DEVELOPMENT OF VALIDATED ANALYTICAL METHODS

FOR THE SIMULTANEOUS ESTIMATION OF

SILDENAFIL CITRATE AND DAPOXETINE HYDROCHLORIDE
BY UV SPECTROMETRY AND RP-HPLC

Dissertation submitted to

KERALA UNIVERSITY OF HEALTH SCIENCES,

THRISSUR
In partial fulfillment of the requirements for the award of degree of

MASTER OF PHARMACY
PHARMACEUTICAL ANALYSIS

Submitted by

ALBIN. P. T., B. Pharm.,

Reg. NO: 103290003

Under the guidance of

Prof. Dr. Y. HARIBABU., M .Pharm., Ph.D.

Professor cum Principal

CONTENTS
Literature Review
Drug Profile
Materials and Instruments Used
Plan Of Work
Method Development by UV- Spectrometry
and its Validation
Method Development by RP-HPLC and its
Validation
Summary And Conclusion
2

LITERATURE REVIEW
Sl. No

Name of the Publisher

Website Address

Result

American Chemical Society

http://www.pubs.acs.org

Annual Reviews

http://arjournals.annualreviews.org

Blackwell

http://www.blackwell-synergy.com/

Cambridge University Press

http://journals.cambridge.org/

Encyclopaedia Britannica

http://search.eb.com/

Portland Press

http://www.portlandpress.com/

Project Muse

http://muse.jhu.edu/journals/

Royal Society of Chemistry

http://www.rsc.org/

Springer & Kluwer

http://www.springerlink.com

10

Taylor& Franics

http://journalsonline.tandf.co.uk

11

Euclid

http://projecteuclid.org

12

Oxford University Press

http://www.oup.co.uk/

13

Knimbus

http://www.knimbus.com

Cont.
Sl.
No.

Journals

Result

International Journal of Pharmaceutical Research

And Bio-science

Inventi Journal

Journal of Pharmacy Research

Journal of Applied Pharmaceutical Science

Pakistan Journal of Science

E-journal of Chemistry

Author

Method

Journal

Year

Ashutosh Pandya et al.,

HPTLC

IJPRBS

27/04/2012

D M Dhaduk et al.,

UV

Inventi

27/04/2012

Reported Analytical Methods of Sildenafil & Dapoxetine

LOGO
in Combination

no RP-HPLC method!

HPTLC
-1
UV Spectroscopy -1
5

Sildenafil Citrate

Drug Profile

Sildenafil Citrate

Drug
Chemical Name

1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3d]pyrimidin-5-yl)phenylsulfonyl]-4-methylpiperazine

Molecular Formula

C22H30N6O4S

Half-life

3 to 4 hours

Molecular weight

474.6 g/mol

Appearance
Solubility

pKa

White to off-white crystalline powder

Methanol-Freely soluble
Water -3.5 mg/mL
Ethyl ether - Slightly soluble
8.2

Dosage

30 mg to 100 mg per day

Activity

Selectively inhibits the PDE-5A that hydrolyzes cGMPcGMP by

preventing it from breaking down Vasodilation and increased Blood
flow.
Headache, nasal congestion and impaired vision.

ADR

Dapoxetine HCl

Drug Profile
Drug
Chemical Name
Molecular Formula
Half-life
Molecular weight

Dapoxetine HCl
(S)-N,N-dimethyl-3-(naphthalen-1-yloxy)-1-phenylpropan-1-amine
C21H23NO.HCl
1.5-1.6 h
305.413 g/mol

Appearance

White crystalline powder

Solubility

Methanol-Freely soluble
Acetone-Insoluble
Ether-Insoluble
Chloroform-Insoluble

pKa
Dosage

9
30-100mg/day

Activity

Inhibition of neuronal reuptake of serotonin potentiation of

serotonin activity increase ejaculation time.

ADR

Facial flushing , headache, stomach pain, nasal congestion, nausea &

diarrhea.
7

MATERIALS AND
INSTRUMENTS USED
Drug Samples:

Sildenafil Citrate (99%) and Dapoxetine HCl (99%)

Erectem tablets

Chemicals and Solvents Used For Estimation:

Ortho Phosphoric Acid (AR Grade)
Acetonitrile ( HPLC Grade)
Methanol (HPLC Grade)
Water (HPLC Grade)
Ammonium acetate(AR Grade)

Instruments used:

Shimadzu balance AY 220

Sonica Ultrasonic cleaner.
Shimadzu UV Spectrophotometer 1800 A
Shimadzu LC-20 AT HPLC.
Pump
Prominence LC 20 AT (double pump)
Column Phenomex Luna 5 C18 (12) 100A,(250 4.6m.m, 5)
Injector Rheodyne 7725i type injector with 20l loop capacity
Detector SPD M20A, Prominence Diode Array Detector
Cuvettes quartz cells
Laboratory glass wares - Borosilicate
8

PLAN OF WORKS

Drugs:- Sildenafil Citrate and Dapoxetine HCl in Combination(tablet)

Method Development by
UV- SPECTROMETRY
and its Validation

Method Development by
RP-HPLC
and its Validation

METHOD DEVELOPMENT BY
UV- SPECTROMETRY
AND ITS
VALIDATION

10

METHOD DEVELOPMENT
Stock Solutions
Solvent Used- Methanol
200g/ml for Sildenafil Citrate &
120g/ml for Dapoxetine HCl
Wavelength Study
Dilutions:2,4,6,8,10,12,14,16,18 & 20g/ml for Sildenafil Citrate from
Stock and
1.2,2.4,3.6,4.8,6,7.2,8.4,9.6,10.8 & 12g/ml for Dapoxetine
HCl from Stock
11

SPECTRUMS OF
STANDARD DRUGS

Sildenafil + Dapoxetine

Sildenafil

SILDENAFIL CITRATE &

DAPOXETINE HCl

Sildenafil + Dapoxetine (Overlay)

Dapoxetine
12

Analysis of Tablet Mixture

I.

Sildenafil Citrate ~50mg

Dapoxetine HCl

~30mg

Vierodts method

CSil = (A2ay1 A1ay2 / ax2ay1 ax1ay2)

CDap = (A1ax2 A2ax1 / ax2ay1 ax1ay2)
II. Double point standardization method

Ctest = [(A test A std1) (C std1-C std2) +C std1 (A std1-A std2)] / (A std1-A std2)

Method

II

Tablet
Formulation

Labelled
Amount
(mg)

Estimated
amount
(mg)

%
Estimated

Sildenafil

50

49.625

99.25

0.023

0.234

Dapoxetine

30

29.724

99.08

0.000

0.000

Sildenafil

50

49.05

98.10

0.004

0.041

Dapoxetine

30

29.748

99.16

0.004

0.068

SD

%RSD
*

*Mean of six observations

13

VALIDATION OF
METHOD
14

LINEARITY OF SILDENAFIL CITRATE

Sildenafil Citrate (292nm)
1.4
1.2

f(x) = 0.06x + 0.02

R = 1

1
0.8

Absorbance (nm)
Linear (Absorbance (nm))

Absorbance(nm)
0.6
0.4
0.2
0
0

5
10
15
Concentration (g/ml)

20

25

Sildenafil Citrate
Concentration
(g/ml)

Absorbance
292nm

0.039

0.091

0.145

0.172

10

0.206

12

0.239

14

0.297

16

0.327

18

0.365

20

0.405
15

Linearity of Dapoxetine
Hydrochloride
Dapoxetine HCl

Dapoxetine (231nm)

Concentration
(g/ml)

0.9
0.8

f(x) = 0.04x + 0.01

R = 1

0.7
0.6

Absorbance (nm)
Linear (Absorbance
(nm))

0.5
Absorbance
0.4 (nm)
0.3

1.2

0.158

2.4

0.253

3.6

0.2
0.1

0.356

4.8

0
0

5
10
Concentration

15

20

25

Absorbance
231nm

0.523

0.613

7.2

0.731

8.4

0.849

9.6

0.955

10.8

1.069

12

1.182
16

Specificity Parameters
Method

Specificity

Tablet

Parameters

Formulation

Lactose

Sildenafil

98.56

Dapoxetine

98.67

Sildenafil

98.56

Dapoxetine

98.67

Sildenafil

98.11

Dapoxetine

99.13

Sildenafil

98.11

Dapoxetine

99.13

Vierodt
Starch
Lactose
Double point
standardization

Starch

%Estimated*

*Mean of six observations

17

ROBUSTNESS
Method

Robustness

Tablet

Parameters

Formulation

pH(2.051)

Sildenafil

98.95

Dapoxetine

94.67

Temperature

Sildenafil

98.51

2-80C

Dapoxetine

94.67

pH(2.051)

Sildenafil

99.00

Dapoxetine

99.28

Sildenafil

98.05

Vierodt

Double point
standardization

Temperature

%Estimated*

2-80C

Dapoxetine
*Mean of six observations

99.28
18

Precision & Recovery

Intra-day Precision

Inter-day Precision
*%RSD

Intra-day Precision

Inter-day Precision

Sildenafil Citrate

Dapoxetine HCl

Sildenafil Citrate

Dapoxetine HCl

0.17809

0.0045

0.049633

0.00315

*Mean of three observations

Level

% RSD*

% Recovery
Sildenafil

Dapoxetine

Tab Mixture+80%

99.20

100.14

Tab Mixture+100%

100.91

99.33

Tab Mixture+120%

99.35

99.72

Sildenafil

Dapoxetine

0.0057

0.011

19

METHOD DEVELOPMENT
BY RP-HPLC AND ITS
VALIDATION

20

METHOD DEVELOPMENT
# Selection of Stationary Phase
# Selection of the Solvent
# Selection of Detection Wavelength
# Selection of mobile phase
# Selection of Mobile phase Ratio
# Selection of flow rate
# Fixed Chromatographic Condition
# Analysis of Mixture
# Method Validation
21

Selection of Stationary Phase,

Solvent & Detection Wavelength

C-18 column, Methanol &

239nm

Since both Sildenafil Citrate and Dapoxetine hydrochloride are polar in nature.
RP-HPLC method with C-18 column was used for the development of the
method.
Stationary phase
: Luna 5u, C-18 column (250mm4.6mm, 5m)
The drugs were readily soluble in methanol and showed good stability. Hence,
methanol was selected as solvent

Overlain spectrum of standard Sildenafil Citrate and Dapoxetine hydrochloride

22

Selection of mobile phase

1%OPA~pH

Mobile phase

Observation

Acetonitrile : water

Asymmetric Peaks

Ammonium Acetate buffer : Methanol

Asymmetric Peaks

Ammonium Acetate buffer(pH-4.4) :

Good separation with tailing

Acetonitrile
Ammonium Acetate buffer(pH-4.3) :
Acetonitrile

Good separation with symmetrical

peaks

Ammonium Acetate buffer(pH-4.2) :

Good separation with fronting

Acetonitrile

Ammonium Acetate buffer 10mM (pH-4.3) : Acetonitrile

23

SELECTION OF MOBILE PHASE

RATIO & FLOW RATE
Ammonium
Acetate buffer(pH4.3) : Acetonitrile
6:94
5:95
4:96

Flow rate
(ml/ min)
1.6
1.7
1.8

Ratio
Flow Rate

Retention time (min)

Sildenafil
Dapoxetine HCl
Citrate
2.898
2.601
3.803

4.256
3.581
4.592

Observation

Broad Peaks
Symmetrical Peaks
Asymmetrical Peaks

Retention time (min)

Sildenafil
Dapoxetine HCl
Citrate
2.843
2.601
2.651

3.415
3.581
3.754

~ 5:95
~ 1.7

Observation

Fronting
Symmetrical Peaks
Tailing
24

Fixed Chromatographic
Condition
Chromatographic method
: RP-HPLC
Selection of wavelength
: 239nm
Stationary phase
: Luna 5u, C18 column (250mm4.6mm, 5m)
Selection of mobile phase
: Ammonium Acetate buffer 10mM(pH-4.3) :
Acetonitrile
Solvent ratio
: 05: 95
Flow rate
: 1.7ml/minute
Temperature
: Room temperature

Standard Chromatogram

25

ANALYSIS OF MIXTURE
Drug

Amount of tablet mixture

Sildenafil Citrate ~50mg

Dapoxetine HCl
%Estimated

(mg)
Amount taken
Estimated

~30mg

Assay(%)
%RSD*

Sildenafil Citrate

50

49.67

99.34

0.0378

Dapoxetine

30

29.76

99.23

0.000035

hydrochloride

26

VALIDATION OF RP-HPLC
METHOD
# Linearity and range
# Precision
# Accuracy
# System suitability studies
# Robustness
# Specificity
27

Linearity and range of

Sildenafil Citrate
Sl. No

Sildenafil Citrate
Concentration

Peak Area

(g/ml)
1

350000
313324

300000

f(x) = 15614.58x - 3279.95

R = 1

280397

250000

31097

200000
Peak Area
150000

60284

90260

118312

10

146128

12

178710

14

216675

16

246337

18

280397

10

20

313324

246337
216675
178710
146128
118312

100000

Area
Linear (Area)

90260
60284

50000

31097
0

0
0

10
15
Concentration

20

25

Calibration graph of Sildenafil Citrate

(2-20g/ml)

28

Linearity and range of

Dapoxetine Hydrochloride
Sl.
No

Dapoxetine hydrochloride
Concentration

Peak Area

(g/ml)
1.2

32323

2.4

65736

3.6

98200

4.8

325085

f(x) = 26899.49x - 386.86

R = 1

300000

288210
256432

250000

350000

123394

225484
200000
Peak Area
150000
100000

157749

7.2

198498

8.4

225484

9.6

256432

10.8

28210

10

12

325085

157749
123394

50000

198498

Area

98200
65736
32323
0
0

4Concentration
6
8

10

12

14

Calibration graph of Dapoxetine hydrochloride

(1.2-12g/ml)

29

Linearity of Sildenafil Citrate(2-20g/ml) and Dapoxetine HCl (1.2-12g/ml)

Linearity of Sildenafil
Citrate & Dapoxetine HCl

30

Precision

Intra-day Precision

Repeatability of injection

Inter-day Precision
%RSD*
Intra-day precision

Inter-day precision

Repeatability of
injection

Sildenafil
Citrate

Dapoxetine HCl

Sildenafil
Citrate

Dapoxetine
HCl

Sildenafil
Citrate

Dapoxetine
HCl

0.00027

0.00009

0.0000065

0.0

0.00000056

0.00026

31

Accuracy & System

Suitability Studies
Accuracy
Level

% Recovery

%RSD*

Sildenafil

Dapoxetine

Sildenafil

Dapoxetine

Citrate

hydrochloride

Citrate

hydrochloride

50%

99.4

99.47

0.0266

0.000000056

100%

99.5

99.4

0.000056

0.0026

System Suitability
Drugs

Rs

Sildenafil Citrate
Dapoxetine
Hydrochloride

4.352

Pi

2992.381

0.9990

1.375

2735.261

0.9990

1.078

32

Robustness and Specificity

# Robustness

Chromatographic

Sildenafil
Citrate

Dapoxetine
HCl

4:96

142124

154568

6:94

142202

153580

4.2

146215

167264

4.4

146368

1.6
1.8

condition
Mobile phase ratio
pH of Water
Flow rate

*Peak area

*Average Peak Area

166487

Sildenafil
Citrate

Dapoxetine
HCl

147895

166987

146153.83

157702.5

147465

166745

# Specificity
Sildenafil

Sildenafil

Dapoxetine

Dapoxetine

Citrate

hydrochloride +

Citrate

hydrochloride

+ Starch

Starch

+ Lactose

+ Lactose

146133.33

157746.83

146354.33

157785.66
33

SUMMARY AND CONCLUSION

UV Spectrophotometric Method development and Validation
Parameters

Values
Sildenafil Citrate
292nm

Working max
Solvent

Dapoxetine HCl
231nm
Methanol

Linearity (g/mL)

2-20

1.2-12

Analysis of Mixture (Vierodts)

99.25%

99.08%

Analysis of Mixture (Double point

98.10%

99.16%

standardization)

RP-HPLC Method development and validation

Values
Parameters

Working max
Solvent
Mobile Phase
Linearity (g/mL)
Resolution
Theoretical Plates
Tailing Factor
Peak Purity Index
Analysis of Mixture

Sildenafil Citrate

Dapoxetine HCl

239nm
Methanol
Ammonium acetate (pH: 4.3) + Acetonitrile (5:95)
2-20
1.2-12
4.352
2992.381
2735.261
1.375
1.078
0.999
0.999 34
99.34%
99.23%

REFERENCES
Garry D.Christian, Analytical Chemistry, 4th edition, University of Wellington, A.W.Sons,
London, 1-4,469-475.
Willard, H.H., Merritt, L.L., Dean, J.A. and Settle, F.A., Instrumental methods of Analysis, 7 th
Edn., CBS Publishers & Distributors, New Delhi, 1986, 169-180.
A. Rajasekaran, Hand book of UV Visible and IR Spectroscopy, RUPI Publications 2010. 74-77
Ramesh L. Sawant *, Manish A. Raskar, Raihan Ahmed and Sameer Pawar, Validated
spectrophotometric methods for simultaneous estimation of telmisartan and indapamide in
pharmaceutical dosage form, Journal of Chemical Metrology, ISSN 0975-413X, 633-638
(2012).
Jinendra M. Sonpetkar*, Deval V. Joshi, Nakul B. Patel And Mansi J. Wagdarikar. UvSpectrophotometric Method For Estimation Of Secnidazole In Bulk And Tablet Dosage Form.
Sonpetkar et al., International Journal of Pharmaceutical Sciences and Research, Vol. 3(2): 536538 (2012).
ICH Harmonised Tripartite Guideline, Validation Of Analytical Procedures: Text And
Methodology Q2(R1), 2005. 6-13
Ashutosh Kar, Pharmaceutical Drug Analysis, 2nd Edn., New Age International (P) Ltd.,
Publishers, 2005. 452-455
Lloyd R. Snyder., Joseph J. Kirkland., Joseph l. Glajch., Practical HPLC Method Development,
2nd ed: 3-4, 234-242, 351-352, 25-27, 42, 653-656. Michael. E. Swartz, Ira. S. Krall. Analytical
Method development and validation. Sourabh printers pvt ltd .: India ; 2009.25-67
35

ANNEXURE
Albin PT*, Y Haribabu, SosammaCicy Eapen, SheejaVelayudhan Kutty, Kumar P,
Nithyamol P, Validated Spectrophotometric Methods for Simultaneous

Estimation of Sildenafil Citrate and Dapoxetine Hcl in Tablet Dosage Form,

The Pharma Innovation Journal, Vol. 2 No. 4 2013
Albin PT*, Y Haribabu, SosammaCicy Eapen, SheejaVelayudhan Kutty,
Kumar P, Nithyamol P, Validated RP-HPLC Method for Simultaneous
Estimation of Sildenafil Citrate and Dapoxetine HCL in Tablet Dosage Form,
International Journal of Pharmaceutical and Chemical Sciences, Volume 2,
Issue 3, July- September, 2013

36

LOGO

thank you.
Presented by,
ALBIN. P. T., B. Pharm.,
Reg. NO: 103290003

aln.albin@gmail.com

Sildenafil + Dapoxetine
(Overlay of Formulation)

38

Ammonium Acetate buffer(pH-4.3) :

Acetonitrile (6:94)

39

Ammonium Acetate buffer(pH-4.3) :

Acetonitrile (4:96)

40

Ammonium Acetate buffer(pH-4.4) :

Acetonitrile

41

Ammonium Acetate buffer(pH-4.2) :

Acetonitrile

42

Flow rate (1.6 ml/ min)

43

Flow rate (1.8 ml/ min)

44

System Suitability

45

46

OVERLAY SPECTRUMS OF
FORMULATION

47

Absorbance and absorptivity of Sildenafil

Citrate at selected Wavelength

292nm
Concentration

Abs.

Absorptivity

0.039
0.091
0.145
0.172
0.206
0.239
0.297
0.327
0.365
0.405

0.01950
0.02275
0.02416
0.02150
0.02060
0.09916
0.02121
0.02043
0.02027
0.02025

(g/ml)
2
4
6
8
10
12
14
16
18
20

231nm

Avg.
Absorptivity

0.028983

Abs.

Absorptivity

0.075
0.187
0.298
0.349
0.421
0.490
0.612
0.674
0.753
0.837

0.03750
0.04675
0.04966
0.04362
0.04210
0.04083
0.04371
0.04212
0.04183
0.04185

48

Avg.
Absorptivity

0.042997

Absorbance and absorptivity of

Dapoxetine HCl at selected wavelengths

Concentration

292nm

(g/ml)

Abs.

Absorptivity

1.2

0.032

2.4

231nm
Avg.
Absorptivity

Abs.

Absorptivity

0.0266666

0.158

0.131667

0.051

0.02125

0.253

0.105417

3.6

0.071

0.0197222

0.356

0.098889

4.8

0.104

0.0216666

0.523

0.108958

0.122

0.0203333

0.613

0.102167

0.731

0.101528

0.0210845

7.2

0.146

0.0202778

8.4

0.173

0.0205952

0.849

0.101071

9.6

0.196

0.0204166

0.955

0.099479

10.8

0.216

0.02

1.069

0.098981

12

0.239

0.0199166

1.182

0.0985

Avg.
Absorptivity

0.104665

#
49

Specificity Chromatorams

50

COMPARISON OF
OVERLAY SPECTRUMS

Sildenafil + Dapoxetine (Formulation)

Sildenafil + Dapoxetine (API)

51

Precision Study
Sl
No

Peak area of Intra-day precision

Sildenafil Citrate

Dapoxetine

Sildenafil

Dapoxetine

(10g/ml)

hydrochloride

Citrate

hydrochloride

(6g/ml)

(10g/ml)

(6g/ml

0.00027

0.00009

146205

157814

146204

157935

146173

157806

4
5

146312
146301

157874
157898

146209

157891

Day

%RSD*

Peak area of Inter-day precision

Sildenafil Citrate

Dapoxetine

146101

hydrochloride
157698

146124

157580

146104

157624

%RSD*
Sildenafil Citrate

Dapoxetine
hydrochloride

0.0000065

0.0

52