Simultaneous Determination of Rosuvastatin and Ezetimibe in pharmaceutical formulations by Stability Indicating Liquid Chromatographic Method

Ezetimibe (EZT) chemically [1] designated as (3R, 4S) 1 (4 fluorophenyl) 3 [(3S) 3 (4 fluorophenyl) 3 hydroxypropyl] 4 (4 hydroxyphenyl) azetidin 2 one (Figure 1A). It is a selective cholesterol absorption inhibitor, used for the treatment of hyperlipidemia, which potentially inhibits the absorption of biliary and dietary cholesterol. Ezetimibe prevents intestinal absorption of cholesterol without affecting absorption of triglycerides, fatty acids, bile acids and fat-soluble vitamins [2-4].


Reagents and solutions
Reference standards of Rosuvastatin (purity 99%) and Ezetimibe (purity 99.5%) were obtained from Glenmark Pharmaceuticals Ltd., India. The combination of Rosuvastatin and Ezetimibe is available as film-coated tablets (10 mg of Rosuvastatin and 10 mg of Ezetimibe) with brand names RAZEL-EZ ® (Glenmark Pharmaceuticals Ltd., India) and ROSUVAS-EZ ® (Ranbaxy Laboratories Ltd., India) and were procured from the local pharmacy store. Acetonitrile (HPLC grade), tetra butyl ammonium hydrogen sulphate (TBAHS) sodium hydroxide and hydrochloric acid and hydrogen peroxide were purchased from Merck (India).

Preparation of rosuvastatin and ezetimibe stock solutions (1 mg/ml)
Stock solutions of Rosuvastatin (1000 µg/ml) and Ezetimibe (1000 µg/ml) were prepared by accurately transferring 25 mg of Rosuvastatin and Ezetimibe separately in two 25 ml volumetric flasks and the volume was made up to volume with mobile phase. Working solutions for HPLC injections were prepared on a daily basis from the stock solution with mobile phase containing tetra butyl ammonium hydrogen sulphate and acetonitrile (32:68, v/v). Solutions were filtered through a 0.45 μm membrane filter prior to injection.

HPLC instrumentation and conditions
Chromatographic separation was achieved by using a Shimadzu Model CBM-20A/20 Alite HPLC system, equipped with SPD M20A prominence photodiode array detector (250 mm × 4.6 mm, 5 µm particle size) maintained at 25ºC. Isocratic elution was performed using tetra butyl ammonium hydrogen sulphate-acetonitrile (32:68, v/v) with a flow rate of 1.0 ml/min (UV detection 254 nm). 20 µl of sample was injected into the HPLC system and all chromatographic conditions were performed at room temperature (25°C ± 2°C).

Method validation
The method was validated for the following parameters: linearity, precision, accuracy, selectivity, robustness, limit of quantitation (LOQ), limit of detection (LOD) and system suitability [21].

Linearity
Linearity test solutions for the assay method were prepared from a stock solution at different concentration levels and 20 µL of each solution was injected in to the HPLC system and the peak area of the chromatogram obtained was noted.
The solutions extracted from the marketed formulations were also injected into the HPLC system and the peak area of the chromatograms was noted. A calibration curve was plotted by taking concentration of the drug solution on the x-axis and the corresponding peak area on the y-axis.

Precision
The intra-day precision of the assay method was evaluated at three concentration levels (10, 50 and 100 µg/ml) (n=3) against a qualified reference standard. The inter-day precision study was performed on three different days i.e. day 1, day 2 and day 3 at three different concentration levels (10, 50 and 100 µg/ml) (n=3). The %RSD of the obtained assay values at three different concentration levels was calculated.

Accuracy
The method accuracy was proved by the recovery test. The accuracy of the assay method was evaluated in triplicate at three concentration levels (80, 100 and 120%), and the percentage recoveries were calculated. A known amount of Rosuvastatin and Ezetimibe standards (10 μg/ml) were added to aliquots of sample solutions and then diluted to yield the total concentrations of 18, 20 and 22 μg/ml.

LOQ and LOD
The LOQ and LOD were based on the standard deviation of the response and the slope of the constructed calibration curve (n=3), as described in International Conference on Harmonization guidelines Q2 (R1) [21].

Robustness
The robustness of the assay method was established by introducing small changes in the HPLC conditions which included wavelength (252 and 256 nm), percentage of acetonitirile in the mobile phase (34 and 30) and flow rate (0.9 and 1.1 ml/min). Robustness of the method was studied using six replicates at a concentration level of 100 µg/ml of Rosuvastatin and Ezetimibe.

Forced degradation studies/Specificity
Stress studies were performed to evaluate the specificity of the method [22]. All samples were diluted with mobile phase to give a final concentration 100 µg/ml and filtered through 0.45 μm nylon filter before injection.
Acidic conditions: Acidic degradation was performed by treating the drug solution mixture (containing each of 1 mg/ml Rosuvastatin and Ezetimibe) with 0.1 N hydrochloric acid for 30 min in a thermostat maintained at 60ºC. The drug solution mixture was cooled, neutralized with 0.1 N sodium hydroxide and then diluted with mobile phase as per the requirement and 20 µL of the solution was injected in to the HPLC system.  Oxidation conditions: Oxidation degradation was performed by treating the drug solution mixture (containing each of 1 mg/ml Rosuvastatin and Ezetimibe) with 30% H 2 O 2 for 30 min in a thermostat maintained at 60ºC. The drug solution mixture was cooled and then diluted with mobile phase as per the requirement and 20 µL of the solution was injected in to the HPLC system.

Photolytic conditions:
The drug solution mixture (containing each of 1 mg/ml Rosuvastatin and Ezetimibe) was exposed to UV light (365 nm) for 3 hours, diluted with mobile phase as per the requirement and 20 µL of the solution was injected in to the HPLC system.

Thermal conditions:
The drug solution mixture (containing each of 1 mg/ml Rosuvastatin and Ezetimibe) was in a thermostat maintained at 60ºC for 8 hours, cooled and 20 µL of the solution was injected in to the HPLC system after necessary dilution with mobile phase.

Solution stability and mobile phase stability
The solution stability of Rosuvastatin and Ezetimibe in the assay method was carried out by leaving both the sample and reference standard solutions in tightly capped volumetric flasks at room temperature for 48 h. The same sample solutions were assayed at 12 h intervals over the study period. The mobile phase stability was also assessed by assaying the freshly prepared sample solutions against freshly prepared reference standard solutions at 12 h intervals up to 48 h. The prepared mobile phase remained constant during the study period. The % RSD of the Rosuvastatin and Ezetimibe assay was calculated for the mobile phase and solution stability experiments. An additional study was carried out using the stock solution by storing it in a tightly capped volumetric flask at 4ºC.

Analysis of tablet formulation
Twenty tablets from each brand (RAZEL-EZ ® and ROSUVAS-EZ ® ) were procured, weighed and crushed to a fine powder. Powder equivalent to 25 mg 25 mg of Rosuvastatin and Ezetimibe was accurately weighed into a 25 mL volumetric flask and made up to volume with mobile phase. The contents of the volumetric flask were sonicated for 30 min to enable complete dissolution of both the drugs. The solution was filtered and diluted with mobile phase as per the requirement. 20 μL of these solutions were injected into the system after filtering through 0.45 μm membrane and the peak area was recorded from the respective chromatogram.

HPLC method development and optimization
Initially the stressed samples were analyzed using a mobile phase consisting of TBAHS: acetonitrile (30:70, v/v) at a flow rate of 1.2 ml/min. Under these conditions, the resolution was not satisfactory. Therefore the mobile phase composition was changed to 32: 68, v/v with a flow rate 1.0 ml/min under which the resolution was good. Therefore mobile phase consisting of TBAHS: acetonitrile (32:68, v/v) with a flow rate of 1.0 ml/min was chosen as the best chromatographic response for the simultaneous determination of Rosuvastatin and Ezetimibe. UV detection was carried out at 254 nm (PDA detector). The present proposed method was compared with the previously published HPLC methods in the literature and discussed in Table 1.

Method validation
Linearity: The combination of Rosuvastatin and Ezetimibe shows linearity over a concentration range of 0.1-200 µg/ml ( Table 2) and the linear regression equations were found to be y = 34716x + 3830.3 (r 2 =0.9998) and y = 44508x + 25845 (r 2 =0.9998) for Rosuvastatin and Ezetimibe respectively. The chromatograms of the mobile phase (blank) and that of the combination of Rosuvastatin and Ezetimibe were shown in Figure 2A-B respectively.

Accuracy:
The accuracy study was repeated over three consecutive days and the resultant % RSD was found to be 0.10-0.14 and 0.10-0.63 for Rosuvastatin and Ezetimibe indicating that the method is precise. The recovery of for Rosuvastatin and Ezetimibe was found to be 99.38-99.95 % and 98.97-99.80 % respectively (Table 3).

Precision:
The % RSD for intra-day precision was found to be 0.41-0.94 and 0.31-0.59 for Rosuvastatin and Ezetimibe respectively whereas the inter-day precision was found to be 0.68-0.95 and 0.68-1.02 for Rosuvastatin and Ezetimibe respectively (Table 4).

Sensitivity/ Limit of quantification (LOQ) and limit of detection (LOD)
The LOD and LOQ were found to be 0.0282 µg/ml and 0.0853 µg/ ml for Rosuvastatin and the LOD and LOQ for Ezetimibe were 0.0297 µg/ml and 0.0901 µg/ml respectively.

Robustness:
Usually a slight change in flow rate, mobile phase composition etc. affects the chromatographic response such as retention time, tailing factor and theoretical plates etc. During this study the theoretical plates were found to be more than 2000 for both the drugs and at the same time the % RSD was found to be < 2.0% (0.52-0.95 % and 0.71-1.38 % for Rosuvastatin and Ezetimibe respectively) indicating that the proposed method is robust. The results were shown in Table 5.

Solution stability and mobile phase stability:
The % RSD for the solution stability was found to be 0.0149 and 0.0153 and for mobile  phase stability was 0.0875 and 0.0843 (< 2 %) for Rosuvastatin and Ezetimibe respectively indicating that the mobile phase as well as the sample solutions used during the assays were stable up to 48 h at room temperature ( Table 6).

Analysis of commercial formulations:
The proposed method was applied for the determination of Rosuvastatin and Ezetimibe in marketed formulations available (RAZEL-EZ ® and ROSUVAS-EZ ® ). The % recovery was found to be 98.57-98.71 and 96.02-96. 19 for Rosuvastatin and Ezetimibe respectively ( Table 7). The resultant chromatograms obtained from the extraction of marketed formulations were shown in Figure 2C-D.
Forced degradation studies: Rosuvastatin is highly resistant towards acidic, alkaline, oxidation, thermal and photolytic degradations as the percentage of degradation was found to be less than 10%. Ezetimibe has extensively undergone alkaline degradation (94.73%) and the phenolic hydroxyl group present in the chemical structure may be responsible for this. The resolution was found to be 6.272 and 5.123 (degradation product, R t 5.950 min) which is greater than 2. During the oxidation an extra peak was observed at 2.502 min. and the resolution was found to be 7.261. The resultant chromatograms obtained during the forced degradation studies were shown in Figure 3A-E. The system suitability parameters for all the degradation studies were shown in Table 8.

Discussion
The present proposed method can be successfully applicable to perform long-term and accelerate stability studies of Rosuvastatin and Ezetimibe formulations. The complete separation of the analytes was accomplished in less than 10 min. In literature no suitable robust and validated stability indicating methods are available with wide linearity range and instead a mixture of solvents were employed as mobile phase.
The system suitability parameters such as the number of theoretical plates (N) is used to determine the performance and effectiveness of the column. The efficiency of a column can be measured by the number of theoretical plates per meter. It is a measure of band spreading of a peak. Smaller the band spread, higher is the number of theoretical plates, indicating good column and system performance. Columns with N ranging from 5,000 to 100,000 plates / meter are ideal for a good system. In the present method during all the stress studies, the   Table 2: Linearity of Rosuvastatin and Ezetimibe. factor was less than <1.5-2 or <2 indicating that the method is more selective and specific.
The specificity of the developed method can be determined from the stress studies and the percentage drug recovery was calculated from the peak area of the resultant chromatograms. During the stress studies it was observed that ezetimibe is highly sensitive towards alkaline degradation. The phenolic hydroxyl group present in its structure may be responsible for the major degradation during alkaline stress conditions.