Simple and Sensitive Spectrophotometric Determination of Sildenafil Citrate in Pharmaceutical Formulations

 

Amir Alhaj Sakur*, Shaza Affas

Analytical and Food Chemistry Department, Faculty of Pharmacy, University of Aleppo, Syria

 

ABSTRACT:

Two simple, rapid and sensitive spectrophotometric methods have been developed for the determination of Sildenafil Citrate (SIL) in pure form and pharmaceutical formulations. The spectrophotometric methods are based on the formation of binary complexes (ion-pair complexes)in dichloromethane (DCM) medium between the drug and tow acidic sulfonephthalein dyes, bromocresol purple (BCP)(method A) and bromophenol blue (BPB) (method B) with absorption maxima at 404 and 410 nm, respectively.

The effects of analytical parameters on the reported systems were investigated. The complexation reactions were extremely rapid at room temperature and t­he absorption values remain unchanged up to 24h. Beer’s law was obeyed in the concentration ranges of 0.6 - 20 and 0.8 -48 µg/ml, detection limits were 0.097 and 0.105μg/ml and the molar absorptivity coefficients were 2.77×10⁴ and 2.60 ×10⁴ L .moL^-1.cm^-1for BCP and BPB respectively.

The proposed methods were successfully applied to the analysis of commercial tablets containing the drugs. Two methods were free-extractive, rapid, and have high sensitivity compared with other previous direct spectrophotometric methods. Interferences of the other ingredients and excipients were not observed.

 

 

 

INTRODUCTION:

Sildenafil (SIL) is a compound of the pyrazolo-pyrimidinylmethylpiperazine class. It is 5-[2-ethoxy-5-(4-methylpiperazin-1-yl sulphonyl) phenyl] -1- methyl-3-propyl-1, 6-dihydro-7H pyrazolo [4,3-d] pyrimidin-7-one, having empirical formula C₂₂H₃₀N₆O₄S and molecular weight 661.71[1].It is a white to off-white, crystalline, odorless powder with a bitter, salty taste [2], having λmax 290 nm in 30% methanolic water and pKa of 8.7 [3].

 

Erectile dysfunction (ED) is one of the most common chronic diseases affecting men and its prevalence increases with aging. It is also the most frequently diagnosed sexual dysfunction in the older male population. Treatment of erectile dysfunction is based on phosphodiesterase type 5 (PDE-5) inhibitors including sildenafil [4].

 

Sildenafil can also be efficient as therapy for a range of cardiovascular diseases, such as pulmonary arterial hypertension (PAH) [5–10]. The mode of action of sildenafil in the erection of the penis involves the release of nitric oxide (NO) in the corpus cavernous during sexual stimulation. The produced(NO) activates the enzyme guanylatecyclase, which results in increased levels of cGMP, producing smooth muscle relaxation of the penile in the corpus cavernous and therefore having the potential to improve penile erectile function by allowing inflow of blood [11].

 

Figure 1: The chemical structure of SIL

 

The assay of SIL in pure and dosage forms, as far as we know, is not official in any pharmacopoeia, and therefore, requires much more investigation. The different analytical methods that have been reported for its determination include LC[10-12], electrochemical [13] and spectrophotometric determination [14-19].

 

There is a lot of studies that used reagent to determine drugs via complex formation like BTB [20] and BCG [21, 22], Calcon[23] and other reagents[24-25].

 

MATERIALS AND METHODS:

Apparatus:

UV-Visible spectrophotometer (JASCO, model V650, Japan)with 1.00 cm quartz cells. Ultrasonic processor (Powersonic, Model 405, Korea) was used to sonicate the sample solutions. Adjustable micro pipettes covering a volume range from 20 to 5000 μL (ISO-LAB, Germany), were used for preparation of the experimental solutions. Analytical balance (Sartorius, model 2474, Germany).

 

Materials and reagents:

Pharmaceutical grade Sildenafil (SIL 99%) was received from Xuhuang, China.

 

BCP and BPB (Riedel-de-Haën, Germany), All reagents and solvents were of analytical grade.

 

A working standard solution of SIL (0.1 mg/mL) was prepared by dissolving 50 mg of SIL in 2mL of dimethylsulfoxide DMSO and the volume was diluted to the mark in a 25 ml calibrated flask with dichloromethane, then 5 ml of this solution was transferred into 100 mL volumetric flask, and diluted with dichloromethane up to mark.

 

Solutions of reagents BCP (5.10^-3M) and BPB(1.10^-3 M)  were prepared by dissolving suitable weight of the reagent in 10 mL dichloromethane to dissolve and  made up to mark with dichloromethane in a 100 ml calibrated flask.

 

All solutions are stable for a period of 2 days when refrigerated (4°C).

 

Procedure for the assay of bulk sample:

Increasing volumes of SIL working standard solution were transferred into series of 10 mL volumetric flasks that contain 2 mL of BCP (5.10^-3M) for method A and 2ml of BPB(1.10^-3M⁾ for method B. Solutions were mixed gently and allowed to stand at room temperature for 2 minutes. Volumes were made up to mark with dichloromethane and mixed before the spectra were recorded at 404 nm and 410nm, for method (A and B), respectively, against reagent blank that had been treated similarly the standard calibration plot was prepared to calculate the amount of the analyzed drug in bulk samples. All measurements were carried out at room temperature (25 ±2°C)

 

Procedure for pharmaceutical samples:

Twenty individual tablets were weighed and pulverized carefully. An accurately weighed amount of the powder equivalent to 50 mg of SIL was transferred into 25 mL volumetric flask and dissolved in 2mL of DMSO. The content of the flask was sonicated for 20 min then diluted to volume with dichloromethane. A portion of this solution was centrifuged at 5000rpm.5 mL of the supernatant was then transferred into 100mL volumetric flask. Then suitable volume was transferred into 10 mL volumetric flask and procedure was continued to use for the analysis of SIL by the spectrophotometric method after 2 min.

 

RESULT:

Preliminary investigations have been shown that SIL react with BCP and BPB in dichloromethane to give ion-association complexes have maximum absorptions at 404 and 410 nm, respectively. Whereas (BCP) and (BPB) solutions have small absorption at this wavelengths.(Figure2)

 

Figure 2: U.V-VIS spectra of: 1) Blank BPB, 2) Blank BCP, 3) SIL complex with BCP4) SIL complex with BPB.

 

The optimum reaction conditions for quantitative determination of the ion pair complexes were established via a number of preliminary experiments.

 

Solvent effect:

In methods A and B, dichloromethane was best solvent for the preparation of BCP and BPB solutions because in this medium the reagent blank gave negligible absorbance and the formed ion-pair complex was found to exhibit higher sensitivity. In other solvents like chloroform, methanol, and acetone the reagent blank yielded high absorbance values.

 

the DMSO solvent was found to be the ideal solvent to dissolve SIL, the effect of the diluting solvent was studied for the methods and the results showed that dichloromethane formed sensitive and stable colored species in methods (A and B). Therefore, dichloromethane was used for dilution throughout the investigation. Dichloromethane was preferred as the most suitable solvent

 

The Amount of dye effect:

The optimum volume of the reagent necessary for the assay of drugs was studied. 2ml of (5.10^-3M) BCP and2ml of (1.10^-3M) BPB were sufficient for complete color development for SIL/dye complex when we use 10 mL volumetric flask.

 

The temperature effect:

The effect of temperature on the complexes formation was studied within the ranges 15-40⁰C. It was found that the temperature had negative effect of formed complex of (SIL-BCP) since the absorbance was decreasing during the heating of sample more than 30⁰C. On the other hand, the formed complex (SIL-BPB) wasn't affected by temperature at this range.

 

The time effect

The time effect on complexes formation has been studied within the 2 min to 24 hours. It was found that the formed complexes of BCP and BPB weren't affected by time at this range. Complexes are formed immediately and remain stable for 24 hours.

 

Composition of SIL: DYE complex:

The composition of SIL:DYE complexes was determined by the molar ratio method and Job's method of continuous variation. The ratio of (SIL) to each of the reagents BCP and BPB was 1:1 for SIL: BCP and SIL:BPB

 

The optimum conditions for spectrophotometric determination of SIL through ion-pair complex formation using BCP and BPB in dichloromethane were shown in table 1.

 

Table (1): the optimum conditions for spectrophotometric determination of SIL by complex formation with BCP and BPB in dichloromethane

Parameters	Operating modes
	BCP	BPB
Temperature of solution	25±2o C	25±2o C
Volume of reagent	2 ml	2 ml
Concentration of reagent	0.005 M	0.001 M
Solvent	dichloromethane	dichloromethane
Stability (h)	24 hours	24 hours
λmax of SIL: DYE complex	404 nm	410

 

 

Linearity and range:

The Beer’s law limits, molar absorptivity, linear regression equation, correlation coef­ficient and detection limits which are deter­mined for each method are given in Table 2.

 

The graphs show negligible intercept and are described by the regression equation, A=mC+b (where A is the absorbance of 1 cm layer, m is the slope, b is the intercept and C is the concen­tration of the measured solution in μg.ml^-1) obtained by the least-squares method [26]. The high molar absorptivities of the resulting colored complexes indicate the high sensitivity of the methods (2.77×10⁴) L.mol^-1 .cm^-1 and (2.60×10⁴⁾L. mol^-1 .cm^-1for method A and B, respectively.

 

Table (2): The parameters established for spectrophotometric determination of SIL by complex formation with BCP and BPB

Parameters	BCP	BPB
Stoichiometric relationship	1:1	1:1
Beer's law limit (µg. ml-1)	0.6 - 20	0.8-48
Molar absorptivity (L.mol-1.cm-1)	2.77 ×104	2.60×104
Linear Regression equation	y = 0.04x + 0.003	y = 0.037x + 0.003
Correlation coefficient, r	0.999	0.999
limit OF Detection (μg.ml-1)	0.097	0.105
Limit if Quantification (μg.ml-1)	0.32	0.36
Range of Error	±4.67 %	±4.75%

 

Accuracy and Precision:

The low values of relative standard deviation (RSD) indicate good precision and reproduc­ibility of the methods. The average percent recoveries obtained were 99.79 – 104.42% for method A and98.80 – 104.05% for method B, indicating good accuracy of the methods. The results obtained are sum­marized in Table 2.

 

 

 

 

 

 

 

Table(3):  Evaluation of precision and accuracy of the proposed methods for determination of SIL in pure form.

Average of five determinations.

 

Application to the pharmaceutical dosage forms:

The proposed procedures were applied to determine the studied substances in their pharmaceutical formulations. The results in Table 3 indicate the high accuracy and precision. As can be seen from Table 3, the proposed method has the advantages of being virtually free from interferences by excipients and common degradation products.

 

The results obtained were compared statistically by the student’s t-test (for accuracy) and the variance ratio F-test (for precision) with those obtained by the reference method [12] on samples of the same batch (Table 3). The values of t- and F-tests obtained at 95% confidence level and four degrees of freedom did not exceed the theoretical tabulated value indicating no significant difference between the proposed method and reference method.

Table (4):Results of the estimation of Sildenafil Citrate in tablets

*Average of five determinations.

 

At 95% confidence limit the theatrical t- and  F value at five degrees of freedom are t=2.776 and f=6.26.

 

DISCUSSION:

Two methods were carried out to determine Sildenafil citrate in pure form and in pharmaceutical preparationsusing BCP and BPB. Unlike GC and HPLC techniques, spectrophotometry is simple and inexpensive, the proposed methods require only dyes as reagents which are cheaper and readily available, no pH-adjustment is required and the procedures do not involve any critical reaction conditions or tedious sample preparation. Moreover, both methods are simple, fast, accurate, extraction-free, adequately sensitive and free from interference excipients. Therefore, they can be easily used for a reliable simultaneous determination of sildenafil in tablet formulation.

 

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Accepted on 20.09.2017      © RJPT All right reserved

Research J. Pharm. and Tech 2017; 10(12): 4242-4246.