INTRODUCTION:

Rosuvastatin Calcium (Fig.-1) (E)-(3R,5S)-7-{4-(4-fluorophenyl)-6-isopropyl 2-{methyl (methyl sulphonyl amino)] pyrimidin-5-yl}3, 5dihydroxyhepten-6-oic acid calcium, is statin (or HMG-CoA reductase inhibitor) a class of drug used to lower cholesterol levels by competitively inhibiting the enzyme HMG-CoA reductase, which plays a central role in the production of cholesterol in the liver. Increased cholesterol levels have been associated with cardiovascular diseases (CVD), high LDL (low density lipoprotein), cholesterol (dyslipidemia), total cholesterol (hypercholesterolemia), and/or triglycerides (hypertriglyceridemia). Rosuvastatin Calcium is therefore used in the prevention and treatment of these diseases ^[1].

Fig.1. Structure of Rosuvastatin Calcium

Fenofibrate, Isopropyl (Fig.-2) 2-[4-(4-chlorobenzoyl) phenoxy]-2-methylpropanoate is a fibric acid derivative whose lipid modifying effects in humans are mediated via activation of peroxisome proliferator activated receptor type alpha(PPAR_).In addition, through modulation of the synthesis and catabolism of VLDL (very low density lipoprotein) fractions. Fenofibrate increases the LDL clearance and reduces small and dense LDL, the levels of which are elevated in the atherogenic lipoprotein phenotype, a common disorder in patients at risk for coronary heart disease. Treatment with fenofibric acid plus rosuvastatin improved HDL (high density lipoprotein) and triglyceride levels significantly better than statin monotherapy and improved LDL levels better than fenofibric acid monotherapy ^[2].

Fig.2. Structure of fenofibrate

Literature survey reveals that a variety of spectrophotometric ^[3-7] and chromatographic ^[8-13] methods have been reported for determination of rosuvastatin calcium and fenofibrate in pharmaceutical dosage forms in single and in combination with other drugs. Stability indicating HPLC methods for rosuvastatin ^[14-16]and fenofibrate ^[17-18]have been reported in literature survey. No stability indicating HPLC method has been reported for simultaneous estimation of rosuvastatin calcium and fenofibrate in the combined dosage form. Hence need was felt to develop stability indicating HPLC method for the combination. This validated method may be used for routine analysis of this combination.

MATERIALS AND METHODS:

Instruments

Chromatographic separation was performed on a Jasco chromatographic system equipped with a Jasco HPLC pump Model PU2080 plus, Jasco MD-2010 plus PDA detector, Rheodyne injector with 20μl loop volume and Elga water system for HPLC grade water.

Chemical s and Reagent:

Analytically pure samples of rosuvastatin calcium and fenofibrate were procured from Jubilant Lifesciences, Noida, India and A to Z Pharmaceuticals Pvt. Ltd. Chennai, India, respectively as gift sample and were used as working standards. Acetonitrile (HPLC grade), and ophosphoric acid (AR grade) were purchased from S.D.Fine-Chemicals and HPLC grade water ware used in mobile phase preparation. Commercially available tablets containing rosuvastatin calcium and fenofibrate in combination were purchased from local market.

General procedure:

The chromatographic parameters were given below

Chromatographic conditions-

Mobile Phase- Acetonitrile: Water (pH-4.0 adjusted with o-phosphoric acid) (70:30 v/v)

Flow rate -1.5 mL min^-1

Injection volume- 20 μl

Elution type- Isocratic elution

Detection wavelength -287 nm

Column- HiQ sil C18 HS column (250 Χ4.6 mm i.d, 5 μm particle size)

Preparation of working Stock and Standard Solution:

25mg each of rosuvastatin calcium and fenofibrate was weighed and transferred to 25ml volumetric flask. Acetonitrile was added to dissolve the drug and final volume was made with the same solvent to obtain a concentration of 1000 μg/ml of each drug. Appropriate amounts of stock solutions were diluted with mobile phase to obtain concentrations 1, 2, 3, 4 and 5 μg /ml of rosuvastatin calcium and fenofibrate.

Sample Preparation:

Marketed sample containing 10 mg rosuvastatin calcium and 160 mg fenofibrate were weighed and powdered. An amount of powder equivalent to 10 mg rosuvastatin calcium and 160 mg fenofibrate was transferred to a 25 ml calibrated volumetric flask. After addition of 20 mL acetonitrile and sonication (10 min) the solution was diluted to make up volume with the same solvent and filtered through a Whatman filter paper No.1. Two separate solutions were obtained by appropriate dilution with mobile phase to get 4μg/ml of rosuvastatin calcium in one and 3.2μg/ml of fenofibrate concentration in another.

Stress degradation studies

Stress degradation studies were carried under condition of acid/ base/ neutral hydrolysis, oxidation, dry heat and photolysis as per ICH guidelines ^[19]. For each study, two samples were prepared: the blank subjected to stress in the same manner as the drug solution. Working standard solution of each was subjected to stress conditions and both solutions was mixed in 1:1 ratio. Dry heat and photolytic degradation were carried out in solid state. Then the study was extended to formulation.

Degradation under alkali catalysed hydrolytic condition.

5.0ml each of rosuvastatin and fenofibrate standard solution (10μg/ml) was mixed with 5.0ml of 0.2N NaOH and kept overnight. Both the solution was mixed and 10μg/ml solution was then injected.

Degradation under acid catalysed hydrolytic condition.

5.0ml each of rosuvastatin and fenofibrate standard solution (10μg/ml) was mixed with 5.0ml of 0.1N HCL and kept overnight. Both the solution was mixed and 10μg/ml solution was then injected.

Degradation under neutral hydrolytic condition

5.0ml each of rosuvastatin and fenofibrate standard solution (10μg/ml) was mixed with 5.0ml of water and kept overnight. Both the solution was mixed and 10μg/ml solution was then injected.

Degradation under oxidative condition

5ml of working standard solution of both the drug was mixed with 5ml 30% solution of H₂O₂. The solution was diluted to 50 ml with acetonitrile and refluxed for 1 hr at 70⁰C. The solution was cooled to room temperature and volume was made to 50ml if required. Both the solution was mixed and 10μg/ml solution was then injected.

Degradation under dry heat

Dry heat studies were performed by keeping drug sample in oven (50⁰ C) for a period of 48 hours. Samples were withdrawn after appropriate time, dissolved in acetonitrile and diluted to get 10μg/ml and injected.

Photo-degradation studies

Photolytic studies were also carried out by exposure of drug to UV light up to 200 watt hours/square meter and subsequently to cool fluorescent light to achieve an illumination of1.2 million Lux.Hr. Sample was weighed, dissolved and diluted get 10μg/ml and injected.

Validation

The method was validated as per ICH guideline ^[20].

Linearity and Range

The linearity of an analytical procedure is its ability (within a given range) to obtain test result which are directly proportional to the concentration (amount) of analyte in the sample. It was studied by analyzing five concentrations of the drug and process was repeated five times.

Precision

Precision of the system was evaluated by analyzing six independent standard preparations and % RSD value obtained was calculated to determine any intra-day variation. These studies were also repeated on different days to determine inter-day variation.

Accuracy

To check accuracy of the method, recovery studies were carried out by addition of standard drug solution to pre-analyzed sample solution at three different levels 80, 100 and 120 %. Mean percentage recovery was determined.

Limit of detection

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample, which can be detected but not necessarily quantitated as an exact value. Based on the Standard Deviation of the Response and the Slope, detection limit (DL) may be expressed as:

3.3 σ

DL = ---------

Limit of quantitation

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample, which can be quantitatively determined with suitable precision and accuracy. Based on the Standard Deviation of the Response and the Slope, The quantitation limit (QL) may be expressed as:

10 σ

QL = ---------

Where,

σ = the standard deviation of the response for the lowest conc. in the range

S = the slope of the calibration curve.

Specificity

The specificity of the method was ascertained by peak purity profiling studies. Purity of the drug peak was ascertained by analyzing the spectrum at peak start, middle and at peak end. The peak purity was determined.

Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. Robustness of the method was determined by carrying out the analysis under conditions during which mobile phase ratio(±2% Acetonitrile), flow rate (±0.05 ml/min)and pH (±0.05)were altered.

RESULTS AND DISCUSSION:

Optimization studies were aimed at achieving separation of rosuvastatin and fenofibrate. The main focus of study to obtain retention time of fenofibrate within a reasonable period of 15 min. The experiments which resulted in a retention time of around 4 min for rosuvastatin led to a very high retention time for fenofibrate. Mixture of acetonitrile and water in different proportions was used to achieve run time of not more than 15 min per injection. So acetonitrile 70% and water 30% (adjusted pH to 4.0 with o- phosphoric acid) was the finally optimized mobile phase (Fig.-3).

Fig.3. Typical Chromatogram of rosuvastatin calcium (Rt 2.28±0.02min) and fenofibrate (Rt 13.06±0.02min) at 287nm.

Stress degradation study:

Hydrolysis at basic pH:

76.80% Rosuvastatin was recovered with no peaks of degradation and 73.82% fenofibrate was recovered with no peak of degradation.

Hydrolysis at acidic pH:

82.00% Rosuvastatin was recovered with no peaks of degradation and for fenofibrate with 100% percent recovery.

Under neutral hydrolysis:

98.70% Rosuvastatin was recovered with no peaks of degradation and 99.68% fenofibrate was recovered with no peak of degradation.

After oxidative condition:

75.80% Rosuvastatin was recovered with no peaks of degradation and 73.50% fenofibrate was recovered with one peak of degradation at 3.37 (Figure 4)

Fig 4: Chromatogram of rosuvastatin and fenofibrate after oxidation (P: Degradation product of fenofibrate, H: H₂O₂ peak, R:Rosuvastatin, F:Fenofibrate )

After the dry heat:

No peak of degradation was observed for rosuvastatin with 84.90% recovery and fenofibrate with 72.81% recovery respectively.

After the photo degradation:

85.18% Rosuvastatin was recovered with two peaks of degradation and 93.54% fenofibrate was recovered with no peak of degradation. (Figure 5)

Fig 5: Chromatogram of rosuvastatin and fenofibrate after photo degradation (R: Rosuvastatin, P₂ andP₃: Photodegradation products of Rosuvastatin, F: Fenofibrate)

Literature survey revealed that no stability indicating HPLC method has been reported for the determination of rosuvastatin and fenofibrate in combination. Stress degradation studies were carried out under condition of acid/ base/ neutral hydrolysis, oxidation, dry heat and photolysis. For acidic and basic hydrolysis various normalities was tried. The exposure to 0.1 N HCL and 0.2N NaOH for a period of 12 hrs was optimized condition for degradation. Since it resulted in % degradation not more than 30%.For neutral hydrolysis, oxidation, dry heat and photolysis not more than 30% degradation was achieved. The goal is to obtain about not more than 30% degradation and not complete degradation of active compound. Acheiving 100% degradation would be too strenuous and could possibly cause secondary degradation, giving degradation products of the product(s) which are not likely to formed under normal storage conditions ^[21].

Stress degradation results are summarized in Table 1 and results of method validation are given in Table 2.

CONCLUSION:

The developed method was found to be simple, sensitive and selective, accurate, precise, and repeatable for analysis of rosuvastatin and fenofibrate in combined market formulation without any interference from the excipients. The method was successfully used for determination of drugs in a pharmaceutical formulation. The results indicated the suitability of the method to study stability of rosuvastatin and fenofibrate under various forced degradation conditions like acid, base, dry heat, neutral, oxidative and photolytic degradation. It can be concluded that as the method could separate the drugs from their degradation products. The extent of degradation for both the drugs fairly matched the ones reported in the stability-indicating methods for the individual drugs.

ACKNOWLEDGEMENTS:

The authors are thankful to Jubilant Life sciences, Noida and A to Z Pharmaceuticals Pvt. Ltd. Chennai, India for providing a working standard of rosuvastatin calcium and fenofibrate respectively. The authors are also thankful to the management, AISSMS College of pharmacy for providing necessary facilities and constant encouragement. There is no funding received for this work.

REFERENCES:

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2. http://en.wikipedia.org/wiki/Fenofibrate .

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4. Gajjar AK, Shah VD. Simultaneous UV spectrophotometric estimation of rosuvastatin and ezetimibe in their combined dosage forms. International Journal of Pharmacy and Pharmaceutical Sciences. 2(1); 2010:131‐138.

5. Rajkondwar VV , Maini P, Vishwakarma M. Characterization and method development for estimation and validation of rosuvastatin calcium by UV – visible spectrophotometry. International Journal of Theoretical and Applied Sciences. 1(1); 2009: 48-53.

6. Dhabale PN, Gharge DS. Simultaneous spectrophotometric estimation of atorvastatin and fenofibrate in bulk drug and dosage form by using simultaneous equation method. IJCTR. 2(1): 2010: 325-328.

7. Gupta KR et al .Validated spectrophotometric determination of fenofibrate in formulation. Der Pharmacia Sinica. 1 (1); 2010: 173-178.

8. Pandya CB et al. Development and validation of RP-HPLC method for determination of rosuvastatin calcium in bulk and pharmaceutical dosage form. International Journal of Pharmaceutical Sciences Review and Research. 5(1); 2010:82-86.

9. Jain N, Raghuwanshi R, and Jain D. Development and validation of RP-HPLC method for simultaneous estimation of atorvastatin calcium and fenofibrate in tablet dosage forms. IJPS. 70(2); 2008:263-265.

10. Sankar GD et al. RP- HPLC method for the estimation of rosuvastatin calcium in bulk and pharmaceutical dosage form. Acta Ciencia Indica Chem. 33; 2007:1–4.

11. Smita S et al. Micellar liquid chromatographic method development for determination of rosuvastatin calcium and ezetimibe in pharmaceutical combination dosage form. Der Pharma Chemica. 2(1); 2010: 371‐377.

12. Kaila HO et al. A new improved RP-HPLC method for assay of rosuvastatin calcium in tablets. Indian Journal of Pharmaceutical Sciences. 72(5); 2010: 592-598.

13. Choudhari VP, Nikalje AP. Simultaneous estimation of atorvastatin, ezetimibe and fenofibrate in pharmaceutical formulation by RP-LC-PDA. Pharmaceutica Analytica Acta. 1(2); 2010:1-5.

14. Gomes F et al. Development and validation of stability indicating HPLC methods for quantitative determination of pravastatin, fluvastatin, atorvastatin and rosuvastatin in Pharmaceuticals. Anal Lett. 42(1); 2009: 784–804.

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19. ICH, Q1A (R2): Stability Testing of New Drug Substances and Products, International Conference on Harmonization, Geneva. 2003.

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Received on 29.03.2012 Modified on 15.04.2012

Asian J. Research Chem. 5(5): May 2012; Page 606-610

*Sr. no.*	*Stress Condition*		*% Degradation by HPLC*
*Sr. no.*	*Conditions*	*Reflux time*	*Rosuvastatin*	*Fenofibrate*
1	Acid hydrolysis ( 0.1N HCL )	-	18.0	-
2	Basic Hydrolysis (0.1N NaOH)	-	23.20	26.18
3	Neutral Hydrolysis	-	1.30	0.32
4	Oxidation(30 % H₂O₂)	1 Hr.	24.20	26.50
5	Dry Heat (50⁰ C, 24Hrs.)	-	15.1	27.19
	PHOTOLYSIS
6	UV (200 watt hours./square meter)	-	14.82	6.46

*Parameter*		*Results*
*Parameter*		*Rosuvastatin*	*Fenofibrate*
Range		1-10 µg mL^-1	1-10 µg mL^-1
Linearity		Y=5285.1x + 4398.7	Y=46951x+9522
r²		0.9901	0.9967
Precision (%RSD)*	Intraday	0.52%	0.82%
Precision (%RSD)*	Interday	0.79%	1.04%
Recovery Levels	80%	81.14	81.26
	100%	101.06	101.0
	120%	120.89	120.56
LOD †		0.031 µg mL^-1	0.029 µg mL^-1
LOQ ‡		0.096 µg mL^-1	0.090 µg mL^-1
Robustness		More sensitive to pH of mobile phase	More sensitive to pH of mobile phase