B Y T

TRNG I HC DC H NI

TH HA

XY DNG PHNG PHP NH

LNG CC NG PHN I

QUANG CA ATENOLOL TRONG

CH PHM BNG IN DI MAO

QUN

KHA LUN TT NGHIP DC S

H NI -2014

B Y T

TRNG I HC DC H NI

TH HA

XY DNG PHNG PHP NH

LNG CC NG PHN I

QUANG CA ATENOLOL TRONG

CH PHM BNG IN DI MAO

QUN

KHA LUN TT NGHIP DC S

Ngi hng dn:

1. Th.S. V Ngn Bnh

2. DS. Ng Minh Thy

Ni thc hin ti:

1. B mn Ha phn tch & c cht

Trng i hc Dc H Ni

2. Vin kim nghim an ton v sinh

thc phm Quc gia

H NI - 2014

LI CM N

Vi lng knh trng v bit n su sc, em xin gi li cm n su sc ti

ThS. V Ngn Bnh B mn Ha phn tch v c cht- Trng i hc Dc

H Ni, DS. Ng Minh Thy v PGS.TS. Nguyn Th Kiu Anh Phng Qun

l khoa hc Trng i hc Dc H Ni, ngi tn tnh hng dn, ch bo,

gip em trong qu trnh thc hin v hon thnh kha lun.

Em xin gi li cm n n thy c gio, cc anh/ ch k thut vin ti B

mn Ha phn tch v c cht- Trng i hc Dc H Ni cng nh ton th

anh/ ch cn b, cng nhn vin ti Labo Ha Vin Kim nghim an ton v sinh

thc phm Quc gia to iu kin tt nht em hon thnh kha lun ny.

Em xin chn thnh cm n cc thy c trong Ban Gim hiu, Phng o to

v cc thy c gio Trng i hc Dc H Ni truyn t cho em nhng kin

thc qu bu trong sut nm nm em hc tp ti trng.

Cui cng em xin cm n gia nh, bn b, v nhng ngi lun theo st

chm lo, ng vin, kch l em trong hc tp v cuc sng.

H Ni, ngy 14 thng 05 nm 2014

Sinh vin

Th Ha

MC LC

DANH MC CC K HIU, CH VIT TT

DANH MC CC BNG

DANH MC HNH V V TH

T VN ......................................................................................................... 1

CHNG 1. TNG QUAN ................................................................................... 3

1.1. TNG QUAN V ATENOLOL .............................................................. 3

1.1.1. Tnh cht l, ha .................................................................................. 3

1.1.2. Dc l v c ch tc dng .................................................................. 4

1.2. TNG QUAN V K THUT IN DI MAO QUN ......................... 6

1.2.1. Khi nim ............................................................................................ 6

1.2.2. Nguyn l ca qu trnh in di mao qun............................................ 7

1.2.3. in di mao qun vng ........................................................................ 9

1.3. VI NT V PHN TCH NG PHN QUANG HC .................... 10

1.3.1. ng phn quang hc ......................................................................... 10

1.3.2. Phng php phn tch ng phn i quang ..................................... 10

1.3.3. ng dng in di mao qun trong phn tch ng phn quang hc ..... 12

1.4. TCH NG PHN I QUANG BNG CE S DNG

CYCLODEXTRIN L TC NHN CHN LC I QUANG ...................... 13

1.4.1. Tng quan v Cyclodextrin ................................................................ 13

1.4.2. Tch ng phn i quang s dng Cyclodextrin l tc nhn chn lc

i quang ....................................................................................................... 15

1.5. MT S NGHIN CU PHN TCH NG PHN I QUANG

CA ATENOLOL ............................................................................................ 16

1.5.1. Nghin cu trong nc ....................................................................... 16

1.5.2. Nghin cu trn th gii ..................................................................... 17

CHNG 2. I TNG V PHNG PHP NGHIN CU ....................... 19

2.1. NGUYN VT LIU, THIT B .......................................................... 19

2.1.1. i tng nghin cu ......................................................................... 19

2.1.2. Dung mi v ha cht ........................................................................ 19

2.1.3. Trang thit b ...................................................................................... 19

2.2. NI DUNG NGHIN CU .................................................................. 20

2.2.1. Hon thin phng php nh lng ng phn i quang ca Atenolol

bng in di mao qun ................................................................................... 20

2.2.2. Thm nh phng php nh lng ng phn i quang ca Atenolol

bng in di mao qun ................................................................................... 20

2.2.3. ng dng phng php ny nh lng ch phm c cha Atenolol

trn th trng ................................................................................................ 21

2.3. PHNG PHP NGHIN CU .......................................................... 21

2.3.1. Chun b cc dung dch chun, dung dch mu v cc dung dch lm

vic 21

2.3.2. Kho st v la chn iu kin in di ............................................... 22

2.3.3. Thm nh phng php phn tch ..................................................... 23

2.3.4. ng dng nh lng Atenolol trong cc ch phm trn th trng .... 23

2.3.5. Phng php x l s liu ................................................................... 23

CHNG 3. THC NGHIM, KT QU V BN LUN ............................... 25

3.1. KHO ST V LA CHN IU KIN IN DI ............................ 25

3.1.1. La chn ct mao qun ...................................................................... 25

3.1.2. Kho st nh hng ca in th p vo hai u mao qun. ............... 26

3.2. THM NH PHNG PHP PHN TCH ....................................... 28

3.2.1. ph hp h thng .......................................................................... 28

3.2.2. Khong nng tuyn tnh ................................................................ 29

3.2.3. c hiu ........................................................................................ 31

3.2.4. lp li ........................................................................................... 33

3.2.5. ng ............................................................................................. 34

3.3. NG DNG NH LNG ATENOLOL TRONG CH PHM. ....... 36

BN LUN .......................................................................................................... 38

KT LUN V KIN NGH ............................................................................... 41

TI LIU THAM KHO

PH LC

DANH MC CC K HIU, CH VIT TT

BGE (Background Electrolyte) Dung dch in ly nn

CE (Capillary Electrophoresis) in di mao qun

CGE (Capillary Gel Electrophoresis) in di mao qun gel

CIEF (capillary isoelectric focusing) in di mao qun hi t

ng in

CITP (capillary isotachophoresis) in di mao qun ng tc

CM--CD Carboxymethyl beta

cyclodextrin

CZE (Capillary Zone Electrophoresis) in di mao qun vng

DM- -CD Dimethyl beta cyclodextrin

EOF ( Electro-osmotic flow ) Dng in thm

GC (Gas Chromatography) Sc k kh

HPCE (High Performance Capillary Electrophoresis)

HPLC ( High Performance Liquid Chromatography) Sc k lng hiu nng cao

MEKC (Micellar Electrokinetic Chromatography) Sc k in ng mixen

Rs (Resolution) phn gii

RSD ( Relative Standard Deviation ) lch chun tng i

SD (Standard Deviation) lch chun

SK S ng k

SFC (Supercritical fluid chromatography) Sc k lng siu ti hn

SKS S kim sot

TLC (Thin layer chromatography) Sc k lp mng

Tris Tris(Hydroxymethy)

amimomethan

-CD Beta cyclodextrin

DANH MC CC BNG

Bng 1: Tnh cht ca mt s CD t nhin. ........................................................... 14

Bng 2: ph hp h thng ................................................................................ 28

Bng 3: Chun b dy dung dch chun Atenolol racemic ...................................... 29

Bng 4: Kt qu kho st khong tuyn tnh .......................................................... 30

Bng 5: Kt qu kho st lp li trn mu th M1 ............................................. 34

Bng 6: Kt qu xc nh phn trm tm li trn mu M1(thm 10%) .................... 35

Bng 7: Kt qu xc nh phn trm tm li trn mu M1(thm 20%) .................... 36

Bng 8: Kt qu nh lng mu M2 ..................................................................... 37

DANH MC HNH V V TH

Hnh 1: Cng thc cu to ca Atenolol .................................................................. 3

Hnh 2: S h thng in di mao qun. ............................................................... 7

Hnh 3: S hnh thnh dng EOF ............................................................................. 8

Hnh 4: Gi tr thc ca hiu in th .................................................................... 25

Hnh 5: Gi tr thc ca cng dng in: ........................................................ 26

Hnh 6: in di 3 hiu in th ...................................................................... 27

Hnh 7: th biu din s ph thuc din tch pic ca S-(-)-Atenolol theo nng

S-(-)-Atenolol ........................................................................................................ 30

Hnh 8: th biu din s ph thuc din tch pic ca R-(+)-Atenolol theo nng

R-(+)-Atenolol....................................................................................................... 31

Hnh 9: in di mu placebo ............................................................................ 32

Hnh 10: in i mu Atenolol racemic chun 100 ppm. .................................. 32

Hnh 11: in di mu th 100 ppm. .................................................................. 32

Hnh 12: in di mu M2 ................................................................................. 37

Hnh 13: V tr tng tc lin kt hydro[22]. .......................................................... 39

1

T VN

Hin nay, trong iu tr trn lm sng s dng ngy cng nhiu cc thuc

c cha hot cht l cc dng ng phn quang hc tinh khit, gip nng cao hiu

qu iu tr, gim liu dng v hn ch c nhiu tc dng khng mong mun so

vi khi dng thuc c cha hot cht dng hn hp racemic. V d nh

levofloxacin (ng phn t tuyn ca ofloxacin) c tc dng khng khun mnh gp

hng chc n hng trm ln dextrofloxacin (ng phn hu tuyn ca ofloxacin)

[4], hoc dexchlorpheniramin maleat (ng phn hu tuyn ca chlorpheniramin

maleat) l mt thuc khng Histamin H1 dng iu tr d ng c tc dng mnh gp

hai ln chlorpheniramin maleat (dng racemic) vi cng liu lng do dng ng

phn t tuyn khng c tc dng, do cho php gim liu dng ch cn mt na

[3].

Trong cuc sng hin i ngy nay, xu hng cc bnh v tim mch ngy

cng tng nhanh. i vi nhm thuc tim mch th thi gian iu tr thng ko

di, do vic s dng dng ng phn quang hc tinh khit li cng c ngha

quan trng. Atenolol l dn cht ca benzenacetamid, c tc dng chn chn lc

trn th th 1- adrenergic, dng iu tr tng huyt p, au tht ngc mn tnh n

nh, nhi mu c tim sm (trong vng 12 gi u) v d phng sau nhi mu c

tim, lon nhp nhanh trn tht [2]. Vi mt carbon bt i trong phn t, Atenolol l

hn hp racemic ca hai ng phn i quang: S-(-)-Atenolol v R-(+)-Atenolol.

Mt s nghin cu chng minh v ch ra rng S-(-)-Atenolol ng vai tr chnh

trong chn giao cm khi dng Atenolol dng hn hp racemic, ng thi c tnh

cng thp hn so vi dng R-(+)-Atenolol [20],[21]. Do vic sn xut v s dng

ng phn S-(-)-Atenolol ngy cng c quan tm nhiu hn. iu ny i hi

phi xy dng phng php phn tch v nh lng c tng dng n ng

phn trong hn hp racemic ca Atenolol.

Trong nhng nm gn y, k thut in di mao qun (Capillary

Electrophoresis - CE) l phng php ha l c cc nh phn tch la chn trong

lnh vc tch cc ng phn quang hc vi nhiu l do: hiu lc tch cao, lng

2

mu v ha cht s dng nh, thit b hon ton t ng, c ch tch n gin v d

dng trong pht trin phng php [16], [19], [22].

Ti Vit Nam, vic p dng k thut in di mao qun trong phn tch kim

nghim vn cn hn ch. nng cao nng lc kim tra cht lng thuc ca h

thng kim nghim cng nh hi nhp vi xu hng pht trin ca ngnh Dc trn

th gii, chng ti thc hin ti: Xy dng phng php nh lng cc ng

phn i quang ca Atenolol trong ch phm bng in di mao qun vi hai

mc tiu:

1. Thm nh phng php nh lng ng phn i quang ca Atenolol bng

in di mao qun.

2. ng dng phng php ny nh lng ch phm c cha Atenolol trn th

trng.

3

CHNG 1. TNG QUAN

1.1. TNG QUAN V ATENOLOL

Cng thc cu to

O

NH2

O NH

CH3

CH3

H

OH

R-(+)- Atenolol

NH2

OO NH CH3

CH3

OH

H

S-(-)-Atenolol

Hnh 1: Cng thc cu to ca Atenolol

Cng thc phn t: C14H22N2O3.

Khi lng phn t: 266,3.

Tn khoa hc:

2-[4-[(2RS)-2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenyl]acetamid.

1.1.1. Tnh cht l, ha

Bt kt tinh mu trng hoc gn nh trng, hi tan trong nc, tan trong

ethanol, methanol, kh tan trong methylen clorid.

im chy: 152oC n 155oC.

Gc quay cc (dung dch 0,10 g Atenolol trong 10,0 ml nc) t -0,10o n

+0,10o.

Cc i hp th: dung dch trong methanol c hai cc i hp th 275 nm

v 282 nm. T s hp th cc i o 275 nm v 282 nm t 1,15 n

1,20 [1], [3], [23].

4

1.1.2. Dc l v c ch tc dng

1.1.2.1. Tc dng v c ch tc dng

Atenolol c tc dng chng tng huyt p, l dn cht ca benzenacetamid,

thuc nhm chn chn lc trn th th 1. Thuc c tc dng lm gim lc co c v

gim tn s tim. Atenolol khng c tc dng n nh mng. Atenolol tan trong nc,

do t thm vo h thn kinh trung ng.

iu tr bng Atenolol s c ch tc dng ca catecholamin khi gng sc v

cng thng tm l, dn n lm gim tn s tim, gim cung lng tim v gim huyt

p. iu tr bng Atenolol khng lm tng hoc lm tng rt t sc cn ca mch

ngoi bin. Trong iu kin c stress vi tng gii phng adrenalin t tuyn thng

thn, Atenolol khng lm mt s co mch sinh l bnh thng. liu iu tr, tc

dng co c trn ph qun ca Atenolol km hn so vi nhng thuc chn th th

beta khng chn lc. Tnh cht ny cho php iu tr c nhng ngi c bnh hen

ph qun nh hoc bnh phi tc nghn khc. iu tr nh vy phi c kt hp

vi thuc ch vn th th 2 dng theo ng ht, di s gim st cht ch ca

thy thuc chuyn khoa v hen.

Atenolol t nh hng n gii phng insulin v chuyn ha carbohydrat.

Phn ng tim mch i vi h ng huyt (nh tim p nhanh) khng b nh

hng mt cch c ngha bi Atenolol. Bi vy, Atenolol c th dng c cho

ngi i tho ng. ngi tng huyt p, Atenolol lm gim mt cch c

ngha huyt p c t th ng ln t th nm. iu tr tng huyt p, nu cn,

c th kt hp Atenolol vi thuc chng tng huyt p khc, ch yu l thuc li

niu v/hoc thuc gin mch ngoi bin [2].

Mt nghin cu cho thy liu 50 mg n ng phn S-(-)-Atenolol lm gim

huyt p tm thu, huyt p tm trng, nhp tim cng mc vi s dng liu 100

mg Atenolol racemic. Trong khi liu 50 mg n ng phn R-(+)-Atenolol

khng lm thay i bt k thng s no. Nh vy ng phn S-(-)-Atenolol l ng

phn c hot tnh chn giao cm v ng phn R-(+)-Atenolol l ng phn

khng c hot tnh [20].

5

ng phn R-(+)-Atenolol c th lm tng hoc thm ch gy ra nhng tc

dng ph nghim trng. l l do nn thay th Atenolol racemic bng n ng

phn S-(-)-Atenolol vi liu ch bng mt na liu Atenolol racemic ang c s

dng gim tc dng ph [21].

1.1.2.2. Dc ng hc

Nng ti a trong huyt tng ca thuc t c trong vng t 2-4 gi

sau khi ung. Sinh kh dng ca Atenolol xp x 45%, nhng c s khc nhau ti 3-

4 ln gia cc ngi bnh. Th tch phn b l 0,7 lt/kg.

Atenolol ch c chuyn ha mt lng nh, di 10% ca liu dng c

bi tit l cht chuyn ha. Phn ln liu thuc dng c bi tit qua thn di

dng khng thay i. Thi gian bn thi ca thuc t 6 - 9 gi i vi ngi ln c

chc nng thn bnh thng. Thi gian bn thi ca thuc tng ln i vi ngi c

chc nng thn gim v khng b nh hng bi bnh gan. Nng thuc trong

mu thng tng theo tui. Nu Atenolol c dng cng vi thc n th sinh kh

dng ca thuc gim t nht l 20%. Tuy nhin iu ny khng c ngha lm sng

[2].

1.1.2.3. Ch nh

Tng huyt p, au tht ngc mn tnh n nh, nhi mu c tim sm (trong

vng 12 gi u) v d phng sau nhi mu c tim, lon nhp nhanh trn tht [2].

1.1.2.4. Chng ch nh

Sc tim, suy tim khng b tr, blc nh - tht II v III, chm nhp tim

c biu hin lm sng.

Khng c dng kt hp vi verapamil [2].

1.1.2.5. Dng thuc v hm lng

Vin nn 25, 50 v 100 mg.

Thuc tim tnh mch 5 mg/10 ml.

Mt s ch phm cha atenolol dng racemic trn th trng nh vin nn

Tenormin 50mg v 100 mg, thuc tim Tenormin 5 mg (Astra-Zeneca), Atenolol

6

Stada 50 mg (Cng ty TNHH Lin doanh Stada Vit Nam), Tenocar 50 mg, Teginol

50 mg (Cng ty c phn Dc Hu Giang).

1.2. TNG QUAN V K THUT IN DI MAO QUN

1.2.1. Khi nim

S pht trin ca in di bt u trong khong t nm 1930 n nm1937 khi

Tiselius c gng tch protein huyt thanh gm , albumin v globulin. l

ln u tin in di c s dng cho vic tch cc hp cht c hot tnh sinh hc.

Tuy nhin, s phn tch khng hon chnh do lng mu ln v s dng hiu in

th thp. Nm 1967, Hjerten thc hin in di protein, acid nucleic v cc ion v c

bng mao qun c ng knh trong 300 m, pht hin bng UV. Vo u nhng

nm 1980, Jorgenson v Lukacs cng b pht minh v mt h thng CE n gin v

thu ht s ch ca rt nhiu cc nh nghin cu. Nm 1989, Karger t chc

Hi ngh quc t u tin v in di mao qun hiu nng cao v trong cng nm ,

h thng CE thng mi u tin c sn. K t , s cng b v CE tng ln

nhanh chng do tnh linh hot, n gin v hiu qu cao ca n [17].

in di l hin tng di chuyn ca tiu phn tch in ha tan hay phn tn

trong cht in gii khi c dng in i qua. Cation di chuyn v pha cc m

(catod), anion di chuyn v pha cc dng (anod). Cc phn t khng tch in

khng b ht v pha hai in cc. in di mao qun (CE) l mt k thut tch cc

cht trong mao qun silica di 25 - 100 cm, ng knh trong 25 - 100 m, ng

knh ngoi 300 - 400 m. in th mt chiu p vo hai u mao qun 10 - 30 kV

(cng in trng c th n 500 V/cm) to ra qu trnh chia tch, cc cht

phn tch c pht hin khi di chuyn v mt u mao qun nh mt detector thch

hp [1], [6].

7

Hnh 2: S h thng in di mao qun.

1.2.2. Nguyn l ca qu trnh in di mao qun

Qu trnh vn hnh ca CE in hnh vi mao qun silica cha dung dch

m lm vic, th nhm silanol (SiOH) trn thnh trong ca mao qun s gii

phng ion hydrogen (H+) vo dung dch m v b mt thnh mao qun s tch in

m ngay c pH rt thp. Cation hay cc cht ha tan tch in dng mt phn

trong mi trng b ht tnh in vo thnh mao qun tch in m to nn 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 t th trn chiu di ct 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 c khi

dung dch trong mao qun i v catod. S chuyn ng ca khi dung dch trong

mao qun silica di tc dng ca lc in trng c gi l dng in thm

(electroosmotic flow - EOF).

8

Hnh 3: S hnh thnh dng EOF

Mc ion ha ca nhm silanol trn thnh mao qun ph thuc ch yu

vo pH ca dung dch m lm vic, do tr s EOF thay i theo pH. pH thp,

nhm silanol ni chung c ion ha thp v dng EOF nh. pH cao hn, nhm

silanol b ion ha nhiu hn v dng EOF tng.

Ngoi pH dung dch m, dng EOF s thay i khi:

Lc ion ca dung dch tng ln, lm gim th Zeta nn dng EOF gim.

Trong mt s trng hp, cc dung mi hu c nh methanol hay

acetonitril c cho thm vo dung dch m lm tng tan ca cc

cht ha tan, hng s in mi ca dung dch in di gim xung ko

theo s gim ca EOF.

Detector c t v pha u catod ca mao qun. Dng EOF thng ln

hn linh in di. Do vy, ngay c anion cng b y v pha catod v detector.

Khi s dng m phosphat pH 7,0 mao qun khng c lp bao, thng thng

trnh t xut hin cc cht tan trong in di l cc cation, cc cht trung tnh v

cc anion.

Hin nay, c 5 loi in di mao qun ch yu:

in di mao qun vng (capillary zone electrophoresis - CZE) cn gi l

in di dung dch t do hay in di mao qun dng t do.

Sc k mixen in ng, cn gi l sc k in ng mixen (micellar

electrokinetic chromatography - MEKC).

in di mao qun gel (capillary gel electrophoresis - CGE).

9

in di mao qun hi t ng in (capillary isoelectric focusing - CIEF)

in di mao qun ng tc (capillary isotachophoresis - CITP) [1], [6].

1.2.3. in di mao qun vng

Trong in di mao qun vng, qu trnh tch c kim sot bng s khc

nhau v linh tng i ca tng thnh phn trong mu th hoc dung dch th.

Linh l hm s ca in tch cht phn tch v kch thc trong iu kin nht

nh ca phng php. Chng c ti u ha bng cch kim sot cc thnh phn

ca m, pH v lc ion.

in di mao qun vng s dng nguyn l ca in di v in thm tch

cc tiu phn tch in.

Linh in di ca ion (EP), c biu din bng phng trnh:

EP = q/(6r) (1)

Trong q l in tch ca ion, l nht dung dch v r l bn knh ca

ion hydrat ha.

T mi lin h ny suy ra, cht phn tch kch thc nh, in tch ln c

linh ln. Cht phn tch kch thc ln, in tch nh c linh thp.

Tc di chuyn (EP), tnh bng cm/s, c biu th bng phng trnh:

vEP =EP ( V / L ) (2)

Trong ep l linh in di, V l in th t v L l chiu di (cm) tng

cng ca mao qun.

Tc ca EOF (EO), tnh bng cm/s, c biu din bng phng trnh:

EO = EO (V / L) (3)

Trong EO l linh dng EOF.

Thi gian di chuyn (tm), tnh bng giy, l thi gian cn thit cho cht tan di

chuyn trn ton b chiu di hiu dng (l) ca mao qun (t u vo n detector),

tm c biu th bng h thc sau:

tm = l / E(EP + EO)= l L / V(EP+ EO) (4)

Trong E l cng in trng (E = V/L)

10

Hiu lc ca h thng in di c th lin quan n linh , dng EOF v

c biu th bng s a l thuyt (N), c tnh bng phng trnh:

N = (EP+ EO) E l /2D (5)

Trong D l h s khuch tn ca cht ha tan v cc k hiu khc c

nh ngha nh trn.

phn gii (Rs) ca hai cht ha tan c ra gii k tip nhau, c th xc

nh bng phng trnh:

= 0,18( )/ +

(6)

Trong EP1 v EP2 l linh ca hai cht ha tan, l linh trung

bnh ca hai cht tan v cc k hiu khc c nh ngha nh trn.

1.3. VI NT V PHN TCH NG PHN QUANG HC

1.3.1. ng phn quang hc

ng phn l nhng hp cht khc nhau c cng mt cng thc phn t.

ng phn quang hc l ng phn c kh nng lm quay mt phng nh sng phn

cc theo cc gc nhau.

Dng ng phn quang hc n gin nht xut hin trong trng hp khi

hp cht c th tn ti di hai dng ng phn lp th c cu to khng chng kht

ln nhau c. Hu ht cc tnh cht l hc thng thng ca hai ng phn i

quang l ging nhau, tr kh nng quay mt phng nh sng phn cc theo nhng

gc bng nhau nhng ngc chiu. Xc nh cu hnh tuyt i ca ng phn i

quang theo quy nh ca h thng ca Cahn-Ingold-Prelog. Nu ng phn quay

mt phng nh sng phn cc sang bn phi th n c gi l ng phn quay phi

R (ting La tinh: rectus (phi)). Nu ng phn quang hc quay mt phng nh

sng sang bn tri th n c gi l ng phn quay tri S (ting Latinh: siniter

(tri)).

Hn hp racemic c cha s phn t bng nhau ca mi dng i quang, do

quay cc tng cng bng khng [5], [9], [15].

1.3.2. Phng php phn tch ng phn i quang

1.3.2.1. Phng php phn tch ng phn i quang gin tip

11

Phng php ny da trn mt phn ng ha hc gia hai ng phn i

quang vi mt hp cht bt i to thnh hn hp hai ng phn lp th nhng

khng i quang trc khi c phn tch bng in di. c th hin nh s :

(R,S)-C + (S)-D [(R)-C-(S)-D] + [(S)-C-(S)-D]

Trong :

C: Hn hp racemic cn phn tch

D: Thuc th bt i

R-, S- tng ng l ng phn quay phi v ng phn quay tri.

Hn hp thu c c th c tch bng GC v HPLC. V nguyn tc,c th

dng CE phn tch hn hp trn, s dng dung dch in ly nn khng chn lc

i quang, bi cc thnh phn trong hn hp c tnh cht l ha khc nhau.Tuy

nhin phng php ny rt kh p dng trong CE. So vi phng php phn tch

trc tip th phng php gin tip ny c mt s nhc im sau y:

Tn thi gian x l mu.

Cn c cc nhm hot ng ha hc trong cu trc nh nit, hydroxyl,

carboxylic.

Phi s dng thuc th bt i rt tinh khit v s c thm mt cht i

quang s to ra thm hai ng phn lp th khng i quang na v gy

kh khn cho qu trnh phn tch.

Hai ng phn trong hn hp racemic cn phn tch phi phn ng vi

cng mc v tc .

p ng ca detector vi hai dn xut to thnh phi tng ng nhau.

iu kin phn ng phi thch hp trnh s chuyn dng lp th ca

thuc th bt i, ng phn quang hc ca cht phn tch v dn xut

ng phn lp th khng i quang.

Thng thng, khng phi d dng gii quyt tt c cc vn trong qu

trnh thc nghim nh m t trn. Tuy nhin, phng php gin tip c th c

p dng trong nhng trng hp m phng php trc tip khng th p dng c

hoc khi cn tng nhy. Phng php gin tip trong CE ch yu c p dng

12

cho phn tch ng phn quang hc ca acid amin. Tuy nhin cng c nhng

nghin cu p dng phng php ny vi dc cht [12].

1.3.2.2. Phng php phn tch ng phn i quang trc tip

Phng php tch trc tip c p dng thnh cng trong CE cho vic tch

ng phn i quang ca mt s nhm hp cht, ch yu l dc phm. Tc nhn

chn lc i quang hoc c th c thm vo dung dch in ly nn hoc c gn

ln thnh mao qun, hoc c c nh hay gn ln cc h gel to mt mi

trng chn lc i quang m tng tc vi hai ng phn i quang trong sut qu

trnh in di da trn s hnh thnh phc ng phn lp th khng i quang tm

thi. Cc phc tm thi di chuyn v pha detector vi vn tc khc nhau ch khi

chng c hng s bn khc nhau. Cc tng tc tng i yu tham gia vo qu

trnh hnh thnh ng phn lp th khng i quang gm lin kt hydro, tng tc

k nc, lin kt -, lng cc - lng cc. tinh khit quang hc ca tc nhn

chn lc i quang trong phng php tch trc tip khng phi l mt yu t quan

trng nh trong phng php tch gin tip; trn thc t, cc tp cht ch nh hng

n phn gii v c ch ra rng kt qu tng i tt c th thu c bng

cch s dng tc nhn chn lc i quang c cha ti 10% ng phn i quang

cn li. C ch phn tch ng phn i quang bao gm to phc lng, to mixen

v tng tc i lc. C ch to phc lng c p dng rng ri nhm nng cao

chn lc trong phn tch mt s hp cht bao gm: ng phn hnh hc, ng

phn lp th v ng phn i quang. n nay ba nhm chnh to phc lng

c s dng trong CE gm cc cyclodextrins (CDs), ether vng c

tetracarboxylat ha v khng sinh [9], [12].

1.3.3. ng dng in di mao qun trong phn tch ng phn quang hc

Hu ht cc thuc l hp cht i quang v mi ng phn th hin tc dng

dc l v c tnh ring bit. Do , phn tch ng phn i quang l cn thit

trong phn tch dc phm c c nhng ng phn an ton v c tc dng

mong mun. Cho n nay phng php phn tch c s dng cho vic tch ng

phn i quang bao gm sc k lng hiu nng cao (HPLC), sc k lp mng

13

(TLC), sc k kh (GC), sc k lng siu ti hn (SFC), v in di mao qun(CE).

ng dng ca sc k kh ch yu gii hn trong cc hp cht d bay hi. Phng

php c s dng ph bin trong phn tch cc thuc i quang l HPLC. CE l

mt k thut b sung cho HPLC, c bit i vi vic phn tch hp cht phn cc

v tch in [9]. u im ca CE gm: hiu lc tch cao, lng mu v ha cht s

dng nh, thit b hon ton t ng, c ch tch n gin v d dng trong pht

trin phng php [9], [19]. Vic tch ng phn i quang bng CE c th c

thc hin bng phng php gin tip hoc trc tip. Cc phng php trc tip

thun li hn cc phng php gin tip do s n gin v tnh sn c ca mt lot

cc tc nhn chn lc i quang nh cyclodextrins (CDs), ether vng c

tetracarboxylat ha v khng sinh to phc lng vi cc ng phn i quang.

Trong s , CD l tc nhn chn lc i quang c s dng ph bin nht. Ngoi

cc CD t nhin, cc dn xut CD khc nhau c thng mi ha v chng

mang li chn lc cao trong tch ng phn i quang. Bn cnh c ch to phc

lng, cc c ch khc dng tch ng phn i quang gm trao i phi t,

tng tc i lc (v d: vic s dng protein, khng sinh l tc nhn chn lc i

quang), v tng tc mixen (v d: s dng cc b mt t nhin hay tng hp l tc

nhn chn lc i quang)[17].

CE hin ang l mtphng php c thit lp phn tch dc phm v

c khuyn khch mt s Dc in. V d, Dc in Anh (BP) xut vic

xc nh erythropoietin v levocabastin hydrochlorid bi MEKC, trong khi Dc

in M (USP) c mt chng chung v CE t nm 2002 [19].

1.4. TCH NG PHN I QUANG BNG CE S DNG

CYCLODEXTRIN L TC NHN CHN LC I QUANG

1.4.1. Tng quan v Cyclodextrin

Cyclodextrin (CD) l oligosaccharid mch vng, gm cc n v glucose lin

kt vi nhau bng lin kt -(1,4)-glucosid v c to ra t phn ng thy phn

tinh bt bng enzym. Mc d trong thc t, CD hnh thnh t 6 ln n 12 n v

14

glucose c phn lp, tuy nhin ch CD c 6,7,8 n v glucose (tng ng vi

, , -CD) l hin ang c s dng trong ha phn tch.

Hnh dng ca CD tng t nh mt hnh nn ct rng vi mt khoang

tng i k nc, c th lu gi cht phn tch v mt ngoi thn nc do s c

mt ca cc nhm hydroxyl (v tr 2, 3 v 6 ca glucopyranose). Vnh rng hn c

cha cc nhm hydroxyl th cp i quang, trong khi vnh hp hn cha nhm

hydroxyl s cp khng i quang. Tnh cht vt l ca cc CD t nhin kh khc

nhau, v d nh v chiu rng ca khoang, tan, trng lng phn t, tuy nhin

chng c cng su (Bng 1).

Bng 1: Tnh cht ca mt s CD t nhin.

Tnh cht Loi CD

S n v glucose 6 7 8

Trng lng phn t 972 1135 1297

ng knh trong (nm) 0,57 0,78 0,95

su (nm) 0,78 0,78 0,78

tan (g/100ml H2O) 14,5 1,85 23,2

pKa 12,33 12,20 12,08

Cc nhm hydroxyl c mt trn vnh ca CD c th d dng c thay th

bi cc phn ng ha hc c c cc dn xut CD vi cc mc th khc

nhau, cc thnh phn CD bin i ph thuc vo mt s thng s nh iu kin

phn ng, loi v t l thuc thMt s lng ln cc dn xut ca CD hin ang

c s dng trong CE phn tch ng phn i quang nh dn xut methyl,

hydroxyethyl, hydroxypropyl, acetyl ha khng tch in v cc dn xut

methylamino, sulphobutylether, carboxymethyl, sulfat, photphat ha tch in. Cc

dn xut ca CD c th biu hin tnh cht rt khc so vi nhng CD t nhin, c

th c s dng d dng ci thin chn lc trong tch ng phn i quang,

v d nh tng tan, tng kh nng to ra cc lin kt th cp khc nhau, tim

15

nng trong phn tch nhng hp cht khng tch in, mc k nc khc nhau

ca hc k nc. V d, so snh - CD v DM- - CD, c th thy rng s c mt

ca cc nhm methoxy lm tng hoc su hoc tan ca tc nhn chn lc i

quang. Nhm th tch in hoc khng tch in thay th cho nhm hydroxyl trn

cu trc CD c th rt c ch cho ti u phng php CE. Trong thc t, ng phn

khng tch in c th c chuyn n detector nh cht phn tch tch in do s

hnh thnh phc lng vi dn xut ca CD c nhm th tch in. Hn na s

chuyn ng ca tc nhn chn lc i quang theo hng ngc li vi cht phn

tch l iu kin l tng t c phn gii tt v lm tng s khc bit v

linh gia cht phn tch t do v cht phn tch trong phc lng [9], [10], [13].

1.4.2. Tch ng phn i quang s dng Cyclodextrin l tc nhn chn lc

i quang

Nhiu dn xut CD c pht trin tng kh nng ha tan trong nc

v thay i hnh dng khoang. Tch ng phn i quang bng CZE vi CD l

tc nhn chn lc i quang ln u tin c gii thiu bi Fanali v l k thut

thnh cng nht trong s cc k thut tch ng phn i quang ca CE; s

lng cc bi bo m t k thut ny gia tng ng k [19].

Trong c ch to phc lng, hp cht lng kht vo khoang CD vi ton b

phn t hoc vi phn k nc ca phn t; do , loi CD c vai tr rt quan trng

trong qu trnh tch. Tng tc k nc vi mnh khoang k nc khng cho

php tch cc ng phn i quang, lin kt yu gia cc nhm nhm th vi trung

tm bt i ca cht phn tch v cc nhm hydroxyl th cp v / hoc s cp ca

CD ng vai tr trong nhn dng i quang [9], [12], [19].

CD l mt hp cht trung ha v in v do chng di chuyn vi tc

ca dng EOF trong CZE. Khi mt cht tan tch in c lng vo khoang ca

CD, phc lng to thnh c in tch ging vi cht tan t do nhng khi lng

phn t tng ln, v do linh in di thp hn so vi cht tan t do. Trong tch

ng phn i quang, cc ng phn c linh in di ng nht v cc ng phn

trong phc lng c th c linh tng t nhau. V vy, nguyn tc ca CZE s

16

dng CD l tc nhn chn lc i quang (CD - CZE) tch ng phn i quang

l s khc bit gia cc hng s bn ca phc lng to ra gia cc ng phn i

quang vi CD. ng phn to phc lng bn hn th c linh hiu dng thp hn.

Linh hiu dng l linh trung bnh hoc biu kin ca cht tan trong iu kin

th nghim, nhng trong hu ht cc trng hp, chng ta s dng n gin l linh

[19].

Phn tch ng phn i quang c th b nh hng bi cc yu t nh: loi

v nng CD, pH, thnh phn ca BGE, nhit mao qun, hiu in th[13]

1.5. MT S NGHIN CU PHN TCH NG PHN I QUANG

CA ATENOLOL

1.5.1. Nghin cu trong nc

Nhm tc gi T Mnh Hng, Trnh Vn Lu, Nguyn Th Thu Hin,

Nguyn Th Kiu Anh [8], tch v nh lng ng phn i quang ca

Atenolol bng phng php sc k lng hiu nng cao HPLC, s dng:

Ct: Chirex 3022 ((S)-indoline-2-carboxylic acid v (R)-1-(-

naphtyl)ethylamin) (4,0 x 250 mm; 5 m).

Pha ng: n-hexan : 1,2 dicloromethan : methanol : acid trifluoroacetic =

57,5 : 35 : 7,5 : 0,2 (tt/tt/tt/tt).

Bc sng pht hin : 229 nm.

Th tch tim mu: 20 L.

Nng cht phn tch: 0,05 mg/ml (racemic)

Tc dng: 1 ml/ pht.

Nhm tc gi T Mnh Hng, Trnh Vn Lu, inh Th Thanh, V Ngn

Bnh, Nguyn Th Kiu Anh (2013) [7], kho st v la chn a ra

iu kin in di bng CE nh sau:

Ct mao qun silica nung chy: chiu di tng 58,5 cm (chiu di hiu

dng 50 cm), ng knh trong 75 m.

Dung dch in ly nn: Tris 50 mM, CM--CD 8 mM, pH 4,0.

Nhit : 200C.

17

Hiu in th: 16 kV

Ch tim mu: 50 mbar x 3s.

Bc sng pht hin: 194 nm

Nng cht phn tch: 100 ppm (kl/tt) Atenolol racemic.

Nghin cu ny mi s b tch, nh tnh cc ng phn. Chng ti s tip

tc nghin cu pht trin, hon thin phng php ng dng vo nh lng cc

ng phn quang hc ca Atenolol.

1.5.2. Nghin cu trn th gii

Mt s nghin cu nh lng ng phn quang hc ca Atenolol bng

HPLC nh sau:

Nghin cu ca Santoro M.I, Cho H.S (2000) [18], s dng iu kin sc k

nh sau:

Ct: Chiralcel OD (250 x 4,6 mm; 10 m).

Pha ng: Hexan - ethanol - diethylamine = 75: 25: 0,1 (tt/tt/tt).

Tc dng: 0,7 ml/ pht.

Bc sng pht hin: 276 nm.

Nghin cu ca Eaga M.C (2010) [11], s dng iu kin sc k nh sau:

Ct: Chiralcel AGP (150 x 4,0 mm; 5 m).

Pha ng: Dung dch m Natri phosphat 10 mM (pH 7,0) - methanol

= 95: 5 (tt/tt).

Tc dng: 0,9 ml/ pht.

Bc sng pht hin: 225 nm.

Khong nng tuyn tnh: 10-100 g/ml.

CE cng c ng dng trong phn tch ng phn i quang ca Atenolol.

Nghin cu ca Wuhong Li, Changhai Liu, Guangguo Tan, Xinrong Zhang,

Zhenyu Zhu, Yifeng Chai [22] sau khi kho sat v la chn, a ra iu kin

in di nh sau:

Ct mao qun silica nung chy, chiu di tng cng 48,5 cm, chiu di

hiu dng 40 cm, ng knh trong 50 m.

18

Tim mu: 50 mbar 3s.

Hiu in th: 24 kV.

Bc sng pht hin : 214 nm.

Nhit mao qun: 20oC.

Dung dch in ly nn (BGE): Tris 50mM v CM--CD 8 mM, c iu

chnh bng acid phosphoric ti pH 4,0.

Dung dch chun gc: ha tan trong dung dch m Tris 50 mM nng

10 mM. Dung dch phn tch c chun b bng cch pha long dung

dch chun gc vi m Tris ti nng 0,2 mM. Tt c cc dung dch

c lc qua mng lc 0,22 m.

19

CHNG 2. I TNG V PHNG PHP NGHIN CU

2.1. NGUYN VT LIU, THIT B

2.1.1. i tng nghin cu

Ch phm vin nn c cha Atenolol 50 mg (Atenolol dng hn hp

racemic):

Vin nn Atenolol STADA 50 mg, SK: VD-12619-10, s l:040913,

hn dng:10/09/2018, nh sn xut: cng ty TNHH lin doanh STADA-

VN, cha 50mg Atenolol.(Mu M1)

Vin nn Tenormin 50 mg, SK: VN-1585-06, s l: JA462, hn dng:

06/06/2016, nh sn xut: Astra Zeneca, Anh, cha 50mg Atenolol.(Mu

M2)

2.1.2. Dung mi v ha cht

2.1.2.1. Cht chun

Cht chun quc gia ca Vin Kim nghim thuc trung ng B Y t:

Atenolol 200mg/l; 100,17% C14H22N2O3 (khan); m: 0,11%; SKS: 0102093.

2.1.2.2. Cc ha cht khc

Ha cht, thuc th t tiu chun tinh khit phn tch ca Merck, c.

Natri hydroxyd

Tris(hydroxyl- methyl)amino methan (Tris)

Acid phosphoric m c.

Methanol.

Acid hydrocloric 0,1M

Mui Natri Carboxymethyl--cyclodextrin (CM--CD): t tiu chun phn

tch ca Sigma-Aldrich, M.

Nc ct 2 ln c kh ion.

Mu placebo.

2.1.3. Trang thit b

My in di mao qun Agilent, model: G1600AX, s seri: DE1603499, ca

Agilent, M.

20

My o pH (744 pH meter), ca Metrohm, Thy S.

Cn phn tch Mettler Toledo AL2004, seri: 1228350002, d= 0,0001g, sn

xut ti Thy S.

My siu m Ultrasonic LC 30, hng cung cp: Elma.

My ct nc hai ln Hamilton, Anh.

H thng lc nc siu sch, c.

Mng lc cellulose acetat vi kch thc l lc 0,2 m ca Sartorius, c.

B l nha ng mu v np y.

Cc dng c chnh xc: cc pipet chnh xc vi th tch khc nhau, bnh nh

mc vi th tch khc nhau.

Cc dng c khc: cc c m, giy lc, phu thy tinh

2.2. NI DUNG NGHIN CU

2.2.1. Hon thin phng php nh lng ng phn i quang ca Atenolol

bng in di mao qun

La chn ct mao qun.

La chn hiu in th.

2.2.2. Thm nh phng php nh lng ng phn i quang ca Atenolol

bng in di mao qun

ph hp h thng.

Khong nng tuyn tnh.

c hiu.

lp li.

ng.

21

2.2.3. ng dng phng php ny nh lng ch phm c cha Atenolol trn

th trng

2.3. PHNG PHP NGHIN CU

2.3.1. Chun b cc dung dch chun, dung dch mu v cc dung dch lm

vic

Dung dch m Tris 50mM: cn khong 1,5 g Tris vo cc c m, ha tan

bng nc, chuyn vo bnh nh mc 250 ml, b sung nc ti vch, lc

u.

Dung dch in ly nn Tris 50 mM cha CM--CD 8mM, pH 4,0: pha dung

dch m Tris 50 mM, iu chnh bng acid phosphoric c ti pH 4,0. Cn

chnh xc khong 0,0616 g CM--CD cho vo bnh nh mc 5 ml, b sung

m Tris 50 mM, pH 4,0 ti vch, lc u.

Dung dch NaOH 1 M: cn khong 4 g NaOH cho vo cc c m 100ml, ha

tan bng nc, thm nc ti vch.

Dung dch NaOH 0,1 M: cn khong 0,4 g NaOH cho vo cc c m 100 ml,

ha tan bng nc, thm nc ti vch.

Dung dch chun gc Atenolol 1000 ppm: cn chnh xc khong 25 mg

Atenolol khan vo bnh nh mc 25 ml, thm khong 20 ml dung dch m

Tris 50 mM, siu m 15 pht, sau b sung m va . Bo qun 4oC,

trnh nh sng. Cc dung dich phn tch c chun b bng cch pha long

dung dch chun gc bng dung dch m Tris 50 mM n nng yu cu.

Vi mi ch phm vin nn cha Atenolol 50mg: cn chnh xc 20 vin tnh

khi lng trung bnh vin, nghin mn. Cn chnh xc lng bt vin tng

ng vi 25 mg Atenolol cho vo bnh nh mc 25 ml, cho khong 20 ml

m Tris, siu m 15 pht, sau b sung m va , lc thu ly dch lc.

Ht 500 l dch lc cho vo bnh nh mc 5 ml, b sung m Tris 50 mM

n vch, lc u.

Dung dch placebo: cn khong 0,1 g placebo cho vo bnh nh mc 25 ml,

cho khong 20 ml m Tris, siu m 15 pht, sau b sung m va ,

22

lc thu ly dch lc. Ht 500 l dch lc cho vo bnh nh mc 5 ml, b

sung m Tris 50 mM n vch, lc u.

Tt c cc dung dch u c lc qua mng lc 0,2 m trc khi in di.

2.3.2. Kho st v la chn iu kin in di

T Mnh Hng [7] kho st cc yu t nh hng ti qu trnh in di

gm: thnh phn dung dch in di, loi tc nhn chn lc i quang, pH dung dch

m, hiu in th v nng Atenolol trong mu phn tch. Da vo kt qu ca

ti ny, chng ti a ra iu kin in di nh sau:

Phng php c tin hnh trn h thng in di mao qun vi detector

mng diod.

Nhit mao qun 250C, tim mu 50mbar x 3s, bc sng pht hin 194

nm.

Nng Atenolol racemic trong mu phn tch khong 100ppm (tng ng

vi khong 50ppm mi n ng phn).

Dung dch in ly nn: m Tris 50mM, pH 4,0 cha CM--CD 8mM.

Trc khi s dng ln u tin, ct mao qun mi c ra sch vi

methanol trong 15 pht, tip theo l dung dch acid hydrocloric 0,1 M trong

15 pht, v dung dch NaOH 1M trong 30 pht.

Hng ngy, vo thi im u ngy cc mao qun c ra sch bng dung

dch NaOH 0,1 M 15 pht, nc 5 pht, v vi dung dch in ly nn (BGE)

15 pht.

Gia mi ln chy, chng trnh luyn ct (preconditioning) c thc hin

vi nc trong 5 pht, dung dch NaOH 0,1 M trong 4 pht, nc 2 pht, v

BGE 4 pht.

Thi im cui ngy, mao qun c ra vi dung dch NaOH 0,1 M trong

30 pht, nc 30 pht, sau thi kh 2 pht.

2.3.2.1. La chn ct mao qun

23

Chng ti tin hnh in di dung dch chun Atenolol racemic 100 ppm vi

iu kin in di trn, s dng cc ct mao qun khc nhau vi chiu di hiu dng

v ng knh trong khc nhau.

2.3.2.2. La chn hiu in th p vo hai u mao qun

V trong ti ny sau khi la chn c ct mao qun chng ti phi tin

hnh kho st li hiu in th p vo hai u mao qun.

Thay i hiu in th p vo hai u mao qun t 15 n 25 kV (E= 5 kV)

v tin hnh phn tch mu, la chn hiu in th ph hp.

2.3.3. Thm nh phng php phn tch

Sau khi kho st, la chn c ct mao qun, hiu in th p vo hai u

mao qun, tin hnh thm nh m bo phng php xy dng c l ph hp.

Cc tiu ch cn thm nh bao gm: ph hp ca h thng, c hiu, khong

nng tuyn tnh, lp li, ng ca phng php.

2.3.4. ng dng nh lng Atenolol trong cc ch phm trn th trng

Tin hnh in di cc mu chun Atenolol racemic v mu th. Da vo din

tch pic ca mu th v mu chun, hm lng Atenolol racemic ca mu chun

xc nh hm lng tng ng phn Atenolol trong mu th.

Hm lng Atenolol, C14H22N2O3, phi t c t 90,0 n 110,0% so vi

lng ghi trn nhn.

2.3.5. Phng php x l s liu

Tnh ton, x l v thng k s liu trn phn mm Microsoft Excel 2007.

2.3.5.1. Cng thc tnh ton cc gi tr thng k

Gi tr trung bnh:

X =1

nX

Phng sai:

S = X X

n 1

lch chun:

24

S = X X

n 1

lch chun tng i:

RSD(%) =S

X 100%

Trong :

n: s php th.

Xi: gi tr ca php th th i.

2.3.5.2. Cng thc tnh kt qu

Hm lng ca mi ng phn trong mt vin (mg/vin):

HL =1

2Sth mchun Cchun (1 %H2O)

Schun mthm

Khi lng thu hi ca mi ng phn (mg):

m =1

2Sth mchun Cchun (1 %H2O)

Smth

HL

m

T l phn trm tm li ca mi ng phn (%):

% =

100%

Trong :

Sth: din tch pic ca mi ng phn trong mu th

Schun: din tch pic ca mi ng phn trong mu chun

mchun: khi lng Atenolol racemic chun

mth: khi lng bt vin.

mth: khi lng thu hi ca mi ng phn.

mthm: khi lng chun thm ca mi ng phn.

: Hm lng trung bnh ca mi ng phn trong mt vin.

Cchun: hm lng Atenolol racemic khan trong cht chun (100,17%)

%H2O: hm m ca cht chun (0,11%).

25

CHNG 3. THC NGHIM, KT QU V BN LUN

3.1. KHO ST V LA CHN IU KIN IN DI

3.1.1. La chn ct mao qun

Tin hnh in di dung dch Atenolol racemic chun 100 ppm vi iu kin

in di la chn, s dng cc ct mao qun sau:

Chiu di hiu dng

(cm)

ng knh trong

(m)

Mao qun 1 40,5 75

Mao qun 2 56 50

Gi tr hiu in th v cng dng thc ca my in di mao qun khi

tin hnh in di th 25 kV c th hin hnh 4 v hnh 5.

(a)

(b)

Hnh 4: Gi tr thc ca hiu in th

(a) Mao qun 1; (b) Mao qun 2.

min5 10 15 20

kV

2

4

6

8

10

12

HPCE1 V, Voltage (ATENOLOL\130518000055.D)

min2 4 6 8 10 12

kV

0

5

10

15

20

25

HPCE1 V, Voltage (ATENOLOL\130518000063.D)

26

(a)

(b)

Hnh 5: Gi tr thc ca cng dng in:

(a) Mao qun 1; (b) Mao qun 2.

Nhn xt:

Tn hiu ca cng dng in c hai mao qun tng i n nh xung

quanh gi tr 50 A.

Gi tr thc ca hiu in th mao qun 1 dao ng ln m khng t c

gi tr t 25 kV. Cn mao qun 2 th n nh, t c gi tr th t 25 kV.

Nh vy, chng ti la chn s dng mao qun 2: chiu di hiu dng 56

cm, ng knh trong 50 m.

3.1.2. Kho st nh hng ca in th p vo hai u mao qun.

min5 10 15 20

uA

10

20

30

40

50

HPCE1 C, Current (ATENOLOL\130518000055.D)

min2 4 6 8 10 12

uA

0

10

20

30

40

HPCE1 C, Current (ATENOLOL\130518000063.D)

27

Chng ti tin hnh kho st cc hiu in th 15kV, 20kV, 25kV vi iu

kin in di la chn, s dng mao qun 2, thu c cc in di sau (Hnh 6).

(a)

(b)

(c)

Hnh 6: in di 3 hiu in th

(a) 15kV; (b) 20 kV; (c) 25 kV

min5 10 15 20 25 30 35

mAU

-10

0

10

20

30

DAD1 D, Sig=194,2 Ref=off (ATENOLOL\130518000132.D)

Are

a: 1

83.8

22

33.

854

Are

a: 1

87.9

35

35.

958

28

Nhn xt:

Vi c ba gi tr hiu in th th cc ng phn u c tch ra tt.

hiu in th 15kV thi gian di chuyn trn 40 pht, hiu in th 20 kV

thi gian di chuyn gn 40 pht dn n ko di thi gian phn tch.

Trong khi hiu in th 25 kV thi gian di chuyn khong 33 pht, thi

gian phn tch ngn hn.

Do chng ti la chn hiu in th 25 kV thc hin ti ny.

3.2. THM NH PHNG PHP PHN TCH

3.2.1. ph hp h thng

nh gi ph hp h thng l php th nhm nh gi n nh ca

ton h thng phn tch bao gm bi cc yu t nh: my mc, thit b, cch tin

hnh phn tch, mu th

Tin hnh tim lp li 6 ln cng mt dung dch chun atenolol racemic

100ppm trong cng iu kin in di.

Kt qu ph hp h thng c th hin trong bng 2:

Bng 2: ph hp h thng

STT

S-(-)-Atenolol R-(+)-Atenolol

tm (pht)

Din tch pic (mAU.s)

tm (pht)

Din tch pic (mAU.s)

1 33,146 184,3 35,357 193,7

2 33,854 183,8 35,958 187,9

3 32,763 183,0 35,167 189,5

4 33,214 192,7 35,618 194,2

5 31,706 185,8 33,542 190,0

6 31,638 187,5 33,710 195,7

Trung bnh 32,720 186,2 34,892 191,8

RSD (%) 2,70 1,92 2,92 1,62

Nhn xt:

29

RSD i vi thi gian di chuyn ca hai ng phn S-(-)-Atenolol v R-(+)-

Atenolol ln lt l 2,70 % v 2,92 %, u ln hn 2%.

RSD i vi din tch pic ca hai ng phn S-(-)-Atenolol v R-(+)-

Atenolol ln lt l 1,92% v 1,62%, u nh hn 2% nn phng php t yu cu

v ph hp h thng i vi din tch pic.

3.2.2. Khong nng tuyn tnh

m bo php nh lng cho kt qu chnh xc, cn xc nh khong

nng m c s tng quan tuyn tnh gia p ng phn tch (din tch pic)

vi nng cht phn tch.

Tin hnh in di dy dung dch chun Atenolol racemic c nng t 50

ppm n 150 ppm. Dy dung dch chun Atenolol c chun b bng cch pha

long dung dch chun gc Atenolol 1000 ppm bng dung dch m Tris 50mM nh

bng 3:

Bng 3: Chun b dy dung dch chun Atenolol racemic

STT Nng

(ppm)

H s hiuchnh

(k)

Chun gc

(l)

m Tris

(ml)

1 50

0,972

250

Va 5ml

2 75 375

3 100 500

4 125 625

5 150 750

Tin hnh phn tch bng chng trnh in di xy dng, kt qu c

trnh by trong bng 4.

30

Bng 4: Kt qu kho st khong tuyn tnh

STT Nng n ng phn

(ppm)

Din tch pic (mAU.s)

S-(-)-Atenolol R-(+)-Atenolol

1 25,0 107,5 106,9

2 37,5 147,5 146,9

3 50,0 182,0 184,5

4 62,5 216,5 216,4

5 75,0 252,4 254,0

Kt qu s ph thuc ca din tch pic ca S-(-) Atenolol v R-(+) Atenolol

vo nng ca mi ng phn Atenolol c th hin trong hai hnh 7 v 8 di

y:

Hnh 7: th biu din s ph thuc din tch pic ca S-(-)-Atenolol theo

nng S-(-)-Atenolol

y = 2,870x + 37,66r = 0,999

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80

Sp

ic(m

AU

.s)

C (ppm)

31

Hnh 8: th biu din s ph thuc din tch pic ca R-(+)-Atenolol theo

nng R-(+)-Atenolol

Nhn xt:

Din tch pic ca S-(-)-Atenolol tng quan vi nng S-(-)-Atenolol theo

phng trnh y = 2,870x + 37,66 vi h s tng quan r = 0,9995. Do trong

khong nng 25-75 ppm, din tch pic ca S-(-)-Atenolol c quan h tuyn tnh

vi nng S-(-)-Atenolol.

Din tch pic ca R-(+)-Atenolol tng quan vi nng Atenolol racemic

theo phng trnh y = 2,909x + 36,26 vi h s r = 0,9990. Do trong khong

nng 25-75 ppm, din tch pic ca R-(+)-Atenolol c quan h tuyn tnh vi

nng S-(-)-Atenolol.

3.2.3. c hiu

dc hiu ca phng php phn tch l kh nng phng php c th

xc nh c mt cch chnh xc, c hiu cht cn phn tch, khng b nh hng

bi s c mt ca cc cht khc trong mu th.

Tin hnh in di ln lt dung dch Atenolol racemic chun, dung dch th,

dung dch placebo trong cng mt iu kin.

Kt qu thu c th hin hnh 9,10,11:

y = 2,909x + 36,26r = 0,998

0

50

100

150

200

250

300

0 10 20 30 40 50 60 70 80

Sp

ic(m

AU

.s)

C (ppm)

32

Hnh 9: in di mu placebo

Hnh 10: in i mu Atenolol racemic chun 100 ppm.

Hnh 11: in di mu th 100 ppm.

Nhn xt:

min5 10 15 20 25 30 35

mAU

-10

0

10

20

30

40

Are

a: 1

84.3

45

33.

146

Are

a: 1

93.7

26

35.

357

33

Thi gian di chuyn ca pic trn in di ca mu th tng ng vi thi

gian di chuyn ca pic trn in di mu chun.

Trn in di ca mu placebo khng xut hin pic thi im xut hin

pic c mu chun v mu th.

Nh vy phng php c c hiu cao.

3.2.4. lp li

lp li ca mt phng php phn tch l mc thng nht gia cc kt

qu ring bit khi quy trnh phn tch c tin hnh lp i lp li nhiu ln trn

cng mt mu ng nht, trong cng mt ngy. lp li c biu th bng

lch chun tng i (RSD).

lp li ca phng php c xc nh trn 6 php th song song trn

cng mt mu vi cng mt iu kin in di, trong cng mt ngy. Kho st lp

li trn mu M1.

Chun b dung dch chun: cn chnh xc khong 25 mg Atenolol

racemic chun vo bnh nh mc 25 ml, thm 20 ml m Tris 50 mM,

siu m 15 pht, b sung m n vch, lc u. Lc, b 5 ml dch lc

u. Ht chnh xc 500 l dch lc vo bnh nh mc 5 ml, b sung m

n vch, lc u. Lc qua mng lc 0,2 m.

Chun b dung dch th: cn chnh xc 20 vin, tnh khi lng trung

bnh vin, nghin mn. Cn chnh xc lng bt vin tng ng vi

khong 25 mg Atenolol racemic vo bnh nh mc 25 ml, thm 20 ml

m Tris 50 mM, siu m 15 pht, b sung m n vch, lc u. Lc,

b 5 ml dch lc u. Ht chnh xc 500 l dch lc vo bnh nh mc 5

ml, b sung m n vch, lc u. Lc qua mng lc 0,2 m.

Khi lng trung bnh vin: = 0,15051 g.

Kt qu kho st lp li trn mu th M1 c trnh by trong bng 5:

34

Bng 5: Kt qu kho st lp li trn mu th M1

STT Khi lng cn (g)

S-(-)-Atenolol R-(+)-Atenolol Tng hm lng

(mg/vin)

Din tch pic

(mAU.s)

Hm lng

(mg/vin)

Din tch pic

(mAU.s)

Hm lng

(mg/vin)

1 0,0754 190,5 25,4 193,8 25,5 50,9

2 0,0758 185,4 24,6 187,3 24,5 49,1

3 0,0743 184,0 24,9 190,5 25,4 50,3

4 0,0767 186,9 24,5 193,9 25,1 49,6

5 0,0751 182,3 24,4 188,4 24,9 49,3

6 0,0756 190,1 25,3 189,3 24,8 50,1

Chun 26,1 mg 195,5 198,2

Trung bnh 24,8 25,0 49,9

RSD (%) 1,69 1,50 1,37

Nhn xt:

Hm lng trung bnh ca ng phn S-(-)-Atenolol v R-(+)-Atenolol trong

mu M1 ln lt l 24,8 mg v 25,0 mg vi lch chun tng i u nh hn

2% (tng ng l 1,69%, 1,50%).

Nh vy vi iu kin in di la chn, phng php nh lng ng

phn Atenolol trong ch phm t yu cu lp li.

3.2.5. ng

ng ca phng php c xc nh bng t l tm li ca mt mu th

c cho thm nhng lng chun nht nh (chng ti cho thm 10% v 20%

so vi lng c trong mu). Mu th ny c xc nh hm lng. Kho st

ng trn mu M1.

Hm lng ca mi ng phn S-(-)-Atenolol v R-(+)-Atenolol trong mu

M1 c xc nh trong mc 3.2.4:

() = 24,8 (mg/vin)

() = 25,0 (mg/vin)

35

Chun b cc dung dich:

Dung dch chun thm: cn chnh xc khong 50 mg Atenolol racemic

chun vo bnh nh mc 50 ml, thm 30 ml m Tris 50 mM, siu m 15

pht, b sung m n vch, lc u.

Dung dch th xc nh phn trm tm li: Cn chnh xc lng bt

vin tng ng vi khong 25 mg Atenolol racemic vo bnh nh mc

25 ml, thm 2,5 ml, 5,0 ml (tng ng vi thm 10%, 20%) dung dch

chun thm, thm 15 ml m Tris 50 mM, siu m 15 pht, b sung m

n vch, lc u. Lc, b 5 ml dch lc u. Ht chnh xc 500 l dch

lc vo bnh nh mc 5 ml, b sung m Tris 50 mM n vch, lc u.

Lc qua mng lc 0,2 m.

Dung dch chun xc nh phn trm tm li: Ht chnh xc 500 l

dung dch chun thm vo bnh nh mc 5ml,b sung m Tris 50 mM

n vch, lc u. Lc qua mng lc 0,2 m.

Tin hnh in di dung dch th v dung dch chun vi iu kin in di

la chn.

Bng 6: Kt qu xc nh phn trm tm li trn mu M1(thm 10%)

STT KL cn (g)

KL thm (mg)

S-(-)-Atenolol R-(+)-Atenolol

KL thc (mg)

KL thu hi

(mg)

% tm li

KL thc (mg)

KL thu hi

(mg)

% tm li

1 0,0756 1,26 12,48 1,27 101,0 12,57 1,28 101,2

2 0,0749 1,26 12,36 1,24 98,6 12,46 1,29 102,1

3 0,0765 1,26 12,63 1,30 103,4 12,72 1,24 98,3

4 0,0783 1,26 12,92 1,25 99,2 13,02 1,30 102,8

5 0,0779 1,26 12,86 1,26 100,3 12,96 1,31 103,9

6 0,0773 1,26 12,76 1,28 101,8 12,86 1,28 101,3

Trung bnh

100,7

101,6

RSD (%)

1,73

1,88

36

Bng 7: Kt qu xc nh phn trm tm li trn mu M1(thm 20%)

STT KL cn (g)

KL thm (mg)

S-(-)-Atenolol R-(+)-Atenolol

KL thc (mg)

KL thu hi

(mg)

% tm li

KL thc (mg)

KL thu hi

(mg)

% tm li

1 0,0739 2,52 12,20 2,52 99,9 12,29 2,51 99,6

2 0,0752 2,52 12,41 2,57 102,2 12,51 2,48 98,6

3 0,0776 2,52 12,81 2,54 100,9 12,91 2,56 101,6

4 0,0748 2,52 12,35 2,58 102,4 12,44 2,58 102,3

5 0,0747 2,52 12,33 2,49 98,6 12,42 2,51 99,8

6 0,0778 2,52 12,84 2,59 102,7 12,94 2,51 99,5

Trung bnh

101,1

100,2

RSD (%)

1,60

1,41

Nhn xt:

T l tm li trung bnh khi thm khong 10% so vi lng c trong mu ca

mi ng phn S-(-)-Atenolol v R-(+)-Atenolol ln lt l 100,7% v 101,6% vi

lch chun tng i tng ng l 1,73% v 1,88%, u nh hn 2%.

T l tm li trung bnh khi thm khong 20% so vi lng c trong mu ca

mi ng phn S-(-)-Atenolol v R-(+)-Atenolol ln lt l 101,1% v 100,2% vi

lch chun tng i tng ng l 1,60% v 1,41%, u nh hn 2%.

Tuy nhin, c hai mc thm chun 10% v 20% u c nhng gi tr t l

phn trm tm li hi cao, nm ngoi khong quy nh 98-102%.

Nh vy phng php cha t yu cu v ng.

3.3. NG DNG NH LNG ATENOLOL TRONG CH PHM.

Trong nghin cu ny chng ti p dng phng php xy dng nh

lng tng ng phn Atenolol ca mt ch phm trn th trng (Mu M2).

Lm 3 ln tnh kt qu trung bnh.

Kt qu thu c nh sau:

Khi lng trung bnh vin: =0,21549 (g)

37

Bng 8: Kt qu nh lng mu M2

STT

Khi

lng cn

(g)

Din tch pic

(mAU.s)

Hm lng

(mg/vin)

Tng hm

lng

(mg/vin)

S(-) R(+) S(-) R(+)

1 0,1083 208,0 209,8

25,9

0,30

25,8

0,25 51,7

2 0,1097 210,5 211,7

3 0,1078 205,8 208,2

Trung

bnh 0,1086 208,5 210,5

Chun 25,5 mg 205,1 207,4

Hm lng so vi lng ghi trn nhn: % Atenolol racemic = ,

100% = 103,4%.

Hnh 12: in di mu M2

38

BN LUN

So vi HPLC, CE c nhc im l nhy thp hn do s dng detector

pht hin cht phn tch trn qung ng ngn hn v lng mu tim t hn. Tuy

nhin, CE ang c nghin cu v ng dng ngy cng nhiu trong phn tch cc

ng phn i quang do c nhiu u im vt tri hn so vi HPLC nh: hiu lc

tch cao hn, tn t dung mi ha cht, tc nhn chn lc i quang. c bit l CE

khng cn phi s dng ct tch i quang t tin bi n gin ch cn cho thm

tc nhn chn lc i quang vo dung dch m. Kh nng thay i linh hot loi v

nng tc nhn chn lc i quang, th tch mu nh cho php s dng cc thuc

th t tin, do m d dng trong qu trnh sng lc tc nhn chn lc i quang,

iu kin in di, pht trin v ti u ha phng php [9].

Atenolol c mt carbon bt i trong phn t tn ti di hai dng ng

phn S-(-), v ng phn R-(+). Nhiu nghin cu ch ra rng trong hai ng

phn th ng phn S-(-) ng vai tr ch yu trong tc dng dc l ca Atenolol

v c tnh ca n th li t hn so vi ng phn R-(+). Do trong thc t i hi

phi sn xut v a vo s dng dng n ng phn S-(-).

Nhm tc gi T Mnh Hng, Trnh Vn Lu, inh Th Thanh, V Ngn

Bnh, Nguyn Th Kiu Anh (2013) [7] nghin cu kho st cc yu t nh

hng n qu trnh in di v a ra iu kin nh sau:

Ct mao qun silica nung chy: chiu di tng 58,5 cm (chiu di hiu

dng 50 cm), ng knh trong 75 m.

Dung dch in ly nn: Tris 50 mM, CM--CD 8 mM, pH 4,0.

Nhit : 200C.

Hiu in th: 16 kV

Ch tim mu: 50 mbar x 3s.

Bc sng pht hin: 194 nm

Nng cht phn tch: 100 ppm (kl/tt) Atenolol racemic.

xc nh th t ra gii, nhm tc gi tin hnh in di hai dung dch:

Atenolol racemic chun 100 pppm v S-(-)-Atenolol chun 50 ppm vi iu kin

39

in di trn th thy rng thi gian di chuyn ca n ng phn S-(-)-Atenolol ph

hp vi thi gian di chuyn ca pic th nht ca hn hp racemic.

Sau tin hnh in di dung dch Atenolol racemic chun 100 ppm thm

S-(-)-Atenolol chun 100 ppm tng ng vi mu tim c t l R(+) /S(-) = 1/3

cng vi iu kin nh trn th thy din tch ca pic th nht ln hn ca pic th

hai khong 3 ln.

Do c th kt lun c th t ra gii ca hai ng phn: ng phn S-(-

)-Atenolol c ra gii ra trc, tc thi gian di chuyn ngn hn ng phn R-

(+)-Atenolol.

iu c th c gii thch bng c ch to phc lng gia hai ng phn

ca Atenolol vi CM--CD.

Hnh 13: V tr tng tc lin kt hydro[22].

ng phn R-(+) c 3 v tr c tng tc lin kt hydro vi CM--CD (v tr

1,2 v 3), trong khi ng phn S-(-) ch c mt v tr c tng tc lin kt hydro

(v tr 3). Do i lc ca ng phn R-(+) vi CM--CD ln hn ca ng phn

S-(-) dn n phc lng ca ng phn R-(+) bn hn. V cui cng dn n linh

in di ca ng phn R-(+) thp hn v c ra gii ra sau ng phn S-(-)

[22].

Nghin cu ca chng ti k tha nghin cu trn v tip tc hon thin

phng php nh lng ng phn i quang ca Atenolol.

V tr 1

V tr 2

V tr 3

Tng tc k nc

40

Sau khi nghin cu v lm thc nghim, chng ti xy dng phng php

nh lng ng phn i quang ca Atenolol trong ch phm bng in di mao

qun.

Phng php c thm nh trn cc ch tiu sau:

ph hp h thng: din tch pic ca hai ng phn S(-), R(+) Atenolol

c lch chun tng i u nh hn 2% (tng ng l 1,92% v

1,62%) nn phng php t yu cu v ph hp h thng v din tch

pic. Tuy nhin lch chun tng i ca thi gian di chuyn ca hai

ng phn S(-), R(+) Atenolol u ln hn 2% (tng ng l 3,04% v

3,17%)

Din tch pic ca mi ng phn S(-), R(+) Atenolol c quan h tuyn

tnh ng bin vi nng ca mi ng phn trong khong 25-75 ppm,

vi h s tng quan r xp x 1 (tng ng l 0,9995 v 0,9990).

c hiu cao: in di ca mu placebo khng xut hin pic ti v

tr tng ng vi pic ca hai ng phn trn in di ca c mu th v

mu chun.

lp li cao: lch chun tng i khi nh lng 6 mu song song

c khi lng cn ch phm khc nhau tng ng vi mi ng phn S(-

), R(+) Atenolol l 1,69% v 1,50%, u nh hn 2%.

ng: T l phn trm tm li c hai mc thm chun u c nhng

gi tr nm ngoi khong quy nh 98-102% nn phng php cha t

yu cu v ng.

Quy trnh tin hnh n gin, tn t ha cht v dung mi, t c hi.

Nh vy phng php nh lng ng phn i quang ca Atenolol m

chng ti xy dng t cc tiu chun ca mt phng php nh lng, c th p

dng nh lng cc ng phn i quang ca Atenolol trong ch phm.

Chng ti ng dng phng php ny vo nh lng mt ch phm trn

th trng (Mu M2) th hm lng ca Atenolol racemic so vi hm lng ghi trn

nhn l 103,4%, t yu cu v hm lng theo Dc in Vit Nam IV.

41

KT LUN V KIN NGH

KT LUN

Chng ti xy dng phng php nh lng ng phn i quang ca

Atenolol bng in di mao qun nh sau:

Kiu in di: in di mao qun vng (CZE).

Ct mao qun: Ct Agilent HPCE c chiu di hiu dng 56cm, ng

knh trong 50m.

Hiu in th: 25kV

Nhit : 25oC.

Bc sng pht hin: 194nm

Dung dch in ly nn: Tris 50mM, iu chnh ti pH 4,0 bng acid

H3PO4 c, cha CM--CD 8mM.

Tim mu: 50mbar 3s.

Trc khi s dng ln u tin, ct mao qun mi c ra sch vi

methanol trong 15 pht, tip theo l dung dch acid hydrocloric 0,1 M

trong 15 pht, v dung dch NaOH 1M trong 30 pht.

Hng ngy, vo thi im u ngy cc mao qun c ra sch bng

dung dch NaOH 0,1 M 15 pht, nc 5 pht, v vi dung dch in ly

nn (BGE) 15 pht.

Gia mi ln chy, chng trnh luyn ct (preconditioning) c thc

hin vi nc 5 pht, dung dch NaOH 0,1 M 4 pht, nc 2 pht, v

BGE 4 pht.

Thi im cui ngy, mao qun c ra vi dung dch NaOH 0,1 M 30

pht, nc 30 pht, sau thi kh 2 pht.

Vi iu kin in di nh trn, phng php xy dng t yu cu v ph

hp h thng, c hiu, lp li. Khong nng tuyn tnh vi mi n ng

phn l t 25 ppm 75 ppm.

Phng php tin hnh n gin, tt t dung mi v ha cht, t c hi.

42

Kt qu nh lng ch phm trn th trng Mu M2 t yu cu nh

lng theo Dc in Vit Nam IV.

KIN NGH

Sau khi thc hin ti ny, chng ti c mt s xut nh sau:

Tip tc hon thin phng php nh lng cc ng phn i quang ca

Atenolol bng in di mao qun.

Sau khi hon thin phng php, ng dng phng php vo kim nghim

thuc, nh gi th ha tan.

TI LIU THAM KHO

1. B Y t (2009), Dc in Vit Nam IV, Nh xut bn Y hc, 72-73

2. B Y t (2009), Dc th Quc gia Vit Nam, Nh xut bn Y hc, 160-162.

3. B Y t (2007), Ha dc tp 1, Nh xut bn Y hc, 156- 277.

4. B Y t (2007), Ha dc tp 2, Nh xut bn Y hc, 100.

5. B Y t (2007), Ha hu c tp 1, Nh xut bn Y hc, 66.

6. B Y t (2007), Ha phn tch tp 2, Nh xut bn Y hc, 225-247.

7. T Mnh Hng, Trnh Vn Lu, inh Th Thanh, V Ngn Bnh, Nguyn Th

Kiu Anh (2013), Bc u nghin cu nh lng ng phn i quang ca

atenolol bng phng php in di mao qun, Tp ch kim nghim thuc,

11(42), 14-19.

8. T Mnh Hng, Trnh Vn Lu, Nguyn Th Thu Hin, Nguyn Th Kiu Anh

(2013), Nghin cu tch cc ng phn quang hc ca Atenolol bng sc k

lng hiu nng cao vi mt s pha tnh i quang, Tp ch Nghin cu dc v

Thng tin thuc, 6, 218-224.

9. Benno A (1997), Chiral separations using capillary electrophoresis,

Technische Universiteit Eindhoven.

10. Blanco M., Valverde I (2003), Choice of chiral selector for enantioseparation

by capillaryelectrophoresis, Trends in Analytical Chemistry, 22(7+8), 428-439.

11. Eaga M.C, Somagoni M.J, Maddi R.S, Reddy S, Yamsani M.R (2010), New

chiral reverse phase HPLC method for determination of atenolol enantiomers in

pharmaceutical formulations, International Journal of Pharmaceutical

Sciences Review and Research, 4(1), 22-26.

12. Fanali S (1996), Identification of chiral drug isomers by capillary

electrophoresis, Journal of Chromatography A, 735, 77-121.

13. Fanali S (2000), Enantioselective determination by capillary electrophoresis

with cyclodextrins as chiral selectors, Journal of Chromatography A, 875, 89

122.

14. Gubitz G, Schmid M (1997), Chiral separation principles in capillary

electrophoresis, Journal of Chromatography A, 792, 179225.

15. INTERNATIONAL UNION OF PURE AND APPLIED CHEMISTRY, (1997)

Analytical chiral separation methods, Pure and Applied Chemistry, 69,1469-

1474.

16. Jie Zhou, Hanchun Yao, Hong Shao, Yinghui Li, Zhenzhong Zhang (2012),

Enantioseparation of -agonists with Carboxymethyl--Cyclodextrin by CE,

Journal of Liquid Chromatography & Related Technologies, 35, 5058.

17. Leena S (2007), Capillary Electrophoresis in Pharmaceutical Analysis: A

Survey on Recent Applications, Journal of Chromatographic Science, 45,

559-577.

18. Santoro M.I, Cho H.S (2000), Enantiomeric separation and quantitative

determination of atenolol in tablets by chiral high-performance liquid

chromatography, Drug development industrial pharmacy, 26(10), 1107-1110.

19. Shigeru Terabe, Koji Otsuka, Hiroyuki Nishi (1994), Separation of

enantiomers by capillary electrophoretic techniques, Journal of

Chromatography A, 666, 295319.

20. Stoschitzky K, Eggingere G, Zernig G, Klein W, Lindner W (1993),

Stereoselective features of (R)- and (S)-Atenolol: Clinical pharmacological,

pharmacokinetic, and radioligand binding studies, Chirality, 5, 15-19.

21. Stoschitzky K, Lindner W, Zernig G (1998), Racemic beta-blockersfixed

combinations of different drugs, Journal of Clinical and Basic Cardiology, 1

(1), 15-19.

22. Wuhong Li, Changhai Liu, Guangguo Tan, Xinrong Zhang, Zhenyu Zhu,

Yifeng Chai (2012), Molecular Modeling Study of Chiral Separation and

Recognition Mechanism of -Adrenergic Antagonists by Capillary

Electrophoresis, International Journal of Molecular Sciences, 13, 710-725.

23. http://pubchem.ncbi.nlm.nih.gov

PH LC

in di kho st ph hp h thng

min5 10 15 20 25 30 35

mAU

-10

0

10

20

30

40

Are

a: 1

84.3

45

33.

146

Are

a: 1

93.7

26

35.

357

min5 10 15 20 25 30 35

mAU

-10

0

10

20

30

DAD1 D, Sig=194,2 Ref=off (ATENOLOL\130518000132.D)

Are

a: 1

83.8

22

33.

854

Are

a: 1

87.9

35

35.

958

min5 10 15 20 25 30

mAU

-10

0

10

20

30

40

50

DAD1 D, Sig=194,2 Ref=off (ATENOLOL\130518000143.D)

Are

a: 1

85.7

78

31.

706

Are

a: 1

89.9

7

33.

542

min5 10 15 20 25 30 35

mAU

-20

-10

0

10

20

30

40

DAD1 D, Sig=194,2 Ref=off (ATENOLOL\130518000141.D)

Are

a: 1

92.7

41

33.

214

Are

a: 1

94.2

12

35.

618

min5 10 15 20 25 30 35

mAU

-20

0

20

40

60

80

100

120

DAD1 D, Sig=194,2 Ref=off (ATENOLOL\130518000142.D)

Are

a: 1

87.4

86

31.

638

Are

a: 1

98.6

6

33.

710

DANH MC CC K HIU, CH VIT TTDANH MC CC BNGDANH MC HNH V V THT VN CHNG 1. TNG QUAN1.1. TNG QUAN V ATENOLOL1.1.1. Tnh cht l, ha1.1.2. Dc l v c ch tc dng 1.1.2.1. Tc dng v c ch tc dng1.1.2.2. Dc ng hc1.1.2.3. Ch nh1.1.2.4. Chng ch nh1.1.2.5. Dng thuc v hm lng

1.2. TNG QUAN V K THUT IN DI MAO QUN1.2.1. Khi nim1.2.2. Nguyn l ca qu trnh in di mao qun1.2.3. in di mao qun vng

1.3. VI NT V PHN TCH NG PHN QUANG HC1.3.1. ng phn quang hc1.3.2. Phng php phn tch ng phn i quang1.3.2.1. Phng php phn tch ng phn i quang gin tip1.3.2.2. Phng php phn tch ng phn i quang trc tip

1.3.3. ng dng in di mao qun trong phn tch ng phn quang hc

1.4. TCH NG PHN I QUANG BNG CE S DNG CYCLODEXTRIN L TC NHN CHN LC I QUANG1.4.1. Tng quan v Cyclodextrin1.4.2. Tch ng phn i quang s dng Cyclodextrin l tc nhn chn lc i quang

1.5. MT S NGHIN CU PHN TCH NG PHN I QUANG CA ATENOLOL1.5.1. Nghin cu trong nc1.5.2. Nghin cu trn th gii

CHNG 2. I TNG V PHNG PHP NGHIN CU2.1. NGUYN VT LIU, THIT B2.1.1. i tng nghin cu2.1.2. Dung mi v ha cht2.1.2.1. Cht chun2.1.2.2. Cc ha cht khc

2.1.3. Trang thit b

2.2. NI DUNG NGHIN CU2.2.1. Hon thin phng php nh lng ng phn i quang ca Atenolol bng in di mao qun2.2.2. Thm nh phng php nh lng ng phn i quang ca Atenolol bng in di mao qun2.2.3. ng dng phng php ny nh lng ch phm c cha Atenolol trn th trng

2.3. PHNG PHP NGHIN CU2.3.1. Chun b cc dung dch chun, dung dch mu v cc dung dch lm vic2.3.2. Kho st v la chn iu kin in di2.3.2.1. La chn ct mao qun 2.3.2.2. La chn hiu in th p vo hai u mao qun

2.3.3. Thm nh phng php phn tch2.3.4. ng dng nh lng Atenolol trong cc ch phm trn th trng2.3.5. Phng php x l s liu2.3.5.1. Cng thc tnh ton cc gi tr thng k2.3.5.2. Cng thc tnh kt qu

CHNG 3. THC NGHIM, KT QU V BN LUN3.1. KHO ST V LA CHN IU KIN IN DI3.1.1. La chn ct mao qun3.1.2. Kho st nh hng ca in th p vo hai u mao qun.

3.2. THM NH PHNG PHP PHN TCH3.2.1. ph hp h thng3.2.2. Khong nng tuyn tnh3.2.3. c hiu3.2.4. lp li3.2.5. ng

3.3. NG DNG NH LNG ATENOLOL TRONG CH PHM.

BN LUNKT LUN V KIN NGHTI LIU THAM KHOPH LC