METHOD DEVELOPMENT AND VALIDATION OF SILDENAFIL IN BULK AND

PHARMACEUTICAL DOSAGE FORM BY RP-HPLC

Sabyasachi Das*, Bera Venkata Varaha Ravi Kumar
Department of Pharmaceutical Analysis and Quality Assurance,
Roland Institute of Pharmaceutical Sciences, Berhampur-760 010, Odisha, India
ABSTRACT

Solubility: Its solubility is 3.5 mg/ml in water. Sildenafil citrate is twice more soluble in
methanol than in water. Its solubility decreases with pH up to 9 when it starts to increase
again.
Dosage and directions for use: Sildenafil citrate is a phosphodiesterase type-5 inhibitor used
in the management of erectile dysfunction and pulmonary arterial hypertension. It is given
orally as the citrate although doses are expressed in terms of the base; 14 mg of Sildenafil
citrate is equivalent to about 10 mg of Sildenafil. In erectile dysfunction the usual dose is
equivalent to Sildenafil 50 mg about one hour before sexual intercourse. The dose may be
increased or decreased depending on response. The maximum recommended dose is 100 mg,
and Sildenafil should not be taken more than once in 24 hours.

EXPERIMENTAL METHODS
Instrumentation:
Quantitative HPLC was performed on Shimadzu HPLC with LC- 20AT pumps
besides SPD- 20A UV-Visible detector. Shimadzu Spinchrom-CFR software is used
along with inertsil shield RP-C 8 (150 mm × 3.9 mm), 5 µm column for the
separation. A calibrated electronic single pan of Mettler Toledo was used.
Preparation of Buffer Solution: Accurately weighed 6.8 gm of potassium
dihydrogen phosphate and 1.3219 gm of di-potassium hydrogen phosphate dissolved
in 2.5 litre of water and pH was adjusted to 7.4 with potassium hydroxide solution.
Preparation of Mobile phase: The mobile phase was prepared by mixing 500 ml of
buffer with 500 ml of acetonitrile. The mobile phase was sonicated for 10 minutes
and then it was filtered through a 0.45 µ membrane filter.
Preparation of Standard stock solution: An accurately weighed quantity of 50 mg
of Sildenafil was transferred to 100 ml volumetric flask, which was then dissolved
and made up to volume with mobile phase. Then 25 ml of this solution was diluted
with mobile phase up to 50 ml to get concentration of 250µg/ml.
Optimized chromatographic conditions: RP-HPLC analysis was performed by
isocratic elution with flow rate of 1 ml/min. The mobile phase was prepared with
buffer and acetonitrile in the ratio of 50:50 to obtain well-resolved peak of Sildenafil
citrate (Rt = 11.625 min). An inertsil C 18 column was used as stationary phase. Run
time of 15 minutes and temperature of 30 0C for the HPLC system were found to be
the optimum for the analysis. The drug shows reasonably good response at 290 nm.
Calibration curve for Sildenafil citrate: Aliquots of standard stock solution 1, 2, 3,
4, 5 and 6 ml were taken in six different 10 ml volumetric flasks and diluted up to the
mark with mobile phase to obtain final concentrations of 25, 50, 75, 100, 125 and
150 µg/ml of Sildenafil citrate respectively. The solutions were injected using a 20µl
fixed loop system and chromatograms were recorded. Calibration curve was drawn
by plotting peak area versus concentration.
Analysis of the marketed formulations: Twenty tablets were weighed accurately

1
and crushed to form fine powder. Accurately weighed quantity of powder equivalent
to 50 mg of Sildenafil was dissolved in 100 mL of volumetric flask with the mobile
phase. The flask was sonicated for 20 min and then the solution was filtered using
0.45µ filter. Then from the filtrate, 10 mL of tablet sample solutions were transferred
into five different 50 mL volumetric flasks and the volume was made up to the mark
with mobile phase to obtain 100 µg/mL of Sildenafil. The solution was sonicated for
10 min and injected under above chromatographic conditions and peak areas were
measured.
Linearity And Range: The linearity range was found 25-150µg/ml. The regression
equation for Sildenafil citrate was found to be y = 14271x -13655 and Correlation
coefficient(r2=0.999).
Precision: Intra-day and inter-day precision of the assay samples containing
Sildenafil citrate (100 µg/ml) were analysed five times in the same day (intraday)
and for three consecutive days (inter-day). Precision was calculated as intra and
inter-day Coefficient of variation or %RSD [% C.V. = (S. D. / mean) x100]
Accuracy: It was found out by recovery study using standard addition method.
Known amount of standard Sildenafil citrate was added to pre-analysed samples at a
level from 80 % to 120% and then subjected to the proposed HPLC method.
Robustness: By introducing small but deliberate changes in the flow rate (± 1%),
mobile phase pH (± 0.1), temperature (± 2 0C), detection wavelength (± 2.0 nm) and
mobile phase composition (± 2%) robustness of the described method were studied.
The robustness of the method was assessed for 100 µg/ml concentration.
Sensitivity: The sensitivity of the method was determined with respect to LOD and
LOQ. The LOD and LOQ were separately determined based on the standard
calibration curve. LOD = (3.3 x S.D /S and LOQ = 10 x S.D/S, where, S.D is the
standard deviation of the Y- intercepts of regression line and S is the average slope
of the calibration curve. The Lower limit of detection and limit of quantification were
found to be 0.1224 and 0.4014 µg/ml respectively.
Results And Conclusion
Optimizations of the method: A mobile phase consisting of buffer (pH 7.4) and
Acetonitrile in the ratios of 50:50 gave a well resolved and sharp peak for Sildenafil
citrate with a retention time of 11.625 min. The flow rate was 1 ml/min and effluent
was monitored at 290 nm. Inertsil C18 column, run time of 15 min and 300C
temperatures were found to be optimum for the analysis. The results of specificity
studies indicated no interference from excipients, impurities and assured that the
peak response was due to a single component only. The % RSD was less than 1 in
intraday, inter-day precision and in each parameter of robustness. So the proposed
method was more precise and robust. The percentage of average recoveries was obtained
in the range of 99.448 to 99.992. The absence of additional peaks in the chromatogram
indicates non-interference of the common excipients used in the tablets. The lower limit of
detection and the limit of quantification were found to be 0.1224 and 0.4014 µg/ml
respectively.
Acknowledgement: The author sincerely thanks to Prof. B.V.V.Ravi Kumar and Roland
Institute of Pharmaceutical Sciences Berhampur, Orissa, India for providing all experimental
facilities to carry out this work.
Bibliography:
1. The Merck Index: an encyclopaedia of chemical, drugs and biological. 13th ed. Merck and
Co. Inc. New York, 2001, 1523.
2. Sweetman, S. C. (2002). Martindale: The Complete Drug Reference, 33rd ed.
Pharmaceutical Press London, pp. 1662.