Direct Spectrophotometric Determination of Sildenafil Citrate in Pharmaceutical Preparations Via Complex Formation with Two sulphonphthalein acid dyes

 

Amir Alhaj Sakur*, Shaza Affas

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

 

ABSTRACT:

Objective: Simple, sensitive and free- extraction spectrophotometric methods have been developed for the determination of Sildenafil citrate (SIL) both in pure and in pharmaceutical forms.

Methods: Two spectrophotometric methods are based on the formation of yellow colored ion-pair complexes between the studied drug and two sulphonphthalein acid dyes, bromocresol green (BCG)(method A) and bromothymol blue (BTB) (method B) with absorption maxima at 414 and 409 nm, respectively.

Results: 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. Various analytical parameters have been evaluated and the results have been validated by statistical. Beer’s law was obeyed in the concentration ranges of (1.33 – 50) µg/ml for method A and (0.8 -27) µg/ml for method B, the limits of detection were 0.093 and 0.059 μg/ml and the molar absorptivity coefficients were 2.28×10⁴ and 3.05 ×10⁴ L .moL^-1.cm^-1  for method A and B respectively.

Conclusion: 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 comparing 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-pyrimidinyl methylpiperazine 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].

 

SIL widely used in oral therapy for erectile dysfunction, is the citrate salt of sildenafil, a selective inhibitor of cyclic guanosine monophosphate (cGMP) specific phosphodiesterase type 5 (PDE5) ^[4–6].

 

This drug can also be efficient as therapy for a range of cardiovascular diseases, such as pulmonary arterial hypertension (PAH) ^[7–12]. 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 guanylate cyclase, 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 ^[13].

 

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 HPLC^[14], electrochemical^[15] and  spectrophotometric determination^[16-21].

 

There is a lot of studies that used reagent to determine drugs via complex formation like BTB ^[22] and  BCG ^{[23, 24]}, Calcon^[25] and other reagents^[26-30].

 

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. BCG (BDH, ENGLAND), BTB (Merck, 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 2 mL 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. All solutions are stable for a period of 2 days when refrigerated (4°C).

 

Solutions of reagents BCG and BTB were prepared with a concentration of (1.10^-3 M) 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.

 

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 BCG (1.10^-3M) for method A and 4 ml of BTB (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 414 nm and 409 nm, for method (A and B), respectively, against reagent blank that had been treated similarly.(Figure2).

 

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)

 

Figure 2: U.V-VIS spectra of :1) Blank BCG, 2) Blank BTB, 3) SIL complex with BCG

4) SIL complex with BTB. SIL concentration is (4×10^-5) mol/l.

 

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 5000 rpm. 5 mL of the supernatant was then transferred into 100 mL volumetric flask, and diluted with dichloromethane up to mark.

 

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.

 

RESULTS:

Preliminary investigations have been shown that SIL react with BCG and BTB in dichloromethane to give ion-association complexes. The optimum reaction conditions for quantitative determination of the ion pair complexes were established via a number of preliminary experiments.

 

Solvent effect:

In order to select a suitable solvent for preparation of the reagent solutions used in the study, the reagents were prepared separately in different solvents such as, chloroform, aceton, n-propanol, methanol, dichloroethane and dichloromethane, and the reaction of SIL with BCG and BTB was followed. In methods A and B, dichloromethane was best suited for the preparation of BCG and BTB solutions. the DMSO solvent was found to be the ideal solvent to dissolve SIL, the effect of the diluting solvent was studied for two methods and the results showed that none of the solvents except 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 because in this medium, the reagent blank gave negligible absorbance and the formed ion-pair complex was found to exhibit higher sensitivity and stability. In other solvents, the reagent blank yielded high absorbance values.

 

Effect of Amount of dye:

The optimum volume of the reagent necessary for the assay of drugs was studied. 2ml of (1.10^-3M) BCG and 4ml of (1.10^-3M) BTB were sufficient for complete color development for SIL/dye complex when we use 10 mL volumetric flask. (Figure3)

 

Figure3: Effect of the amount of dye.

 

Stachiometric Ratios Determination of SIL-Dye complexes:

The ratio of (SIL) to each of the reagents BCG and BTB was 1:1 for SIL:BCG and SIL:BTB  (Figure.4 and Figure.5)

 

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 Table1. A linear relationship was found between the absorbance at λ_max and the concentration of the drug in the ranges 1.33-66 and 0.8-27 µg/ml for method A and B, respectively.(Fig.6 and Fig.7)

 

Figure 4: Molar ratio method for (SIL- Dye) complexes

 

Figure5: Continuous variation method for SIL-Dye complexes

 

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 [31]. The high molar absorp­tivities of the resulting colored complexes indicate the high sensitivity of the methods (2.28×10⁴ L.mol^-1.cm^-1 and 3.05×10⁴ L.mol^-1 .cm^-1) for method A and B, respectively.

 

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 99.09 – 101.29% for method A and 99.15 – 101.75% for method B, indicating good accuracy of the methods. The results obtained are sum­marized in Table 2.

 

Application to the pharmaceutical dosage forms

The proposed methods have been successfully applied to the determination of Sildenafil Citrate in pharmaceutical preparations (table 3).

 

The ingredients in the tablets did not interfere in the experiments.

 

Figure 6: calibration plot of SIL-BCG                                                               Figure 7: calibration plot of SIL-BTB

 

Table (1) Spectral characteristics of SIL-Dye complexes

 

Table(2) Evaluation of precision and accuracy of the proposed methods for determination of SIL in pure form.

·                      Average of five determinations.

 

Table (3): Results of the estimation of Sildenafil Citrate in tablets

formulation	Claim (mg/tab)	Recovery* ± SD		Official method[14] (HPLC)
		Method A (BCG)	Method B (BTB)
VIGRAVID	25	100.25% ± 1.97 t= 1.32      F=1.34	101.08% ± 2.76 t= 1.13      F=2.09	100.76% ± 0.52 t= 1.22
	50	101.05% ± 1.05 t= 1.22      F=2.13	100.92% ± 1.65 t= 1.52      F=1.76	100.66% ± 0.92 t= 1.32
	100	101.04% ± 0.73 t= 1.27      F=1.24	101.00% ± 1.87 t= 1.06      F=1.89	101.35% ± 0.89 t= 1.02
VIGRAN	25	99.55% ± 1.81 t= 1.32      F=1.34	100.05% ± 0.94 t= 1.97      F=3.34	100.25% ± 0.97 t= 1.83
	50	99.87% ± 2.47 t= 1.12      F=1.06	98.92% ± 0.07 t= 1.87      F=2.98	100.05% ± 0.37 t= 0.76
	100	101.34% ± 1.18 t= 1.4      F=1.76	100.95% ± 1.97 t= 1.32      F= 0.34	101.15% ± 1.25 t= 0.62

*Average of five determinations.

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

DISCUSSION:

In this study, two methods which are free-extractive, rapid, and have high sensitivity comparing with other previous direct methods, are achieved. The proposed methods can be carried out in a short time at room temperature in contrast with a lot of previous direct spectrophotometric methods that need heating and long time to achieved. A comparative summarized study between the proposed methods and previous complex formation spectrophotometric methods for determination of Sildenafil has been provided in table 4.

 

Table 4 :Comparison of the proposed methods with the existing spectrophotometric methods for the determination of sildenafil citrate.

Reagent	Spectrophotometric Method	Solvent	Temp.°C	Concentration rang. µg.mL-1	L.mol-1cm-1	Reference
Iodine	Direct	1,2-Dichloroethane	25±1	15.0–160	3.75×103	[16]
TCNQ	Direct	Acetonitrile	50±2	15.0–220	2.58×103	[16]
DDQ	Direct	Methanol	50±2	20.0–260	2.41×103	[16]
TCNE	Direct	Acetonitrile	50±2	10.0–210	3.05×103	[16]
TNF	Direct	1,2-Dichloroethane	60±2	15.0–240	2.25×103	[16]
Chloranilic acid	Direct	Acetonitrile	60±2	20.0–180	3.26×103	[16]
Chloranil	Direct	Acetonitrile	60±2	28.0–150	3.42×103	[16]
Bromanil l	Direct	Methanol	60±2	15.0 –170	2.90×103	[16]
BCG	Extractive	Chloroform	25±2	1.2 – 25.0	1.58×104	[17]
CCR	Extractive	Chloroform	25±2	1.5– 60.0	9.79×103	[17]
chromotrope 2B	Extractive	Methylene chloride	25±2	3.3– 87.0	1.02×104	[18]
chromotrope 2R	Extractive	Methylene chloride	25±2	3.3 – 96.0	8.30×103	[18]
3-phenylazo-6-o-carboxyphenylazo-chromotropic acid	Extractive	Methylene chloride	25±2	5.0 - 115.0	6.83×103	[18]
bis-3,6-(ohydroxyphenylazo)- chromotropic acid	Extractive	Methylene chloride	25±2	2.5 - 125.0	5.42×103	[18]
bis-3,6-(p-N,N-dimethylphenylazo)-chromotropic acid	Extractive	Methylene chloride	25±2	8.3 - 166.7	3.35×103	[18]
3-phenylazo-6-o-hydroxyphenylazo-chromotorpic acid	Extractive	Methylene chloride	25±2	0.8 - 15.0	2.32×104	[18]
BTB	Extractive	Chloroform	40±2	1.0- 40.0	20.12×103	[19]
PBP	Extractive	Chloroform	25±2	1.0 - 50	44.40×103	[19]
EPPR	Extractive	Chloroform	25±2	3.0 - 70.0	24.00×103	[19]
MCP	Extractive	Chloroform	25±2	3.0 -70.0	7.86×103	[20]
Proposed methods
BCG	Direct	Dichloromethane	25±2	1.3-50.0	2.28×104
BTB	Direct	Dichloromethane	25±2	0.8 – 27.0	3.05×104

 

CONCLUSION:

Two methods were carried out to determine Sildenafil citrate in pure form and in Pharmaceutical Preparations using BCG and BTB.

 

The proposed methods are advantageous over many of the reported spectrophotometric methods, Table4, due to their sensitivity, high molar absorptivity value (2.28 ×10⁴ L.mol⁻¹.cm⁻¹ and 3.05×10⁴ L.mol⁻¹.cm⁻¹), high percentage of recovery, wide application range and low relative standard deviation, they are sufficiently sensitive to permit determination even down to 1.5 μg ml^-1.

 

The methods are free-extractive, rapid and can be carried out in a short time at room temperature, Unlike HPLC methods, the proposed methods do not need expensive sophisticated apparatus, all the analytical reagents are inexpensive,  have excellent shelf life, and are available in many analytical laboratories. Therefore, the methods are practical and valuable for routine application in quality control laboratories for analysis of SIL.

 

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Accepted on 25.03.2017           © RJPT All right reserved