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Praktikum Bioanalisis II
PROTOKOL VALIDASI METODE ANALISIS AMITRIPTILINDALAM PLASMA SECARA KROMATOGRAFI GAS
KELAS : B
KELOMPOK : IIIANGGOTA KELOMPOK : 1. Astri Y.Permana (2010210033)
2. Astria ita Lin!i" (201021003#)
3. $ae%i ia Arsi (20102100#&)
#. 'enn Ni% a $. (20102100*1)
FAKULTAS FARMASI
UNIVERSITAS PANCASILA
AKARTA
!"#$
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PROTOKOL
No% ""#& !"#$
PROTOKOL VALIDASI METODE ANALISIS AMITRIPTILIN DALAM
PLASMA SECARA KROMATOGRAFI GAS
Dia'ukan ol()*
La+oratorium ,-La+
Fakultas Farmasi Uni.(rsitas Pan/asila
Dis0onsori ol() *
PT% Pan/ain1ra Farma
akarta2 Okto+(r !"#$
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u1ul
P+OTOKOL ,ALI'ASI METO'E ANALISIS AMIT+IPTILIN 'ALAM PLASMA
SE$A+A K+OMATOG+A-I GAS
Nomor Protokol
N . 001 2013
S0onsor
Nama : PT. Pan%ain/ra -arma
A amat : . +a a La an n 13 a arta Se atan
T(m0at P(n(litian
Nama : La4 rat ri!m Pen !5ian 67 La4 -a ! tas -armasi 8ni9ersitas Pan%asi a
A amat : . Sren sen Sa a" a a arsa a arta Se atan 12*#0
P(rsonil
Pene iti 8tama : $ae%i ia Arsi S.Si. M.Si. A;t
Pene iti Pen/am;in : Astri Y.P S.-arm A;t
Ana is : 'enn $"i in < S. -arm. A;t
' ter Penan !n 5a a4 : /r. Astria ita Lin!i"
Tan33al P(laksanaan
O t 4er 2013
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I% PENDA4ULUANAmitri;ti in s!at! /eri9ate /i4en= /i "e;ta/in mer!;a an anti/e;resan
asi an arena str! t!r imian a /ise4!t se4a ai anti/e;resan trisi i . O4at
ini 4an a /i !na an !nt! tera;i /e;resi /an 4 e" /ian a; se4a ai ;en anti
;en "am4at MAO an ti/a 4an a /i !na an a i. 'eri9at
/i4en= /i "e;ta/ine te a" /i4! ti an /a;at men !ran i ea/aan /e;resi
ter!tama /e;resi en/ en. Per4ai an 4er !5!/ se4a ai ;er4ai an s!asana
;erasaan (m /) 4ertam4a"n a a ti>itas >isi e as;a/aan menta ;er4ai an
na>s! ma an /an ; a ti/!r an e4i" terat!r.G n an 4at ini 4e er5a men "am4at am4i an em4a i ne!r transmit r
/i ta .
Amitri;ti in terse/ia /a am 4ent! ta4 et 10 /an 2& m /an /a am 4ent! ar!tan
s!nti 100 m 10 m . ' sis ;erm! aan ?& m se"ari. ' sis ini em!/iaan
/itin i an sam;ai menim4! an e>e tera;e!ti 4iasan a antara 1&0 m @ 300
m se"ari.
Amitri;ti in terse/ia /a am 4ent! aram amitri;ti in "i/r ri/a an
memi i i e ar!tan m!/a" ar!t /a am air /an etan .Amitri;ti in 4er!;a ser4!
"a4 !r ata! "a4 !r e%i ;!ti" ata! "am;ir ;!ti" ti/a 4er4a! ata! "am;ir ti/a
4er4a! .
O4at ini /i ntrain/i asi an ;a/a ;asien an "i;ersensiti> ter"a/a;
/eri9at /i4en= /i "e;ta/in. Pasien ma /is rasia /ara" /e;resi s!ms!m
t! an er!sa an "ati er!sa an ta s!4 rti a /i ntrain/i asi an
men !na an Amitri;ti in..
Ber/asar an Ba/an Pen a asan O4at /an Ma anan +e;!4 i In/ nesia
Amitri;ti in mer!;a an 4at % ; an a5i4 !nt! !5i 4i e !i9a ensi. 8nt! it!
/i;er ! an s!at! met /e ana isis 4at an ter;er%a a /a am matri s 4i is
an ses!ai. Pen ! !ran ana it /a am matri s 4i is "ar!s /i9a i/asi. ,a i/asi
met /e 4i ana isis men%a !; sem!a ;r se/!r an men!n5! an 4a" a met /e
"!s!s an /i !na an !nt! ;en ! !ran !antitati> ana it an 4erasa /a am
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'i !m;! an ; asma 4 an an /i !na an !nt! men/a;at an a i4rasi
Sam;e /i;er e" /ari s! are a an an se"at. E'TA sam;e /ara" (& mL)
/i !m;! an !nt! en ti; /anAna isis >arma ineti . Sem!a sam;e
/isim;an ;a/a 720 C $ an /i%air an se4e !m /i !na an.
C% Pr(0arasi Sam0(l8nt! &00 !L ; asma /i a%a7t!t!; 10 mL ta4!n sentri>!s &0 !L ar!tan
er5a 4a ! interna (2 m mL71) &00 !L 4!>>er Tris ; D & /an & mL "e sana:
is ;r ; i% a " (D&:& 9 9) /itam4a" an. Sete a" ;!saran ;en%am;!ran
(1 menit) %am;!ran /isentri>! asi ;a/a 3&00 se ama 10 menit /an >ase
r ani /i;in/a" an e se e as ta4!n t!t!; an 1&0 !L > s>at asam 0 1 (9
9) se4e !mn a /itam4a" an. $am;!ran ini /i9 rteF se ama 1 menit /an
em!/ian /isentri>! asi ;a/a 3&00 se ama 10 menit. La;isan r ani
/i4!an /an 100 !L >ase 4erair /i;in/a" an ;a/a mi%r 9ia !nt! a!t sam; er
/an &0 !L /is!nti .
D% O0timasi M(to1(-r nt in etS; it m /e (/i4a i rasi 20:1) /en an ;er;e%a"an /ari 23 1 mL
menit /an t ta /ari 2? 2 mL menit. Tem;erat!r a a 300 $
/en an te anan 12 ;si.$arier Gas
e i!m.O9en tem;erat!reS!"! 9en is rati an /i !na an m! ai /ari 2#0 $ /an 2*0 $.
Ana isis
!nt! ana it /a am eti asetat /i a ! an ;a/a s!"! 9en is rati 2*0
$. Nam!n an !an "a/ir 4er9ariasi /en an masin 7masin set
sam;e an /iana isis. O e" arena it! /i !na an s!"! s;esi>i !nt!
setia; set sam;e 4er9ariasi antara 2#0 $ /an 2*0 $ Pa/a /asarn a
9en s!"! /i9ariasi an 8nt! men5a a ;emisa"an /asar /ari ana it
4ai /ari sat! sama ain /an ainn a$ !mnSe4!a" 30 0 m P7& (& P"en meti si san) m a;i er
/en an /iameter
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320 m m ete4a an 0 2& M 'an s!"! ma sim!m 32& $. K nstan
te anan 12 ;si /an a a rate /ari 1 2 mL menit.'ete%t r
Nitr en7> s> r /ete t r /en an s!"! 2#0 $ Gas "i/r en /ari # 0 mL
menit /an !/ara me/is /ari *0 0 mL menit. e i!m rate /ari 1& mL menit.+!n time& menit+e ati9e Ana te +etenti n Time !nt! amitri;t ine /an n rtri;t ine /i"it!n /a am
re>erensi /en an a t! retensi stan/ar interna ma;r t ine1
!nt! amitri;t ine an 0 H0 menit /an retensi re ati>
a t! !nt! n rtri;t ine an 0 H2 menit.
E% Vali1asi M(to1(,a i/asi met /e1. Se e ti9itasSa a" sat! met /e !nt! men e9a !asi se e ti9itas met /e r mat ra>i
a/a a" mem4an/in an a t! retensi ;!n%a "a/ir /a am r mat ram
sam;e /en an a t! retensi an /i"asi an /en an met /e an sama.
Nam!n met /e ini "an a % % !nt! menent! an se e ti9itas /a amas!s7 as!s tertent! arena met /e ini san at ti/a 4isa /ian/a an.
Biasan a masa a" ini /ise esai an /en an /ata i/entitas ;!n%a
men !na an met /e ain.
Se e ti9itas met /e !nt! amitri;t ine n rtri;t ine /an ma;r t ine
/a am matri s eti asetat /itent! an /en an men !nti an /an
men ana isis seran aian s !si ana it stan/ar ;a/a *HD0 P r mat ra>i
as. a ini /i a ! an !nt! memasti an 4a" a sem!a ;!n%a ana it an
te;at /ise esai an. Lar!tan stan/art a/a a" eti asetat s n . Se an5!tn a
stan/ar ter;isa" amitri;t ine n rtri;t ine /an ma;r t ine /iana isis
!nt! menent! an a t! retensi masin 7masin . Sete a" a t! retensi
!nt! masin 7masin /ari ti a sen a a /itent! an stan/ar %am;!ranan men an/!n nsentrasi an sama amitri;t ine n rtri;t ine /an
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ma;r t ine /is!nti an !nt! menent! an a;a a" ti a 4at /a;at
/i;isa" an an ses!ai res !si . Se4!a" r mat ram men am4ar an
4ase ine ;emisa"an ti a m; nen amitri;t ine n rtri;t ine /an
ma;r t ine a/a a" /isa5i an ;a/a Gam4ar A.1 ( Lam;iran A ) .2. A !rasiPresisi /an a !rasi /i;eri sa /en an menam4a" an se5!m a" ana it ;a/a
; asma s n /en an a "ir nsentrasi 1& 200 /an #00 n mL71
masin 7masin ana it. Sam;e ntr !a itas /iana isis /a am ran a;
ti a ;a/a setia; * "ari. 1H nsentrasi ter! !r ;er nsentrasi tin at
men5a/i sasaran ana isis 9arians (ANO,A) men in at "ari se4a ai
9aria4e ;en e m; an. A !rasi /i"it!n /en an mem4an/in an rata7
rata /ari 1H nsentrasi /i! !r ;er tin at nsentrasi /en an nsentrasin mina .
3. Presisia. Presisi intra/a+S' ( ) /i;er ira an a an tin i ;a/a sam;e an s n arena a%a
> ! t!asi. Ber/asar an ;en!r!nan %e;at /a am +S' antara sam;e s n
/an & m L stan/ar tam;a 4a" a 4atas /ete si /an 4atas !antitasi
!nt! amitri;t ine a/a a" setara /en an 4e4era;a nsentrasi antara 0
/an & m L. Nam!n nsentrasi stan/ar memi i i +S' an !ran /ari
am4an 4atas 10 /iteta; an !nt! sem!a nsentrasi ain /a am rentan
nsentrasi an 4e er5a !nt! ana it. 4. Presisi inter/a+asi tin i ;!n%a !nt! sem4i an s!nti an setia; stan/ar a i4rasi
se !r!" ;eri /e 9a i/asi an /is!s!n /an stan/ar masin 7masin re ati9e
;en im;an an /i"it!n . +S' !nt! 0 m L & m L /an 10 m L
amitri;t ine a i4rasi stan/ar san at tin i /an me e4i"i riteria
;enerimaan ;resisi 1& ;a/a 4atas nsentrasi /an 10 !nt! sem!a
nsentrasi ainn a. Se ain ti a stan/ar nsentrasi an e4i" ren/a" ini
+S's !nt! sem!a ainn a amitri;t ine stan/ar a i4rasi memen!"i
riteria ;enerimaan ;resisi arena masin 7masin +S's sem!a !ran
/ari 10 .
#. LinearitasPers aratan inearitas !nt! ;e er5aan ana itis %! !; etat. Men!r!t Sa/e
(2000) e>isien re asi /ari 0.DD& ata! e4i" /i;er ! an !nt! e iatan
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ana isis an %! !; ritis. 8nt! ;e er5aan !ran ritis K e>isien
re asi /ari 0 DD0 ata! !ran m!n in /i;er4 e" an (Sa/e 2000).
Linearitas e4!t!"an /a am "a e>isien re asi a an ter ant!n ;a/a
t!5!an /ari met /e an /i!5i /an se4a ai "asi n a a an /i4atasi e"
;ertim4an an e!an an /an a t!. 8nt! ;er% 4aan ini e>isien
re asi /ari 0 DH ata! e4i" 4esar an /a;at /iterima !nt! men ata an
4a" a met /e a/a a" inier se ama rentan nsentrasi an /i"ara; an.
'a am sit!asi an i/ea eren /ari !r9a a i4rasi "ar!s % nstant /an
ni ai 7inter%e;t "ar!s sama /en an n . i a !r9a a i4rasi ti/a 4er5a an
me a !i titi asa nstan esa a"an sistematis an "a/ir /an esa a"an
sistematis ;r ; rsi na a/a eti a eren ti/a nstan (Me er 1DDH).Pen ! !ran LO' /an LLO6K!r9a a i4rasi masi" /i;er ! an !nt! ;enent!an LO' /an LO6 /an
arena it! sam;e /i!5i termas! ti/a "an a s n teta;i 5! a a i4rasi
stan/ar. Sem4i an stan/ar a i4rasi /i4!at /ari %am;!ran 0 1 m mL
amitri;t ine /an 0 1 m mL n rtri;t ine ar!tan st (/a am metan ).
Masin 7masin /ari sem4i an stan/ar a i4rasi /isia; an /a am 10 mL
9 !metri 4ertan a.Masin 7masin stan/ar sem4i an a i4rasi /isia; an /en an
menam4a" an &0 L /ari 0 1 m m ma;r t ine (stan/ar interna ) /ii !ti
e" 9 !me an ses!ai /ari %am;!ran amitri;t ine /an ar!tan st
n rtri;t ine /an em!/ian meni;is an e tan/a ;a/a 9 !metri% /en an
eti asetat. nsentrasi stan/ar interna /i masin 7masin sem4i an stan/ar
a i4rasi a/a a" 0 & m L. 'a am ran a !nt! menent! an tin at
e4isin an /asar 0 & m L ma;r t ine stan/ar /en an ti/a a/an a
amitri;t ine /an n rtri;t ine 5! a /isia; an. Sete a" ;en%am;!ran
men e !r!" se4!a" a i ! t /ari setia; stan/ar a i4rasi /i;i;et e
a!t in5e%t r 4 t 4ersi" /an /it!t!;.Stan/ar a i4rasi /i5a an an ;a/a P *HD0 r mat ra>i as /en an
/ete t r nitr en7> s> r ses!ai /en an met /e an /i em4an an
se4e !mn a. Stan/ar a i4rasi /is!nti /ari nsentrasi teren/a" (0 1 m
L) e nsentrasi tertin i (1 & m L) /an se !r!" ran aian stan/ar
a i4rasi /ari teren/a" e nsentrasi tertin i /is!nti t!5!" a i
4ert!r!t7t!r!t. Tar et 5!m a" ana isis !nt! setia; stan/ar a/a a" se;! !".
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Nam!n a!t in5e%t r 5ar!m men5a/i ter;asan se ama men5a an an /an
se4a ai "asi n a "an a t!5!" set en a;. 'ata a i4rasi /i;er e".
'!a ;! !" s!nti an ! an an /ari L ma;r t ine stan/ar 0 & m /i a ! an
!nt! mem;er ira an e4isin an /asar ;a/a a t! retensi amitri;t ine/an n rtri;t ine. Se ain it! 10 s!nti an ! an an /ari a i4rasi stan/ar
;e ar!t eti asetat /i a ! an !nt! mem;er ira an e4isin an a a /i
4a a" ma;r t ine an ;!n%a rata7rata ( S') /a am sat!an ;!n%a
tin i ( ;A ) ;a/a a t! retensi /i"it!n /ari 20 s!nti an ! an an /ari
0 & m L ma;r t ine stan/ar. Ini 0 & m L stan/ar ma;r t ine
/ise4!t se4a ai sam;e s n se4a ai men an/!n ti/a amitri;t ine
ata! n rtri;t ine . Se;erti /ise4!t an se4e !mn a LO' /i"it!n /en anmen !na an met /e statisti a/a a" 4erarti ;!n%a tin i ( ;A ) ;a/a
setia; a t! retensi /ari stan/ar s n /itam4a" ti a stan/ar /e9iasi
( S' ) . 'a am ran a !nt! menent! an nsentrasi setara /en an LO'
!nt! setia; ana it etin ian ;!n%a rata7rata an ses!ai /itam4a"
ti a S' "ar!s /i n9ersi /en an rasi tin i ;!n%a /en an
mem4a in a /en an ma;r t ine rata ( stan/ar interna ) ;!n%a tin i
!nt! 20 s!nti an ! an an /ari sam;e s n . K nsentrasi masin 7
masin setara /en an ana it LO' em!/ian /a;at /i"it!n /en an
memas! an rasi tin i ;!n%a /i"it!n ( ) e /a am ;ersamaan !nt!
!r9a a i4rasi an te;at /an ;eme%a"an !nt! nsentrasi (F).
F% Analisis Statistik
M /e ANO,A (A inear miFe/7e>>e%ts ana sis > 9arian%e)
/i !na an !nt! ana isa / sis n rma /en an ;arameter A8$ ast A8$ in> /an
$ maF. M /e ini men a4!n an >a t r7>a t r !nt! ;en 4atan ;eri /e
!r!tan /an s!45e an /i !m;! an se4a ai s! are a an se"at /a am st!/i
%r ss7 9er /an ;en 4atan /an s!45e ;asien an ti/a r!mit !nt! st!/i
;ara e . +esi/!a err r (err r mean s !are esa a"an rata7rata !a/rat)
/i !na an !nt! mem4an !n se an e;er%a aan D0 . 85i n n7;arametri
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;er4e/aan ;en 4atan !nt! /ata T maF ti/a 4er;asan an an /i;er e" ;a/a
st!/i ;asien an ti/a r!mit. Bi e i9a ensi /isim;! an 5i a se an
e;er%a aan D0 A8$ 4era/a /a am rentan H0712& . Tara> si ni>i an
0 0&. essi na ,ersi &.3 (P"arsi "t $ r; rati n M !ntain
,ie $A 8SA) /i !na an !nt! >arma ineti /an ana isis statisti
;arametri . SPSS !nt!
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