New Simple Spectrophotometric Method for the Simultaneous Estimation of the Mixtures of Sildenafil and some Serotonin Reuptake Inhibitors

 

Shaza Affas*, Amir Alhaj Sakur

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

 

ABSTRACT:

This work presents a simple, reproducible, sensitive, generic and efficient method for simultaneous estimation. Vierordt’s method or simultaneous equation method was developed and validated for simultaneous estimation of two mixtures: Sildenafil and Duloxetine, Sildenafil and Fluoxetine, in the pure and pharmaceutical dosage form. The method was based on the measurement of absorbance at two wavelengths for each mixture in methanol. Calibration curves of all drugs were found to be linear. With good correlation coefficient values more than 0.99. The RSD values were found within limits and the recoveries ranging between 98 to 102% that confirms that the proposed method is accurate. This method for simultaneous estimation of these four mixtures is simple and economical and can be applied successfully for the simultaneous estimation of PDEs inhibitors and their combinations with some serotonin reuptake inhibitors in the pure and pharmaceuticals dosage forms.

 

 

 

1. INTRODUCTION:

Erectile dysfunction and premature ejaculation are the most common male sexual dysfunctions. Treatment of erectile dysfunction is based on phosphodiesterase type5 inhibitors, which include Sildenafil (SIL). PDE-5 inhibitors are effective and safe in general, even in difficult-to-treat conditions such as patients with diabetes mellitus.While, a variety of medical therapies is available for premature ejaculation, including tricyclic antidepressants, serotonin reuptake inhibitors, centrally acting opiates, PDE-5 inhibitors and topical desensitizing creams [1–3]. Daily SSRIs such as Fluoxetine, although effective, do have substantial and prolonged side effects. Past trials with DPX have documented successful outcomes without serious side effects [1–5].

 

 

As mentioned earlier, either SSRIs or PDE-5 inhibitors used as monotherapy can delay time to ejaculation. Some clinical trials were conducted to compare the efficacy of SSRI monotherapy versus thecombination of SSRI and PDE-5 inhibitor therapy for the treatment of premature ejaculation. A statistically significant delay in time to ejaculation was found in patients using both an SSRI and PDE-5 inhibitor when compared to those on an SSRI alone. It has been concluded that combination therapy may be prescribed for patients who fail SSRI monotherapy or have concomitant erectile dysfunction [6]

 

Several pharmaceutical preparations containing PDE-5 inhibitor/SSRI binary combinations can be easily found and purchased through online pharmacies in spite of not all of them yet approved by most international drug organizations. Few analytical methods were reported for some PDE-5 inhibitor/ SRI inhibitor binary combinations. there are reported HPLC methods to determine the expected combination of PDE-5 inhibitors with SRI [7] and RP-HPLC method for simultaneous estimation of SIL and FLX [8].

 

This work discusses a simple, rapid, and reliable spectrophotometric method for the simultaneous determination of SIL+DLX and SIL+FLX. The Simultaneous equation or Vierordt’s method was not reported for these new combinations. This method is typically applied to determine drug combinations that contain two drugs or more than two drugs in combined dosage form. The procedures involved in this method are few compared to other UV methods. The developed method was fully validated and applied successfully for the simultaneous estimation of mentioned mixtures. The obtained results were compared with results obtained by reference method by applying static tests, which demonstrate that there is no sufficient difference between mentioned results. 

 

2. MATERIALS AND METHODS:

2.1. Apparatus:

Uv-visible spectrophotometer (JASCO, model V650, Japan) with 1.00 cm quartz cells.

 

2.2. Chemicals and Reagents:

Pure samples of the three investigated drugs (Purity: 99.3–99.7%): Sildenafil citrate, Duloxetine hydrochloride and Fluoxetine hydrochloride (Fig.1). Reference standards were kindly obtained from Syrian Pharmaceutical Factories. (Shifa, Ibn al-Haytham, AL-Balsam).

 

Analytical grade methanol used as a solvent was purchased from Panreac, (E.U).

 

2.3. Preparation of standard solutions:

The standard stock solutions of SIL, DLX and FLX were prepared by dissolving 25 mg of each drug in 25 ml of methanol and final volume was adjusted with methanol to get solutions containing 1µg/ml, series of standard solutions were prepared to have concentration ranges mentioned in table 1. The absorbance of solutions was measured at λmax of each drug, individually. The calibration curves were plotted. All the drugs obeyed linearity in the concentration range under study. All solutions are stable for a period of 7 days when stored at (4°C).

 

 

 

 

 

 

Figure 1: Chemical structures of the studied drugs.

 

3. THEORETICAL BACKGROUND:

3.1 Simultaneous equation method:

This method of analysis was based on the absorption of both drugs in the binary mixture at the wavelength maximum of each other. The absorptivity values A (1%, 1cm) were determined for both drugs at the selected wavelengths [9].

If a sample contains two absorbing drugs (x and y) each of them absorbs at the λ max of the other one, both drugs may be possible to determine by the technique of simultaneous equation (Vierordt’s method) if certain criteria apply.

 

The concentration of both drugs in the mixture can be calculated by using following equations:

·      The absorptivities of x at λ ₁ and λ ₂, a_x1 and a_x2 respectively

·      The absorptivities of y at λ ₁ and λ ₂, a_y1 and a_y2 respectively

·      The absorbance of the diluted samples at λ ₁ and λ ₂, A₁ and A₂ respectively.

 

Let C_x and C_y be the concentration of x and y respectively in the diluted samples.

 

Two equations are constructed depending on the fact:  at λ ₁, the absorbance of the mixture is the sum of the individual absorbance of x and y [10].

 

A₁= a_x1 b C_x + a_y1 b c_y                                   eq. (1)

A₂= a_x2 b C_x + a_y2 b c_y                                   eq. (2)

For measurements in 1 cm cells, b =1cm. Rearrange  Eq.                    (2)

            A₂-ax₁ C_x

C_y=---------------                                                             eq. (3)

              ^ay2

Substituting for Cy in eq. (1) and rearranging gives:

 

                    A₂ ay1 – A¹ a _y2

C_X=------------------------------

                    a_x2 ay₁ – a_x1 a_y2                                                 eq. (4)

 

               A₁ a_x2 – A₂ a_x1

C_Y=----------------------

              a_x2 a_y1 – a_x1 a_y2                                                        eq. (5)

 

For the selection of analytical wavelengths, standard solutions of each drug were prepared separately by appropriate dilutions of standard stock solutions with methanol and scanned in in the UV range 200 – 400 nm to determine λ max of each drug. The overlay spectra of both drugs in each combination were recorded, from overlain spectra; wavelengths were selected for analysis of both drugs using simultaneous equation method. The λmax were found to be 292 nm, 288 nm and 227 nm for SIL, DLX, FLX, respectively.

 

In this work, the Simultaneous equation method was applied to determine mixture of SIL+DLX, but the determination of SIL+FLX was differentas it explained latter.

 

 

 

 

Figure 2:The overlay spectra of both drugs in each mixture.

 

3.2. Determination of SIL+FLX:

The method to determine the concentration of SIL+ FLX in their mixture is deferent because FLX has no absorbance at λ _max of Sildenafil, so we can find the concentration of SIL directly, that Absorbance of sil = Absorbance of the mixture at 292. Whereas we can calculate the concentrations of FLX and SIL from equations:

 

A₁ = a_x1C_sil                                                                                eq. (6)

 

A₂ = a_x2C_sil + a_y2C_flx                                                         eq. (7)

A₁: the absorbance of mixture at 292 nm, A₂: the absorbance of mixture at 227 nm, a_x1: the absorpitivity of sil at 292, a_x2: the absorpitivity of sil at 227, a_y2: the absorpitivity of FLX at 227.

 

By rearranging the equations 6,7:

          A₁

C_SIL= -----                                                  eq. (8)

           a_x1

 

            A₂ ^aX1 - A₁ a_X2

C_FLX = ---------------------                                      eq. (9)

                  a_Y2 a_X1

 

3.3. Determination of Absorptivity Value:

The absorptivity value of each drug was calculated using following formula [10]

 

                                     Absorbance

Absorbitivity=-------------------------------------

                         Concentration (gm ⁄ 100ml)

 

4. RESULTS AND DISCUSSION:

4.1. Selection of solvent:

After assessing, the solubility of both drugs, that form each binary mixture, in different solvents, methanol was selected as a common solvent for developing spectral characteristics. In addition, it gives us the pest sensitivity for analysis.

 

4.2. Analytical parameters:

4.2.1. Mixture 1 (SIL+DLX):

Linearity of SIL and DLX: series of the slandered solutions of SIL and DLX were prepared in the ranges of concentrations (1 – 60) µg/ml and (1-40) µg/ml, respectively. The absorbance of solutions was measured at λmax of SIL and λmax of DLX. The calibration curves were constructed by plotting absorbance versus concentration and the regression equations were resulting.

 

The equation regression of SIL: y = 0.0203x + 0.0103 at 292 nm

 

The equation regression of DLX:y = 0.0259x + 0.0036 at 288 nm

The apsorpitivities of SIL and DLX were calculated at two wavelengths 292, 288 nm to construct the simultaneous equations

 

 

 

 

Whereas

A₁, A₂ are the absorbance of mixture solution at 292 nm, 288 nm, respectively

 

4.2.2. MIXTURE 4 (SIL+FLX):

Linearity of SIL and FLX: series of the slandered solutions of SIL and FLX were prepared in the ranges of concentrations (2–20) µg/ml and (1-20) µg/ml, respectively.

 

The absorbance of SIL solutions was measured at λmax of FLX and λmax of FLX, whereas the absorbance of FLX solutions was measured at λmax of FLX (227 nm) only, because FLX solutions do not have absorbance at λmax of SIL.

 

The calibration curves were constructed by plotting absorbance versus concentration and the regression equations were resulting.

 

The equation regression of SIL: y = 0.0233x + 0.0084 at 292 nm

 

The equation regression of FLX:   y = 0.0411x + 0.0065 at 227 nm

 

The apsorpitivities of SILwere calculated at two wavelengths 292, 227 nm and the apsorpitivity of FLXwas calculated at 227 to construct the simultaneous equations:

 

 

 

 

Whereas

A₁, A₂ are the absorbance of mixture solution at 292 nm, 227 nm, respectively.

 

The results were summarized in Tables 1, A₁% values were mean of five estimations.

 

4.3. Analytical Method Validation:

4.3.1. Specificity:

No interference was observed at wavelengths indicating in the method for each mixture.

 

4.3.2. Linearity:

The linearity of the methods was estimated by plotting the absorbance values of six concentrations of each drug and each concentration was repeated five times. Results show that there is a good correlation between the concentration of the sample and their absorbance. (Table1).

 

4.3.3. Precision and Accuracy:

Repeatability: five different concentrations of each drug in the binary laboratory-prepared mixtures were evaluated five times, intraday; using the proposed methods and the RSD was calculated. Also, the among day (inter-day) precision and accuracy were tested by analyzing the same concentrations using replicate determinations repeated on five days. RSD% values were less than 2.0%, thus confirming the high precision and accuracy of the developed method for estimation of the investigated drugs in their mixture (Table 2).

 

Accuracy was determined also by matching measured concentrations of each drug with the real values and by statistical comparison between the results obtained by the proposed spectrophotometric methods and the reported method (HPLC method) for the drugs determination in the pharmaceutical dosage form. The values of calculated t and F tests are less than the arranged onesat 95% confidence level. (Table 4)

 

 

Table1: Analytical parameters of spectrophotometric method

Mixture		A1% λ1	A1% λ2	Linearity µg/ml	Correlation coefficient	LOD µg/ml	LOQ µg/ml
1	SIL (292 nm)	202.96	198.59	1-60	0.9996	0.21	0.71
	DLX (288 nm)	245.51	259.17	1-40	0.9999	0.86	0.26
2	SIL (292 nm)	233.21	547.03	2-20	0.9996	0.25	0.69
	FLX (227 nm)	-	410.82	1- 20	0.9992	0.23	0.75

 

 

Table 2: Evaluation of precision and accuracy of the proposed methods for Determination of binary laboratory-prepared mixtures of the studied drugs:

	Taken µg/ml		INTERDAY				INTRADAY
			Recovery*	RSD (%)	Recovery*	RSD (%)	Recovery*	RSD (%)	Recovery*	RSD (%)
Mixture 1	SIL	DLX	SIL		DLX		SIL		DLX
	1	1	101.33	1.68	101.20	1.98	102.00	1.18	101.00	1.98
	10	3	99.93	1.42	99.47	1.68	100.80	0.32	102.00	0.88
	20	6	99.80	0.93	101.33	1.53	99.40	0.75	101.17	1.27
	30	9	100.34	1.67	98.20	1.85	101.40	0.67	101.20	1.80
	5	5	101.20	1.52	101.00	1.52	100.30	1.50	100.80	0.99
	10	10	100.94	1.60	100.45	0.85	101.15	0.44	100.55	0.30
	20	20	101.33	1.68	101.20	1.98	102.00	1.18	101.00	1.98
Mixture 2	SIL	FLX	SIL		FLX		SIL		FLX
	2	1	101.60	1.28	101.90	1.88	101.50	0.99	102.00	0.98
	10	4	100.30	1.95	99.45	1.91	101.20	2.00	101.50	1.97
	20	8	99.23	1.61	100.90	1.93	100.90	1.59	101.75	1.97
	5	5	100.55	0.93	101.08	1.66	100.65	0.60	99.88	0.95
	10	10	101.20	1.72	99.70	0.80	101.20	1.38	101.48	1.54
	20	20	100.48	0.97	100.76	0.99	100.90	0.34	101.58	0.63

*Recovery is mean of five determinations. RSD= (SD/Mean)*100

Table 3: Results observed by changing the wavelength ± 1 nm.

	Wavelength nm	Taken	Recovery*	RSD (%)	Wavelength nm	Taken	Recovery*	RSD (%)
Mixture 1	SIL				DLX
	291	10	99.93	0.86	288	3	99.47	1.34
		20	99.80	0.28		6	101.33	0.82
		30	100.10	0.18		9	99.98	0.78
	293	10	100.80	0.32	290	3	102.33	1.76
		20	99.40	0.75		6	101.17	1.27
		30	99.43	0.18		9	100.33	0.78
Mixture 2	SIL				FLX
	291	10	99.90	0.60	226	4	101.00	1.49
		20	100.50	0.45		8	101.13	0.99
	293	10	100.90	1.59	228	4	101.75	1.97
		20	100.65	0.60		8	99.88	0.95

*Found is mean of five determinations.

 

4.3.4. Robustness:

Robustness of the method was determined by changing the wavelength ±1 nm. The results were offered in The RSD% value calculated from the robustness study was found to be less than 2%, indicating that the method is robust.

 

4.4. Application of Developed Method to Marketed Dosage Forms:

Twenty individual tablets were weighed and pulverized. An accurately weighed amount of the powder equivalent to one tablet was transferred into 100 ml volumetric flask and dissolve in 50 ml of methanol. The content of the flask was sonicated for 10 min then diluted to the volume with methanol. A portion of this solution was centrifuged for 20 min at 5000 rpm then 150 µl of the supernatant was transferred into 10 ml volumetric flask. The absorbance of the resulting solution was measured at the selected wavelengths.

 

The developed new simple simultaneous equation method was applied successfully to the marketed tablet dosage form and the assay results were indicating that this method could be effectively used for the estimation of both drugs in combined dosage form.

 

Table4: Results obtained for the determination of the studied drugs in their co-formulated tablets by the proposed method and reported Method

product		Claim (mg/tab)	Proposed method		Reference method [7]		t-test	f-test
			R* %	sd	R* %	sd
MlegraDxt **	SIL	100	99.97	0.16	99.80	0.23	1.36	2.07
	DLX	30	100.25	0.02	100.27	0.08	1.75	4.00
MlegraFxt**	SIL	100	99.43	0.43	100.06	0.48	0.14	1.31
	FLX	40	99.35	0.36	99.65	0.11	0.79	1.49

At 95% confidence limit the theatrical t- and F value at five degree of freedom are t=2.776 and f=6.26. *Recovery is mean of five determinations. ** Products name.

 

5. CONCLUSION:

Simultaneous equation method has been developed and validated for analysis of Four binary mixtures. The developed method is simple, precise, economical, and accurate. Statistical analysis proved that the method was repeatable and selective for the simultaneous estimation of drugs in some binary combinations of phosphodiesterase type 5 (PDE-5) inhibitors/ Serotonin reuptake inhibitors without any interference with the excipients. This method is superior to chromatographic separation methods in terms of the short time required for analysis, ease of action and low cost. In addition, this method does not require a spectra library or standard material as some spectrophotometric derivation methods. The simultaneous equation method is a new simple method can be used routinely for an estimate of mixtures.

 

6. CONFLICT OF INTERESTS:

The authors declare that there is no conflict of interests regarding the publication of this paper.

 

7. REFERENCES:

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Accepted on 30.11.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(2):711-716.