Analytical Method Development and Validation of Antihypertensive Drug: Rosuvastatin Calcium using Reverse Phase-High Performance Liquid Chromatogrhaphic Method

 

Monika Puri¹, Yogesh K. Walia²

¹Research Scholar, Career Point University, Hamirpur (H.P.), India.

²Professor, Department of Chemistry, Career Point University, Hamirpur (H.P.), India.

 

ABSTRACT:

A liquid chromatography method was developed to estimate Rosuvastatin calcium in bulk and pharmaceutical dosage forms accurately. The developed method demonstrates high accuracy, specificity, and reliability, making it particularly suitable for pharmaceutical research. The chromatographic analysis was performed using a C-18 column (250 x 4.6mm, 5µm) with a mobile phase consisting of Acetonitrile and acidified water (pH 3.0 adjusted with orthophosphoric acid) at a ratio of 55:45 v/v. Detection was achieved at a wavelength of 237nm. The retention time for Rosuvastatin Calcium was observed at approximately 7.0 minutes.

 

 

 

INTRODUCTION:

Hypertension significantly increases the risk of cardiovascular diseases, making its management a critical health objective¹. Lifestyle interventions combined with pharmacological treatments form the cornerstone for effective hypertension management^2,3. It's crucial to understand that high blood pressure can develop gradually over time without any obvious causes. However, it's important to rule out any underlying medical conditions first, such as kidney disease, thyroid problems, certain medications, and adrenal dysfunction.

 

 

This type of hypertension without an identifiable cause is called benign or essential hypertension, and it can worsen over time^4,5,6. As individuals age, blood pressure also tends to increase. Being overweight is a significant risk factor for developing pre-hypertension, and other lifestyle factors such as a sedentary lifestyle, high-sodium meals, smoking, and excessive alcohol consumption can also contribute to high blood pressure⁷. While there may not be a clear genetic pattern, it's worth noting that blood pressure levels may run in families. It's essential to monitor blood pressure levels regularly and take steps to manage hypertension to prevent the risk of developing more serious health problems in the future^8,9.

 

Rosuvastatin calcium, a potent statin, works by competitively inhibiting the HMG-CoA reductase enzyme, thereby lowering cholesterol synthesis and reducing cardiovascular events. Its structural formula (C₂₂H₂₇FN₃O₆S)₂Ca highlights its complex nature, making accurate quantification crucial for its clinical application (Fig.1.). It is a safe and effective way to improve heart health^10,11.

 

 

Fig. 1 Rosuvastatin Calcium

 

MATERIALS AND METHODS:

Chemicals and reagents:

All chemicals, including acetonitrile, orthophosphoric acid, and HPLC-grade water, were of analytical grade. The rosuvastatin calcium reference standard was traceable to NIST. Test sample of - Dosage form with Rosuvastatin, - 10/20mg.

 

Instrumentation:

Analysis was conducted using RP-HPLC equipped with a UV detector. A C-18 analytical column (250mm × 4.6 mm, 5µm) was employed.

 

Determination of λ_max:

By running the spectrum of the drug solution in a double-beam UV-spectrophotometer, the λ_max of drugs was determined.

 

Selection and procedure for preparation of mobile phase:

Different mobile phase ratios were evaluated to optimize the estimation of rosuvastatin calcium in pharmaceutical dosage forms. The most effective mobile phase was determined to be acetonitrile mixed with water adjusted to pH 3.00±0.05 using 0.1% orthophosphoric acid (V/V) in a ratio of 45:55.

 

Selection of Diluent:

The selected mobile phase served as the diluent for preparing standards and samples. The same diluent was also utilized as a blank solution during analysis.

 

Standard Solution Preparations:

Standard Stock Solution: Weigh 5mg of Rosuvastatin calcium and transfer it to a 50mL volumetric flask. Add 25mL of diluent and mix well. Sonicate for five minutes. Cool the solution to room temperature and make volume with diluent.

 

Standard Working Solution:

Pipet 5mL of the Standard stock solution and dilute to 50mL with diluent.

 

Concentration of working standard:

Rosuvastatin - 10ppm (10µg/mL). Filter the working standard solution using the 5-10mL disposable syringe and PTFE filters into a 2mL Autosampler vials and seal with the caps.

SAMPLE SOLUTION PREPARATION:

Sample Preparation:

Transfer dosage form with composition Rosuvastatin – 10/20mg into 100/200 mL volumetric flask. Fill the volumetric flask to 50% of capacity and mix well. Sonicate the sample till complete dissolution. Shake vigorously to ensure complete dissolution of the sample. Cool the solution to room temperature and makeup the volume with diluent. Pipette 5ml of the above solution and dilute to 50mL with diluent. Concentration of sample standard Rosuvastatin - 10ppm (10µg/mL). Filter the sample working solution using the 3ml disposable syringe and PTFE filters into a 2ml Autosampler vials and seal with the caps.

 

OPERATING CONDITIONS:

Table 1: Typical instrument Settings

Detector	237 nm
Flow rate	1.0ml/min
Injection volume	20µL
Mobile phase	acetonitrile with water adjusted to pH 3.00 ±0.05 using 0.1% orthophosphoric acid (V/V) in a ratio of 45:55
Running time	20min
Retention Time	Retention time for peaks in standard solution for Rosuvastatin calcium is 7 min.
Tailing Factor	Not more than 1.2 for all analyte in standard solution
Theoretical plates	Not less than 2500 for all analyte in standard solution.
Resolution	Not less than 2.0
Capacity Factor	To observe the value of retention factor.

 

EXPERIMENTAL PROCEDURE:

The instrument was initially allowed to equilibrate under the optimized chromatographic conditions prior to analysis. A blank solution was injected initially to confirm the system specificity. Subsequently, the retention time of Rosuvastatin calcium was determined by injecting the working standard solution. System suitability was assessed by performing five replicate injections of the standard solution, where the acceptance criterion for the relative standard deviation (RSD) of peak areas was established as not more than 2.0%. Continued system suitability was verified by injecting the working standard solution after every set of ten or fewer sample injections. The global RSD for all standard injections throughout the analysis was maintained below 2.0%. Finally, each sample solution was injected twice, and the concentration of Rosuvastatin calcium in samples was calculated based on the average peak area from five replicate standard injections.

 

RESULTS AND DISCUSSION:

Specificity

Specificity refers to the ability of an analytical method to accurately distinguish and detect the target analyte in the presence of potential interfering substances^12,13.

 

 

Table 2: Results of Specificity

S. No.	Solution	Retention Time
1	Blank	0.2
2	Standard – Rosuvastatin Calcium	7.2
	Sample
1	Rosuvastatin Calcium	7.2
2	Capacity Factor -Rosuvastatin Calcium	35.0

 

Observations and Conclusion:

The retention period of the peak of rosuvastatin calcium in standard solution is not detected in the blank (Table 2, Fig. 3(a,b)). A unique analytical approach is used to determine the therapeutic dose of rosuvastatin calcium.

 

System Suitability:

The precision of an instrument is the degree of agreement among the replicate injection of the standard^14,15.

 

Table 3: System Suitability Data

S. No.	Injection	Area of Rosuvastatin Calcium
1	1	630523
2	2	631422
3	3	629823
4	4	630501
5	5	630943
6	Mean	630642
7	Standard Deviation	592.41
8	Relative Standard Deviation (%)	0.09
9	RT	7.2
10	Theoretical Plates	3260
11	Tailing Factor	1.05

 

Observation and Conclusion:

The observed RSD of the replicate five standard injections is less than 2.0% which meets the system suitability parameter (Table 3). Hence Analytical method for the determination of Rosuvastatin Calcium in therapeutic dosage is meeting the system precision criteria.

 

Precision (Repeatability):

Precision in an analytical method refers to the consistency of test results when the procedure is repeatedly applied to multiple preparations of a uniform sample^16,17.

 

Table 4: Results of Precision(Repeatability)

S. No.	Test Preparation	Assays of Rosuvastatin Calcium
1	1	100.2%
2	2	100.8%
3	3	99.7%
4	4	100.3%
5	5	100.9%
6	6	99.9%
7	Mean	100.3%
8	Standard Deviation	0.005
9	Relative Standard Deviation (%)	0.48

Observation and Conclusion:

The six distinct determinations showed a Relative Standard Deviation less than 2.0%, which satisfies the requirement for acceptance (Table 4). Hence The technique precision (Repeatability) requirements for the analysis of rosuvastatin calcium in therapeutic dose are being satisfied.

 

Precision (Reproducibility):

The degree of agreement between individual test findings when a method is applied repeatedly to different preparations of a homogenous sample is what is known as an analytical method's precision¹⁸.

 

Table 5: Results of precision(Reproducibility)

S. No.	Test Preparation	Assays of Rosuvastatin Calcium
1	1	100.4%
2	2	99.9%
3	3	99.6%
4	4	100.4%
5	5	100.5%
6	6	100.0%
7	Mean	100.1%
8	Standard Deviation	0.004
9	Relative Standard Deviation (%)	0.36

 

Table 6: Summarised Results of precision

% Rosuvastatin Calcium     First Set	% Rosuvastatin Calcium Second Set	% Difference between the mean
100.3%	100.1%	0.2%

 

Observation and Conclusion:

The average difference between repeatability and reproductivity is less than 2.0%, and the noticed Relevant Standard deviation of the six individual determinations is less than 2.0%, both of which fulfil the acceptance standards. Hence The analytical procedure for determining the therapeutic doses of rosuvastatin calcium satisfies the technique precision (reproducibility) requirements (Table 5, 6).

 

Accuracy (Recovery):

The degree to which test results produced using the suggested approach are near to the actual value defines how accurate a method is. Applying the procedure to a placebo that has had known amounts of the analyte added at three different concentration levels—80%, 100%, and 120% of the test concentration—will allow you to assess the method's accuracy^19,20.

 

Table 7: Accuracy Data

Test ID	Analyte	Weight of Standard (gm)	Weight of Placebo (gm)	Amount of Std added (mL)	Added %	Recovered%	Recovery %
80%-1	Rosuvastatin Cal	0.005	0.221	4	3.62	3.63	100.3%
80%-2	RosuvastatinCal	0.005	0.222	4	3.60	3.62	100.5%
80%-3	RosuvastatinCal	0.005	0.225	4	3.56	3.57	100.4%
Test ID	Analyte	Weight of Standard (gm)	Weight of Placebo (gm)	Amount of Std added (mL)	Added %	Recovered%	Recovery %
100%-1	RosuvastatinCal	0.005	0.218	5	4.59	4.51	98.3%
100%-2	RosuvastatinCal	0.005	0.220	5	4.55	4.53	99.7%
100%-3	RosuvastatinCal	0.005	0.221	5	4.52	4.49	99.2%
Test ID	Analyte	Weight of Standard (gm)	Weight of Placebo (gm)	Amount of Std added (mL)	Added %	Recovered%	Recovery %
120%-1	RosuvastatinCal	0.005	0.223	6	5.38	5.41	100.5%
120%-2	RosuvastatinCal	0.005	0.218	6	5.50	5.52	100.3%
120%-3	RosuvastatinCal	0.005	0.221	6	5.43	5.48	100.9%

 

 

Table 8: Summarised Accuracy Data

S. No.	Test ID	Analyte	Average Recovery
1	80%-Level	Rosuvastatin Cal	100.4%
2	100%-Level	Rosuvastatin Cal	99.1%
3	120%-Level	Rosuvastatin Cal	100.6%

 

Observation and Conclusion:

The observed individual percentage recovery at each level is between 99.1 and 100.6%, and the mean recovery at each level is between 100.4 and 103%, all of which are within the acceptable range. Hence The analytical technique for determining the therapeutic dose of rosuvastatin calcium satisfies the Accuracy (Recovery) criterion (Table 7, 8).

 

Linearity:

Linearity refers to the ability of an analytical method to generate test results that maintain a direct proportional relationship with the analyte concentration within a defined range or follow a consistent mathematical correlation^21,22.

 

 

Fig. 2 Linearity curve of Rosuvastatin Calcium.

 

Table 9: Linearity Data

Linearity - Rosuvastatin Cal
S. No.	Injection	Area	Average	Conc.
1	1	504354	504514	80%
2	2	504674
3	1	567398	567578	90%
4	2	567758
5	1	630442	630642	100%
6	2	630842
7	1	693486	693706	110%
8	2	693926
9	1	756530	756770	120%
10	2	757010
Correlation coefficient			1.0000

 

Observation and Conclusion:

The observed correlation's coefficient, which is 1.000, satisfies the requirements for acceptance. Hence, The analytical technique used to determine the therapeutic dose of rosuvastatin calcium satisfies the linearity requirement (Table 9).

 

Range:

The distance between the upper and lower levels of an analyte (including these levels) that can be determined using the standard technique with a sufficient level of precision, accuracy, and linearity is known as the range of an analytical method. To determine the method's range, analyse the data from the linearity study, precision study, and accuracy study^23,24.

 

Observation and Conclusion:

The linearity, accuracy, and precision all fulfill the standards for acceptance. As a result, the analytical technique for determining rosuvastatin calcium in therapeutic dose is given a range of 80-120% of test concentration.

 

Robustness:

A technique's robustness, which measures its ability to be unaffected by modest but intentional changes in method parameters, gives a clue as to how reliable it will be under typical conditions²⁵. The robustness of the procedure is assessed by altering the mobile phase's composition, the column's lot, and the flow rate.

 

Table 10: Robustness Data

Test ID	Analyte	% Assay First Set- Method Precision	% Assay First Set – Robustness	% Difference
Buffer pH-2.95	Rosuvastatin Cal	100.30%	99.90%	0.40%
Buffer pH-3.05	Rosuvastatin Cal	100.30%	99.70%	0.60%
Mobile Phase Composition Change (Buffer:CAN :: 40:60)	Rosuvastatin Cal	100.30%	99.20%	1.10%
Mobile Phase Composition Change (Buffer:CAN :: 50:50)	Rosuvastatin Cal	100.30%	99.00%	1.30%
Change in Column Lot	Rosuvastatin Cal	100.30%	99.43%	0.87%
Change in Column - Particle Size C18, 5u, 250 x 4.0	Rosuvastatin Cal	100.30%	100.00%	0.30%
Flow Rate Change 0.9 ml	Rosuvastatin Cal	100.30%	98.90%	1.40%
Flow Rate Change 1.1 ml	Rosuvastatin Cal	100.30%	99.70%	0.60%

 

 

Table 11: Results of Solution Stability

Test ID	Analyte	Initial	6 (hrs.)	12(hrs.)	18 (hrs.)	24 (hrs.)	36(hrs.)
Solution Stability	Rosuvastatin Cal	100.30%	99.70%	100.60%	100.20%	100.00%	99.98%
% Difference	Rosuvastatin Cal	N/A	0.60%	-0.30%	0.10%	0.30%	0.32%

 

 

Observation and Conclusion:

The system appropriateness parameter was reached with all the adjustments, and the observed % fluctuation in the result produced with purposeful change is less than 2%, which satisfies the acceptance criterion. Hence The analytical technique used to determine the therapeutic dose of rosuvastatin calcium satisfies the Robustness requirement (Table 10).

 

Solution Stability:

The term "solution stability" refers to the consistency of the extracted sample solution (ready to inject) from the sample or matrix and the standard solution^26,27. Depending on the stability of the sample and standard solution, it should be stored properly at room temperature or in a refrigerator. (Table-11).

 

Observation and Conclusion:

A 36-hour solution stability investigation found that there was less than 2% variance in the results, which fulfils the standards for acceptance. Hence a 24-hour period can be employed with an analytical solution created for the measurement of rosuvastatin calcium in therapeutic dose (Table 11.

 

Reference Chromatogram:

 

 

 

Fig. 3 Chromatogram of (a) Blank (b) Rosuvastatin Calcium.

 

SUMMARY:

The validated RP-HPLC method presented here is simple, robust, and highly precise for the routine quantification of rosuvastatin calcium in bulk and pharmaceutical formulations. Compliance with ICH guidelines underscores the reliability and accuracy of this analytical method, making it highly suitable for quality control laboratories and pharmaceutical industries.

 

ACKNOWLEDGEMENT:

The authors would like to express their sincere gratitude to Career Point University, Hamirpur (H.P.), for providing resources and support throughout this research. We also wish to extend special thanks to our colleagues for their valuable insights and to our families for their constant support and understanding. Their contributions have been invaluable.

 

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Received on 26.01.2026      Revised on 02.03.2026 Accepted on 03.04.2026      Published on 21.04.2026 Available online from April 24, 2026 Res.  J. Pharma. Dosage Forms and Tech.2026; 18(2):115-120. DOI: 10.52711/0975-4377.2026.00018 ©AandV Publications All Right Reserved
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