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

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

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

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

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.

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.

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

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

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

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.

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

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

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

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

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

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:

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

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

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.

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:

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:

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

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:

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