Analysis Of Dapoxetine In Bulk

Published Date: 02 Nov 2017

1Sri Vishnu College of Pharmacy, Bhimvaram, West Godavari, A.P.

2IST, Center for Pharmaceutical Sciences ,JNTU,Hyderabad

3St Peter’s institute of Pharmaceutical Sciences, Vidyanagar, Hanamkonda, Warangal, A.P.

4Sri Venkateshwara College of Pharmacy, Madhapur, Hyderabad, A.P.

ABSTRACT:

A simple, rapid, selective and sensitive reverse phase high performance liquid chromatographic method has been developed for the estimation of Dapoxetine in its pure form as well as in pharmaceutical dosage form. Chromatography was carried out on BDS Hypersil, C-18 column (150x 4.6mm), 5µ particle size using a mixture of Phosphate buffer (pH 2.5) and Acetonitrile (50:50 v/v) as the mobile phase at a flow rate of 1ml/min. The detection was monitored out by UV detector at 292nm. The retention time of the drug was 3.711±0.001 min. The method was successfully validated in accordance to ICH guidelines acceptance criteria for specificity, linearity, accuracy, precision and robustness. The specificity of the method was evaluated by performing stress studies. The degradation products formed were well separated from the main peak. The drug was labile in acid, base and photo conditions and it is stable in all the other conditions. The method produced linear responses in the concentration range of 10-80µg/ml of Dapoxetine. The method was found to be reproducible for analysis of the drug in tablet dosage forms.

Keywords: Dapoxetine, RP-HPLC method development, Validation.

Corresponding author: E-mail: [email protected]

INTRODUCTION

Chemically Dapoxetine is (S)-N,N-[dimethyl-3-(naphthalen-1-yloxy)-1-phenylpropan-1-

amine] hydrochloride. Its empirical formula is C21H23NO. Dapoxetine is a white coloured

powder with molecular mass of 305.413 g/mol. It is partially soluble in water and greatly

soluble in methanol. It is a short-acting selective re-uptake inhibitor1 and the only drug with

regulatory approval for the treatment of premature ejaculation in men2.

A literature survey revealed that only a few HPLC3-5 methods are available for the estimation of Dapoxetine. The authors now propose a new validated, sensitive and reproducible and stability indicating HPLC method for the determination of Dapoxetine and the Tablet dosage forms was also observed.

EXPERIMENTAL

Chromatographic conditions

A prominence isocratic HPLC system (Younglin HPLC YL9000 series) with YL 9110 Pump and with "autochro 3000" software and UV-Vis detector YL9120, BDS Hypersil C18 ODS column (250x4.6mm, 5µ). A 20µL Hamitton injection syringe was used for sample injection. HPLC grade, a freshly prepared potassium dihydrogen ortho phosphate(pH2.5) and Acetonitrile (50:50v/v) was used as the mobile phase. The solvents was filtered through a 0.45µ membrane filter and sonicated before use. The flow rate of the mobile phase was maintained at 1.0mL/min. the column temperature was maintained at room temperature, the detection of the drug was carried out at 290nm. Stress studies were carried by using O.1N Hcl. O.1N NaOH and 3% H2O2. Photo stability studies were carried out by exposing to sunlight for 3 days.

Selection of mobile phase

The solution of Dapoxetine was injected into the HPLC system and run in different solvent systems. Different mobile phases containing methanol, water, acetonitrile and phosphate buffer in different proportions were tried and finally phosphate buffer(pH 2.5) and Acetonitrile (50:50 v/v) was selected as an appropriate mobile phase which gave good resolution and acceptable peak parameters for Dapoxetine.

Preparation of Mobile Phase

Mobile phase comprised of 10 mM Potassium dihydrogen ortho phosphate (Adjusted to PH 2.5±0.05 with Ortho phosphoric acid), and Acetonitrile (50:50 v/v). Mobile phase was filtered through a 0.45-µm membrane filter, degassed with a helium spurge for 20 min and pumped from the respective solvent reservoir to the column (flow rate, 1 ml/min), which yield a column back pressure of 653-750 psi. Run time was set as 6 min, column was equilibrated for 60 min with mobile phase flowing through the system. Eluents were monitored at 290nm and data were acquired, stored and analyzed with the software "Autochro-3000" (Young Lin).

Selection of analytical wavelength

From the standard stock solution, further dilutions were prepared using mobile phase and scanned over the range of 200 – 400 nm and the spectrum was overlain. It was observed that 290nm is the max for Dapoxetine and the wavelength suitable for Dapoxetine was preferred.

Checking the resolution of drug and material standard

The column was saturated with the mobile phase (indicated by constant back pressure at desired flow rate). Standard solution of Dapoxetine was injected to get the chromatogram. The retention time for Dapoxetine was found to be 3.71 min.

Preparation of Standard Solutions:

A stock solution of Dapoxetine was prepared by dissolving Dapoxetine(100 mg) in a volumetric flask(100 ml) containing 25 ml of diluent(Mobile Phase), sonicated for 20 min and then made up to the volume with diluent. Working standard solution of Dapoxetine(100µg/ml) was prepared by suitable dilution of stock solution with diluent(Mobile Phase). Linearity solutions were prepared in diluents(Mobile Phase) containing 10-80µg/ml. Each of these drug solutions (20µl) was injected into the column and the peak area and retention times were recorded.

Estimation of Dapoxetine in Tablets:

Two commercial samples of the Tablets containing the drug were chosen for testing the suitability of the proposed method to estimate Dapoxetine Tablets. For this, weigh accurately quantity of the powdered contents of tablets equivalent to about 100mg of Dapoxetine into 100mL volumetric flask, add about 60mL of diluents, sonicate for about 30min and dilute to 20 ml with water and methanol. Filter through 0.45 micron filter. The contents of the flasks were made up to the volume with the mobile phase and mixed well. From the above stock, 40 µg/mL sample solution was prepared with mobile phase. Twenty micro liters of each of these solutions was then injected five times into the column. The mean peak area ratios of the drug to the five such determinations were calculated and the drug content in the tablets was quantified using the regression equation obtained for the pure sample.

Results & discussion:

The present study was carried out to develop a sensitive, precise and accurate RP-HPLC method for the analysis of Dapoxetine in bulk drug and pharmaceutical dosage forms. The retention time for Dapoxetine was 3.711 minutes for a run period of 6 minutes (Fig.1 and Table1). Each sample was injected five times and the similar retention times were observed in all cases. The peak areas of different concentrations set up as above were calculated and average value for 5 such determinations are shown in Table-2 The peak area for drug solution was reproducible as indicated by low coefficient of variation(Fig.2).

Stress studies were done to establish specificity of the method. The degradation products formed were well separated from the main peak, indicating the method is specific.

A good linear relationship (r2 = 1.000) was observed between the concentration of Dapoxetine and the respective peak areas. The calibration graph was found to be linear in the range of 10-80 g/mL, when the Dapoxetine solution was analyzed by the proposed RP-HPLC method. In intra and inter day variation studies, inter day and intraday precision was determined by analyzing the drug sample at three different concentration levels.

The results are presented in the form of %RSD which is below 1.00 (Tables 3,4 and 5) which shows that the proposed HPLC method was highly precise. The method was robust (Table 7) as observed from insignificant variation in the results of analysis by changes in flow rate, mobile phase composition and temperature separately. The drug content in the Tablet was quantified using the proposed analytical method. The tablet was found to contain an average 101.755 of the labeled amount of drug (Table 6). The proposed reversed phase HPLC method was found to be simple, precise, highly accurate, specific and less time consuming.

Fig: 1. A typical chromatogram for Dapoxetine standard solution (60µg/ml)

Table 1: A parameters for typical chromatogram for Dapoxetine (60µg/ml) standard Solution

S.NO

Drug

RT(min)

Peak Area

Height

Tailing factor

Plates

HETP

1.

Dapoxetine

3.711±0.001

3465.480

410.1589

1.3

2028

0.0035

Table 2: Calibration of the proposed method for Dapoxetine

S.No

Concentration(g/ml)

Retention Time(min)

Peak Area

Peak Height

1

10

3.711

577.450

68.334

2

20

3.712

1155.456

136.710

3

30

3.711

1732.456

205.078

4

40

3.709

2310.453

273.445

5

50

3.712

2887.466

341.798

6

60

3.712

3465.480

410.505

7

70

3.710

4042.536

478.506

8

80

3.710

4620.399

543.86

Fig 2: Linearity curve of Dapoxetine

Table 4: Intra-day precision of Dapoxetine

Concentration(g/ml)

Peak Area

Mean(n=5)

S.D

% RSD

60

3465.222

3461.6372

4.930172481

0.1424

60

3465.256

60

3456.254

60

3456.219

60

3465.235

Table 4: Inter-day precision of Dapoxetine

Concentration(g/ml)

Peak Area

Mean(n=5)

S.D

% RSD

60

3464.456

3459.884

5.228142452

0.151

60

3464.256

60

3454.254

60

3454.219

60

3462.235

Table 5: Recovery data of Dapoxetine:

Amount pure drug Added g/ml

Mean Amount found g/ml, (n=5)

±SD

Mean Percent recovery

Percent RSD

20

20.007

0.0153

100.03

0.765

40

40.016

0.0020

100.04

0.005

60

60.103

0.0122

100.17

0.020

Table 6: Assay of Dapoxetine

Formulation

Label Claim

Amount found

% Amount found

Brand-1

30

29.93

99.766

Brand-1

30

30.04

100.133

Table 7: Results of the Robustness Study

Variables

Chromatographic Parameters

Retention Time(Minutes)

Tailing Factor

Theoretical Plates

% Assay

55% Acetonitrile (More Organic)

3.653

1.3

2002.6

100.04

45% Acetonitrile (Less Organic)

3.753

1.3

2018

100.07

Flow Rate-1.1ml/mint

(more flow)

3.743

1.2

2120

99.989

Flow Rate-0.9ml/mint

(Less flow)

3.754

1.4

2146.2

100.05

Structure of Dapoxetine

C:\Documents and Settings\office\Desktop\800px-Dapoxetine-2D-skeletal.png