International Journal of PharmTech Research

CODEN (USA): IJPRIF ISSN : 0974-4304

Vol.2, No.3, pp 2016-2021, July-Sept 2010

Development and Validation of Reversed-Phase

HPLC Method for Simultaneous Estimation of
Rosuvastatin and Fenofibrate in Tablet Dosage Form
Suresh Kumar GV1*, Rajendraprasad Y2
1
St Johns Pharmacy College, Department of Medicinal Chemistry,6, 2nd stage
Vijaynagar,R.P.C.Layout,Bangalore-560040,Karnataka,India

Telephone: +91-80-23190191,Mobile Phone: +91-9886104039,Fax: +91-80-23350035

2
Department of Pharmaceutical Sciences,Andhra University,Visakhapatnam-
530002,Andhra Pradesh,India

*Corres.Author: gvsureshkumar@yahoo.com
Telephone: +91-80-23190191,Mobile Phone: +91-9886104039,Fax: +91-80-23350035

Abstract: A simple, precise and accurate reversed-phase liquid chromatographic method has been developed for the
simultaneous estimation of Rosuvastatin and Fenofibrate in tablet formulations. The chromatographic separation was
achieved on (Inertsil ODS, 250 x 4.6mm, 5µ column) analytical column. And mobile phase as mixture of Water (pH
adjusted to 2.5 with ortho phosphoric acid) and acetonitrile in ratio (30:70 v/v) at flow rate of 1.0ml/min and dual
detector wavelength 248 nm for Rosuvastatin and 286 nm for Fenofibrate. The retention time of Rosuvastatin and
Fenofibrate was found to be 3.6 and 20.5 minutes respectively. The validation of the proposed method was carried out
for its specificity, linearity, accuracy, precision, limit of detection and quantification for both atorvastatin calcium and
telmisartan. The developed method can be used for routine quality analysis of titled drugs in combination in tablet
formulation.
Key words: Rosuvastatin, Fenofibrate, RP-HPLC, validation, simultaneous estimation.

1. Introduction the risk of atherosclerosis, which in turn can lead to

Rosuvastatin, new member of a class of cholesterol- several cardiovascular complications such as heart
lowering drugs commonly referred to as “statins”, was attack, stroke and peripheral vascular disease. RST
approved for the treatment of dyslipidemia [1–3]. peak plasma concentrations were reached by 3–5 h
Rosuvastatin (RST) is chemically bis [(E)-7-[4-(4- following oral administration in humans [4].
fluorophenyl)-6-isopropyl-2-[methyl-(methyl- Fenofibrate has been widely used drug in the
sulfonyl)amino] pyrimidin-5-yl](3R,5S)-3,5-dihydroxy treatment of dyslipidaemia. The current formulations
hept- 6-enoicacid] calcium salt. RST, a synthetic lipid- of FBT shown an improved bioavailability due to the
lowering agent, is a selective and competitive inhibitor incorporation of a micronized process in product
of 3-hydroxy-3-methylglutaryl-coenzyne A (HMG- development [5,6]. Chemically, FBT is 2-[4-(4-
CoA) reductase, the key rate-limiting enzyme of chlorobenzoyl) phenoxy]-2- methyl-propanoic acid, 1-
cholesterol biosynthesis in liver. RST is used to reduce methylethyl ester. Fenofibric acid (FFA), the active
the amounts of LDL cholesterol, total cholesterol, metabolite of FBT, contributes for the reductions in
triglycerides and apolipoprotein B in the blood. RST total cholesterol, LDL cholesterol, apolipoprotien B,
also modestly increases the level of HDL cholesterol total triglycerides and triglyceride rich lipoprotein
in the blood. These actions are important in reducing [7,8].
Suresh Kumar GV et al /Int.J. PharmTech Res.2010,2(3) 2017

A detailed survey of analytical literature for 2.4. Standard solutions and calibration graphs
estimation of Rosuvastatin revealed several methods Standard stock solution of Rosuvastatin (0.1 mg/ml)
based on varied techniques viz, HPLC [9-11], and Fenofibrate (2.0 mg/ml) was prepared in
Capillary Zone Electrophoresis [12], methanol as diluent. To study the linearity range of
Spectrophotometry [10] and High Performance Thin each component, serial dilutions were made to obtain
Layer Chromatography (HPTLC) [13-14]. Estimation working standards in the concentration range of
of Fenofibrate are reported in bulk and formulations Rosuvastatin (50-150 µg/ml) and Fenofibrate (1000 -
using nuclear magnetic resonance (NMR) 3000 µg/ml). A graph was plotted as concentration of
spectrometry and LC (15), and in human plasma by drugs versus peak area response and results were
LC/tandem mass spectrometry (LC/MS/MS) with found linear for both analytes. From the standard stock
electrospray ionization (16). solution, a mixed standard solution was prepared
However no references have been found for containing Rosuvastatin (100 µg/ml) and Fenofibrate
simultaneous determination of Rosuvastatin and (2000 µg/ml). The system suitability test was
Fenofibrate in pharmaceutical preparations. The performed from five replicate injections of mixed
present manuscript describes a simple, rapid, precise standard solution.
and accurate isocratic reversed-phase HPLC method
for simultaneous determination of Rosuvastatin and 2.5. Sample preparation
Fenofibrate in the same tablet dosage form. Twenty tablets were weighed and finely powdered.
The average weight of tablets was determined with
2. Experimental weight of 20 tablets. A portion of powder equivalent to
2.1. Chemicals the weight of one tablet was accurately weighed into
Rosuvastatin (94.51%) and Fenofibrate (94.41%) were 100 ml A-grade volumetric flask and 70 ml diluent
obtained from Biocon Limited, Bangalore, India and was added. The volumetric flasks were sonicated for
Troikaa Pharmaceuticals Ltd. respectively as gift about 20min to effect complete dissolution of the
samples. Acetonitrile (HPLC Grade) and Methanol telmisartan and atorvastatin calcium, the solutions
(HPLC Grade) were purchased from E. Merck (India) were then made up to volume with diluent. The
Ltd. Worli, Mumbai, India. The 0.45-μm nylon filters solution was filtered through 0.45 µ m nylon filter.
were purchased from Advanced Micro Devices Pvt. The aliquot portion of the filtrate was further diluted to
Ltd. Chandigarh, India. Mili-Q water was used get final concentration of 100 µg/ml of Rosuvastatin
throughout the experiment. and 2000 µg/ml of Fenofibrate. Five microlitres of the
test solution was injected and chromatogram was
2.2. Equipments recorded for the same, and the amounts of the drugs
Analysis was performed on a chromatographic system were calculated.
Agilent 1200 series separation module (Japan)
equipped with an auto injector (G1329A), Diode array 2.6. Method validation
detector (DAD) SL (G1315C), Quaternary pump The HPLC method was validated in terms of precision,
(G1311A) and column thermostat (G1316A). A accuracy and linearity according to ICH guidelines
chromatographic separation was achieved by [17]. Assay method precision was determined by using
Symmetry C-18, 250 x 4.6mm, 5µ analytical column. nine-independent test solutions. The intermediate
Data acquisition was made with Chemstation software. precision of the assay method was also evaluated using
The peak purity was checked with the DAD detector. different analyst on three different days. The accuracy
of the assay method was evaluated with the recovery
2.3 Liquid chromatographic conditions of the standards from excipients. Three different
Chromatographic conditions were obtained using a quantities (low, medium and high) of the authentic
stainless steel column (Inertsil ODS, 250 x 4.6mm, 5µ standards were added to the placebo. The mixtures
column), which was maintained at 25o C. The dual were extracted as described in Section 2.5 and
analytical was set, 248 nm for Rosuvastatin and 286 analyzed using the developed HPLC method. Linearity
nm for Fenofibrate and samples of 5µl were injected to test solutions were prepared as described in Section 2.4.
HPLC system. The mobile phase was a mixture of The LOD and LOQ for analytes were estimated by
water (pH 2.5 adjusted with ortho-phosphoric acid) injecting a series of dilute solutions with known
and acetonitrile in ratio of 30:70 (v/v) at a flow rate of concentration. To determine the robustness of the
1.0ml/min. The mobile phase was filtered through method, the final experimental conditions were
0.45µm filter (Sartorius, Germany) and degassed for purposely altered and the results were examined. The
10 minutes by sonication. flow rate was varied by (±) 0.1 ml/min. The percentage
of organic modifier was varied by (±) 5% and pH of
mobile phase was varied by (±) 0.1.
Suresh Kumar GV et al /Int.J. PharmTech Res.2010,2(3) 2018

Table 1: Results of the recovery analysis of Rosuvastatin and Finofibrate

Compound Wt spiked Wt recovered Recovery (%) RSD (%)
(mg) (mg) N=3
Rosuvastatin 5.08 4.96 97.64 0.82
10.13 10.02 98.91 0.53
15.19 15.09 99.34 0.94
Finofibrate 100.24 100.14 99.90 0.51
200.19 200.07 99.94 0.72
300.21 300.18 99.99 0.84
R.S.D.: relative standard deviation Wt: weight.

Table 2: System suitability parameters.

Parameters Rosuvastatin Finofibrate
a
Theoretical plates 8836 15389
a
USP resolution 41.37 --
peak symmetrya 1.20 0.98
% RSD 0.82 0.97
a
USP-NF 29 section 621, pp.2135

Table 3: Intra and Inter-day assay precision data (n=9)

Actual Concentration Measured concentration (µg/ml), RSD. (%)
Intra -day Inter-day
Rosuvastatin (µg/ml)
5.14 5.02 (0.54) 5.09 (0.30)
10.35 10.36(0.34) 10.21 (0.42)
15.19 15.04 (0.91) 15.03 (0.78)
Finofibrate (µg/ml)
100.43 100.07 (0.23) 100.21 (0.61)
200.01 199.38 (0.49) 200.03 (0.67)
300.33 300.01 (0.51) 300.35 (0.42)
Data expressed as mean for “measured concentration” values.

Table 4:Results of robustness study

Factor Level
Rosuvastatin Finofibrate
Mean % assay (n=3) Mean % assay (n=3)
(% R.S.D.) (% R.S.D.)
pH of mobile phase 2.4 100.1 (0.31) 99.7(0.53)
2.6 99.6(0.76) 100.2(0.91)
Flow rate (ml/min) 0.9 99.7(0.54) 100.7(0.47)
1.1 100.4(0.62) 99.1(0.44)
% of acetonitrile 25 99.2(0.21) 99.3(0.71)
35 99.5(0.84) 99.0(0.39)
Suresh Kumar GV et al /Int.J. PharmTech Res.2010,2(3) 2019

Fig 1. A typical chromatogram of sample solution containing 100 µg/ml of rosuvastatin

and 200 µg/ml of fenofibrate

3. Results and discussion 3.2. Validation of method

3.1. Optimization of the chromatographic 3.2.1. Specificity

conditions The specificity of the HPLC method is illustrated in
In order to develop RP-HPLC method for combination Figures-1 which depicts complete separation of
of cardiovascular drugs Rosuvastatin and Fenofibrate Rosuvastatin and Fenofibrate in presence of tablet
in single formulation. The chromatographic conditions excipients. And no interfering peaks of endogenous
were optimized for better resolution by using water compounds observed at the retention time of the
and various buffers like phosphate, acetate and citrate analytes. In peak purity analysis with DAD detector,
for mobile phase preparation. After a series of purity angle was less than purity threshold for both the
screening experiments, it was concluded that water analytes, which implies that both analytes are pure and
(pH at 2.5 with ortho phosphoric acid) gave better excipients in the formulation doesn’t interfere the
peak shapes than different buffer at different pH. With analytes.
methanol as solvent both the peaks shows less
theoretical plates and bad peak shapes, on changing to 3.2.2. Accuracy
acetonitrile the peak shape improved along with Accuracy of the method was calculated by recovery
theoretical plates. Further optimization experiments studies at three levels by standard addition method
were carried out with varying percentage of (Table 1). The mean percentage of recoveries obtained
acetonitrile in mobile phase. The best peak shape and for Rosuvastatin and Fenofibrate was found to be
maximum separation was achieved with mobile phase 98.63 and 99.94% respectively.
composition comprising mixture of water-acetonitrile
(30:70 v/v). 3.2.3. Precision
The best separation, peak symmetry and Precision is the degree of repeatability of an analytical
reproducibility were obtained with Inertsil ODS, 250 x method under normal operational conditions. The
4.6mm, 5µ column compared to Zorbax C18, 250mm system precision is a measure of method variability
x 4.6mm, 5µm and Waters symmetry C18, 250mm x that can be expected for a given analyst performing the
4.6mm, 5µm column. The optimum wavelength for analysis and was determined by performing five
detecting both the analytes was ascertained and found replicate analysis of the same working solution. The
to be dual detector wavelength 248 nm for relative standard deviation (R.S.D.) obtained for
Rosuvastatin and 286 nm for Fenofibrate. Peak tailing Rosuvastatin and Fenofibrate are 0.82 and 0.97%
was observed for rosuvastatin when the flow rate was respectively (Table 2).
0.8ml/min using optimized mobile phase conditions. The intra- and inter-day variability or precision data
However, a flow rate of 1.0ml/min yielded optimum are summarized in Table 3. The intra-day precision of
separation and peak asymmetry. the developed LC method was determined by
preparing the tablet samples of the same batch in nine
Suresh Kumar GV et al /Int.J. PharmTech Res.2010,2(3) 2020

determinations with three concentrations and three LOD and LOQ for Rosuvastatin were 0.24 and 0.81
replicate each. The R.S.D. of the assay results, µg/ml respectively and for Fenofibrate were 0.14 and
expressed as percentage of label claim, was used to 0.45 µg/ml, respectively.
evaluate the method precision. The inter-day precision 3.2.6. Robustness
was also determined by assaying the tablets in The robustness of an analytical procedure is measure
triplicate per day for consecutive 3 days. The results of its ability to remain unaffected by small, but
indicated the good precision of the developed method deliberate variations in method parameters. Robustness
(Table 3). of the method was investigated by varying
experimental conditions such as changes in
3.2.4. Linearity wavelength, flow rate, pH and composition of mobile
Linearity was determined for telmisartan in the range phase. The mixed standard solution is injected in five
of Rosuvastatin 50–150µg/ml and for Fenofibrate replicates and sample solution of 100% concentration
1000–3000µg/ml. The correlation coefficient (‘r’) is prepared and injected in triplicate for every
values for both the drugs were >0.999. condition and % R.S.D. of assay was calculated for
each condition. The degree of reproducibility of the
3.2.5. Limit of detection (LOD) and limit of results obtained implies method is robust for routine
quantitation (LOQ) quality analysis (Table 4).
The limit of detection (LOD) is defined as the lowest
concentration of an analyte that an analytical process 4. Conclusion
can reliably differentiate from background levels [17]. A simple, specific, linear, precise and accurate RP-
The limit of quantification (LOQ) is defined as the HPLC method has been developed and validated for
lowest concentration of the standard curve that can be quantitative determination of Rosuvastatin and
measured with acceptable accuracy, precision and Fenofibrate in new tablet formulation. The method is
variability. The LOD and LOQ were calculated as very simple and specific as both peaks are well
separated from its excipient peaks, which makes the
LOD = 3.3 x Syx developed method suitable for routine quality control
b analysis.

LOQ = 10.0 x Syx Acknowledgements

b We thank management of St. Johns Pharmacy College,
Where Syx is residual variance due to regression; b is Bangalore, for providing necessary facilities. We are
slope. grateful to Dr. Wilkin Einstein, Director of Infant
Jesus Academy and Research Centre, Bangalore, India,
for providing the chemicals and necessities.

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