analysis sildenafil citrate dapoxetin hplc uv

7/24/2019 Analysis Sildenafil Citrate Dapoxetin HPLC UV

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Dissertation submitted to

KERALA UNIVERSITY OF HEALTH SCIENCES,

THRISSURIn partial fulfillment of the requirements for the award of degree of

MASTER OF PHARMACYPHARMACEUTICAL ANALYSIS

Submitted by

ALBIN. P. T., B. Pharm.,Reg. NO: 103290003

Under the guidance of

Prof. Dr. Y. HARIBABU., M .Pharm., Ph.D.Profeor !"# Pr$%!$&'(

DEVELOPMENT OF VALIDATED ANALYTICAL METHODS

FOR THE SIMULTANEOUS ESTIMATION OF

SILDENAFIL CITRATE AND DAPOXETINE HYDROCHLORIDE

BY UV SPECTROMETRY AND RP-HPLC


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CONTENTS

Drug Profile

Materials and Instruments Used

Plan Of Work

Method Development by UV- Spectrometryand its Validation

Method Development by RP-HPLC and itsValidation

Summary And Conclusion

Literature Review

2


3/52

LITERATURE

REVIEW

Sl. No Name of the Publisher Website Address Result

1 American Chemical Society http://www.pubs.acs.org X

2 Annual Reviews http://arjournals.annualreviews.org X

!lac"well http://www.blac"well#synergy.com/ X

$ Cambri%ge &niversity 'ress http://journals.cambri%ge.org/ X

( )ncyclopae%ia !ritannica http://search.eb.com/ X

* 'ortlan% 'ress http://www.portlan%press.com/ X

+ 'roject ,use http://muse.jhu.e%u/journals/ X

- Royal Society o Chemistry http://www.rsc.org/ X

Springer 0 luwer http://www.springerlin".com X

1 3aylor0 4ranics http://journalsonline.tan%.co.u" X

11 )ucli% http://projecteucli%.org X

12 56or% &niversity 'ress http://www.oup.co.u"/ X

1 nimbus http://www."nimbus.com X3


4/52

Cont.

Sl.No. Journals Result

1 7nternational 8ournal o 'harmaceutical ResearchAn% !io#science

2 7nventi 8ournal

8ournal o 'harmacy Research

$ 8ournal o Applie% 'harmaceutical Science

( 'a"istan 8ournal o Science

* )#journal o Chemistry

Author Method Journal Year

Ashutosh 'an%ya et al.9 '3;C 78'R!S 2+/$/212

< ,


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LOGO

HPTLC -1UV Spectroscopy-1

no RP-HPLC method!

Reported Analytical Methods of Sildenafil & Dapoetine

in !ombination

5


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Drug Profile

Dru"Sildenafil !itrate

Chemical >ame 1#?$#etho6y##@*9+#%ihy%ro#1#methyl#+#o6o##propyl#1#pyraolo?$9#%Bpyrimi%in#(#ylphenylsulonylB#$#methylpiperaine

,olecular 4ormula C22>*5$S

al#lie to $ hours

,olecular weight $+$.* g/molAppearance Dhite to o#white crystalline pow%er

Solubility ,ethanol#4reely solubleDater #.( mg/m;)thyl ether # Slightly soluble

pa -.2


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Drug Profile

Dru" Dapoetine #!l

Chemical >ame@S#>9>#%imethyl##@naphthalen#1#ylo6y#1#phenylpropan#1#amine

,olecular 4ormula C212>5.Cl

al#lie 1.(#1.* h

,olecular weight (.$1 g/mol

Appearance Dhite crystalline pow%er

Solubility ,ethanol#4reely soluble

Acetone#7nsoluble

)ther#7nsoluble

Chloroorm#7nsoluble

pa


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MATERIALS ANDINSTRUMENTS 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 220Sonica Ultrasonic cleaner.Shimadzu UV Spectrophotometer 1800 AShimadzu 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 capacityDetector- SPD M20A, Prominence Diode Array DetectorCuvettes quartz cells

Laboratory glass wares - Borosilicate$


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PLAN OF )ORKS

,etho%


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*ETHO+ +EVELOP*ENT Y

UV- SPECTRO*ETRY

AN+ ITSVALI+ATION

&'


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*ETHO+ +EVELOP*ENT

So!/ So("$o%

Sol(ent Used) *ethanol

2''+g,ml for Sildenafil Citrate -

&2'+g,ml for Dapo!etine "Cl

)'e(e%g S"

Dilutions.)

2/4//$/&'/&2/&4/&/&$ - 2'+g,ml for Sildenafil Citrate from

Stoc0 and

&12/214/31/41$//#12/$14/%1/&'1$ - &2+g,ml for Dapo!etine

"Cl from Stoc0

&&


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SPECTRU*S OF

STAN+AR++RU4S

Sil%enail I


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A%'($ of T'5(e *$6"re

I. V$ero7 #eo CS$( < =2ay& > &ay2 , a!2ay& > a!&ay2?

C+'&< =&a!2 > 2a!& , a!2ay& > a!&ay2?

II. +o"5(e &o$% '%'r$8'$o% #eo Ce< @= test > std&? =C std&)C std2? AC std& = std&) std2?B = std&) std2?Method $ablet

%ormulationabelledAmount

'm"(

)stimatedamount

'm"(

*)stimated

SD *RSD

+

7 Sil%enail ( $.*2( .2( .2 .2$


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VALIDATION OF

METHOD

&4


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LINEARITY OF SIL+ENAFIL CITRATE

' 5 &' &5 2' 25

'

'12

'14

'1

'1$

&

&12

&14

f=!? < '1'! A '1'28 < &

S$(e%'f$( C$r'e 292%#;

bsorbance =nm?

inear =bsorbance =nm??

Co%!e%r'$o%


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L$%e'r$ of +'&o6e$%e

Hro!(or$e

' 5 &' &5 2' 25

'

'1&

'12

'13

'14

'15

'1

'1#

'1$

'1%

f=!? < '1'4! A '1'&8 < &

+'&o6e$%e 231%#;

bsorbance =nm?

inear =bsorbance=nm??

Co%!e%r'$o%

A5or5'%!e %#;

Dapoetine #!l!oncentration

',"-ml(

Absorbance

01nm

1.2.1(-

2.$.2(

.*.(*

$.-.(2

*.*1

+.2

.+1-.$.-$

.*.((

1.-1.*

12

1.1-2&


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S&e!$f$!$ P'r'#eer

Method Specificity

Parameters

$ablet

%ormulation

*)stimated+

2ierodt

;actose Sil%enail -.(*


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ROUSTNESS

Method Robustness

Parameters

$ablet

%ormulation

*)stimated+

2ierodt

p@2.(1 Sil%enail -.(


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Pre!$$o% = Re!oer

+*RSD

4ntra5day Precision 4nter5day Precision

Sil%enail Citrate


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*ETHO+ +EVELOP*ENT

Y RP-HPLC AN+ ITSVALI+ATION

2'


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METHOD DEVELOPMENT

7 Selection of Stationary Phase

7Selection of the Sol6ent

7Selection of Detection Wa6elen"th

7Selection of mobile phase

7Selection of Mobile phase Ratio

7Selection of flo8 rate

7%ied !hromato"raphic !ondition

7Analysis of Miture

7Method 2alidation

2&


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Se(e!$o% of S'$o%'r P'e,

So(e% = +ee!$o% )'e(e%g

Since both Sil%enail Citrate an%


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Se(e!$o% of #o5$(e &'e

Mobile phase 9bser6ation

Acetonitrile : water Asymmetric 'ea"s

Ammonium Acetate buer : ,ethanol Asymmetric 'ea"s

Ammonium Acetate buer@p#$.$ :

Acetonitrile

Eoo% separation with tailing

Ammonium Acetate buffer'p#5:.0( ;

Acetonitrile

mM 'p#5:.0( ; Acetonitrile

&E:9p"

23


24/52

SELECTION OF *OILE PHASE

RATIO = FLO) RATE

4low rate@ml/ min

Retention time @min 5bservation

Sil%enailCitrate

1 0.B1 Symmetrical Pea=s

1.- 2.*(1 .+($ 3ailing

AmmoniumAcetate buer@p#$. : Acetonitrile

Retention time @min 5bservationSil%enailCitrate


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F$6e Cro#'ogr'&$!

Co%$$o%

Chromatographic metho% : R'#';C

Selection o wavelength : 2nm

Stationary phase : ;una (u9 C1- column @2(mmL$.*mm9 (Mm

Selection o mobile phase : Ammonium Acetate buer 1m,@p#$. :

Acetonitrile

Solvent ratio : (: (

4low rate : 1.+ml/minute

3emperature : Room temperature

S'%'r Cro#'ogr'# 25


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ANALYSIS OF *I?TURE

Dru" Amount of tablet miture

'm"(

*)stimated Assay'*( C

*RSD+Amount ta=en )stimated

Sil%enail Citrate ( $.*+ .$ .+-


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VALI+ATION OF RP-HPLC

*ETHO+

7inearity and ran"e

7Precision

7Accuracy

7System suitability studies

7Robustness

7Specificity

2#


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L$%e'r$ '% r'%ge of

S$(e%'f$( C$r'e

S(. No S$(e%'f$( C$r'e

Co%!e%r'$o%


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L$%e'r$ '% r'%ge of

+'&o6e$%e Hro!(or$e

S(.No

+'&o6e$%e ro!(or$e

Co%!e%r'$o%


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L$%e'r$ of S$(e%'f$(

C$r'e = +'&o6e$%e HC(

L$%e'r$ of S$(e%'f$( C$r'e2-20


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Pre!$$o%

@RS+

I%r'-' &re!$$o% I%er-' &re!$$o% Re&e''5$($ of$%Be!$o%

SildenafilCitrate

Dapo!etine "Cl SildenafilCitrate

Dapo!etine"Cl

SildenafilCitrate

Dapo!etine"Cl

'1'''2# '1''''% '1'''''5 '1' '1''''''5 '1'''2

4ntra5day Precision Repeatability of inection

4nter5day Precision

3&


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A!!"r'! = Se#

S"$'5$($ S"$e

Lee( @ Re!oer @RS+

Sildenafil

Citrate

Dapo!etine

hydrochloride

Sildenafil

Citrate

Dapo!etine

hydrochloride

5'E %%14 %%14# '1'2 '1'''''''5

&''E %%15 %%14 '1''''5 '1''2

+r"g R N P$ T

Sildenafil Citrate

41352

2%%213$& '1%%%' &13#5

Dapo!etine"ydrochloride

2#3512& '1%%%' &1'#$

System Suitability

Accuracy

32


33/52

Ro5"%e '% S&e!$f$!$

Cro#'ogr'&$!

!o%$$o%

Pe'/ 're'

S$(e%'f$(C$r'e

+'&o6e$%eHC(

*obile phase ratio 4.% &42&24 &545$

.%4 &422'2 &535$'

p" of Fater 412 &42&5

414 &43$ &4$#

6low rate &1 &4#$%5 &%$#

&1$ &4#45 -

Sildenafil

Citrate

S'r!

Dapo!etine

hydrochloride

S'r!

Sildenafil

Citrate

L'!oe

Dapo!etine

hydrochloride

L'!oe

&4&33133 &5##41$3 &4354133 &5##$51

33

7 Specificity

Aer'ge Pe'/ Are'

S$(e%'f$(C$r'e

+'&o6e$%eHC(

1D13.>3 1GG02.

Robustness

SUMMARYANDCONCLUSION


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SUMMARY AND CONCLUSIONE2 Spectrophotometric Method de6elopment and 2alidation

Parameters 2alues

Sildenafil !itrate Dapoetine #!l

Dor"ing ma6 22nm 21nm

Solvent ,ethanol

;inearity @Mg/m; 2#2 1.2#12

Analysis o ,i6ture @2ierodtFs .2(K .-K

Analysis o ,i6ture @Double pointstandardi3ation(

-.1K .1*KRP5#P! Method de6elopment and 6alidation

Parameters2alues

Sildenafil !itrate Dapoetine #!l

Dor"ing ma6 2nm

Solvent ,ethanol,obile 'hase Ammonium acetate @p: $. I Acetonitrile @(:(;inearity @Mg/m; 2#2 1.2#12Resolution $.(23heoretical 'lates 22.-1 2+(.2*13ailing 4actor 1.+( 1.+-

'ea" 'urity 7n%e6 . .Analysis o ,i6ture .$K .2K

34


35/52

REFERENCESGEarry +(#$1X9 *#*-

@212.

G8inen%ra ,. Sonpet"arJ9 a"ul !. 'atel An% ,ansi 8. Dag%ari"ar. &v#

Spectrophotometric ,etho% 4or )stimation 5 Secni%aole 7n !ul" An% 3ablet


36/52

ANNE?URE

Albin '3J9 H aribabu9 SosammaCicy )apen9 Sheeja=elayu%han utty9 umar '9>ithyamol '9 =ali%ate% Spectrophotometric ,etho%s or Simultaneous

)stimation o Sil%enail Citrate an%


37/52

LOGO

'%/ o".

aln1albinHgmail1com

Presented by,ALBIN. P. T., B. Pharm.,

Reg. NO: 103290003


38/52

Sildenafil G Dapoetine

'96erlay of %ormulation(

3$


39/52

A##o%$"# A!e'e5"ffer&H-D.3; :

A!eo%$r$(e :9D;

3%


40/52

A##o%$"# A!e'e 5"ffer&H-D.3; :

A!eo%$r$(e D:9;

4'


41/52

A##o%$"# A!e'e 5"ffer&H-D.D; :

A!eo%$r$(e

4&


42/52

A##o%$"# A!e'e 5"ffer&H-D.2; :

A!eo%$r$(e

42


43/52

F(oJ r'e 1. #( #$%;

43


44/52

F(oJ r'e 1.> #( #$%;

44


45/52

Se# S"$'5$($

45


46/52

4


47/52

OVERLAY SPECTRU*S OF

FOR*ULATION

4#


48/52

A5or5'%!e '% '5or&$$ of S$(e%'f$(

C$r'e ' e(e!e )'e(e%g

4$

Concentration

=g,ml?

2%2nm 23&nm

bs1 bsorpti(ity (g1

bsorpti(ity

bs1 bsorpti(ity (g1

bsorpti(ity

2 '1'3% '1'&%5'

'1'2$%$3

'1'#5 '1'3#5'

'1'42%%#

4 '1'%& '1'22#5 '1&$# '1'4#5

'1&45 '1'24& '12%$ '1'4%

$ '1 '1'2&5' '134% '1'432

&' '12' '1'2'' '142& '1'42&'

&2 '123% '1'%%& '14%' '1'4'$3

&4 '12%# '1'2&2& '1&2 '1'43#&

& '132# '1'2'43 '1#4 '1'42&2&$ '135 '1'2'2# '1#53 '1'4&$3

2' '14'5 '1'2'25 '1$3# '1'4&$5


49/52

A5or5'%!e '% '5or&$$ of

+'&o6e$%e HC( ' e(e!e J'e(e%g

4%

Concentration

=g,ml?

2%2nm 23&nm

bs1 bsorpti(ity (g1

bsorpti(ity

bs1 bsorpti(ity (g1

bsorpti(ity

&12 '1'32 '1'2

'1'2&'$45

'1&5$ '1&3

'1&'45

214 '1'5& '1'2&25'1253 '1&'54

31 '1'#& '1'&%#222 '135 '1'%$$$%41$ '1&'4 '1'2& '1523 '1&'$%5$ '1&22 '1'2'3333 '1&3 '1&'2

#12 '1&4 '1'2'2##$ '1#3& '1&'&52$$14 '1 '1'2'5%52 '1$4% '1&'&'#&

%1 '1&% '1'2'4& '1%55 '1'%%4#%&'1$ '12& '1'2 &1'% '1'%$%$&&2 '123% '1'&%%& &1&$2 '1'%$5

J


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S&e!$f$!$ Cro#'or'#

5'


51/52

CO*PARISON OF

OVERLAY SPECTRU*S

5&

Sil%enail I


52/52

Precision Study

Sl

o

Pe'/ 're' of I%r'-' &re!$$o% @RS+

S$(e%'f$( C$r'e

10

analysis sildenafil citrate dapoxetin hplc uv

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