Determination of Fenofibrate and the Degradation Product Using Simultaneous UV-Derivative Spectrometric Method and HPLC

Two new selective, precise, and accurate methods were developed for the determination of fenofibrate in the presence of its basic degradation product. In the first method fenofibrate was determined using an algorithm bivariate calibration derivative method, in which an optimum pair of wavelengths was chosen for the determination of different binary mixtures. In the second method (HPLC), separation was achieved on RESTEK Pinnacle II phenyl column (5 μm, 250 × 4.6 mm) and Pinnacle II phenyl (5 μm, 10 × 4 mm) guard cartridge using a mobile phase consisting of methanol –0.1% phosphoric acid (60:40, v/v) at a flow rate 2 mL·min, and the column oven temperature was set at 50 ̊C. The UV detector was time programmed at 302 nm and 289 nm for the internal standard (I.S.) and fenofibrate, respectively. The proposed methods were successfully applied for the determination of fenofibrate and its degradation product in the laboratory-prepared mixture and in pharmaceutical formulation. The assay results obtained using the bivariate method were statistically compared to those of the HPLC method and good agreement was observed.

Different methods for analysis of fenofibrate have been reviewed.Fenofibrate was assayed in British Pharmacopeia (BP) by a liquid chromatography method [1].However, several chromatographic methods have been
To the best of our knowledge, none of the reported procedures describe stability-indicating method for the determination of fenofibrate using an algorithm bivariate calibration derivative method.For HPLC method; the most considerable difference of the proposed method in comparison to the reported stability indicating HPLC methods [21,22], is the addition of I.S, which reduces the expected analytical errors and improve the accuracy, precision, and robustness.
The present work aims to develop simple, selective, precise and stability-indicating procedures for the analysis of fenofibrate in the presence of its basic degradation product.Adaptation of the proposed procedures to the analysis of the available dosage forms is also an important task in order to solve problems encountered in the quality control and analysis of expired samples.Moreover, accelerated stability experiments to predict expiry dates of pharmaceutical products necessitate such methods.

Materials, Chemicals and Reagents
Fenofibrate was kindly provided by Sigma Pharmaceutical Company, Egypt.Lipolex tablets (labeled to contain 300 mg fenofibrate per tablet) were purchased from the Egyptian market.Organic solvent for chromatography were of HPLC grade.Internal standard (salicylic acid), reagents and chemicals used were of analytical grade and all were purchased from Sigma-Aldrich (Steinheium, Germany).Glass distilled water was further purified using Milli-Q water purification system (Millipore, Bedford, MA, USA).

2.2.1.
Bivariate Method.Individual stock solutions of fenofibrate and fenofibric acid were prepared by dissolving appropriate amounts of ~40 mg in 50 mL methanol.The final volume of each solution was then diluted to 100 mL with methanol.Working solutions 40 µg•mL -1 for both fenofibrate and fenofibric acid were prepared from the above stock solutions in methanol for assay determination.Calibration standards were prepared by diluting the working solutions with methanol.

Liquid Chromatographic Method
Individual stock solutions of fenofibrate and salicylic acid as (I.S.) were prepared by dissolving appropriate amounts of ~50 mg in 50 mL methanol.The final volume of each solution was then diluted to 100 mL with methanol.Working solutions 50 µg•mL -1 for both fenofibrate and fenofibric acid were prepared from the above stock solutions in mobile phase for assay determination.Calibration standards were prepared by diluting the working solutions with the mobile phase and spiked with a constant concentration 10 µg•mL -1 of internal standard.

Degradation of Fenofibrate:
One gram of fenofibrate was dissolved in 25 mL methanol and refluxed with 25 mL of 0.2 M sodium hydroxide at 100˚C for 2 h.During reflux, small portions were cooled and spotted on a TLC plate and then developed using acetone: n-hexane (10:20, v/v) as a developing system.After complete degradation, the solution was allowed to cool, adjusted to pH 6 with 1 M hydrochloric acid using pH-meter, and evaporated to dryness under vacuum.The residue was extracted 3 times, each with 30 mL chloroform. 2 g anhydrous sodium sulphate was added to the chloroformic extract to remove the traces of water and then filtered.The filtrate extract was evaporated to dryness under a vacuum.The dried residue was analyzed by IR, 1 H NMR, and 13 C NMR and found to be 2-[4-(4-chlorobenzoyl) phenoxy]-2-methylpropanoic acid (fenofibric acid).

2.4.2.
Bivariate Method 2 D 2.4.2.1.Linearity.Different aliquots ranging from 0.5 -5 mL of both fenofibrate and fenofibric acid were transferred separately into 10 mL volumetric flasks from their respective working standard solutions (40 µg•mL -1 ) and completed to volume with methanol.The spectra of fenofibrate and its degradation product were recorded between 200 and 400 nm and stored on a computer.The second derivative spectra ( ) for both fenofibrate and its degradation product were obtained at 2 D   = 10 nm and scaling factor equal to 1000.The amplitude of the second derivative peak for both fenofibrate and its degradation product was measured at the optimum wavelengths found by the Kaiser's method (293 and 306 nm).into the HPLC system.The procedure was carried out in triplicate for each concentration.The analyte/I.S. peak area ratios obtained (dimensionless numbers) were plotted against the corresponding concentration of the analyte (expressed as µg•mL -1 ).The detector was time programmed to be set at 302 nm for 3 minutes from the beginning of the run time for detection of I.S. then exchanged to 289 nm for detection of fenofibrate.Chromatographic separation was achieved using RESTEK Pinnacle II phenyl column (5 µm, 250 × 4.6 mm) and Pinnacle II phenyl (5 µm, 10 × 4 mm) guard cartridge using a mobile phase consisting of methanol -0.1% phosphoric acid (60:40, v/v), and the column oven temperature was set at 50˚С.Mobile phase was filtered through a 0.45 µm nylon membrane filter, degassed and pumped at a flow rate 2 mL•min -1 .

Analysis of Laboratory Prepared Mixtures
Containing Different Ratios from Fenofibrate and its Degradation Product.2.4.4.1.

Identification of the Degradation Product
Identification was made by scanning the FTIR spectra on KBr discs and NMR spectra in deutrated chloroform for both fenofibrate and its degradation product.
The FTIR spectrum of pure fenofibrate (Figure 1(a)) shows two absorption peaks at 1728 and 1651 cm -1 which indicates the presence of two carbonyl frequencies of ester and ketone, respectively.The ester peak is confirmed by its characteristic absorption at 1178 and 1246 cm -1 .The appearance of the absorption peaks at 2800 -3400 cm -1 are associated with carbon-hydrogen (C-H) stretching vibrations.On the other hand, the FTIR of degraded fenofibrate (Figure 1(b)) shows broad absorption band at 3000 -2500 cm -1 which indicates hydrogen bonded (O-H) of a carboxylic acid dimer.Peaks at 1664 and 1305 cm -1 are also indicative of this group and peak corresponding to ketone functional group is shifted to 1643 cm -1 .Moreover, there is a complete disappearance of the ester peak at 1728 cm -1 and disappearance of some peaks of (C-H) stretching which indicating the removal of isopropyl moiety.
1 H NMR spectrum of fenofibrate in (Figure 2  Copyright © 2011 SciRes.AJAC For the selection of the two most appropriate wavelengths with respect to their sensitivity for the simultaneous determination of the substances, we applied Kaiser's [28][29][30] method, which consists of resolving the determinant from the so called selectivity matrix, K.   , correspond to the sensitivity parameters of component B, in this case considered as calibration curve slopes for each component at two given wavelengths. 13C NMR spectra of fenofibrate and its degraded products in (Figures 3(a) & 3(b)) show identical carbon peaks except in the degraded product there is a complete disappearance of carbon peaks at δ 21.52 and at 69.34 corresponding to the aliphatic carbon of the two methyl groups of [-O-CH-(CH 3 ) 2 ] and the methine carbon of [-O-CH-(CH 3 ) 2 ], respectively.And this is considered as a further confirmation of the removal of the isopropyl moiety.The degradation pathway is illustrated in Scheme 1.

Bivariate Method
The resolution of two components by the bivariate calibration has been recently proposed [23][24][25][26][27].The concentration of two components A and B in a mixture can be determined according to Lambert-Beer's law, through a system of four calibration curves: that is, using the second two derivative spectra calibration curves for each component at two different wavelengths: C as follows: Equation 1: The "bivariate calibration method" was applied to the second derivative spectrum for the resolution of the binary mixture of fenofibrate and its degradation product (Figure 4(a)).The main advantage of the derivative method is the presence of a large number of maxima and minima, which in turn, provides an opportunity for the determination of active compounds in the presence of other degradation products, which possibly interfere with the analysis.Moreover, in the zero order and first order "bivariate calibration method" a particular case arises when one or both of the analytes present broad or flat bands with no well-defined maximum (Figures 4(b) & 4(c)).In such cases similar consecutive results are expected within the range of wavelengths of the band [31].For these reasons, the spectra for fenofibrate and its degradation product standard solutions were selected.The effect of pH on the absorbance of fenofibrate and its degradation product was studied by using phosphate buffer of different pH, as shown in (Figure 4(d)) neither absorbance nor 2

D
 maxima affected significantly by pH changes.The optimization of the derivative spectra was achieved at   = 10 and scaling factor of 1000.In order to apply the "derivative bivariate calibration method" for the resolution of the binary mixture fenofibrate and its degradation product, the signals of all standard solutions at nine located wavelengths were obtained.The correlation data of their calibration curves are presented in (Table 1).According to Kaiser's method the slope values from these regression lines represent the sensitivity values for each component.The sensitivity value for each wavelength pair was defined (Table 2) by resolving the determinants of the selectivity matrices K proposed by this method.In order to resolve the respective determinants, it is suggested that the value of the slope should be kept (including its sign (±), which is obtained from the calibration curve).It is worth mentioning that, for the model proposed, it is necessary for the calibration curves of the two components to comply with Lambert-Beer's aw at each wavelength, giving a straight line.Otherwise Equation 2:  there will be a great error in determination, as it will not be possible, the contribution of one of the components (to the mixture) to be assessed adequately.In the present investigation, all the calibration curves show a satisfactory linear regression coefficient (>0.999).According to the results, the wavelength pairs with the highest absolute sensitivity values were 293 and 306 nm.By using the correlation data of the above wavelength pairs and the two Equations 1 and 2, the recoveries of synthetic mixtures were calculated (Table 3).

Liquid Chromatographic Method
In order to affect the simultaneous analysis of the two component peaks under isocratic conditions, the mixtures of methanol or acetonitrile with a buffer or 0.1% phosphoric acid in different combinations were assayed as the mobile phase using phenyl packing a stationary phase.A binary mixture of methanol -0.1% phosphoric acid (60: 40, v/v) proved to be better than the mixture of acetone-trile-buffer for the separation since the chromatographic peaks were better defined and resolved, and free from tailing.Among several flow rates tested (0.5 -2.5 mL•min -1 ), the flow rate of 2 mL•min -1 was the best with respect to location and resolution of analytical peaks.
The temperature was examined in the range of 30˚С to 60˚С using methanol (50 -65%, v/v) -0.1% phosphoric acid (50 -35%, v/v) as a mobile phase.A combination of temperature (50˚С) and methanol (60%) gave a good separation for all of the components.Resolution and separation factors for this system were found 34.71 and 4.22, respectively.Tailing factor and the number of theoretical plates were 1.02 and 11603, respectively.The above described chromatographic conditions allow a resolution between I.S. and fenofibrate in a reasonable time of 2.047 and 10.787 min, respectively.The chromatogram of the standard solution containing fenofibrate and the I.S. is reported in (Figure 5(a)).As can be seen, the peaks are neat, symmetric and well separated and the wavelength changes do not distort in any way the baseline appearance.Degradation product obtained with forced the degradation condition is showen in (Figure 5(b)).The chromatogram of the degradation product showing that peaks of fenofibrate and I.S. were free of interference of the degradation product.Effects of small deliberate changes in the ionic strength of the mobile   (a) y = a + xb where y is the amplitude of the second derivative peak in case of bivariate method or the analyte/I.S. peak area ratios in case of HPLC method; x is the concentration; S y/x is the standard deviation of the residuals; S a is the standard deviation of the intercept; S b is the standard deviation of the slope phase, pH, percentage of organic phase, flow rate and wavelength detection were evaluated as a part of testing for method robustness.Each result is the average of three separate determination case of bivariate method, and 1 -25 µg•mL -1 for the HPLC method.The analytical parameters of the proposed methods are summarized in (Table 4).The detection limit and the quantification limit were calculated using the following equation [32]: where F: factor of 3.3 and 10 for DL and QL, respecttively.SD: standard deviation of the intercept and b: slope of the regression line.The estimated limits were verified by analyzing a suitable number of samples containing the analyte at the corresponding concentrations.

Precision and Accuracy
Precision was evaluated at three different concentrations   within the same day to obtain repeatability (intraday precision) and over three different days to obtain intermediate precision (inter-day precision), both expressed as RSD% values.RSD% values for intraday precision were lower than 0.18% and 0.54% for bivariate and HPLC method, respectively.RSD% values for inter-day precision were lower than 0.30% and 0.63% for bivariate and HPLC method, respectively.Precision results of the validation are summarized in (Table 5).To ascertain the accuracy of the proposed procedures, they were successfully applied for the determination of fenofibrate in Lipolex capsules as presented in (Table 6).

Selectivity and Specificity
The selectivity and specificity of the proposed methods were verified by determination of fenofibrate in laboratory prepared mixtures containing different ratios of the drug and its degradation product within the linearity range and analyzing the mixtures following the prescribed conditions.The analysis was valid up to 90% of the degradation product for both bivariate and chromatographic methods (Table 3), indicating the high selectivity and specificity of the proposed methods.

Robustness of the Liquid Chromatographic
Method.Robustness is an important aspect of method validation for chromatographic methods.The influence of small changes in the operations (variables) of the analytical procedure is evaluated on measured or calculated responses.The changes introduced when performing a robustness test reflect the changes that can occur when a method is transferred between different laboratories.The robustness of the method was investigated under a variety of conditions including ionic strength of the mobile phase, percentage of organic phase, column temperature, flow rate and wavelength detection.The values of the chromatographic variables are listed in (Table 7).The measured response variables were the capacity factor (k') and the retention time (Figure 6(a)).The figures show that the parameters, detection wavelength, column temperature and flow rate, do not significantly affect on the capacity factor.A decrease in methanol concentration (%) increases the capacity factor of fenofibrate.The capacity factor of fenofibrate was negatively influenced by an increase of percent phosphoric acid concentration.Also (Figure 6(b)) shows how the retention time corresponds to fenofibrate change with respect to the concentration of methanol (%), the percent of phosphoric acid, and column temperature.The degree of reproducibility of the results obtained as a result of small deliberate variations in the method parameters and by changing analytical operators has proven that the method is robust.

Statistical Analysis of the Results
The results of the analysis of the bivariate method were compared statistically by the Student's t -test and the variance ratio F-test with those obtained by the proposed HPLC method.The Student's -values at 95% confidence level did not exceed the theoretical values, indicating that there was no significant difference between the bivariate method and the proposed HPLC method.It was also noticed that the variance ratio t F -values calculated for = 0.05 did not exceed the theoretical values, indicating that there was no significant difference between the precision of the proposed methods.The results are given in (Table 8).

Conclusions
The proposed procedures are simple, sensitive, selective and stability indicating.The methods can be used for the routine analysis of fenofibrate either in bulk powder or in pharmaceutical dosage forms.The proposed methods can be applied in laboratories lacking sophisticated instruments such as GC-MS or LC-MS.It was concluded that the developed methods are equally accurate, sensitive and precise and could be applied directly and easily to the pharmaceutical formulation with a good recovery.

2. 4 . 5 .
Analysis of Fenofibrate in Lipolex Capsules.The powder of 10 Lipolex capsules, after unpacking, was weighed.An amount of powdered mass equivalent to 40 mg or 50 mg of fenofibrate was weighed and transferred to 50 mL conical flask, the drug from powder was dissolved and extracted with methanol.The extract was filtered, and residue was washed with methanol.The extract and washing were pooled and transferred to a 100 mL volumetric flask and volume was made with methanol.Working solutions 40 µg•mL -1 or 50 µg•mL -1 were prepared in methanol by appropriate dilution and subjected to analysis as mentioned under (section 2.4.2.1 & 2.4.3.1)
protons of the two methyl group of [O=C-C(CH 3 ) 2 -O-] and multiplet at  5.08 -5.13 of the methine proton [-O-CH-(CH 3 ) 2 ].In addition to the presence of a doublet aromatic protons at  6.87 -6.89,  7.45 -7.47 and  7.70 -7.76.While 1 H NMR spectrum of degraded fenofibrate in (Figure 2(b)) shows a complete disappearance of a doublet and multiplet signals at  1.21 and  5.08 -5.13, respectively.This gives an evidence of the removal of the isopropyl moiety.parameters of component A at the two selected wavelengths ( 1

1 int AB , 2 int
tion curve slope values of the second derivative spectra, A C and B C the concentration of components A and B, respectively and AB represent the sum of the intercept of the calibration curves of the two components at the two given wavelengths.The solution of this algorithm system of equations allows the determination of A C and B

Figure 6 .
Figure 6.(a) Effect of chromatographic variables on the capacity factor (k') of fenofibrate.(b) Effect of chromatographic variables on the retention time of fenofibrate. p

Table 8 . Statistical analysis of fenofibrate by the proposed methods.
aThe figures in parenthesis are the corresponding tabulated values at P = 0