Stability Indicating Liquid Chromatographic Method for the Determination of Fenofibrate and its Application to Kinetic Studies

Transcription

1 1866 Int J Pharm Sci Nanotech Vol 5; Issue 4 January March 2013 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 5 Issue 4 January March 2013 Research Paper MS ID: IJPSN RAO Stability Indicating Liquid Chromatographic Method for the Determination of Fenofibrate and its Application to Kinetic Studies K.S. Rao a *, N.K. Keshar a, N. Jena a, M.E.B. Rao a and A.K. Patnaik b a Roland Institute of Pharmaceutical Sciences, Berhampur , Orissa, India; and b Khallikote College, Berhampur , Odisha, India. Received May 4, 2012; accepted July 25, 2012 ABSTRACT A stability-indicating LC assay method was developed for the quantitative determination of fenofibrate (FFB) in pharmaceutical dosage form in the presence of its degradation products and kinetic determinations were evaluated in acidic, alkaline and peroxide degradation conditions. Chromatographic separation was achieved by use of Zorbax C18 column ( mm, 5 μm). The mobile phase was established by mixing phosphate buffer (ph adjusted 3 with phosphoric acid) and acetonitrile (:70 v/v). FFB degraded in acidic, alkaline and hydrogen peroxide conditions, while it was more stable in thermal and photolytic conditions. The described method was linear over a range of μg/ml for determination of FFB (r= ). The precision was demonstrated by relative standard deviation (RSD) of intra-day (RSD= ) and inter-day studies (RSD= 1.47). The mean recovery was found to be %. The acid and alkaline degradations of FFB in 1M HCl and 1M NaOH solutions showed an apparent zero-order kinetics with rate constants and min 1 respectively and the peroxide degradation with 5% H 2 O 2 demonstrated an apparent first-order kinetics with rate constant k = per min. The t 1/2, t 90 values are also determined for all the kinetic studies. The developed method was found to be simple, specific, robust, linear, precise, and accurate for the determination of FFB in pharmaceutical formulations. KEYWORDS: Fenofibrate; stability-indicating assay; stress study; kinetics of degradation. Introduction Fenofibrate (FFB) is 2-[4-(4-chlorobenzoyl)phenoxy]- 2-methyl-propanoic acid, 1-methylethyl ester. It is indicated for the treatment of hypercholesterolemia and mixed dyslipidemia (Jain et al., 2008; Maryadele, 2001 ). Literature reveals that UPLC determination of fenofibrate (FFB) (Kadav and Vora, 2008), square-wave voltammetric determination of fenofibrate in pharmaceutical formulations and biological fluids (Ceren and Nuran, 2004; Korany et al., 2008), specctrophotometric methods (Dhabale and Gharge, 2010; Vipul et al., 2007), simultaneous densitometric TLC analysis (Jain et al., 2008; Masnatta et al., 1996), HPLC methods for the estimation of FFB in combination with other drugs (Shirkhedkar and Surana, 2009; Komsta and Misztal, 2005) has been reported. El-Gindy and colleagues (El-Gindy et al., 2005) have reported spectrophotometric and liquid chromatographic determination of fenofibrate and vinpocetine and their hydrolysis products while Lacroix and colleagues (Lacroix et al., 1998) described a HPLC method for fenofibrate assay and purity verification. Another method has been reported by Gupta and colleagues (Gupta et al., 2010) dealing with validated spectrophotometric determination of fenofibrate in formulation. The main objective of this study was to develop and validate a simple stability-indicating LC method for the determination of FFB in tablet dosage form and to determine the kinetics of degradation describing the concentration changes of FFB in acid, alkaline, and peroxide degradations. Materials and Methods Drugs and Chemicals Fenofibrate hydrochloride reference standard (91.1% of FFB free base) was obtained from Dr Reddy labs (Hyderabad, India). Fenofibrate tablets (Fenolip 160 mg) were purchased from the market. The excipients contained in the pharmaceutical dosage form (hypromellose, hydroxypropyl methylcellulose acetate succinate, sucrose, talc, titanium dioxide, and triethyl citrate) were all of pharmaceutical grades and acquired from different distributors. Acetonitrile LC grade was purchased from Tedia (Fairfield, CA, USA), and o- 1866

2 Rao et al: Stability Indicating Liquid Chromatographic Method for the Determination of Fenofibrate 1867 phosphoric acid and potassium dihydrogen phosphate were obtained from Merck (Germany). Apparatus and chromatographic conditions The LC system, used for method development, forced degradation studies and kinetics studies was on a waters separation module 2695, with an auto injector, and waters 2696 PDA detector. The output signal was monitored and integrated using Empower software. Zorbax Eclipse XDB-C mm X 4.6 mm, 5 µm column was used. The mobile phase was prepared by mixing a degassed mixture of 1 liter of 50 mm Phosphate buffer adjusted the ph to 3 with o-phosphoric acid (% v/v) and acetonitrile (70% v/v). Separation was achieved by using Zorbax XDB C18 column (250 x 4.0 mm, 5 μm) and the flow rate was 1.0 ml/min. The HPLC column was maintained at 25 ºC and the detector was set at a wavelength of 286 nm. The injection volume was 20 μl. Preparation of standard and sample solutions Stock standard solution of FFB (100 μg/ml) was prepared in acetonitrile. An aliquot of this solution was diluted in the mobile phase to obtain the final concentration of 10 μg/ml of FFB. To prepare a sample solution, twenty weighed tablets of Fenolip (160 mg of FFB) were ground and an amount of powder equivalent to 10 mg of active compound was diluted with acetonitrile. The sample solution was filtered and the appropriate aliquot was diluted in the mobile phase to obtain a final solution containing 10 μg/ml of FFB. Method validation The validation procedure for the analysis of FFB by LC method followed the International Conference on Harmonization (ICH) guideline and United States Pharmacopoeia (ICH, 2005; ICH, 2003). The performance parameters evaluated in this method were specificity, robustness, linearity, limit of detection (LOD), limit of quantitation (LOQ), precision and accuracy. Specificity and forced degradation studies The specificity of the LC method was evaluated to ensure that there was no interference from the excipients contained in pharmaceutical product or from products resulting from forced degradation. A stability-indicating method is the one that accurately quantifies the active ingredients without interference from degradation products, process impurities, excipients, or other potential impurities (Alsante KM et al., 2007; Bakshi M and Singh S, 2002). This greatly contributes to the possibility of improving drug safety (Gorog S, 2008). Solutions containing 1 mg/ml of the drug were prepared in acetonitrile for the stress degradation studies. An appropriate aliquot was transferred into a volumetric flask and the volume was completed with 0.75 M NaOH, 1 M NaOH, 1.5 M NaOH, 0.75 M HCl, 1 M HCl, 1.5M HCl, 3% H2O2, 5% H2O2 and 10% H2O2 to give a final concentration of 100 μg/ml of FFB. These solutions were subjected to heating at 70 ºC. The hydrolytic study was carried out in 0.75 M NaOH for 1 h, 1 M NaOH for 45min, 1.5 M NaOH for min, 0.75 M HCl for 1 h, 1 M HCl for 45min, 1.5M HCl for min. The oxidative reaction was performed with 3% H2O2 for 1 h, 5% H2O2 for min and 10% H2O2 for 15 min at room temperature. For LC analyses, 1.0ml aliquots of the above solutions were transferred to 10ml volumetric flasks, neutralized as needed, and each sample diluted to the mark with mobile phase. The stress degradation study under UV radiation was performed by exposing the FFB solution in acetonitrile (1 mg/ml) for min and 1 h at room temperature, resulting in an overall illumination of 210 Wh/m 2 with UV radiation at nm in a photostability chamber (1.0 x 0.17 x 0.17 m) with mirrors and equipped with UV lamps and UV cuvettes, were used as a container for samples. Samples submitted to identical conditions, but protected from light, were used as a control. Peak purity test performed by photodiode array detector (PDA) was useful to show that the analyzed chromatography peak did not contain more than one substance. Similarly the samples for thermal studies are exposed to a controlledtemperature oven at 80 o C for min and 1 h. Robustness Chromatographic parameters (peak retention time, theoretical plates, tailing factor, retention factor, and repeatability) were evaluated using both samples and reference substance solutions (10 μg/ml) changing wavelength (284 and 288 nm), column temperature (23 and 27 ºC), flow rate (0.8 and 1.2 ml/min), acetonitrile concentration (68 and 72%), and buffer ph (2.8 and 3.2). Linearity Linearity was established by least squares linear regression analysis of the calibration curve. The constructed calibration curves (n=3) were linear over the concentration range of μg/ml. Peak areas of FFB was plotted against their respective concentrations and linear regression analysis was performed on the resultant curve. LOD and LOQ LOD and LOQ were determined by reducing the concentration of a standard solution until the FFB peak response was approximately three or ten times, greater than the noise, respectively. Precision The precision of the reported method for the determination of FFB was studied using the parameters repeatability (intra-day) and intermediate precision (inter-day). It was expressed as relative standard deviation (RSD) at series of measurements. The repeatability of this method was verified on the same day, at the same concentration and under the same experimental conditions for each of the samples evaluated. The intermediate precision, which is the interday variation at the same concentration level, was

3 1868 Int J Pharm Sci Nanotech Vol 5; Issue 4 January March 2013 determined on three consecutive days. In order to evaluate the precision of the method, six solutions were prepared (10 μg ml -1 ) and relative standard deviation was determined Accuracy The accuracy of the method was determined through the recovery test of the samples, using known amounts of FFB reference standard. For LC method, aliquots of 0.8, 1.0 and 1.2 ml of a FFB standard solution (100 μg/ml) were added to three sample solutions containing a fixed amount of FFB (100 μg) in mobile phase, respectively. Therefore, this recovery study was performed at a final concentration solution of 80, 100 and 120 μg/ml FFB. All solutions were prepared in triplicate and analyzed. System suitability test System suitability tests were performed to ensure that the LC system and procedure are capable of providing quality data based on USP 31 requirements (USP, 2008). The system suitability parameters include FFB retention time, tailing factor and number of theoretical plates, as well as the peak area relativestandard deviation (RSD, n= 6) of reference standard. Kinetic determinations The kinetics of acid degradation of FFB was evaluated in 1M HCl at 70 ºC for different times. Tablets of Fenolip 160mg were ground and an amount of powder equivalent to 25 mg of FFB was dissolved in 100 ml acetonitrile. The solution was filtered and an aliquot was transferred into 50 ml volumetric flask and diluted with 1M HCl to 100 μg ml -1 FFB. This solution was placed at 70 ºC and evaluated for time intervals of 0, 9, 18, 27, 36 and 45 min. Three samples were analyzed for each time interval. After the required time, aliquots of 1 ml were transferred to a 10 ml volumetric flask then neutralized with 1 ml 1 M NaOH. This solution was diluted with mobile phase to 10 μg ml -1 for the LC determinations. Similarly, kinetics of alkaline degradation was also studied on 100 μg ml -1 FFB solution with 1M NaOH placed at 70 ºC and neutralized with 1M HCl, followed by evaluation at different time intervals of 0, 9, 18, 27, 36 and 45 min. Stock solutions (100 μg/ml) of FFB tablets in acetonitrile were prepared. The kinetics of peroxide degradation was performed by exposing the solutions to 5% H2O2 for min at room temperature. The evaluation was carried out at 0, 5, 10, 15, 20, 25 and min. After the required time, aliquots of 1 ml were transferred to a 10 ml volumetric flask and diluted with mobile phase to 10 μg ml -1 for the LC determinations. Three samples were analyzed for each time interval. The concentrations of the remaining FFB determined at the different time intervals in kinetics determination were used in the plots. The plots were (a) values of concentration against time (zero-order kinetics), (b) ln of concentration versus time (first-order kinetics), and (c) reciprocal of concentration versus time (second-order kinetics). The kinetic parameters such as apparent order degradation rate constant (k), half-life (t1/2) and t90 were obtained. Results and Discussion Development of LC Method Regulatory agencies recommend the use of stabilityindicating methods (FDA, CDER and CBER, 2000) for the analysis of stability samples. Thus, stress studies are required in order to generate the stressed samples, method development and method validation (Swartz M and Krull I, 2005). In order to separate FFB and degradation products produced under stressed conditions, different mobile phases were used and adjusted to obtain a rapid and simple assay method with a reasonable run time, suitable retention time and the sharpness of the peak. Distinct proportions of organic solvent (methanol and acetonitrile) were evaluated and the acetonitrile was chosen because it improved the retention time and symmetry of FFB peak. Buffer ph played a major role in separating all the degradation products of FFB. When the ph of the buffer was adjusted to 7.0 with phosphoric acid solution, the tailing factor of the FFB peak was 1.9 and the retention time of FFB was around 22 min. Besides, some of its degradation products were co-eluted. When ph increased towards acidic side (ph 3.0), the symmetry of the FFB peak and retention time were improved. The resolution among all its degradation products was also improved. Thus, the mobile phase was established by mixing aqueous 50 mm phosphate buffer (ph 3.0) and acetonitrile (:70, v/v). The literature has demonstrated a stability-indicating LC method for determination of FFB using the complex mixture of aqueous 0.1% trifluoroacetic acid, methanol, tetrahydrofuran (60:20:20, v/v/v) as mobile phase. However, the mobile phase established in our study is very simple. Method Validation Specificity and forced degradation studies Forced degradation or stress testing is undertaken to demonstrate specificity when developing stabilityindicating methods, particularly when little information is available about potential degradation products. The ICH guideline entitled Stability Testing of New Drug Substances and Products requires the stress testing to be carried out to elucidate the inherent stability characteristics of the active substances. Stress testing of the drug substance can help identify the likely degradation products, which can in turn help establish the degradation pathways and the intrinsic stability of the molecule and validate the stability-indicating power of the analytical procedures used. The use of an ideal stability-indicating method quantifies the drug per se and also resolves its degradation products. Stress degradation studies on FFB revealed the drug behavior, as summarized in chromatograms (Fig. 1-5).

4 Rao et al: Stability Indicating Liquid Chromatographic Method for the Determination of Fenofibrate 1869 Upon heating the drug in HCl and NaOH (0.75M, 1M, 1.5M) for different times (1h, 45min, min) at 70 C, fall in the original drug peak areas were observed and one additional peak was observed in all the chromatograms (Fig-1-2). Photolytic degradation and thermal degradation resulted in slight decrease of the peak area and did not produce any detectable eluting degradation product (Fig-3-4). FFB was rapidly degraded in acidic and alkaline media and the observed degradation products were shown in chromatograms. The increase concentration of acid and alkaline raised the formation of main degradation product and the area of FFB decreased substantially. Fig.1. Representative chromatogram of FFB in acid degradation. Fig. 2. Representative chromatogram of FFB in alkaline degradaion. Fig. 3. Representative chromatogram of FFB under UV light.

5 1870 Int J Pharm Sci Nanotech Vol 5; Issue 4 January March 2013 Fig.4. Representative chromatogram of FFB under thermal degradation. Fig. 5. Representative chromatogram of FFB in peroxide degradation. The FFB solution was exposed to chemical oxidation with 3% H2O2 for 1h and 5% H2O2 for min and 10% H2O2 for 15 min, the oxidative hydrolysis with H2O2 exhibited a significant decrease of peak area and only one eluting peak was observed in all the chromatograms (Fig.5). The results of the stress conditions are presented in table 1. Thus, our study demonstrated that FFB was degraded in acidic, alkaline media and in the presence of hydrogen peroxide too. Nevertheless, it was observed FFB degradation only under acid hydrolysis, and determined that under other stress conditions this drug was found to be stable. The excipients of Fenolip tablets did not cause interference in the FFB analysis, which indicated the specificity of the method. The peak purity was determined with a PDA detector under the optimized chromatographic conditions; the peak purity of FFB was satisfactory in all stressed samples tested. Assay studies were carried out for stress samples against qualified reference standard and the mass balance (% assay + % impurities + % degradation products) was calculated. This demonstrated that it was pure in all cases indicating that no additional peaks were co-eluting with the FFB and evidencing the ability of the method to assess unequivocally the drug of interest in the presence of potential interferences. In order to consider an assay method specific, it should demonstrate that it could separate and quantify the drug from a physical mixture of the drug, degradation products and excipients. Robustness The robustness of the method was examined by small variations of critical parameters, and percent of FFB, retention time (Rt), number of theoretical plates (N), tailing factor (T), and retention factor (k) were evaluated (Table 2). The robustness study has been proved that in every employed condition, the chromatographic parameters agreed with established values and the assay data remained acceptable. A tailing factor of 1 refers to a symmetric peak. The calculated values for the tailing factor for each chromatographic condition were in the acceptable range of 0.8 T 1.5. The retention factor should be in the range of 0.5 < k < 10. The k values obtained around 2.0 indicating optimum separation and the number of theoretical plates demonstrated the measure the column efficiency in different conditions. Flow rate (0.8 and 1.2 ml min -1 ) and percent of acetonitrile (68 and 72%) resulted in changes in the retention time in comparison with the proposed normal condition. However, no significant changes were observed regarding quantification of FFB.

6 Rao et al: Stability Indicating Liquid Chromatographic Method for the Determination of Fenofibrate 1871 TABLE 1 Summary of forced degradation results. Stress condition Acid Hydrolysis (70 o C) 0.75M HCl 1M HCl 1.5M HCl Base Hydrolysis (70 o C) 0.75M NaOH 1M NaOH 1.5M NaOH Oxidation (Room temp) 3% H2O2 5% H2O2 10% H2O2 Time (Min) Thermal (80 o C) 60 Light (Photolytic degradation) at Room temp 60 % Assay of active substance Mass balance (% assay+% impurities+% degradation products) Remarks One major degradation product was formed in all the conditions One major degradation product was formed in all the conditions One major degradation product was formed in all the conditions No degradation product was formed in both the conditions No degradation product was formed in both the conditions TABLE 2 Robustness experiments of LC method for determination of FFB. Chromatographic parameter Condition FFB (%) Rt a FFB (min) N b T c k d Wavelength (nm) , , Temperature ( ºC) , , Flow rate (ml min -1 ) , , Acetonitrile (%) , , ph of buffer solution , , Normal e , art: retention time bn: number of theoretical plates ct: tailing factor dk: retention factor enormal condition (mobile phase): Zorbax -18 column ( mm, 5 μm); 50 mm potassium phosphate buffer (ph 3.0) and acetonitrile (:70, v/v), flow rate 1.0 ml min -1 ; UV detection at 286 nm Linearity The standard curves for FFB were constructed and demonstrated to be linear in the concentration range of μg/ml. The representative linear equation was y = 58138x , where x is the concentration (μg/ml) and y is the peak area. The correlation coefficient was r= Linearity data were validated by the analysis of variance (ANOVA), which demonstrated significant linear regression and no significant linearity deviation (p < 0.05). LOD and LOQ The limit of quantitation (LOQ) of the present method was found to be 0.9 µg/ml with a resultant %RSD of 0.41% (n = 5). The limit of detection (LOD) was found to be 0.2 µg/ml. These low values obtained were indicative of the high sensitivity of the method. Precision Precision values obtained for the determination of FFB in samples with their RSD are shown in table 3. The variability of the results for determination of FFB was low. RSD values were lower than 0.91 for intra-day, and values for inter-day were 1.47 for FFB in pharmaceutical dosage form, confirming the good precision of the method. Accuracy Accuracy was evaluated by the simultaneous determination of the analyte in solutions prepared by the standard addition method. Three different concentrations of FFB standard were added to Fenolip 160 mg tablet solution. The mean recovery was found to be 100% (Table 4) and this value showed that the method was accurate.

7 1872 Int J Pharm Sci Nanotech Vol 5; Issue 4 January March 2013 System suitability test The system suitability parameters evaluated, under the experimental conditions, showed a single peak of the drug around min, tailing factor (T= 1.15) and number of theoretical plates (N= 13,000), as well as the peak area relative-standard deviation (RSD= 0.8%, n = 6). Assay The validated method was applied to the determination of FFB in commercially available Fenolip 160mg tablets. Fig-6 and Fig-7 illustrates two typical HPLC chromatograms obtained from FFB standard solution and from the assay of Fenolip tablets respectively. The results of the assay (n = 9) undertaken yielded % (%RSD = 0.39%) of label claim for FFB. The observed concentration of FFB was found to be ±0.640µg/ml (mean±sd). The mean retention time of FFB was min. The results of the assay indicate that the method is selective for the analysis of FFB without interference from the excipients used to formulate and produce these tablets. TABLE 3 Inter and intra-day assay variation of FFB by proposed method. Sample (mg/tablet) Days Experimental amount a (mg) Mean±SD % FFB obtained %RSD Intra-day %RSD Inter-day ± ± ± amean of six samples in triplicate (n=6) ± Standard Deviation TABLE 4 Recovery of standard solution added to commercially available sample. Amount added (μg ml -1 ) Amount found (μg ml -1 ) % Recovery a ± RSD Mean % Recovery ± ± ± 0.26 aeach value is a mean of three determinations. Fig. 6 Representative chromatogram of FFB in reference standard. Fig. 7 Representative chromatogram of FFB in pharmaceutical formulations.

8 Rao et al: Stability Indicating Liquid Chromatographic Method for the Determination of Fenofibrate 1873 Kinetic determinations Most of the degradation reactions of pharmaceuticals occur at finite rates and are chemical in nature. These reactions are affected by conditions such as solvent, concentration of reactants, temperature, ph of the medium, radiation energy, and the presence of catalysts. The order of the reaction is described based on the reaction rate and on the concentration of the reactant. The degradation of most pharmaceuticals can be classified as zero order, first order or pseudo-first order (Ahuja, 2007). Thus, kinetic studies of decomposition of drugs using stability testing techniques are essential for the quality control of such products. In our study, the kinetic investigation of acid, alkaline and peroxide degradation was carried out. The values of concentration, ln of concentration and reciprocal of concentration of the remaining drug versus time for kinetic determination under acidic and alkaline conditions are shown in table 5 and 6 respectively. Through the evaluation of the correlation coefficients, it could be concluded that the acidic and alkaline degradations of FFB in 1M HCl and 1M NaOH solutions showed an apparent zero-order kinetics (r= and respectively) under the experimental conditions applied. The calculated zero-order acid degradation rate constant was k = min 1, t1/2= 68.0 min and t90= 13.6 min and the calculated zero-order alkaline degradation rate constant was k = min 1, t1/2= min and t90= 14.3 min were obtained. The solutions employed in the kinetic determination of the peroxide degradation were found to be around 20.0% of FFB, degraded in 20 min with 5%H2O2. The values of concentration, ln of concentration and reciprocal of concentration of the remaining drug versus time are shown in table 7. The correlation coefficient (r) observed under the experimental conditions applied in peroxide degradation study was It was obtained in the course of this kinetic determination when the ln of concentration of drug remaining versus time was plotted. Therefore, the kinetic model for first-order degradation process was confirmed. The calculated first-order degradation rate constant was carried out and k = min 1, t1/2= 34.3 min and t90= 5.19 min were obtained. TABLE 5 Kinetics of acid degradation in 1M HCl for FFB solution. Time (min) Concentration of FFB (μg ml -1 ) Ln Concentration 1/Concentration R * *Zero-order kinetics (k = min 1, t1/2= 68.0 min and t90= 13.6 min) TABLE 6 Kinetics of alkaline degradation in 1M NaOH for FFB solution. Time (min) Concentration of FFB (μg ml -1 ) Ln Concentration 1/Concentration R * *Zero-order kinetics (k = min 1, t1/2= min and t90= 14.3 min) TABLE 7 Kinetics of peroxide degradation in 5% H2O2 for FFB solution. Time (min) Concentration of FFB (μg ml -1 ) Ln Concentration 1/Concentration r * *First-order kinetics (k = min 1, t1/2= 34.3 min and t90= 5.19 min)

9 1874 Int J Pharm Sci Nanotech Vol 5; Issue 4 January March 2013 Conclusions In this study, we optimized a simple analytical method for quantitative determination of FFB in pharmaceutical dosage form. The developed and validated LC method according to guidelines was simple, specific, linear, precise, accurate, and stabilityindicating. FFB was rapidly degraded in acidic and alkaline medium and in the presence of hydrogen peroxide too, while it was more stable in UV radiation and thermal conditions. The acid and alkaline degradations showed apparent zero-order kinetics and the peroxide degradation demonstrated apparent firstorder kinetics. Kinetic parameters of degradation rate constant, t1/2 and t90 could be predicted. The proposed LC method presented the ability to separate FFB from all its degradation products and therefore can be applied in stability testing of the commercially available FFB tablets. References Ahuja SS (2007). Assuring quality of drugs by monitoring impurities. Adv Drug Del Rev 59: Alsante KM, Ando A, Brown R, Ensing J, Hatajik TD, Kong W and Tsuda Y (2007). The role of degradant profiling in active pharmaceutical ingredients and drug products. Adv Drug Deliv Rev 59: Bakshi M and Singh S (2002). Development of validated stabilityindicating assay methods-critical review. J Pharm Biomed Anal 28: Ceren Y and Nuran O (2004). Electrochemical studies and squarewave voltammetric determination of fenofibrate in pharmaceutical formulations. Anal Bioanal Chem 378: Dhabale PN and Gharge DS (2010). Simultaneous spectrophotometric estimation of atorvastatin and fenofibrate in bulk drug and dosage form by using simultaneous equation method. Int J ChemTech Res 2: El-Gindy A, Emara S, Mesbah MK and Hadad GM (2005). Spectrophotometric and liquid chromatographic determination of fenofibrate and vinpocetine and their hydrolysis products. Farmaco 60: FDA, Center for Drug Evaluation and Research (CDER), Center for biologics evaluation and research (CBER). Guidance for Industry. Analytical Procedures and Methods Validation. Rockville, USA, Gorog S (2008). Industrial and compendial steroid analysis. J Pharm Biomed Anal 48: Gupta KR, Askarkar SS, Rathod PR and Wadodkar SG (2010). Validated spectrophotometric determination of fenofibrate in formulation. Der Pharmacia Sinica 1: Jain N, Raghuwanshi R and Jain D (2008). Development and validation of RP-HPLC method for simultaneous estimation of atorvastatin calcium and fenofibrate in tablet dosage forms. Indian J Pharm Sci 70: Kadav AA and Vora DN (2008). Stability indicating UPLC method for simultaneous determination of atorvastatin, fenofibrate and their degradation products in tablets. J Pharm Biomed Anal 48: Komsta L and Misztal G (2005). Determination of fenofibrate and gemfibrozil in pharmaceuticals by densitometric and videodensitometric thin-layer chromatography. J AOAC Int 88: Korany MA, Hewala II and Abdel-Hay KM (2008). Determination of etofibrate, fenofibrate, and atorvastatin in pharmaceutical preparations and plasma using differential pulse polarographic and square wave voltammetric techniques. J AOAC Int 91: Lacroix PM, Dawson BA, Sears RW, Black DB, Cyr TD and Ethier JC (1998). Fenofibrate raw materials: HPLC methods for assay and purity and an NMR method for purity. J Pharmaceut Biomed Anal 18: Maryadele JO (2001). Merck index. 13th ed. Merck Research Lab, NJ (USA). Masnatta LD, Cuniberti LA, Rey RH and Werba JP (1996). Determination of bezafibrate, ciprofibrate and fenofibric acid in human plasma by high-performance liquid chromatography. J Chrom Biomed Appl 687: Nagaraj, Vipul K and Rajshree M (2007). Simultaneous quantitative resolution of atorvastatin calcium and fenofibrate in pharmaceutical preparation by using derivative ratio spectrophotometry and chemometric calibrations. Anal Sci 23: Shirkhedkar AA and Surana SJ (2009). Simultaneous densitometric TLC analysis of atorvastatin calcium and fenofibrate in the bulk drug and in pharmaceutical formulations. J Planar Chromatog- Mod TLC 22: Swartz M and Krull I (2005). Analytical method validation for biotechnology proteins, peptides, and antibodies. LC-GC 23: The United States Pharmacopeia. Rockville, MD (2008). Topic Q1A (R2) Stability testing of new drug substances and products. International Conference on Harmonization (ICH), Geneva, Switzerland Address correspondence to: K. Srinivasa Rao, Professor, Roland Institute of Pharmaceutical Sciences, Berhampur , Odisha, India. ksrao108@gmail.com