1. A stability indicating RP-HPLC method was developed for the simultaneous quantification of simvastatin and fenofibrate. 2. The method used a C18 column with a mobile phase of acetonitrile and ammonium acetate buffer at pH 4.3 to achieve separation of the drugs from their degradation products. 3. The drugs were subjected to stress conditions and the degraded products were identified using LC-MS to prove the stability indicating capability of the developed method.

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eISSN: 2231-0541 CAS CODEN: PHARN8 An EMBASE, EMCare Covered Journal
PHARMANEST
An International Journal of Advances in Pharmaceutical Sciences
Volume 6|Issue 1|January- February 2015|Pages 2697-2703
Original Research Article
STABILITY INDICATING STUDIES USING RP-HPLC METHOD FOR SIMULTANEOUS
ESTIMATION OF SIMVASTATIN AND FENOFIBRATE IN ACTIVE PHARMACEUTICAL
INGREDIENTS AND DEGRADANTS IDENTIFICATION BY LC-MS
a,b
YEGGINA M RAGHAVENDRA*, a
A. K.M PAWAR, b
V. JAYATHIRTHA RAO
a
Department of Pharmaceutical Analysis and Quality Assurance, Andhra University, Visakhapatnam-003, A.P, India
b
Crop Protection Chemicals, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad-007, T.S, India
Author for Correspondence: meharraghavendra.yeggina@gmail.com
ABSTRACT
A simple, sensitive and accurate stability indicating RP-HPLC method for the simultaneous estimation of Simvastatin and Fenofibrate in active
pharmaceutical ingredients was developed on a Macherey-Nagel EC 250 x 4.6 mm nucleosil 100-5 C18 column and PDA detector operated at 247
nm wavelength. The acetonitrile-ammonium acetate buffer (8 mM, pH 4.3, 70:30, v/v) was used as mobile phase in isocratic mode with a flow rate
of 0.7 mL/min and run time of 18 min. The combination of Simvastatin and Fenofibrate was subjected to stress degradation and the degraded
products were separated and identified using LC-MS. Excellent separation of analytes form their degradation products proved the effectiveness of
the developed stability indicating method. The developed method was validated according to ICH guidelines and can be applied for the
quantification of Simvastatin and Fenofibrate in 10-60 µg/ml and 30-180 µg/ml range, respectively.
Key Words: Simvastatin, Fenofibrate, Stability indicating, RP-HPLC, LC-MS.
INTRODUCTION
Simvastain (SIM), (1S, 2S ,6R, 8S, 8aR)
- 1, 2, 6, 7, 8, 8a-hexahydro-1-(2-((2R,4R) –
tetrahydro - 4 - hydroxy-6-oxo - 2H – pyran -2-
yl) ethyl) -2,6-dimethylnaphthalen-8-yl 2,2-
dimethylbutanoate (fig. 1), is a semi-synthetic1
derivative of a Lovastatin, useful for the
treatment of hypercholesterolemia.
SIM is administered orally as a pro-drug
(inactive lactone form) to control the synthesis
of cholesterol by undergoing enzymatic
hydrolysis with the production of its
corresponding β-hydroxy acid, which is a potent
competitive inhibitor of microsomal enzyme 3-
hydroxy-3-methylglutaryl-coenzyme-A
(HMGCoA) reductase2-3
which leads to the
induction of hepatic LDL (Low density
lipoprotein) receptors4
causes the increase in
breakdown of LDL cholesterol.
Stability indicating studies for the SIM
alone under hydrolytic conditions has been
reported previously using liquid
chromatography5
and simultaneous estimation
of SIM in combination with other drugs in fixed
dosage forms has been reported using RP-
HPLC6-7
, RP-UPLC8
and HPTLC9
. Identification of
the impurities of SIM has been reported using
liquid chromatography/tandem mass
spectrometry10
.
O
OHO
H
O
O
Figure 1. Chemical Structure of Simvastatin.
Fenofibrate (FB), 2-[4-(4-chlorobenzoyl)
phenoxy]-2- methyl-propanoic acid, 1-
methylethyl ester (fig. 2), is a lipid lowering
agent. After oral administration, FB converts to
Fenofibric acid through the hydrolysis of ester
bond11
Fenofibric acid is the active metabolite
which lowers plasma triglycerides by inhibiting
triglyceride synthesis12-13
resulting in the
reduction of VLDL (Very low density lipoprotein)
and triggers the catabolism of VLDL.
It decreases serum uric acid levels by
increasing the urinary excretion of uric acid.
Simultaneous estimation of FB in different fixed
dosage combinations has been reported using
UV Spectrophotometry14
and RP-HPLC15-17
.
Stability indicating studies of FB in combination
with other drugs has been reported by HPLC18
.
Assay and purity of the FB raw materials has
been reported using HPLC and NMR19
. FB and its
degradation products have been reported using
simultaneous UV-Derivative Spectrometric
Method and HPLC20
.
Cl
O
O
O
O
Figure 2. Chemical Structure of Fenofibrate.
Recently, the combination of SIM and FB
in the treatment of Primary Mixed
hyperlipidaemia was reported21
. Since these
results are positive and no reports are available
regarding to their stability studies, herewith we

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have developed a new stability indicating RP-
HPLC method for the simultaneous estimation of
Simvastatin (SIM) and Fenofibrate (FB). The
present method was validated as per the ICH
guidelines. Excellent separation of analytes (SIM
and FB) from their degradation products was
achieved using this method.
MATERIALS AND METHODS
Pure form of SIM and FB were obtained
from Piramal Healthcare Laboratories,
Hyderabad. Acetonitrile (HPLC grade),
ammonium acetate, sodium hydroxide, Glacial
acetic acid, hydrochloric acid and hydrogen
peroxide (analytical reagent grade) were
purchased from Sigma Aldrich. Milli-Q water was
used for all the experiments.
Solutions:
8 mM ammonium acetate buffer was
prepared freshly by dissolving 185 mg of
ammonium acetate in 300 ml of de-ionized
water and the pH was adjusted to 4.3 using
20% glacial acetic acid. Mobile Phase was
prepared by mixing acetonitrile and 8 mM
ammonium acetate buffer, pH 4.3 in the ratio of
70:30 v/v.
Preparation of Standard and working
standard solutions:
Standard solutions of SIM and FB of
lmg/ml were prepared in acetonitrile.
Preparations of working standard solutions
were performed from the standard solutions
with the mobile phase having SIM and FB in the
ratio of 1:3 respectively. These two drugs
having the concentration ranges of SIM 10-60
µg/ml and FB 30-180 µg/ml were prepared.
The column was equilibrated at least for 30-45
min with mobile phase before injecting the
sample solutions.
INSTRUMENTATION
HPLC Instrumentation and
Chromatographic conditions:
Analysis of samples were performed on
Waters HPLC system, integrated with a model
series of 515 binary HPLC pumps, PC2 Pump
control module-II, DG2 in-line degasser AF,
7725i Rheodyne injection valve with 10 µL
sample loop and variable wavelength
programmable 2998 PDA detector.
The processing and data acquisition was
done using Empower software. Macherey-Nagel,
Nucleosil 100-5 C18 column was used as
stationary phase maintained at ambient
temperature. Isocratic elution with mobile phase
composition of acetonitrile and 8 mM ammonium
acetate buffer, pH 4.3 (70:30, v/v). The mobile
phase was pumped through the column with
flow rate of 0.7 mL/min. Injection volume of 20
µl was used in all experiments. The optimum
wavelength selected was 247 nm, which
represents the wavelength of maximum
response for both SIM and FB.
LC-MS Instrumentation and Conditions:
LC-MS 2020 with Shimadzu HPLC was
used for the study of SIM and FB. The
degradation studies were carried out on
Macherey-Nagel, Nucleosil 100-5 C18 column.
The degradants in the stressed sample were
separated using isocratic program. The mobile
phase used was acetonitrile and ammonium
acetate buffer (pH 4.3) in the ratio of 70:30
(v/v). For LC-MS analysis, Shimadzu HPLC
system coupled to quadrupole mass analyzer
equipped with an ESI source. Lab solutions
software was used for data acquisition. The
typical operating source conditions for MS scan
of SIM and FB in ESI were optimized as follows:
nebulizing gas flow rate was 1.5 l/min, Heat
block temperature was 200 °C, Detector voltage
0.95 kV. Autosampler was set at following
conditions: oven temperature at 30 °C, PDA
wavelength at 247 nm, cell temperature at 30
°C and scan speed 1000 µs.
Linearity:
Calibration curves were constructed by
six different concentrations of working standard
solutions for SIM 10–60 µg/ml (10, 20, 30, 40,
50 and 60 µg/mL) and FB 30–180 µg/mL (30,
60, 90, 120, 150 and 180 µg/ml). Triplicate
preparations of each sample solution were done
to inject into the column. Standard calibration
curves were plotted by taking mean peak area
on Y-axis and concentrations of drug on X-axis.
Accuracy:
To demonstrate the accuracy of the
proposed method a standard addition method
was used for analyzing the samples. For this
purpose, known amounts of SIM and FB were
supplemented to the working standard sample
solution which was previously analyzed and then
compared the obtained experimental values to
the true values. Each solution was injected in six
times and the percentage recovery was
calculated.
Precision:
Method precision was determined in
terms of repeatability. It was studied by
determination of intra-day and inter-day
precision. For intra-day precision, three different
concentration levels SIM (10 μg/ml) and FB (30
μg/ml), SIM (30 μg/ml) and FB (90 μg/ml) and
SIM (60 μg/ml) and FB (180 μg/ml) with six
injections of the each sample solution (n=6) on
the same day and inter-day precision was
determined by injecting the same solutions for
three consecutive days. The relative standard
deviation (RSD %) was calculated from peak
area to represent precision.
Robustness:
The method robustness was studied by
evaluating the premeditated variations under
the experimental conditions of the proposed
method. The different variations such as
variation of pH of the buffer solution, flow rate,
wavelength and mobile phase composition.
These changes affect the chromatographic
parameters such as retention time, purity angle,
purity threshold, tailing factor and theoretical
plate count was then measured.
Limit of Detection (LOD) and Limit of
Quantitation (LOQ):
The LOD and LOQ are the lowest
concentrations of the standard curves in which
LOD is typically determined where the signal to
noise ratio is 3 and LOQ can be quantified with
acceptable accuracy and precision.

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Specificity (stress testing):
Samples were allowed to hydrolyze in
different strengths of stress conditions such as
acid, base, oxidative, thermal and photolytic
stresses. All these studies were performed in 10
ml volumetric flasks.
Acid degradation studies:
To perform acid degradation studies, 0.1
N HCl was used at a temperature condition of
40 o
C. For this purpose, accurately measured 7
ml of standard stock solution was taken in 10 ml
volumetric flask. 1mL of 0.1 N HCl was added in
the flask and kept at 40 o
C for 24 hrs. After
completion of the test, the solution was
neutralized by using 0.1 N NaOH and the
solution was completed up to the mark with the
mobile phase.
Base degradation studies:
To perform base degradation studies,
0.1 N NaOH was used at a temperature
condition of 40 o
C. For this purpose, accurately
measured 7 ml of standard stock solution was
taken in 10 mL volumetric flask. 1mL of 0.1 N
NaOH was added in the flask and kept at 40 o
C
for 24 hrs. After completion of the test, the
solution was neutralized by using 0.1 N HCl and
the solution was completed up to the mark with
the mobile phase.
Oxidative degradation studies:
To perform oxidative degradation
studies, 3% H2O2 was used at a room
temperature. For this purpose, accurately
measured 7 ml of standard stock solution was
taken in 10 ml volumetric flask. 1mL of 3% H2O2
was added in the flask and kept at room
temperature for 24 hrs. After completion of the
test, the solution was completed up to the mark
with the mobile phase.
Thermal degradation studies:
To perform thermal degradation studies,
two different temperature conditions were used.
For this purpose, accurately measured 7 ml of
standard stock solution was taken in two
different 10 ml volumetric flasks. At room
temperature one of the volumetric flasks was
placed for 24 hrs and another at 40 o
C for 6 hrs.
After completion of the test, the solution was
completed up to the mark with the mobile
phase.
Photolytic degradation studies:
To perform photolytic degradation, 7 ml
of the standard stock solution was placed in
direct sunlight for 1 hour.
RESULTS AND DISCUSSION
Method development and optimization:
In the present work, the RP-HPLC
method compatible to LC-MS was found to be
simple, sensitive and accurate for the
simultaneous estimation of SIM, FB and
identification of their stress induced degradation
products by LC-MS. To optimize the
chromatographic conditions, number of trails
was performed i.e. change in composition of
mobile phase, adjusting the pH of the mobile
phase and selection of the suitable column.
Optimization of the conditions:
To optimize the chromatographic
conditions such as change in mobile phase
compositions, different pH conditions of the
mobile phase and different analytical columns
were used. The novelty of the proposed method
was the identification of co-eluting degradation
products of the SIM and FB by LC-MS in the
stability indicating studies of their simultaneous
estimation. The method development process
was initiated with different ratios (80:20, 75:25,
70:30 and 65:35) of acetonitrile and water (pH
adjusted to 3.0, 4.0, and 5.0). In order to get
symmetrical peaks, different stationary phases
(Agilent Zorbax CN, Agilent eclipse XDB C18 and
Macherey-Nagel, Nucleosil 100-5 C18 column) at
different pH values (3.0, 4.0 and 5.0) were tried
with acetonitrile and water mobile phase. With
this mobile phase composition Macherey-Nagel,
Nucleosil 100-5 provided peaks with reliable
separation but the tailing is observed in both
peaks of drugs SIM (tailing more than 2.67) and
FB (tailing more than 1.4) at pH values of 3.0,
4.0 and 5.0. Further the method was developed
using different molarities of the ammonium
acetate buffer were tried to obtain symmetrical
peaks of the analytes. Finally 8 mM ammonium
acetate buffer with acetonitrile gave good
separation of analytes.
Acetonitrile and 8 mM ammonium
acetate buffer (70:30, v/v) with glacial acetic
acid, pH 4.3 mobile phase was finally selected to
reduced peak tailing and acceptable peak purity
index of SIM and FB using Macherey-Nagel,
Nucleosil 100-5 C18 as the stationary phase.
Under these conditions, SIM and FB peaks are
symmetrical and sharp in acidic, basic and
photolytic (fig. 3) studies. But there was no
degradation observed due to oxidation under
specified condition.
Figure 3.Chromatograms of simvastatin and fenofibrate
under A. Acidic. B. Basic C. Photolytic Conditions.
LC-MS:
The LC-MS analysis directly gave the
molecular weight of the analytes eluted. To
identify the degradation products we transferred
the method to LC-ESI/MS. The first degraded
product full mass spectra revealed the presence
of a molecular ion peak at m/z 475 because of

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the addition of acetonitrile [M+ CH3CNH]+
indicating a molecular weight for DP1 at 433
m/z (fig .4). The second and third degraded
product full mass spectra revealed the presence
of a molecular ion peaks at m/z 575 and 487
because of the addition of acetonitrile
[M+CH3CNH]+ and [M+CH3CNH]- indicating a
molecular weight for DP2 and DP3 at 533 (fig
.5) and 446 m/z (fig .6). The fourth degraded
product full mass spectra revealed the presence
of a molecular ion peak at m/z 391 because of
the removal of proton [M+H] - indicating a
molecular weight for DP4 at 392 m/z (fig .7).
The fifth degraded product full mass spectra
revealed the presence of a molecular ion peak at
m/z 191 because of the addition of water
[M+H20]+ indicating a molecular weight for DP5
at 172 m/z (fig .8). The sixth degraded product
full mass spectra revealed the presence of a
molecular ion peak at m/z 231 because of the
removal of proton [M+H] - indicating a
molecular weight for DP6 at 232 m/z (fig .9).
Proposed structures for the degradation
products of Simvastatin and Fenofibrate were
cited (fig .10 and fig .11).
Figure 4. Mass Spectrum of Degradation Product 1
(DP1).
Figure 5. Mass Spectrum of Degradation Product 2
(DP2).
Figure 6. Mass Spectrum of Degradation Product 3
(DP3).
Figure 7. Mass Spectrum of Degradation Product 4
(DP4).
Figure 8. Mass Spectrum of Degradation Product 5
(DP5).
Figure 9. Mass Spectrum of Degradation Product 6
(DP6).
O
OHO
H
O
O
0.1N HCl / NaOH
400
C for 24hr
Sunlight for 1hr
O
OO
H
O
O
Simvastatin
Exact Mass: 432.29
Mol. Wt.: 432.59
DP1
Figure 10. Proposed structure for the degradation
Product of Simvastatin.

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Cl
O
O
O
O
Cl
O
O
O
O
O
Cl
Cl O
O
O
O
O
O
Cl O
O
O
O
Cl OH
O
0.1N HCl
40
0 C for 24hr
0.1N HCl 400C for 24hr
/Sunlight for 1hr
0.1N NaOH
400C for 24hr
Sun light
for
1hr
Sun light
for
1hr
Fenofibrate
DP-2
DP-3
DP-4
DP-6
Exact Mass: 532.08
Mol. Wt.: 533.4
Exact Mass: 446.15
Mol. Wt.: 446.92
Exact Mass: 332.08
Mol. Wt.: 332.78
Exact Mass: 232.03
Mol. Wt.: 232.66
Cl COOH
O
Exact Mass: 171.99
Mol. Wt.: 172.57
DP-5
Figure 11. Proposed structures for the degradation
Products of Fenofibrate.
Analytical method validation:
The proposed method was validated
using ICH guidelines22
includes linearity,
accuracy, precision, robustness, specificity, and
limit of detection and quantification.
Linearity calibration plots were obtained
in the concentration range of 10-60 mg mL-1
for
SIM (10, 20, 30, 40, 50 and 60 µg/ml) and 30
to 180 µg/mL for FB (30, 60, 90, 120, 150 and
180 µg/ml). The linear regression equation for
SIM was found to be Y = 89148x-19998 with
the correlation coefficient equal to 0.999,
whereas for FB, it was Y= 60007x-55319 with
the correlation coefficient equal to 0.999. The
LOD and LOQ were determined by making serial
dilutions. The LOD for SIM and FB was found to
be 0.009 µg/ml and 0.025 µg/ml respectively
(signal to noise ratio of 3: 1). The LOQ for SIM
and FB was found to be 0.03 µg/ml and 0.082
µg/ml respectively (signal to noise ratio of
10: 1).
To determine the accuracy of the
method standard addition technique was
performed. Three levels of nominal analytical
concentrations (80, 100 and 120%) were
prepared. For each analyte (n = 6) percentage
recoveries along with standard deviation and
relative standard deviations were represented in
Table 1. Accuracy and suitability of the proposed
method was proved from these recovery
studies.
Table 1.Accuracy results of Simvastatin and Fenofibrate.
Drugs
Selected
Concentration
(µg/mL)
Spiked
Concentration
(µg/mL)
Measured
Concentration
(µg/mL)
Recovery (%)
± S.D
RSD (%)
SIM
FB
20
20
20
40
40
40
16
20
24
32
40
48
36.32
39.81
43.72
72.10
79.89
87.68
100.90±0.18
99.54±0.32
99.37±0.35
100.14±0.29
99.87±0.12
99.63±0.31
0.5
0.81
0.8
0.40
0.15
0.36
Intra-day precision was determined by injecting
three standard solutions of three different concentrations on
the same day (n = 6) and inter-day precision was determined
by injecting the same solutions for three consecutive days
were displayed in Table 2.
To represent the precision relative standard
deviation (RSD %) of the peak area was calculated.
The different variations such as variation of pH of
the buffer solution, flow rate and mobile phase composition
were discussed in Table 3. According to the ICH guidelines,
system suitability parameters were showed in Table 4.
Table 2.Intra-day and Inter-day precision values of Simvastatin and Fenofibrate.
Drugs
Actual
Concentration
(µg/mL)
Intra-day Precision Inter-day Precision
Measured
Concentration± S.D
(%RSD)
Measured
Concentration± S.D
(%RSD)
SIM
FB
10
30
60
30
90
180
9.91±0.06(0.64)
30.58±0.27(0.89)
59.47±0.29(0.49)
29.75±0.87(2.93)
90.71±0.51(0.57)
180.79±1.58(0.87)
9.69±0.18(1.89)
30.42±0.71(2.37)
59.54±1.12(1.89)
29.59±0.71(2.42)
89.84±0.87(0.96)
180.87±1.00(0.55)
Table 3.Effect of deliberated changes on the system suitability parameters.
Description Condition
Retention time(min) Tailing factor
Resolution
Plate count
SIM FB SIM FB SIM FB
Flow Rate (mL/min)
Buffer Conc (mM)
pH of buffer
0.5
0.9
5
11
4.5
4.1
15.57
11.19
13.05
12.43
13.57
13.39
17.13
13.49
15.07
14.56
15.44
15.51
1.353
1.341
1.362
1.358
1.299
1.347
1.563
1.572
1.593
1.565
1.559
1.552
4.21
2.06
2.53
2.58
2.17
2.08
5576
5464
5523
5556
5682
5521
6485
6671
6586
6550
6464
6524

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Table 4.System suitability parameters.
Parameter
Retention time Tailing factor Plate count
SD %RSD SD %RSD SD %RSD
SIM
FB
DP1
DP2
DP3
DP4
DP5
DP6
0.141
0.165
0.231
0.044
0.189
0.053
0.047
0.086
1.367
1.368
4.865
0.634
2.305
0.705
1.320
1.383
0.008
0.010
0.043
0.024
0.026
0.019
0.024
0.019
0.991
1.117
4.513
2.442
2.846
2.138
2.345
2.046
263.18
249.25
143.86
36.662
30.798
32.037
22.151
24.490
4.428
4.450
4.423
0.788
1.236
0.785
0.381
0.766
Specificity of the developed method was
carried out in different stress conditions (acid,
base, oxidation, thermal and photolytic) to
Simvastatin and Fenofibrate in a combination
form. From the results of the stress except
acidic and basic applied at 40 0
C (hot plate)
were enough to degrade both the drugs within
24 hours. Both the drugs were remaining intact
in oxidative stress condition. The stress induced
degradation products (impurities) were unique
to acidic (7.025 and 7.861 min) and basic
(7.622 min) stress conditions. Comparison of
the two drugs showed that Simvastatin was
more stable than Fenofibrate.
CONCLUSION
A simple, sensitive, accurate and
isocratic reverse phase LC-MS compatible RP-
HPLC method has been described for
simultaneous determination of Simvastatin and
Fenofibrate in active pharmaceutical ingredients.
The proposed method was validated in
accordance with ICH guidelines by testing its
parameters include linearity, accuracy,
precision, robustness, LOD and LOQ. The
method was very specific to separate the peaks
of active pharmaceutical ingredients from the
degradation products. That allows the
identification of degradation products using LC-
ESI-MS. In sum, stress induced studies proves
the effectiveness of the proposed stability
indicating method.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the Dr.
Lakshmi Kantam, Director, Indian Institute of
Chemical Technology for her constant support in
providing excellent instrumentation facility in
their laboratories, Dr. Ganga Rao, Principal, AU
College of Pharmaceutical Sciences, Andhra
University for his kind help and encouragement
and also Piramal Healthcare Laboratories for gift
samples. VJR thanks to IICT-ORIGIN project for
funding.
CONFLICT OF INTEREST
Authors declare no Conflict of Interest.
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Available online: http://www.pharmanest.net
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HOW TO CITE THIS ARTICLE
Yeggina M Raghavendra*, A. K.M Pawar, V. Jayathirtha Rao.
(2015 February 1) Stability Indicating Studies Using RP-HPLC
Method for Simultaneous Estimation of Simvastatin and
Fenofibrate in Active Pharmaceutical Ingredients and
Degradants Identification by LC-MS.
PHARMANEST,6(1),2697-2703.http://www.pharmanest.net