 mL/min at the isosbestic wavelength of 233 nm detected using UV detectors. With the optimized method, well-resolved peaks were obtained for Rosuvastatin around 3.39 min and Ezetimibe around 4.58 min. The method was validated according to the guidelines outlined by ICH Q2(R2) and was found to be linearly in the range of 6-30 µg/mL for Rosuvastatin and 3-15 µg/mL for Ezetimibe with correlation coefficient near unity. The accuracy was found between 98.96 to 100.29% for Rosuvastatin and 98.46–100.74% for Ezetimibe and repeatability, intraday precision and interday precision showed %RSD values less than 2% proving the precision of the method. The developed method was determined to be specific, sensitive, rugged and robust with good limits of detection and limits of quantitation and validated successfully for the determination of marketed tablet formulations, giving a high accuracy of 99.81% for Rosuvastatin, 99.68% of Ezetimibe. Forced degradation studies conducted in acidic, alkaline, oxidative and photolytic conditions proved that the method was suitable for identifying degradation products from Rosuvastatin and Ezetimibe, the latter being relatively more susceptible to degradation than Rosuvastatin. The developed method of RP-HPLC is simple, economical, reliable, routine QC method and can be used for stability study of model compound and simultaneous estimation of Rosuvastatin and Ezetimibe in combined pharmaceutical dosage formulation.

 

INTRODUCTION:

Hyperlipidemia (Dyslipidemia) is the abnormal increase, either quantitatively or qualitatively, of plasma lipoproteins: high total cholesterol, low-density lipoprotein cholesterol (LDL-C), triglycerides, or a decrease in the amount of high-density lipoprotein cholesterol (HDL-C) [1-3]. Hyperlipidemia, which is can be divided into primary (hereditary) and secondary, then classified again as isolated hypercholesterolemia, isolated hypertriglyceridemia, mixed hyperlipidemia or low HDL‑C phenotypes [3-4]. Emerging evidence from population‑based studies suggest that dyslipidemia has become very common across the world and is one of the most important modifiable risk factors for atherosclerotic cardiovascular disease, including coronary artery disease and stroke. [3]

The epidemiology of dyslipidemia in India has changed dramatically over recent decades due to social and lifestyle changes associated with rapid urbanization, sedentary lifestyles, dietary transitions along with large regional variation [4]. The atherogenic risk attributable to typical Indian dyslipidemia pattern is also realized through borderline‑high LDL‑C, raised triglycerides and low HDL‑C [4]. Classical risk factors including obesity, type 2 diabetes mellitus, hypertension, smoking and metabolic syndrome act synergistically with dyslipidemia to accelerate atherosclerosis by aggravating development of endothelial dysfunction and increasing oxidative stress and inflammatory activation in the arterial wall [3-4].

Atherosclerosis risk is conferred by the entrapment and oxidation of LDL within the subendothelial environment, which leads to foam cell formation, plaque expansion and plaque rupture with clinical sequelae such as myocardial infarction, ischemic stroke or peripheral arterial disease [3]. Many trials have established that LDL-C reduction, in particular, but effective lipid-lowering therapy in general, reduces cardiovascular morbidity and mortality by up to 60% in both primary and secondary prevention [5-6]. Consequently, currently existing dyslipidemia management strategies focus on the optimization of pharmacotherapy by use of agents that affect distinct pathways in cholesterol homeostasis [5].

To evaluate a selective cholesterol absorption inhibitor, ezetimibe as an important adjunctive agent in lipid management with emphasis on patients not being able to achieve LDL‑C targets with statin therapy alone [5-7]. Chemically, ezetimibe is (3R,4S)‑1‑(4‑fluorophenyl)‑3‑[(S)‑3-(4-fluorophenyl)‐3‐hydroxypropyl]‐4-(4-hydroxyphenyl)azetidine-2‑one, has the empirical molecular formula C₂₄H₂₁F₂NO₃ and approximate molecular weight of 409.4 g/mol [8]. The drug is an intestinal cholesterol absorption inhibitor.

Ezetimibe is a selective inhibitor of Niemann–Pick C1‑like 1transporter in the intestinal mucosa, dietary and biliary cholesterol as well as phytosterols are absorbed from the intestinal lumen by enterocytes in an evolvatively conserved fashion and mediated primarily by the diffusion of fatty acids across cell membranes or (in principal) via specific transporters that include NPC1L1. This, in turn, results in reduced delivery of cholesterol to the liver, up‑regulation of hepatic LDL receptors and increased clearance of circulating LDL‑C [5, 10]. After oral administration, ezetimibe is quickly absorbed and rapidly conjugated by phase II metabolism to its pharmacologically active glucuronide conjugate, which then undergoes enterohepatic recycling that gives it a terminal half‑life of around 22 hours enabling once‑daily dosing [11-12]. It is predominantly eliminated by feces, with only a small part excreted by the urine as glucuronide. [11-12]

On the therapeutic side, ezetimibe is indicated for primary hypercholesterolemia, mixed dyslipidemia, homozygous familial high serum cholesterol and homozygous sitosterolemia. This medicine is usually administered orally as 10 mg film-coated tablets [7]. Ezetimibe generally has a good safety profile with mild adverse effects such as soft stool and headache along with increased liver transaminases, especially in combination with statins. It is also associated with rare reports of myopathy or rhabdomyolysis[13]. The limiting feature for ezetimibe therapy is the modest amount by which it lowers LDL‑C when used alone and the need for careful monitoring when combined with other lipid‑lowering drugs [5, 7].

Rosuvastatin is a highly potent statin belonging to the class of synthetic HMG‑CoA reductase inhibitors with an attractive pharmacokinetic profile. Chemically, rosuvastatin calcium is (E)‑7‑[4‑(4‑fluorophenyl)−6−isopropyl-2-methyl(methylsulfonyl)amino]pyrimidin-5-y1-3,5-dihydroxyhept‐6‐enoic acid calcium salt [6]. Its empirical formula is (C₂₂H₂₇FN₃O₆S)2Ca and molecular weight 1001.14 g/mol approximately. It is a member of the statin family and the molecular structure for rosuvastatin. It is classified as a statin and the detailed chemical structure of rosuvastatin calcium is provided separately in the dissertation.

Rosuvastatin is a competitive inhibitor of HMG-CoA reductase, which is the rate-limiting enzyme involved in hepatic cholesterol biosynthesis; it has been shown to reduce intrahepatic cholesterol, up-regulate LDL receptors and therefore increase the clearance of circulating LDL-C [6, 15]. The hepatoselective drug has a limited ability to invade extrahepatic tissues, and undergoes less metabolism via cytochrome P450 isoenzymes (as this is the case for other lipid-lowering drugs), which contribute to its intensive LDL‑C and triglyceride lowering effects and comparatively lower risk of drug–drug interactions [6-7]. Rosuvastatin is given orally with an effective half‑life of ~19 hours allowing once‑daily dosing; it is eliminated mainly via fecal excretion [6, 15].

Rosuvastatin is approved for treatment of primary hypercholesterolemia, mixed dyslipidemia, and hypertriglyceridemia, as well as to reduce cardiovascular risk in appropriate high‑risk patients[6, 15]. It is sold under the trade name in film-coated tablets in strengths of 5 mg, 10 mg, 20 mg and 40 mg. Myalgia, headache, gastroenteritis and asymptomatic elevation of hepatic transaminases represent some of the frequent adverse effects; however rare but serious events like rhabdomyolysis and severe myopathy make clear that patient selection and monitoring is essential. The limitations of rosuvastatin therapy include statin intolerance in some [6, 15].

Combining ezetimibe with rosuvastatin is scientifically rational because the two agents act on distinct yet complementary pathways of cholesterol homeostasis [5, 10]. Because the cholesterol-lowering effect of statins occurs by two mechanisms as inhibition of both endogenous production and receptor-mediated clearance of LDL-C; ezetimibe reduces intestinal absorption of exogenous and biliary cholesterol, thereby providing dual inhibition of cholesterol influx and production. [5, 10] Clinical studies have demonstrated that co‑administration of ezetimibe plus statins, including rosuvastatin, yields larger reductions in LDL‑C and non‑HDL‑C compared with statin monotherapy — together with beneficial effects on triglycerides and HDL‑C. [5-6]

This dual-action therapy approach has specific utility in patients with high baseline LDL‑C, familial hypercholesterolemia, or statin intolerance at higher doses and allows attaining target lipid levels with lower statin dosages [5]. Ezetimibe–rosuvastatin functional combinations improve surrogate markers of atherosclerosis and also prevent the incidence of major cardiovascular events with additional benefit in high-risk populations when compared to statin monotherapy, improving lipid control through additive effects on low-density lipoprotein (LDL) particle size and non-HDL cholesterol levels. Consequently, fixed-dose combination formulations of ezetimibe and rosuvastatin have become one of the mainstay therapeutic options; strong analytical procedures are necessary for routine quality control and efficacy studies [1-2].

For many chemically diverse drugs, reversed-phase HPLC (RP‑HPLC), with a non‑polar stationary phase, such as C18 and a relatively polar mobile phase containing aqueous mobile phase and organic modifiers, is the best available method for separating and quantifying components, in particular because of its robustness and suitability for both the aqueous and organic solvents common to many drug formulations, as well as for compatibility with UV detection. [18-19] The selection of stationary phase, mobile phase composition and pH, column flow rate, column temperature, and detection wavelengths are very important to achieve best resolution, peak symmetry, and optimum reproducibility while simultaneously estimating multiple drugs in complex matrices in HPLC systems.

RP-HPLC is very well suited for pharmaceutical analysis of fixed-dose combinations including the combination of ezetimibe and rosuvastatin, which contains aromatic chromophores and moderate lipophilicity of both drugs [19, 21]. RP-HPLC is well suited to the pharmaceutical analysis of fixed dose combinations such as ezetimibe and rosuvastatin, which contains aromatic chromophores and a moderate lipophilicity of both drugs.

A stability indicating analytical method is defined, in line with regulatory and ICH guidelines, as a validated analytical method that quantifies the pharmaceutical product or drug substance accurately and precisely and demonstrates for the assay profile that the product is free from process impurities, excipients and degradation products, both normal storage conditions and stress induced ones. [18,23] These methods are extremely essential for establishing the intrinsic stability of a pharmaceutical product, to support shelf life development, and for assuring quality, safety and efficacy throughout the product lifecycle. [24-25] For combination products, the method should be able to resolve and quantify each active pharmaceutical ingredient (API), their respective degradants and additionally the combination product itself, which can prove to be challenging during method development and optimization. [18, 23] For a combination product, an analytical method should be able to separate and quantify each active pharmaceutical ingredient, their respective degraded forms, and also the combination product itself, which can be challenging in the development and optimization of the method. [1, 26]

Most of the essential validation parameters recommended in the revised ICH Q2(R2) Guideline for the Validation of Analytical Procedures include the following for assay methods used for quantitative chromatographic analysis such as specificity, selectivity, linearity, accuracy, precision, range, limit of detection (LOD), limit of quantification (LOQ), robustness, ruggedness (where applicable), and system suitability testing.

Specificity and selectivity describe the ability of the method to measure the analyte unequivocally in the presence of other components such as impurities, excipients, and degradants, which is particularly important for stability‑indicating assays.[23, 27] Linearity refers to the proportional relationship between analyte concentration and detector response over a defined range, while accuracy expresses the closeness of measured values to the true value, often assessed via recovery studies. [21, 27] Precision encompasses repeatability and intermediate precision, evaluating the degree of agreement between replicate measurements under the same and varying conditions, respectively.[27, 30-31]

LOD and LOQ represent the smallest amounts of analyte that can be reliably detected and quantified, which are critical for impurity and degradation product profiling.[23, 27] Robustness reflects the method’s capacity to remain unaffected by small deliberate variations in parameters such as mobile phase composition, pH, flow rate, and column temperature, thereby indicating its reliability during routine use. [22, 27] Ruggedness, when assessed, extends this concept to inter‑laboratory changes, different analysts, or instruments. [25, 27] System suitability tests, including parameters such as theoretical plates, tailing factor, resolution, and repeatability of peak response, are performed prior to analysis to verify that the chromatographic system is functioning properly.[20, 27]

Several chromatographic methods for the simultaneous determination of ezetimibe and rosuvastatin in bulk drug and combination tablet dosage forms have been reported, a few of which contained stability-indicating element(s), however, most of the above mentioned methods involve relatively complex mobile phases, longer run time, limited evaluation of forced degradation conditions and incompleteness of validation meeting current ICH Q2(R2) recommendation for analytical procedure. [1-2,26] Further, the increased use of ezetimibe–rosuvastatin fixed-dose products in human clinical practice requires simple, rapid, accurate, precise, sensitive, robust and economical method to be adopted in routine quality control laboratories. [5-6] The present study is of immense importance since it aims to develop and validate a novel, stability indicating RP‑HPLC method for the simultaneous estimation of ezetimibe and rosuvastatin in pharmaceutical dosage form, which not only meets current regulatory requirement and analytical science but will also offer a well captured stability indication data for the assay of ezetimibe and rosuvastatin [27, 29]  simultaneously in longer period of time under the ICH recommended stress and storage conditions which will facilitate formulation development, shelf life assignment and submission to regulatory authority.

MATERIALS & METHODS

Rosuvastatin and Ezetimibe active pharmaceutical ingredients (APIs) were procured from Swapnroop Drugs and Pharmaceuticals, Aurangabad, India. The marketed formulation, Novastat EZ tablets, was obtained from Lupin Ltd. HPLC-grade methanol and 0.1 N citric acid were purchased from Merck Ltd., India, while purified water was obtained from Research Lab Fine Chem Industries. All chemicals and reagents used in the study were of analytical or HPLC grade and were used without further purification.

Determination of Absorption Spectra

The Rosuvastatin and Ezetimibe stock solutions were separately dissolved in methanol to make the concentration of 1000 µg/mL from accurately weighted quantities of each drug. Working standard drug solutions of 10µg/mL were prepared as appropriate dilutions of the drug solutions. The UV absorption spectra were recorded over the wavelength range of 200–400 nm using methanol as the blank. The individual absorption maxima (λmax) of Rosuvastatin and Ezetimibe were determined from the spectra, and the overlain spectra were used to identify the common isosbestic wavelength, which was selected for subsequent RP-HPLC analysis.

HPLC Instrumentation and Chromatographic Conditions

Chromatographic analysis was performed using an Agilent 1100 Gradient High-Performance Liquid Chromatography (HPLC) system equipped with a G1310A ISOPUMP, a Photodiode Array (PWD) detector, and ChemStation software for instrument control, data acquisition, and analysis. Separation was achieved on an Agilent RP-C18 column (4.6 × 250 mm, 5.0 µm particle size) using a mobile phase consisting of methanol and 0.1 N citric acid (60:40, v/v). The mobile phase was delivered at a flow rate of 1.0 mL/min, and analytes were detected at a wavelength of 233 nm. The column temperature was maintained at 25°C, and the injection volume was 20 µL. The G1310A ISOPUMP is a high-pressure reciprocating pump with a maximum operating pressure of 400 bar, a flow rate range of 0.001–5.0 mL/min, pressure display accuracy of ±5%, four-solvent capability, and a programmable mixing ratio of 0–100%, providing precise and reproducible mobile phase delivery throughout the chromatographic analysis.

Method Development and Optimization

Chromatographic conditions were optimized by evaluating different mobile phase compositions consisting of methanol with 0.1% acetic acid and citric acid in various proportions. A variety of combinations were subjected to flow rates suitable for the application (0.7 to 1.0 mL/min) to obtain good peak resolution, retention time, peak shape, and theoretical plate count. The optimized mobile phase of Methanol : Citric acid (60:40, v/v) was chosen based on the chromatographic performance. The optimized method gave well-resolved and symmetrical peaks for both analytes having satisfactory system suitability parameters.

 

 

 

Table 01 Method Development Trials for Optimization of RP-HPLC Conditions

 

Preparation of Standard Stock Solution

Accurately weighed quantities of Rosuvastatin (30 mg) and Ezetimibe (15 mg) were transferred into separate 100 mL volumetric flasks. About 70 mL of methanol was then added to each solution and sonicated for 15 minutes to allow them to completely dissolve. The concentrations of Rosuvastatin and Ezetimibe were then adjusted to be 300 µg/mL and 150 µg/mL in methanol to form stock solutions.

Preparation of Working Standard Solutions (Linearity)

Standard stock solutions were suitably diluted with the optimized mobile phase to make the calibration standards. Rosuvastatin working solutions were diluted in the range of 6–30 µg/mL, and Ezetimibe solutions in the range of 3–15 µg/mL. All solutions were sonicated for the removal of dissolved gases, and filtered through a 0.45 µm membrane filter before the injection.

Accuracy

Standard addition method was used to assess the developed RP-HPLC method of the accuracy up to 80, 100 and 120% of the target concentration. Previously analysed tablet sample solutions were spiked with known quantities of Rosuvastatin and Ezetimibe standard solutions and analyzed under the optimized chromatographic conditions. To determine the accuracy of the analytical method, the percentage recovery was calculated for each level.

Repeatability

The repeatability of the method was determined by processing six replicate injections of a mixed standard solution, containing Rosuvastatin (24 µg/mL) and Ezetimibe (12 µg/mL), under similar conditions. The repeatability of the proposed RP-HPLC method was evaluated by calculating the mean, standard deviation and percentage relative standard deviation (%RSD) from the peaks areas obtained from the replicate injections.

Intraday Precision

Intra-day precision was evaluated by analyzing a mixed standard solution with three different concentrations of Rosuvastatin (12, 18 and 24 µg/mL) and three different concentrations of Ezetimibe (6, 9 and 12 µg/mL) at various timepoints within the same day. All concentrations were analyzed in duplicate and conditions were the same on the chromatograph for all. The precision was reported as the percent relative standard deviation (%RSD) of the peak areas.

Interday Precision

Interday precision was assessed by analyzing freshly prepared mixed standard solutions containing 12, 18, and 24 µg/mL of Rosuvastatin and 6, 9, and 12 µg/mL of Ezetimibe on three consecutive days using the same chromatographic conditions. The consistency of the analytical method was evaluated by calculating the mean peak area, standard deviation, and percentage relative standard deviation (%RSD).

Limit of Detection (LOD) and Limit of Quantification (LOQ)

The developed RP-HPLC method was evaluated for its sensitivity and the limits of detection (LOD) and limits of quantification (LOQ) were determined as per ICH Q2(R1). The parameters can be computed from the standard deviation of the response (σ) and the slope (S) of the calibration curve as follows:

The calculated LOD and LOQ values showed that the developed analytical method was sensitive enough for the simultaneous estimation of Rosuvastatin and Ezetimibe.

Specificity

The developed HPLC method was validated for specificity by analyzing the blank and standard solutions as well as sample solutions under the optimized HPLC conditions. The chromatograms were checked for the presence of any excipient peak, solvent peak or impurity peak at the retention time of Rosuvastatin and Ezetimibe. The two peaks, namely the interfering peaks, were not seen in the background, indicating that the background was contained and not interfering with the method developed.

Ruggedness

The developed method was evaluated for ruggedness by analyzing the samples by the same method, but on different days and different analysts. A standard solution of 18µg/mL for Rosuvastatin and 9µg/mL for Ezetimibe was analyzed and the obtained results were compared by the following parameters: peak area, percentage label claim. The %RSDs were small, which indicated the ruggedness of the method.

Robustness

To test the robustness of the RP-HPLC method the following deliberate changes were introduced in the chromatographic conditions: mobile phase composition ±1% and wavelength of detection (243 and 245 nm), with all other experimental conditions remaining the same. Under these modified conditions, standard solutions of 18 µg/mL of Rosuvastatin in 9 µg/mL of Ezetimibe were analyzed. Areas and the %RSD values generated were examined to assess the effect of these minor variations on the method.

Assay of Marketed Tablet Formulation

Twenty marketed tablets of Novastat EZ tablets containing Rosuvastatin and Ezetimibe were accurately weighed and finely powdered. Both powders contained the same mass on a label as each other and the same mass was transferred into a volumetric flask and extracted with methanol, sonicated for 15 minutes after which each was filtered through a 0.45 µm membrane filter. The optimized mobile phase was used to prepare suitable dilutions in order to obtain the final concentration of rosuvastatin and ezetimibe 18 µg/mL and 9 µg/mL, respectively. The optimal chromatography conditions were used to analyze the prepared solution and the percentage label claim was determined.

Forced Degradation Studies

Acid Degradation

Acid degradation was performed by adding appropriate amount of 0.1 N HCl to the standard solution mixture. The solution was left to stand for 2 hours and then for 24 hours and before the chromatographic analysis the solution was neutralized and diluted with the mobile phase appropriately.

Alkaline Degradation

The mixed standard solution was treated with 0.1 N sodium hydroxide to degrade it in an alkaline condition and then kept for 2 hours and 24 hours at room temperature. The solution was subjected to the mobile phase after neutralization and filtered with 0.45 µm membrane filter and then analyzed by RP-HPLC.

 Oxidative Degradation

Oxidative degradation test was carried out by reacting the mixed standard solution with 3% hydrogen peroxide (H₂O₂). The solution was stabbed for 2 h and 24 h under ambient laboratory conditions and analyzed by analysis of chromatography to get the solution. The areas of the sample and the standard were compared to find the percentage degradation.

Photolytic Degradation

To simulate the photo-degradation, the mixture standard solution was subjected to UV radiation for the mentioned exposure period. The solution exposed was analysed by the optimized RP-HPLC method and the percentage of degradation was calculated by comparing the peak areas of the exposed sample with the peak areas of the untreated sample.

RESULTS AND DISCUSSION:

Determination of Absorption Spectrum and Iso-absorptive Point

The UV absorption spectra of Rosuvastatin and Ezetimibe were recorded individually in methanol over the wavelength range of 200–400 nm to determine their respective absorption maxima and identify a suitable analytical wavelength for simultaneous estimation. The maximum absorbance of rosuvastatin and ezetimibe were found to be 236 nm and 227 nm respectively. The two drugs had an isosbestic curve point at 233nm between the overlain spectra of both drugs, indicating that these two analytes have the same absorptivity at this wavelength. Thus, the detection wavelength of 233 nm was chosen for further RP-HPLC analysis, which allowed the determination with proper sensitivity and simultaneous determination of both drugs without significant spectral interferences.

Fig. 01 UV absorption spectrum of Rosuvastatin (10 µg/mL) in methanol

Fig. 02 UV absorption spectrum of Ezetimibe (10 µg/mL) in methanol

Fig. 03 Isosbestic point of Rosuvastatin and Ezetimibe at 233 nm

Mobile Phase Optimization

Optimization of the chromatographic conditions was carried out through systematic evaluation of different mobile phase compositions consisting of methanol combined with 0.1% acetic acid and 0.1% citric acid at various proportions and flow rates. During the first trials, a methanol–acetic acid system was employed, resulting in wide peaks, poor peak shape and poor resolution of Rosuvastatin and Ezetimibe. Optimization with methanol–citric acid mixtures gave better chromatographic performance. The optimized chromatographic condition were found to be with Methanol : Citric acid (60:40, v/v) with flow rate of 1.0 mL/min where retention time of Rosuvastatin and Ezetimibe were around 3.39 and 4.58 min, with their sharp peak shape and symmetrical peak with good resolution. The optimized chromatogram showed good theoretical plates, acceptable peak symmetry, and complete baseline resolution required for the simultaneous estimation and further validation studies and the mobile phase was found to be suitable.

Fig. 04 Chromatogram of Trial-11 optimized mobile phase condition

Table 02 Chromatographic performance data of mobile phase trials

Linearity

The linearity of developed RP-HPLC method was investigated by analyzing standard solution of Rosuvastatin in the range of 6–30 µg/mL and Ezetimibe in the range of 3–15 µg/mL. For both drugs, a linear relationship between peak area and concentration was determined over the given concentration ranges. Rosuvastatin showed a regression equation of y = 113.1175x + 73.687 with a correlation coefficient (R² = 0.9998), and Ezetimibe showed an excellent linearity with a correlation coefficient very close to unity. The obtained low standard deviation and percentage relative standard deviation (%RSD) showed good repeatability in the responses of detector at all the concentrations. These results verified the method developed is useful for the intended analytical concentration range and meets the ICH guidelines for the linearity.

Table 03 Linearity Data for Rosuvastatin (ROSU)

 

Table 04 Linearity Data for Ezetimibe (EZET)

 

Fig. 04 Calibration curve of Rosuvastatin

Fig. 05 Calibration curve of Ezetimibe

Accuracy

The developed method was checked for the accuracy by standard addition method at 80%, 100% and 120% concentration levels. The mean percentage recoveries of Rosuvastatin ranged between 98.96% to 100.29% whilst the Ezetimibe mean percentage recoveries ranged between 98.46% to 100.74%. The RSD percentage value for both the drugs was below 2%, which also indicates excellent reproducibility of the recovery results. The recovery values were observed to be approaching 100%, indicating that the developed analytical method was effective in quantifying both the analytes without getting interfered by the formulation excipients. The results indicated that proposed RP-HPLC method was highly accurate and useful for routine pharmaceutical analysis.

Table 05 Summary of Accuracy Studies of Rosuvastatin (ROSU)

 

Table 06 Summary of Accuracy Studies of Ezetimibe (EZET)

Sample (% level)	Conc. of marketed formulation (µg/mL)	Conc. of API added (µg/mL)	Absorbance (Area)	Amount Recovered (µg/mL)	% Recovery	Mean	SD	%RSD
80%	6	4.8	1173.28	4.74	98.72	98.46	0.37	0.38
			1170.57	4.71	98.19
100%		6	1307.60	5.99	99.86	99.60	0.36	0.37
			1304.29	5.96	99.34
120%		7.2	1445.47	7.28	101.08	100.74	0.48	0.48
			1440.24	7.23	100.40

 

Intraday Precision

Intraday precision of the developed RP-HPLC method was evaluated by analyzing three different concentrations of Rosuvastatin (12, 18, and 24 µg/mL) and Ezetimibe (6, 9, and 12 µg/mL) multiple times within the same day under identical chromatographic conditions. The assay values obtained in the process were found to be close to the nominal value, with the %RSD of the values in the range of 0.05-0.27% for Rosuvastatin and 0.00-0.38% for Ezetimibe. The %RSD data were indicative of high repeatability and consistency between replicate analyses performed on the same day. As a result these results were found to be in accordance with the developed RP-HPLC method having high intraday precision and also fulfilling the acceptance criteria fixed by ICH guidelines.

Interday Precision

Interday precision was assessed by analyzing freshly prepared standard solutions of Rosuvastatin (12, 18, and 24 µg/mL) and Ezetimibe (6, 9, and 12 µg/mL) on different days using the same chromatographic conditions. The values obtained at the various time points of the study were comparable indicating the time stability of the developed method. . The %RSD values for Ezetimibe ranged from 0.12% to 0.78%, while Rosuvastatin exhibited acceptable precision across all concentration levels, confirming reliable analytical performance between different days. The results obtained confirmed the developed HPLC method yielded acceptable intermediate precision for the simultaneous RP-HPLC analysis of Rosuvastatin and Ezetimibe in tablet formulation, which can be used for routine quality control analysis.

Table 07 Intraday Precision for Rosuvastatin (ROSUVA)

 

Table 08 Intraday Precision for Ezetimibe (EZET)

Table 09 Interday Precision for Rosuvastatin (ROSUVA)

 

Table 10 Interday Precision for Ezetimibe (EZET)

 

LOD and LOQ:

The sensitivity of the developed RP-HPLC method was determined by calculating the Limit of Detection (LOD) and Limit of Quantification (LOQ). The obtained values proved that developed chromatographic method has excellent analytical sensitivity, which allows detecting and accurate quantifying of very low concentration levels of Rosuvastatin and Ezetimibe. The results of the proposed method with low detection limits and low quantitative limits revealed that the method has great potential applications in quality control routine applications, stability studies and can be used for the determination of trace level in pharmaceutical formulations.

Repeatability

Repeatability was assessed after six injections of standard solutions were made, with the same chromatographic conditions. The mean peak area for Rosuvastatin was 98.61% assay at 24 µg/mL, whereas that of Ezetimibe was 99.74% assay at 12 µg/mL with a fortuitously low %RSD of 0.02%. The low overall variability of each injection proved the precision of the instrument and the uniformity of the developed chromatographic method. These findings demonstrated the good repeatability of the method for general quality control.

Table 11 Repeatability for Rosuvastatin and Ezetimibe

Drug	Concentration (µg/mL)	Mean Area	Amount Found (µg/mL)	% Amount Found	SD	%RSD
Rosuvastatin	24	2750.41	23.67	98.61	22.86	0.83
Ezetimibe	12	1305.11	11.97	99.74	0.30	0.02

 

Specificity

Specificity studies were performed, and the developed RP-HPLC method was able to selectively estimate Rosuvastatin and Ezetimibe in the presence of formulation excipients, without any chromatographic interference between the two and any excipients. For both drugs, well-resolved peaks were generated with good peak purity, and consistent peak retention times. The assay of Rosuvastatin was an average of 100.75%, and Ezetimibe, 99.59% with very low %RSD values of 0.065% and 0.056%, respectively. These results indicated that there was no interference of the common excipients and other formulation components during the determination of analytes and the developed analytical method was considered to be specific.

Table 12 Specificity for Rosuvastatin and Ezetimibe

Drug	Concentration (µg/mL)	Mean Area	Amount Found (µg/mL)	% Label Claim	SD	%RSD
Rosuvastatin	18	2124.75	18.14	100.75	0.012	0.065
Ezetimibe	9	982.93	8.96	99.59	0.005	0.056

 

Ruggedness

Ruggedness was checked by comparing the same sample tested under varying analytical parameters with different analysts and different operating days. Rosuvastatin had an average assay of 100.79% whereas Ezetimibe exhibited 99.57%, substantially low %RSD of 0.016% and 0.085% respectively. The method being developed was also significantly rugged and reproducible as minimal difference was seen between the different operating conditions.

Table 13 Ruggedness Study for Rosuvastatin and Ezetimibe

Drug	Conc (µg/mL)	Amount Found (Mean, µg/mL)	% Label Claim (Mean)	SD	%RSD
Rosuvastatin	18.00	18.14	100.79	0.003	0.016
Ezetimibe	9.00	8.96	99.57	0.008	0.085

 

Robustness

The robustness of the developed analytical method was investigated by deliberately introducing minor variations in chromatographic parameters, including mobile phase composition and detection wavelength. All deliberate changes did not result in any noticeable changes in retention time, peak shape, peak area, or chromatographic resolution. The %RSD for each value obtained were less than 2%, showing that variations in the experimental conditions caused little effect on the analytical performance. Hence, the developed method RP-HPLC proved to be sufficiently robust and reliable method and hence could be used in regular pharmaceutical analysis.

 

Table 14 Robustness Study for Rosuvastatin and Ezetimibe

Parameter	Drug	Condition	Conc (µg/mL)	Area	Mean	SD	%RSD
Mobile Phase Change	Ezetimibe	39:61 MeOH	9	1029.43	1027.20	3.10	0.30
				1025.05
		41:59 MeOH		1024.64	1024.42	0.32	0.03
				1024.19
	Rosuvastatin	39:61 MeOH	18	2150.00	2149.20	0.88	0.04
				2148.40
		41:59 MeOH		2142.49	2142.32	0.17	0.01
				2142.15
Wavelength Change	Ezetimibe	243 nm	9	1008.53	1020.90	17.45	1.71
				1033.21
		245 nm		314.01	315.73	2.43	0.77
				317.44
	Rosuvastatin	243 nm	18	2161.89	2158.50	3.39	0.16
				2155.11
		245 nm		3016.09	3015.79	0.30	0.01
				3015.49

 

Assay of Tablet Formulation

The RP-HPLC method was validated and successfully applied to determine the quantitative content of Rosuvastatin and Ezetimibe in the Novastat EZ tablets. Rosuvastatin had a mean assay value of 100.81% of the label claim, while Ezetimibe showed 99.68% of the label claim. This was reflected in the low SD and %RSD values that indicated high assay repeatability as well as accuracy. The developed procedure proved to be appropriate for quality control application of the combined formulation of tablet dosage forms of Rosuvastatin and Ezetimibe on daily basis.

Table 15 Assay of Rosuvastatin and Ezetimibe (% Label Claim)

 

Fig 06: Chromatogram of Marketed formulation

Force degradation:

Acid Degradation

The developed RPHPLC method was evaluated in acidic stress conditions to check the stability of Rosuvastatin and Ezetimibe in the acidic environment. The rate of degradation of Ezetimibe and Rosuvastatin by acid hydrolysis was 3.83% and 6.79%, respectively, after 2 h, suggesting that Rosuvastatin is relatively highly susceptible to hydrolysis by acid. With a longer exposure time of 24 hours degradation rose to 7.09 % for Ezetimibe and 9.62 % for Rosuvastatin. The developed RP-HPLC method is also highly specific and showed high stability indicating ability, as the peaks due to the formed degradation products were well separated from the analyte peaks under the acidic stress conditions without any interference.

Alkaline (Basic) Degradation

Alkaline stability of the both drugs was studied by exposing the samples to alkaline hydrolysis. Ezetimibe exhibited 3.97% degradation and Rosuvastatin exhibited 6.85% degradation after 2 hours indicating Rosuvastatin is more susceptible to alkaline degradation. After 24 hours, degradation has reached 6.78% and 7.64% for Ezetimibe and Rosuvastatin, respectively. There was no significant change in chromatographic separation during the course of the study with the degradation products being well separated from the parent drug peaks. The results revealed the developed method can accurately estimate both drugs in the presence of any alkaline degradation products.

Oxidative Degradation

Hydrogen peroxide was used to investigate the oxidative stability of Rosuvastatin and Ezetimibe during the oxidative degradation studies. In cases of oxidative stress, Ezetimibe was oxidized by 4.51% after 2 hours compared with Rosuvastatin which was degraded by 6.99% in the same period, suggesting that Rosuvastatin was more sensitive to oxidation. Ezetimibe degradation also rose after 24 hours, reaching 7.32%, and this degradation also increased for Rosuvastatin reaching 8.99% after 24 hours. The chromatograms under oxidative condition indicated complete separation of all the degradation products from the drug peaks, and the developed RP-HPLC method was found to be specific and oxidative stress indicative.

Table 16 Forced Degradation Study of Ezetimibe (9 µg/mL) After 2 Hours

 

Table 17 Forced Degradation Study of Rosuvastatin (18 µg/mL) After 2 Hours

Sr. No.	Degradation Condition	Area of Standard	Area of Degraded Sample	Degraded up to (%)	Actual % Degradation
1	Acid Degradation	2121.88831	1977.79907	93.21	6.79
2	Basic Degradation	2121.88831	1976.52637	93.15	6.85
3	H₂O₂ Degradation	2121.88831	1973.59595	93.01	6.99

 

Table 18 Forced Degradation Study of Ezetimibe (9 µg/mL) After 24 Hours

Sr. No.	Degradation Condition	Area of Standard	Area of Degraded Sample	Degraded up to (%)	Actual % Degradation
1	Acid Degradation	989.60168	919.42950	92.91	7.09
2	Basic Degradation	989.60168	922.53821	93.22	6.78
3	H₂O₂ Degradation	989.60168	917.17700	92.68	7.32

 

Table 19 Forced Degradation Study of Rosuvastatin (18 µg/mL) After 24 Hours

Sr. No.	Degradation Condition	Area of Standard	Area of Degraded Sample	Degraded up to (%)	Actual % Degradation
1	Acid Degradation	2121.88831	1917.74927	90.38	9.62
2	Basic Degradation	2121.88831	1959.70227	92.36	7.64
3	H₂O₂ Degradation	2121.88831	1931.08215	91.01	8.99

 

Photolytic Degradation Study (UV Light)

Under the conditions of UV light-induced stress, the drug samples have been exposed to the UV light and subjected to photolytic degradation studies as an assessment of the stability of the drug. Both of the drugs, Ezetimibe and Rosuvastatin, showed less degradation with 1.65% for Ezetimibe and 1.79% for Rosuvastatin suggesting their relative stability under photolytic conditions. The chromatograms revealed that there was no interference from the degradation products and there was sharpness and good separation of the drug peaks. As per these observations, developed HPLC method proved to be good stability-indicating HPLC method as it was able to selectively estimate Rosuvastatin and Ezetimibe even after exposure to UV light.

Table 20 Photolytic Degradation Study of Ezetimibe and Rosuvastatin

Sr. No.	Drug	Area of Standard	Area of Degraded Sample	Degraded up to (%)	Actual % Degradation
4	Ezetimibe	989.60168	973.30475	98.35	1.65
5	Rosuvastatin	2121.88831	2083.81470	98.21	1.79

 

CONCLUSION:

A Stability-Indicating RP-HPLC method for the simultaneous determination of Rosuvastatin and Ezetimibe in bulk drug and pharmaceutical dosage forms has been developed and validated successfully which could be used for separation, quantitative estimation of both the compounds under the study in real samples with pretreatment method. The proposed method has good linearity, sensitivity, accuracy, precision, specificity, ruggedness and robustness with all validation parameters in accordance with the guideline of ICH Q2 (R2). The proposed method was successfully employed in the assay of market tablet formulations and satisfactory recovery and reproducible obtained. In addition, forced degradation experiments under acidic, alkaline, oxidative and photolytic stress conditions showed that the method is stability indicating since the peaks of the degradation products have been separated from the analyte peak. Hence the proposed RP-HPLC method could be applied to routine quality control, stability and simultaneous estimation of Rosuvastatin and Ezetimibe in combined pharmaceutical dosage form.

REFERENCES:

1.        Mukthinuthalapati MA, Bukkapatnam V, Bandaru SP. Stability indicating liquid chromatographic method for the simultaneous determination of rosuvastatin and ezetimibe in pharmaceutical formulations. Adv Pharm Bull. 2014 Dec; 4(4):405-11.

2.        Varghese SJ, Ravi TK. Determination of rosuvastatin and ezetimibe in a combined tablet dosage form using high-performance column liquid chromatography and high-performance thin-layer chromatography. J AOAC Int. 2010 Jul-Aug; 93(4):1222-7.

3.        Sharma S, Gaur K, Gupta R. Trends in epidemiology of dyslipidemias in India. Indian Heart J. 2024 Mar; 76(Suppl 1):S20-S28.

4.        Phan BA, Dayspring TD, Toth PP. Ezetimibe therapy: mechanism of action and clinical update. Vasc Health Risk Manag. 2012; 8:415-27.

5.        Bajaj T, Patel P, Giwa AO. Rosuvastatin [Internet]. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2026 [updated 2026 Jan 31; cited 2026 Jul 16]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK539883/

6.        Sizar O, Nassereddin A, Talati R. Ezetimibe [Internet]. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2026 [updated 2023 Aug 28; cited 2026 Jul 16]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK532879/

7.        Davidson MH, et al. Ezetimibe coadministered with simvastatin in patients with primary hypercholesterolemia. J Am Coll Cardiol. 2002; 40(12):2125.

8.        Sudhop T, et al. Inhibition of intestinal cholesterol absorption by ezetimibe in humans. Circulation. 2002; 106(15):1943-8.

9.        Sweeney ME, Johnson RR. Ezetimibe: an update on the mechanism of action, pharmacokinetics and recent clinical trials. Expert Opin Drug Metab Toxicol. 2007 Jun; 3(3):441-50.

10.    Kosoglou T, Statkevich P, Johnson-Levonas AO, Paolini JF, Bergman AJ, Alton KB. Ezetimibe: a review of its metabolism, pharmacokinetics and drug interactions. Clin Pharmacokinet. 2005; 44(5):467-94.

11.    Simard C, Turgeon J. The pharmacokinetics of ezetimibe. Can J Clin Pharmacol. 2003 Winter; 10(Suppl A):13A-20A.

12.    LiverTox: Clinical and Research Information on Drug-Induced Liver Injury [Internet]. Bethesda (MD): National Institute of Diabetes and Digestive and Kidney Diseases; 2012-. Ezetimibe [updated 2021 Dec 1; cited 2026 Jul 16]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK548095/

13.    Luvai A, Mbagaya W, Hall AS, Barth JH. Rosuvastatin: a review of the pharmacology and clinical effectiveness in cardiovascular disease. Clin Med Insights Cardiol. 2012; 6:17-33.

14.    Dongala T, Katari NK, Palakurthi AK, Jonnalagadda SB. Development and validation of a generic RP-HPLC PDA method for the simultaneous separation and quantification of active ingredients in cold and cough medicines. Biomed Chromatogr. 2019 Nov; 33(11):e4641.

15.    Shabir GA. Development and Validation of a Reversed-phase HPLC Method for the Determination of Hydroxybenzene in a Cream Formulation. Indian J Pharm Sci. 2010 May; 72(3):307-11.

16.    Rani GT, Sankar DG, Kadgapathi P, Satyanarayana B. A Validated RP-HPLC Method for Simultaneous Estimation of Atenolol and Indapamide in Pharmaceutical Formulations. J Chem. 2011; 8:121420.

17.    Shamim A, Ansari MJ, Aodah A, Iqbal M, Aqil M, Mirza MA, et al. QbD-Engineered Development and Validation of a RP-HPLC Method for Simultaneous Estimation of Rutin and Ciprofloxacin HCl in Bilosomal Nanoformulation. ACS Omega. 2023 Jun 8; 8(24):21618-21627.

18.    Draft Guidance for Industry on Analytical Procedures and Methods Validation: Chemistry, Manufacturing, and Controls Documentation. Fed Regist. 2000 Aug 30.

19.    Chaitali Dhale, Onkar Doke, Abhijeet B. Survase, Uniket Gosavi, Saundarya S. Kurde. Stability Indicating Hptlc Method Development And Validation For Simultaneous Estimation Of Rosuvastatin Calcium And Ezetimibe In Bulk Form And Pharmaceutical Formulation. J ReAttach Ther Dev Divers. 2023; 6(6s):737-747.

20.    International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). ICH Harmonised Guideline: Validation of Analytical Procedures: Text and Methodology Q2(R2). Geneva: ICH; 2023.

21.    Snorek SM, Bauer JF, Chidambaram N, et al. PQRI Recommendations on Particle Size Analysis of Drug Substances Used in Oral Dosage forms. J Pharm Sci. 2007; 96(6):1451-1467.

22.    Wang X, Li W, Rasmussen HT. Orthogonal method development using hydrophilic interaction chromatography and reversed-phase high-performance liquid chromatography for the determination of pharmaceuticals and impurities. J Chromatogr A. 2005 Aug 12; 1083(1-2):58-62.

23.    Jawhari AH, Mohamed ZH. Developing and Validating a Robust RP-HPLC Method for Metoclopramide and Camylofin Simultaneous Analysis Using Response Surface Methodology. Int J Anal Chem. 2025 Oct 15; 2025:5543392.

24.    Cauchon N, Pelletier MB. Overview of ICH Q2(R2)/Q14: Development and Validation of Analytical Procedures [Internet]. 2023 [cited 2024 Jan]. Available from: https://www.casss.org/docs/default-source/consultants'-network/2023/cauchon-nina-amgen-inc.-and-pelletier-mary-beth-biogen-2023.pdf?sfvrsn=aba028c1_5

25.    Azarian S, Shaghaghi M, Dehghan G, Sheibani N. A rapid, simple and ultrasensitive spectrofluorimetric method for the direct detection of metformin in real samples based on a nanoquenching approach. J Lumin. 2021; 36(3):658-667.

26.    Usha Rani N, Nagoor Ankani. New stability-indicating RP-HPLC method development and validation for the determination of rosuvastatin (calcium) in pharmaceutical dosage forms. Int J Pharm Sci Drug Res. 2014; 6(4):283-290.

27.    Ramu K, Aleti P, Venisetty RK. Analytical method development and validation of simultaneous estimation of ezetimibe and rosuvastatin in tablet dosage form by RP-HPLC. Int J Pharm Biol Sci. 2012; 3(4).

28.    Sharma S, Sharma MC, Kohli DV, Chaturvedi SC. Simultaneous estimation of Rosuvastatin calcium and Ezetimibe in tablet dosage form by reverse phase high performance liquid chromatography. Optoelectron Adv Mater Rapid Commun. 2010 Feb; 4:238-241.

29.    Kurbanoglu S, Esim O, Kose Ozkan C, Uslu B, Savaser A, Ozkan SA. Stability-indicating liquid chromatographic method for the simultaneous determination of rosuvastatin and ezetimibe from pharmaceuticals and biological samples. JOTCSA. 2020; 7(3):865-874.

30.    Gupta A, Mishra P, Shah K. Simple UV spectrophotometric determination of Rosuvastatin calcium in pure form and in pharmaceutical formulations. E J Chem. 2009; 6:89-92.

31.    Hasumati AR, Rajput SJ, Dave JB, Patel CN. Development and validation of two chromatographic stability-indicating methods for determination of Rosuvastatin in pure form and pharmaceutical preparation. Int J Chem Tech Res. 2009; 1:677-689.