High Performance Liquid Chromatographic Method for Determination of Sildenafil Citrate ( Viagra ) in Pharmaceutical Formulation and Industrial Effluent Sample

A simple, precise, rapid and accurate reversed–phase high performance liquid chromatographic method has been developed for the determination of sildenafil citrate in a pure form, pharmaceutical formulations and industrial effluent samples. Chromatography was carried out on supelco L7 reversedphase column (25cm × 4.6mm), 5 microns, using a mixture of methanol: water: triethylamine (630: 370 :2 v/v/v) and adjusted to pH 4.0 with dilute phosphoric acid as a mobile phase at a flow rate of 1.0 ml.min.Uv detection was performed at 220 nm at ambient temperature (25 °C). The retention time for sildenafil citrate found to be 4.6 minutes. The calibration curve was linear (r= 0.9998) over a concentration range from 10 to 70 μg/ml. Limit of detection (LOD) and limit of quantification (LOQ) were 60 ng/ml and 180 ng/ml respectively. The method was validated for its linearity, precision and accuracy. The proposed method was successfully applied to the determination of sildenafil citrate in tablets and industrial effluent samples. Keyword: HPLC, Sildenafil citrate, Viagra, Pharmaceutical preparations, Industrial effluent. ــــــــــــــــــــــــــــــــــــــــــــــــــــــ مادختسا هقيرط لئاسلا ايفارغوتامورك يلاع ءادلأا تيرتس ليفانيدليسلا ريدقتل ) اركيفلا ( يف هايملا يفو ةينلاديصلا هتارضحتسم ةيعانصلا ةحورطملا


EXPERIMENTAL Apparatus
The chromatographic apparatus is a Shimadzu HPLC model LC-20AT with UV detector model SPD-20A and L 7 Supelco column (25cm×4.6mm), 5 microns.HPLC conditions are given in Table (1).

Chemicals and reagents
All chemicals used were of analytical or pharmaceutical grade and deionized water was used throughout.
Sildenafil citrate stock solution was prepared by dissolving accurately weighed quantity of 100 mg of the drug in 100 ml of methanol (final concentration,1mg/ml).From this stock solution, standard solution containing 100 µg/ml sildenafil citrate was prepared by suitable dilution of the stock solution with methanol.

Recommended procedure
A series of standard solutions containing10-70 µg /ml of sildenafil citrate and the sample solution of pharmaceutical preparation were applied, respectively.20 µl aliquot of each solution was injected into the column in a duplicate and the chromatograms were recorded.Calibration graph was constructed by plotting the mean peak area versus concentration of sildenafil citrate.The concentration of the unknown was read from the calibration graph or calculated from the regression equation derived from the concentration and peak area data.

Procedure for pharmaceutical preparations (tablet):
To minimize a possible variation in the composition of the tablet, the mixed content of 10 tablets, were weighed and grounded, then the powder equivalent to 50 mg of sildenafil citrate was stirred well with 100 ml methanol for 30 min.and then filtered through Whatman No. 42 filter paper.The filtrate was made up to 250 ml with methanol in a volumetric flask and the filtrate solution was further diluted to get 30 µg/ml and then determination of sildenafil citrate as described under recommended procedure.

Procedure for industrial waste water
To demonstrate the practical applicability of the proposed method, industrial waste water samples from the state company for drug industries and medical appliances, Mosul-Iraq, were collected in polyethylene container cleaned with nitric acid, and filtered through Whatman No.41 filter paper.Filtered samples were stored at 4 C º until analyzed by spiking with the concentrations ranging from 20-60 µg.ml -1 of sildenafil citrate and aliquot of this solution was treated as described above for recommended procedure.

RESULTS AND DISCUSSION
The chromatographic conditions were adjusted in order to provide a good performance of the assay.The method involved a mobile phase consisting of methanol: water: triethylamine 630:370:2 (v/v/v), adjusted to pH 4 by adding phosphoric acid (5 M), accomplished at 220 nm.The retention time was 4.6 minutes at a flow-rate of 1.0 ml /min and the injection volume was 20 µl.The mobile phase was chosen after several trials with other solvent combinations.Mobile phase selection was based on peak parameters (symmetry, tailing), run time, ease of preparation and cost.Fig. 1 shows a typical chromatogram obtained from the analysis of a standard of sildenafil citrate using the proposed method.Under the described experimental conditions, the analyte peaks were well-defined and free from tailing.
Sildenafil citrate was determined by measuring the peak area.A plot of peak area against concentration gave a linear relationship (r 2 = 0.999) over the concentration range 10-70 µg/ml.Using regression analysis, the linear equation Y=21996×-99461 was obtained where Y is the mean peak area and X is the concentration in µg/ml (Fig. 2).

Determination of limit of detection and limit of quantification ( sensitivity )
A series of dilute solutions were prepared in the rang of 0.1%, 0.5% and 1% of the assay concentration (30 µg/ml) using the standard solutions.20 µl of each of the above solutions were injected in 6 times and the area were calculated due to sildenafil citrate peak.The standard deviation for the 6 injections for each concentration was calculated.The standard deviation at concentration zero was calculated and this value was used for the calculation of the limit of detection and limit of quantification.The limits of detection (LOD) and quantification (LOQ) were calculated using the following formulae: LOD= (3.3σ/s) and LOQ=(10 σ/s) where σ is the standard deviation of the response and s is the slope of the regression line (Laxman et al., 2011).Limit of detection (LOD) and limit of quantification (LOQ) were found 60 ng/ml and 180 ng/ml, respectively.The results indicate that the method was sensitive enough to detect a concentration of 60 ng/ml and able to quantify at a concentration of above 180 ng/ml.

Precision and accuracy
The precision of the calibration graph was established by carrying out the analysis of sildenafil citrate (n=6) using the proposed method.The low value of standard deviation showed that the method was precise.The results obtained are presented in Table 2. To ensure the reliability and accuracy, recovery studies were carried out at five different levels.The results of recovery studies were found to be accurate, mean recoveries being 100.653±1.9(n=6) as shown in Table 2.

APPLICATIONS
The proposed method was successfully applied to the assay of sildenafil citrate in tablet and industrial waste water sample.The result of analysis of pharmaceutical formulation (Table 3) reveals that there is a close agreement between the results obtained by the proposed method and label claim, and the results of the industrial waste water samples.Table 4 shows that the recovery values obtained were close to 100%.

CONCLUSION
In this study, a simple, rapid, accurate HPLC method was developed and validated for the determination of sildenafil citrate in tablets and industrial waste water samples.The method was selective using L 7 analytical column and applicable to pharmaceutical preparations.Thus, the developed method is recommended for control throughout the entire manufacturing process of drugs as well as quality control of the finished product in view of its high recovery and precision.

Table 3 : Determination of sildenafil citrate in tablets.
Mean of six determinations.