A simple and validated RP-HPLC method for the simultaneous determination of Ezetimibe and Fenofibrate in bulk and pharmaceutical dosage forms

Yamini Mangalagiri, Siva Sreelakshmi Mamidala, Sathish Kumar Konidala*

Department of Pharmaceutical Analysis and Quality Assurance,

Aditya Institute of Pharmaceutical Sciences and Research, Surampalem, Kakinada, EG, AP, India - 533 437

*Corresponding Author E-mail: sathishkonidala@gmail.com

ABSTRACT:

A reverse phase high performance liquid chromatographic method was developed for the simultaneous determination of Ezetimibe and Fenofibrate in bulk and pharmaceutical dosage forms. The determination was performed by using Waters Symmetry Inertsil ODS (250×4.6mm×5µ) as stationary phase and Methanol: Acetonitrile: Water in the ratio of (80:10: 10 %v/v/v) as mobile phase. The flow rate of mobile phase was optimized as 1mL/min and effluents were monitored at 251nm. The retention time of Ezetimibe and Fenofibrate were found as 3.23min and 6.48min respectively. The method shows linearity in the concentration range of 6-14 μg/mL and 87-203 μg/mL respectively. The developed method was validated for specificity, precision, linearity, accuracy, robustness, Ruggedness, LOD and LOQ. Recovery of Ezetimibe and Fenofibrate in formulations was found to be in the range of 98.07-101.94% and 98.45-101.40% respectively conforms the non-interferences of the excepients in the formulation. Due to its simplicity, rapidness and high precision, the proposed RP- HPLC method can be used for the simultaneous determination of these two drugs in Quality control department for regular analysis. .

KEYWORDS: RP-HPLC, Ezetimibe and Fenofibrate.

INTRODUCTION:

Ezetimibe¹ belongs to a new class of an anti-hyper lipidemic medication which is used to lower cholesterol levels. Ezetimibe localizes and appears to act at the brush border of the small intestine and inhibits the absorption of cholesterol. This leads to a decrease in the delivery of intestinal cholesterol to the liver. Ezetimibe is chemically known as (3R, 4S)-1-(4-fluoro phenyl)-3-[(3S)-3-(4-fluoro phenyl)-3-hydroxy propyl]-4-(4-hydroxy phenyl)azetidin-2-one. The Molecular Formula is C₂₄H₂₁F₂NO₃ and Molecular weight is 409.42.

Fig. 1: Structure of Ezetimibe

FENOFIBRATE:

Fenofibrate² is a anti-lipemic agent which reduces both cholesterol and triglycerides in the blood. Fenofibrate exerts its therapeutic effects through activation of peroxisome proliferator activated receptor a (PPARa). This increases lipolysis and elimination of triglyceride-rich particles from plasma by activating lipoprotein lipase and reducing production of a poprotein C-III. The resulting fall in triglycerides produces an alteration in the size and composition of LDL from small, dense particles, to large buoyant particles. These larger particles have a greater affinity for cholesterol receptors and are catabolized rapidly. Fenofibrate is chemically known as propan-2-yl 2-{4-[(4-chlorophenyl) carbonyl] phenoxy}-2-methylpropanoate. The Molecular Formula is C₂₀H₂₁ClO₄and Molecular weight is 360.83.

Fig.2: Structure of Fenofibrate

From the literature survey, we found that Ezetimibe and Fenofibrate were estimated by different analytical methods like RP-HPLC i.e. A simple, precise, rapid and accurate reversed-phase liquid chromatographic method has been developed for the simultaneous estimation of Simvastatin, Ezetimibe and Fenofibrate in their ternary mixture form³, A simple, rapid and precise reversed-phase liquid chromatographic method is developed for simultaneous determination of Atorvastatin, Ezetimibe and Fenofibrate in their ternary mixture of commercial pharmaceutical preparations⁴, A simple, specific and accurate reverse phase liquid chromatographic method was developed for the estimation of Rosuvastatin Calcium (ROS) and Fenofibrate (FEN) in combination⁵, A simple, precise, accurate and rapid high-performance thin-layer chromatographic method has been developed and validated for the estimation of atorvastatin calcium and ezetimibe simultaneously in combined dosage forms⁶, A simple, selective, robust and sensitive reversed phase high performance liquid chromatography method has been developed and validated for the simultaneous estimation of atorvastatin and ezetimibe in bulk drug and pharmaceutical formulations⁷, high-performance liquid chromatography (HPLC) method for the simultaneous determination of atorvastatin calcium, ezetimibe and fenofibrate in a tablet formulation⁸ and by spectrophotometry i.e. A simple, accurate, sensitive, selective, precise and robust spectrophotometric method of analysis was suggested for determination of antilipidemic drugs; Fenofibrate and Ezetimibe⁹.The availability of an HPLC method for simultaneous estimation of above mention cardiovascular drugs will be very much useful for the determination in bulk and pharmaceutical formulations. This study aimed to develop a simple, precise, accurate and validated Reversed-Phase HPLC method for the simultaneous estimation of Amlodipine Besylate and Telmisartan in bulk and pharmaceutical dosage form as per ICH guidelines^10-11. The statistical analysis proved that method is reproducible and selective for the simultaneous analysis of Amlodipine Besylate and Telmisartan in bulk and formulations.

MATERIALS AND METHODS:

A Waters Shimadzu (LC 20 AT VP), connected with Prominence UV-Vis detector and Spinchrom Software, pH meter (Global digital) and Electronic balance (Shimadzu) were used for method development.

Standard drugs of Ezetimibe and Fenofibrate were purchad from market. Acetonitrile (HPLC grade) and methanol (HPLC grade) were procured from E. Merck. Di Potassium mono hydrogen phosphate analytical reagent grade was supplied by Fischer Scientific Chemicals. Water HPLC grade was obtained from in-house Milli-QR water purification system.

Ezetimibe and Fenofibrate tablets are available in the market as Satnlip EZ (contains Fenofibrate 145 mg and Ezetimibe 10 mg). The samples were properly checked for their manufacturing license numbers, batch numbers, production, expiry dates and stored properly.

Preparation and Selection of mobile phase:

The preliminary isocratic studies on a reverse phase C₁₈ column with different mobile phases like 0.1M di Potassium mono hydro phosphate and Acetonitrile, Mthanol and Distilled water. After some trials the mobile phase was optimized as follows.

A mixture of 80 volumes of Methanol, 10 volumes of Acetonitrile and 10 volumes of Water was used as mobile phase. The mobile phase was filtered through 0.45µ membrane filter to remove all fine particles and ultra-sonicated for 10min to remove dissolved gases.

Determination of Working Wavelength (λmax):

In simultaneous estimation of two drugs, the responce of anlyte was measured at their Isobestic point by Spectrophotometrically. “Isobestic point is the wavelength where the molar absorptivity is the same for two substances that are interconvertible”. So this Isobestic point or wavelength was determined as 251 nm by scanning the diluted solutions of both the drugs in UV regien againest blank solution .

Preparation of standard solution:

Preparation of standard stock solution of Fenofibrate:

Weigh accurately 10 mg of Fenofibrate (Working standard) in to a 100 ml volumetric flask and dissolved in Methanol and then dilute up to the mark with methanol and prepare 10 µg /ml of solution by diluting 1ml to 10ml with methanol.

Preparation of standard stock solution of Ezetimibe:

Weigh accurately 10 mg of Ezetimibe (Working standard) in to a 100 ml volumetric flask and dissolved in Methanol and then dilute up to the mark with methanol and prepare 10 µg/ml of solution by diluting 1ml to 10ml with methanol.

Preparation of mixed standard solution:

Weigh accurately 145 mg of Fenofibrate and 10 mg of Ezetimibe and transfer to 100ml volumetric flask. Then add sufficient amount of mobile phase to dissolve the content and make up the final volume up to the mark with mobile phase to get resulting solution having concentration of 1.45 mg/ml of Fenofibrate and 0.10 mg/ml of Ezetimibe (Solution-A).

From the Solution-A pipette out required aliquots and dilute with mobile phase to prepare the solution containing the concentration of 145 μg/ml of Fenofibrate and 10μg/ml of Ezetimibe. This solution was used for recording the chromatogram.

Note: Maintain the temperature in during sonication between 15°C to 20°C

Preparation of Sample Solution:

Weigh accurately powder equivalent to 14.5 μg/ml of Fenofibrate and 1mg of Ezetimibe and transfer to 100ml volumetric flask. Then add sufficient amount of mobile phase to dissolve the content by vigorous shaking or subjected to sonication and make up the final volume up to the mark with mobile phase. Then filter the resulting solution through whattman filter paper.

From the above solution pipette out required aliquots and dilute with mobile phase to prepare the solution containing the concentration of 145 μg/ml of Fenofibrate and 10μg/ml of Ezetimibe. This solution was used for recording the chromatogram. The Assay results were shown in Table 1.

Chromatographic Conditions:

The mobile phase methanol: Acn: Water in the ratio of 80: 10: 10 v/v/v was pumped at a flow rate of 1 mL /min through the Inertsil ODS (250×4.6×5µ) column at 35ºC.The mobile phase was degassed prior to use under vacuum by filtration through a 0.45μ membrane filter. Both drugs showed high absorbance values at 251 nm, which was selected as wavelength for further analysis.

VALIDATION:

Present study was conducted to obtain a new, affordable, cost-effective and convenient RP-HPLC method for simultaneous determination of Ezetimibe and Fenofibrate in tablet dosage form. The experiment was carried out according to the official specifications of ICH- 1996 and Global Quality Guidelines-2002.The method was validated for the parameters like system suitability , selectivity, linearity, accuracy, precision, LOD, LOQ, Ruggedness and robustness.

System Suitability:

System suitability study of the method was carried out by six replicate analysis of solution containing 100% target concentration of Ezetimibe and Fenofibrate. Various chromatographic parameters such as retention time, peak area tailing factor, theoretical plates (Tangent) of the column and resolution between the peaks were determined and the method was evaluated by analyzing these parameters. The results were shown in Table 2.

Acceptance criteria:

The % RSD for the peak area responses of Ezetimibe and Fenofibrate peaks from 6 replicate injections of each standard solution should be not more than 2.0%.

The number of theoretical plates (N) for the Ezetimibe and Fenofibrate peaks is not less than 2000.

The Tailing factor (T) for the Ezetimibe and Fenofibrate peak is not more than 2.0.

Linearity:

Linearity of the method was determined by constructing calibration curves. Standard solutions of Ezetimibe and Fenofibrate of different concentrations level (50%, 75%, 100%, 125%, and 150%) were used for this purpose. Each measurement was carried out in six replicates and the peak areas of the chromatograms were plotted against the concentrations to obtain the calibration curves and correlation coefficients were 1.000 for both the drugs which prove that the method is linear. The calibration curves were shown in Fig 6 and 7. The results were shown in table 3.

Acceptance criteria:

The relationship between the concentration (in %) of Ezetimibe and Fenofibrate and area of Ezetimibe and Fenofibrate should be linear in the specified range and the correlation should not be less than 0.99.

Accuracy (Recovery Studies):

To check the degree of accuracy of the method, recovery studies were performed in triplicate by standard addition method at 80%, 100% and 120%. Known amounts of standard Ezetimibe and Fenofibrate were added to pre-analyzed samples and were subjected to the proposed HPLC method. The measured value was obtained by recovery test. Spiked amount of both the drugs were compared against the recovery amount. % Recovery was 99.52% for Ezetimibe and 100.39% for Fenofibrate. The results were shown in Table 4 and 5.

Acceptance criteria:

The % recovery of Ezetimibe and Fenofibrate should lie between 98% and 102%.

Precision:

Precision was evaluated by carrying out six independent sample preparation of a single lot of formulation. The sample solution was prepared in the same manner as described in sample preparation. Percentage relative standard deviation (%RSD) was found to be less than 2% for within a day and day to day variations, which proves that method is precise. The results were shown in Table 6.

Acceptance criteria:

The % Relative standard deviation of Assay preparations of Fenofibrate and Ezetimibe should be not more than 2.0%.

Robustness of Method:

To evaluate the robustness of the developed RP-HPLC method the prepared solution as per the test method was injected at different variable conditions like using different conditions flow rate and wavelength. The results of robustness testing were reported in Table 7.

Ruggedness:

The ruggedness test of analytical assay method is defined as degree of reproducibility of assay results obtained by the successful applications of the assay over time and among multiple laboratories and analyst. The result of ruggedness testing was reported in Table 8.

Acceptance criteria:

The % Relative standard deviation of Assay preparations of Fenofibrate and Ezetimibe should be not more than 2.0%.

Limit of detection and limit of quantification:

The limit of detection (LOD) and limit of quantification (LOQ) of the drug were calculated using the following equations as per International Conference of Harmonization (ICH) guidelines. The results were shown table 9.

LOD = 3.3 X α /S

LOQ = 10 X α /S

Observation:

Test results for Ezetimibe and Fenofibrate of LOD and LOQ were within limits.

RESULTS AND DISCUSSION:

The developed method was validated as per the parameters like System suitability parameters, linearity, precession, accuracy, robustness, ruggedness and the values all above parameters are within the limit so the developed method was validated according to ICH guidelines. The results were shown below tables.

Table 1: Assay Results

Drug	Label Claim (mg/tab)	Amount Recovered*	Percentage Purity (%w/w)	%RSD
Ezetimibe	10	9.95	99.53	0.245
Fenofibrate	145	145.25	100.17	0.645

*Mean of Six readings

Table 2: Results for system suitability of Fenofibrate

Drug		Retention time (min)	Peak area	Theoretical plates	Tailing factor	Resolution
Ezetimibe	Mean	3.2428	405.692	2084.5	1.88	9.70
Ezetimibe	%RSD	0.17	0.88	-	-
Fenofibrate	Mean	6.463	5192.429	4642.33	1.384
Fenofibrate	% RSD	0.39	0.13	-	-

*Mean of Six readings

Observation:

The % RSD for the retention times and peak area of Ezetimibe and Fenofibrate were found to be less than 2%. The plate count and tailing factor results were found to be satisfactory and were found to be within the limit.

Table 3: Linearity of Ezetimibe and Fenofibrate

% Concentration	Area*
% Concentration	Ezetimibe	Fenofibrate
50	213.809	3104.819
75	309.977	4096.687
100	428.237	5123.828
125	526.955	6057.348
150	639.222	7122.666
Slope	999.6	53.39
Intercept	2102	110.2
r²	0.999	0.999

*Mean of three readings

Observation:

The correlation coefficient for linear curve obtained between concentration vs. Area for standard preparations of Ezetimibe and Fenofibrate is 0.999 and 0.999. The relationship between the concentration of Ezetimibe and Fenofibrate and area of Ezetimibe and Fenofibrate was linear in the range examined since all points lie in a straight line and the correlation coefficient was well within limits.

Fig. 6: Linearity graph for Ezetimibe Fig. 7: Linearity graph for Fenofibrate

Table 4: Results of Accuracy for Ezetimibe:

Amount of sample taken (µg/ml)	Amount of standard added (µg/ml)	% of Std. added	Amount recovered (µg/ml)^*	% Amount recovered^*	% RSD
12	10	80	10.19	101,94	0.43 0.28 0.39
12	12	100	11.77	98.08
12	14	120	13.80	98.57

*Mean of three readings

Table 5: Results of Accuracy for Ezetimibe:

Amount of sample taken (µg/ml)	Amount of standard added (µg/ml)	% of Std added	Amount recovered (µg/ml)^*	% amount recovered^*	% RSD
174	145	80	146.95	101,94	0.43 0.28 0.39
174	174	100	171.30	98.08
174	203	120	205.84	98.57

*Mean of three readings

Observation:

The percentage mean recovery of Ezetimibe and Fenofibrate was 99.52% and 100.39% respectively

Table 6: Method precision of Ezetimibe and Fenofibrate

Drug		Retention time (min)	Peak area
Ezetimibe	Mean	3.232	408.12
Ezetimibe	%RSD	0.75	0.16
Fenofibrate	Mean% RSD	6.496	520.43
Fenofibrate	%RSD	0.25	0.72

*Mean of Six readings

Table 7: Results of Robustness

Robustness		Ezetimibe	Fenofibrate
% RSD (Rt)		0.37	0.28
Area*	Change in Flow rate	405.27	5193.67
Area*	Change Wavelength	400.89	5189.54

* Mean of three readings

Observation:

The % RSD peak area was found to be within limit. So the method was robust.

Table 8: Ruggedness data of Ezetimibe and Fenofibrate

Ruggedness		Ezetimibe	Fenofibrate
% RSD (Rt)		0.05	0.24
Assay*	Analyst-1	97.89	99.16
Assay*	Analyst-2	99.83	99.66

* Mean of three readings

Observation:

Test results for Ezetimibe and Fenofibrate are showing that the %RSD of Assay results were within limits.

Table 9: Results of LOD and LOQ

Parameter	Ezetimibe	Fenofibrate
LOD	0.22	0.636
LOQ	0.66	1.92

CONCLUSION:

The new Reverse phase HPLC method developed and validated for simultaneous determination of Ezetimibe and Fenofibrate in pharmaceutical dosage forms assured the satisfactory precision and accuracy and also determining lower concentration of each drug in its dosage forms. This method was doing in simple manor but founded rapidly accurate values. So, this method will be use full for quality control department, formulation and also stability determination of selected drugs.

REFERENCES:

1. http://www.drugbank.ca/drugs/DB00973 retrived on April 15 for Ezetimibe.

2. http://www.drugbank.ca/drugs/DB01039 retrived on April 15 for Fenofibrate

3. M.S. Kondawar, K.G. Kamble, et al.. UV Spectrophotometric estimation of Ezetimibe and Fenofibrate in Bulk drug and Dosage form using Simultaneous Equation Method. Int. J. Chem.Tech. Res. 3 (2); 2011: 749-754.

4. Deepak Kumar Jain, Pratibha Patel, et al. Development and Validation of RP-HPLC Method for Simultaneous Estimation of Simvastatin, Ezetimibe and Fenofibrate in Ternary Mixtures. International Journal of Pure and Applied Chemistry. 7 (2); 2012:116-125

5. Vishnu P. Choudhari and Anna Pratima Nikalje. Simultaneous Estimation of Atorvastatin, Ezetimibe and Fenofibrate in Pharmaceutical Formulation by RP-LC-PDA. Pharm Anal Acta. 1; 2010: 111.

6. Jajam Thriveni, R. Rambabu, et al. Development and Validation of RP-HPLC Method for Simultaneous Estimation of Rosuvastatin Calcium and Fenofibrate in Bulk and Pharmaceutical Dosage Forms. International Journal of Research in Pharmacy and Chemistry. 3 (2); 2013: 29-32

7. BG Chaudhari, NM Patel, et al. Development and validation of a HPTLC method for the simultaneous estimation of atorvastatin calcium and ezetimibe. Indian J. Pharma. Sci. 68; 2006: 793-796.

8. Ganesh Mani, Hemalatha Pushparaj, et al. Simultaneous Estimation of Atorvastatin and Ezetimibe in Combined Formulation by RP-HPLC. Asian Journal of Chemistry. 24 (4); 2012: 1867-1871.

9. Patel Archita, Macwana Chhaya, et al. Simultaneous Determination of Atorvastatin Calcium, Ezetimibe, and Fenofibrate in a Tablet Formulation by HPLC. Journal of AOAC International. 95 (2); 2012: 419-426.

10. ICH-Q2B, Validation of Analytical Procedures: Methodology. ICH Harmonized Tripartite Guideline, Geneva, 1996: pp. 1-8.

11. ICH Guidelines, Q2 (R1) Validation of Analytical Procedures Text and Methodology, 2005; pp. 1-6.

Received on 16.04.2015 Accepted on 25.05.2015

Asian J. Pharm. Ana. 5(2): April-June 2015; Page 93-99

DOI: 10.5958/2231-5675.2015.00015.0