An Experimental Design Approach for Method Development and Impurity Profiling of Simvastatin by UV Spectrophotometric and RP–HPLC Methods

202
* Corresponding author: Prakash Katakam
E-mail address: pkatakam9@gmail.com
IJPAR |Volume 3 | Issue 2|April-June-2014 ISSN: 2320-2831
Available Online at: www.ijpar.com
[Research article]
An Experimental Design Approach for Method Development and
Impurity Profiling of Simvastatin by UV Spectrophotometric and
RP–HPLC Methods
Prakash Katakam1
*, Baishakhi Dey2
, Nagiat T. Hwisa1
, Fathi H. Assalah1
, Narayan N.
Rao3
, Babu R. Chandu1
, Analava Mitra2
1
Faculty of Pharmacy, University of Zawia, Az Zawiyah, Libya.
2
School of Medical Science & Technology, IIT Kharaghpur, India.
3
Department of Mechanical Engineering, Vignan University, Guntur, India.
ABSTRACT
Analytical method development is a vital part of pre-formulation and formulation development research.
Development of validated, robust, cost-effective methodologies for routine drug estimations is the urgent need
of the pharmaceutical R&Ds. Quality is an essential attribute in any pharmaceutical product and impurity
profiling offers a broad scope with the changed perspectives of the research scenario. The present work aims to
devise a validated UV-spectroscopic and RP–HPLC method for estimations of SVN in bulk and formulations
and impurity profiling of SVN related impurities at the specification limit with the aid of response surface
methodology. The percent assay for SVN determined by UV method was 100.4±0.15 and mean %recovery was
achieved to be 99.87±0.19. The percent assay of SVN by RP-HPLC method was found to be 100.14±0.1. The
mean percent recovery at different spike levels (50–150%) ranged from 97.3±0.01–100.5±0.02 and the %RSD
of assays at lower and higher spike levels were 1.4 and 0.3 respectively. The linearity data of impurities (A–G)
at different spike levels (25, 50, 100, 125 and 200 µg) showed a high correlation coefficient of 0.99 in all cases.
Percent mean recovery of impurities (A–G) at different spike levels comply the acceptance criterion. All other
validation parameters also comply within the range of acceptable limits. The impurities were well separated
with good resolution and peak shape, good retention times. The robustness of the developed HPLC method and
that of impurity profiling was optimized applying Box-Benkhen experimental design approach.
Keywords: Method development, Impurity profiling, Validation, Box-Benkhen, Experimental design.
INTRODUCTION
Analytical method development plays a pivotal role
in statuary certification of drugs and their
formulations either by the industry or by the
regulatory authorities. It is an integral part of
preformulation and formulation development
research. Quality assurance and quality control
departments of pharmaceutical industries are
largely responsible in bringing out safe, effective
dosage formulations. The current good
manufacturing practices (CGMP) and the Food
Drug Administration (FDA) guidelines insist for
adoption of analytical methodologies which are
simple, rapid, cost effective, and robust thus
providing results with great accuracy and precision.
Sophisticated hyphenated techniques are in vogue.

PrakashKatakam, et al / Int. J. of Pharmacy and Analytical Research Vol-3(2) 2014 [202-213]
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But cost factor, analyte extraction from respective
sample matrices and complicated sample
preparation steps, time consumption, difficulty in
operation and error in recovery limit their routine
applications[1-3].Impurity profiling is a broad term
which encompasses identification, quantitative
determination and structural elucidation of
impurities with the aid of spectroscopic or
chromatographic techniques or the utilizations of
latest developed hyphenated methods. ICH defines
impurities as “any chemical compound of the
medicinal product which is not the chemical entity
defined as the active substance or as an excipient in
the product.” According to its guidelines, the
threshold limit for any impurity is lower at 0.1%
for drugs used in dosages more than 2 gm/day and
those dosed at less than 2 gm/day, the limit is
below 0.05%.
Quality is an essential attribute in any
pharmaceutical product greatly determined by the
content of active ingredient present in it. In the
pharmaceutical world, impurities are considered as
any material other than the active pharmaceutical
ingredient (API) or excipients; may be of organic
or inorganic origin, may arise from varied sources
like process related drug substances (starting
material, intermediate or drug product), impurity in
starting materials, due to degradation of drug
substances, or unwanted excipient-interactions,
contaminations of some reagents and catalysts,
presence of enantiomeric impurities and some
impurities may be due to environmental factors.
The presence of these unwanted chemicals, even in
small amount, may influence the efficacy and
safety of the pharmaceutical products and can
precipitate adverse and toxic drug reactions in
patients after consumption.[1-5] With the
tremendous advancements of analytical
technologies and changed perspectives of the
research scenario, not only detection of active
constituents but a detailed profiling of impurities
offers a broad scope of research in pharmaceutical
and other bio-allied fields.
Simvastatin (SVN), chemically butanoic acid, 2,2-
dimethyl-,1,2,3,7,8,8a hexahydro-3,7-dimethyl-8-
[2(tetrahydro-4-hydroxy-6-oxo-2H pyran-2-yl)-
ethyl]-1-napthalenyl ester (Fig.1); a lipid lowering
agent derived synthetically from fermentation
products of Aspergillusterreus. Pharmacologically,
SVN on oral ingestion is an inactive lactone,
hydrolyzed to β-hydroxy acid leading to the
inhibition of HMG-CoA reductase (3-hydroxy-3-
methyl glutaryl coenzyme A) which catalyzes the
conversion of HMG-CoA to mevalonate which is
the early rate limiting step in cholesterol
biosynthesis. SVN can be obtained in various
synthesis pathways. During synthesis apart from
the main reaction, unwanted side reactions are one
of the major causes of impurities. In case of SVN,
lovastatin and analogues are one of the major
sources of impurities. The possible degradation
pathways of SVN are provided in Fig.2. The major
process related impurities of SVN which may be
either due to synthetic or degradation pathways are
presented in Fig.3. The impurities of simvastatin
are: (A) (3R,5R)-7-[(1S,2S,6R,8S,8aR)-8-[(2,2-
dimethylbutanoyl)oxy]-2,6-dimethyl-1,2,6,7,8,8a-
hexahydronaphthalen-1-yl]-3,5-dihydroxy
heptanoic acid (hydroxyacid); (B) (1S,3R,
7S,8S,8aR)-8-[2-[(2R,4R)-4-(acetyloxy)-6-oxotetra
hydro-2H-pyran-2-yl]ethyl]-3,7-dimethyl-1,2,3,7,
8,8a-hexahydronaphthalen-1-yl2,2-dimethyl
butanoate (acetate ester);(C) (1S,3R,7S,8S,8aR)-
3,7-dimethyl-8-[2-[(2R)-6-oxo-3,6-dihydro-2H-
pyran-2-yl]ethyl]-1,2,3,7,8,8a-hexahydro
naphthalen-1-yl2,2-dimethylbutanoate (anhydro
simvastatin); (D) (2R,4R)-2-[[(1S,2S,6R,8S,8aR)-8-
[(2,2-dimethylbutanoyl)oxy]-2,6-dimethyl-1,2,6,
7,8,8a-hexahydronaphthalen-1-yl]ethyl]-6-
oxotetrahydro-2H-pyran-4-yl(3R,5R)-7-[(1S,2S,
6R,8S,8aR)-8-[(2,2-dimethylbutanoyl)oxy]-2,6-
dimethyl-1,2,6,7,8,8a-hexahydronaphthalen-1-yl]-
3,5-dihydroxyheptanoate (dimer); (E) R1=CH3,
R2=H: (1S,3R,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy
-6-oxotetrahydro-2H-pyran-2-yl]ethyl]-3,7-
dimethyl-1,2,3,7,8,8a-hexahydronaphthalen-1-
yl(2S)-2-methylbutanoate (lovastatin);(F) R1=H,
R2=CH3:(1S,3R,7S,8S,8aR)-8-[2-[(2R,4R)-4-
hydroxy-6-oxotetrahydro-2H-pyran-2-yl]ethyl]-
3,7-dimethyl-1,2,3,7,8,8a-hexahydronaphthalen-1-
yl(2R)-2-methylbutanoate (epilovastatin); and (G)
(1S,7S,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6-oxo
tetrahydro-2H-pyran-2-yl]ethyl]-7-methyl-3-
methylene-1,2,3,7,8,8a-hexahydronaphthalen-1-
yl2,2-dimethylbutanoate.
Fig.1: Structure of Simvastatin
Factorial experiments find extensive applications in
all research fields which allows investigation of
multiple factors simultaneously and also
examination of one factor at different levels of the
other factor or factors. The Box-Behnken
experimental design (BBD), developed by Box and
Behnken in 1980 is a useful response surface
methodology, where the level of one of the factors
is fixed at centre level while combinations of all
levels of the other factors are applied.[6-8]
Extensive literature surveys have reported some of

204
the spectroscopic and chromatographic methods for
estimations of SVN in bulk and formulations.[9-15]
There are reporting’s of impurity profiling of SVN
in USP (United States Pharmacopeia) and EP
(European Pharmacopeia). Some researchers
reported about FTIR, UPLC and hyphenated
techniques.[16,17] But current research aims to
devise a validated UV-spectroscopic and RP–
HPLC method for estimations of SVN in bulk and
formulations and impurity profiling of SVN related
impurities at the specification limit with the aid of
response surface methodology.
Fig.2: Possible degradation pathways of Simvastatin
Fig.3: Impurities of simvastatin: (A) hydroxyacid (B) acetate ester; (C) anhydrosimvastatin (D) dimer; (E) lovastatin;
(F) epilovastatin; and (G) dimethylbutanoate
MATERIALS AND METHODS
Reagents
Simvastatin (SVN) was a kind gratis of Salius
Pharma Pvt. Ltd. Mumbai, India. Impurities (A–G)
were obtained from Sigma Aldrich, Mumbai, India.
Di Sodium hydrogen phosphate, potassium
dihydrogen phosphate, acetonitrile, triple distilled
water, methanol and other reagents used were
either AR grade or HPLC grade, purchased from
Merck (Mumbai) and Sigma (India).
Instruments
HPLC (Equipped with a Agilent technologies 1100
series VWD detector); UV-visible spectrophoto-
meter (Thermo Scientific, Aquamate Plus, India),
Electronic balance (Shimadzu, Japan), Sonicator
(Cyber labs, India), pH meter (Datla instruments,
DI-45, India), Syringe Filters (ZodiacLife Sciences,
India), Nylon filters; water bath (Lab Companion,
Mumbai, India).

205
Software
Experimental design, data analysis and surface
plots were performed by using Design Expert Trial
version 7.0 and Matlab version 12.0.
UV method development
For the UV estimation of SVN methanol was used
as the main solvent for preparing standard and
stock solutions. A buffer solution was used as the
diluent. The buffer solution was prepared by
mixing a solution of 13.6 gm of Potassium
dihydrogen phosphate in 1000 mL of distilled
water designated as solution A. Another solution-B
was prepared by dissolving 35.8 gm of disodium
hydrogen phosphate in 1000 mL distilled water.
Next, 20 mL of solution-B and 1000 mL of
solution-A was mixed which was used as diluent.
The standard and stock solutions of SVN were
prepared in methanol and further dilutions of
working standards were achieved by dilutions with
the above diluent. A solution of SVN of 10 µg/mL
concentration was scanned in the wavelength range
of 200–400 nm and the drug showed absorbance
maxima at 238 nm. The standard calibration curve
was prepared with aliquots in the concentration
range of 2.5–15µg/mL using reagent as the blank.
Validation of the method
The above method was validated as per ICH
guidelines in terms of linearity, accuracy, precision,
Limit of Detection (LOD) and Limit of
Quantification (LOQ).
The linearity range for the estimation of SVN by
UV was determined by preparing aliquots in the
concentration range of 2.5–15 µg/mL and
absorbances measured at 238 nm. Calibration
curves (concentrations vs absorbance) were plotted
and R2
value not less than 0.99 was regarded as
acceptance criterion.
Accuracy of the proposed method was ascertained
by recovery studies using standard addition method
where known quantity of standard drug was mixed
with formulation sample at three different levels of
50, 100 and 150% and percent recovery for SVN in
the range of 98102% were regarded as acceptance
criterion.The precision was studied by inter and
intra-day variations in the test method of SVN and
expressed as percent relative standard deviation
(%RSD) where the values should not be greater
than 2%.The LOD and LOQ parameters were
determined from the calibration curves basing on
the formulae:
LOD = 3.3 σ / S and LOQ = 10 σ / S
Assay of SVN
Ten tablets were weighed accurately and then
triturated thoroughly. Accurately weighed portion
of powder equivalent to 5 mg of SVM was
transferred to 1000 mL volumetric flask; dissolved
in 20 mL of methanol and diluted with diluent. It
was centrifuged for 20 min. Absorbance was
determined at 238 nm. Amount of SVN was
determined using the formula:
O.D.Standard
factornDissolutioconc.StandardO.D.Test
presentAmount


100
claimLabel
presentAmount
Purity% 
Estimation of SVN by RPHPLC
The current research represents a new stability
indicating, validated RPHPLC method
development. The buffer was prepared by
dissolving 35.49 gm of disodium hydrogen
phosphate in 1000 mL of distilled water, pH
adjusted to 4.5 with dilute ortho phosphoric acid,
filtered through 0.45µm nylon membrane filter and
degassed. The diluents consisted of a mixture of
methanol: water: buffer: BHA in the ratio of
63:35:1:1 well sonicated and degassed.
Optimization of chromatographic conditions
Before proceeding to the optimized RPHPLC
chromatographic conditions for SVN estimations,
three trails were conducted with varying ratios of
methanol: water: buffer mobile phase
compositions. Amongst three trials injection
volume (10 µL), flow rate (1 mL/min), column
temperature (40ο
C), and detector wavelength (238
nm) were kept constant. Run time varied between
15–18 min. With mobile phase ratio of 40:40:20
(methanol: water: buffer), a comparatively long
retention time was observed. On changing the ratio
to 50:45:5, peak tailing was observed with delayed
retention time. With mobile phase ratio of 55:40:5,
peak tailing was observed. Satisfactory results were
obtained with a mobile phase ratio of 60:36:4 with
a run time of 15 min.
Thus the optimized chromatographic conditions for
SVN estimations were obtained with isocratic
separation mode in a C18 column (150 × 3.9 mm,
5) using a degassed mixture of methanol: water:
buffer in the ratio of 60:36:4; injection volume (10
µL), flow rate (1 mL/min) and run time (15 min), at
column temperature (40ο
C), and detector
wavelength (238 nm).
The stock solution was prepared by dissolving 100
mg of SVN in 100 mL of methanol. From this
solution, 10 mL was pipette into 100 mL
volumetric flask, mixed well with 30 mL diluents
and volume adjusted with the same. This served as
the working standard solution.
Twenty tablets were accurately weighed and the
average weight was calculated. The tablets were
triturated well and power equivalent to 10 mg were
transferred to 100 mL volumetric flask containing

206
30 mL of diluent. The solution was sonicated for
about 20 min and volume adjusted with the diluent.
Next the solution was centrifuged at 4000 rpm for
15 min. This centrifuged solution served as the test
solution.
System suitability
The solution for testing system suitability was
prepared by transferring 1 mg of Lovastatin (LVN)
standard in 100 mL volumetric flask to which 10
mL of standard stock solution and 40 mL of
diluents was added. The mixture was sonicated
well and volume adjusted with diluents.
Acceptance criteria lies in the fact that the % RSD
for the retention times of principal peak from 10
replicate injections of system suitability solution
should be not more than 2.0 %; resolution between
SVN and LVN should not be less than 3;
theoretical plate number and tailing factor of SVN
should not be more than 2000 and not less than 2
respectively.
Assay of SVN
The percent assay of SVN was determined using
the formula:
100
claimLabel
WtAvg.
100
P
WT
DT
DS
WS
AS
AT
%Assay 
where AT is area of the test substance, as is the
area of standard substance, WS and WT are the
weights of standard and test substance respectively
and DS, DT are the dilutions of standard and test
respectively; P is the percent purity. The assay of
SVN should be not less than 97.0% and not more
than 103.0%.
Method validation
The proposed RPHPLC method was validated as
per ICH guidelines. Linearity of the method was
determined using solutions prepared in the
concentration range of 50–150% from SVN
working standard. Accuracy of the proposed
method was ascertained by recovery studies using
standard addition method, where the spike level of
drug substance with placebo were in concentrations
of 50, 75, 100, 125 and 150 µg and the acceptable
criteria of percent recovery for SVN were set
between 95105%. Precision was studied in terms
of repeatability (system precision), where six
samples prepared on SVN tablets as per test
procedure were assayed and %RSD of assay results
were calculated which should not be more than
2.Intermediate precision study or ruggedness of
experimentation was carried out by different
analyst, on different instrument, using different
columns and on different days. The %RSD of assay
results should not be more than 2. Specificity of the
method is tested by observing placebo interference
at the retention time of SVN.
For measurement of robustness, BBD approach of
RSM was employed to evaluate the effect of three
independent factors, viz. flow rate (X1), mobile
phase composition (X2) and pH of mobile phase
(X3) on responses like tailing factor of SVN peak
(Y1), resolution between LVN and SVN peak (Y2),
%RSD of peak areas of SVN after five replicate
injections (Y3) [8-10]. Basing on previous
experimentations and knowledge, the ranges of
values used in the design are as follows: flow rate
(X1): 0.8–1.2 mL/min; mobile phase composition
(X2): 90% and 110%; and buffer pH (X3): 4.3–4.7.
Compatibility with system suitability values under
altered conditions was tested to check if they meet
the acceptance criteria. The polynomial equation
generated for the study was:
whereX1= mobile phase composition, X2= flow
rate, X3= pH of mobile phase, X4= variation in
column oven temperature.
Bench top stability of SVN standard and test
solutions were determined at initial, first, second
and seventh day against freshly prepared standard
each time. Difference in percent assay results not
more than 3 from initial value are regarded as
acceptable criteria. Similarity factors for standard
preparations in range of 0.98–1.02 are the
acceptance limit. Bench top stability of mobile
phase tested initially, first and second day is
considered stable if system suitability criteria are
within acceptable limits.
Interference from the products of forced
degradation studies conducted by acid hydrolysis,
base hydrolysis, peroxide oxidation, degradation by
sun light and UV radiations, heat degradation,
humidity and water stress conditions were
evaluated for peak purity. Acceptable criterion is
satisfied if purity angle be less than purity
threshold of SVN peak and should not have any
flag in purity results.
Impurity profiling by RP–HPLC method
The optimized chromatographic conditions for
impurity profiling include gradient elution through
Lichrosphere RP18 column (250 × 4.6mm, 5µm)
using o-phosphoric acid as mobile phase A and
acetonitrile (ACN) as mobile phase B. A degassed
mixture of buffer: ACN (20:80 v/v) was used as
diluents. Injection volume was 20μL with a flow
rate of 1.5 mL/min. Column temperature was
maintained at 30ο
C, the detector wavelength set at
238 nm with a total run time of 50 min.
The buffer solution was prepared by dissolving
1.41 gm of potassium dihydrogen phosphate in
1000 mL water, well sonicated and the pH was

207
adjusted to 4 with dilute orthophosphoric acid. It
was filtered through 0.45µ membrane filter. For
preparation of sample solution, 20 tablets were
weighed accurately and triturated well. A 50 mg of
SVN was transferred to 50 mL of volumetric flask,
20 mL of diluent added, sonicated for 10 min and
volume adjusted with diluent. The solution was
centrifuged at 4000 rpm for 10 min and the
supernatant was used for analysis.
The standard solution was prepared by dissolving
20 mg of SVN working standard in 15 mL of
diluents taken in 100 mL of volumetric flask,
sonicated for dissolution of SVN and volume
adjusted with the diluent. The solution for placebo
was prepared by dissolving placebo powder
equivalent to 50 mg of SVN in 20 mL of diluent
taken in 50 mL volumetric flask, sonicated for 10
min and volume adjusted with diluent. The solution
was centrifuged at 4000rpm for 10min and the
supernatant was used for further analysis.
The impurities stock solution were prepared by
dissolving accurately weighed 20 mg of impurities
in 20 mL of diluents taken in 50 mL of volumetric
flask, sonicated well for 10 min and volume
adjusted with the diluent. The spiked sample
solution was prepared by transferring 20 mg of
SVN working standard in 100 mL volumetric flask
to which 3 mL of impurities stock solution was
added, sonicated and volume adjusted with diluent.
Next to inject separately 20μL of diluents (as
blank) twice and standard, test and placebo
preparations six times in to the HPLC system and
the relative response factors (RRF) and relative
retention time (RRT) of known impurities with
respect to SVN were determined.
System suitability
The solution for system suitability was prepared by
dissolving 20 mg of SVN working standard in 40
mL of diluent taken in 100 mL volumetric flask;
sonicated for 10 min and volume adjusted with
diluent. In another 25 mL volumetric flask, 12.5
mg of lovastatin working standard was dissolved in
diluent, sonicated and volume adjusted with the
same. A 1 mL of each of the two solutions were
pipetted into another 25 mL volumetric flask,
mixed well and volume adjusted with the diluent.
The solution is injected into the HPLC system. The
acceptance criteria complies when theoretical plate
count of LVN and SVN peak is not less than 8000;
resolution between SVN and LVN is not less than 6
and %RSD of LVN and SVN after six replicate
injections should not be more than 2.
Method validation
The developed RP–HPLC method for detection and
segregation of impurities A–G was validated as per
ICH guidelines [4–7]. The linearity of the method
for impurity profiling was studied by injecting
impurities and SVN with concentration ranging
from LOQ to 200% and R2
value not less than 0.99
and %RSD of peak areas of the solution not more
than 2% was regarded as the acceptance criterion.
Accuracy of the proposed method ascertained by
recovery studies was carried out in triplicate using
standard addition method where the test
preparation was spiked with impurities stock
solutions in the concentrations of 50, 75, 100, 125
and 200 µg and the acceptable criteria for percent
recovery of impurities were set at 85–115%.
System precision or repeatability of the method
was evaluated by analyzing six samples prepared
by spiking test preparations with impurity blend
solutions to get each impurity target concentration
and the %RSD of relative retention times(RRT)
should not be more than 2%. Intermediate precision
or ruggedness was carried out by different analyst,
on different instrument and on different days.
The specificity of the method was studied to see if
there is any interference of placebo or interferences
from known impurities at the retention times of test
impurities.Interference of degradation products due
to acid hydrolysis, base Hydrolysis, peroxide
oxidation, degradation by Sun light and UV
radiations, thermal and humidity degradations were
also conducted.
For measurement of robustness, BBD approach of
RSM was employed to evaluate the effect of
independent factors, viz.mobile phase composition
(90% and 110%); flow rate (1.3 mL/min and 1.7
mL/min); and pH (3.8 and 4.2) on responses like
resolution between LVN and SVN peak (Y1); RSD
of peak areas of LVN from six replicate injections
(Y2); RSD of peak areas of SVN from six replicate
injections (Y3). The polynomial equation generated
for the study was:
whereX1=flow rate, X2=mobile phase composition,
X3=pH of mobile phase composition.
LOD and LOQ for SVN impurities and SVN were
determined basing on signal to noise ratio and the
S/N ratio near to 3.0 and 10.0 was considered for
LOD and LOQ respectively.
RESULTS AND DISCUSSION
UV method development
SVN showed absorption maxima at 238 nm and
linearity was achieved in the concentration range of
2.5–15 µg/mL with R2
value of 0.99
(y=0.06x+0.000). The percent assay for SVN was
determined to be 100.4±0.15. The mean %recovery
was achieved to be 99.87±0.19. Precision studies of

208
intra and inter day assays expressed in terms of
%RSD were found to be 0.2 and 0.3 respectively.
The LOD and LOQ were found to be 0.165 µg/mL
and 0.5 µg/mL respectively.
RP–HPLC method development
The linearity of the method was demonstrated in
the concentration range of 18–225 µg/mL with R2
value of 0.99 (Fig.4). The percent assay of SVN
was found to be 100.14±0.1. Results of system
suitability parameters have shown that tailing
factor of SVN peak was 1.1; resolution between
SVN and lovastatin (LVN) peak was 4.2 and
%RSD of peak areas of SVN from five replicate
injections were 0.1; values of all parameters falling
within the acceptance criterion.
The mean percent recovery at different spike levels
(50–150%) ranged from 97.3±0.01–100.5±0.02 and
the %RSD of assays at lower and higher spike
levels were 1.4 and 0.3 respectively. Regarding
precision, the system precision or repeatability of
the method showed %RSD of peak areas to be 0.2
and it is 0.1 in case of intermediate precision or
ruggedness. In both cases the values are less than
2%. Results of method robustness and specificity
due to physical and chemical degradation studies
are presented in Table 1 and 2. The surface and
contour plots of the method robustness is provided
in Fig.5-6.
Bench top stability of SVN standard and test
preparations have shown the initial and final assay
results to be 102.3 and 103.0 respectively and the
similarity factors for standard preparations is 0.99–
1.00; all values coming within acceptance criterion.
Considering the bench top stability of the mobile
phase, initial and final values of %RSD of peak
areas of SVN after five replicate injections were
0.1 and 0.2 respectively, the tailing factor value of
1.1 was same in both cases and the resolution of
SVN and LVN peaks were 4.2 initially and 4.0
after 2 days. Thus all the system suitability
parameters of bench top stability of mobile phase
falls within the acceptance criterion.
Impurity profiling by RP–HPLC method
Results of system suitability parameters have
shown that resolution between LVN and SVN
peaks was 10, %RSD of peak areas of LVN after
six replicate injections were 0.1 and the same
following SVN injection was also 0.1; all values
meeting the acceptance criterion. The linearity data
of impurities (A–G) at different spike levels (25,
50, 100, 125 and 200 g) showed a high correlation
coefficient of 0.99 in all cases. Percent mean
recovery of impurities (A–G) at different spike
levels (50%, 75%, 100%, 150% and 200%) carried
out in triplicate were found to be 106.25 ± 0.01,
108.19 ± 0.02, 107.31 ± 0.01, 107.81 ± 0.01,
105.05 ± 0.01 for impurity A at respective spike
levels; for impurity B it was 100.65 ± 0.01, 100.66
± 0.01, 98.18 ± 0.01, 98.29 ± 0.01, 97.96 ± 0.01;
for impurity C values are 97.05 ± 0.01, 97.68 ±
0.01, 96.11 ± 0.02, 97.10 ± 0.02, 94.65 ± 0.02; for
impurity D values are 91.44 ± 0.01, 92.88 ± 0.01,
90.85 ± 0.02, 94.32 ± 0.01, 91.26 ± 0.01; for
impurity E it was 101.03 ± 0.01, 101.17 ± 0.01,
100.65 ± 0.01, 100.15 ± 0.01,100.68 ± 0.01; for
impurity F it was 100.03 ± 0.01, 100.19 ± 0.01,
99.65 ± 0.01, 99.15 ± 0.01, 99.68 ± 0.01; for
impurity G values are 92.65 ± 0.01, 91.15 ± 0.01,
91.74 ± 0.01, 92.78 ± 0.01, 93.15 ± 0.01. The
values of the recovery studies in all cases comply
within the range of acceptance criterion (85–
115%).
The %RSD values of System precision or
repeatability of the method for impurities (A–G) in
terms of relative retention times (RRT) and
%impurities were 0.02 and 0.98 respectively for
impurity A; 0.116 and 0.47 respectively for
impurity B; 0.119 and 0.32 respectively for
impurity C; 0.153 and 0.43 respectively for
impurity D; 0.033 and 0.4 respectively for impurity
E; 0.114 and 0.41 respectively for impurity F;
0.017 and 0.5 respectively for impurity G.
Intermediate precision or ruggedness with two
different analyst have shown that resolution
between LVN and SVN peaks were 10 and 9 for
analyst 1 and 2 respectively; %RSD of peak areas
of LVN with two analysts were 0.1 in both cases;
and %RSD of peak areas of SVN with two analysts
were 0.1 in both cases. Thus in all cases the values
comply with the standard acceptance criterion.
The LOD and LOQ values of impurities with
corresponding signal to noise ratio is provided in
Table 3. Specificity of the method in terms of
interferences of the impurities and physical and
chemical degradation studies are provided in
Tables 4 and 5. Results of robustness of the
impurity profiling method are presented in Table 6
and the corresponding surface and contour plots in
Fig.7-8.
Fig.4: Calibration curve for HPLC method
development

209
Table 1: Robustness of the RP–HPLC method development of simvastatin
Robustness parameters System suitability parameters
TF of SVN
peak
Resolution
between SVN
and LVN peak
%RSD of peak
areas of SVN
Mobile phase composition
90% 1.1 5.0 0.2
100% 1.1 4.2 1.0
110% 1.1 3.0 0.2
Flow rate (mL/min)
0.8 1.1 4.8 0.1
1.0 1.1 4.1 0.1
1.2 1.1 4.5 0.1
pH of mobile phase
4.3 1.1 3.0 0.2
4.5 1.1 4.1 0.1
4.7 1.1 3.0 0.2
Table 2: Specificity of the method by physical and chemical degradation studies
Stress condition Purity
angle
Purity
Threshold
Purity
Flag
UV light stress 7days 0.171 0.413 No
Acid Degradation
(0.1N HCl at 60°C for 30 min)
0.132 0.331 No
Base degradation
(0.1N NaOH at 60°C for 30 min)
0.126 0.325 No
Peroxide degradation
(1% H2O2 at 60°C for 30 min)
0.107 0.309 No
Thermal degradation
(at 105°C for 6 hr)
0.178 0.399 No
Humidity degradation
(90% RH for 7 days)
0.191 0.419 No
Table 3: LOD and LOQ values of impurities
Name of the
impurities
Parameters Signal to Noise ratio
LOD
% Impurity
LOQ
% Impurity
LOD LOQ
Impurity-A 1.1 1.2 4.097 11.28
Impurity-G 0.0028 0.0093 4.499 11.68
Impurity-E 0.0016 0.0053 4.413 12.09
Impurity-B 0.0013 0.0042 4.692 11.33
Impurity-C 0.0014 0.0048 4.169 10.54
Impurity-D 0.0026 0.0088 4.022 9.03
Simvastatin 0.0005 0.004 2.349 10.517

210
Fig.5: Contour plots of HPLC method robustness
showing the responses of independent variables like
mobile phase composition, flow rate and pH of mobile
phase
Fig.6: Surface plots of HPLC method robustness
showing the responses of independent variables
like mobile phase composition, flow rate and pH
of mobile phase

211
Fig.7: Contour plots of impurity profiling
showing the responses (resolution between LVN
and SVN peak; RSD of peak areas of LVN and
RSD of peak areas of SVN) of independent
variables (mobile phase composition, flow rate
and pH of mobile phase).
Table 4: Specificity of the method due to
impurity interference
Name of impurity RT
Impurity A 8.207
Impurity-E 10.867
Impurity-F 11.178
Impurity-G 12.316
Impurity-B 23.520
Impurity-C 23.687
Impurity-D 37.123
Simvastatin 14.669
Table 5: Specificity of the impurity profiling
method by physical and chemical degradation
studies
Stress condition Purity
angle
Purity
Thres-
hold
Purity
Flag
UV light stress
(7 days)
0.318 0.761 No
Acid Degradation
(0.1N HCl at
60°C for 30 min)
0.312 0.635 No
Base degradation
(0.1N NaOH at
60°C for 30 min)
0.375 0.754 No
Peroxide
degradation
(1% H2O2 at 60°C
for 30 min)
0.294 0.452 No
Thermal
degradation
(at 105°C for 6 h)
0.374 0.547 No
Humidity
degradation
(90% RH for 7
days)
0.302 0.712 No

212
Table 6: Robustness of the RP–HPLC method
for impurity profiling
Robustness
parameters
System suitability parameters
%RSD
of peak
areas of
SVN
Resolution
between
SVN and
LVN peak
%RSD
of peak
areas of
LVN
Mobile phase
composition
90% 0.2 9.0 0.12
100% 0.1 10 0.1
110% 0.1 9.3 0.15
Flow rate
(mL/min)
1.3 0.3 11 0.2
1.5 0.1 10 0.1
1.7 0.2 9 0.3
Mobile phase
pH
3.8 0.1 10 0.1
4.0 0.1 9 0.1
4.2 0.2 9 0.15
CONCLUSION
The UV and the RP-HPLC method developed were
found to be simple, rapid and economical. The two
validated methods were found suitable for the
routine quality control of Simvastatin in bulk drugs
and pharmaceutical dosage forms both by small
and large scale pharmaceutical industries. Impurity
profiling is of crucial importance in drug synthesis,
quality control and storage, as depicted by ICH
guidelines and stated in different Pharmacopeia
specifications. The developed and optimized RP-
HPLC methods by BBD approach was found to be
simple, accurate, robust, precise, specific and
suitable in terms of tailing factor, theoretical plate
count. The impurities were well separated with
good resolution and peak shape, good retention
times finding applications in impurity profiling of
simvastatin related substances.
CONFLICT OF INTEREST: Conflict of interest
is nil.
Fig.8: Surface plots of impurity profiling showing the
responses (resolution between LVN and SVN peak;
RSD of peak areas of LVN and RSD of peak areas of
SVN) of independent variables (mobile phase
composition, flow rate and pH of mobile phase

213
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An Experimental Design Approach for Method Development and Impurity Profiling of Simvastatin by UV Spectrophotometric and RP–HPLC Methods

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An Experimental Design Approach for Method Development and Impurity Profiling of Simvastatin by UV Spectrophotometric and RP–HPLC Methods