Xây dựng phương pháp định lượng đồng thời Lumivudin Zidovudin và Nevirapin trong...
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Transcript of Xây dựng phương pháp định lượng đồng thời Lumivudin Zidovudin và Nevirapin trong...
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B Y T
TRNG I HC DC H NI
VNG LAN HNG
XY DNG PHNG PHP NH
LNG NG THI LAMIVUDIN,
ZIDOVUDIN & NEVIRAPIN TRONG
CH PHM VIN NN BNG MEKC
KHA LUN TT NGHIP DC S
H NI, 2013
-
B Y T
TRNG I HC DC H NI
VNG LAN HNG
XY DNG PHNG PHP NH
LNG NG THI LAMIVUDIN,
ZIDOVUDIN & NEVIRAPIN TRONG
CH PHM VIN NN BNG MEKC
KHA LUN TT NGHIP DC S
GV hng dn: Nguyn Th Thy Linh
Ni thc hin: B mn Ha phn tch - c cht
Trng i hc Dc H Ni
H NI, 2013
-
LI CM N
Kha lun tt nghip ny c hon thnh ti b mn Ha phn tch -
c cht trng i hc dc H Ni.
Em xin chn thnh gi li cm n su sc ti c Nguyn Th Thy
Linh, ngi tn tm du dt, ch bo em trong sut qu trnh nghin cu v
hon thnh kha lun.
Em cng xin chn thnh cm n Ban gim hiu, cc thy c gio
trng i hc Dc H Ni cng nh cc thy c gio, cc anh ch k thut
vin b mn Ha phn tch - c cht to iu kin tt nht cho em trong
qu trnh hc tp v nghin cu.
Cui cng em xin gi li cm n n gia nh, bn b, nhng ngi
ht lng gip v ng vin em hon thnh kha lun ny
H Ni, thng 02 nm 2013
Sinh vin
Vng Lan Hng
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MC LC
T VN ......................................................................................................... 1
PHN I: TNG QUAN .......................................................................................... 3
1.1 Tng quan v Lamivudin, Zidovudin & Nevirapin .................................... 3
1.1.1 Tng quan chung ................................................................................... 3
1.1.2 Dc ng hc ...................................................................................... 4
1.1.3 ng dng Lamivudin, Zidovudin & Nevirapin trong iu tr HIV ........ 6
1.2 C s l thuyt in di mao qun .............................................................. 8
1.2.1 C ch ca qu trnh in di trong mao qun ....................................... 8
1.2.2 Cc kiu in di mao qun ................................................................... 10
1.2.3 Cc yu t nh hng n qu trnh in di ........................................ 12
1.2.4 in di mao qun micelle (MEKC) ...................................................... 12
1.2.5 u - nhc im ca MEKC ................................................................ 17
1.3 Mt s nghin cu gn y v tch v nh lng ng thi 3TC, AZT
& NVP ............................................................................................................... 18
1.3.1 Phng php HPLC ............................................................................. 18
1.3.2 Phng php CE .................................................................................. 20
PHN II: I TNG, NI DUNG & PHNG PHP NGHIN CU ....... 21
2.1 iu kin nghin cu ............................................................................... 21
2.1.1 Cc cht chun i chiu ..................................................................... 21
2.1.2 Ha cht ............................................................................................... 21
2.1.3 Thit b v dng c ............................................................................... 23
2.2 i tng nghin cu .............................................................................. 23
2.3 Ni dung v phng php nghin cu .................................................... 24
2.3.1 Ni dung nghin cu ............................................................................ 24
2.3.2 Phng php nghin cu ..................................................................... 24
3.1 La chn iu kin in di ti u ............................................................ 27
3.1.1 La chn bc sng ............................................................................. 27
3.1.2 iu kin in di ................................................................................... 28
3.1.3 La chn phng php bm mu ........................................................ 29
3.1.4 La chn kiu in di ........................................................................... 30
3.1.5 La chn dung dch in ly nn ........................................................... 31
3.1.6 Kho st iu kin pH ca dung dch in ly nn ................................ 32
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3.1.7 Kho st nng dung dch in ly nn .............................................. 35
3.1.8 Kho st nng cht to micelle SDS ............................................... 37
3.1.9 Kho st th t vo hai u mao qun ............................................... 39
3.1.10 Kho st p sut tim mu ................................................................ 41
3.1.11 nh tnh Lamivudin, Zidovudin, Nevirapin trong iu kin in
di c thit lp ........................................................................................ 43
3.1.12 Kt lun ............................................................................................. 45
3.2 Thm nh phng php .......................................................................... 45
3.2.1 Kho st tnh tng thch h thng ...................................................... 45
3.2.2 Tnh c hiu ........................................................................................ 46
3.2.3 Xc nh khong tuyn tnh .................................................................. 48
3.2.4 lp li ca phng php ................................................................. 52
3.2.5 ng ca phng php ................................................................... 54
3.3 ng dng nh lng trong ch phm .................................................... 56
PHN IV: KT LUN V XUT .................................................................. 58
4.1 Kt lun .................................................................................................... 58
4.2 xut ..................................................................................................... 59
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DANH MC CH VIT TT
AIDS Hi chng suy gim min dch mc phi ngi ( Acquired
immunodeficiency syndrome)
AZT Zidovudin
C Nng
CE in di mao qun (Capillary Electrophoresis)
CEC in sc k mao qun (Capillary Electrochromatography)
CGE in di mao qun gel (Capillary Gel Electrophoresis)
CZE in di mao qun vng (Capillary Zone Electrophoresis)
Cmax Nng nh ca thuc trong huyt tng
DAD Detector mng diod (Diod Array Detector)
EOF Dng in thm (Electro osmotic Flow)
HPLC Sc k lng hiu nng cao (High Performance Liquid
Chromatography)
HIV Virus gy bnh suy gim min dch ngi ( Human
Immunodeficiency Virus)
NVP Nevirapin
MEKC Sc k in ng micelle (Micellar Electrokinetic
chromatography)
RSD % lch chun tng i (Relative Standard Deviation)
Spic Din tch pic
SD lch chun (Standard Deviation)
-
STT S th t
T1/2 Thi gian bn thi
Tmax Thi gian t Cmax
Tm Thi gian di chuyn
Tnc Nhit chy
UV VIS Bc sng t ngoi - kh kin (Ultra Violet Visiable)
WHO T chc Y t th gii (World Heal Organization)
3TC Lamivudin
ddC Zalcitabine
ddI Didanosine
-
DANH MC CC BNG
STT bng Tn bng
1 Tng quan chung v Lamivudin, Zidovudin & Nevirapin
2 Dc ng hc ca Lamivudin, Zidovudin & Nevirapin
3 Cc kiu in di, c ch tch v ng dng
4
Kt qu kho st tnh tng thch h thng ca phng php
(n=6)
5 Din tch pic trn in di v nng 3TC tng ng
6 Din tch pic trn in di v nng AZT tng ng
7 Din tch pic trn in di v nng NVP tng ng
8 Kt qu xc nh lp li ca phng php MEKC trong nh
lng 3TC
9 Kt qu xc nh lp li ca phng php MEKC trong nh
lng AZT
10 Kt qu xc nh lp li ca phng php MEKC trong nh
lng NVP
11 Kt qu kho st ng ca phng php trn thm chun
Lamivudin
12 Kt qu kho st ng ca phng php trn thm chun
Zidovudin
13 Kt qu kho st ng ca phng php trn thm chun
Nevirapin
14 Kt qu nh lng vin nn Avocomb-N
-
15 Kt qu nh lng vin nn Lamivudin 150mg & Zidovudin
300mg
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DANH MC CC HNH
STT hnh Tn hnh
1 S nguyn tc hot ng ca CE
2 S di chuyn ca ion v phn t trong mao qun
3 Th t ra gii trn in di
4 Cu trc ca cc micelle v dng EOF trong MEKC
5 Micell hnh cu
6 Ph hp th ca Lamivudin
7 Ph hp th ca Zidovudin
8 Ph hp th ca Nevirapin
9 in di chun Lamivudin, Zidovudin, Nevirapin ba dung
dch in ly nn khc nhau
10 in di hn hp ba chun vi dung dch in ly nn Na2B4O7
10mM/ SDS 50mM cc pH khc nhau
11 in di hn hp 3 chun vi dung dch in ly nn cc nng
borat khc nhau
12 in di hn hp 3 chun vi dung dch in ly nn cc nng
SDS khc nhau
13 in di hn hp 3 chun vi dung dch in ly nn borat
10mM/SDS 50mM cc th khc nhau
14 in di hn hp chun vi thi gian tim mu khc nhau
15 in di hn hp th
-
16 in di mu th thm Lamivudin chun
17 in di mu th thm Zidovudin chun
18 in di mu th thm Nevirapin chun
19 in di hn hp chun diu kin in di ti u
20 in di mu trng
21 in di hn hp chun Lamivudin, Zidovudin, Nevirapin
22 in di hn hp ch phm lm vic
23 in di hn hp ch phm thm chun
24 th biu din s ph thuc gia din tch pic v nng 3TC
tng ng
25 th biu din s ph thuc gia din tch pic v nng AZT
tng ng
26 th biu din s ph thuc gia din tch pic v nng NVP
tng ng
27 in di ch phm vin nn Avocomb-N
28 in di ch phm vin nn Lamivudin 150mg & Zidovudin
300mg
-
1
T VN Vo thng 6 nm 2011, ti Hi ngh cp cao v HIV/AIDS New
York, M, Vit Nam khng nh li quyt tm ca mnh trong cng cuc
phng, chng HIV/AIDS v cam kt thc hin nhng mc tiu mi thng qua
vic k Tuyn b Chnh tr nm 2011 v HIV/AIDS: Tng cng n lc
loi tr HIV/AIDS. c th thc hin mc tiu , ngoi nhng bin php
x hi cn c thc hin th vic kim sot cht lng thuc dng trong iu
tr cng gp phn quan trng khng km. [10]
Hin nay, trn th trng c rt nhiu thuc dng iu tr HIV. Tuy
nhin, theo khuyn co ca t chc y t th gii WHO, iu tr HIV nn
dng phi hp hai thuc NRTIs hoc cng phi hp vi mt thuc NNRTIs
m khng nn n tr liu v c th dn n khng thuc, lm gim hiu qu
iu tr. [3], [11], [13]
Xut pht t nhu cu , trn th trng bt u xut hin nhng thuc
dng hn hp a thnh phn. Ch phm chng ti dng nghin cu cng l
mt trong s . V vy, vic kim tra cht lng cng nh xc nh cc
thnh phn trong ch phm hn hp ng mt vai tr ht sc quan trng trong
vic m bo cht lng thuc, em li s an ton, hiu qu cho ngi s
dng.
Hin nay, sc k lng hiu nng cao (HPLC) l phng php phn tch
c a s cc Dc in quc t la chn trong kim nghim thuc iu tr
HIV. u im ni bt ca phng php ny l lp li, chnh xc cao,
kh nng tch tt, nhy cao v gii hn pht hin nh. Tuy nhin, phng
php ny i hi phi s dng mt lng ln dung mi tinh khit, t tin,
nhiu loi dung mi c th gy c vi ngi phn tch v nhim mi
trng. ng thi phng php ny tng i hn ch trong vic phn tch
-
2
cc cht c kh nng ion ha, nht l sc k lng hiu nng cao pha o RP
HPLC (Reversed phase liquid chromatography). [16], [17]
Nhm mc ch xy dng mt phng php nghin cu phn tch t tn
km v t c hi hn, chng ti hng n phng php in di mao qun.
c nhiu nghin cu phn tch 3TC, AZT & NVP bng phng php
HPLC. Tuy nhin, cha thy mt nghin cu no phn tch nh lng ng
thi ba cht bng phng php CE. Mt khc, CE tuy khng cn mi l
trn th gii, nhng Vit Nam, vic ng dng CE vo trong kim nghim
dc phm vn cn rt hn ch. Vi nhng mc ch trn, ng thi nhm
nng cao cht lng vic xy dng tiu chun v qun l cc thuc iu tr
HIV ang c s dng ti Vit Nam; gip kim nghim vin c thm nhiu
la chn khi tm phng php kim nghim ph hp vi iu kin ni lm
vic nht, chng ti tin hnh kha lun Xy dng phng php nh
lng ng thi Lamivudin, Nevirapin & Zidovudin bng MEKC nhm
hai mc tiu
- Xy dng phng php ph hp v n nh tch v nh lng
ng thi Lamivudin, Zidovudin & Nevirapin.
- ng dng phng php xy dng c kim tra mt s ch
phm cha Lamivudin, Zidovudin & Nevirapin c dng trong
phc iu tr HIV/AIDS.
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3
PHN I: TNG QUAN
1.1 Tng quan v Lamivudin, Nevirapin v Zidovudin
1.1.1 Tng quan chung [4], [5], [12]
Bng 1: Tng quan chung v Lamivudin, Zidovudin & Nevirapin
c im Lamivudin Zidovudin Nevirapin
CTHH
CTPT C8H11N3O3S C10H13N5O4 C15H14N4O
PTK 229,3 g / mol 267,2 g/mol 266,3 g/mol
Tn KH
4-Amino-1-
[(2R,5S)-2-
(hydroxymethyl)-
1,3-oxathiolan-5-
yl]pyrimidin-2(1H)-
one.
1-(3-Azido-2,3-
dideoxy-b-D-erythro-
pentofuranosyl)-5-
methylpyrimidine-
2,4(1H,3H)-dione.
11-Cyclopropyl-4-
methyl-5,11-
dihydro-6H-dipyrido
[3,2-b:2,3-
e][1,4]diazepin-6-
one.
Tnh cht
Bt kt tinh mu
trng hoc gn nh
trng
Tinh th trng hoc
hi nu.
Bt kt tinh mu
trng hoc gn nh
trng
Tnc 160 162 0C 120 122 0C 242 246 0C
tan
D tan trong nc,
tan trong cn, kh
tan trong cc dung
Kh tan trong nc,
EtOH
Thc t khng tan
trong nc, t tan
trong CH3Cl.
-
4
mi hu c khng
phn cc
pKa 4,3 9,68 2,8
Nng
sut quay
cc
- 1350 (C = 0,38%
trong MeOH)
+ 60,50 + 63,00 ( C
= 1% trong EtOH)
+ 990 (C = 0,5%
trong nc)
max 270nm 264,5 nm 268,5 nm
1.1.2 Dc ng hc [3], [6], [11]
Bng 2: Dc ng hc ca Lamivudin, Zidovudin & Nevirapin
c im Lamivudin Zidovudin Nevirapin
Sinh kh
dng
~ 80%, khng b
nh hng bi thc
n
60 70 %, b nh
hng khi n nhiu
cht bo
>90%, khng b nh
hng bi thc n
hoc thuc khng
acid
Tmax 1h sau khi ung 0,5 1h sau khi
ung
4h sau khi dng liu
duy nht
Cmax 1,5 g/ml (liu
150mg)
1,2 mg/l ( liu
250mg )
5,74 g/ml (liu
200mg)
Phn b Vd = 1,3 0,4
lit/kg Vd = 1,6 0,6 lit/kg
Nevirapin i qua
c nhau thai v
hng ro no ty,
phn b trong dch
no ty v sa m.
Vd = 1,21 0,09
lit/kg
-
5
Lin kt
protein
huyt
tng
70% thi tr qua
nc tiu di dng
chuyn ha.
thanh
thi Cl
0,37 0,05
lit/gi/kg 0,2 0,3 lit/gi/kg
0,017 0,032
lit/gi/kg
-
6
1.1.3 ng dng Lamivudin, Zidovudin & Nevirapin trong iu tr HIV
1.1.3.1 Cc nhm thuc iu tr HIV
- Nhm 1: Thuc c ch men sao chp ngc c gc nucleoside
(nucleoside reverse transcriptase inhibitors = NRTIs)
VD: Zidovudin; Didanosin; Zalcitabin; Stavudine; Lamivudin; Abacavir;
Tenofovir disoproxil fumarate.
- Nhm 2: Thuc c ch men sao chp ngc khng c gc nucleoside
(non-nucleoside reverse transcriptase ninhibitors =NNRTIs)
VD: Nevirapin; Delavirdin; Effavirenz
- Nhm 3: Thuc c ch proteases (protease inhibitors = PIs)
VD: Indinavir; Ritonavir; Saquinavir; Nelfinavir; Amprenavir; Lopinavir.
Qua nghin cu, ngi ta thy khng nn n ha tr liu m nn phi
hp cc thuc nhm 1 vi nhau v vi mt thuc nhm 2 hoc vi mt thuc
nhm 3 t c hiu qu iu tr cao v trnh khng thuc.
1.1.3.2 Phc phi hp trong iu tr HIV [3], [11], [13], [20]
Trong liu php khng retrovirus, thuc c chn lc l nhng thuc
tng t nucleoside. Hin nay cha bit r lc no l thi im tt nht bt
u iu tr vi thuc khng retrovirus. Liu php khng retrovirus cng lm
tng thi gian sng st ngi bnh c s lng t bo CD4 di 500 trong
1mm3. Liu php ny cng c th dng cho ngi bnh c mt HIV trn
30.000/ml huyt tng, khng ph thuc vo s lng CD4, v mt HIV l
mt yu t tin lng s tin trin ca bnh. Mt virus cao hn dn n
gim t bo CD4 nhanh hn. Mc tiu iu tr l t mt HIV mc
khng th pht hin c.
Liu php chun hin nay gm 2 thuc tng t nucleoside khng
retrovirus, cng vi mt thuc c ch protease.
-
7
Ngi bnh iu tr khng c kt qu (tng gp 3 ln mt virus, hoc
gim s lng t bo CD4, hoc tin trin thnh bnh AIDS) phi chuyn
sang dng kt hp vi mt thuc khng retrovirus khc. La chn thuc sao
cho nguy c khng cho vi nhng thuc dng l ti thiu. Khi liu php
c khng c tc dng cn thm thuc mi, nguyn tc l cho thm khng ch
mt thuc m kt hp 2 thuc mi.
Lamivudin v Zidovudin c cu trc tng t nucleoside c tc dng
khng retrovirus bao gm c HIV-1 v HIV-2 do c ch enzyme phin m
ngc ca virus. Hai thuc ny c s dng phi hp trong liu php khng
retrovirus iu tr HIV.
Nevirapin l cht c ch enzyme phin m ngc non-nucleosid c hot
tnh khng retrovirus HIV-1.
Lamivudin, Zidovudin v Nevirapin l ba thuc phi hp c t chc Y
t th gii WHO khuyn co dng trong iu tr HIV.
Liu dng:
- Nevirapin 200mg/ln/ngy (trong hai tun u), sau 200mg/2 ln/ngy
- Zidovudin 300mg/ 2 ln/ngy. Bnh nhn b suy gim chc nng thn
hoc phi iu chnh liu do b nhim c t khng c s dng vin
phi hp.
- Lamivudin 150mg/2 ln/ngy
Nevirapin c khuyn co nn dng na liu trong vng 14 ngy u
lm gim nguy c pht ban. Theo lu trn, trong sut thi k ny, khng nn
s dng vin nn phi hp 3 thnh phn trn. Khng nn c gng s dng
mt vin/ ngy, v iu c th dn n vic khng Lamivudin v iu tr
tht bi. Thay v , nn s dng nhng vin ring bit. Sau 14 ngy, liu ca
Nevirapin nn c tng ln 200mg/2 ln/ ngy hoc c th s dng vin
thuc phi hp.
-
8
Vin phi hp Zidovudin/Lamivudin khng ph hp vi nhng ngi b
suy thn hoc nhng bnh nhn s dng liu Zidovudin thp hn do tc dng
ph
1.2 C s l thuyt in di mao qun [1], [2], [9], [14], [15]
in di mao qun l mt k thut tch phn tch c Vit ha t thut
ng Capillary Electrophoresis, thng c gi tt l CE.
1.2.1 C ch ca qu trnh in di trong mao qun
Qu trnh in di c thc hin trn mao qun silica di 25-100 cm,
ng knh trong 25 - 100 m, ng knh ngoi 300 - 400 m. S dng p
sut a dung dch mu v dung dch m ln mao qun. in th mt
chiu t vo hai u mao qun to ra qu trnh tch. Di tc dng ca lc
in trng E (Electric Field Force: EFF) v dng in di thm thu (Electro-
Osmotic Flow: EOF), cc phn t cht tan s di chuyn vi tc khc nhau
v tch ra khi nhau do in tch, kch thc v linh ng ca chng khc
nhau. Cc cht phn tch c pht hin nh mt detector thch hp khi di
chuyn v mt u mao qun.
Detector hay dng nht l detector UV hay detector mng diod DAD. V tr
pht hin nm ngay trn mao qun gi l ca s.
Hnh 1: S h thng in di
-
9
Dng in thm (EOF)
Trong CE, dng in thm (EOF: electro-osmotic flow) l mt thnh phn
c bn, to nn s di chuyn ca ton b dung dch trong mao qun bt ngun
t in tch b mt trong ca thnh mao qun.
Ngun gc EOF
Khi in di vi mao qun silica, cc nhm silanol (SiOH) trn thnh
trong ca mao qun s gii phng ra H+ vo dung dch v b mt thnh mao
qun s mang in tch m. Qu trnh gii phng H+ ph thuc vo hng s
cn bng K, nhng thc nghim ch r rng dng EOF c gi tr ng k khi
pH dung dch trn 4.
Do b mt thnh trong mao qun mang in tch m, nn i ion (ch yu
l cation) do lc ht tnh in s to thnh mt lp in kp cn bng in
tch v to nn mt hiu in th vng st thnh mao qun gi l th Zeta.
Khi p th vo mao qun, cc cation trong lp in kp khuch tn s di
chuyn v pha catod. Nhng cc cation ny b solvat ha nn ko theo c
khi dung dch trong mao qun i v catod. S di chuyn ca khi dung dch
trong mao qun silica di tc dng ca in trng c gi l dng in
thm EOF.
Hai c im ca EOF
- c im u tin ca EOF l to nn mt dng chuyn khi phng trong
mao qun. Bi v lc to ra s di chuyn ca EOF phn b ng nht dc
theo mao qun, cho nn khng c s st p trong mao qun: dng ng
nht t u n cui (mao qun).
Tuy nhin tnh cht to dng chuyn khi phng ca dung dch in di ch
xut hin khi ng knh trong mao qun (di) di 200m. Nu di 200m,
sc cng b mt khng to ra s di chuyn ng nht gia phn cht lng
gia v gn thnh mao qun.
-
10
- c im th hai ca EOF l a tt c cc tiu phn c mt trong dung
dch in di, bt k c in tch hay khng, di chuyn theo cng hng.
Vi iu kin in di thng thng (thnh mao qun tch in m), dng
ny di chuyn v catod. Cc anion cng s di chuyn v pha catod v tc
EOF thng ln hn rt nhiu ln so vi tc in di.
Hnh 2: S di chuyn ca ion v phn t trong mao qun
Cation di chuyn nhanh nht do lc in di v EOF cng hng, tip theo
sau l cc cht khng mang in tch (trn hnh v l N) di chuyn theo EOF
nhng khng tch khi nhau c. Cui cng l anion. Trn in di th t
ra gii theo thi gian nh sau: cation c in tch ln kch thc nh ra trc
nht, anion c in tch ln kch thc nh ra sau cng.
Hnh 3: Th t ra gii trn in di
1.2.2 Cc kiu in di mao qun
Thi gian
in tch ln
kch thc
nh
Cation
Cc cht
trung ha
Anion
in tch ln
kch thc nh
-
11
in di mao qun kh a dng, c th thc hin theo nhiu kiu khc
nhau. Mi kiu c c ch tch ring ca n. Bng di y tm tt bn kiu
in di.
Bng 3: Cc kiu in di, c ch tch v ng dng
STT Kiu in di C ch tch ng dng
1
in di mao qun vng
(Capillary zone
electrophoresis: CZE)
Da trn linh
khc nhau
ca cc ion
trong dung dch
t do
-Sinh hc: phn tch cc
peptid, protein, dc bit l
glucoprotein
-Xc nh dc cht v cc
cht chuyn ha ca chng
-Phn tch cc cht nhim
mi trng
2
Sc k in ng
micelle (Micellar
electrokinetic
chromatography:
MEKC)
Da trn s
tng tc
nc/ion vi
ht micelle
trong dung
dch.
L k thut b sung cho
CZE tch cc ion v
phn t trung ha v in
3
in di mao qun gel
(Capillary gel
electrophoresis: CGE)
Da trn kch
thc v in
tch
Dng phn tch cc
phn on AND, tch
protein
4
in sc k mao qun
(Capilarry
electrochromatography:
CEC)
Da trn s
phn b v linh
khc nhau
ca cc cht.
-Phn tch dc: dng phn
tch dc cht thuc nhiu
nhm khc nhau
-Phn tch cc phn t sinh
hc, cc ng phn quang
hc
-
12
1.2.3 Cc yu t nh hng n qu trnh in di
1.2.3.1 in th
Thi gian phn tch t l nghch vi in th t vo hai u mao qun. Tuy
nhin, nu in th qu cao c th gy ra hiu ng June, to ra nhit trong
mao qun, lm thay i nht tng vng trong mao qun, khin cho cc
phn t cht tan di chuyn khng ng nht, dn n s khuch tn khc nhau
v lm m rng vng mu.
1.2.3.2 Nhit
nh hng ca nhit lm thay i vn tc v dn in ca dung dch
in ly nn. Trong mt s trng hp nu nhit mao qun tng l nguyn
nhn thay i cu trc protein, lm thay i thi gian in di v hiu qu phn
tch.
1.2.3.3 Dung dch in ly
- Loi dung dch in ly v nng : dung dch in ly thch hp cho CE khi
c tc dng tt nht trong dy m chn v gim thp nht s sinh nhit
trong dung dch y
- pH: pH ca dung dch in ly c th tc dng ln kh nng phn tch v
lm thay i EOF.
1.2.3.4 Ct mao qun
Kch thc ct mao qun (chiu di v ng knh) nh hng n thi
gian tch v hiu qu phn tch. Nu tng c hai yu t l chiu di hiu dng
v chiu di ct mao qun c th lm gim in trng v tng thi gian phn
tch.
1.2.4 in di mao qun micelle (MEKC)
MEKC c khi cn c vit tt l MECC (micellar electrokinetic capillary
chromatography) l mt k thut b sung cho CZE: tch c ion v c phn
t trung ha in bng cch thm cc cht hot ng b mt vo dung dch
-
13
in di nng trn nng micelle ti hn (critical micelle concentration
CMC). Cht hot ng b mt thng dng l natri dodecylsulfat (SDS) c
CMC = 9mM. V y cng l cht din hot chng ti s dng trong nghin
cu ny.
Phng php MEKC l mt kiu k thut tch theo bn cht ca s in di
c s dng kt hp c tnh cht ca k thut in di mao qun v sc k lng
c pha tnh. N l mt trong cc kiu tch sc k ca k thut CE c ng
dng rng ri trong sinh hc y hc v dc. MEKC l mt phng php tch
c hiu qu cao, ch yu ph hp phn tch cc hp cht trung ha nh. C
ch tch ca k thut ny da trn s khc bit ca cn bng phn phi ca
cc hp cht mu gia pha ng l dung dch nc v pha tnh gi l cc
micelle. u im ca MEKC l kh nng d dng thay i cc thnh phn
ca h thng,v c bit c th thay i pha tnh gi micelle. Theo , c th
d dng v nhanh chng kim sot c pha ng v ti u ha phn gii.
V vy n b sung v lm phong ph v chng loi ca k thut in di.
Bn cht v trung tm ca s tch y l s hnh thnh cc phn t
Micell ( tiu phn Micell pha tnh gi) trong ng mao qun v cc tiu phn
ny s dn dt v ng gp kh nng ca qu trnh tch sc k in di cc
cht trn nn ca dung dch m v cht in ly trong ng mao qun. S tch
sc k ca cc phn t cht tan trung ha bng k thut MEKC c iu
chnh bi cc cht hot ng b mt nm trong dung dch in ly nn.
Phng php MEKC dng tch cc cht phn tch c in tch v c
trung ha. i vi cc cht c in tch, cc micelle loi ny chuyn ng
hoc l cng chiu hoc l ngc chiu vi dng EOF, l ty thuc vo in
tch ca cht hot ng b mt v cu trc ca micelle. Cc cht hot ng b
mt anion ha, v d SDS, s di chuyn v anod (cc dng), ngha l di
chuyn ngc chiu vi hng ca dng EOF. Trong dung dch m in di
-
14
c pH trung tnh v kim, khi dng EOF di chuyn nhanh hn tc ca
micelle, th s in di thc (net movement) ca micelle l theo hng ca
dng EOF. Trong khi di chuyn nh th, cc micelle c th tng tc vi cc
phn t cht tan (cht phn tch) nh kiu phn b ca sc k ct lng rn (R-
LC) theo c tng tc a nc, tng tc k nc v tng tc tnh in,
to ra qu trnh sc k ca cc cht mu, khi chy qua ct mao qun trong qu
trnh in di.
Hnh 4 : Cu trc ca cc micelle v dng EOF trong MEKC
i vi cc phn t cht tan trung tnh, n ch l s phn b ca cht tan
vo trong micelle v ngoi micelle (pha ng in di) theo mt cn bng
ng hc c hng s phn b Ki nht nh trong iu kin chn. Chnh s
phn b theo kiu cn bng ng hc ny gy ra s lu gi khc nhau ca cc
cht tan, v to ra s tch sc k ca cc cht phn tch theo kiu micelle. V
th cc micelle trong ng mao qun ca k thut tch ny c gi l pha tnh
gi. Nhiu cht tng tc vi micelle kh bn, n tn ti trong micelle di hn
s di chuyn ca n, khi cc micelle mang n mt phn di chuyn ngc
chiu dng EOF.
-
15
Khi cc cht tan khng tng tc vi cc micelle, n c ra gii cng
vi dng EOF. Nhiu hp cht k nc tng tc rt mnh vi cc micelle v
b lu li kh lu trong mao qun, nn lm tng thi gian lu gi ca cht tan.
C ch ca s tch cc phn t trung tnh trong MEKC v c bn vn l
kiu tng tc phn b ca cht tan, tng t nh trong sc k ct lng - rn
(LC), nhng li c iu khin bi lc in trng E ca th cao V gia hai
u mao qun to ra m khng phi bng p sut y pha ng nh trong
HPLC. Trong k thut phn tch MEKC, cc tnh ton v h s dung lng
ki, s a l thuyt N ca ct mao qun, phn gii R,... cng da trn c s
tng t vi cc tnh ton ca h sc k ct LC, v tt nhin c b sung thm
mt s yu t cho thch hp v ng hn cho h pha MEKC.
H s dung lng k:
k = ( 0)
0 (1
) = K.
Trong :
tr : thi gian di chuyn ca cht tan
t0: thi gian di chuyn ca cht khng lu gi, c xc nh bng cch
tim mt cht nh du micelle nh sudan III, khi di chuyn n lun nm
trong micelle.
k: h s phn b ca cht tan trong v ngoi micelle
VS: th tch ca pha micelle
VM: th tch ca pha ng
Ngi ta gi k thut ny l sc k in ng micelle bi v ht micelle
c coi nh mt pha tnh gi (pseudostationary phase). Vic tch cc cht
trung ha da trn c s s phn b ca chng gia dung dch v pha tnh gi.
C th thay i s phn b trn bng nhiu cch khc nhau, nh:
- Thay i pH v nng ca dung dch in di
-
16
- Thay i bn cht vt l ca micelle (kch thc, in tch hoc hnh
dng ca n) bng cch dng cc cht hot ng b mt khc nhau
(tng t thay i pha tnh trong LC)
- Thay i nng ca cht hot ng b mt.
- Thay i nhit in di
- Thm dung mi hu c vo pha ng (dung dch in ly nn)
Cc cht hot ng b mt c dng trong MEKC cng c th tng tc
vi thnh ng mao qun v cng gy nh hng nht nh n dng EOF, lm
thay i s tng tc hp ph gia cht tan v thnh ng mao qun. Hng di
chuyn ca cht tan v ca cc micell cng ph thuc vo in tch ca micell
v tc dng EOF. Ni chung, cc h m in di c pH tng i cao
thng c s dng duy tr dng EOF c tc ln v m bo
hng di chuyn n nh ca cc micell.
1.2.4.1 Cht to micelle SDS
CTHH:
CTPT: CH3(CH2)11 OSO3Na
PTK: 288,372 g/mol
Tn KH: Sodium lauryl sulfate
Tn khc: Sodium monododecyl sulfate; Sodium lauryl sulfate; Sodium
monolauryl sulfate; Sodium dodecanesulfate; Sodium coco-sulfate; dodecyl
alcohol, hydrogen sulfate, sodium salt; n-dodecyl sulfate sodium; Sulfuric
acid monodedocyl ester sodium
H s du/ nc HLB = 40
SDS l mt cht hot ng b mt ainon ha. Khi ha tan vo nc, SDS
phn ly thnh ion m R- v ion Na+ khng c tnh hot ng b mt.
-
17
C ch to micelle:
SDS c mt phn k nc v phn a nc. Khi nng SDS trong dung
dch vt qu nng micelle ti hn (CMC = 9mM), cc phn t SDS tp
hp li vi nhau, u a nc s hng ra ngoi v u k nc s hng vo
bn trong hnh thnh cc micelle hnh cu.
Hnh 5: Micell hnh cu
1.2.4.2 ng dng ca MEKC
MEKC l mt kiu in di c nhiu ng dng bi v c th tch nh
lng cc cht trung ha v mang in tch, c c tnh s nc v thn nc
nh acid amin, nucleotide, vitamin, hydrocarbon thm. Vi ch phm dc
c th dng MEKC xc nh hot cht trong cc loi vin, dung dch tim.
1.2.5 u - nhc im ca MEKC
Phng php MEKC l mt phng php phn tch hin i cho tin cy
v hiu qu tch cao. Phng php c th tin hnh trn nhiu i tng khc
nhau bao gm c cht mang in v cht trung tnh. Phng php c mt s
c im c bn:
- Lng mu phn tch nh, tc phn tch nhanh, thao tc n gin hn so
vi k thut HPLC.
-
18
- Dung dch pha ng cng nh mu phn tch thng c pha trong nc
loi tr ion v s dng t, t tn km, t c hi.
- C hiu lc tch cc cht rt cao
- in th s dng rt cao (10 100kV), c th thay i c thi gian di
chuyn hp l.
- C kh nng t ng ha nn d dng tin hnh vi mt s lng mu ln.
- Ct tch l ng mao qun nh, r, d ti sinh hn so vi HPLC.
Tuy nhin, phng php ny cng c mt s nhc im:
- Do flowcell nm ngay trn mao qun nn nhy ca phng php thp
hn nhiu so vi cc phng php khc. Gii hn ca n c mg/l i vi
detector UV-VIS, DAD. Trong khi cc phng php khc nh HPLC c
gii hn nh hn nhiu.
- My lm vic di in th cao nn phi cn thn khi lm vic.
- Lng mu s dng nh l mt u im ca phng php, ng thi cng
l nhc im ca n. Lng mu nh c th dn n sai s ln khi phn
tch mu c hm lng cao do h s pha long cao. i vi mu c hm
lng nh, khi tng thi gian phn tch th gy ra hin tng dong pic,
hiu sut khng cao.
- Thi gian lu ca dung dch ph thuc rt nhiu vo thnh phn m, dung
dch in ly. V vy i hi phi cn thn v t m.
1.3 Mt s nghin cu gn y v tch v nh lng ng thi
3TC, AZT & NVP
1.3.1 Phng php HPLC
Di y chng ti trnh by mt s nghin cu gn y v tch v nh
lng ng thi Lamivudin, Zidovudin v Nevirapin bng phng php
HPLC.
-
19
- Bin Fan, James T. Stewart nghin cu nh lng ng thi AZT, 3TC
& NVP trong huyt tng ngi bng HPLC vi pha ng l m
phosphate acetonitril (86:14 V/V) c iu chnh n pH 3,2 bng acid
phosphoric. Tin hnh chy sc k trn ct C 8 (150 3,9mm) vi bc
sng pht hin 265nm. Aprobarbital c chn lm cht chun ni. Kt
qu thu c cho thy thi gian lu ca ba cht AZT, 3TC & NVP tng
ng ln lt l: 3,1; 6,1 v 15,0 pht. [16]
- Theo [17], Geetha Ramachandran cng cc cng s dng HPLC
phn tch AZT v NVP. Cc iu kin chy sc k: pha ng: KH2PO4
(15mM, pH = 7,5) : actetonitril = 80 : 20 (V/V); pha tnh: Ct C 18,
150mm x 4,6 mm; chun ni: 3 xanthine 1- methyl isobutyl. Bc sng
pht hin: 260nm. Thi gian lu ca AZT l 2,4 pht; ca NVP l 6,0 pht.
- Phn tch ng thi 3TC, AZT& NVP trong ch phm, Anantha Kurma v
cc cng s s dng phng php RP-HPLC [18]. Cc iu kin chy
sc k c xc nh bao gm: Pha ng: orthophosphate kali dihydrogen
0,015 M(pH 5,0) : acetonitril = 45:55 (v/v); pha tnh: ct hiQ Sil C18V;
tc dng chy: 1,0ml/pht. Bc sng pht hin ti 270 nm. Cht chun
ni: Carbamazipine. Thi gian lu gi i vi 3TC, AZT v NVP ln lt
l 2,45; 2,93 v 3,72 pht.
- Tc gi Bin Fan [21] xy dng quy trnh phn tch ng thi Zidovudin,
Zalcitabin & Nevirapin trong huyt tng ngi bng phng php HPLC.
iu kin chy sc k nh sau: Pha ng: 20 mM sodium phosphate m
(cha 8 mM 1-octanesulfonic axit, mui natri) - acetonitril (86:14, v / v),
pH c iu chnh n 3,2 bng acid phosphoric; pha tnh: Ct C 8, 150
x 3,9mm, = 5m. Tc dng: 1,0ml/pht. Chun ni: Aprobarbital.
Tim mu 20l ba ln vo ct HPLC. Bc sng pht hin: 265nm. Thi
-
20
gian lu ca AZT l 3,1 pht, ca ddC l 5,2 pht v ca NVP l 15,0
pht.
1.3.2 Phng php CE
Gn y, phng php CE c s dng rng ri do tnh u vit v hiu
qu tch cao, thi gian phn tch ngn, lng mu tim tn t. Phng php
ny c ng dng trong nghin cu tch v nh lng Lamivudin,
Zidovudin v Nevirapin trong nhiu hn hp khc nhau.
- Bin Fan v J.T. Stewart s dng phng php in di mao qun vng CZE
vi thnh phn dung dch in ly nn gm c m phosphate 80mM, pH =
2,5 thm 100nM N,N-dimethyloctylamine. Tin hnh phn tch ti th
20kV, nhit mao qun 300C. Ct mao qun silica trn di 52cm, chiu
di hiu dng 30cm, ng knh 50m. Phng php cho php tch 3TC,
ddI & NVP v chng c pht hin bi detector DAD bc sng
210nm. Thi gian lu thu c ca ba cht ln lt l: 3TC l 4,3 pht;
NVP l 6,2 pht v ddI l 9,8 pht.[19]
- Nghin cu tch v nh lng ng thi AZT, ddI & NVP trong huyt
tng ngi bng MEKC, Bin Fan v cng s s dng dung dch in ly
nn l m phosphat 15mM v m borat, pH = 9,0 vi cht to micell l
SDS 18mM. Qu trnh in di din ra trong ct mao qun silica trn di
52cm, chiu di hiu dng 30cm, ng knh 50m. Nhit 30 0C. Bc
sng pht hin 210nm. in th p vo hai u mao qun l15kV. Cng
dng in duy tr trong mao qun 30 A. Cht chun ni:
Aprobarbital. Kt qu thu c vi thi gian lu ca cc cht nh sau: thi
gian lu ca AZT l 3,7 pht; ca ddI l 4,0 pht; ca NVP l 6,0 pht.[20]
-
21
PHN II: I TNG, NI DUNG & PHNG
PHP NGHIN CU
2.1 iu kin nghin cu
2.1.1 Cc cht chun i chiu
- Chun quc gia Lamivudin (200mg/l) hm lng 99,45% C8H11N3O3S
tnh theo nguyn trng, SKS 0312146.03, bo qun nhit 5 0C, trnh
nh sng - Vin kim nghim thuc TW- B Y T.
- Chun quc gia Zidovudin (200mg/l) hm lng 99,64% C10H13N5O4
(HPLC) tnh theo nguyn trng, SKS 0105143, bo qun nhit 5 0C,
trnh nh sng - Vin kim nghim thuc TW - B Y t.
- Chun quc gia Nevirapin khan (200mg/l) hm lng 99,49% C15H14N4O
tnh theo nguyn trng, SKS WS. 0109263, bo qun nhit 2 -8 0C,
trnh nh sng - Vin kim nghim thuc TW - B Y t
2.1.2 Ha cht
- Nc loi ion: l nc ct hai ln mi, c lc qua b lc siu tinh khit
c ct trao i ainon v cation, sau lc qua mng lc 0,2 m.
- NaOH, Na2B4O7, MeOH, SDS
Chun b dung dch chun
- Dung dch gc chun: cn chnh xc khong 30,9 mg Lamivudin, 39,7 mg
Nevirapin v 59,6 mg Zidovudin vo cc c m. Thm vo 10ml MeOH
ha tan, sau thm nc loi ion ha tan. Chuyn ht vo bnh nh mc
100ml, siu m cho tan hon ton, thm nc loi ion n th tch va
v lc u.
- Pha cc dung dch chun (1), (2), (3), (4), (5), (6) c nng Lamivudin
ln lt l 77,25g/ml; 123,6g/ml; 154,5g/ml; 169,95g/ml;
185,4g/ml; 231,7g/ml; nng Zidovudin: 149,0g/ml;238,4g/ml;
-
22
298,0g/ml; 327,8g/ml; 357,6g/ml; 447,0g/ml; nng Nevirapin:
99,25g/ml; 158,8g/ml; 198,5g/ml; 218,3g/ml; 238,2g/ml;
297,7g/ml.
- Dung dch mu trng: chun b tng t mu chun nhng khng c cht
cn kho st
Chun b dung dch th
Cn 20 vin thuc vin, xc nh khi lng trung bnh mt vin l
1,2441g v nghin thnh bt mn. Cn mt lng bt vin tng ng vi
15,00 mg Lamivudin, 20,00 mg Nevirapin, 30,00 mg Zidovudin cho vo
bnh nh mc 100 ml, thm 10ml MeOH, 20ml nc loi ion siu m cho tan
ht, thm nc loi ion n va th tch, lc u. Lc qua giy lc ng
knh 11cm. B 20ml dch lc u. Dch lc tip tc c lc qua b lc ht
chn khng mng lc cellulose kch thc l lc 0,45m. Tip tc b 20ml
dch lc u. Dch lc c lc qua mng lc 0,2 m trc khi tim vo l
ng mu.
Pha dung dch in ly nn:
Cc dung dch in ly nn c pha t ha cht t tiu chun bng nc
loi ion.
- Dung dch in ly nn Na2B4O7 (Natritetraborat) 10mM.
Cn chnh xc khong 0,0953 g Natritetraborat vo bnh nh mc 25ml,
thm nc v siu m ha tan hon ton. iu chnh nc va n
vch, lc u.
- Dung dch in ly nn Na2B4O7 / SDS
Na2B4O7 10mM, SDS 50mM, iu chnh pH bng NaOH 1M v H3BO3
n cc pH 8,75; pH 9,0; pH 9,3; pH 9,6; pH 10,0 bng my o pH.
Cn chnh xc khong 0,1906 g Na2B4O7 v 0,7210 g SDS vo bnh nh
mc 50,0 ml thm nc siu m ui kh v ha tan hon ton. iu chnh
-
23
nc ti vch v lc u. Dung dch in ly nn ny iu chnh pH n pH
8,5 ; pH 9,3 v pH 10,0.
Pha cc dung dch in ly nn Na2B4O7 10mM/ SDS 25mM; Na2B4O7
10mM/ SDS 75mM; Na2B4O7 5mM/ SDS 50mM; Na2B4O7 20mM/ SDS
50mM: tin hnh cn v pha tng t nh trn vo bnh nh mc 25ml.
2.1.3 Thit b v dng c
- My in di mao qun Aligent CE system 9001 c iu khin bng phn
mm Chemstation 100001 (sn xut ti hng Hewlett Packard, c) ca b
mn Ha phn tch - c cht, trng i hc Dc H Ni.
- My lc nc loi ion Satorius arium 611: model SATORIUS AG
GOTYINGEN, p sut ti a 100psi/ 6,9bar.
- My siu m Ultrasonic LC 60H (sn xut ti hng Elma, c).
- My o pH EUTECH Instrument
- Cn phn tch Satorius TE214S.
- Giy lc ng knh 11cm Trung Quc
- Mng lc PTFE vi kch thc l lc 0,2 m.
- B lc ht chn khng mng lc cellulose vi kch thc l lc 0,45 m.
- Cc dng c chnh xc: bnh nh mc, pipet chnh xc vi cc th tch
khc nhau.
- Cc dng c khc: cc c m, ng nghim, a thy tinh, chy v ci.
2.2 i tng nghin cu
Ch phm lm vic Vin nn Avocomb-N c hm lng Lamivudin 150mg,
Zidovudin 300mg & Nevirapin 200mg do cng ty Ranbaxy laboratories
Limited, n sn xut. Ngy sn xut 9/2011. Hn s dng 9/ 2013.
Vin nn Lamivudin 150mg & Zidovudin 300mg do cng ty Matrix
Laboratories Limited n, n sn xut. Ngy sn xut 9/2011. Hn s
dng 10/2013.
-
24
2.3 Ni dung v phng php nghin cu
2.3.1 Ni dung nghin cu
Tham kho v nghin cu cc ti liu v CE, MEKC, cc c tnh l ha
ca hot cht cn phn tch. Trn c s kho st cc iu kin in di
nhm tm ra iu kin ti u nht tch v nh lng ng thi Lamivudin,
Zidovudin v Niverapin trong ch phm vin nn. Thm nh phng php
xy dng c. T phng php , tin hnh kim tra cht lng cc thuc
c cng hot cht ang c s dng trong phc iu tr HIV/AIDS Vit
Nam.
2.3.2 Phng php nghin cu
Phng php c chn y l phng php in di mao qun vi hai
kiu in di CZE v MEKC. Tin hnh kho st cc iu kin :
- Xy dng phng php tch v nh lng ng thi 3TC, AZT &
NVP
- ng dng trong nh lng ch phm vin nn.
2.3.2.1 Thm nh phng php
Phng php phn tch ang xy dng c thm nh theo cc tiu ch
Khong tuyn tnh
m bo s chnh xc ca php nh lng, chng ti tin hnh trn 6
mu hn hp chun cn phn tch cc nng khc nhau, lp mi tng
quan gia nng ca Lamivudin, Zidovudin & Nevirapin vi din tch pic
thu c trn in di bng phng trnh hi quy cng cc h s tng
quan tng ng v th.
Yu cu ng hi quy thu c phi c dng ng thng v gi tr h s
tng quan r 0,99.
lp li ca phng php
-
25
Tin hnh phn tch mc nng xc nh vi 5 mu c lp, kho
st lp li ca din tch pic ca ba cht cn phn tch. nh gi phn tn
cc s liu din tch pic v thi gian lu thu c da vo gi tr lch
chun tng i RSD(%):
% =
100
= ( )2
1
Trong :
SD: lch chun
RSD: lch chun tng i
xi : gi tr tnh c ca ln th nghim th i
: gi tr trung bnh cc ln th nghim
n: s ln th nghim
Yu cu RSD(%) khng c qu 5%.
ng ca phng php
ng ca phng php c xc nh bng t l tm li ca mt mu
th c cho thm nhng lng chun nht nh. Trong nghin cu ny,
chng ti tin hnh lp li nm ln, chun thm vo khong 20% so vi
lng trong mu. Phng php thm chun vo mu c chun b nh sau:
Mu th: l dung dch ch phm c pha vi nng nm trong khong
tuyn tnh.
Mu thm chun: c tin hnh song song cng mu th. Thm 20%
chun so vi hm lng c trong mu.
Tnh thu hi: Da vo phng trnh chun hi quy tnh ton c nng
ca tng mu (C mu) v nng ca tng mu thm chun (C mu thm chun)
C thu hi = C mu thm chun C mu
T l phn trm thu hi (%) =
-
26
Tnh tng thch h thng
Tnh tng thch h thng c xc nh bng cch tim lp li 6 ln cng
mt mu hn hp ba chun c xc nh nng . nh gi phn tn
ca s liu da vo gi tr lch chun tng i RSD(%) v gi tr phn
gii Rs ca hai pic lin k.
Rs = 2 () ()
( + )
Trong : (tR)A : thi gian dch chuyn ca cht A
(tR)B : thi gian dch chuyn ca cht B
WA ,WB : rng chn pic trn in di ca hai cht A v B
Tnh c hiu ca phng php
Tnh c hiu ca phng php l kh nng xc nh mt cch chc chn
s c mt ca cc cht cn phn tch khi c cc thnh phn khc bng cch
in di ln lt mu trng, mu chun, mu th trong cng mt iu kin in
di la chn. Trong mu trng chnh l dung mi (trong nghin cu ny
chng ti s dng MeOH 10%/ H2O lm dung mi) dng pha mu th
v mu chun.
2.3.2.2 p dng phng php MEKC vo nh lng vin nn
Avocomb-N v vin nn Lamivudin 150mg & Zidovudin 300mg
Trn c s kho st, chn nng nh lng mu th nm trong
khong tuyn tnh.
T ng chun xy dng c khi xc nh khong tuyn tnh, tnh
ra c hm lng cht cn phn tch c trong mu nh lng. Tip tnh
ra hm lng hot cht c trong ch phm.
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27
PHN III: THC NGHIM V KT QU
3.1 La chn iu kin in di ti u
3.1.1 La chn bc sng
la chn bc sng pht hin thch hp, chng ti tin hnh qut ph
tm max ca c ba cht 3TC, AZT & NVP trn my in di mao qun CE vi
nng ba cht tng ng ln lt l 150g/ml; 300g/ml & 200g/ml, vi
dung dch in ly nn Natritetraborat 10mM/ SDS 50mM pH = 9,3, mu trng
l 10% MeOH trong nc loi ion. Tin hnh qut ph trong vng bc sng
t 190nm 600nm.
Ph hp th ca ba cht c th hin trong hnh 6, 7 & 8.
Hnh 6: Ph hp th ca Lamivudin
Hnh 7: Ph hp th ca Zidovudin
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28
Hnh 8: Ph hp th ca Nevirapin
Kt qu thu c cho thy Lamivudin hp th cc i bc sng
270,5 nm, Nevirapin l 268,5 nm, Zidovudin l 264,5nm.
My in di CE s dng detector DAD cho php chng ti c th nh
lng tng thnh phn ti bc sng hp th cc i ca n. V vy chng ti
la chn cc bc sng 270,5nm; 268,5nm & 264,5nm tng ng nh
lng 3TC, AZT v NVP.
3.1.2 iu kin in di
Phng php tin hnh da trn k thut in di mao qun v cn c
xy dng mc n gin nht cho php thu c kt qu phn tch kh quan
nht. Trong qu trnh in di, c mt s iu kin c gi c nh, khng
thay i:
- Ct mao qun silica trn c chiu di hiu dng 60cm, ng knh trong 75
m.
- Detector DAD
- Bc sng pht hin : Lamivudin 270,5 nm; Nevirapin 268,5 nm;
Zidovudin 264,5 nm
- Nhit 25 0C.
- iu kin ra mao qun: trong thi gian tin hnh in di, mao qun u
c ra phc hi v duy tr b mt mao qun n nh, gim s hp ph
-
29
ca cht phn tch ln trn b mt mao qun, lm mi li b mt to iu
kin cho silanol in ly, to b mt mao qun mang in tch.
Thi im bt du: ln lt ra bng nc loi ion 10 pht, dung dch
NaOH 1M 5 pht, nc loi ion 10 pht, dung dch NaOH 0,1M 10 pht v
ra li bng nc loi ion 10 pht (tt c cc dung dch trc khi chy u
c lc qua mng lc 0,2m).
Gia cc ln chy: 2 pht nc loi ion 2 pht NaOH 0,1M 2 pht nc
loi ion 2 pht dung dch in ly nn.
Thi im kt thc: ln lt ra bng nc loi ion 10 pht, dung dch
NaOH 0,1M 10 pht, nc loi ion 10 pht.
Cc iu kin chng ti s tin hnh kho st bao gm:
- La chn phng php bm mu
- La chn kiu in di
- La chn dung dch in ly nn
- Kho st nng dung dch in ly nn
- Kho st nng cht to micelle SDS
- Kho st iu kin pH dung dch in ly nn
- Kho st th p vo hai u mao qun
- Kho st thi gian tim mu
3.1.3 La chn phng php bm mu
Trong k thut in di mao qun, mu c th c a vo mao qun
bng hai phng php chnh l phng php in ng hc (electrokinetic),
phng php thy ng hc (hydrodynamic)
Phng php in ng hc: t mt in th nh hn 3 5 ln so vi
in th t lc in di. Hai u ca mao qun c nhng trong l mu v l
m. Khi , nhng ion mu di chuyn vo mao qun to thnh vng mu
u mao qun. Cht phn tch i vo mao qun bng c di chuyn v lc y
-
30
ca EOF. Nh vy, lng mu tim ph thuc vo linh in di ca cht
phn tch: linh cng ln, lng mu np vo cng nhiu. S sai khc ny
s ln nu thi gian bm mu di. Tuy nhin, c th loi tr sai s ny bng
cch bm mu trong thi gian ngn v nhng iu kin nh nhau trong mi
th nghim nghin cu. Bm mu trong thi gian ngn c nh hng n
nhy ca phng php. Ngoi ra, lng mu bm cn ph thuc vo dn
in ca dung dch mu, c bit nhng mu c nng mui cao m ta
khng th bit trc c nh nhng mu nc tiu.
Phng php xi phng: nng cao bnh ng mu v h thp bnh u
ra. Phng php xi phng n gin nhng khi np mt lng mu c vi nl
th kh chnh xc. Phng php ny ch p dng cho nhng h in di t to
n gin, lp li km.
Phng php thy ng lc hc: tng p sut u mao qun nhng
vo bnh cha mu hoc to chn khng bnh u ra. Do chnh lch p sut
hai u mao qun, mt lng mu s c bm vo u mao qun. Mu
c bm vo s c nng u v ging vi nng ca cht tan trong mu
phn tch. y l phng php tim mu ph bin nht.
Trong kha lun ny, chng ti la chn phng php tim thy ng
lc hc vi p sut l 50mbar.
3.1.4 La chn kiu in di
Vi cc iu kin in di c la chn pha trn, chng ti tin hnh
la chn kiu in di sao cho c th tch c ng thi ba cht.
Cc kiu in di chng ti kho st y gm c:
- in di mao qun vng (CZE) vi dung dch in ly nn l Natritetraborat
10mM.
- in di mao qun micelle (MEKC) vi dung dch in ly nn l
Natritetraborat 10mM / SDS 50mM
-
31
Chy mu hn hp 3 chun Lamivudin, Nevirapin v Zidovudin c nng
tng ng ln lt l 150g/ml; 300g/ml v 200g/ml. Hnh di y
kho st nh hng ca cc dung dch in ly nn trn kh nng tch ca
Lamivudin, Nevirapin & Zidovudin chun.
Hnh 9 : in di chun Lamivudin, Zidovudin, Nevirapin ba dung dch
in ly nn khc nhau
Nhn xt: Nhn vo hnh 9, ta thy, vi kiu in di vng CZE, trn
in di ch thy xut hin 2 pic. Trong khi , tin hnh phn tch bng
MEKC, in di xut hin c 3 pic, cc pic ang c xu hng tch khi
nhau. V vy, chng ti la chn MEKC l phng php tin hnh phn tch
trong kha lun ny.
3.1.5 La chn dung dch in ly nn
Na2B4O7 10mM
Na2B4O710mM
SDS 50mM
-
32
Sau khi la chn c kiu in di l MEKC, chng ti tin hnh la
chn dung dch in ly nn s dng trong phn tch ny.
Dung dch in ly nn ng mt vai tr quan trng i vi kt qu phn
tch thu c trong phng php in di mao qun. Tc ng ca cht in
gii th hin qua vic n nh, duy tr dng in I v dng in thm EOF
trong mao qun, qua s lm nh hng n linh in di hiu dng ca
cht phn tch. Trn thc t, ngi ta thng chn cht m pH cng chnh l
cht in gii ca sc k in di.
Trong h m, cc h m a acid hoc a base thng c hiu lc tt
hn cc h m n acid, n base. Chng c nhiu nc phn ly nn c kh
nng m trong vng pH rng v tnh linh hot cao.
Chng ti tin hnh kho st cc m phosphate, citrate, acid
phosphoric. Tuy nhin, ch n khi kho st vi m Natritetraborat, chng
ti mi thy ba cht ang gn tch khi nhau trn in di . Mt khc, khi
tin hnh phn tch vi dung dch in ly nn l m borat, dng in duy tr
trong mao qun n nh, th p vo hai u mao qun n nh, t gy hiu ng
nhit ln cho ng mao qun. V l do , chng ti la chn m
Natritetraborat lm dung dch in ly nn khi tin hnh kha lun ny.
3.1.6 Kho st iu kin pH ca dung dch in ly nn
pH ca dung dch in ly nn ng mt vai tr ht sc quan trng trong
qu trnh phn tch bng CE do n nh hng n s hnh thnh v tc ca
dng EOF. Thc nghim ch r dng EOF ch c gi tr ng k khi pH
ca dung dch trn 4. Ty tng iu kin in di c th, trong khong pH 2
12 dng EOF c th thay i n 10 ln.
Cc iu kin in di c gi c nh nh la chn pha trn.
Vi dung dch in ly nn l Na2B4O7 10mM/ SDS 50 mM, cc pH c
kho st tng dn t 8 n 10. Chy vi hn hp chun nh chun b phn
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33
2.1.2, nng Lamivudin 150g/ml, Zidovudin 300g/ml, Nevirapin
200g/ml. p th in 20kV, bm mu 50mbar x 5s.
Kt qu:
pH = 8,75
pH = 9,0
pH = 9,3
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34
Hnh 10: in di hn hp ba chun vi dung dch in ly nn Na2B4O7
10mM/ SDS 50mM cc pH khc nhau
Nhn xt: Da vo hnh 10, ta thy:
- Khi pH tng t 8,75 n 9,0, thi gian lu ca c 3 cht u tng, thi
gian phn tch ~ 16 pht, pic th 3 b dong chn pic do thi gian lu
gi lu.
- Khi tng pH n 9,6 c th nhn thy thi gian lu ca c 3 cht u
gim, cc pic xut hin trc pht th 10, pic th ba nhn, cc pic u
tch nhau r rng, pic p.
- Khi tng pH n 10, thi gian lu ca c 3 pic tng, khong cch gia
cc pic trn in di tng, kh nng tch ri ca cc pic tng, thi
gian phn tch di hn tuy nhin pic vn nhn v p.
pH = 9,6
pH = 10,0
-
35
Tuy nhin, khi pH tng t 8,75 n 10,0, dng in trong mao qun tng
ln mt cch ng k. iu d gy hng mao qun, gy ra hin tng sinh
nhit trong mao qun, lm nh hng n kt qu phn tch.
Mt khc, khi pH thay i, n cng s lm thay i s tch in ca thnh
mao qun. V khi pH ca pha ng in di thay i, s lm thay i qu trnh
qu trnh de-hydro, tch ion H+ trong nhm OH trn thnh mao qun, tc l
lm thay i lp in kp v th Zta, qua nh hng n s in di ca
cc cht.
Trong kha lun ny, chng ti la chn pH = 9,3 tin hnh cc qu
trnh phn tch sau. pH ny khng qu cao, m bo dng in trong mao
qun nm trong gii hn cho php (30 70 A), hn ch sai s do hin tng
m rng vng mu khi dng in trong mao qun qu cao dn n sinh nhit,
nhng vn m bo cc pic tch nhau c r rng, thi gian phn tch khng
qu di v thi gian lu ca cc cht hp l.
3.1.7 Kho st nng dung dch in ly nn
Sau khi chn c pH dung dch in ly nn l 9,3, chng ti tin hnh
kho st nng dung dch in ly nn Natritetraborat.
Cng vi pH ca dung dch m, cht in ly trong pha ng cng c vai
tr quan trng v phi c nng ph hp. Trn thc t, ngi ta c gng
chn cht m pH cng ng thi l cht in gii ca sc k in di. Nh
vy thnh phn pha ng s khng phc tp, khng gy kh khn, nh hng
n hiu qu tch sc k.
Khi nng m tng, ngha l nng cc ion tng, thng lm gim
ln lp in kp, nh hng n tng tc tnh in ca cht tan vi thnh
mao qun. Mt khc, n lm tng lc ion ca dung dch, lm lp in kp b
thu hp li kt qu l lm gim th Zta v gim lun tc ca dng EOF.
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36
Lc ion ln li to ra dng in cao v gy ra hiu ng nhit June ln lm
mao qun nng ln v xut hin hin tng m rng vng mu.
Kho st nh hng ca nng m borat bng cch c nh nng
SDS v cc iu kin in di khc, ri tin hnh phn tch hn hp chun
Lamivudin 150g/ml, Zidovudin 300g/ml, Nevirapin 200g/ml cc nng
dung dch natritetraborat khc nhau bao gm: 5mM, 10mM v 20mM. Kt
qu thu c c th hin Hnh 11.
Borat 5mM
SDS 50mM
Borat 10mM
SDS 50mM
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37
Hnh 11: in di hn hp 3 chun vi dung dch in ly nn cc nng
borat khc nhau
Nhn xt: Kt qu thu c cho thy, nng borat tng t 5mM n
20mM th thi gian lu ca cc cht tng dn, ng thi kh nng tch ca
cc cht cng tng. Tuy nhin, y, chng ti la chn nng borat
10mM tin hnh cho cc php phn tch sau. nng borat 10mM, c ba
pic u tch khi nhau, mt khc, thi gian phn tch li khng di (cc pic
u xut hin trc pht th 10), pic nhn, khng b dong chn, dng in
trong qu trnh chy in di ph hp. Khi tin hnh chy vi nng borat
5mM, dng in trong mao qun khng n nh, hay xy ra hin tng st
dng. Tin hnh phn tch vi borat 20mM, dng in li qu cao ~80 A, th
p vo mao qun khng n nh. Phn tch th vi borat 30mM, gn nh
khng tin hnh phn tch c do dng in trong mao qun qu ln, dn
n hin tng st dng v phi dng qu trnh phn tch.
3.1.8 Kho st nng cht to micelle SDS
Cht hot ng b mt SDS c xem nh pha tnh gi ca qu trnh
tch v thc cht n vn chuyn ng cng vi dng in di. Nng SDS
trong dung dch in di c nh hng mnh ti in ly hiu dng ca cht
phn tch do n lin quan n s hnh thnh cc ht micell cng nh nng
micell trong dung dch m. kho st nh hng ca SDS n qu trnh
Borat 20mM
SDS 50mM
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38
in di, chng ti thay i nng SDS ti ba gi tr l 25mM, 50mM v
75mM, gi c nh cc iu kin kho st khc bao gm: m borat 10mM;
pH 9,3; p th 20kV.
Tin hnh phn tch vi hn hp 3 chun c nng Lamivudin
150g/ml, Zidovudin 300g/ml, Nevirapin 200g/ml, chng ti thu c kt
qu nh sau:
Borat 10mM
SDS 25 mM
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39
Hnh 12: in di hn hp 3 chun vi dung dch in ly nn cc nng
SDS khc nhau
Nhn xt: Ta nhn thy khi nng SDS tng, thi gian lu ca ba cht
tng, thi gian phn tch tng, phn gii RS tng (8,62 - 9,52 11,45 i
vi pic 1 & 2; 90,73 - 91,84 94,31 i vi pic 2 & 3). Tuy nhin, khi tng
nng SDS (mt cht din hot anion ha), cng dng in trong mao
qun tng ln r rt v gy ra hiu ng nhit June, lm mao qun nng ln.
cc nng SDS 25mM v 75mM, tuy pic nhn nhng chiu cao pic khng
bng nng SDS 50mM, gy nh hng n nhy. V vy chng ti
chn nng SDS 50mM ti u kho st cc th nghim tip theo.
3.1.9 Kho st th t vo hai u mao qun
Sau khi chn c dung dch in ly nn l Borat 10mM/SDS 50mM pH =
9,3, chng ti tin hnh chy hn hp ba chun c nng Lamivudin
150g/ml, Zidovudin 300g/ml, Nevirapin 200g/ml ln lt th 15kV,
20kV v 22kV. Kt qu thu c nh sau:
Borat 10mM
SDS 50 mM Borat 10mM
SDS 75 mM
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40
Hnh 13: in di hn hp 3 chun vi dung dch in ly nn borat
10mM/SDS 50mM cc th khc nhau
Nhn xt: Khi tng th in di t 15kV n 22kV, ta c th nhn thy, thi
gian lu ca 3 pic u gim, thi gian phn tch gim. th 15kV, thi gian
phn tch kh di ~14 pht, pic th ba khng nhn v b dong chn. Ngc
li, th 22kV, thi gian phn tch c 3 cht kh ngn ~7 pht, do kh
15kV
20kV
22kV
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41
nng tch gia cc pic cng gim. Mc d th 22kV, ta vn nhn thy ba pic
tch nhau r rng, thi gian phn tch ngn, cc pic u nhn v p, tuy
nhin, bi th p vo mao qun qu ln, cng dng in trong mao qun
tng cao, d gy ra hiu ng June, to nhit trong mao qun, lm thay i
nht tng vng trong mao qun, khin cc phn t cht tan di chuyn khng
ng nht, dn n s khuch tn khc nhau v lm m rng vng mu, lm
nh hng n kt qu phn tch. Do , chng ti la chn th 20kV tin
hnh lm phn tch trong kha lun ny. th 20kV, thi gian phn tch
khng qu di ~ 10 pht, cc pic vn tch c nhau, pic thu c trn in
di nhn, khng b dong chn pic.
3.1.10 Kho st p sut tim mu
Khi np mu vo mao qun, lng mu hay vng mu np phi nh hn
gii hn cho php ( 2% chiu di l). Nu vng mu np vo qu ln th s
phn tn (m rng vng mu) s xut hin mnh, do hin tng khuch tn.
Vng mu cng ln th s phn tn cng ln. Lc ny phn gii v hiu
sut tch s b gim mnh theo s phn tn m rng vng mu ca cht phn
tch trong mao qun.
kho st thi gian tim mu, chng ti tin hnh tim mu 50mbar vi
cc thi gian khc nhau, 3s, 5s v 7s. Tin hnh phn tch vi hn hp 3
chun c nng Lamivudin 150g/ml, Zidovudin 300g/ml, Nevirapin
200g/ml. Cc iu kin in di c gi nh trong mc 3.1.2. Th in di
20kV. Kt qu thu c nh sau:
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42
Hnh 14: in di hn hp chun vi thi gian tim mu khc nhau
Nhn xt: Nhn vo hnh trn, ta c th nhn thy, vi thi gian tim mu
l 3s v 7s, thi gian lu ca cc cht l lu hn so vi tim mu 5s. Mt
khc, pic th ba thi gian tim mu 3s v 7s chn pic b dong nhiu, pic
khng nhn. V vy, chng ti la chn p sut tim mu l 50mbar x 5s.
50mbar x 3s
50mbar x 5s
50mbar x 7s
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43
3.1.11 nh tnh Lamivudin, Zidovudin, Nevirapin trong iu kin in di
c thit lp
Chng ti nh tnh ba cht bng phng php thm chun.
Tin hnh chy mu th c nng 3TC, AZT & NVP ln lt l
120g/ml; 240g/ml & 160g/ml. Sau tin hnh chy ln lt lt mu
th thm chnh xc mt lng Lamivudin chun, Zidovudin chun v
Nevirapin chun. Kt qu thu c nh sau:
Hnh 15: in di hn hp th
Hnh 16: in di mu th thm Lamivudin chun
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44
Hnh 17: in di mu th thm Nevirapin chun
Hnh 18: in di mu th thm Zidovudin chun
Nhn xt: Tin hnh chy hn hp ba chun, trn din di ch xut hin
ba pic.
Khi thm chun Lamivudin vo, ta nhn thy, din tch v chiu cao ca
pic u tin tng ln r rt (Spic t 47,4 tng ln 82,3), trong khi din tch v
chiu cao ca 2 pic sau thay i khng ng k. Nh vy, trong iu kin
in di chng ti thit lp, Lamivudin di chuyn n detector trc.
Khi thm chun Zidovudin vo, ta thy din tch v chiu cao ca pic th 2
tng ln r rt (Spic t 93 tng ln 140,7), trong khi din tch v chiu cao ca
2 pic cn li thay i khng ng k. Nh vy, trong iu kin in di ny,
Zidovudin l cht di chuyn n detector th hai, sau Lamivudin.
Thm chun Nevirapin, ta thy din tch v chiu cao ca pic th ba tng
ln (Spic t 71,6 tng ln 107). Nh vy, Nevirapin l cht n detector sau
cng.
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45
3.1.12 Kt lun
Qua qu trnh kho st trn, chng ti la chn c iu kin in di nh
sau:
- Chiu di hiu dng ct mao qun silica trn 60cm, ng knh trong
75m.
- in th t vo hai u mao qun 20kV
- Nhit mao qun 25 0C.
- Tim mu 50mbar x 5s
- Bc sng pht hin 270,5nm i vi Lamivudin; 264,5nm i vi
Zidovudin & 268,5nm i vi Nevirapin.
- Dung dch in ly nn Borat 10mM/SDS 50mM, pH = 9,3
Tin hnh phn tch vi cc iu kin in di ti u chn nh trnvi
hn hp ba chun c nng 3TC, AZT v NVP tng ng l 150g/ml;
300g/ml v 200g/ml thu c in di sau (Hnh 19)
Hnh 19: in di hn hp ba chun iu kin in di ti u
3.2 Thm nh phng php
3.2.1 Kho st tnh tng thch h thng
Tim lp li 6 ln cng mt mu chun trn. Kt qu phn tch c trnh
by bng sau:
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46
Bng 4: Kt qu kho st tnh tng thch h thng ca phng php (n=6)
STT
Lamivudin Zidovudin Nevirapin phn
gii
TM
(pht)
Spic
(mAU.s)
TM
(pht)
Spic
(mAU.s)
TM
(pht)
Spic
(mAU.s)
R12
(3TC
&
AZT)
R23
(AZT
&
NVP)
1 5,154 66,3 5,567 126,2 9,745 125,3 8,69 90,24
2 5,172 66,7 5,586 125,9 9,829 124,7 9,52 91,84
3 5,161 66,4 5,596 126,1 9,766 125,1 9,28 93,51
4 5,202 67,2 5,632 126,6 9,975 126,2 9,01 82,72
5 5,195 66,3 5,651 125,4 9,755 127,0 9,13 80,87
6 5,382 66,0 5,850 126,6 9,613 125,9 9,67 74,07
5,211 66,5 5,647 126,1 9,781 125,7
SD 0,086 0,41 0,104 0,455 0,119 0,837
RSD
(%) 1,6 0,6 1,8 0,4 1,2 0,7
Nhn xt: Kt qu thng k cho thy qua 6 ln in di cng mt mu
chun, thi gian lu v din tch pic c RSD % < 2%, gi tr phn gii ln
hn 1,5. Vy phng php t tnh tng thch h thng.
3.2.2 Tnh c hiu
Chng ti tin hnh in di mu trng, mu chun v mu th theo iu
kin in di chn pha trn. Kt qu thu c nh sau:
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47
Hnh 20: in di mu trng
Hnh 21: in di hn hp chun Lamivudin, Zidovudin, Nevirapin
Hnh 22: in di hn hp ch phm lm vic
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48
Hnh 23: in di hn hp ch phm thm hn hp chun
Nhn xt: Mu trng khng c pic trng vi pic cht phn tch hay pic l.
in di mu chun v mu th u cho kt qu l ba pic vi thi gian lu
tng t nhau. Khi thm chun vo hn hp ch phm, tin hnh in di
khng thy xut hin pic l v din tch pic mu th tng ln. Vy phng
php c tnh c hiu.
3.2.3 Xc nh khong tuyn tnh
Chng ti tin hnh chy chun theo iu kin in di c la chn.
Chun b 6 hn hp dung dch chun c cha Lamivudin c nng t
77,25g/ml n 231,7g/ml; Zidovudin c nng t 149,0g/ml n
447,0g/ml; Nevirapin c nng t 99,25g/ml n 297,7g/ml v tin
hnh kho st. Kt qu thu c nh sau:
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49
Bng 5: Din tch pic trn in di v nng Lamivudin tng ng
Nng
(g/ml) 77,25 123,6 154,5 169,95 185,4 231,7
Din tch pic
(mAU.s) 29,85 47,60 62,10 70,70 78,76 97,80
Phng trnh hi quy tuyn tnh S = 0,4493 x C 6,1032
H s tng quan R2 = 0,9968
Hnh 24: th biu din s ph thuc gia din tch pic v nng 3TC
tng ng
y = 0.4493x - 6.1032
R = 0.9968
20
45
70
95
120
50 70 90 110 130 150 170 190 210 230 250
Di
n t
ch
pic
mA
U.s
Nng Lamivudine g/ml
-
50
Bng 6: Din tch pic trn in di v nng Zidovudin tng ng
Nng
(g/ml) 149,0 238,4 298,0 327,8 357,6 447,0
Din tch pic
(mAU.s) 57,4 94,8 120,6 138,7 149,4 186,5
Phng trnh hi quy tuyn tnh S = 0,4385 x C 8,2912
H s tng quan R2 = 0,9982
Hnh 25: th biu din s ph thuc gia din tch pic v nng AZT
tng ng
y = 0.4385x - 8.2912
R = 0.9982
20
70
120
170
220
100 150 200 250 300 350 400 450 500
Di
n t
ch
pic
mA
U.s
Nng Zidovudine g/ml
-
51
Bng 7: Din tch pic trn in di v nng Nevirapin tng ng
Nng
(g/ml) 99,25 158,8 198,5 218,3 238,2 297,7
Din tch
pic (mAU.s) 58,6 93,7 119,48 136,7 148,6 188,4
Phng trnh hi quy tuyn tnh S = 0,6606 x C 9,0497
H s tng quan R2 = 0,9981
Hnh 26: th biu din s ph thuc gia din tch pic v nng NVP
tng ng
Nhn xt: T kt qu bng 5, 6 & 7 v hnh 24, 25 & 26, ta c th nhn
thy trong khong nng kho st dung dch hn hp chun 3TC, AZT &
NVP vi nng 3TC 77,25 g/ml 231,7 g/ml; nng AZT 149,0 g/ml
447,0 g/ml & nng NVP 99,25 g/ml 297,7 g/ml c mi tng quan
y = 0.6606x - 9.0497
R = 0.9981
20
70
120
170
220
50 100 150 200 250 300 350
Di
n t
ch
pic
mA
U.s
Nng Nevirapine g/ml
-
52
tuyn tnh gia din tch pic v nng , cng vi h s tng quan tuyn tnh
r 1 (0,998; 0,999 & 0,999). Nh vy c ba cht u cho tuyn tnh cao
gia nng v din tch pic ca tng cht khi phn tch bng in di mao
qun trong khong nng chng ti kho st vi cc iu kin in di
c la chn mc 3.1.12.
3.2.4 lp li ca phng php
lp li ca phng php c tnh khi khi php phn tch c lp li
trong cng mt phng th nghim, trong mt khong thi gian ngn do cng
mt ngi phn tch. lp li c biu th bng lch chun tng i
(RSD%).
Tin hnh phn tch c lp 5 ln ba mc nng khc nhau trn dung
dch chun 3TC, AZT & NVP. Kt qu xc nh lp li c th hin
cc bng sau:
Bng 8. Kt qu xc nh lp li ca phng php MEKC trong nh lng
3TC
Ln tim mu
Din tch pic cc mc nng ca 3TC
Thp
77,25g/ml
Trung bnh
150g/ml Cao 231,7 g/ml
1 29,85 63,51 98,83
2 30,10 62,73 97,85
3 29,54 63,32 98,43
4 29,76 62,86 98,62
5 30,55 62,95 97,95
29,96 63,07 98,34
SD 0,386 0,328 0,424
RSD (%) 1,3 0,5 0,4
-
53
Bng 9. Kt qu xc nh lp li ca phng php MEKC trong nh lng
AZT
Ln tim mu
Din tch pic cc mc nng ca AZT
Thp 149 g/ml Trung bnh 298
g/ml Cao 447 g/ml
1 57,65 122,35 190,23
2 58,34 120,60 186,56
3 57,40 119,76 187,49
4 58,63 122,57 191,12
5 58,80 122,20 187,32
58,16 121,50 188,54
SD 0,612 1,244 2,001
RSD (%) 1,1 1,0 1,1
Bng 10. Kt qu xc nh lp li ca phng php MEKC trong nh
lng NVP
Ln tim mu
Din tch pic cc mc nng ca NVP
Thp 99,25
g/ml
Trung bnh
198,5g/ml Cao 297,7 g/ml
1 59,70 120,56 190,67
2 58,63 121,34 190,32
3 58,12 119,48 188,44
4 57,45 120,40 189,95
5 59,36 122,87 188,74
58,65 120,93 189,62
SD 0,912 1,270 0,983
RSD (%) 1,6 1,1 0,5
-
54
Nhn xt: T kt qu bng 8, 9 & 10, ta nhn thy RSD (%) < 2 % i
vi c ba cht 3TC, AZT & NVP ba mc nng khc nhau. V vy,
phng php c tp trung d liu cao.
3.2.5 ng ca phng php
ng ca phng php c xc nh bng t l tm li ca mt mu
th c cho thm nhng lng cht chun nht nh. Trong phn thm
nh ny, chng ti thm khong 20% cht chun so vi lng c trong mu.
T l thu hi l thuyt l 98 102%.
Bng 11. Kt qu kho st ng ca phng php trn thm chun
Lamivudin
S
mu
Din
tch pic
th
(mAu.s)
Nng
th
g/ml
Nng
chun
thm
vo
(g/ml)
Din
tch pic
th
thm
chun
(mAu.s)
Nng
thu
c
g/ml
Nng
thu
hi
(g/ml)
T l
phn
trm
thu
hi
1 48,3 121,08
30,09
61,9 151,35 30,27 100,6
2 47,8 119,97 61,3 150,02 30,05 99,87
3 47,5 119,30 60,8 148,91 29,61 98,40
4 48,4 121,31 62,0 151,58 30,27 100,6
5 48,7 121,97 62,2 152,02 30,05 99,87
6 47,4 119,08 61,1 149,57 30,49 101,3
-
55
Bng 12 .Kt qu kho st ng ca phng php trn thm chun AZT
S
mu
Din
tch pic
th
(mAu.s)
Nng
th
g/ml
Nng
chun
thm vo
(g/ml)
Din tch
pic th
thm
chun
(mAu.s)
Nng
thu
c
g/ml
Nng
thu
hi
(g/ml)
T l
phn
trm
thu
hi
1 95,3 236,24
59,8
121,5 295,99 59,75 99,92
2 94,9 235,33 120,8 294,39 59,06 98,76
3 95,6 236,92 122,0 297,13 60,21 100,7
4 95,8 237,38 121,8 296,67 59,29 99,15
5 96,1 238,06 122,6 298,50 60,43 101,1
6 95,6 236,92 122,3 297,81 60,89 101,8
Bng 13. Kt qu kho st ng ca phng php trn thm chun NVP
S
mu
Din
tch pic
th
(mAu.s)
Nng
th
g/ml
Nng
chun
thm vo
(g/ml)
Din tch
pic th
thm
chun
(mAu.s)
Nng
thu
c
g/ml
Nng
thu
hi
(g/ml)
T l
phn
trm
thu
hi
1 93,9 155,84
39,9
120,7 196,41 40,57 101,7
2 95,3 157,96 121,9 198,23 40,27 100,9
3 94,4 156,60 120,4 195,96 39,36 98,65
4 94,8 157,21 121,3 197,32 40,12 100,6
5 95,0 157,51 120,9 196,71 39,21 98,27
6 94,7 157,05 121,5 197,62 40,57 101,7
-
56
Nhn xt: T bng 11, 12 & 13 ta thy, t l phn trm thu hi ca 3TC,
AZT & NVP nm trong gii hn cho php (98 % - 102 %). Vy phng php
ny c ng cao.
3.3 ng dng nh lng trong ch phm
Phng php xy dng c c ng dng xc nh lm lng
Lamivudin, Zidovudin & Nevirapin trong ch phm vin nn Avocomb-N v
vin nn Lamivudin 150mg & Zidovudin 300mg c s dng trong iu tr
HIV/AIDS.
Tin hnh chy in di mu ch phm iu kin in di ti u chn
(mc 3.1.12), ta thu c cc kt qu sau:
Hnh 27: in di ch phm vin nn Avocomb-N
Bng 14: Kt qu nh lng vin nn Avocomb-N
Mu
th
Hm lng hot cht trong 1
vin (mg) % Hm lng so vi trn nhn
3TC NVP AZT 3TC NVP AZT
T1 152,6 203,5 298,3 101,73 101,8 99,43
T2 148,3 205,0 295,5 98,87 102,5 98,5
T3 154,3 200,8 286,4 102,87 100,4 95,47
TB 151,7 203,1 293,4 101,13 101,6 97,8
-
57
Hnh 28: in di ch phm vin nn Lamivudin 150mg & Zidovudin
300mg
Bng 15: Kt qu nh lng vin nn Lamivudin 150mg & Zidovudin 300
mg
Mu
th
Hm lng hot cht trong 1
vin (mg) % Hm lng so vi trn nhn
Lamivudin Zidovudin Lamivudin Zidovudin
T1 148,5 304,7 99,0 101,57
T2 153,6 297,4 102,4 99,13
T3 151,5 302,5 101,0 100,83
TB 151,2 301,5 100,8 100,5
Nhn xt: Sau ba ln chy in di i vi mi ch phm iu kin ti u
c thit lp, cc ch phm c kho st c hm lng ch phm nm
trong khong gii hn cho php (Lamivudin l 95% - 105%; Zidovudin l
97% - 102%; Nevirapin l 98% - 102%)
-
58
PHN IV: KT LUN V XUT
4.1 Kt lun
Nh vy, sau mt thi gian nghin cu, chng ti hon thnh c hai
mc tiu ra nh sau:
1. Xy dng c phng php tch v nh lng ng thi Lamivudin,
Zidovudin v Nevirapin trong ch phm bng phng php MEKC vi
cc iu kin in di nh sau:
- Chiu di hiu dng ct mao qun silica trn 60cm, ng knh trong
75m.
- in th t vo hai u mao qun 20kV
- Nhit mao qun 25 0C.
- Tim mu 50mbar x 5s
- Bc sng pht hin 270,5nm i vi Lamivudin; 264,5nm i vi
Zidovudin & 268,5nm i vi Nevirapin.
- Dung dch in ly nn Borat 10mM/SDS 50mM, pH = 9,3
Hot ha ct:
Thi im bt u: ln lt ra bng nc loi ion 10 pht, dung dch
NaOH 1M 5 pht, nc loi ion 10 pht, dung dch NaOH 0,1M 10 pht
v ra li bng nc loi ion 10 pht (tt c cc dung dch trc khi chy
u c lc qua mng lc 0,2m).
Gia cc ln chy: 2 pht nc loi ion 2 pht NaOH 0,1M 2 pht
nc loi ion 2 pht dung dch in ly nn.
Thi im kt thc: ln lt ra bng nc loi ion 10 pht, dung dch
NaOH 0,1M 10 pht, nc loi ion 10 pht.
2. Phng php c p dng v thu c kt qu rt kh quan trong
vic nh lng hm lng Lamivudin, Zidovudin, Nevirapin trong ch
phm vin nn Avocomb-N Lamivudin 150mg, Zidovudin 300mg v
-
59
Nevirapin 200mg do cng ty Ranbaxy ca n sn sut ang c
s dng trong phc iu tr HIV/AIDS Vit Nam.
4.2 xut
Kt thc qu trnh thc nghim ny, ti xin php c mt vi xut nh
sau:
1. Tip tc nghin cu, xy dng hon thin v p dng phng php ny
nh lng ng thi Lamivudin, Zidovudin & Nevirapin trong ch
phm ang lu hnh trn th trng, nng cao nng lc qun l cht
lng thuc, m bo s dng thuc an ton hp l.
2. Nn cn nhc vic trang b my in di mao qun vo cc trung tm
kim nghim v cc phng kim nghim ti c s sn xut v y l
phng php c nhiu u im nh t tn dung mi, ha cht, t c
hi, hiu lc tch cao
-
Ti liu tham kho
Ting vit
1. Nguyn Th Kiu Anh (2004), Mt s c im v ng dng ca in
di mao qun, Chuyn chuyn su 3 ca nghin cu sinh, Trng
i hc Dc H Ni.
2. Phm Lun (1999), C s l thuyt v sc k in di mao qun hiu
sut cao, Trng i hc Tng hp Quc gia H Ni, tr. 123 128
3. B Y t (2009), Dc th Quc gia Vit Nam, Nh xut bn Y hc, tr.
715 717; tr. 1179 1182.
4. B Y t (2009), Dc in Vit Nam IV, xut bn ln th t, Nh xut
bn Y hc, tr.346 348; tr. 637 639.
5. B Y t (2007), Ha dc tp II, Nh xut bn Y hc, tr. 224 226; tr.
227 228.
6. B Y t (2007), Dc l hc tp II, Nh xut bn Y hc, tr. 238 241.
7. B Y t (2007), Kim nghim dc phm, Nh xut bn Y hc.
8. B Y t (2007), Ha phn tch tp I, Nh xut bn Y hc, tr. 54 106.
9. B Y t (2007), Ha phn tch tp II, Nh xut bn Y hc, tr. 215
234.
10. y ban quc gia phng chng AIDS (2012), Bo co tin phng,
chng AIDS Vit Nam 2012.
Ting Anh
11. Martindale 36 (2009), vol 1, p. 856 859.
12. US. Pharmacopeia 32 (2009), vol 2, p. 2747 2748; vol 3, p. 3072
3076; p. 3887 - 3891.
-
13. World Heal Organization (2009), Lamivudin, Zidovudin and Nevirapin
tablets: Final text for addition to The International Pharmacopoeia
14. David Heiger (2000), High performance capillary electrophoresis,
Agilent Technologies, p. 6 58
15. Fred Strohmerier (1995), A new High performance capillary
electrophoresis instrument, Hewlett Packard Journal, p. 10 15.
16. Bin Fan, James T. Stewart, Determination of Zidovudine/ Lamivudine/
Nevirapine in human plasma using ion-pair HPLC, Journal of
Pharmaceutical and Biomedical Analysis Volume 28, Issue 5, 1 June
2002, Pages 903908
17. Geetha Ramachandran, A.K. Hemanthkumar, V. Kumaraswami,
Soumya Swaminathan, A simple and rapid liquid chromatography
method for simultaneous determination of Zidovudine and Nevirapine
in plasma, Journal of Chromatography B Volume 843, Issue 2, 7
November 2006, Pages 339344.
18. D. Anantha Kumar, M.V. Naveen Babu, J.V.L.N. Seshagiri Rao and
V. Jayathirtha Rao, 2009, Simultaneous determination of Lamivudine,
Zidovudine and Nevirapine in tablet dosage forms by RP-HPLC
method.
19. Bin Fan, James T. Stewart, Determination of Lamivudine/ Didanosine/
Nevirapine in human serum using Capillary zone electrophoresis, J
Capill Electrophor Microchip Technol. 2002 Sep-Dec;7(5-6):103-6
20. Bin Fan, James T. Stewart, Determinations of zidovudine/ didanosine/
nevirapine and zidovudine/ didanosine/ ritonavir in human serum by
micellar electrokinetic chromatography, Journal of Pharmaceutical and
Biomedical Analysis Volume 30, Issue 4, 7 November 2002, Pages
955960.
-
21. BIN FAN, B.S., Shanghai Medical University, P.R.China, (1992), Part
II, Bioanalysis of Anti-HIV Drugs in Human Serum Using HPLC, CE,
LC-MS-MS (Under the direction of JAMES THOMAS STEWART)
22. Antimicrobial agents and chemotherapy, Feb. 1993, Vol 37 No 2,
Pharmacokinetics of Nevirapine: Initial Single-Rising-Dose, p.178
182.
Trang web
23. http://www.ncbi.nlm.nih.gov/pubmed/12546158
24. http://www.fip.org/files/fip/BPS/BCS/Monographs/Lamivudine.pdf