International Journal for Pharmaceutical Research … and Validation of Analytical Method for Simultaneous Estimation of Vardenafil and Dapoxetine HCl in Bulk and in Combined Dosage

International Journal for Pharmaceutical

Research Scholars (IJPRS) V-3, I-2, 2014 ISSN No: 2277 - 7873

RESEARCH ARTICLE

© Copyright reserved by IJPRS Impact Factor = 1.0285 17

Development and Validation of Analytical Method for Simultaneous Estimation of

Vardenafil and Dapoxetine HCl in Bulk and in Combined Dosage Form Patel PD, Makwana CJ, Patel MD, Dey S, Upadhyay UM

Sigma Institute of Pharmacy Vadodara, Gujarat, India.

Manuscript No: IJPRS/V3/I2/00158, Received On: 01/04/2014, Accepted On: 05/04/2014

ABSTRACT

A simple, economic, selective, precise, and stability-indicating Reverse phase High Performance Liquid

Chromatography method for analysis of Vardenafil and Dapoxetine HCl was developed and validated

according to ICH guidelines. The quantification of the drug was carried out using Hypercil BDS C18

250mm × 4.6mm × 5μm or its equivalent in isocratic mode, with mobile phase compressing of Buffer

(KH2PO4): Acetonitrile: Triethylamine (55: 45: 0.1 v/v/v) the flow rate was 1ml/min and the detection

was carried at 266 nm. The retention time for Vardenafil and Dapoxetine HCl was found to be 3.673 and

5.070min respectively. The percent assay for Vardenafil and Dapoxetine HCl was found to be 100.22%

and 99.44% respectively. The method was also applied for the determination of Vardenafil and

Dapoxetine HCl in the presence of their degradation products formed under variety of stress conditions.

Proposed method was validated for precision, accuracy, linearity range, specificity and robustness.

KEYWORDS

Vardenafil, Dapoxetine HCl, RP-HPLC, Stability Indicating, Validation

INTRODUCTION

Dapoxetine Hydrochloride

Category: Selective serotonin reuptake inhibitor.

Empirical Formula C21H23NO ·HCl, IUPAC

Name (S)-N,N-dimethyl-3-(naphthalen-1-

yloxy)-1-phenylpropan-1-amin.

Mechanism of Action Dapoxetine hydrochloride

is a SSRI in development for the management of

premature ejaculation. Its mechanism of action

is to increase the serotonin level in central

nervous system.

Vardenafil

Category: Vasoconstrictor Agents,

Phosphodiesterase Inhibitors, Anti-Impotence

Agents

*Address for Correspondence:

Paritosh D. Patel

Sigma Institute of Pharmacy, Bakrol, Ajwa-Nimeta Road,

Vadodara- 390019, Gujarat, India .

E-Mail Id: [email protected]

Development and Validation of Analytical Method for Simultaneous Estimation of Vardenafil and Dapoxetine HCl in Bulk and in Combined Dosage Form


Empirical Formula C23H32N6O4S, IUPAC Name

2-[2-ethoxy-5-(4-ethylpiperazine-1-

sulfonyl)phenyl] -5-methyl- 7-propyl-1H,

4Himidazo[4,3-f][1,2,4]triazin-4-one.

Mechanism of Action Vardenafil inhibits the

cGMP specificphosphodiesterase type 5 (PDE5)

which is responsible for degradation of cGMP

in the corpus cavernosum located around the

penis. Penile erection during sexual stimulation

is caused by increased penile blood flow

resulting from the relaxation of penile arteries

and corpus cavernosal smooth muscle. This

response is mediated by the release of nitric

oxide (NO) from nerve terminals and

endothelial cells, which stimulates the synthesis

of cGMP in smooth muscle cells. Cyclic GMP

causes smooth muscle relaxation and increased

blood flow into the corpus cavernosum. The

inhibition of phosphodiesterase type 5 (PDE5)

by vardenafil enhances erectile function by

increasing the amount of Cgmp.

MATERIALS AND METHOD

Materials

Gift sample of Vardenafil was procured from

Centurion Laboratory Pvt. Ltd. Vadodara.

Dapoxetine HCl was kindly gifted by Centurion

Laboratory Pvt. Ltd., Vadodara both the drugs

are used as a standard without further

purification. HPLC grade Methanol,

Acetonitrile, KH2PO4, Ortho-phosphoric acid

and Double distilled water were used.

Method

Identification of Drugs

Identification of both the drug should be done

by following parameter:

Melting point

IR identification

Solubility

Selection of Wavelength

The sensitivity of HPLC method that uses UV

detection depends upon proper selection of

detection wavelength. An ideal wavelength is

the one that gives good response for the drugs

that are to be detected. In the present study 10

μg/ml of VAR and 30 μg/ml of DAPO. These

drug solutions were than scanned in the UV

region of 200-400 nm and the overlay spectrum

were recorded.

Selection of Chromatographic Condition

Proper selection of the HPLC method depends

upon the nature of the sample (ionic, ionizable

or neutral molecule), its molecular weight and

solubility. The drugs selected for the present

study are polar in nature and hence either

reversed phase or ion-pair or ion exchange

chromatography can be used. Reversed phase

HPLC was selected for the initial separations

because of its simplicity and suitability. To

optimize the chromatographic conditions, the

effect of chromatographic variables such as

mobile phase, pH, flow rate, and solvent ratio

were studied. The resulting chromatograms

were recorded and the chromatographic

parameters such as capacity factor, asymmetric

factor, and resolution and column efficiency

were calculated. The conditions that gave the

best resolution, symmetry and capacity factor

were selected for estimation.

Effect of Ratio of Mobile Phase

Mixed standard solution containing 10 ppm

Vardenafil and 30 ppm Dapoxetine HCl was

chromatographed using different type of mobile

phases and optimized mobile phase was

finalised.

Preparation of Mobile Phase

A mixture of 45 ml Acetonitrile and 55 ml

0.02m Phosphate Buffer (pH 3.5+0.1% Tea) of

HPLC grade filtered through 0.75 μm filter

paper, Sonicate for 15minutes to degas the

mixture and used as mobile phase.

Preparation of Standard Stock Solution

Vardenafil Standard Stock Solution

(100μg/ml)

A 10 mg of standard Vardenafil was weighed

and transferred to a 100 ml volumetric flask and

dissolved in 25 ml mobile phase. The flask was

shaken and volume was made up to the mark

with mobile phase to give a solution containing

100μg/ml Vardenafil. Dapoxetine HCl standard



stock solution: (300μg/ml) A 30 mg of standard

Dapoxetine HCl was accurately weighed and

transferred to a 100 ml volumetric flask and

dissolved in 25 ml mobile phase. The flask was

shaken and volume was made up to the mark

with mobile phase to give a solution containing

300μg/ml Dapoxetine HCl.

Calibration Curve for Vardenafil and

Dapoxetine HCl

Calibration curve for the Vardenafil (5-15

μg/ml) and Dapoxetine HCl (15-45 μg/ml)

Appropriate volume of aliquots from standard

Vardenafi and Dapoxetine HCl stock solutions

weretransferred to same volumetric flasks of 10

ml capacity. The volume was adjusted to the

mark with mobile phase give a solution

containing 5, 7.5, 10, 12.5 and 15 μg/ml

Vardenafil and 15, 22.5, 30, 30.7, and 45 μg/ml

Dapoxetine Hcl.

The mixed standard solution was

chromatographed for 10minutes using mobile

phase at a flow rate of 1.0 ml/min. The graphs

were plotted for peak area vs. concentration for

both thedrugs.

Determination of Vardenafil and Dapoxetine

HCl from its Marketed Formulation

Ten tablets were weighed and average weight

was calculated. The tablets were crushed to

obtain fine powder. Tablet powder Weighted

equivalent to 25mg was transferred to 100 ml

volumetric flask. Mobile Phase was added to

dissolve the drugs and sonicated for 10 minutes

and then volume was made up to the mark with

same.

The solution was then filtered through

Whatmann filter paper (No. 41). From that 1 ml

of aliquots was taken and diluted up to 10 ml for

getting concentration of 100 μg/ml. From this

stock solution, working standard solution of 10

μg/ml of VAR and 30 μg/ml of DAPO was

prepared and diluted it up to 10 ml with mobile

phase. This solution was used for the estimation

of VAR and DAPO in their combined dosage

form.

Sample Preparation

Estimation of Vardenafil and Dapoxetine HCl in

its Marketed formulation: The prepared sample

solution was chromatographed for 10 minutes

using mobile phase at a flow rate of 1.0 ml/min.

From the peak area obtained in the

chromatogram, the amounts of both the drugs

were calculated by fitting peak area responses

into the equation of the straight line representing

the calibration curves for Vardenafil and

Dapoxetine HCl.

Validation of the Development HPLC

Method

Accuracy

Preparation of sample solution for % recovery

10 mg Vardenafil and 30 mg Dapoxetine HCl

was accurately weighed and transferred to

volumetric flask of 100ml capacity and aliquot

them to make final concentration10μg/ml

Vardenafil and 30μg/ml Dapoxetine HCl. The

resulting solution was filtered through Whatman

filter paper. Then chromatogram of each sample

solutions was taken at selected wavelength for

Vardenafil and Dapoxetine HCl and

concentration is calculated which is known as

pre-analyzed sample.

Table 1: Spiking of Std. solution in pre-analyzed

solution

Spiking

Concent

ration

Concentration

Of Preanalysed

Sample (μg/ml)

Concentratio

n of Spiked

Std. Solution

(μg/ml)

Total

Concentrati

on (μg/ml)

VAR DAPO VAR DAPO VAR DAPO

80 10 30 8 24 18 54

100 10 30 10 30 20 60

120 10 30 12 36 22 66



Precision

Repeatability

6 replicates of standard mixture solution having

Vardenafil (10 μg/ml) and Dapoxetine HCl

(30μg/ml) were prepared and chromatograms

were recorded and RSD was calculated.

Intraday Precision

Standard solutions containing 5, 10 and 15

μg/ml Vardenafil and 15, 30 and 45 μg/ml

Dapoxetine HCl were analyzed 3 times on the

same day as per the procedure. Chromatogram

of each sample was taken. SD and RSD were

calculated.

Inter day precision

Standard solutions containing 5, 10 and 15

μg/ml Vardenafil and 10, 30 and 60 μg/ml

Dapoxetine HCl were analyzed on three

different daysas per the procedure.

Chromatogram of each sample was taken. SD

and RSD were calculated.

Linearity and Range

The linearity peak area response was determined

by analyzing solutions having concentrations in

the range of 5-15μg/ml and 15-45 μg/ml for

Vardenafil and Dapoxetine HCl respectively

from same solution.

Limit of Detection

Calibration Curve was repeated for 6 times and

the SD of the Intercept was calculated then LOD

was calculated.

Limit of Quantitation

Calibration Curve was repeated for 6 times and

the SD of the Intercept was calculated then LOD

was calculated.

Robustness

Robustness study was performed in following

altered chromatographic conditions:

• Variation in Analyst

• Variations in the mobile phase (± 1)

• Variation in pH (± 0.1)

Forced Degradation Study (Stress Testing)

by RP-HPLC

Preparation of Stock Solution from Marketed

Formulation

Twenty capsules were taken and drug content

was collect by empty gelatin shell. Capsule

powder equivalent to 10 mg VAR and 30 mg of

DAPO was taken in 100 ml volumetric flask.

Mobile phase (50 ml) was added to the above

flask and was sonicated for 15 minutes. The

solution was filtered using 0.45 μm whatman

filter paper and volume was made up to the

mark with the mobile phase. It was

concentration of 100 μg/ml of VAR and 300

μg/ml of DAPO.

Forced degradation study was carried out by

Acid Hydrolysis, Alkali Hydrolysis, Oxidative

Hydrolysis, Thermal Hydrolysis and Photolytic

Hydrolysis.

RESULTS AND DISCUSSION

Identification of Drugs

Melting Point

Table 2: Melting Point of API

Name of

Drug

Observed

M.P (0C)

Reported

M.P (0C)

Vardenafil 170-174 (0C) 175-179 (

0C)

Dapoxetine

HCl 228-233 (

0C) 230-235 (

0C)

IR Identification by FTIR Spectra

Figure 1: FTIR spectrum of Vardenafil



IR spectrum of Vardenafil sample was

interpreted. From these, we can say that the

given sample may be Vardenafil

Figure 2: FTIR spectrum of Dapoxetine HCl

IR spectrum of Dapoxetine HCl sample was

interpreted. From these, we can say that the

given sample may be Dapoxetine HCl.

Solubility Study

Solubility

Vardenafil Dapoxetine HCl

Soluble in Distilled

water, Ethanol (98%),

Methanol, Phosphate

Buffer and

Acetonitrile

Soluble in Methanol,

Acetonitrile, Ethanol,

Phosphate Buffer and

Distilled water

Selection of Wavelength

Vardenafil and Dapoxetine HCl were scanned in

UV as well different trails were taken in RP-

HPLC at different wavelength in which both

Vardenafil and Dapoxetine HCl show

reasonably good response at 266.0nm.

Figure 3: Spectra of Vardenafil and Dapoxetine

HCl at 266 nm

Selection of Mobile Phase

Different type of mobile phase were tried and

from chromatogram optimized mobile phase

was finalised having the composition as below.

Buffer (0.02m KH2PO4 (pH-3.5+0.1% TEA)-

Acetonitrile (55-45 v/v)

Figure 4: Buffer 0.02m KH2PO4 (pH

3.5+0.1%TEA): CAN (55:45) (Finalized Mobile

phase)

Calibration Curve for the Vardenafil and

Dapoxetine HCl

Figure 5: Chromatograms of Vardenafil and

Dapoxetine HCl for Linearity



Calibration Curve for the Vardenafil (5-15

μg/ml)

Figure 6: Graph of Calibration curve for

Vardenafil

Table 4: Calibration curve for Vardenafil

Concentration

(μg/ml)

Peak Area of

Vardenafil

5 684.863

7.5 1023.604

10 1366.508

12.5 1657.954

15 2046.418

Linearity range for Vardenafil was found to be

5-15 μg/ml in Mobile Phase. Regression

Equation for Vardenafil 266 nm: Y=134.3x +

12.88. r2 value: 0.998.

Calibration Curve for the Dapoxetine HCl

(15-45 μg/ml)

Linearity range for Dapoxetine HCl was found

to be 15-45 μg/ml in mobile phase. Regression

Equation for Dapoxetine HCl at 266 nm: Y=

81.25x + 1.993 r2 value: 0.999.

Figure 7: Graph of Calibration curve for

Dapoxetine HCl

Calibration curve for the Dapoxetine HCl (15-

45 μg/ml)

Table 5: Calibration curve for Dapoxetine HCl

Concentration

(μg/ml)

Peak Area of

Dapoxetine Hcl

15 1221.746

22.5 1826.255

30 2438.193

37.5 3060.792

45 3651.643

% Assay of Vardenafil (10µg/ml) and

Dapoxetine HCl (30µg/ml) in their Marketed

Product

% Assay of Vardenafil and Dapoxetine HCl was

found in an acceptance limit as shown in table 6.

So this method could be used for analysis of this

combination.

Validation of the developed HPLC method

Accuracy (% Recovery Study)

Vardenafil:

Concentration of Preanalysed sample of

Vardenafil: 10μg/ml.



Result obtained reveals that % recovery of

Vardenafil was within acceptance criteria given

in ICH i.e. 98-102%.

Dapoxetine HCl:

Concentration of Preanalysed sample of

Dapoxetine HCl: 30 μg/ml

Result obtained reveals that % recovery of

Dapoxetine HCl was within acceptance criteria

given in ICH i.e. 98-102%.

Table 6: % Assay of Vardenafil and Dapoxetine HCl

Drug

Concentration

in Marketed

Formulation

Concentration

taken for %

Assay

Peak

Area of

Sample

Solution

Concentration

found from

marketed

Formulation

% Assay ±

SD

Vardenafil 10 mg 10 μg/ml 1463.61 10.80 100.22 ± 0.42

Dapoxetine

Hcl 30 mg 30 μg/ml 2427.08 29.80 99.44 ± 0.32

Table 7: % Recovery of Vardenafil

Level of

Recovery

Amount of

Std. VAR

Spiked (μg/ml)

Total

Amount of

VAR (μg/ml)

Amount of

VAR

recovered (μg/ml)

% Recovery

Mean %

Recovery ±

SD

80 %

8 18 7.98 99.856

99.762 ± 0.70 8 18 8.03 100.415

8 18 7.92 99.015

100 %

10 20 9.97 99.796

99.862 ± 0.84 10 20 9.89 98.998

10 20 10.00 100.684

150 %

12 22 11.90 99.810

99.946 ± 0.75 12 22 12.00 100.312

12 22 11.96 99.717



Table 8: % Recovery of Dapoxetine HCl

Level of

Recovery

Amount of

Std. DAPO

Spiked (μg/ml)

Total

Amount of

DAPO (μg/ml)

Amount of

DAPO

recovered (μg/ml)

% Recovery Mean %

Recovery ± SD

80 %

24 54 24.03 100.130

99.926 ± 0.64 24 54 24.10 100.450

24 54 23.80 99.199

100 %

30 60 29.99 99.973

100.108 ± 1.0 30 60 29.75 99.175

30 60 30.35 101.177

150 %

36 66 36.19 100.548

100.295 ± 0.37 36 66 36.16 100.471

36 66 35.95 99.866

Precision

Repeatability

Table 9: Repeatability data for Vardenafil and Dapoxetine HCl

Standard

Drug

Target

Concentration

(μg/ml)

Peak

Area of

Sample

Found

Concentration

(μg/ml)

Mean SD % RSD

Vardenafil

10 1352.853 9.9

10 0.12 1.2

10 1381.578 10.1

10 1352.450 9.9

10 1385.690 10.2

10 1351.490 9.9

10 1358.773 10.0

Dapoxetine

Hcl

30 2465.094 30.3

29.9 0.22 0.73

30 2413.817 29.7

30 2423.541 29.8

30 2472.380 30.0

30 2411.389 29.7

30 2433.313 29.9



The SD and % RSD for Repeatability of both

the drugs was found to be less than 1 and less

than 2 respectively.

So, it was concluded that proposed method for

estimation of Vardenafil and Dapoxetine HCl is

précised in nature.

Intraday Precision

The SD and % RSD for Intraday precision of

both the drugs was found to be less than 1 and

less than 2 respectively. So, it was concluded

that proposed method for estimation of

Vardenafil and Dapoxetine HCl is précised in

nature.

Table 10: Intraday precision data for Vardenafil Dapoxetine HCl

Standard

Drug

Target

Concentration

(μg/ml)

Peak Area

of Sample

Found

Concentration

(μg/ml)

Mean SD % RSD

Vardenafil

5 686.218 5.09

4.97 0.03 0.60 5 680.072 4.97

5 677.161 4.95

10 1385.69 10.2

10.06 0.12 1.19 10 1351.49 9.97

10 1358.77 10.02

15 2050.46 15.17

15.06 0.11 0.73 15 2019.84 14.94

15 2037.27 15.07

Dapoxetine

HCl

15 1224.19 15.04

14.97 0.07 0.46 15 1213.19 14.90

15 1219.31 14.98

30 2472.38 30.40

29.99 0.37 1.2 30 2411.39 29.65

30 2433.31 29.92

45 3658.94 45.00

44.72 0.34 0.76 45 3604.27 44.33

45 3644.38 44.83



Interday Precision

The SD and % RSD for Interday precision of

both the drugs was found to be less than 1 and

less than 2 respectively. So, it was concluded

that proposed method for estimation of

Vardenafil and Dapoxetinel HCl is précised in

nature.

Limit of Detection

The proposed method can detect Vardenafil and

Dapoxetine HCl at very low level .So, it was

concluded that the proposed method is very

sensitive in nature.

Limit of Quantitation

The proposed method can quantify small

amount of drugs with precisely. So, it was

concluded that the proposed method is very

sensitive in nature.

Table 11: Interday precision data for Vardenafil and Dapoxetine HCl

Standard

Drug

Target

Concentratio

n (μg/ml)

Peak Area

of Sample

Found

Concentration

(μg/ml)

Mean SD % RSD

Vardenafil

5 681.44 4.97

4.95 0.05 1.01 5 689.63 5.0

5 670.74 4.9

10 1381.58 10.1

10.01 0.07 0.69 10 1352.85 9.97

10 1352.45 9.97

15 2066.81 15.29

15.14 0.15 0.99 15 2025.97 14.98

15 2047.57 15.15

Dapoxetine

HCl

15 1215.66 14.93

14.96 0.13 0.86 15 1230.32 15.11

15 1208.31 14.84

30 2465.09 30.31

29.93 0.33 1.10 30 2413.82 29.68

30 2423.54 29.80

45 3688.13 45.36

44.99 0.46 1.02 45 3615.25 44.47

45 3669.94 45.14

Table 12: LOD data for Vardenafil and Dapoxetine HCl

Parameters Vardenafil Dapoxetine HCl

Mean Slope (n=5) 134.92 81.228

SD of Y-intercept (n=5) 7.684 2.0748

LOD (μg/ml) 0.18 0.10



Results of forced degradation study by RP-

HPLC:

Acid Hydrolysis

Figure 8: Chromatogram of Acid degradation of

Blank

Figure 9: Chromatogram of standard Drug

Table 13: LOQ data for Vardenafil and Dapoxetine HCl

Parameters Vardenafil Dapoxetine HCl

Mean Slope (n=5) 134.92 81.228

SD of Y-intercept (n=5) 7.684 2.0748

LOQ (μg/ml) 0.56 0.32

Robustness

Table 14: Robustness data for Vardenafil and Dapoxetine HCl

Parameters

(n+5) Variation

Average Peak Area % RSD

Vardenafil Dapoxetine

HCl Vardenafil

Dapoxetine

HCl

Flow rate 1.1 1292.40 2310.41 1.20 1.11

0.9 1424.29 2551.01 1.24 1.02

Mobile phase 56:44 1361.76 2434.14 0.69 0.95

54:46 1359.79 2430.08 1.05 1.00

pH 3.6 1361.56 2431.72 1.23 1.20

3.4 1360.03 2430.00 1.29 1.21



Figure 10: Chromatogram of Acid degradation

of drug product

Impurities generated from acid degradation

were well separated by this method with good

resolution.

Alkali Hydrolysis

Figure 11: Chromatogram of Alkali degradation

of Blank

Figure 12: Chromatogram of Alkali degradation

of drug product

Impurities generated from alkali degradation


resolution.

Oxidative Degradation

Figure 13: Chromatogram of Oxidative

degradation of Blank

Figure 14: Chromatogram of Oxidative

degradation of drug product

Impurities generated from oxidative degradation


resolution.

Thermal Hydrolysis

Figure 15: Chromatogram of Thermal

degradation of drug product



Impurities generated from Thermal degradation


resolution.

Photolytic Degradation

Figure 16: Photolytic degradation of drug

product

Impurities generated from Photolytic

degradation were well separated by this method

with good resolution.

CONCLUSION

A simple, specific, accurate and precise RP-

HPLC method has been developed and validated

for simultaneous estimation of Vardenafil and

Dapoxetine HCl in its dosage form. Vardenafil

and Dapoxetine HCl were estimated on

HYPERSIL BDS C18 column using Buffer

0.02m KH2PO4:Acetonitrile (pH 3.5+0.1%TEA)

(55:45 v/v) as mobile phase and detection was

carried out at 266 nm. The linearity range was

found to be 5-15 μg/ml for Vardenafil and 15-45

μg/ml for Dapoxetine HCl. The co-relation

coefficient was found to be 0.998 and 0.999 for

Vardenafil and Dapoxetine HCl respectively.

The assay value for Vardenafil and Dapoxetine

HCl was found to be 100.22% and 99.44%

respectively. A validated stability-indicating

HPLC analytical method has been developed for

the determination of Vardenafil & Dapoxetine

HCl in bulk and in dosage forms. Vardenafil

and Dapoxetine HCl does not undergo any

degradation so it can be said that Vardenafil and

Dapoxetine HCl is stable to forced degradation

conditions.

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Documents

International Journal for Pharmaceutical Research … and Validation of Analytical Method for Simultaneous Estimation of Vardenafil and Dapoxetine HCl in Bulk and in Combined Dosage