A NOVEL RP- HPLC METHOD DEVELOPMENT AND VALIDATION OF ATORVASTATIN AND FENOFIBRATE IN BULK AND PHARMACEUTICAL DOSAGE FORMS

ABOUT AUTHORS:
¹Vinjam Swathi* ²Nanda Kishore Agarwal
¹M pharmacy, Department of Pharmaceutical Analysis and Quality Assurance, Nimra College of Pharmacy, Jupudi, Vijayawada, A.P, India
²Professor and head of the department of pharmaceutical chemistry, Nimra College Of Pharmacy, Jupudi, Ibrahimpatnam, Vijayawada, A.P, India
swathi.vinjam90@gmail.com

ABSTRACT
The present investigation describes about a simple, economic, selective, accurate, precise reverse phase high performance liquid chromatographic method for the simultaneous estimation of Atorvastatin and Fenofibrate in pure and pharmaceutical dosage forms. Atorvastatin and Fenofibrate were well separated using a Thermohypersil BDS C18column of dimension 100 × 4.6, 5µm and Mobile phase consisting of Methanol: Water (Adjusted with orthophosphoric acid to pH-2) in the ratio of 40:60v/v at the flow rate 1 ml/min and the detection was carried out at 274nm with PDA detector. The Retention time for Atorvastatin and Fenofibrate were found to be 1.438, 2.949 respectively. The developed method was validated for recovery, specificity, precision, accuracy, linearity according to ICH guidelines. The method was successfully applied to Atorvastatin and Fenofibrate combination pharmaceutical dosage form.

REFERENCE ID: PHARMATUTOR-ART-2001

1. INTRODUCTION
Atorvastatin ((3R, 5R)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3, 5-dihydroxyheptanoic acid) is a member of the drug class known as statins. It is used for lowering cholesterol. Atorvastatin is a competitive inhibitor of hydroxymethylglutaryl-coenzyme-A (HMG-CoA) reductase, the rate-determining enzyme in cholesterol biosynthesis via the mevalonate pathway. HMG-CoA reductase catalyzes the conversion of HMG-CoA to mevalonate. It acts primarily in the liver. Decreased hepatic cholesterol levels increases hepatic uptake of cholesterol and reduces plasma cholesterol levels.

Fenofibrate (propan-2-yl 2-{4-[(4-chlorophenyl) carbonyl] phenoxy}-2 methyl propanoate) is a drug of the fibrate class. It is mainly used to reduce cholesterol levels in patients at risk of cardiovascular disease. Like other fibrates, it reduces low-density lipoprotein (LDL) and very low density lipoprotein (VLDL) levels, as well as increasing high-density lipoprotein (HDL) levels and reducing triglycerides level. It is used alone or in conjunction with statins in the treatment of hypercholesterolemia and hypertriglyceridemia. It lowers lipid levels by activating peroxisome proliferator-activated receptor alpha (PPARα). PPARα activates lipoprotein lipase and reduces apoprotein CIII, which increases lipolysis and elimination of triglyceride-rich particles from plasma.

Literature survey revealed that very few methods have been reported for the analysis of Atorvastatin and Fenofibrate combinational dosage forms which include UV spectroscopy, Reverse Phase High performance Liquid Chromatography, Densitometric method, HPTLC methods. The present study illustrate development and validation of simple, economical, selective, accurate, precise RP-HPLC method for the determination Aof torvastatin and Fenofibrate in bulk and Pharmaceutical dosage forms as per ICH guidelines.

The goal of this study is to develop rapid, economical HPLC method for the analysis of Atorvastatin and Fenofibrate in combined dosage form using most commonly employed column (C18) and simple mobile phase preparation.

In the present proposed work a successful attempt had been made to develop a method for the simultaneous estimation of Atorvastatin and Fenofibrate pharmaceutical dosage form and validate it. From the economical point of view and for the purpose of routine analysis, it was decided to develop a more economical RP-HPLC method with simple mobile phase preparation for the estimation of Atorvastatin and Fenofibrate combinational dosage form. The method would help in estimate of drugs in single run which reduces the time of analysis and does not require separate method for each drug. Thus, the paper reports an economical, simple and accurate RP-HPLC method for the above said pharmaceutical dosage forms.

2. MATERIALS AND METHODS
Quantitative HPLC was performed on a high performance liquid chromatograph -Waters e2695Alliance HPLC system connected with PDA Detector 2998 and Empower2 Software. The drug analysis data were acquired and processed using Empower2 software running under Windows XP on a Pentium PC and Thermohypersil BDS C18column of dimension 100 × 4.6, 5µm particle size. In addition an analytical balance (DENVER 0.1mg sensitivity), digital pH meter (Eutech pH 510), a sonicator (Unichrome associates UCA 701) were used in this study.

Standards and chemicals used:Pharmaceutical grade Atorvastatin and Fenofibratewere kindly supplied as a gift sample by Dr.Reddy’s Laboratory, Hyderabad, and Andhra Pradesh, India. Methanol was of HPLC grade and Purchased from E. Merck, Darmstadt, Germany. Ortho Phosphoric Acid was analytical reagent grade supplied by Fischer Scientific Chemicals. Water HPLC grade was obtained from a Milli-QRO water purification system. Atorvastatin and Fenofibrate Tablets available in the market as Lipicure-TG (intas pharmaceuticals, Sikkim, India.) in composition of Atorvastatin (10mg), Fenofibrate (150mg).

Preparation of mobile phase: Transfer water into 1000ml of beaker dissolve and diluted volume with water. Then adjust its pH to 2 with Ortho Phosphoric Acid (OPA). The Water adjusted pH to 2 with OPA: Methanol (60:40 v/v) and filtered through 0.45µ membrane filter and degassed by sonication.

Preparation of calibration standards:10mg Atorvastatin and 16mg Fenofibrate was taken into a 10, 50 ml of volumetric flask and add 10ml ofDiluent and sonicated for 10 minutes and made up with Diluent. It was further diluted to get stock solution of Atorvastatin and Fenofibrate (To get 0.02 ppm and 0.32 ppm solution respectively). This is taken as a 100% concentration. Working standard solutions of Atorvastatin and Fenofibrate was prepared with mobile phase. To a series of 10 ml volumetric flasks, standard solutions of Atorvastatin and Fenofibrate in the concentration range of 0.01-0.03µg/ml and 0.16-0.48µg/ml were transferred respectively.

System suitability: System suitability are an integral part of chromatographic system. To ascertain its effectiveness, certain system suitability test parameters were checked by repetitively injecting the drug solutions at 100% concentration level for Atorvastatin and Fenofibrate to check the reproducibility of the system. At first the HPLC system was stabilized for 40 min.One blank followed by six replicate analysis of solution containing 100% target concentration of Atorvastatin and Fenofibrate were injected to check the system suitability. To ascertain the system suitability for the proposed method, a number of parameters such as theoretical plates, peak asymmetry, and retention time were taken and results were presented in Table 1.

Recommended procedure:

Calibration curves for Atorvastatin and Fenofibrate: Replicate analysis of solution containing 0.01-0.03µg/mL, 0.16-0.48µg/mL of Atorvastatin and Fenofibrate sample solutions respectively were injected into HPLC according to the procedure in a sequence and chromatograms were recorded. Calibration curves were constructed by plotting by taking concentrations on X-axis and ratio of peak areas of standards on Y-axis and regression equation were computed for both drugs and represented in Table .6

Analysis of marketed formulation: The content of ten tablets was weighed accurately. Their average weights were determined. Powder of tablets equivalent to one tablet weight (521.1mg) were weighed and taken in a 50 ml volumetric flask, dissolved in diluents, shaken and sonicated for about 20 minutes then filtered through 0.45µ membrane filter. The filtered solution was further diluted (5 to 50ml) in the diluents to make the final concentration of working sample equivalent to 100% of target concentration.The prepared sample and standard solutions were injected into HPLC system according to the procedure. from the peak areas of Atorvastatin and Fenofibrate the amount of the drugs in the sample were computed. The contents were calculated as an average of six determinations and experimental results were presented in Table 4. The representive standard and sample chromatograms were shown in fig.2 and fig.3.

Validation study of Atorvastatin and Fenofibrate: An integral part of analytical method development is validation. Method validation is the process to confirm that the analytical procedure employed for a specific test is suitable for its intended use. The newly developed RP-HPLC method was validated as per International Conference on Harmonization (ICH) guidelines for parameters like specificity, system suitability, accuracy, linearity, precision (repeatability), limit of detection (LOD), limit of Quantification (LOQ) and robustness.

Specificity: The effect of wide range of excipients and other additives usually present in the formulation of Atorvastatin and Fenofibrate in the determination under optimum conditions were investigated. The specificity of the RP-HPLC method was established by injecting the mobile phase and placebo solution in triplicate and recording the chromatograms. The common excipients such as lactose anhydrous, microcrystalline cellulose and magnesium state have been added to the sample solution injected and tested.

Precision: precision study of sample (Losartan potassium and Amlodipine) was carried out by estimating corresponding responses 6 times on the same day for the 100% target concentration. The percent relative standard deviation (%RSD) is calculated which is within the acceptable criteria of not more than 2.0.

Linearity: The linearity graphs for the proposed assay methods were obtained over the concentration range of 0.01-0.03µg/ml and 0.16-0.48µg/ml (50-150%) Atorvastatin and Fenofibrate respectively. Method of least square analysis is carried out for getting the slope, intercept and correlation coefficient, regression data values and the results were presented in Table 2. The representative chromatograms indicating the sample were shown in fig.2&3. A calibration curve was plotted between concentration and area response and statistical analysis of the calibration curves were shown in fig. 6&7.

Accuracy (Recovery studies): The accuracy of the method is determined by calculating recovery of Atorvastatin and Fenofibrate by the method of addition. Known amount of Atorvastatin and Fenofibrate at 50%, 100%, 150% is added to a pre quantified sample solution. The recovery studies were carried out in the tablet in triplicate each in the presence of placebo. The mean percentage recovery of Atorvastatin and Fenofibrate at each level is not less than 99% and not more than 101%.

Robustness: The robustness is evaluated by the analysis of Atorvastatin and Fenofibrate under different experimental conditions such as making small changes in flow rate (±0.2 ml/min), λmax (±5), column temperature (±5), mobile phase composition (±5%), and pH of the buffer solution.

LOD and LOQ: Limit of detection is the lowest concentration in a sample that can be detected but not necessarily quantified. Under the stated experimental conditions. The limit of quantification is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy. Limit of detection and limit of quantification were calculated using following formula LOD=3.3(SD)/S and LOQ=10(SD)/S, where SD= standard deviation of response (peak area) and S= average of the slope of the calibration curve.

3. RESULTS AND DISCUSSION
Reverse phase HPLC method was preferred for the determination of Atorvastatin and Fenofibrate. Preliminary experiments were carried out to achieve the best chromatographic conditions for the simultaneous determination of the drug substances. Several column types and lengths were tried considering other chromatographic parameters. C18 column with a 4.6 mm inner diameter and 5µm particle size was chosen. The detection wave length was selected as 274nm with PDA detector. Chromatographic conditions were optimized by changing the mobile phase composition and buffers used in mobile phase. Different experiments were performed to optimize the mobile phase but adequate separation of the drugs could not be achieved. By altering the pH of buffer results a good separation. Different proportions of solvents were tested. Eventually the best separation was obtained by the isocratic elution system using a mixture of water (adjusted the pH to 2 with OPA): methanol (60:40, v/v) at a flow rate of 1 ml/min. a typical chromatogram for simultaneous estimation of the two drugs obtained by using a above mentioned mobile phase. Under these conditions Atorvastatin and Fenofibrate were eluted at 1.438min and 2.949minutes respectively with a run time of 5 minutes. The representative chromatogram of this simultaneous estimation shown in fig. 3 & 4 and results were summarized in Table 1.

The Methanol and water (pH 2 with OPA) (40:60, v/v) was chosen as the mobile phase. The run time of the HPLC procedure was 5 minutes at flow rate of 1ml/min was optimized which gave sharp peak, minimum tailing factor. The system suitability parameters were shown in Table 1 were in within limit, hence it was concluded that the system was suitable to perform the assay. The method shows linearity between the concentration range of 0.01-0.03µg/ml for Atorvastatin and 0.16-0.48µg/ml for Fenofibrate. The experimental results were shown in table 6 and fig.6&7. The % recovery of Atorvastatin and Fenofibrate was found to be in the range of 99.5 to 100 % & 99 to 100.33% respectively. As there was no interference due to excipients and mobile phase, the method was found to be specific. As both compounds pass the peak purity, the method was found to be specific. The method was robust and rugged as observed from insignificant variation in the results of analysis by changes in Flow rate, column oven temperature, mobile phase composition and wave length separately and analysis being performed by different analysts. The results were shown in Table 5.The LOD and LOQ values were calculated based on the standard deviation of the response and the slope of the calibration curve at levels approximately the LOD and LOQ. The limit of detection was obtained as 0.00013µg/mLfor Atorvastatin and 0.00210µg/mL for Fenofibrate. The limit of quantitation was obtained as 0.00046µg/mLfor Atorvastatin and 0.0070µg/mLfor Fenofibrate which shows that the method is very sensitive. The results were shown in Table.7.

4. CONCLUSION
A new validated RP-HPLC method has been developed for the quantitative and Qualitative determination of Atorvastatin and Fenofibrate in tablet dosage forms in bulk and pharmaceutical dosage forms was established. The method was completely validated shows satisfactory results for all the method validation parameters tested and method was free from interferences of the other active ingredients and additives used in the formulation. Infact results of the study indicate that the developed method was found to be simple, reliable, accurate, linear, sensitive, economical and reproducible and have short run time which makes the method rapid. Hence it can be concluded that the proposed method was a good approach for obtaining reliable results and found to be suitable for the routine analysis of Atorvastatin and Fenofibrate in Bulk drug and Pharmaceutical formulations.

5. ACKNOWLEDGEMENT
The authors would like to thank beloved parents and all my well wishers, one and all who have helped me directly and indirectly in completing this project work.

Table 1: optimized chromatographic conditions and system suitability parameters for proposed method

S.NO	Parameter	Chromatographic conditions
1.	Instrument	Waters e2695 Alliance HPLC with Empower2 software
2.	Column	thermohypersil C18, (5μ, 150 x 4.6mm)
3.	Detector	PDA Detector 2998
4.	Diluents	Methanol
5.	Mobile phase	Water(adjusted pH 2.0 with OPA): methanolo (60:40 v/v)
6.	Flow rate	1ml/min
7.	Detection wavelength	274nm
8.	Temperature	35°c
9.	Injection volume	5µl
10.	Retention time
	Atorvastatin	1.438
	Fenofibrate	2.949
11.	Theoretical plate count
	Atorvastatin	2552
	Fenofibrate	3000
12.	Tailing factor
	Atorvastatin	1.17
	Fenofibrate	1.59
13.	Resolution factor	9.38

Table 2: Specificity study

S.NO.	Name of the solution	Retention time in min
1.	Blank	No peaks
2.	Atorvastatin	1.438
3.	Fenofibrate	2.949

Table 3: results of precision study

S.NO	Sample	Injection number	precission
S.NO	Sample	Injection number	RT	Peak area
1.	Atorvastatin	1	1.443	2054782
		2	1.438	2054809
		3	1.442	2065165
		4	1.382	2060368
		5	1..441	2041300
		6	1.444	2043795
		Mean		2053370
		%RSD(NMT 2.0)		0.50
2.	Fenofibrate	1	2.977	3515822
		2	2.961	3503597
		3	2.973	3509064
		4	2.970	3507668
		5	2.973	3511717
		6	2.877	3513906
		Mean		3510296
		%RSD(NMT 2.0)		0.1

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Table 4: Recovery data of the proposed Atorvastatin and Fenofibrate

S.NO	Sample	Spiked Amount (µg/ml)	Recovered Amount (µg/ml)	%Recovered	%Average recovery
1.	Atorvastatin	10.04	10.02	100.16	100%
		19.983	19.94	100
		29.937	29.85	99.3
2.	Fenofibrate	158.47	158.82	100.33	100.33%
		316.538	317.59	100.33
		474.20	476.32	100.33

Table 5: Robustness results of Atorvastatin and Fenofibrate

S.NO	sample	Linearity level (µg/ml)	Peak area	Slope	Y-intercept	r²
1.	Atorvastatin	0.1	1024859	20379	1640.2	0.999
		0.15	1530720
		0.2	2028343
		0.25	2552721
		0.3	3061292
2.	Fenofibrate	0.01	1756257	34837	15635	0.999
		0.015	2634474
		0.02	3501613
		0.025	4352056
		0.03	5252030

S.NO	sample	parameters	Optimized	Used	RT	Peak area	Plate count
1.	Atorvastatin	Flow rate (±0.2)	1ml/min	0.8	1.799	2187263	3021
				1	1.436	6100287	2723
				1.2	1.209	1639328	2856
		Temperature (±5°C)	35°C	30	1.357	1565593	2943
				35	1.438	2043083	2552
				40	1.211	1639328	2734
2.	Fenofibrate	Flow rate (±0.2)	1ml/min	0.8	2.508	3198139	3037
				1	2.940	3507208	3234
				1.2	2.495	2378287	2810
		Temperature (±5°C)	35°C	30	2.488	2519528	2853
				35	2.949	3527161	3222
				40	3.684	2643655	2958

Table 6: linearity data of the Atorvastatin and Fenofibrate

Table 7: Limit of Detection and Limit of Quantification

Parameter	Atorvastatin	Fenofibrate
Limit of detection(LOD)	0.00013µg/mL	0.00210µg/mL
Limit of Quantification(LOQ)	0.00046µg/mL	0.0070µg/mL

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