analysis sildenafil citrate dapoxetin hplc uv
TRANSCRIPT
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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
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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
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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!$$o% = Re!oer
+*RSD
4ntra5day Precision 4nter5day Precision
Sil%enail Citrate
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*ETHO+ +EVELOP*ENT
Y RP-HPLC AN+ ITSVALI+ATION
2'
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METHOD DEVELOPMENT
7 Selection of Stationary Phase
7Selection of the Sol6ent
7Selection of Detection Wa6elen"th
7Selection of mobile phase
7Selection of Mobile phase Ratio
7Selection of flo8 rate
7%ied !hromato"raphic !ondition
7Analysis of Miture
7Method 2alidation
2&
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Se(e!$o% of S'$o%'r P'e,
So(e% = +ee!$o% )'e(e%g
Since both Sil%enail Citrate an%
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Se(e!$o% of #o5$(e &'e
Mobile phase 9bser6ation
Acetonitrile : water Asymmetric 'ea"s
Ammonium Acetate buer : ,ethanol Asymmetric 'ea"s
Ammonium Acetate buer@p#$.$ :
Acetonitrile
Eoo% separation with tailing
Ammonium Acetate buffer'p#5:.0( ;
Acetonitrile
mM 'p#5:.0( ; Acetonitrile
&E:9p"
23
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SELECTION OF *OILE PHASE
RATIO = FLO) RATE
4low rate@ml/ min
Retention time @min 5bservation
Sil%enailCitrate
1 0.B1 Symmetrical Pea=s
1.- 2.*(1 .+($ 3ailing
AmmoniumAcetate buer@p#$. : Acetonitrile
Retention time @min 5bservationSil%enailCitrate
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F$6e Cro#'ogr'&$!
Co%$$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!$$o% Re&e''5$($ of$%Be!$o%
SildenafilCitrate
Dapo!etine "Cl SildenafilCitrate
Dapo!etine"Cl
SildenafilCitrate
Dapo!etine"Cl
'1'''2# '1''''% '1'''''5 '1' '1''''''5 '1'''2
4ntra5day Precision Repeatability of inection
4nter5day Precision
3&
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A!!"r'! = Se#
S"$'5$($ S"$e
Lee( @ Re!oer @RS+
Sildenafil
Citrate
Dapo!etine
hydrochloride
Sildenafil
Citrate
Dapo!etine
hydrochloride
5'E %%14 %%14# '1'2 '1'''''''5
&''E %%15 %%14 '1''''5 '1''2
+r"g R N P$ T
Sildenafil Citrate
41352
2%%213$& '1%%%' &13#5
Dapo!etine"ydrochloride
2#3512& '1%%%' &1'#$
System Suitability
Accuracy
32
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Ro5"%e '% S&e!$f$!$
Cro#'ogr'&$!
!o%$$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
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REFERENCESGEarry +(#$1X9 *#*-
@212.
G8inen%ra ,. Sonpet"arJ9 a"ul !. 'atel An% ,ansi 8. Dag%ari"ar. &v#
Spectrophotometric ,etho% 4or )stimation 5 Secni%aole 7n !ul" An% 3ablet
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ANNE?URE
Albin '3J9 H aribabu9 SosammaCicy )apen9 Sheeja=elayu%han utty9 umar '9>ithyamol '9 =ali%ate% Spectrophotometric ,etho%s or Simultaneous
)stimation o Sil%enail Citrate an%
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LOGO
'%/ o".
aln1albinHgmail1com
Presented by,ALBIN. P. T., B. Pharm.,
Reg. NO: 103290003
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Sildenafil G Dapoetine
'96erlay of %ormulation(
3$
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A##o%$"# A!e'e5"ffer&H-D.3; :
A!eo%$r$(e :9D;
3%
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A##o%$"# A!e'e 5"ffer&H-D.3; :
A!eo%$r$(e D:9;
4'
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A##o%$"# A!e'e 5"ffer&H-D.D; :
A!eo%$r$(e
4&
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A##o%$"# A!e'e 5"ffer&H-D.2; :
A!eo%$r$(e
42
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F(oJ r'e 1. #( #$%;
43
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F(oJ r'e 1.> #( #$%;
44
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Se# S"$'5$($
45
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4
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OVERLAY SPECTRU*S OF
FOR*ULATION
4#
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A5or5'%!e '% '5or&$$ of S$(e%'f$(
C$r'e ' e(e!e )'e(e%g
4$
Concentration
=g,ml?
2%2nm 23&nm
bs1 bsorpti(ity (g1
bsorpti(ity
bs1 bsorpti(ity (g1
bsorpti(ity
2 '1'3% '1'&%5'
'1'2$%$3
'1'#5 '1'3#5'
'1'42%%#
4 '1'%& '1'22#5 '1&$# '1'4#5
'1&45 '1'24& '12%$ '1'4%
$ '1 '1'2&5' '134% '1'432
&' '12' '1'2'' '142& '1'42&'
&2 '123% '1'%%& '14%' '1'4'$3
&4 '12%# '1'2&2& '1&2 '1'43#&
& '132# '1'2'43 '1#4 '1'42&2&$ '135 '1'2'2# '1#53 '1'4&$3
2' '14'5 '1'2'25 '1$3# '1'4&$5
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A5or5'%!e '% '5or&$$ 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'
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CO*PARISON OF
OVERLAY SPECTRU*S
5&
Sil%enail I
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Precision Study
Sl
o
Pe'/ 're' of I%r'-' &re!$$o% @RS+
S$(e%'f$( C$r'e
10