A Validated Reversed-Phase HPLC Analytical Method for the Analysis of Fenofibrate in Bulk Drug and Tablet Dosage Formulation

Aims: A accurate, precise, and stability-indicating Reversed-Phase HPLC technique has been established for the estimation of fenofibrate in tablet formulation. The chromatographic separation was attained on RP Princeton column (C18) (250 mm x 4.6 mm, 5 µ) with mobile solvent system as a mixture of water (pH 3.0 along o-phosphoric acid) and acetonitrile in the proportion (40:60) v/v, flow rate 1.0 ml per minute, at 240 nm. The retention time of fenofibrate was 3.905 minutes. Results: The method demonstrated linearity in the concentration range of 87-232 µg/ml with a coefficient of correlation (r 2 ) of 0.9994. The % RSD was ˂2% and percentage recovery was found to be 99.13-100.74%. The assay of marketed tablet formulations was found to be 99.98%. Conclusion: The developed and validated technique as per ICH rules for specificity, accuracy, precision, linearity, and system suitability. Reverse Phase-HPLC technique was utilized to the market formulation.


Chemicals and Reagents
Analytical grade Fenofibrate were procured from pharmaceutical company Cadila Pharmaceuticals Limited., Ahmedabad, Zyrova F-10 a tablet marketed formulation.
Acetonitrile, methanol, o-phosphoric acid of analytical grade was used.

Instrumentation
Shimadzu HPLC system and PDA detector with Lab Solution software were used.

Optimized Chromatographic Conditions
Chromatographic isolation was attained on a RP column Princeton C-18 (5 μ, 250 mm × 4.6 mm) at ambient temperature using a mobile phase containing of a mixture of buffer (3.0 pH, with ophosphoric acid) & ACN in the proportion of (40:60) v/v, flow rate 1.0 ml per minute, at 240 nm. The pH of the solvent system at 3.0, Injection volume 10 μl (Table 1). A typical chromatograph of a mixture of standard and sample fenofibrate is summarized by Fig. 2 and Fig. 3 respectively.

Preparation of Sample Solution of Fenofibrate
20 tablets were weighed and finely powdered and a precisely weighed quantity of powder equal to 14.5 mg of fenofibrate was conveyed into a 10.0milliliter volumetric flask. The powder was added with 5-milliliter of methanol. The resulting solution was dilute up to the level with diluent and filtered over filter paper (Whatman Grade-I). One milliliter filtrate was conveyed into a 10-milliliter flask and the quantity was made up to the level with diluent to provide a sample solution consist of 14.5 μg per milliliter of fenofibrate. Six replicate tablet solutions consist of 14.5 μg per milliliter of fenofibrate solutions were prepared similarly.

Assay Procedure
After equilibration of stationary phase, 3 replicate injections of each of sample solutions were made solely and chromatograms were reported. By using peak zone of a quantity of drug existing in the average weight of tablet as percent labeled claim was calculated by using formula given below.

Specificity
The developed technique was specified by correlating the chromatograph of the standard and sample solution ( Table 2).

LOD and LOQ
The limits of detection are the lowermost sample concentration that can be noticed and limits of quantification is the lowest analyte concentration, evaluated along adequate accuracy and precision. LOQ and LOD were settled, under ICH guidelines, by the purpose of the equations Limit of Detection = 3.3σ/S and Limit of Quantification = 10σ/S, where σ is the standard deviation of the regression line, and S is the slope of the calibration plot.

Linearity
Linearity test solutions of fenofibrate were arranged by diluting the stock solution at concentration levels of 116-174 µg/ml. Linearity was settled by the least-squares linear regression analysis obtained. Peak areas versus linear regression analysis and corresponding concentrations were achieved on the resulting curves. The linear curve of fenofibrate was shown in Fig.4.

Precision
The measurement of the precision an area of 6 qualified working standards for fenofibrate calculating the % RSD. The assay technique precision was estimated by operating six independent assays of test samples of fenofibrate across qualified working standards and considering the %RSD. The intermediate precision of the technique was also proved using different analysts and different days.

Accuracy
The analytical accuracy operation suggests the adjacency of covenant midway the value, which is confirmed either as an ideal correct value or a received mention value. It was computed at 3 different levels (80%, 100%, and 120%) of the label claim (Table 4).

Robustness
To specify the robustness study of the validated chromatographic technique, the chromatographic conditions were consciously variation and the resolution for fenofibrate was estimated. To survey the outcome of wavelength on the assessment, and the wavelength variation by ± 2 nm, i.e., 238 and 242 nm from the original wavelength, 240 nm. To survey the outcome of flow rate on the assessment, the flow rate was varied by ± 0.1 millimeters per minute i.e., 0.9 and 1.1 millimeters per minute from the certain flow rate, 1.0 millimeters per minute (Table 5).

Stability of Solution
The stability of the sample was noticed for 24 hours. % Relative standard deviation of 0.9 indicates the stability of the technique for 24 hours. Thus, the technique was found to be specific

High Performance Liquid Chromatography Development and Optimization
Initially, pure drugs solution was chromatographed using a solvent system containing a combination of buffer (3.0 pH, with o-phosphoric acid) & acetonitrile in the proportion of (40:60) v/v, flow rate 1.0 ml/min gives wellresolved peaks of drugs, at 240 nm. The retention time 3.905 minutes.

Limit of Quantification and Limit of Detection
The Limit of Quantification and Limit of Detection of fenofibrate were 70.08 and 23.12 respectively.

Linearity
Linearity by the least-squares linear regression analysis of the calibration data.

Precision
The results of interday precision and intraday precision were 0.8 and 0.4 for fenofibrate. The % RSD of method, system, and intermediate precision results within ±2.0%, indicate that the technique was precise.

Accuracy
The percentage of recoveries was 100.15± 0.5016% for fenofibrate.

Robustness
To estimate the robustness of the developed technique, the chromatographic conditions were deliberately altered, and determined. To study the effect of wavelength flow rate on the estimation (Table 6).

Analysis of Fenofibrate from marketed tablets
Percentage analysis of the marketed formulation was erect to be 99.98 for fenofibrate.
Hence, an attempt was made to develop RP-HPLC which is short runtime and high economical with a less proportion of organic phase, specific, accurate, precise, and, economical technique for the determination of fenofibrate in combined tablet and bulk dosage form

CONCLUSION
The technique enables simple, rapid, accurate, precise, specific, economical, and sensitive estimation of fenofibrate in combined tablet and bulk dosage formulations. The technique therefore, utilized for regular estimation of fenofibrate in tablet and bulk dosage form.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.