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STABILITY INDICATING NEW RP-HPLC METHOD FOR THE DETERMINATION OF ROSUVASTATIN CALCIUM IN PURE AND TABLETS DOSAGE FORMS


Mohamed El-Kassem M Hassouna*, Hafsa Omar Salem
Chemistry Department, Faculty of Science 62514, Beni-Suef University, Beni-Suef , Egypt

 

ABSTRACT

 A new, specific, precise, simple, and accurate RP-HPLC method is developed and validated for the determination of Rosuvastatin calcium (ROS-Ca) in pure and tablets dosage forms. The method is performed on the Agilent Eclipse XDB C8 column (250 mm X 4.6 mm, 5μm particle size, using buffer solution of pH 4.5 containing 0.05M sodium dihydrogen phosphate: acetonitrile (50:50 v/v) as the mobile phase at a flow rate of 1.2 mL/min, injection volume 10 µL and UV detection at 245 nm. The total run time is 5.0 min. Linear relationship are obtained in the range 5-100 µg/mL and Rt values of 3.684 min for ROS-Ca with correlation coefficient (r) = 0.9995, limit of detection 1.50 µgmL-1 and limit of quantitation is 4.56 µgmL-1 for ROS-Ca. The overall recovery is 100 ± 2 % ; the relative standard deviation for precision and intraday precision is less than 2.0 %. Also, the forced degradation studies as acidity, alkalinity, oxidation, heat and photo degradation are performed according to ICH guidelines.  The method is validated according to ICH guidelines and USP requirements for new methods, which include accuracy, precision, specificity, LOD, LOQ, robustness, ruggedness, linearity and range. Hence this RP-HPLC method is suitable for quality control of raw materials and finished products.

Key words: Rosuvastatin Calcium; Stability; Assay; tablets dosage form; ICH; USP; HPLC

INTRODUCTION

Rosuvastatin Calcium (ROS-Ca); is chemically known as [(3R,5S,6E)-7-[4-(4-fluorophenyl)-6-(1-methyleythyl) -2- [methyl (methyl sulfonyl) amino] pyrimidine-5- yl]-3,5-dihydroxyhept-6-enoate]. It has a molecular formula of C44H45CaF2N6O12S2 and a molecular weight of 1001 [1, 2]. (Fig 1).

Rosuvastatin Calcium is a white or almost white, hygroscopic powder, slightly soluble in water, freely soluble in methylene chloride, practically insoluble in anhydrous ethanol. Store in airtight container, protected from light, at temperature of 2°C to 8°C.[1,2].

Rosuvastatin is available in tablet form containing 5 mg, 10 mg, 20 mg and 40 mg of the active ingredient, Rosuvastatin calcium. The excipients are: microcrystalline cellulose NF, lactose monohydrate NF, tribasic calcium phosphate NF, crospovidone NF, magnesium stearate NF, hypromellose NF, triacetin NF, titanium dioxide USP, yellow ferric oxide, and red ferric oxide NF. [3].

 Figure 1: Chemical structures of Rosuvastatin Calcium.

Rosuvastatin selectively and competitively inhibits HMG-CoA reductase, the rate-limiting enzyme that converts HMG-CoA to mevalonate, a precursor of cholesterol. Rosuvastatin has been shown to have high uptake and selectivity in the liver, the target organ for cholesterol lowering. Rosuvastatin produces its lipid-modifying effects in two ways: it increases the number of hepatic LDL receptors on the cell-surface to enhance the uptake and catabolism of LDL, and it inhibits hepatic synthesis of VLDL, which reduces the total number of VLDL and LDL particles [3].

Literature review revealed that various analytical methods have been described for the determination of Rosuvastatin Calcium including spectrophotometric [4-15], thin layer chromatography (TLC) [16], capillary electrophoresis [17],mass spectrometry [18-24],liquid chromatography (HPLC, UPLC)[25–50], electrochemical methods [51,52] and complexometric titration [53] have been developed for the estimation of ROS-Ca in pure or in dosage forms.

The aim of the present work is to develop a new, simple, sensitive, short retention time and accurate RP-HPLC method for the determination of ROS-Ca in pure or in dosage forms and application to assay dosage forms with high sensitivity, selectivity that are required to be in routine quality control analysis and validate the developed methods according to ICH guidelines [54].

 MATERIALS AND METHOD

 Chemicals and Reagents

Acetonitrile, Methanol HPLC-grade, Sodium dihydrogen phosphate monohydrate analytical grade, Triethylamine HPLC grade, Hydrochloric Acid analytical grade, Sodium Hydroxide reagent grade and distilled Water are procured from (scharlau, Spain).

 Pure samples

Pure samples of Rosuvastatin Calcium were kindly supplied by SPIMACO Pharmaceutical Company, Alexandria, Egypt with claimed purity of 100.5%. The content of Rosuvastatin Calcium in BP Pharmacopeia is in the range 97.0 to 102.0 percent.

Pharmaceutical dosage form

Six pharmaceutical preparations viz., Cholerose 10 mg Tab, Crestore 10 mg Tab, Estero-map 10 mg Tab, Advochol 10 mg Tab, Justechol 10 mg Tab and Crestolip 10 mg Tab are purchased from the local Egyptian market. Each tablet is claimed to contain 10.4 mg of Rosuvastatin Calcium.

Mobile phase preparation: Buffer: Acetonitrile (50:50)

Sodium dihydrogen buffer was prepared by dissolving 6.85 gm of sodium dihydrogen phosphate monohydrate in 700 mL distilled water and sonicated to dissolve, adjust pH to 4.5 by orthophosphoric acid solution or triethylamine. Make up to 1000 mL with distilled water, filter and degass mixtures of buffer and acetonitrile (50:50) through 0.45μ membrane filter under vacuum pump.

 Diluent:

Methanol HPLC-grade

Apparatus

HPLC system (Shimadzu LC SPD 20 A) with a detector (dual wavelength), equipped with a binary pump, Autosampler, oven CTO-20A/20AC with temperature range (10-85◦C), LC Solution software.

Mettler pH Meter.

Shimadzu analytical balance.

Ultra-sonic bath.

 HPLC Chromatographic Conditions

Chromatographic separation is performed on column Agilent Eclipse XDB- C8 (250 X 4.6 mm i.d, 5 µm particle size) .Using a mobile phase mixture of sodium dihydrogenphosphate buffer and acetonitrile in the ratio of 50:50 % v/v at ambient temperature, flow rate of 1.2 mL/min, UV detection is performed at 245 nm, injection volume is 10 μL and run time is 5.0 min.

 Preparation of standard and samples solution

Stock solutions of Rosuvastatin Calcium (1000 μg /mL)

100mg of ROS-Ca of the working standard are weighed accurately, transferred into 100 mL volumetric flask, add 70 mL of diluent and sonicate to dissolve. Complete the volume to the mark with the same diluent and mix well.

Working standard solutions of Rosuvastatin Calcium (50 μg /mL)

Transfer 5 mL from the stock solution of ROS-Ca into 100 mL volumetric flask, add 70 mL of the diluent and sonicate to dissolve, complete to the mark with the same diluent and mix well. The obtained chromatogram is shown in Figure 2.

Figure 2:  HPLC chromatogram of standard solution of (50μg/mL) of Rosuvastatin Calcium.

 Application to pharmaceutical formulations

Weigh not less than 10 tablets from products mentioned above and determine the average weight of one tablet. Grind to fine powder. Transfer an accurately weighed weight from powdered tablets equivalent to the average weight of one tablet into 200-mL volumetric flask. Add about 100 mL diluent, sonicate till dissolve and complete to volume with the same diluent and mix well. Filter through 0.45 μm syringe filter and inject into the chromatographic system. The obtained chromatogram is shown in (Figure 3). Also, the standard addition technique has been carried out to assess the validity of the method by spiking the pharmaceutical formulation with known amount of standard solution of ROS-Ca. The recovery of the added standards is then calculated after applying the proposed method.

Construction of calibration curves:

Different concentrations of ROS-Ca equivalent to (5–100) μg /mL, are separately withdrawn from their respective stock standards into separate series of 100 mL volumetric flasks, and the volumes are made up to the mark with the diluent. Duplicate 10 µL injections are made for each concentration maintaining the flow rate at 1.2 mL/min and the effluent is UV- scanned at 245 nm. The chromatographic separation is performed following the procedure under chromatographic conditions. The chromatograms are recorded, peak areas of ROS-Ca are determined and the calibration curves relating the obtained integrated peak areas to the corresponding concentrations are constructed and the regression equations are performed

.RESULTS AND DISCUSSION

 The goal of this study is to develop HPLC assay for the analysis of Ros.Ca drug in its pure form and in pharmaceutical formulation. Although, the literature is full of a number of HPLC methods for the determination of rosuvastatin calcium [43-50] due to the high accuracy and precision obtained by HPLC technique, the present method is devoted, also, for the same goal with aim of achieving better results under much simpler conditions. Chromatograms are obtained with Rt value of 3.684 min for ROS-Ca with correlation coefficients (r) =0.9995, limit of detection 1.50 µg mL-1 and limit of quantitation is 4.56 µg mL-1 for ROS Ca. No occurrence of interfering peaks.

Methods development and optimization

Different developing systems of different compositions and ratios are tried including: methanol: water (50:50, v/v), acetonitrile: water (50:50, v/v), but no peak was observed for ROS-Ca. Different flow rates are tried (0.7, 1.0, 1.2 and 1.5 mL/min), scanning wavelengths (200 -400 nm) are also tried. Preliminary studies involved trying C18, C8 reversed-phase columns. The best developing system proved to be sodium dihydrogen phosphate buffer pH (4.5): ACN (50:50, v/v) at flow rate of 1.2mL/min and at wavelength of 245.0 nm using column Agilent Eclipse XDB- C8 (250 mm X 4.6 mm i.d., 5μm).  This selected developing system allows good separation and sharp peak with good Rt values without tailing of the separated bands and good theoretical plates.

Validation of the Analytical Method

The method is validated, in accordance with ICH guidelines (ICH Q2R1), for system suitability, precision, accuracy, linearity, specificity, ruggedness, robustness, LOD and LOQ [54].

 Linearity and range

The linearity of the proposed methods is obtained in the concentration range (5.0 -100.0 μg/mL) for Rosuvastatin Calcium. Calibration curves are composed by plotting peak areas against the corresponding concentrations. The obtained coefficients of regression are 0.9995 for ROS-Ca. Linearity results are shown in Table 1.

 Repeatability

Repeatability of the method is evaluated by calculating the RSD of the peak areas of six replicate injections for the standard concentration (50.0 μg/mL) of ROS-Ca. Results are examined as % RSD values of the concentrations of the determined drug. Low values of % RSD (less than 2) indicate high precision of the method as shown in Table 1.

 Table 1: Regression and validation parameters of the proposed HPLC method for determination of ROS-Ca

ROS-Ca Parameter
   Linear
5-100 range (µg/mL)
32348.6829 Slope
8420.0893 Intercept
0.9995 Correlation coefficient
1.50 LOD a (µg/mL)
4.56 LOQ a (µg/mL)
0.013 Repeatability b

 aLimit of detection (3.3× σ /Slope) and limit of quantitation (10× σ /Slope).

b Repeatability for n≥5, RSD ≤2.

Table 2: Data of Accuracy for ROS-Ca.

ROS-Ca. Standard (μg/mL) ROS-Ca
μg/mL (Injected) μg/mL (found) Recovery%
25 25 25.37 101.48%
25 25.33 101.33%
25 25.30 101.20%
50 50 50.03 100.06%
50 50.04 100.09%
50 50.07 100.13%
80 80 79.39 99.24%
80 78.78 98.47%
80 78.64 98.30%
Accuracy (Mean)                                       100.04

 

Table 3: Determination of ROS-Ca in pharmaceutical formulation by the proposed HPLC method and application of standard addition technique

Recovery % Added(µg/mL)   Pharmaceutical formulation
ROS-Ca ROS-Ca
99.13 10 Crestolip 10 mg Tab

ROS Ca, 10.4 mg(claimed)

 

Mean ± RSD

100.31 20
98.53 30
99.32±0.91

 Table 4: Assay result for the determination of Rosuvastatin Calcium in pharmaceutical formulation by the proposed HPLC method.

limit % Recovery % Conc.(µg/mL)   Pharmaceutical formulation
ROS-Ca ROS-Ca ROS-Ca
(90 -110) 104.75% 50 Cholerose 10 mg Tab

ROS Ca, 10.4 mg(claimed)

 

 

 

 

Mean ± RSD

104.72%
104.52%
104.81%
104.87%
104.92%
104.76±0.13

 Table 5: Assay result for the determination of Rosuvastatin Calcium in pharmaceutical formulation by the proposed HPLC method.

limit % Recovery % Conc.(µg/mL)   Pharmaceutical formulation
ROS-Ca ROS-Ca ROS-Ca
(90 -110) 105.30% 50 Crestore 10 mg Tab

ROS Ca, 10.4 mg(claimed)

 

 

 

 

Mean ± RSD

106.69%
106.39%
107.76%
107.26%
103.43%
106.14±1.47

 Table 6: Assay result for the determination of Rosuvastatin Calcium in pharmaceutical formulation by the proposed HPLC method.

limit % Recovery % Conc.(µg/mL)   Pharmaceutical formulation
ROS-Ca ROS-Ca ROS-Ca
(90 -110) 105.48% 50 Estero-map 10 mg Tab

ROS Ca, 10.4 mg(claimed)

 

 

 

 

Mean ± RSD

105.29%
105.56%
105.61%
105.55%
105.86%
105.55±0.17

Table 7: Assay result for the determination of Rosuvastatin Calcium in pharmaceutical formulation by the proposed HPLC method.

limit % Recovery % Conc.(µg/mL)   Pharmaceutical formulation
ROS-Ca ROS-Ca ROS-Ca
(90 -110) 100.52% 50 Advochol 10 mg Tab

ROS Ca, 10.4 mg(claimed)

 

 

 

 

Mean ± RSD

100.58%
100.32%
100.48%
100.41%
100.55%
100.47±0.09

Table 8: Assay result for the determination of Rosuvastatin Calcium in pharmaceutical formulation by the proposed HPLC method.

limit % Recovery % Conc.(µg/mL)   Pharmaceutical formulation
ROS-Ca ROS-Ca ROS-Ca
(90 -110) 104.27% 50 Justechol 10 mg Tab

ROS Ca, 10.4 mg(claimed)

 

 

 

 

Mean ± RSD

104.31%
104.29%
104.75%
104.30%
104.31%
104.37±0.18

Detection and Quantitation limits

These approaches are based on the Standard Deviation of the Response and the Slope.  A specific calibration curve should be studied using samples containing an analyte in the range of LOD and LOQ. The residual standard deviation of a regression line or the standard deviation of y-intercepts of regression lines may be used as the standard deviation. LOD= 3.3 × σ /slope and LOQ =10 × σ /slope, where σ = the standard deviation of the response Table 1.

Accuracy and recovery

Accuracy of the proposed method is calculated as the percentage recoveries of pure samples of the studied drugs. Accuracy is assessed using three different concentrations (25, 50 & 80 μg/mL) for ROS-Ca within the linearity range (i.e. three concentrations and three replicates). Concentrations are calculated from the corresponding regression equations. The mean % recoveries for ROS-Ca are between 98.0 % to 102 %. These data are shown in Table 2. Accuracy is further assessed by applying the standard addition technique to Crestolip® 10 mg Tab, where good recoveries are obtained revealing that there is no interference from excipients, Table 3.

Formulation assay

The validated method is applied to the determination of ROS-Ca in commercially available pharmaceutical preparations available in the local Egyptian market. The results of the assay indicate that the method is selective for the analysis of all mentioned products without interference from the excipients. The results are displayed in Tables 4 – 9.

Intermediate precision (ruggedness)

Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment’s, etc. Good results are obtained and presented in Table10.

 Robustness

The robustness of the proposed method is evaluated in the development phase where the effects of different factors on the method are studied to obtain the optimum parameters for complete separation. Robustness of the method is studied by deliberately varying parameters like flow rate (±0.1 mL/min) and studying the effect of changing mobile phase pH by (± 0.2), acetonitrile composition (±5%) and column temperature changed (±5°c). The low values of the % RSD, as given in Table11, indicate the robustness of the proposed method.

 System suitability

System suitability testing is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such. System suitability is checked by calculating tailing factor (T), column efficiency (N), resolution (Rs) factors. All calculated parameters are within the acceptable limits indicating good selectivity of the method and ensuring system performance, Table 12.

Stability of the analytical solution

To demonstrate the stability of standard solution during analysis, solution is analyzed over a period of 24 h at room temperature and refrigerator. The results showed that for all the solutions, the retention times and peak areas of ROS-Ca remained almost unchanged (RSD<2.0%) indicating that no significant degradation occurred within this period, i.e. solutions are stable for at least 24 h, which is sufficient to complete the whole analytical process. The results are displayed in Table 13.

Specificity

Placebo interference:

Method specificity is determined for samples of studied compounds and placebo matrix containing all excipients present in the finished product. No interferences are detected at the retention time of ROS-Ca. Also, the inactive ingredients content of the pharmaceutical preparations do not interfere with HPLC procedure.

 Forced degradation:

Forced degradation of the active pharmaceutical ingredient (API) was carried out according to ICH guidelines (ICH, Q2B) in acid, base, oxidation, photo and heat.

Table 9: Assay result for the determination of Rosuvastatin Calcium in pharmaceutical formulation by the proposed HPLC method.

limit % Recovery % Conc.(µg/mL)   Pharmaceutical formulation
ROS-Ca ROS-Ca ROS-Ca
(90 -110) 105.53% 50 Crestolip 10 mg Tab

ROS Ca, 10.4 mg(claimed)

 

 

 

 

Mean ± RSD

105.47%
105.59%
105.80%
105.53%
105.53%
105.57±0.11

 Table 10: Ruggedness of the method

Parameter(%RSD) ROS-Ca
Intraday 0.159
Interday 0.989
Analyst to Analyst 0.168
Column to Column 0.353

Table 11: Robustness of the method

Parameter(%RSD) ROS-Ca
Flow rate change (±0.1 mL/min) 1.253
pH change of mobile phase (±0.2) 1.425
Wave length change (±0.2nm) 0.889
Column temperature change(±5C) 0.643

 

Table 12: System suitability testing parameters of the developed method

Item Obtained Value Reference values
ROS-Ca  
Tailing factor 1.110 T ≤ 2
Resolution Rs>2
Selectivity 3.5 k’ > 2
Injection precision 0.178 RSD ≤1%
Retention time (Rt) 0.122 RSD ≤1%
Number of theoretical plates(N) 5530.772 N > 2000

 

Table 13: Result of stability of analytical solution

Condition ROS-Ca
Fridge (2-8°C) 100.69%
Room temperature (25°C) 100.67%

Table 14: Results of analysis for forced degradation study samples using the proposed method, indicating percentage degradation of ROS-Ca

Parameter Rosuvastatin Calcium
Effect Observed tR Peak Area Degradation %
Test Without Effect(control) 3.684 1668239
Oxidation Effect 3.705 220023 86.81
Alkali Effect 3.198 1550541 7.05
Acid Effect 3.857 1494943 10.38
Light Effect (Sun light) 3.687 1621340 2.81
Heat Effect 3.682 1656890 0.68
Placebo No peak observed No area observed

 

Photo degradation:

The powder of working standard ROS-Ca is kept under sunlight for 48 hours. From this powder, accurately weigh 10.0 mg and transfer to 200-mL volumetric flask. Add 100 mL of diluent and sonicate to dissolve. Make up to the mark with water and mix well. Filter through 0.45 μm membrane filter, reject the first portion then analyze by HPLC. No interference is found at the retention time of ROS-Ca and no degradation products appeared after light degradation.

Heat degradation:

Keep the powder sample of the working standard ROS-Ca in dry oven at 80ºC for 6 hours. Similarly, accurately weigh 10.0 mg and transfer to 200-mL volumetric flask. Add 100 mL of diluent and sonicate to dissolve. Make up to the mark with water and mix well. Filter through 0.45 μm membrane filter, reject the first portion then analyze by HPLC. No interference is found at the retention time of ROS-Ca and no degradation products appeared after heat degradation.

Acid degradation:

Accurately weigh 10.0 mg of the working standard ROS-Ca powder and transfer into 200-mL conical flask. Add 100 mL of diluent and sonicate to dissolve, add 10 mL of 0.1 N HCl then keep the acidified solutions in room temperature for one day. Then complete to the mark with diluent and mix well. Filter and analyze by HPLC. No interference is found at the retention time of ROS-Ca and no degradation products appeared after acid degradation.

 

 

Figure 3.Typical HPLC chromatogram of sample solution (50μg/mL) of each (a)Cholerose 10 mg Tab, (b)Crestore 10 mg Tab, (c)Estero-map 10 mg Tab, (d) Advochol 10 mg Tab, (e) Justechol 10 mg Tab and (f) Crestolip 10 mg Tab.

 

Figure 4: HPLC chromatograms of 50 µg/ml solution of (a) Rosuvastatin Calcium after exposure to (b) heat degradation, (c) photo degradation, (d) acid degradation, (e) base degradation and (f) oxidative degradation.

Base degradation:

Similarly, weigh 10.0 mg of the working standard ROS-Ca powder and transfer it into 200-mL conical flask. Add 100 mL of diluent and sonicate to dissolve, add 10 mL of 0.1 N NaOH then keep the alkaline solutions in room temperature for one day. Then complete to the mark with diluent and mix well. Filter and analyze by HPLC. No interference is found at the retention time of ROS-Ca but its peak area is affected after base degradation. The degradation percent is calculated and displayed in (Table 14).

H2O2 degradation:

Finally, weigh 10.0 mg of the working standard ROS-Ca powder and transfer into 200-mL conical flask. Add 100 mL of diluent and sonicate to dissolve, add 15 mL of 3.0 % H2O2 then keep at room temperature for one day. Then complete to the mark with the diluent and mix well. Filter and analyze by HPLC. No interference is found at the retention time of ROS-Ca but some degradation products appeared after the H2O2 treatment. The degradation percent is calculated and displayed in (Table 14).  The corresponding chromatogram obtained is shown in (Figure 4).

 

CONCLUSION

 The proposed RP-HPLC method for determination of Rosuvastatin Calcium in pure and tablets dosage form is, precise, specific, accurate and less time consuming for analysis, low cost and rapid. The results of forced degradation undertaken according to the International Conference on Harmonization (ICH) guidelines reveal that the method is selective. Based on the above results, the analytical method is valid, fit for use and can be used for regular routine analysis and stability study.

 Acknowledgement

The authors are thankful to chemist Mahmoud A Mohamed, QQC Supervisor at EPCI part of HIKMA group, Beni-Suef, Egypt for his valuable help and providing training and guidance for the second author throughout the performance of the present work.

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