INTRODUCTION:

Sildenafil citrate (Fig.1) is chemically as 1-[[3-(6,7-Dihydro -1-methyl- 7-oxo-3-propyl -1H-pyrazolo [4,3-d] pyrimidin-5-yl) -4-ethoxyphenyl]sulphonyl]-4-methyl piperazine citrate (Figure 1) is a compound of the pyrazolo-pyrimidinyl-methyl piperazine class, and is used to treat male erectile dysfunction. Sildenafil citrate, sold as Viagra, Revatio and under various other trade names, is a drug used to treat erectile dysfunction and pulmonary arterial hypertension (PAH). It acts by inhibiting cGMP-specific phosphodiesterase type5, an enzyme that promotes degradation of cGMP, which regulates blood flow in the penis. It relaxes the arterial wall, leading to decreased pulmonary arterial resistance and pressure. Various analytical methods have been reported for the assay of Sildenafil in pure form as well as in pharmaceutical formulations. They include spectrophotometric methods^1-7,Simultaneous estimation with Dapoxetine⁸, HPLCmethods ^9-14 and TLC¹⁵methods.

Figure-1 Sildenafil

So far, no derivative spectrophotometric method has been reported for the estimation of Sildenafil from pharmaceutical dosage form with. This paper deals with validation and development of a method by derivative spectrophotometry for the assay of Sildenafil from its bulk drug and in pharmaceutical dosage forms.

EXPERIMENTAL:

Instrumentation:

A Lab India model 1885 double beam UV/Visible spectrophotometer with spectral width of 2 nm, wavelength accuracy of 0.5 nm and a pair of 10 mm matched quartz cell was used to measure absorbance of all the solutions. Spectra were automatically obtained by UV win system software.

MATERIALS AND METHODS:

Sildenafil was a gift sample by Pharmatrain Pvt. Ltd. Hyderabad, India and was used without further purification. All chemicals and reagents used were of analytical grade and were purchased from Merck Chemicals, India.

Preparation of standard and sample solutions:

Stock solution of 1000 μg.mL^-1 of Sildenafil was prepared in methanol, for zero order and first order derivative spectrophotometric analysis. The standard solutions were prepared by accurately weigh and transfer 10 mg of Sildenafil working standard into a 10 mL volumetric flask add about 7mL of Methanol and sonicate to dissolve it completely and make volume up to the mark with the same solvent (Stock solution). Further pipette 0.4ml of the Sildenafil stock solution into a 10ml volumetric flask and dilute up to the mark with methanol. Methanol was used as a blank solution.

Assay procedure:

Accurately weigh and transfer equivalent to 10 mg of tablet powder Sildenafil working standard into a100 mL volumetric flask add about 70 mL of methanol and the solutions were filtered through a 0.45 μm nylon filter and sonicated for about 15 min and then volume made up with methanol. This solution was filtered to remove any insoluble matter. The filtrate was collected in a clean flask and makes volume up to the mark with the same solvent (Stock solution). Appropriate dilutions were made with methanol from stock solution for both zero order and first order derivative spectrophotometric methods

Development of the methods:

Method A: Zero order spectroscopic method

The solutions were scanned in the range from 400-200 nm, and the peak was observed and gives maximum absorbance at 292 nm (Figure – 4). So, the wavelength selected for the analysis of the drug was 292 nm. The drug followed the Beer’s- Lamberts law in the range of 30-50 μg/ml.

Method B: First order derivative spectroscopic method

The standard drug solution was diluted so as to get the final concentration in the range of 30-50 μg/ml and scanned in the first order derivative spectral mode. The first order derivative spectra at showed a maxima and minima at 274 and 306 nm respectively (Figure-5). The amplitude of absorbance was measured at 274 nm (peak maxima) and at 306 nm (peak minima) and was plotted against concentration to give calibration curve, and regression equation was calculated. The amplitude was linear in the concentration range of 30-50 μg/ml.

VALIDATION OF THE PROPOSED METHODS:

The proposed method is validated according to the International Conference on Harmonization (ICH) guidelines.

Linearity and Range:

From the stock solution different aliquots of 0.3, 0.35, 0.4, 0.45, 0.5ml were taken in volumetric flask and diluted up to 10 ml with methanol to obtain a concentrations of 30,35,40,45,50 µg/ml. Under the experimental conditions described, the graph obtained for zero order and first order derivative spectra showed linear relationship. Regression analysis was made for the slope, intercept and correlation coefficient values. The regression equations of calibration curves were y = Y= 0.017x + 0.067 (r² = 0.999) at 292 nm for zero order and Y= 0.000468x - 0.00204 (r² =0.999) for first order derivative spectrophotometric method. The range was found to be 30-50 μg.mL^-1 for both zero order and first order derivative spectrophotometric methods. The regression data is shown in Table – 1.

Table-1 Linearity data for proposed method:

Concentration (μg/ml)	Absorbance
Concentration (μg/ml)	Zero order	First order
30	0.5931	0.0121
35	0.6822	0.0143
40	0.7711	0.0166
45	0.8620	0.0189
50	0.9423	0.0215
Correlation Coefficient	0.999	0.999
slope	0.017	0.000468
intercept	0.067	-0.00204

Figure – 2: Linearity curves

Figure – 3: Linearity curves

Precision:

To determine the precision of the method, Sildenafil solutions at a concentration of 40 μg /L were analyzed each five times for both zero order and first order derivative spectrophotometric methods. Measure the absorbance and calculate the %RSD. The %RSD for the five replicates absorbance was found to be within the specified limits. The % RSD for the area of five standard injections results should not be more than 2%. The precision data is shown in Table – 2.

Sensitivity:

The limit of detection (LOD) and limit of quantification (LOQ) were calculated by using the equations LOD = 3.3 σ / S and LOQ = 10 σ /S, where σ is the standard deviation of intercept, S is the slope. The LOD and LOQ were found to be 0.17μg/ml and 0.52 μg /ml respectively for zero order derivative method and 0.5 μg /ml and 1.7μg/ml for first order derivative method.

Recovery:

To study the accuracy of the proposed methods, and to check the interference from excipients used in the dosage forms, recovery experiments were carried out by the standard addition method. This study was performed by addition of known amounts of Sildenafil to reanalyzed solutions of commercial tablets. Measure the Absorbance of the standard solution, Accuracy 50%, Accuracy 100% and Accuracy 150% solutions, Calculate the amount found and amount added for Sildenafil, calculate the individual recovery and mean recovery values (Table – 3).

Analysis of the marketed formulation:

There was no interference from the excipients commonly present in the tablets. The drug content was found to be 100% and 99.8% for zero order and first order derivative spectrophotometric methods respectively. It may therefore be inferred that degradation of Sildenafil had not occurred in the marketed formulations that were analyzed by this method. The low % R.S.D. value indicated the suitability of this method for routine analysis of Sildenafil in pharmaceutical dosage form.

Table - 2 Intra and Inter day precision Results:

	Intraday Precision		Interday Precision
Replicate	Zero order	First order	Zero order	First order
1	0.7821	0.0161	0.7892	0.0161
2	0.7823	0.0163	0.7861	0.0164
3	0.7831	0.0162	0.7863	0.0162
4	0.7841	0.0162	0.7861	0.0163
5	0.7820	0.0163	0.7871	0.0162
Average	0.78272	0.01622	0.78696	0.01624
S.D	0.000884	0.000083	0.001318	0.000114
%RSD	0.11	0.51	0.16	0.70

Table- 4 Assay results for determination of Sildenafil in pharmaceutical Formulation

Parameters	Amount of Tablet label claim	Amount found mg	Drug content %
Zero order	100mg	100	100
First order	100mg	99.8	99.8

The summary of the validation parameters is depicted in (Table - 5).

Figure-4 Zero order derivative spectrum

Figure-5 First order derivative spectrum

Table- 5 Regression analysis data and summary of validation parameters for the proposed methods

Parameter	Zero order	First order
Absorption maxima and minima (nm)	292	274
Beer’s-Lamberts range (μg/ml)	30-50	30-50
Regression equation y=mx+c	Y= 0.017x + 0.067	Y= 0.000468x - 0.00204
Slope(m)	0.017	0.000468
Intercept(c)	0.067	-0.00204
Correlation coefficient (r²)	0.999	0.999
Mean Recovery %	99.7	99.8
Precision (% RSD)	0.11	0.51
Intermediate precision	0.51	0.70
LOD (μg/ml)	0.17	0.5
LOQ (μg/ml)	0.52	1.7

CONCLUSION:

No UV or derivative spectrophotometric methods have been described for the determination of Sildenafil. Therefore simple, fast and reliable derivative spectrophotometric methods were developed for the routine determination of Sildenafil. The developed methods can be concluded as accurate, sensitive and precise and does not involve the use of complex instrument such as HPLC, which is expensive in both the hardware and chromatographic reagents hence can be employed for routine analysis in quality control laboratories.

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Received on 24.09.2013 Modified on 07.10.2013

Asian J. Research Chem. 6(12): December 2013; Page 1116-1120

Zero order derivative method
Accuracy level	Absorbance	Amount added (mg)	Amount found (mg)	% recovery	Mean recovery
50%	0.387	5	4.99	99.8%	99.7%
100%	0.772	10	9.95	99.5%
150%	1.161	15	14.97	99.8%
First order derivative method
50%	0.008	5	4.99	99.8%	99.8%
100%	0.016	10	9.98	99.8%
150%	0.024	15	14.9	99.8%