American Journal of Anal yt ical Chemistry, 2011, 2, 332-343
doi:10.4236/ajac.2011.23042 Published Online July 2011 (http://www.scirp.org/journal/ajac)
Determination of Fenofibrate and the Degradation Product
Using Simultaneous UV-Derivative Spectrometric Method
and HPLC
Fathy M. M. Salama1, Mohamed W. I. Nassar1, Mohie M. K. Sharaf El-Din2, Khalid A. M. Attia1,
Mohamed Yousri Kaddah3*
1Department of Analytical Chemistry, College of Pharmacy, Al-Azhar University, Cairo, Egypt
2Department of Analytical Chemistry, College of Pharmacy, Mansoura University, Mansoura, Egypt
3Department of Pharmaceutical Chemistry, College of Pharmacy, King Khalid University, Abha, Saudi Arabia
Received November 25, 2010; revised March 30, 2011; accepted May 16, 2011
Abstract
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 deter-
mination 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–1,
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.
Keywords: Fenofibrate, Stability, Degradation Product, UV Derivative Spectrometric Method, HPLC
1. Introduction
Fenofibrate, 1-methylethyl 2-[4-(4-chlorobenzoyl) phe-
noxy]-2-methylpropanoate, is used as antihyperlipidemic
drug [1]. Fenofibrate activates lipoprotein lipase, which
reduces triglycerides and increases HDL cholesterol. It
exerts a variable but generally modest LDL cholesterol-
lowering effect [2].
Different methods for analysis of fenofibrate have
been reviewed. Fenofibrate was assayed in British Phar-
macopeia (BP) by a liquid chromatography method [1].
However, several chromatographic methods have been
Fenofibrate
reported for the determination of fenofibrate, in pharma-
ceutical formulations and or in biological fluids, includ-
ing HPLC [3-11], stability indicating HPLC method for
simultaneous determination of fenofibrate with other
drugs from their combination products [12,13], LC-MS
[14-17], and capillary electrophoresis [18,19]. In addition,
there are other methods reported for the determination of
fenofibrate, including voltammetry, polarography [20,21],
and derivative spectrophotometry [22].
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,

F. M. M. SALAMA ET AL. 333
2D
precise and stability-indicating procedures for the analy-
sis 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 impor-
tant task in order to solve problems encountered in the
quality control and analysis of expired samples. More-
over, accelerated stability experiments to predict expiry
dates of pharmaceutical products necessitate such meth-
ods.
2. Experimental
2.1. Materials, Chemicals and Reagents
Fenofibrate was kindly provided by Sigma Pharmaceuti-
cal 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 us-
ing Milli-Q water purification system (Millipore, Bed-
ford, MA, USA).
2.2. Standard Solutions
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 solu-
tions in methanol for assay determination. Calibration
standards were prepared by diluting the working solu-
tions with methanol.
2.2.2. 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 vol-
ume of each solution was then diluted to 100 mL with
methanol. Working solutions 50 µg·mL–1 for both feno-
fibrate 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.
2.3. Apparatus
A Shimadzu UV-2550 UV-visible spectrophotometer
(Japan) with 1 cm quartz cells was used for all absorb-
ance measurements. Spectra were automatically obtained
by Shimadzu UV-Probe software, version 2.1. Bruker
500 MHz NMR spectrometer. A pH-meter (Mettler-To-
ledo GmbH, Switzerland) was used for pH adjustment.
The HPLC system consists of solvent delivery module
(LC-20 AT) Prominence Liquid Chromatography, a sys-
tem controller (CBM-20A) Prominence Communication
BUS Module, (SPD-20 A) Prominence UV-VIS Detector,
(DGU-20 A5) Prominence Degasser and (CTO-20 A)
Prominence Column Oven, all from Shimadzu, Japan.
2.4. Procedures
2.4.1. Degradation of Fenofibrate:
One gram of fenofibrate was dissolved in 25 mL metha-
nol 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 evapo-
rated to dryness under a vacuum. The dried residue was
analyzed by IR, 1H NMR, and 13C NMR and found to be
2-[4-(4-chlorobenzoyl) phenoxy]-2-methylpropanoic acid
(fenofibric acid).
2.4.2. Bivariate Method
2D
2.4.2.1. Linearity.
Different aliquots ranging from 0.5 - 5 mL of both feno-
fibrate and fenofibric acid were transferred separately
into 10 mL volumetric flasks from their respective work-
ing 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
2D


= 10 nm and scaling fac-
tor 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 Kai-
ser’s method (293 and 306 nm).
2.4.3. Liquid Chromatographic Method
2.4.3.1. Linearity.
Aliquots of 10 µL of analyte standard solution at seven
different concentrations (1 - 25 µg·mL–1) containing the
I.S. at constant concentration (10 µg·mL–1) were injected

F. M. M. SALAMA ET AL. 334
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 plot-
ted against the corresponding concentration of the ana-
lyte (expressed as µg·mL–1). The detector was time pro-
grammed to be set at 302 nm for 3 minutes from the be-
ginning of the run time for detection of I.S. then ex-
changed to 289 nm for detection of fenofibrate. Chro-
matographic 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 tem-
perature 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.
2.4.4. Analysis of Laboratory Prepared Mixtures
Containing Different Ratios from Fenofibrate
and its Degradation Product.
2.4.4.1. Bivariate method.
2D
Aliquot portions equivalent to 18 - 2 µg mL–1 of fenofi-
brate were transferred into a series of 10 mL volumetric
flask containing 2 - 18 µg·mL–1 of fenofibrate degrada-
Figure 1. The FTIR spectra of (a) Fenofibrate (b) Degrada-
tion product.
tion product and diluted to the volume by methanol.
Continue as under linearity (section 2.4.2.1.)
2.4.4.2. Liquid Chromatographic Method.
Aliquot portions equivalent to 20 - 2.5 µg·mL–1 of feno-
fibrate and its basic degradation products of 5 - 22.5
µg·mL–1 containing salicylic acid (as I.S.) at constant
concentration (10 µg·mL–1) were transferred into a series
of 10 mL volumetric flasks. Ten µL of the prepared
mixtures were injected into HPLC under the adopted
operating conditions (section 2.4.3.1.).
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 dis-
solved 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 metha-
nol. Working solutions 40 µg·mL–1 or 50 µg·mL–1 were
prepared in methanol by appropriate dilution and sub-
jected to analysis as mentioned under (section 2.4.2.1 &
2.4.3.1)
3. Results and Discussion
3.1. 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 con-
firmed 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 absorp-
tion 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.
1H NMR spectrum of fenofibrate in (Figure 2(a))
shows doublet at

1.21 of the six protons of the two
ethyl groups of [–O–CH–(CHm3)2], singlet at δ 1.68 of

F. M. M. SALAMA ET AL. 335
(a)
(b)
Figure 2. 1H NMR spectra of (a) Fenofibrate (b) Degradation product.

F. M. M. SALAMA ET AL. 336
(a)
(b)
Figure 3. 13C NMR spectra of (a) Fenofibrate (b) Degradation product.

F. M. M. SALAMA ET AL. 337
For the selection of the two most appropriate wave-
lengths with respect to their sensitivity for the simulta-
neous determination of the substances, we applied Kai-
ser’s [28-30] method, which consists of resolving the
determinant from the so called selectivity matrix, K.
Scheme 1. The degradation pathway of fenofibrate.
11
22
AB
AB
K





the six protons of the two methyl group of [O=C–
C(CH3)2–O–] and multiplet at

5.08 - 5.13 of the me-
thine proton [–O–CH–(CH3)2]. In addition to the pres-
ence of a doublet aromatic protons at

6.87 - 6.89,

7.45 - 7.47 and

7.70 - 7.76. While 1H NMR spec-
trum of degraded fenofibrate in (Figure 2(b)) shows a
complete disappearance of a doublet and multiplet sig-
nals at

1.21 and

5.08 - 5.13, respectively. This
gives an evidence of the removal of the isopropyl moi-
ety.
where 1
A

, 2
A

represent the sensitivity parameters of
component A at the two selected wavelengths (1

, 2

)
and 1
B

, 2
B

, correspond to the sensitivity parameters
of component B, in this case considered as calibration
curve slopes for each component at two given wave-
lengths.
13C NMR spectra of fenofibrate and its degraded prod-
ucts 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–(CH3)2] and the methine carbon of
[–O–CH–(CH3)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.
3.2. Bivariate Method
The resolution of two components by the bivariate cali-
bration has been recently proposed [23-27]. The concen-
tration 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 sec-
ond two derivative spectra calibration curves for each
component at two different wavelengths:

222
11111 1
int
ABABAA BBAB
DDD CC   

222
intDDD CC   
1
2 22222 2ABABAABBAB
where 1
A

, 2
A

and 1
B

, 2
B

represent the calibra-
tion curve slope values of the second derivative spectra,
A
C and
B
C the concentration of components A and B,
respectively and 1 int
A
B, 2
int
A
B represent the sum of the
intercept of the calibration curves of the two components
at the two given wavelengths. The solution of this algo-
rithm system of equations allows the determination of
A
C and
B
C as follows:
Equation 1:
2
211 122
21 12
(((int)((int))
AABABA ABAB
B
AB AB
D
C

 
 

2
D
The “bivariate calibration method” was applied to the
second derivative spectrum for the resolution of the bi-
nary 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 ex-
pected 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
2D

maxima affected significantly by pH
changes. The optimization of the derivative spectra was
achieved at


= 10 and scaling factor of 1000. In or-
der to apply the “derivative bivariate calibration method”
for the resolution of the binary mixture fenofibrate and
its degradation product, the signals of all standard solu-
tions 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 sensitiv-
ity 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 deter-
minants, 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:
2
111
1
int
A
BABB
A
A
DC
C



B l

F. M. M. SALAMA ET AL. 338
(a) (b)
(c) (d)
Figure 4. UV-spectra of (a) Zero-order spectra of 10 µg·mL–1 fenofibrate (······), 10 µg·mL–1 degradation product (―), and
their mixture (·-·-·-). (b) First-derivative spectra of fenofibrate (······), degradation product (―), and their mixture (·-·-·-). (c)
second-derivative spectra of fenofibrate (······), degradation product (―), and their mixture (·-·-·-). (d) Effect of pH on the
absorbance of fenofibrate and its basic degradation at pH 4.0 (······), pH 7.0 (―), pH 9.0 (·-·-·-).
Table 1. Correlation data of calibration curves to 2D spectrum obtained for the fenofibrate and its degradation product, at
the selected wavelengths and considered as sensitivity parameters in Kaiser’s matrix.
Fenofibrate Degradation product

nm 
Slope Intercept Correlation coefficient Slope Intercept Correlation coefficient
289 –0.107 0.025 0.999 –0.0470.027 0.999
291 –0.112 0.045 0.999 –0.0680.051 0.999
293 –0.112 0.06 0.999 –0.0880.069 0.999
298 –0.08 0.067 0.999 –0.1250.088 0.999
302 –0.032 0.045 0.999 –0.1290.075 0.999
306 0.021 0.019 0.999 –0.1040.048 0.999
308 0.045 0.011 0.998 –0.0820.037 0.999
318 0.08 0.003 0.999 0.04 –0.006 0.999
320 0.074 –0.001 0.999 0.054 –0.009 0.999
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 satisfac-
tory linear regression coefficient (>0.999). According to
the results, the wavelength pairs with the highest abso-
lute 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).

F. M. M. SALAMA ET AL. 339
Table 2. Values of the selectivity matrix determinants cal-
culated according to Kaiser’s method (K × 106) for the mix-
ture of fenofibrate and its degradation product.

nm  289 291 293 298302 306 308 318320
289 0 2012 4152 961512299 12115 10889 –520–2300
291 0 2240 856012272 13076 12244 960–1016
293 0 696011632
13496 13144 2560464
298 0 6320 10945 12185 68004930
302 0 6037 8429 90407818
306 0 2958 91608830
308 0 83608498
318 01360
320 0
3.3. 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% phos-
phoric 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 al-
low a resolution between I.S. and fenofibrate in a rea-
sonable time of 2.047 and 10.787 min, respectively. The
chromatogram of the standard solution containing feno-
fibrate 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
Table 3. Determination of fenofibrate in laboratory prepared mixtures by the proposed methods.
Concentration taken (μg·ml–1) Percentage Recovery (% )
Method Fenofibrate Degradation productFenofibrate Degradation product Fenofibrate
1- Bivariate method
Mix. 1 18 2 90 10 101.88
Mix. 2 16 4 80 20 101.71
Mix. 3 14 6 70 30 101.65
Mix. 4 12 8 60 40 102.34
Mix. 5 11 9 55 45 102.66
Mix. 6 10 10 50 50 101.59
Mix. 7 8 12 40 60 101.2
Mix. 8 6 14 30 70 101.52
Mix. 9 4 16 20 80 99.95
Mix. 10 2 18 10 90 101.83
Mean ± S.D. 101.63 ± 0.72
N 10
S.D. 0.72
RSD (%) 0.22
2- HPLC method
Mix. 1 20 5 80 20 101.50
Mix. 2 15 10 60 40 99.14
Mix. 3 10 15 40 60 100.87
Mix. 4 5 20 20 80 102.47
Mix. 5 2.5 22.5 10 90 102.53
Mean ± S.D. 101.30 ± 1.25
N 5
S.D. 1.25
RSD (%) 0.55
Each result is the average of three separate determination

F. M. M. SALAMA ET AL. 340
Figure 5. The representative chromatograms of: (a) 10 µl
injection of 15 µg·mL–1 fenofibrate and 10 µg·mL–1 of I.S, (b)
10 µl injection of laboratory-prepared mixture containing
20 µg·mL–1 fenofibrate, 5 µg·mL–1 basic degradation of
fenofibrate, and 10 µg·mL–1 I.S.
Table 4. Analytical parameters of the proposed methods.
Bivariate method
Parameters 293 nm 306 nm
HPLC
method
Linearity range
(µg·mL–1) 2 - 20 2 - 20 1 - 25
Limit of detection
(µg·mL–1) 0.11 0.18 0.11
Limit of quantification
(µg·mL–1) 0.32 0.55 0.36
Regression equation(a)
Slope (b) –0.112 0.064 0.336
Intercept (a) 0.045 0.007 –0.080
Correlation coefficient
(r) 0.999 0.999 0.999
Sy/x 0.0083 0.0026 0.11
Sa 0.0036 0.0012 0.013
Sb 0.0635 0.0124 0.0015
(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 o
f
HPLC method; x is the concentration; Sy/x is the standard deviation o
f
the residuals; Sa is the standard deviation of the intercept; Sbis 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.
3.4. Validation of the Method
3.4.1. Linearity, LOD and LOQ
Satisfactory linearity (r > 0.999) was obtained for feno-
fibrate over the concentration range 2 - 20 µg·mL–1 in
Table 5. Precision and accuracy results of the validation.
Bivariate method HPLC method
Known concen-
tration
(µg·mL–1)
Recovery (%)
Known concen-
tration
(µg·mL–1)
Recovery (%)
Intra-day Intra-day
8 99.11 5 101.28
12 99.40 10 99.65
16 99.72 15 99.66
Mean ± S.D. 99.41 ± 0.31 100.20 ± 0.94
N 3 3
S.D. 0.31 0.94
RSD (%) 0.18 0.54
Inter-day Inter-day
8 99.03 5 101.89
12 99.83 10 99.76
16 99.99 15 100.39
Mean ± S.D. 99.62 ± 0.51 100.68 ± 1.10
N 3 9
S.D. 0.51 1.10
RSD (%) 0.30 0.63
Each result is the average of three separate determination
Table 6. Tablet recovery by the proposed methods.
Bivariate method HPLC method
Method Known concen-
tration (µg·mL–1)
Recovery
(%)
Known concen-
tration (µg·mL–1)
Recovery
(%)
8
98.33
5 101.76
12
98.32
10 100.57
16
98.36
15 99.96
Mean ± S.D.
98.34 ±
0.02 100.76 ± 0.91
N 3 3
S.D. 0.02 0.91
RSD (%) 0.01 0.52
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 pro-
posed methods are summarized in (Table 4). The detec-
tion limit and the quantification limit were calculated
using the following equation [32]:
;
F
SD
DL QLb


where F: factor of 3.3 and 10 for DL and QL, respect-
tively. 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 con-
taining the analyte at the corresponding concentrations.
3.4.2. Precision and Accuracy
Precision was evaluated at three different concentrations

F. M. M. SALAMA ET AL. 341
Table 7. Robustness study: nominal values corresponding
with low, central, and upper levels.
Chromatographic variable Low value Central value Upper value
UV detection (nm) 288 289 290
Column temperature (˚C) 49 50 51
Ionic strength (% of phos-
phoric acid) 0.09 0.1 0.11
% of methanol 58 60 62
Flow rate 1.9 2 2.1
(a)
(b)
Figure 6. (a) Effect of chromatographic variables on the
capacity factor (k') of fenofibrate. (b) Effect of chroma-
tographic variables on the retention time of fenofibrate.
Table 8. Statistical analysis of fenofibrate by the proposed
methods.
Items
The proposed HPLC
method Bivariate method
Mean 100.76 ± 0.91 98.34 ± 0.02
N 3 3
V 0. 84 0.0004
S.D. 0.91 0.02
RSD (%) 0.52 0.01
F-test 0.001 (19.0)a
Student's
t-test 0.05 (2.776)a
aThe figures in parenthesis are the corresponding tabulated values at P
= 0
within the same day to obtain repeatability (intraday pre-
cision) and over three different days to obtain intermedi-
ate 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 preci-
sion were lower than 0.30% and 0.63% for bivariate and
HPLC method, respectively. Precision results of the vali-
dation are summarized in (Table 5). To ascertain the
accuracy of the proposed procedures, they were success-
fully applied for the determination of fenofibrate in
Lipolex capsules as presented in (Table 6).
3.4.3. Selectivity and Specificity
The selectivity and specificity of the proposed methods
were verified by determination of fenofibrate in labora-
tory prepared mixtures containing different ratios of the
drug and its degradation product within the linearity
range and analyzing the mixtures following the pre-
scribed conditions. The analysis was valid up to 90% of
the degradation product for both bivariate and chroma-
tographic methods (Table 3), indicating the high selec-
tivity and specificity of the proposed methods.
3.4.4. 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 re-
sponses. 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 vari-
ety 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 tem-
perature 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 col-
umn 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.

F. M. M. SALAMA ET AL. 342
3.5. 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% confi-
dence level did not exceed the theoretical values, indi-
cating 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 calcu-
lated for = 0.05 did not exceed the theoretical values,
indicating that there was no significant difference be-
tween the precision of the proposed methods. The results
are given in (Table 8).
p
4. 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 instru-
ments 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.
5. References
[1] British Pharmacopoeia Commission Secretariat of the
Medicines and Healthcare products Regulatory Agency,
“British Pharmacopoeia,” Stationary Office, London,
2004, p. 639.
[2] M. A. Koda-Kimble, L. Y. Young, W. A. Kradjan, B. J.
Guglielmo, B. K. Alldredge and R. L. Corelli, “Applied
Therapeutics the Clinical Use of Drugs CD-ROM,” 8th
Edition, Lippincott Williams &Wilkins, Philadelphia,
2005, pp. 50-77.
[3] T. V. Madureira, M. J. Rocha, Q. B. Cass and M. E. Tiri-
tan, “Development and Optimization of a HPLC-DAD
Method for the Determination of Diverse Pharmaceuti-
cals in Estuarine Surface Waters,” Journal of Chroma-
tographic Science, Vol. 48, No. 3, 2010, pp. 176-182.
[4] R. J. Straka, R. T. Burkhardt and J. E. Fisher, “Determi-
nation of Fenofibric Acid Concentrations by HPLC after
Anion Exchange Solid-Phase Extraction from Human
Serum,” Therapeutic Drug Monitoring, Vol. 29, No. 2,
2007, pp. 197-202. doi:10.1097/ftd.0b013e318030860a
[5] A. W. Meikle, J. Findling, M. M. Kushnir, A. L. Rock-
wood, G. J. Nelson and A. H. Terry, “Pseudo-Cushing
Syndrome Caused by Fenofibrate Interference with Uri-
nary Cortisol Assayed by High-Performance Liquid
Chromatography,” Journal of Clinical Endocrinology &
Metabolism, Vol. 88, No. 8, 2003, pp. 3521-3524.
doi:10.1210/jc.2003-030234
[6] A. Lössner, P. Banditt and U. Tröger, “Rapid and Simple
Method for Detection of Fenofibric Acid in Human Se-
rum by High-Performance Liquid Chromatography,”
Pharmazie, Vol. 56, No. 1, 2001, pp. 50-51.
[7] B. Streel, P. Hubert and A. Ceccato, “Determination of
Fenofibric Acid in Human Plasma Using Automated So-
lid-Phase Extraction Coupled to Liquid Chromatogra-
phy,” Journal of Chromatography B: Biomedical Sciences
and Applications, Vol. 942, No. 2, 2001, pp. 391-400.
[8] P. M. Lacroix, B. A. Dawson, R. W. Sears, D. B. Black,
T. D. Cyr and J. C. Ethier, “Fenofibrate Raw Materials:
HPLC Methods for Assay and Purity and an NMR
Method for Purity,” Journal of Pharmaceutical and Bio-
medical Analysis, Vol. 18, No. 3, 1998, pp. 383-402.
doi:10.1016/S0731-7085(98)00051-X
[9] S. Abe, K. Ono, M. Mogi and T. Hayashi, “High-Per-
formance Liquid Chromatographic Method for the De-
termination of Fenofibric Acid and Reduced Fenofibric
Acid in Human Blood, Plasma and Urine,” Yakugaku
Zasshi, Vol. 118, No. 10, 1998, pp. 447-455.
[10] L. D. Masnatta, L. A. Cuniberti, R. H. Rey and J. P.
Werba, “Determination of Bezafibrate, Ciprofibrate and
Fenofibric Acid in Human Plasma by High-Performance
Liquid Chromatography,” Journal of Chromatography B :
Biomedical Sciences and Applications, Vol. 687, No. 2,
1996, pp. 437-442. doi:10.1016/S0378-4347(96)00254-X
[11] K. Doser, R. Guserle, V. Nitsche and G. Arnold, “Com-
parative Steady State Study with 2 Fenofibrate 250 mg
Slow Release Capsules. An Example of Bioequivalence
Assessment with a Highly Variable Drug,” International
Journal of Clinical Pharmacology, Therapy and Toxi-
cology, Vol. 34, No. 8, 1996, pp. 345-348.
[12] A. A. Kadav and D. N. Vora, “Stability Indicating UPLC
Method for Simultaneous Determination of Atorvastatin,
Fenofibrate and their Degradation Products in Tablets,”
Journal of Pharmaceutical and Biomedical Analysis, Vol.
48, No. 1, 2008, pp. 120-126.
doi:10.1016/j.jpba.2008.05.018
[13] A. El-Gindy, S. Emara, M. K. Mesbah and G. M. Hadad,
“Pectrophotometric and Liquid Chromatographic Deter-
mination of Fenofibrate and Vinpocetine and their Hy-
drolysis Products,” Farmaco, Vol. 60, No. 5, 2005, pp.
425-438. doi:10.1016/j.farmac.2005.01.013
[14] D. Bhavesh, S. Shah and Shivprakash, “Determination of
Genofibric Acid in Human Plasma by Ultra Performance
Liquid Chromatography-Electrospray Ionization Mass
Spectrometry: Application to a Bioequivalence Study,”
Biomedical Chromatography, Vol. 23, No. 9, 2009, pp.
922-928. doi:10.1002/bmc.1203
[15] A. Liu, A. D. Patterson, Z. Yang, X. Zhang, W. Liu, F.
Qiu, H. Sun, K. W. Krausz, J. R. Idle, F. J. Gonzalez and
R. Dai, “Fenofibrate Metabolism in the Cynomolgus
Monkey Using Ultraperformance Liquid Chromatogra-
phy-Quadrupole Time-of-Flight Mass Spectrometry-
Based Metabolomics,” Drug Metabolism and Disposition,
Vol. 37, No. 6, 2009, pp. 1157-1163.
doi:10.1124/dmd.108.025817
[16] M. D. Hernando, M. Petrovic, A. R. Fernández-Alba and
D. Barceló, “Analysis by Liquid Chromatography-Elec-

F. M. M. SALAMA ET AL. 343
trospray Ionization Tandem Mass Spectrometry and
Acute Toxicity Evaluation for Beta-Blockers and Lipid-
Regulating Agents in Wastewater Samples,” Journal of
Chromatography A, Vol. 1046, No. 1-2, 2004, pp. 133-
140.
[17] P. Vazquez-Roig, R. Segarra, C. Blasco, V. Andreu and
Y. Picó, “Determination of Pharmaceuticals in Soils and
Sediments by Pressurized Liquid Extraction and Liquid
Chromatography Tandem Mass Spectrometry,” Journal
of Chromatography A, Vol. 1217, No. 16, 2010, pp. 2471
-2483. doi:10.1016/j.chroma.2009.11.033
[18] Ł. Komsta, G. Misztal, E. Majchrzak and A. Hauzer,
“Separation of Fibrate-Type Antihyperlipidemic Drugs
by Capillary Electrophoresis and their Quantitation in
Pharmaceuticals,” Journal of Pharmaceutical and Bio-
medical Analysis, Vol. 41, No. 2, 2010, pp. 408-414.
doi:10.1016/j.jpba.2005.11.032
[19] Z. K. Shihabi, “Fenofibrate and Fenofibric Acid Analysis
by Capillary Electrophoresis,” Electrophoresis, Vol. 25,
No. 10-11, 2004, pp. 1648-1651.
doi:10.1016/j.jpba.2005.11.032
[20] M. A. Korany, I. I. Hewala and K. M. Abdel-Hay, “De-
termination of Etofibrate, Fenofibrate, and Atorvastatin in
Pharmaceutical Preparations and Plasma Using Differen-
tial Pulse Polarographic and Square Wave Voltammetric
Techniques,” Journal of AOAC (Association of Official
Agricultural Chemists) International, Vol. 91, No. 5,
2008, pp. 1051-1058.
[21] C. Yardimci and N. Ozaltin, “Electrochemical Studies
and Square-Wave Voltammetric Determination of Feno-
fibrate in Pharmaceutical Formulations,” Analytical and
Bioanalytical Chemistry, Vol. 378, No. 2, 2004, pp. 495-
498. doi:10.1007/s00216-003-2246-x
[22] Nagaraj, K. Vipul and M. Rajshree, “Simultaneous Quan-
titative Resolution of Atorvastatin Calcium and Fenofi-
brate in Pharmaceutical Preparation by Using Derivative
ratio Spectrophotometry and Chemometric Calibrations,”
Analytical Sciences, Vol. 23, No. 4, 2007, pp. 445-451.
doi:10.2116/analsci.23.445
[23] P. L. Lopez-de-Alba, L. Lopez-Martinez, K. Wrobel-
Kaczmarczyk, K. Wrobel-Zasada, J. Amador-Hernandez
and M. L. Yepez-Murrieta, “Application of the Bivariate
Spectrophotometric Method for the Determination of
Metronidazole, Furazolidone and Di-Iodohydroxyqui-
noline in Pharmaceutical Formulations,” Journal of Phar-
maceutical and Biomedical Analysis, Vol. 16, No. 2,
1997, pp. 349-355. doi:10.1016/S0731-7085(97)00068-X
[24] L. Lopez-Martinez, P. L. Lopez-de-Alba and V. Cedra-
Martin, “Bivariate Calibration as an Alternative for Zero-
Crossing Technique in the Resolution of Binary Mixtures
by Dderivative Spectrophotometry,” Analytical Letters,
Vol. 34, No. 14, 2004, pp. 2563-2583.
doi:10.1016/S0731-7085(97)00068-X
[25] C. K. Markopoulou, E. T. Malliou and J. E. Koundourel-
lis, “Chemometric and Derivative Methods as Flexible
Spectrophotometric Approaches for Dissolution and As-
Saying Tests in Multicomponent Tablets,” Farmaco, Vol.
59, No. 8, 2004, pp. 627-636.
doi:10.1016/j.farmac.2004.02.006
[26] F. H. Metwally, “Simultaneous Determination of Nifu-
roxazide and Drotaverine Hydrochloride in Pharmaceuti-
cal Preparations by Bivariate and Multivariate Spectral
Analysis,” Spectrochemica Acta, Vol. 69, No. 2, 2008, pp.
343-349. doi:10.1016/j.saa.2007.04.004
[27] C. K. Markopoulou and J. E. Koundourellis, “Two De-
rivative Spectrophotometric Methods for the Simultane-
ous Determination of Lovastatin Combined with Three
Antioxidants,” Pharmaceutical and Biomedical Analysis,
Vol. 33, No. 5, 2003, pp. 1163-1173.
doi:10.1016/S0731-7085(03)00429-1
[28] D. L. Massart, B. G. M. Vandeginste, S. N. Deming, Y.
Michotte and L. Kaufman, “Chemometrics,” Elsevier,
Amsterdam, 1988, p. 124.
[29] J. Karpin´ska, A. Wiszowata and M. Skoczylas, “Simul-
taneous Determination of Levomepromazine Hydrochlo-
ride and Its Sulfoxide by UV-Derivative Spectropho-
tometry and Bivariate Calibration Method,” Analytical
Letters, Vol. 39, No. 6, 2006, pp. 1129-1141.
doi:10.1080/00032710600620609
[30] P. L. Lopez-de-Alba, L. Lopez-Martinez, K. Wrobel-
Kaczmarczyk, K. Wrobel-Zasada and J. Amador-Hernan-
dez, “The Resolution of Dye Binary Mixtures by Bivari-
ate Calibration Using Spectrophotometric Data,” Ana-
lytical Letters, Vol. 29, No. 3, 1996, pp. 487-503.
[31] P. L. Lopez de Alba, L. Lopez Martinez, L. I. Michelini-
Rodriguez, K. Wrobel and J. Amador-Herandez, “Extrac-
tion of Sunset Yellow and Tartrazine by Ion-Pair Forma-
tion with Adogen-464 and their Simultaneous Determina-
tion by Bivariate Calibration and Derivative Spectropho-
tometry,” Analyst, Vol. 122, No. 12, 1997, pp. 1575-1579.
doi:10.1039/a702268i
[32] J. Ermer and J. H. M. Miller, “Method Validation in
Pharmaceutical Analysis. A Guide to Best Practice,”
WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim,
2005.