Asian Journal of Chemistry Vol. 21, No.

6 (2009), 4294-4300

Gradient Stability Indicating RP-HPLC Method for

Impurity Profiling of Simvastatin in Tablet Dosage Forms

D. GOWRI SANKAR*, R. KONDAVENI, T.V. RAGHAVA RAJU and M. VAMSI KRISHNA

Department of Pharmaceutical Analysis and Quality Assurance, University College of
Pharmaceutical Sciences, Andhra University, Visakhapatnam-530 003, India
E-mail: [email protected]

Gradient, reversed phase high performance liquid chromatographic

(RP-HPLC) method was developed for quantitative estimation and valida-
tion of simvastatin impurities which are generated during formulation
and storage of simvastatin in tablet dosage forms. The chromatographic
separation was achieved on column intersil ODS (150 mm × 4.6 mm, 5 µm)
by following gradient flow using mobile phase A and B containing 0.1 %
phosphoric acid, acetonitrile in the ratio of 47:53 and 90:10, respec-
tively. Flow rate was 1.0 mL/min. The photo diode array detector was
operated at 238 nm. Forced degradation studies were performed on tablets
powder which contain simvastatin using acid hydrolysis, base, peroxide,
water and UV, thermal, sunlight, humidity degradations. The method
was validated for specificity, linearity, precision, accuracy and limit of
quantification. The degree of linearity of the calibration curves, the
recoveries of simvastatin impurities, the limit of detection and quantifi-
cation for the HPLC method were determined. The method was found
to be simple, specific, precise, accurate and reproducible. The method
was applicable for the quality control of commercial simvastatin tablets
to quantify the drug and its related substances and to check the formulation
content uniformity.

Key Words: Simvastatin, Impurity profiling, Reversed phase HPLC.

INTRODUCTION
Simvastatin1 is chemically [(1S,3R,7R,8S,8aR)-8-[2-[(2R,4R)-4-hydroxy-6-
oxo-oxan-2-yl]ethyl]-3,7-dimethyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl]-2,2-
dimethylbutanoate) is a hypolipidemic drug belonging to the class of pharmaceuticals
called ‘statins’. Simvastatin is a synthetic derivate of a fermentation product of
Aspergillus terreus. It is used to control hypercholesterolemia (elevated cholesterol
levels) and to prevent cardiovascular disease. The active ingredient simvastatin has
five impurities (Fig. 1) in this process. These impurities may be present in small
quantities and reduce the quality of simvastatin. Therefore separation and quantification
of simvastatin and its impurities are quiet important not only for quality assurance
but also for monitoring reactions involved in process development. Literature survey
revealed that different UV2,3, HPLC4-6 and GC-MS7,8 methods for determination of
impurities of simvastatin were reported. The proposed method is simple, fast, accurate
and precise for estimation of simvastatin impurities in tablets.
Vol. 21, No. 6 (2009) RP-HPLC Method for Impurity Profiling of Simvastatin 4295

Simvastatin Hydroxy acid Lovastatin

Acetate ester Dehydro simvastatin

Fig. 1. Structures of simavastatin and its impurities

EXPERIMENTAL
The waters LC system with a photo diode array detector was used for method
development and forced degradation studies. The output signal was monitored and
processed using EMPOWER software.
The waters LC system, used for method validation was water alliance HPLC
consisted of 2695 separation module, variable wavelength programmable UV detector
waters 2996, EMPOWER software and intersil ODS 3V column 150 mm × 4.6 mm,
5 µm particle size was used.
Chromatographic conditions: The mobile phase A was made by first preparing
a 0.1 % v/v orthophosphoric acid (1 mL of orthophosphoric acid into a 1000 mL of
milli-Q-water). 470 volumes of this 0.1 % v/v orthophosphoric acid in water was
mixed with 530 volumes of acetonitrile to yield mobile phase A.
The mobile phase B was made by first preparing a 0.1 % v/v orthophosphoric
acid (1 mL of orthophosphoric acid into a 1000 mL of acetonitrile). 900 volumes of
this 0.1 % v/v orthophosphoric acid in acetonitrile was mixed with 100 volumes of
Milli Q water to yield mobile phase B.
Separations were performed on a intersil ODS 3V column 150 mm × 4.6 mm,
5 µm particle size. The mobile phase flow rate was 1.0 mL/min (gradient) with 27 ºC
column temperature, 20 µL injection volume and UV detection at 238 nm. Run
time of 75 min.
4296 Gowri Sankar et al. Asian J. Chem.

Gradient program:
Time Mobile phase A% Mobile phase B%
00.0 100 000
15.0 100 000
35.0 016 084
40.0 016 084
42.0 000 100
62.0 000 100
64.0 100 000
75.0 100 000

Procedure
Diluent preparation: Mix acetonitrile and phosphate buffer (dissolve 1.36 g
of potassium dihydrogen phosphate in 1000 mL milli Q water and adjust pH of
solution to 4.0 with phosphoric acid) in the ratio of 60:40. Adjust pH of the solution
to 4.0 with orthophosphoric acid.
Standard solution: Weigh accurately 80 mg of simvastatin working standard
in a 100 mL volumetric flask. Dissolve and dilute to volume with diluent. Transfer
5 mL of above solution to 50 mL volumetric flask and dilute to volume with diluent.
Transfer 5 mL of above solution into a 100 mL volumetric flask, add 10 mL 0.1 %
phosphoric acid and dilute to volume with diluent.
Sample preparation: The test solution was prepared by taking 10 tablets (400 mg)
in 500 mL volumetric flask. Add 50 mL of 0.1 % phosphoric acid and sonicate.
Dilute to volume with diluent and mix well to get 0.8 mg/mL of simvastatin.
Method validation: The precision of test method was evaluated by analyzing
6 samples prepared by spiking test preparation with simvastatin impurities blend
solution to get 0.5 % of acetate ester, 1.0 % lovastatin, 1.5 % of simvastatin dimer,
dehydro simvastatin and 2.5 % of hydroxy acid. The relative standard deviation
was calculated for the response of each impurities.
Limit of detection for simvastatin impurities were established by identifying
the concentration, which gives signal to noise ratio of about 3. Limit of quantification
for simvastatin impurities were established by identifying the concentration, which
gives signal to noise ratio of about 10. Precision and accuracy studies were also
carried out at the limit of quantification (LOQ) level by injecting six individual
preparations of impurities and calculated the % RSD of the area.
Linearity test solution for related substance method was prepared from the
impurities stock % of the specification. Plotting the peak areas solution at 10 concen-
tration levels from LOQ to 150 of impurities versus its corresponding concentration.
Calculate the slope, Y-intercept and correlation coefficient for each impurity.
An accurate study of simvastatin impurities from spiked samples of simvastatin
test preparation was conducted. Sample were prepared in triplicate by spiking test
preparation with 25, 50, 75, 100 and 150 % to the target concentration of simvastatin
Vol. 21, No. 6 (2009) RP-HPLC Method for Impurity Profiling of Simvastatin 4297

impurities blend solution. (0.5 % of acetate ester, 1.0 % lovastatin, 1.5 % of

simvastatin dimer, dehydro simvastatin and 2.5 % of hydroxy acid). % Recoveries
of individual impurities were calculated by external standard method.
Specificity is the ability of the method to measure the analyte response in the
presence of its potential impurities. According to ICH guidelines forced degradation
studies were performed on simvastatin tablets powder to provide an indication of
the stability indicating property and specificity of the proposed method. Intentional
degradation was attempted to stress conditions of UV light, heat, acid, base, oxidation,
water, sunlight and humidity to evaluate the ability of the proposed method to separate
simvastatin from its degradation products.
RESULTS AND DISCUSSION
The present study was carried out to develop a simple, fast, accurate and precise
HPLC method for analysis of simvastatin impurities in tablets. In this process hydroxy
acid, lovastatin, acetate ester, dehydro simvastatin, dimer are potential impurities
for simvastatin. The chromatographic separation was achieved by following gradient
flow using mobile phase A and mobile phase B containing 0.1 % orthophosphoric
acid and acetonitrile in the ratio of 47:53 and 90:10. A typical chromatogram was
shown in Fig. 2. The retention time for simvastatin and its impurities hydroxy acid,
lovastatin, acetate ester, dehydro simvastatin, dimer are about 25.93 and 16.35,
21.55, 34.16, 35.24, 59.66 min, respectively. The relative retention time for
simvastatin and its impurities hydroxy acid, lovastatin, acetate ester, dehydro
simvastatin, dimer were about 1.00 and 0.63, 0.83, 1.32, 1.36, 2.30, respectively.
The results are given Table-1.

Fig. 2. Representative chromatogram for simvastatin and its impurities

Linearity results shows that good correlation existed between the peak area
and concentration of impurities. The results are summarized in Table-2.
4298 Gowri Sankar et al. Asian J. Chem.

TABLE-1
RETENTION TIME, RELATIVE RETENTION TIME AND RELATIVE RESPONSE
FACTOR’S OF SIMVASTATIN AND ITS IMPURITIES
Relative retention Relative response
Name of the impurity Retention time
time factor
Hydroxy acid 16.35 0.63 0.90
Lovastatin 21.55 0.83 1.02
Simvastatin 25.93 1.00 –
Acetate ester 34.16 1.32 0.87
Dehydro simvastatin 35.24 1.36 0.83
Dimer 59.66 2.30 0.84

TABLE-2
LINEARITY DATA OF SIMVASTATIN IMPURITIES
Concentration Coefficient of
Name of the impurity Slope (b) Intercept (a)
range (µg/mL) correlation (r)
Hydroxy acid 0.199-34.849 0.99983 58791.20904 1187.9535
Lovastatin 0.160-14.040 0.99982 65040.32015 -3864.4606
Acetate ester 0.081-7.069 0.99980 57096.95345 -152.6960
Dehydro simvastatin 0.115-20.185 0.99987 54935.09170 -2635.6283
Dimer 0.717-14.947 0.99974 19406.18333 -3550.6907

The % RSD of response of hydroxy acid, lovastatin, acetate ester, dehydro

simvastatin, dimer during precision and intermediate precision was found to be
less than 15.0 %. The results are summarized in Table-3.

TABLE-3
PRECISION OF THE PROPOSED HPLC METHOD
Simvastatin impurities
Sample No. Hydroxy Dehydro
Lovastatin Acetate ester Dimer
acid simvastatin
01 3.156 1.563 0.617 1.765 1.621
02 3.179 1.560 0.615 1.763 1.620
03 3.363 1.659 0.658 1.880 1.732
04 3.182 1.571 0.625 1.773 1.636
05 3.171 1.571 0.625 1.772 1.635
06 3.227 1.596 0.635 1.798 1.662
Average 3.213 1.587 0.629 1.792 1.651
% RSD 2.400 2.400 2.500 2.500 2.600

The recovery studies were performed from 25 to 150 % of target concentration

(0.5 % of acetate ester,1.0 % lovastatin, 1.5 % of simvastatin dimer, dehydro
simvastatin and 2.5 % of hydroxy acid). The % mean recoveries simvastatin in
simvastatin tablets are satisfactory. The results are summarized in Table-4.
Vol. 21, No. 6 (2009) RP-HPLC Method for Impurity Profiling of Simvastatin 4299

TABLE-4
ACCURACY OF HPLC METHOD FOR DETERMINATION OF
SIMVASTATIN IMPURITIES IN TABLETS
Average Average Mean
Sample No. Spike level (%)
‘µg/mL’ added ‘µg/mL’ found % recovery
050 10.1661 10.8160 106.4
Hydroxy acid 100 20.3322 26.3680 110.0
150 30.4983 31.4213 103.0
050 4.0320 4.1760 103.6
Lovastatin 100 8.0640 8.5786 106.4
150 12.0960 12.5120 103.4
050 2.1351 2.2213 104.3
Acetate ester 100 4.2702 4.2740 100.1
150 6.4053 5.8320 091.1
050 5.7554 6.2180 108.1
Dehydro
100 11.5108 12.4020 107.7
simvastatin
150 17.2662 18.5013 107.1
050 5.8890 6.5120 105.0
Dimer 100 11.7780 11.6300 098.8
150 17.6670 17.7440 100.4

The limit of detection and limit of quantification for all impurities were estab-
lished by signal to noise method and precision and accuracy as verified at LOQ
level. The results are summarized in Table-5.
TABLE-5
LIMIT OF DETECTION AND LIMIT OF QUANTIFICATION OF
SIMVASTATIN IMPURITIES
Test name Signal to noise ratio
Limit of Limit of % Impurity
Name of the impurity
detection quantification LOD LOQ at LOQ level
(µg/mL) (µg/mL)
Hydroxy acid 0.056 0.176 3.10 09.70 0.022
Lovastatin 0.056 0.168 2.81 10.20 0.021
Acetate ester 0.024 0.088 2.73 10.20 0.011
Dehydro simvastatin 0.032 0.120 2.70 09.70 0.015
Dimer 0.248 0.776 3.22 09.93 0.097

From forced degradation studies peak purity has been verified and purity angle
is found to be less than purity threshold. The results are summarized in Table-6.
Conclusion
The RP-LC method developed for related substance determination of simvastatin
in simvastatin tablets are precise, accurate, specific and selective. The method was
validated shown satisfactory data for all the method validation parameters tested.
The developed method is stability indicating and can be used for assessing the
simvastatin impurities in simvastatin tablets.
4300 Gowri Sankar et al. Asian J. Chem.

TABLE-6
FORCED DEGRAGATION STUDIES OF SIMVASTATIN
Simvastatin
Stress condition
% Degradation Purity angle Purity threshold
Acid degradation 5.13 0.177 0.518
Base degradation 2.07 0.100 0.363
Peroxide degradation 2.43 0.137 0.434
Water degradation 1.47 0.123 0.412
UV degradation 0.52 0.137 0.422
Thermal degradation 3.54 0.101 0.353
Sunlight degradation 1.15 0.173 0.518
Humidity degradation 0.89 0.172 0.510

ACKNOWLEDGEMENTS
The authors are grateful to Andhra University for providing the laboratory facilities
and to A.I.C.T.E., New Delhi, India for providing financial assistance.
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(Received: 14 April 2008; Accepted: 6 March 2009) AJC-7328