Varaprasad Bobbarala et al.

/ Journal of Pharmacy Research 2010, 3(3),631-635

Research Article
ISSN: 0974-6943 Available online through
www.jpronline.info
RP-HPLC method development and validation for determination of dissolution and assay of
sildenafil citrate tablets
Useni Reddy Mallu1 K. Hussain Reddy 1, Varaprasad Bobbarala2*, Somasekhar Penumajji 2
1
Department of Chemistry, Sri Krishnadevaraya University, Anathapur-515003, A.P. India
2
Vivimed labs Limited, 2nd, 4th Floor, Veeranag towers, Habsiguda, Hyderabad, A.P. India
Received on: 20-11-2009; Revised on: 16-12-2009; Accepted on:19-02-2010
ABSTRACT
The aim of this study is to development and validation of a stability indicating RP-HPLC method for the determination of dissolution and assay of Sildenafil
citrate tablets 50mg and 100mg using RP-HPLC with UV detector. Sildenafil citrate and related substances were baseline separated and quantitated on a C18
reverse phase column (4.6mm × 250mm, 5.0µm), using a mobile phase composed of a phosphate buffer-acetonitrile (40:60v/v,) delivered at a flow rate of
1.0mL/min, and with UV detection (λexcitation = 225nm). The method was proven to be linear over a Sildenafil citrate concentration range of 13 to 83µg/mL with
a mean correlation coefficient of 0.9999.

Keywords: Sildenafil citrate,pulmonary arterial hypertension, RP-High performance liquid chromatography (RP-HPLC).

INTRODUCTION

Sildenafil citrate is a drug used to treat erectile dysfunction ence of the impurities with the Sildenafil citrate peaks needs to be
and pulmonary arterial hypertension (PAH). It acts by inhibiting cGMP assured. The following are the structures of Sildenafil and its three
specific phosphodiesterase type 5, an enzyme that regulates blood impurities.
flow in the penis. The mechanism of action of Sildenafil citrate in-
volves the release of nitric oxide (NO) in the corpus cavernosum of
the penis. Nitric oxide binds to the receptors of the enzyme guanylate
cyclase, which results in increased levels of cyclic guanosine mono-
phosphate (cGMP), leading to smooth muscle relaxation (vasodila-
tion) of the intimal cushions of the helicine arteries, resulting in in-
creased inflow of blood and an erection1. Sildenafil is metabolized by
Sildenafil citrate Structure Structure of Impurity A:
liver enzymes and excreted by both the liver and kidneys. If taken
with a high-fat meal, absorption is reduced; the time taken to reach
the maximum plasma concentration increases by around one hour,
and the maximum concentration itself is decreased by nearly one-
third 2. The primary indication of sildenafil is treatment of erectile dys-
function (inability to sustain a satisfactory erection to complete inter-
course). Its use is now standard treatment for erectile dysfunction in
all settings, including diabetes 3. People on antidepressants may ex-
perience sexual dysfunction, either as a result of their illness or as a
Structure of Impurity B
result of their treatment. A 2003 study showed that sildenafil improved
sexual function in men in this situation4. Following up to earlier re-
ports from 19995 the same researchers found that sildenafil was able
to improve sexual function in female patients on antidepressants as
well6. During the manufacturing process of Sildenafil Citrate three
different impurities are produced for Sildenafil Citrate. The method
validation has a criterion called specificity in which the non interfer-

*Corresponding author.
Dr. Varaprasad Bobbarala M.Sc., Ph.D.
# 36-92-248/23, Sreenivasa Nagar, Structure of Impurity C
Kancharapalem, Visakhapatnam-530008,
Andhrapradesh, India.
Mobile No: 91-9949129539
E-mail: varaprasadphd@rediffmail.com,bsitalaxmi@yahoo.com The objective of validation of an analytical procedure is to
Journal of Pharmacy Research Vol.3.Issue 3.March 2010 631-635
 Varaprasad Bobbarala et al. / Journal of Pharmacy Research 2010, 3(3),631-635
demonstrate that it is suitable and stability indicating method for its
intended purpose. A tabular summation of the characteristics appli-
cable to identification, control of impurities and assay procedures is
included in the study. validation of analytical procedures is directed
to the four most common types of analytical procedures are identifi-
cation tests, quantitative tests for impurities content, limit tests for
the control of impurities, Quantitative tests of the active moiety in
samples of drug substance or drug product or other selected compo-
nents in the drug product. Typical validation characteristics which
should be considered are Accuracy, Precision, Specificity, Detection
Limit, Quantitation Limit Linearity and Range. Specificity is the ability
to assess unequivocally the analyte in the presence of components
which may be expected to be present. Typically these might include
impurities, degradants, matrix, etc. The accuracy of an analytical pro-
cedure expresses the closeness of agreement between the value which
is accepted either as a conventional true value or an accepted refer-
ence value and the value found. This is sometimes termed trueness.
The precision of an analytical procedure expresses the closeness of
agreement (degree of scatter) between a series of measurements ob-
tained from multiple sampling of the same homogeneous sample un-
der the prescribed conditions. Precision may be considered at three
levels: repeatability, intermediate precision and reproducibility. Preci-
sion should be investigated using homogeneous, authentic samples.
However, if it is not possible to obtain a homogeneous sample it may
be investigated using artificially prepared samples or a sample solu-
tion. The precision of an analytical procedure is usually expressed as
the variance, standard deviation or coefficient of variation of a series
of measurements. Precision has two parameters Repeatability and
Intermediate Precision. Repeatability expresses the precision under
the same operating conditions over a short interval of time. Repeat-
ability is also termed intra-assay precision. Intermediate precision
expresses within-laboratories variations: different days, different ana-
lysts, different equipment, etc. The detection limit of an individual
analytical procedure is the lowest amount of analyte in a sample
which can be detected but not necessarily quantitated as an exact
value. The quantitation limit of an individual analytical procedure is
the lowest amount of analyte in a sample which can be quantitatively
determined with suitable precision and accuracy. The quantitation
limit is a parameter of quantitative assays for low levels of compounds
in sample matrices, and is used particularly for the determination of
impurities and/or degradation products. The linearity of an analytical
procedure is its ability (within a given range) to obtain test results
which are directly proportional to the concentration (ppm) of analyte
in the sample. The range of an analytical procedure is the interval
between the upper and lower concentration (ppm) of analyte in the
sample (including these concentrations) for which it has been dem-
onstrated that the analytical procedure has a suitable level of preci-
sion, accuracy and linearity. The robustness of an analytical proce-
dure is a measure of its capacity to remain unaffected by small, but
deliberate variations in method parameters and provides an indica-
tion of its reliability during normal usage.
MATERIALS AND METHOD
Tablet Dissolution Conditions:
Journal of Pharmacy Research Vol.3.Issue 3.March 2010
Dissolution was performed using by dropping six tablets in
six unit Lab India Disso 2000 instrument. The dissolution medium
used was 900mL 0.01NHCl with USP apparatus-I (Basket) with 100
RPM for 45 minutes and water bath temperature of 37°C ± 0.5°C.
Chromatographic Conditions:
Chromatographic separations were performed using isocratic
elution at ambient temperature. The Waters HPLC system (Waters
Corporation, Milford, MA, U.S.A) consisted of a model 2695 with UV
detector, and a computer running Waters Empower 3.2 software. The
mobile phase was composed of a mixture of Buffer (8.70g of K2HPO4
in to 1000ml of HPLC water) and acetonitrile (40:60v/v) and degassed.
Sildenafil was separated on a reversed phase C18 column (4.6X250mm,
5.0µ). The flow rate was set at 1.0mL/min and the injection volume
was 20µL. UV measurements were made at a wavelength of 225nm.
Standard Preparation:
Prepare Sildenafil Citrate Standard with a known concentra-
tion of 55.55ppm
Test solution preparation for Assay
Take 20 tablets weigh and crush. Prepare the test sample to
get the known concentration of Sildenafil citrate is 55.55ppm.
Test solution preparation for Dissolution
Take six tablets and introduce one tablet each into six differ-
ent dissolution flask containing 900mL of dissolution medium (Previ-
ously equilibrated at 37°C ±0.5°C). Stir it for 45 minutes, withdraw
10mL sample from dissolution vessel, and further dilute 5ml in to 10ml
with medium.
Calculation:
Percentage of Sildenafil Citrate = At X DS X P
As X DT
Where in As is the Area of standard solution, At is the Area of test
sample solution, DT is the dilution factor for test solution prepara-
tion, DS is the Sildenafil citrate working standard dilution factor for
standard solution preparation and P is the Potency of Sildenafil cit-
rate.
RESULTS AND DISCUSSION
For Validation Q2B guidelines of ICH were followed and the
results area as follows.
Specificity:
The Relative standard deviation for Sildenafil Citrate peak areas for
five replicate injections of standard should be not more than 2.0per-
cent(%) and the tailing factor for Sildenafil Citrate peak should be not
631-635
 Varaprasad Bobbarala et al. / Journal of Pharmacy Research 2010, 3(3),631-635
mAU

Sildenafil/6.083
Detector A:225nm
500

450

400

350

300

250

200

150

Impurity-B/14.720
Impurity-A/4.264
Impurity-C/4.564

100

50

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min

Chromatogram 1: Sildenafil Citrate standard along with impurity standards

0.40
6.094
AU

0.20

0.00
0.40
6.096
AU

0.20

0.00
0.40
6.117
AU

0.20

0.00
0.40
6.036
AU

0.20

0.00
0.40
6.046
AU

0.20

0.00
0.40
6.002
AU

0.20

0.00
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
Minutes

Chromatogram 2: System Suitability solution chromatograms of Sildenafil Citrate

Journal of Pharmacy Research Vol.3.Issue 3.March 2010 631-635

 Varaprasad Bobbarala et al. / Journal of Pharmacy Research 2010, 3(3),631-635
more than 2.0 and the observed value for percent RSD was 0.6per- to be linear from 13.9ppm to 83.3ppm with a correlation coefficient of
cent(%) and the tailing factor was 1.1. Chromatogram-1 represents 0.9999. The above chromatogram-3 and table-1 represents the differ-
the spiked Sildenafil citrate with the three impurities and it reveals ent linearity level concentrations (ppm) of sildenafil citrate.
that impurity retention times has no interference with the sildenafil Table-2: Repeatability (precision)
citrate peak. Chromatogram-2 represents the five replicate injections
of system suitability solution. Sample. Percent (%) Assay (percent)
No. Dissolution

Selectivity is testing the blank, placebo and the impurity 1 95.8 98.7
interference with the Sildenafil citrate peak. As a norm of selectivity a 2 95.2 98.7
3 95.0 98.1
study to establish the interference of placebo was conducted. Disso- 4 95.3 98.6
lution was performed on Placebo (Placebo contains without Sildenafil 5 95.4 95.9
6 95.2 94.9
Citrate) in duplicate equivalent to about the weight of placebo present MEAN 95.3 97.5
in portion of test preparation as per test method. Chromatograms of Percent (%) RSD 0.3 1.7

placebo had shown no peaks at the retention time of Sildenafil Cit- (Limit NMT 5.0%)

rate. And impurities also have no interference. This indicates that the Precision has two factors one is the Repeatability and the
excipients used in the formulation do not interfere in estimation of other Reproducibility. The precision of test method was evaluated by
Sildenafil Citrate in Tablets. dissolution the six tablets of the Sildenafil Tablets and analysed as
per test method. The percent (%) dissolution and the Relative stan-
Linearity: dard deviation of Sildenafil Citrate were found to be within the limits.
0.55
Table-2 represents the detailed results of precision samples. Repro-
ducibility is the Analyst to Analyst variability study. It was con-
6.100 6.098

0.50 ducted by two different analysts by dissolution of six tablets of

0.45
Sildenafil Citrate tablets as per the test method. The percentage of
dissolution and relative standard deviation was found to be within
0.40 the limits by both the analysts. The system suitability parameters
6.100

0.35
were evaluated as per the test method by both the analysts and
found to be within the limits. Comparison of the results obtained by
6.102 6.101

0.30
two different analyst’s shows that the dissolution method was meet-
AU

0.25
ing the Intermediate precision (Reproducibility) acceptance criteria.
Accuracy:
0.20
Accuracy is the study of recovery of the Sildenafil Citrate.
0.15
Samples were prepared, with Sildenafil Citrate and placebo equiva-
lent to about 50percent, 60percent, 75percent, 100percent and 125per-
0.10
cent of the target assay concentration of the Sildenafil Citrate Tab-
6.100

0.05
lets. Sample solutions were prepared in lower level and higher level as
six preparations and other levels triplicate for each spike level and
0.00
Dissolution as per test method. The percent (%) recovery was found
5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 to be within the limits. Six replicate samples of lower spike level (at
Minutes
50percent (%) spike level) and higher spike level (at 125percent (%)
spike level) and analyzed as per test method as a part of Precision in
Chromatogram 3: Linearity of Sildenafil Citrate Accuracy. Relative Standard Deviation of percent (%) dissolution
results should be not more than 5.0percent (%). The dissolution of
Sildenafil Linearity Sildenafil Citrate Tablets should be NLT 80.0percent (%). The mean
percentage dissolution and the relative standard deviation of Sildenafil
Linearity Concentration Area
Citrate were found to be within the limits.
Level (ppm)
Table-3: ACCURACY
01 13.9 1001177
02 27.77 2202354 Sample Spike level Percent (%) Mean percent (%)
No. (percent) Recovery Recovery
03 41.66 3303530
04 55.55 4404707 1 50 98.7 98.8
05 69.43 5505884 2 50 100.2
06 83.3 6607082 3 50 97.5
1 60 98.5 98.5
Correlation co-efficient 0.9999 2 60 98.6
3 60 98.5
1 75 98.6 99.4
Linearity was established by plotting a graph between 2 75 100.2
3 75 99.4
sildenafil citrate concentrations (ppm) versus peak area and deter- 1 100 99.0 98.5
mined the correlation coefficient. Six different concentrations of stan- 2 100 98.2
3 100 98.4
dard solutions were prepared in the concentration range of about 1 125 101.8 100.4
2 125 98.9
25% to 150% of the target concentration of Sildenafil citrate tablets 3 125 100.7
and injected into the HPLC system. The detector response was found
Journal of Pharmacy Research Vol.3.Issue 3.March 2010 631-635
 Varaprasad Bobbarala et al. / Journal of Pharmacy Research 2010, 3(3),631-635
Sample solutions were prepared in triplicate for each spike level (50per-
cent, 60percent, 75percent, 100percent and 125percent) and assayed
as per test method. Table-3 represents the mean percent (%) recov-
ery at each spike level was found to be within the limits. Hence it was
concluded that the method performance at extremes of Range (50per-
cent (%) to 125percent (%) is linear, precise and accurate.
Ruggedness:
The stability study of Sildenafil Citrate tablets in Standard
preparation and test preparation on bench top was conducted at
Initial, after 1 day and 2 days. The dissolution of Sildenafil Citrate
tablets test preparation and standard preparation were estimated
against freshly prepared standard each time. The difference in per-
centage assay of test preparation from initial to up 2days results were
found to be within the accepted criteria. The similarity factor for
standard preparation from initial to 2 days should be in the range of
0.98 to 1.02 and was found to be within the limits. The percent (%)
dissolution of Sildenafil Citrate tablets in Standard and Test prepara-
tion should not deviate by more than 3.0percent from initial value and
is found with in the limits. A study to establish the Bench top stability
of mobile phase was conducted at Initial, after 1 day and after 2 days.
Standard solution was prepared as per test method and system suit-
ability parameters were evaluated by using the same lot of Mobile
phase each time. The system suitability was found to be within the
Limits.
Robustness:
A study was conducted to determine the effect of Deaera-
tion of Dissolution medium, conducted as per test procedure. The
system suitability parameters were evaluated as per test method with
both the flow rates. The Difference in average percentage (%) of
Source of support: Nil, Conflict of interest: None Declared
Journal of Pharmacy Research Vol.3.Issue 3.March 2010
dissolution is not more than 5.0percent. The Relative Standard De-
viation of dissolution should be not more than 5.0percent. The result
was found to be within the limits. A study to establish the suitability
of filters was conducted using two different filters namely, 0.45µm
Durapore hydrophilic membrane filter (PVDF) (manufactured by M/s.
Millipore) and 0.45µm Nylon 66 filter (manufactured by M/s. Pall Life
Sciences). Portion of test preparation was filtered through different
filters and calculated dissolution results for different filters against
centrifuged sample solution. The percent (%) of dissolution differ-
ence between filtered samples and centrifuged sample is not more
than 3.0percent and was found to be within the limits. Based on the
above results the method was proven to pass the parameters de-
scribed as per Q2 guidelines of ICH and can be used for analysis.
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