B Y T TRNG I HC DC H NI

NGUYN VN C

NGHIN CU NH LNG

RIFAMPICIN TRONG HUYT TNG

BNG SC K LNG HIU NNG CAO

KHA LUN TT NGHIP DC S

H NI - 2013

B Y T

TRNG I HC DC H NI

NGUYN VN C

NGHIN CU NH LNG

RIFAMPICIN TRONG HUYT TNG

BNG SC K LNG HIU NNG CAO

KHA LUN TT NGHIP DC S

Ngi hng dn: 1. ThS: Trn Th Lan Hng 2. ThS: T Mnh Hng Ni thc hin: Trung tm nh gi tng ng

sinh hc - Vin kim nghim thuc TW

H NI 2013

LI CM N

Sau mt thi gian thc hin ti vi nhiu n lc v c gng, thi im

hon thnh kha lun l lc em xin php c by t lng bit n chn thnh

ca mnh vi nhng ngi dy d, hng dn v gip em trong sut thi

gian qua.

Trc ht, em xin by t lng bit n su sc v chn thnh nht ti ThS.

Trn Th Lan Hng v ThS. T Mnh Hng, nhng ngi thy dnh rt

nhiu thi gian v tm huyt hng dn, quan tm gip em hon thnh kha

lun tt nghip ny.

ng thi, em xin chn thnh cm n cc anh ch ti trung tm nh gi

tng ng sinh hc Vin kim nghim thuc Trung ng tn tnh gip ,

ch bo em trong sut qu trnh thc hin ti.

Trong sut 5 nm hc tp ti trng i hc Dc H Ni, em nhn c

s du dt, dy bo tn tnh ca cc thy c. Em xin chn thnh cm n!

Vi tt c tnh thng yu, em xin gi li cm n su sc n b m, anh ch

em v nhng ngi bn nhng ngi lun ng vin, khch l em trong sut thi

gian qua.

Mt ln na, em xin chn thnh cm n!

H Ni, ngy 19 thng 5 nm 2013

Sinh vin

Nguyn Vn c

MC LC

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

CHNG 1: TNG QUAN ...................................................................................2

1.1.Tng quan v Rifampicin (RIF) .........................................................................2

1.1.1.Cu trc ha hc..........................................................................................2

1.1.2.Tnh cht ha l...........................................................................................2

1.1.3.Dc ng hc ............................................................................................3

1.1.4.Ph tc dng v c ch ................................................................................3

1.1.5.Ch nh.......................................................................................................3

1.1.6.Mt s ch phm trn th trng..................................................................4

1.1.7.Mt s phng php nh lng RIF trong dch sinh hc bng HPLC.........5

1.2.Tng quan v phng php sc k lng hiu nng cao ......................................7

1.2.1.nh ngha...................................................................................................7

1.2.2.Mt s thng s c bn ca qu trnh sc k................................................7

1.2.3.Nguyn tc cu to ca h thng my HPLC ...............................................8

1.2.4.C s l thuyt ca vic la chn iu kin sc k ......................................9

1.2.5.Phng php nh lng bng HPLC ........................................................13

1.3.Thm nh phng php phn tch trong dch sinh hc....................................14

CHNG 2: NGUYN VT LIU, THIT B, NI DUNG V PHNG PHP

NGHIN CU......................................................................................................17

2.1. Nguyn vt liu, thit b..................................................................................17

2.2. Ni dung nghin cu ......................................................................................18

2.3.Phng php nghin cu .................................................................................18

2.3.1.Chun b mu trong nghin cu.................................................................18

2.3.2. Xy dng phng php phn tch .............................................................20

2.3.3. Thm nh phng php phn tch............................................................21

CHNG 3: THC NGHIM, KT QU V BN LUN...............................24

3.1. Xy dng phng php nh lng rifampicin trong huyt tng:..................24

3.1.1. Kho st v la chn iu kin sc k v ni chun..................................24

3.1.2. Kho st v tm iu kin x l mu c Rifampicin..................................28

3.1.3. Qui trnh phn tch RIF trong huyt tng ................................................31

3.2. Thm nh phng php phn tch..................................................................31

3.2.1. Tnh chn lc............................................................................................32

3.2.2. ng chun v khong tuyn tnh ..........................................................34

3.2.3. Gii hn nh lng di .........................................................................37

3.2.4. ng lp li trong ngy v khc ngy ..........................................37

3.2.5. T l thu hi ca phng php .................................................................40

3.2.6. n nh ca mu huyt tng ..............................................................42

3.3. Bn lun .........................................................................................................45

3.3.1. Phng php x l mu huyt tng c Rifampicin .................................45

3.3.2. Phng php nh lng rifampicin trong huyt tng.............................45

CHNG 4: KT LUN V KIN NGH..........................................................47

TI LIU THAM KHO .....................................................................................49

DANH MC CC K HIU, CC CH VIT TT

CC : ng chun

HPLC : Sc k lng hiu nng cao (High Performance Liquid

Chromatography)

HQC : Mu kim sot cht lng nng cao (High Quality Control

Sample)

HT : Huyt tng

IS : Ni chun (Internal Standard)

LLOQ : Gii hn nh lng di (Lower Limit Of Quantification)

LQC : Mu kim sot cht lng nng thp (Lower Quality Control

Sample)

MeCN : Acetonitril

MeOH : Methanol

MQC : Mu kim sot cht lng nng trung bnh (Middle Quality

Control Sample)

QC : Mu kim sot cht lng (Quality Control Sample)

RIF : Rifampicin

SK : Sc k

SPE : Chit pha rn (Solid-phase extration)

ULOQ : Nng gii hn nh lng trn (Upper Limit Of Quantification)

US-FDA : Cc qun l thuc v thc phm M (The United States Food and

Drug Administration)

UV-VIS : T ngoi kh kin (Ultraviolet - Visible)

WHO : T chc y t th gii (World Health Organization)

DANH MC CC BNG Bng Ni dung Trang

1.1 Cc ch phm rifampicin trn th trng 4

1.2 Mt s nghin cu nh lng rifampicin trong dch sinh hc 5

2.1 Cc cht chun dng trong nghin cu 17

2.2 Cc ha cht dng trong nghin cu 18

2.3 Chun b ng chun 19

2.4 Chun b mu kim tra 20

2.5 Chun b mu gii hn nh lng di 20

3.1 Kt qu xc nh nh hng ca mu trng ti thi im trng

thi gian lu ca rifampicin v ni chun 34

3.2 Kt qu s tng quan gia nng rifampicin trong huyt

tng v t l p ng pic 35

3.3 Kt qu thm nh ng chun 36

3.4 Kt qu thm nh gii hn nh lng di 37

3.5 Kt qu thm nh ng, lp li trong ngy 38

3.6 Kt qu thm nh ng, lp li gia cc ngy 39

3.7 Kt qu thm nh t l thu hi ni chun 40

3.8 Kt qu thm nh t l thu hi rifampicin 41

3.9 Kt qu thm nh n nh ca mu huyt tng sau 3 chu

k ngr 42

3.10 Kt qu thm nh n nh ca mu huyt tng nhit

phng trong thi gian ngn 43

3.11 Kt qu thm nh n nh ca mu huyt tng nhit -

800C trong thi gian di 44

DANH MC CC HNH V Hnh Ni dung Trang

1.1 Cu trc ct LC DB 11

1.2 Cu trc ct c gc isopropyl 11

3.1 Ph hp th UV-VIS ca rifampicin 24

3.2 Sc chun rifampicin trong pha ng: methanol - m phosphat

(65:35) 26

3.3 Sc chun ni chun trong pha ng: methanol - m phosphat

(65:35) 26

3.4 Sc chun rifampicin trong pha ng: methanol - acetonitril -

m phosphat (48,8: 16,2: 35) 27

3.5 Sc chun ni chun trong pha ng: methanol - acetonitril - m

phosphat (48,8: 16,2: 35) 27

3.6 Sc chun rifampicin v ni chun vi tc dng khc nhau 28

3.7 Sc mu huyt tng trng ta protein bng methanol 29

3.8 Sc mu huyt tng trng thm rifampicin v ni chun ta

protein vi 1,5 mL methanol 29

3.9 Sc mu huyt tng trng thm rifampicin v ni chun ta

protein vi 1,0 mL methanol 29

3.10 Sc chun rifampicin trong methanol c Vitamin C 1,0 mg/mL 30

3.11 Sc mu huyt tng trng thm nh chn lc 32

3.12 Sc mu huyt tng trng c pha chun rifampicin (nng 0,2

g/mL) v ni chun 33

3.13 Sc mu huyt tng trng c pha chun rifampicin (nng 9,0

g/mL) v ni chun 33

1

T VN

Lao l bnh truyn nhim nguy him v ph bin. Bnh do vi khun lao

Mycobacterium tuberculosis gy ra. C quan chu tc ng ph bin nht ca vi

khun lao l phi. Ngoi ra, cc v tr khc c th gy bnh ca vi khun lao l

xng, khp, hch v no. Theo t chc y t th gii, lao l mi e da ln trn

ton cu v vn sc khe [21].

Vit Nam nm trong danh sch cc nc c s ca mc lao cao trn th gii,

ng th 12/23 nc c s lng bnh nhn lao cao trn ton cu. c bit, nhng

s liu gn y cho thy t l s bnh nhn lao ti pht v tht bi iu tr c xu

hng gia tng [20].

Rifampicin l mt trong nm thuc ca thuc iu tr lao nhm 1 gm c

isoniazid, rifampicin, pyrazinamid, ethambutol, streptomycin. Vi khun lao khng

thuc hin nay ang c xu hng gia tng v l mt vn ln gy tht bi trong

iu tr. C nhiu yu t khc nhau nh hng n kt qu trong iu tr trong

nng thuc chng lao trong mu c lin quan cht ch n hiu qu iu tr thc

t. nh gi chnh xc vic iu tr c t yu cu hay khng, vic nh lng

nng thuc trong mu l mt yu cu cn thit c t ra. iu ng ch ,

nm 2010 B Y t ra quy nh cc thuc cha hot cht rifampicin phi c bo

co, s liu nh gi tng ng sinh hc invivo mi c cp php ng k v

lu hnh trn th trng Vit Nam [7]. Do vy, xc nh nng rifampicin

trong huyt tng nhm phc v cho cng tc iu tr v nh gi tng ng sinh

hc cc ch phm cha hot cht rifampicin, chng ti tin hnh ti: Nghin

cu nh lng rifampicin trong huyt tng bng sc k lng hiu nng cao

vi 2 mc tiu:

1. Kho st, xy dng phng php nh lng rifampicin trong huyt tng

bng sc k lng hiu nng cao vi detector UV.

2. Thm nh phng php xy dng theo qui nh ca US-FDA.

2

CHNG 1: TNG QUAN 1.1.TNG QUAN V RIFAMPICIN (RIF)

1.1.1.Cu trc ha hc

- Cng thc phn t: C43H58N4O12 ; M=822,95 g/mol

- Cng thc cu to:

- Tn khoa hc:

(12Z,14E,24E) (2S,16S,17S,18R,19R,20R,21S,22R,23S) 5,6,9,17,19-

pentahydroxy 23 methoxy 2,4,12,16,18,20,22 heptamethyl 8 [N-(4-

methyl-1-pipeainyl) pentadecatrienoimino] -1,2-dihydronaphtho [2,1-b] furan -21-

yl acetat. [2], [4], [13], [19]

1.1.2.Tnh cht ha l

- Bt kt tinh mu cam, hoc nu, khng bn khi b m.

- D tan trong cloroform; tan trong methanol; tan t trong nc, ethanol, ether.

- Dung dch RIF khng bn v bin i ty thuc vo pH v nhit . pH

kim, RIF d b oxy ha bi oxy khng kh lm chuyn RIF sang dng quinon;

pH acid, RIF b thy phn ra hp cht 3-formyl rifamycin SV. Cc nhm ester b

thy phn ngay pH trung tnh. [2], [4], [13], [19]

3

1.1.3.Dc ng hc

RIF c hp thu nhanh qua ng tiu ha, sinh kh dng trn 90%. Khi

ung liu 600 mg, sau 2-4 gi t nng nh trong huyt tng l 7-9 g/ml.

Thc n lm chm v gim hp thu thuc. Khong 80% thuc lin kt khng bn

vi protein huyt tng.

Thuc phn b rng ri vo cc m v dch c th, c bit l phi v dch

ph qun. Thuc vo c nhau thai, sa m v dch no ty khi mng no b vim,

th tch phn b l 1,6 L/kg.

RIF chuyn ha gan bng phn ng acetyl ha, thi tr khong 65% qua

phn v khong 30% qua nc tiu, phn cn li qua m hi, nc bt, nc mt.

Sn phm thi tr c mu , thi gian bn thi 3-5 gi. [1], [3]

1.1.4.Ph tc dng v c ch

RIF c tc dng tt vi cc chng vi khun Mycobacterium c bit l

Mycobacterium tuberculosis, vi khun phong Mycobacterium laprae v cc vi

khun c hi M.bovis, M.avium. Nng c ch ti thiu vi trc khun lao l 0,1 -

2 g/ml.

C ch: RIF gn vo cc tiu phn n v ca ARN-polymerase, lm sai

lch thng tin ca cc enzym ny, do c ch s khi u ca qu trnh tng hp

ca ARN mi. Thuc c tc dng dit khun. Vi khun lao khng thuc l do s

thay i cu trc tiu n v ca enzym ARN polymerase. RIF khng khng

cho vi cc thuc chng lao khc nhng khi s dng n c v lm dng dn n

tng t l khng thuc. Theo s liu chng trnh chng lao quc gia, khong 3,6%

bnh nhn lao c trc khun khng RIF. Do s dng RIF cn c s gim st

nghim ngt m bo hiu qu iu tr. [1], [3]

1.1.5.Ch nh

- iu tr tt c cc th lao bao gm c lao mng no (phi hp vi cc thuc

khc theo phc ).

- iu tr phong (theo phc ).

- Phng v iu tr vim mng no do H.influenzae v N.meningitidis.

4

- iu tr nhim khun nng do Staphylococus khng Methicillin. [1], [3]

1.1.6.Mt s ch phm trn th trng

RIF l khng sinh bn tng hp, ch yu dng dng vin ung hm lng

150 mg hoc 300 mg. Cc ch phm trn th trng c th cha thnh phn duy

nht l RIF hoc dng kt hp RIF vi cc thuc chng lao khc thuc nhm thuc

iu tr lao nhm 1 nh l Isoniazid (H), Pyrazinamid (Z), Ethambutol(E). Mt s

ch phm cha RIF trn th trng Vit Nam c trnh by c th ti bng 1.1 sau:

Bng 1.1: Cc ch phm RIF trn th trng [23]

STT Bit dc Thnh phn

dc cht

Dng thuc Nh sn xut

1

-Eremfat 150 v

Eremfat 300

-Rifampicin

150mg

-Rifampicin 300

mg

RIF

-Vin nn bao

phim 150mg v

300mg

-Vin nang

-Vin nang

cng

-Fatol Arzneimittel

GmbH c

-CTCP Dc phm

TW2

-CTCP DP Nam H

2 -Rifa H 250

-Rifampicin

INH

RIF + Isoniazid

- Vin nn bao

phim

-Vin nang

-CTCP Dc phm

TW2

-CTCP ha DP

Mekophar-VN

3 Turbezid

RIF + Isoniazid

+ Pyrazinamid

Vin nn bao

phim CTCP DP Nam H

4

Rimstar 4 FDC

RIF + Isoniazid

+ Pyrazinamid

+ Ethambutol

Vin nn bao

phim

Novartis

Bangladesh

5

1.1.7.Mt s phng php nh lng RIF trong dch sinh hc bng HPLC

Hin nay, i vi RIF nguyn liu c th nh lng bng phng php o

quang v HPLC. Nhng trong dch sinh hc do c nhiu thnh phn nn m bo

chnh xc v gim thiu sai s nh lng RIF trong huyt tng th phng php

HPLC thng c chn tin hnh. Mt s phng php nh lng RIF trong

dch sinh hc bng HPLC c trnh by bng 1.2 [2], [13], [19].

Bng 1.2: Mt s nghin cu nh lng rifampicin trong dch sinh hc.

TT Mu X l mu iu kin sc k Detector Ni chun Ti

liu

1 HT

ngi

Chit pha rn

SPE

Ct C18, pha ng:

Amonium acetat (0,02

M; pH 4,0) : MeCN.

UV,

334 nm

Papaverin

hydrochlorid

12

2 HT

ngi

SPE, thm

Vitamin C

1,0 mg/mL

Ct C8, pha ng:

KH2PO4 0,05 M :

MeCN (55: 45)

UV,

340 nm

Sulindac 15

3 HT

ngi,

nc

tiu

Ta protein

bng MeOH,

thm

Vitamin C

(0,5 mg/mL)

Ct: C18 (250x 4,6

mm, 5 m). Pha ng:

m phosphat (0,05

M; pH 2,6) : MeCN

(55: 45); tc dng:

1,2 mL/pht; th tch

tim mu: 20 L.

UV,

254 nm

Rifapentine 11

4 HT

ngi

Ta protein

bng dung

mi MeCN.

Ct C18 (25x 4,6 mm;

5 m); pha ng:

MeOH m

phosphat pH 4,5 (65 :

35); tc dng 1,0

mL/pht; th tch tim

mu: 30 L.

UV,

254 nm

9

6

Nhn xt:

Phng php 1, 2: c chnh xc cao, khong nng tuyn tnh t 0,5

20 g/mL v 0,5 35 g/mL. Tuy nhin, c 2 phng php trn u s dng quy

trnh x l mu bng SPE tng i phc tp v chi ph cao. Trong iu kin phng

th nghim nc ta hin nay, vic p dng phng php ny gp nhiu kh khn

do khng kinh ph mua ct chit v trang thit b chit pha rn. V vy, 2 phng

php ny kh c th p dng ngay nc ta hin nay.

Phng php 3: qui trnh x l mu bng phng php ta protein trong

dung mi MeOH n gin, d thc hin. Tuy nhin, phng php s dng cht ni

chun l rifapentine, l 1 cht hin ti trong iu kin phng th nghim chng ti

khng c nn kh c th p dng phng php vo trong nghin cu.

Phng php 4: qui trnh x l mu bng phng php ta protein trong

dung mi MeCN n gin, d thc hin. Khong nng tuyn tnh ca phng

php t 1,0 30 g/mL. Tuy nhin phng php khng s dng bt k cht ni

chun no nh lng RIF trong huyt tng do kh m bo c chnh

xc v khng gim c sai s trong qu trnh x l mu.

Da trn s tham kho cc ti liu c cng b v da vo tnh cht l

ha ca RIF, chng ti d kin xy dng phng php nh lng RIF trong huyt

tng bng phng php HPLC vi detector UV-VIS c s dng cht ni chun

ph hp p ng yu cu ng, chnh xc cao ca phng php phn tch

trong dch sinh hc, d thc hin vi iu kin hin c ti cc phng kim nghim

ca Vit Nam.

7

1.2.TNG QUAN V PHNG PHP SC K LNG HIU NNG CAO

1.2.1.nh ngha

Sc k lng hiu nng cao (HPLC) l k thut phn tch da trn c s ca

s phn tch cc cht trn mt pha tnh cha trong ct nh dng di chuyn ca pha

ng lng p sut cao. Pha tnh c th l cht rn c bao trn b mt mt cht

mang rn, hoc l cht mang rn c bin i bng lin kt ha hc vi cc

nhm hu c. Qu trnh sc k xy ra theo c ch: hp ph, phn tn, trao i ion,

loi c hay tng tc ha hc trn b mt.[5]

1.2.2.Mt s thng s c bn ca qu trnh sc k

Thi gian lu tR:

Thi gian lu ca mt cht l thi gian cn mt cht di chuyn t ni tim

mu qua ct sc k, ti detertor v cho pic trn sc k tnh t lc tim n lc

xut hin nh ca pic. Thi gian lu l thng tin v mt nh tnh ca sc k , n

l hng s i vi mt cht nht nh khi tin hnh sc k trong iu kin khng

i. Ngoi ra trong mt php phn tch cn thi gian lu hiu chnh, c tnh bng

cng thc sau: tR = tR tM. Vi: tM l thi gian lu ca 1 cht khng b lu gi bi pha tnh.

Nu tR qu nh th s tch km, cn nu tR qu ln th pic b long, thi gian

phn tch ko di.[5]

H s phn b k: l h s phn b trng thi cn bng xc nh tc trung

bnh ca mi vng cht tan do pha ng vn chuyn khi n qua ct

k = Trong : CS l nng mol ca cht tan trong pha tnh.

CM l nng mol ca cht tan trong pha ng

H s dung lng k: cho bit kh nng phn b ca cht phn tch trong hai

pha v c tnh theo sc cha ca ct:

k = k x = = =

k: ti u trong khong 1 k 8 [5]

8

H s chn lc :

c trng cho tc di chuyn t i ca 2 cht A v B, ngi ta dng h

s chn lc :

= = =

Qui c : Cht B l cht b lu gi mnh hn cht A nn >1

tch ring 2 cht thng chn 1,05 2,0 [5]

H s i xng ca pic F:

F = Trong : W: chiu rng ca pic o 1/20 chiu cao ca pic

a: l khong cch t chn ng vung gc h t nh

pic n mp ng cong pha trc ti v tr 1/20 chiu cao pic. [5]

S a l thuyt v hiu lc ct N:

Hiu lc ct c o bng thng s: S a l thuyt N ca ct

N = 16 hoc N = 5,54

Trong : W1/2h l chiu rng pic o na chiu cao ca nh pic.

W l chiu rng ca pic o y pic. [5]

phn gii Rsca ct:

Rs = hoc

Trong :

t(R)A; t(R)B : thi gian lu ca 2 pic lin k nhau (B v A).

WB; WA : rng pic o cc y pic.

W1/2A; W1/2B : rng pic o na chiu cao pic.

Yu cu: RS> 1; gi tr ti u Rs = 1,5. [5]

1.2.3.Nguyn tc cu to ca h thng my HPLC

H thng HPLC n gin v lm vic theo k thut HPLC bao gm

cc b phn chnh sau:

9

Bm cao p:

bm pha ng vo ct sc k, bm ny phi iu chnh c p sut

(thng 0 400 bar) y pha ng qua h thng vi mt tc dng n nh,

ph hp vi qu trnh sc k.

Bnh cha dung mi v h thng x l dung mi:

Bnh cha dung mi thng bng thy tinh i khi bng thp khng g. Dung

mi chy sc k c lc qua mng lc (thng dng mng lc c 0,45 m) v

ui kh ha tan.

H tim mu:

a mu phn tch vo ct c th tim mu bng tay hay tim mu bng

h thng tim t ng.

Ct sc k:

Qu trnh tch cc cht din ra ct. Ct c ch to bng thp c bit tr

vi ha cht, chu c p sut cao n vi trm bar. Trong ct c nhi pha tnh.

Ct sc k c nhiu c khc nhau ty thuc vo mc v mc ch ca qu trnh

sc k. Ni chung, cc ct phn tch thng c chiu di t 10 25 cm, ng knh

2 5 mm.

Detector:

L b phn pht hin cht phn tch. Ty theo bn cht ca cc cht cn phn

tch m s dng detector thch hp bao gm cc loi sau: Detector hp th UV

VIS, detector ph pht x nguyn t, detector hunh quang.[5]

1.2.4.C s l thuyt ca vic la chn iu kin sc k

1.2.4.1.La chn pha tnh cho HPLC

Pha tnh trong HPLC l cht nhi ct, l yu t quan trng quyt nh s tch

ca mt hn hp nhiu cht. Ty vo phn cc ca pha tnh v pha ng sc k

c chia thnh 2 loi:

Sc k pha thun: pha tnh phn cc, pha ng khng phn cc, cht phn

tch thng l cc cht phn cc hoc l cc cht c th phn cc.

10

Sc k pha o: pha tnh t phn cc, pha ng phn cc, cht phn tch

thng l cc cht c th phn cc hoc t phn cc.

Pha tnh thng c ch to trn nn Silica (SiO2), nn oxyd nhm (Al2O3),

nn hp cht cao phn t (cellulose) hay trn mch carbon. Trong sc k hp ph

pha o: pha tnh trn nn silica c nhiu u vit c s dng nhiu nht.

C th phn loi cht nhi ct theo gc Siloxan:

R l cc nhm phn cc (a nc): nhm OH hoc cc alkylamin CH2-

NH2, alkyl nitril CH2 CN, s dng lm pha tnh trong sc k hp ph pha thun

phn tch cc cht t hoc khng phn cc.[22]

Vi R l cc nhm t phn cc nh octyl, octadecyl, phenyl, c iu ch

bng cch alkyl ha cc nhm OH b mt silica trung tnh bng cc gc alkyl-R ca

mch carbon (C2;C8;C18) hay cc gc carbon vng phenyl. Do cc nhm OH thn

nc c thay th bng cc gc R k nc nn b mt tr nn t phn cc. Silica

alkyl ha c s dng trong sc k hp ph pha o phn tch cc cht phn

cc, t phn cc hay sc k to cp ion.[5]

Tuy nhin do hiu ng lp th nn trong cu trc ca pha tnh cn nhng

nhm OH gy nh hng xu n qu trnh tch sc k ty theo mi trng pH

phn tch.

Trong mi trng qu acid, pH < 2 th c s phn ly cc nhm ether ra khi

nn. Lc ny ct s mt hot tnh dn n cht cn phn tch khng th tng tc

vi ct.

11

Trong mi trng Base (pH>7), cc nhm OH trong ct s tng tc vi

cht tan c tnh base. Tng tc ny khc vi kiu tng tc ca cht tan vi nhm

Si-C18, dn n hin tng cng mt cht nhng s c nhng nh ra khc nhau

hay l hin tng ko ui. Kt qu gim i chnh xc.

Mt trong nhng cch khc phc hin tng ny l dng cc hp cht nh

trimethylclorosilan ClSi(CH3)3 hoc hexametyldisizan (t s dng hn) tng tc

vi nhm OH ny. Cu trc pha lin kt c trnh by hnh 1.1

Hnh 1.1: Cu trc ct LC-DB

C mt cch khc loi tr bt s nh hng ca nhm OH m khng cn

tng tc vi n l thay nhng nhm methyl ca Si(CH3)2-C18 bng nhng nhm

th ln hn nh isopropyl nhng nhm ny s che chn i nhng nhm OH,

c trnh by hnh 1.2.

Hnh 1.2 : Cu trc ct c gc Isopropyl

Ngoi ra, trong mt s trng hp cn ghp ln dy C18 mt s nhm phn

cc tng thm phn cc ca dy C18 lm ct c kh nng tch chn lc hn

i vi nhng hp cht phn cc mnh (Ct EPS Expended Polar Selectivity)[22]

12

1.2.4.2.La chn pha ng cho HPLC

Pha ng l dung mi ra gii cht phn tch ra khi ct sc k, l yu t

th hai quyt nh hiu sut phn tch ca mt hn hp. Trong sc k pha o, pha

ng l dung mi phn cc nh: nc, methanol, acetonitril hoc hn hp ca

chng. Cc dung mi ny c th ha tan thm mt lng nh acid hay base hu c.

Pha ng trong HPLC nh hng ti rt nhiu thng s ca qu trnh sc k

nh: chn lc , thi gian lu tR, phn gii Rs, rng ca y pic nn vic

phn tch, la chn pha ng thch hp l rt quan trng.

Yu cu ca 1 pha ng:

Tr vi pha tnh v bn vng theo thi gian.

Ha tan cht cn phn tch.

C tinh khit cao.

Nhanh t cn bng trong qu trnh sc k.

C tnh kinh t v m bo mi trng.

C cc yu t quan trng ca pha ng nh hng n kt qu:

Bn cht ca dung mi pha pha ng.

Thnh phn cc cht trong pha ng.

pH ca pha ng.

Tc dng ca pha ng.

Chn m pH:

Trong sc k hp ph m cht tan c tnh cht acid hoc base thng phi s

dng m pha ng n nh pH cho qu trnh sc k. Gi tr pH thch hp s

lm tng hiu lc tch ca sc k.

Tc dng ca pha ng:

Sau khi c pha tnh, pha ng, pH thch hp, tng hiu lc tch ca qu

trnh phi chn tc pha ng ph hp, m bo vic tch cc cht phn tch c

tt nht m t hiu qu cao v thi gian v kinh t. [22]

13

1.2.4.3.La chn Detector

Khi pha ng ra gii cc cht ra khi ct s ghi nhn bi detector chuyn

thnh tn hiu v c ghi trn sc k . Detector ph bin nht l Detector UV-

VIS. Ty theo cht cn phn tch m ngi ta t cc bc sng pht hin khc

nhau.[22]

1.2.5.Phng php nh lng bng HPLC

1.2.5.1.Cch nh gi pic

nh gi din tch pic: tnh din tch pic c th dng tch phn k hoc

dng my tnh ci t sn chng trnh.

nh gi chiu cao ca pic vi iu kin cc ch s k l hng nh. o chiu

cao ca pic ch thch hp khi nng mu th t thp n trung bnh.

1.2.5.2.Phng php nh lng v cch tnh kt qu trong HPLC

Tt c cc phng php nh lng bng sc k u da trn nguyn tc:

Nng ca cht phn tch t l vi chiu cao hoc din tch pic ca n.

C 4 phng php nh lng hay c s dng trong sc k: phng php

chun ngoi, phng php chun ni, phng php thm chun, phng php

chun ha din tch.

Hin nay, phng php chun ni c s dng nhiu nht nh lng

thuc trong huyt tng. Phng php chun ni khc phc c nhng nhc

im ca phng php chun ngoi, gip hn ch ti a nhng sai s c th gy ra

do my mc, k thut, nht l vi nhng qui trnh x l mu phc tp v cc mu c

hm lng cht cn nh lng thp.

Nguyn tc phng php chun ni: thm c vo mu chun v mu th

nhng lng bng nhau ca mt cht tinh khit (cht chun ni) ri tin hnh sc k

trong cng iu kin.

Nguyn tc la chn cht ni chun:

- Trong cng iu kin sc k, cht IS phi c tch hon ton v c thi

gian lu gn vi thi gian lu ca cht cn phn tch trong mu th.

- C cu trc ha hc tng t nh cht th.

14

- C nng xp x vi nng cht th.

- Khng phn ng vi bt k thnh phn no ca mu th.

- Phi c tinh khit cao v d kim.

C 2 phng php l chun ha 1 im v chun ha nhiu im. Trong

khun kh kha lun ti xin trnh by c th v phng php chun ni chun ha

nhiu im. Chun b mt dy chun t nht l 5 mu c nng cht chun khc

nhau nhng tt c cng cha mt lng chun ni bng nhau. Tin hnh chy sc

k cc mu, ta xy dng ng chun biu din s tng quan gia t s din tch

(hay chiu cao) pic ca cht chun vi chun ni (SS / SIS) so vi nng cht

chun c trong mu (CS). Da vo ng chun tm ra nng cht th. [5], [6],

[10]

1.3.THM NH PHNG PHP PHN TCH TRONG DCH SINH HC

1.3.1.Yu cu chung

Mu sinh hc thng c nhiu tp cht, lng mu t v nng cht phn

tch thng rt thp, do vy phng php phn tch sinh hc phi c thm nh

trc khi p dng vo phn tch mu.

Thm nh mt phng php phn tch sinh hc l qu trnh xc nh bng

nghin cu trong phng th nghim nhng c im c trng ca phng php

m bo phng php t yu cu vi cc ng dng phn tch thc t, nhm

chng minh rng phng php phn tch l ng tin cy v c kh nng thc

hin phn tch nhng mu c kho st. [8], [10], [16]

1.3.2.Ni dung thm nh phng php

1.3.2.1.Tnh chn lc

Tnh chn lc l kh nng nh gi mt cch r rng cht cn phn tch khi c

mt cc thnh phn khc nh tp cht hoc cc cht cn tr khc. Ni cch khc,

tnh chn lc th hin kh nng phng php phn tch c th nhn din cht cn

phn tch v khng b nhm ln bi cc cht khc. Phng php t yu cu v tnh

chn lc khi tha mn iu kin sau:

- Pic ca RIF c nhn din r rng, tch c hon ton khi pic tp.

15

- p ng ca mu trng ti thi im trng vi thi gian lu ca RIF khng c vt qu 20% p ng ca RIF nng LLOQ.

- p ng ca mu trng ti thi im trng vi thi gian lu ca IS khng c vt qu 5% p ng ca IS. [16] 1.3.2.2.ng chun v khong tuyn tnh

ng chun biu din mi quan h gia cc p ng ca pic (din tch hay chiu cao) v nng cht cn phn tch trong dch sinh hc. Khong tuyn tnh l khong nng t thp nht n cao nht trong mt ng chun c p ng tuyn tnh.

Tnh cht tuyn tnh c biu th bng phng trnh hi quy: y = ax + b vi h s tng quan tuyn tnh r.

ng chun phi tha mn cc yu cu sau: + Yu cu v h s tng quan r: r 0,98 + C t nht 75% s im ca ng chun (bao gm c mu c nng

thp nht v mu c nng cao nht) phi c ng nm trong khong t 85% n 115%, ring im thp nht ca ng chun cho php sai s khng qu 20%. [8], [16] 1.3.2.3. chnh xc ca phng php

ng

ng ca phng php l mc gn st ca kt qu phn tch vi gi tr thc ca mu bit.

Yu cu: ng ca phng php ti mi nng phi nm trong khong t 85% n 115%, tr gi tr gii hn nh lng di LLOQ cho php t 80 120%. [8], [16]

lp li

lp li ca 1 phng php phn tch l mc thng nht gia cc kt

qu ring bit khi quy trnh c p dng lp i lp li nhiu ln trn cng 1 mu

ng nht. lp li c biu th bng lch RSD hoc CV (%).

Yu cu: lp li trong ngy: Gi tr CV% phi 15%. lp li gia cc ngy: Gi tr CV% phi 15%. [16]

16

1.3.2.5.Gii hn nh lng di (LLOQ) LLOQ l nng thp nht trn ng chun c th nh lng c vi

tnh ng v tnh chnh xc chp nhn c. Mu chun c nng thp nht trn ng chun c chp nhn l LLOQ nu tha mn: - p ng ca cht phn tch ti LLOQ t nht bng 5 ln p ng ca mu trng ti cng thi gian lu. - ng phi bng 80% 120% so vi nng thc. - chnh xc th hin bng gi tr CV% khi tin hnh phn tch trn t nht 5 mu LLOQ c lp phi nh hn hoc bng 20% [8], [16] 1.3.2.6.T l thu hi

T l thu hi l t l nng hot cht sau khi mu c chit tch theo qui trnh chn so vi nng khng c x l qua chit tch (mu pha trong dung mi pha ng).

Yu cu: - Phng php x l mu c coi l thch hp khi t l thu hi khng qu

110% v khng thp hn 30%. - Gi tr CV% gia cc p ng ca ni chun v gia cc p ng ca cht

phn tch trong cc mu QC c qua x l mi nng phi nh hn hoc bng 15%.

- T l thu hi trung bnh ti cc nng khng khc nhau qu 15% [16]

1.3.2.7. n nh n nh ca mu phn tch trong dch sinh hc cn c nh gi trong

lc ly mu, x l v bo qun mu. Trong thm nh phng php cn xc nh cc loi n nh sau:

n nh ca dung dch chun gc, ni chun gc bao gm: n nh iu kin nhit phng, n nh iu kin bo qun di ngy, n nh ca dung dch pha long (nu c). [8], [16]

n nh ca mu huyt tng bao gm: n nh sau 3 chu k ng r ng, n nh thi gian ngn nhit phng i vi dung dch mu th sau khi x l (chit tch) v n nh thi gian di ng lnh ca mu th trong khong thi gian d nh. [8], [16]

17

CHNG 2: NGUYN VT LIU, THIT B, NI DUNG V

PHNG PHP NGHIN CU 2.1. NGUYN VT LIU, THIT B

2.1.1. My mc, thit b

- H thng my sc k lng HPLC Shimadzu gm: Bm cao p LC-20AT;

b phn tim mu t ng SIL-20AC; bung n nhit ct CTO-20A; Detector UV

SPD-M20A; phn mm iu khin v x l kt qu LC Solution Shimadzu.

- Cn phn tch Sartorius CP-224S, c

- Cn k thut Sartorius E412

- My lc xoy Velp, Italia.

- My ly tm lnh Sartorius Sigma 2 16K, c

- T lnh su 40 0C, Sanyo MDF 236, Nht

- T lnh su 80 0C, Sanyo MDF 192, Nht

- Micropipet, Eppendorf, c

- ng ly tm nha, dung tch 15 mL

- Bm ht chn khng Satorius.

- Bnh nh mc, ng ong, pipet nh mc v cc dng c phn tch cn

thit khc.

2.1.2. Nguyn liu, ha cht, dung mi

Cc ha cht, dung mi dng trong nghin cu c trnh by bng 2.1 v

bng 2.2

Bng 2.1: Cc cht chun dng trong nghin cu

Tn Mc ch

s dng

Ni sn xut S kim sot Hm

lng

m

Rifampicin Chun Vin KN

thuc T 0307018 100,82% 0,40%

Dipyridamol Ni chun Vin KN

thuc T 0102127 99,66% 0,15%

18

Bng 2.2: Cc ha cht dng trong nghin cu

Tn thuc th tinh khit Ni sn xut

Acetonitril HPLC J.T.Baker, Anh

Methanol HPLC J.T.Baker, Anh

Methanol p.a J.T.Baker, Anh

Kali dihydrophosphat p.a Merck, c

Vitamin C p.a Vit Nam

- Huyt tng trng: Vin huyt hc truyn mu TW. S l: 12PN03134,

12PN03110, 12PN03280, 12PN03178, 12PN03209, 12PN03211. Hn dng:

20/5/2013. Bo qun 40 0C

2.2. NI DUNG NGHIN CU

2.2.1. Xy dng phng php nh lng Rifampicin trong huyt tng bng

HPLC

- Quy trnh chit tch RIF: Da trn cc nguyn l chit tch (chit lng

lng, ta loi protein bng dung mi hoc acid), kho st trn cc mu t to la

chn phng php x l mu thch hp.

- La chn iu kin sc k, xy dng phng php phn tch m bo phn

tch RIF khi cc thnh phn tp trong huyt tng cho php xc nh c lng

RIF trong mu vi chnh xc thch hp.

2.2.2. Thm nh phng php phn tch

Thm nh phng php c la chn vi cc ch tiu: Tnh c

hiu/chn lc, ng chun v khong nng tuyn tnh, ng, lp li trong

ngy v gia cc ngy, gii hn nh lng di (LLOQ), t l thu hi, n nh.

2.3.PHNG PHP NGHIN CU

2.3.1.Chun b mu trong nghin cu

Chun b mu chun RIF v IS trong MeOH v trong HT trng:

- Mu trng: Huyt tng trng khng cha RIF

19

- Dung dch chun gc: Nng chnh xc khong 200,0 g RIF/mL MeOH

cha vitamin C (1 mg/mL)

- Dung dch chun ni: Nng chnh xc khong 1,0 mg IS/mL MeOH

cha Vitamin C (1 mg/mL)

- Dung dch chun lm vic: T dung dch chun gc, chun b hai dung dch

chun lm vic WS1 v WS2 ln lt c nng RIF chnh xc khong 20,0 v 2,0

g/mL trong huyt tng trng.

+ Dung dch chun lm vic WS1 (20,0 g/mL): Ly 0,5 mL dung dch

chun gc, bc hi dung mi bng kh N2. Ha tan cn trong 5,0 mL huyt tng

trng cha Vitamin C (1mg/mL)

+ Dung dch chun lm vic WS2 (2,0 g/mL): Ht 1,0 mL WS1 ha tan

trong 10,0 mL huyt tng cha Vitamin C (1mg/mL)

T dung dch chun lm vic tin hnh pha long vi huyt tng trng nh

bng 2.3 thu c mu chun c nng t 0,2 n 20,0 g/mL

Bng 2.3: Chun b ng chun

Nng

(g/mL) 0 0,2 0,5 1,0 2,0 4,0 8,0 16,0 20,0

V WS2 (L) - 50 125 250 500 - - - -

V WS1 (L) - - - - - 100 200 400 500

VHT trng

(L) 500 450 375 250 - 400 300 100 -

Chun b cc mu QC:

Chun b tng t mu chun. Mu QC chun b t dung dch chun gc c

lp vi dung dch chun gc dng pha mu chun. Theo quy nh ca hng

dn, mu QC gm 3 loi: Mu LQC c nng bng khong 2 3 ln nng mu

LLOQ; mu MQC c nng khong 50% nng Cmax v mu HQC c nng

khong 70 80 % mu ULOQ.

20

T dung dch chun gc, chun b hai dung dch chun lm vic WSQC 1 v

WSQC 2 ln lt c nng RIF chnh xc khong 20,0 v 4,0 g/mL trong huyt

tng trng cha Vitamin C (1 mg/mL)

+ Chun b tng t nh mu chun lm vic WS1 c c dung dch

WSQC1 c nng RIF khong 20 g/mL trong huyt tng trng cha Vitamin C

(1 mg/mL)

+ WSQC2 (4,0 g/mL): Ht 1,0 mL WSQC1 ha tan trong 5,0 mL huyt

tng trng cha Vitamin C (1 mg/mL)

Chun b cc mu QC c nng ln lt l: LQC = 0,6 g/mL; MQC = 9,0

g/mL; HQC = 18,0 g/mL c trnh by bng 2.4

Bng 2.4: Chun b mu kim tra

QC Nng (g/mL) V WSQC2 (L) V WSQC1 (L) VHT trng (L)

LQC 0,6 75 - 425

MQC 9,0 - 225 275

HQC 18,0 - 450 50

Chun b mu gii hn nh lng di: Mu LLOQ 0,2 g/mL c chun

b nh bng 2.5

Bng 2.5: Chun b mu gii hn nh lng di

Nng (g/mL) V WS2 (L) VHT trng (L)

LLOQ 0,2 50 450

2.3.2. Xy dng phng php phn tch

2.3.2.1.Xc nh iu kin sc k

Kho st phng php phn tch trn h thng my sc k lng HPLC

Shimadzu detector UV-VIS, vi cc iu kin sc k khc nhau nh:

+ Cc ct pha o khc nhau.

+ Pha ng c thnh phn, t l khc nhau.

+ La chn cht thch hp lm ni chun.

21

T kt qu kho st, la chn nhng iu kin sc k thch hp c th tin

hnh nh lng rifampicin trong huyt tng bng phng php HPLC vi

detector UV-VIS.

2.3.2.2.Xy dng quy trnh x l mu huyt tng

Da vo cc ti liu tham kho, tin hnh kho st phng php x l mu,

chit tch rifampicin t huyt tng trn cc mu chun rifampicin ha tan trong

huyt tng trng bng cc phng php nh:

+ Ta protein vi cc tc nhn gy ta l: MeOH, HClO4.

+ Chit lng - lng.

2.3.3. Thm nh phng php phn tch

2.3.3.1. Tnh c hiu - chn lc

Ch tiu c hiu chn lc c quy nh ti mc 1.3.2.1

Tin hnh phn tch sc k cc mu:

Huyt tng trng vi t nht 6 mu c ngun gc khc nhau

Huyt tng trng c pha chun RIF nng LLOQ (0,2 g/mL) v IS.

Huyt tng trng c pha chun RIF nng khong gia ng chun (9

g/mL) v IS

So snh cc sc k thu c t vic phn tch cc mu trn.

2.3.3.2. ng chun v khong nng tuyn tnh

Pha long dung dch chun lm vic vi huyt tng trng c cc dung

dch c nng nh sau: 0,2 0,5 1,0 2,0 4,0 8,0 16,0 20,0 g/mL

Tin hnh phn tch cc mu. T p ng pic ca RIF v IS ti cc nng

tng ng, xy dng phng trnh hi quy gia t l p ng pic ca RIF/IS v

nng RIF c trong mu v xc nh h s tng quan r. Tnh li nng RIF cc

mu theo phng trnh hi quy, xc nh li ng so vi gi tr thc ca tng

nng .

2.3.3.3. ng

Chun b cc l mu QC bao gm: LQC, MQC v HQC, mi l gm 6 mu

c lp c cng nng .

22

Tin hnh sc k cc l mu QC. Xc nh kt qu nh lng cc mu QC

da vo ng chun pha trong huyt tng trng, tin hnh trong cng iu kin.

Xc nh ng ca phng php bng cch so snh gi tr nh lng c vi gi

tr thc c trong mu.

2.3.3.4. lp li trong ngy v gia cc ngy

Chun b cc l mu QC tng t nh mc xc nh ng. Xc nh kt

qu nh lng cc mu QC theo ng chun pha trong huyt tng trng, tin

hnh trong cng iu kin.

Xc nh lp li trong ngy bng cch tnh ton lch CV% gia gi tr

cc ln nh lng ca mi nng c phn tch trong cng mt ngy

Xc nh lp li gia cc ngy bng cch tnh ton lch CV% gia gi

tr cc ln nh lng ca mi nng c phn tch trong t nht 3 ngy khc nhau.

2.3.3.5. Gii hn nh lng di

Tin hnh sc k 6 mu chun c nng thp nht trong khong nng

tuyn tnh. Ghi li p ng pic ca cc mu chun. Da theo ng chun pha trong

huyt tng trng tin hnh trong cng iu kin, tnh li nng ca cc mu

LLOQ. Xc nh ng ca phng php bng cch so snh gi tr nh lng

c vi gi tr thc c trong mu. Xc nh chnh xc bng cch tnh ton lch

CV% gia gi tr cc ln nh lng.

2.3.3.6. T l thu hi

Tin hnh sc k cc l mu QC bao gm LQC, MQC v HQC, mi l gm

t nht 5 mu c lp. Song song tin hnh sc k cc mu chun pha trong dung mi

pha mu c nng tng ng.

Xc nh t l thu hi ca RIF v IS bng cch so snh kt qu p ng ca

RIF v IS trong cc mu QC c qua x l vi p ng ca RIF v IS trong mu chun

pha trong dung mi pha mu (khng qua x l).

2.3.3.7. n nh ca huyt tng

Xc nh n nh ca RIF trong huyt tng sau ba chu k ng r

ng v trong qu trnh x l mu trn cc mu LQC v HQC.

23

n nh ca mu huyt tng sau 3 chu k ng r ng: Bo qun

mu nhit -800C v r ng nhit phng. Sau khi tan chy hon

ton, mu tr li ng lnh trong 12 24 gi. Lp li chu k ng r ng 2 ln

na. Sau 3 chu k ng r, tin hnh phn tch xc nh nng RIF c trong

mu. So snh vi kt qu xc nh nng RIF c trong cc mu tin hnh phn

tch ngay sau khi pha (nng ban u). Nng RIF trong mu sau 3 chu k ng

- r phi sai khc vi nng ban u khng qu 15% v gi tr CV% gia cc kt

qu nh lng mi nng phi nh hn hoc bng 15%.

n nh ca mu huyt tng nhit phng trong thi gian ngn:

Phn tch mu huyt tng 2 nng LQC v HQC sau khi r ng v

nhit phng mt thi gian nht nh (5 gi), so snh vi nng mu c x l

ngay sau khi r ng. Nng RIF trong mu c x l sau khi mt thi gian

nht nh nhit phng phi sai khc vi nng mu x l ngay khng qu

15% v gi tr CV% gia cc kt qu nh lng mi nng phi nh hn hoc

bng 15%.

n nh ca mu huyt tng nhit -800C trong thi gian di: Bo

qun mu huyt tng 2 nng LQC v HQC nhit -800C, phn tch

mu ti thi im ban u v sau tng khong thi gian bo qun nht nh (6,

14, 21 v 28 ngy). So snh nng ca mu huyt tng sau khi bo qun vi

nng ca mu ti thi im ban u. Nng RIF trong mu sau khi bo qun

mt thi gian nht nh phi sai khc vi nng ban u khng qu 15% v gi tr

CV% gia cc kt qu nh lng mi nng phi nh hn hoc bng 15%.

24

200 300 400 500 600 700 nm

-25

0

25

50

75

100

125

150

175

200

mAU 4.21/ 1.00

212

295

393

743

723

238

336

476

735

769

CHNG 3: THC NGHIM, KT QU V BN LUN 3.1. XY DNG PHNG PHP NH LNG RIFAMPICIN TRONG

HUYT TNG

3.1.1. Kho st v la chn iu kin sc k v ni chun

3.1.1.1. Kho st bc sng hp th

Qut ph hp th ca mu chun RIF dung dch trong nc, ta c ph ca

RIF nh hnh 3.1 sau:

Hnh 3.1: Ph hp th UV-VIS ca RIF

Da vo ph hp th ca RIF trn, RIF c 3 cc i hp th ti = 238

nm, 336 nm v 476 nm. = 238 nm, cc i gn im khng n nh ca ph hp

th. = 476 nm hp th ca RIF thp. Do , kt hp tham kho mt s ti

liu v phng php nh lng RIF, chng ti la chn bc sng ph hp tin

hnh chy sc k l = 336 1 nm. Qua qu trnh kho st iu kin sc k, nhn

thy mu chun p ng tt ti bc sng = 337 nm.

25

3.1.1.2. La chn ni chun

Tham kho phng php nh lng ca cc tc gi Co, B G, Wagan, E G,

Lagdameo, E E, Molinar, Adelaida M tin hnh, s dng dipyridamol lm

cht IS trong nh lng RIF.

Cu trc ha hc ca dipyridamol:

c im ca dipyridamol l mt hp cht thm a vng, c kh nng hp

th t ngoi kh kin v gn vi max ca RIF. ng thi dipyridamol l mt hp

cht c mu, c kh nng tan tt trong dung mi MeOH, thun li cho qu trnh

kho st iu kin sc k nh lng cng RIF pha trong dung mi MeOH [17].

Vi iu kin sn c ca phng th ngim, chng ti la chn dipyridamol ha tan

trong MeOH vi nng chnh xc khong 1 mg/mL lm cht IS nh lng

RIF trong huyt tng bng HPLC.

3.1.1.3. La chn pha tnh

Trong sc k phn b pha o, ct C8 v C18 l hai loi ct hay c s

dng phn tch. Do RIF l mt cht t phn cc nn i hi pha tnh rt t phn

cc ng thi ct C8 phn cc hn ct C18 nn RIF kh tch trn ct C8. Kt hp

tham kho mt s ti liu nh lng rifampicn trong dch sinh hc [9], [11], [12],

[18] chng ti tin hnh kho st trn ct Gemini C18e (250 x 4,6 mm; 5 m) vi

bo v ct Rp18 (4 x 3 mm; 5m) thy ct c kh nng tch tt v chn lc

rifampicin ra khi mu phn tch.

26

3.1.1.4. La chn nhit autosampler

Kho st nhit autosampler nhit phng v 40C. Do RIF khng bn,

d b nh hng bi nhit nn trong qu trnh phn tch phi tin hnh nhit

thp gim kh nng thy phn ca RIF. Qua kho st chng ti thy 40C kh

nng tch v p ng ca rifampicin tt hn so vi nhit phng.

3.1.1.5. La chn pha ng

Do tnh cht ha l ca RIF: pH kim RIF d b oxy ha bi oxy khng kh

v pH acid mnh d b thy phn nn chng ti la chn pH ca dung dch m

trong khong pH acid gn trung tnh (t 4,0 5,0) m bo RIF khng b thy

phn v khng b oxy ha. Kt hp vi tham kho mt s ti liu, chng ti chn

pH = 4,5 l pH ca dung dch m trong ngin cu ny. Chng ti tin hnh kho

st pha ng vi t l khc nhau ca dung mi hu c vi dung dch m phosphate

pH 4,5.

Trong sc k phn b pha o, MeOH l dung mi c s dng ph bin do

gi thnh thp, c kh nng ra gii tt v th bc u chng ti tin hnh kho st

thnh phn pha ng gm MeOH : dung dch m phosphat 0,02M pH 4,5 vi cc

t l khc nhau. t l 65 : 35 chng ti thu c pic ca RIF v IS nhn, cn i.

Sc k ca RIF v IS vi h MeOH : m phosphat pH 4,5 = 65 : 35 c trnh

by hnh 3.2 v 3.3.

0.0 2.5 5.0 7.5 10.0 min

0

10

20

30

40

50

mAU337nm4nm (1.00)

Rifa

mpi

cin/

5.70

9

0.0 2.5 5.0 7.5 10.0 min

-2.5

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

mAU337nm4nm (1.00)

IS/1

0.44

6

27

Hnh 3.2: SK chun RIF Hnh 3.3: SK chun IS

Nh vy, vi h MeOH m phosphat pH 4,5 (65:35) thi gian lu ca

RIF l 5,70 pht, ca IS l 10,40 pht. Thi gian phn tch vi h trn l hi di nn

gim thi gian phn tch, chng ti cho thm MeCN vo thnh phn pha ng ri

tin hnh kho st.

Qua kho st, chng ti nhn thy khi s dng h pha ng MeOH - MeCN -

m phosphat (48,8: 16,2: 35) hai pic RIF v IS u nhn cn xng, xut hin pic

ca RIF vi thi gian lu 5,8 pht v pic ca IS vi thi gian lu 8,8 pht l ph

hp, gim c thi gian phn tch mt mu. Sc k mu chun RIF v IS c

trnh by hnh 3.4 v 3.5

0.0 2.5 5.0 7.5 min

0

25

50

75

100

125

mAU337nm,4nm (1.00)

Rifa

mpi

cin/

5.84

3

0.0 2.5 5.0 7.5 min-10

-5

0

5

10

15

20

25

30

35

mAU337nm,4nm (1.00)

/8.7

56

Hnh 3.4: SK chun RIF Hnh 3.5: SK chun IS

Nh vy, tch hon ton v chn lc RIF v IS ra khi mu phn tch

ng thi m bo thi gian phn tch mt mu khng qu di chng ti thy h

pha ng MeOH - MeCN - dung dch m phosphat pH 4,5 (dung dch KH2PO4

0,02M, iu chnh pH bng dung dch H3PO4 1M) vi t l 48,8: 16,2: 35 l ph

hp.

3.1.1.6. La chn tc dng

Chng ti tin hnh kho st tch RIF v IS 2 tc dng l 1,5 mL/pht

v 2,0 mL/pht thy:

28

- Tc dng 1,5 mL/pht pic RIF xut hin lc 5,70 pht v pic IS xut

hin lc 10,40 pht.

- c thi gian phn tch hp l ng thi cc pic tch ri nhau chng ti

chn tc dng 1,5 mL/pht cho khong thi gian t 0 6,50 pht v tc dng

2,0 mL/pht cho khong thi gian t 6,51 9,0 pht. Kt qu thu c thi gian lu

ca RIF l 5,7 pht v ca IS l 7,8 pht. Sc k ca chun RIF v IS thu c

nh hnh 3.6

0.0 2.5 5.0 7.5 min

-2.5

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

mAU

0

10

20

30

40

50

60

70

80

90

MPaT.Flow(Method)337nm,4nm (1.00)

RIF

A/5.63

8

IS/7.784

Hnh 3.6: SK chun RIF v IS vi tc dng khc nhau

Nh vy, chng ti la chn tc dng 1,5 mL/pht cho khong thi gian 0

6,50 pht v tc dng 2.0 mL/pht cho khong thi gian 6.51 9,0 pht c

thi gian phn tch ph hp.

3.1.2. Kho st v tm iu kin x l mu c Rifampicin

3.1.2.1. Xy dng phng php chit tch

Da trn tnh cht l ha ca RIF, tham kho mt s ti liu v iu kin k

thut hin c, chng ti tin hnh chit tch RIF theo phng php kt ta protein

vi mt s tc nhn thng dng gy ta khc nhau nh cc dung mi: MeOH

hoc MeCN, tc nhn ta l acid HClO4.

- Tc nhn gy ta acid HClO4 l acid mnh. Ch cn s dng mt lng nh

gy kt ta protein trong huyt tng, mu huyt tng khng b pha long.

Nhng theo tnh cht l ha ca RIF, d b thy phn pH acid nn nu s dng tc

29

nhn gy ta ny s lm RIF b bin i, gy nh hng n qu trnh phn tch. Do

khng s dng tc nhn gy ta protein l acid mnh.

- Tc nhn gy ta l MeOH: cn phi s dng mt lng dung mi ln, gp

2,0 3,0 ln lng mu ta ht protein trong mu nn mu b pha long. RIF v

dipyridamol u ha tan tt trong MeOH, mc p ng ca RIF v IS ln nn ta

c th s dng phng php ny tin hnh x l mu.

Tin hnh ta protein bng dung mi MeOH vi cc th tch khc nhau:

Qui trnh 1: 0,5 mL mu thm 50 L dung dch IS, lc xoy. Thm

1,5 mL MeOH. Lc xoy, ly tm 10000 vng/pht trong 5 pht. Ht

lp dung mi, tim sc k.

Qui trnh 2: 0,5 mL mu thm 50 L dung dch IS, lc xoy. Thm

1,0 mL MeOH. Lc xoy, ly tm 10000 vng/pht trong 5 pht. Ht

lp dung mi, tim sc k.

Sc k ca mu huyt tng trng ta bng MeOH v SK mu huyt

tng trng c thm RIF v IS ta protein bng cc th tch MeOH khc nhau c

th hin hnh 3.7, 3.8 v 3.9

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 min-30

-20

-10

0

10

20

30

40

50

mAU337nm,4nm (1.00)

(Rifa

mpi

cin)/

5.70

0

(Dip

yrid

amol

e)/7

.600

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 min-2.5

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

mAU337nm,4nm (1.00)

Rifa

mpi

cin/5

.800

Dip

yrid

amol

/7.6

62

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 min

0.0

2.5

5.0

7.5

10.0

12.5

mA U337nm,4nm (1.00)

Rifa

mpi

cin/5

.736

Dip

yrid

amol

/7.5

78

Hnh 3.7:

SK mu HT trng

Hnh 3.8:

SK mu HT trng thm

RIF v IS ta 1,5 mL MeOH

Hnh 3.9:

SK mu HT trng thm

RIF v IS ta 1,0 mL MeOH

30

Nhn xt: Trong s cc phng php ta protein th phng php ta protein

bng dung mi MeOH u ta ht protein trong qui trnh 2 ta bng 1,0 mL

MeOH cho p ng pic cao hn, ng thi khng lm mu b pha long qu nhiu.

Do , chng ti la chn phng php ta protein bng dung mi MeOH

vi th tch 1,0 mL x l mu va cho pic RIF tch khi cc pic tp va cho

p ng pic tt nht.

3.1.2.2. Kho st s nh hng ca Vitamin C trong qu trnh x l mu

Do RIF nhy cm vi nh sng v d b oxy ha nn chng ti kho st s n

nh ca RIF trong dung mi c cha Vitamin C, l 1 cht chng oxy ha v dung

mi khng cha Vitamin C.

Pha RIF nng 6 g/mL gm:

+ 1 mu pha trong MeOH c cha Vitamin C 1,0 mg/mL.

+ 1 mu pha trong MeOH khng c Vitamin C.

Kt qu: Pic ca mu RIF chun trong MeOH c cha Vitamin C 1,0 mg/mL

nhn v cn i hn mu RIF chun trong MeOH khng c Vitamin C. Sc k

ca chun RIF trong MeOH cha Vitamin C 1,0 mg/mL thu c nh hnh 3.10

0.0 2.5 5.0 7.5 10.0 min

0

10

20

30

40

50

mAU337nm4nm (1.00)

Rifa

mpi

cin/

5.70

9

Hnh 3.10: SK RIF chun trong MeOH cha Vitamin C 1,0 mg/mL

T kt qu kho st chng ti quyt nh s dng Vitamin C trong dung dch

chun RIF pha trong MeOH.

31

3.1.3. Qui trnh phn tch RIF trong huyt tng T kt qu thc nghim, chng ti la chn c qui trnh x l mu v

iu kin sc k c th tch c RIF khi cc pic tp c trong mu huyt tng

nh sau:

- Quy trnh x l mu:

+ Mu huyt tng r ng nhit phng

+ Ngay sau khi r ng, ly 0,5 mL huyt tng thm Vitamin C (nng

khong 1,0 mg/mL), thm 50 L dung dch ni chun v 1,0 mL MeOH

+ Lc xoy 15 giy, ly tm 10000 vng/pht x 5 pht, ht lp dung mi, tim

sc k.

- iu kin sc k:

+ Ct sc k: Rp18, 250 x 4,6 mm, 5 m. Bo v ct Rp18, 4 x 3 mm, 5m.

+ Pha ng: MeOH MeCN m phosphat 0,02M pH 4,5 t l 48,8 : 16,2 :

35

+ Tc dng: 0,01 6,50 pht: 1,5 mL/pht

6,51 9,51 pht: 2,0 mL/pht

+ Detector: UV, = 337 nm.

+ Th tch tim: 50 L.

+ Autosampler: 40C.

+ Ton b qu trnh x l mu v phn tch sc k c tin hnh trong iu

kin trnh nh sng.

3.2. THM NH PHNG PHP PHN TCH m bo phng php kho st trn l thch hp cho nh lng RIF

trong huyt tng, chng ti tin hnh thm nh phng php theo hng dn

thm nh phng php phn tch sinh hc ca FDA v cc qui nh trong dc

in M gm tnh c hiu/chn lc, ng, lp li trong ngy v gia cc

ngy, khong tuyn tnh, gii hn nh lng di, t l thu hi, n nh.

Do rifampicin nhy cm vi nh sng v d b oxy ha nn ton b qu trnh

xy dng v thm nh phng php phn tch u c tin hnh trn cc mu t

to c cha vitamin C trong iu kin trnh nh sng trc tip.

32

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 min

-2.5

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0mAU

0

10

20

30

40

50

60

70

80

90

MPaT.Flow(Method)337nm,4nm (1.00)

RIF

A/5.

670

IS/7

.843

3.2.1. Tnh chn lc Tin hnh nh mc 2.3.3.1 , x l mu v phn tch vi iu kin sc k nh

mc 3.1.3. Kt qu thu c nh sau:

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 min

-2.5

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0mAU

0

10

20

30

40

50

60

70

80

90

MPaT.Flow(Method)337nm,4nm (1.00)

(RIF

A)/5

.600

(IS)/7.

600

Hnh 3.11: Sc k mu huyt tng trng

Hnh 3.12: Sc k mu huyt tng trng c pha chun RIF (nng 0,2

g/mL) v IS.

33

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 min

-2.5

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

mAU

0

10

20

30

40

50

60

70

80

90

MPaT.Flow(Method)337nm,4nm (1.00)

RIF

A/5.

606

IS/7

.831

Hnh 3.13: Sc k mu huyt tng trng c pha chun RIF (nng 9,0

g/mL) v IS.

Nhn xt: Trn sc k ca

- Hnh 3.12 ta thy: trong khong thi gian t 5 6 pht v 7 8 pht trong

mu huyt tng trng khng thy xut hin pic.

- Hnh 3.13 ta thy: mu RIF chun nng 0,2 g/mL trong huyt tng

trng xut hin 1 pic c thi gian lu khong 5,7 pht v mu IS xut hin 1 pic c

thi gian lu khong 7,8 pht

- Hnh 3.14 ta thy: mu RIF chun nng 9,0 g/mL trong huyt tng

trng xut hin 1 pic c thi gian lu khong 5,7 pht, pic ny ln hn so vi pic

RIF chun nng 0,2 g/mL (hnh 3.3) v mu IS xut hin 1 pic c thi gian lu

khong 7,8 pht bng vi pic IS hnh 3.3.

Pic RIF trn cc sc k cn i, r nt, c thi gian lu khong 5,7 pht

v tch ring bit vi IS v cc thnh phn tp c trong mu. Phng php phn

tch m bo nhn din, phn bit c RIF v khng b nh hng bi cc tp

c trong huyt tng.

Theo qui nh, phng php phn tch c c hiu chn lc th ngoi kh

nng nhn din v phn bit c hot cht nh xc nh trn, p ng ca

mu trng ti thi im trng vi thi gian lu ca RIF v IS phi t cc yu cu

34

nh nu mc 1.3.2.1. Kt qu xc nh nh hng ca mu trng ti thi im

trng thi gian lu ca RIF v IS c trnh by bng 3.1

Bng 3.1: nh hng ca mu trng ti thi im trng thi gian lu (tR ) ca RIF v IS

p ng mu trng ti tR = tRIF p ng mu trng ti tR = tIS

TT Mu trng

(mAU.s)

Mu LLOQ

(mAU.s)

Mu trng

(mAU.s)

Mu IS

(mAU.s)

1 0 4453 0 167742

2 0 4309 0 165712

3 0 4007 0 168551

4 0 4252 0 171227

5 0 4253 0 171360

6 0 4327 0 169799

T l p ng pic (%) 0 0

Kt qu cho thy ti thi im trng vi thi gian lu ca RIF (5,7 pht) v

IS (7,8 pht) p ng ca mu trng u l 0%. Nh vy cc s liu thc nghim

chng minh phng php c c hiu v chn lc i vi RIF.

3.2.2. ng chun v khong tuyn tnh

Phn tch cc mu chun RIF trong huyt tng trng c nng 0,2 0,5

1,0 2,0 4,0 8,0 16,0 20,0 g/mL vi 08 mu chun, mi nng phn tch

hai ln, x l mu vi iu kin sc k nh mc 3.1.3. Xc nh s tng quan gia

nng RIF trong huyt tng v t l din tch pic RIF/IS o c. Kt qu c

trnh by bng 3.2

35

Bng 3.2: S tng quan gia nng RIF trong huyt tng v t l p ng pc:

p ng (mAU.s) p ng (mAU.s)

Mu Nng

(g/mL) RIF-

CC1 IS-CC1

T l 1 RIF-

CC2 IS-CC2

T l 2

Std1 0,204 4173 173966 0,024 3969 176090 0,023

Std2 0,510 8931 169136 0,053 10440 175396 0,060

Std3 1,019 20328 169941 0,120 21168 173738 0,122

Std4 2,038 43758 173303 0,252 44773 173677 0,258

Std5 4,076 82046 173304 0,473 86490 177834 0,486

Std6 8,153 168020 175522 0,957 171371 175513 0,976

Std7 16,306 347543 177988 1,953 348275 173508 2,007

Std8 20,382 444528 175277 2,536 450325 173410 2,597

Phng trnh hi

quy

Y1 = 0,119 X1 0,001

R = 0,9998

Y2 = 0,123 X2 - 0,003

R = 0,9998

(Y: t l din tch pic RIF/IS; X: nng RIF)

Kt qu bng 3.2 cho thy, c mi tng quan tuyn tnh gia nng

RIF c trong mu v t l din tch pic RIF/IS thu c (ng chun c h s

tng quan r xp x bng 1 (r = 0,9998). T phng trnh hi quy tuyn tnh va

thit lp xc nh li nng RIF c trong mu. Kt qu thu c khi thm nh

ng chun c trnh by bng 3.3

36

Bng 3.3: Kt qu thm nh ng chun

STT Nng thc

(g/mL)

Nng tm thy(a)

(g/mL) ng(b) (%) t/ Khng t

0,212 104,0 t 1

0,204

0,205 100,4 t

0,454 89,2 t 2 0,510

0,504 99,0 t

1,016 99,7 t 3 1,019

1,010 99,1 t

2,134 104,7 t 4 2,038

2,112 103,6 t

3,993 98,0 t 5 4,076

3,965 97,3 t

8,063 98,9 t 6 8,153

7,937 97,4 t

16,437 100,8 t 7 16,306

16,294 99,9 t

21,346 104,7 t 8

20,382

21,074 103,4 t

(a) tnh t phng trnh hi qui (b) % so vi nng thc

Nh vy, tt c cc im ca ng chun gi tr nng tnh li t ng

chun so vi nng thc t c trong mu u c ng nm trong khong t 85

115%, ring im thp nht ca ng chun sai s khng vt qu 20% (4% v

0,4%). Do trong khong nng t 0,2 20,0 g/mL c s tng quan tuyn

tnh cht ch gia nng RIF trong huyt tng vi t l din tch pic RIF/IS thu

c.

37

3.2.3. Gii hn nh lng di

Tin hnh thm nh vi mu chun RIF nng khong 0,2 g/mL vi 6

mu c lp. Tin hnh tng t nh mc 2.3.3.5. Kt qu xc nh gi tr LLOQ

ca phng php c trnh by bng 3.4:

Bng 3.4: Kt qu thm nh gii hn nh lng di (LLOQ)

Mu trng Mu chun (0,204 g/mL)

Stt p ng

(mAU.s)

RIF

(mAU.s)

IS

(mAU.s)

Nng tm

thy(a) (g

/mL)

ng(b)

(%)

t/

Khng

t

1 0 4453 167742 0,233 93.6 t

2 0 4309 165712 0,229 119.4 t

3 0 4007 168551 0,210 96.0 t

4 0 4252 171227 0,219 116.5 t

5 0 4253 171360 0,219 96.1 t

0 4327 169799 0,225 109.6 t

TB 0,223 109,2

CV (%) 3,7 3,7 t

(a) tnh t phng trnh hi qui (b) % so vi nng thc

Nhn xt: Kt qu cho thy phng php c:

- ng nm trong khong t 80 120% so vi nng thc (93,6

119,4%)

- chnh xc vi gi tr CV% 20% (3,7%)

Nh vy mu chun cha RIF c nng 0,2 g/mL p ng yu cu LLOQ

v ng, chnh xc ca phng php phn tch trong dch sinh hc.

3.2.4. ng lp li trong ngy v khc ngy

3.2.4.1. ng, lp li trong ngy

Pha cc mu LQC, MQC, HQC nh mc 2.3.1. Mi loi QC gm t nht

5 mu c lp c cng nng . Tin hnh x l mu v sc k theo cc iu

38

kin la chn. Kt qu xc nh ng, lp li trong ngy c trnh

by bng 3.5:

Bng 3.5: Kt qu thm nh ng, lp li trong ngy

LQC (0,603g/mL) MQC (9,038g/mL) HQC (18,075g/mL)

Mu

T l

din

tch

pc

RIF/IS

Nng

(a)

(g/mL)

ng(b)

(%)

T l

din tch

pc

RIF/IS

Nng

(a)

(g/mL)

ng(b)

(%)

T l

din

tch pc

RIF/IS

Nng

(a)

(g/mL)

ng(b)

(%)

1 0,078 0,593 98,4 1,185 9,741 107,8 2,320 19,113 105,7

2 0,078 0,593 98,5 1,163 9,557 105,7 2,332 19,214 106,3

3 0,077 0,585 97,1 1,132 9,301 102,9 2,258 18,606 102,9

4 0,078 0,593 98,5 1,140 9,366 103,6 2,283 18,808 104,1

5 0,076 0,579 96,0 1,154 9,480 104,9 2,338 19,260 106,6

6 0,079 0,600 99,5 1,104 9,065 100,3 2,142 17,643 97,6

TB 0,590 98,0 9,419 104,2 18,774 103,9

CV (%) 1,2 2,5 3,2

(a): tnh t phng trnh hi qui (b): % so vi nng thc

Kt qu cho thy phng php nh lng c ng tt nm trong khong

96,0 107,8%, lch nm trong gii hn cho php 15%, lp li trong ngy

vi gi tr CV% nh (1,2 3,2%), p ng yu cu v ng, chnh xc ca

phng php phn tch trong dch sinh hc.

3.2.4.2. ng, lp li khc ngy

Tin hnh phn tch cc mu LQC, MQC, HQC c chun b tng t nh

xc nh ng, lp li trong ngy theo phng php xy dng ti cc ngy

khc nhau. Xc nh nng RIF trong cc mu QC da vo ng chun tin

hnh song song trong tng ngy. Kt qu xc nh ng lp li khc ngy ca

phng php c trnh by bng 3.6

39

Bng 3.6: Kt qu thm nh ng, lp li gia cc ngy

LQC ( 0,6 g/mL) MQC ( 9,0 g/mL) HQC ( 18,0 g/mL)

Ngy Nng (a)

(g/mL)

ng(b)

(%)

Nng (a)

(g/mL)

ng(b)

(%)

Nng (a)

(g/mL)

ng(b)

(%)

0,668 104,1 10,217 106,2 19,591 101,8

0,640 99,8 9,002 93,5 19,588 101,8

0,619 96,5 8,940 92,9 19,682 102,2

0,608 94,7 9,341 97,0 19,568 101,7

0,595 92,7 9,018 93,7 19,537 101,5

I

0,588 91,6 9,340 97,0 19,399 100,8

0,593 98,4 9,741 107,8 19,113 105,7

0,593 98,5 9,557 105,7 19,214 106,3

0,585 97,1 9,301 102,9 18,606 102,9

0,593 98,5 9,366 103,6 18,808 104,1

0,579 96,0 9,480 104,9 19,260 106,6

II

0,600 99,5 9,065 100,3 17,643 97,6

0,550 89,0 8,526 92,0 17,817 96,2

0,583 94,5 8,633 93,2 17,678 95,4

0,625 101,2 8,799 95,0 17,288 93,3

0,544 88,0 9,026 97,4 17,479 94,3

0,591 95,6 8,760 94,6 17,606 95,0

III

0,591 95,7 8,920 96,3 17,424 94,0

TB (%) 96,2 98,6 100,1

CV (%) 4,3 5,4 4,4

40

Kt qu thc nghim cho thy c 3 nng (0,6; 9,0; 18,0 g/mL), phng

php u cho ng trong khong 85% n 115%, lp li khc ngy vi gi

tr CV% 15% (4,3 5,4%) chng t phng php c nghin cu c

ng, chnh xc cao, p ng yu cu i vi phng php dng trong dch

sinh hc.

3.2.5. T l thu hi ca phng php

3.2.5.1.T l thu hi ni chun

X l cc l mu QC theo qui trnh la chn. Tin hnh phn tch xc nh

din tch pic ca IS. Song song, xc nh din tch pic ca IS trong mu chun pha

trong dung mi pha mu (khng qua x l).

Kt qu xc nh t l thu hi ca IS c trnh by bng 3.7

Bng 3.7: Kt qu thm nh t l thu hi IS

p ng IS (mAU.s) STT

Huyt tng Dung mi

1 168477 165217

2 169264 169659

3 169032 170108

4 163877 168898

5 167941 170205

6 169999 168031

TB 168098 168686

CV (%) 1,3 1,1

T l thu hi (%) 99,7

Kt qu thc nghim cho thy, t l thu hi ca ni chun t 99,7% (tha

mn iu kin nm trong khong 30 110%) v CV% nng ca IS u 15%

(1,3 v 1,1%). Do vy qui trnh x l mu huyt tng c nghin cu l ph

hp chit tch cht ni chun.

41

3.2.5.2.T l thu hi RIF

Chun b cc l mu QC nh mc 2.3.1 v x l mu chun RIF pha trong

huyt tng theo phng php xy dng. Tin hnh sc k, xc nh nng

RIF c trong cc mu. Song song, nh lng mu chun pha trong dung mi pha

mu (khng qua x l).

Kt qu xc nh t l thu hi ca RIF c trnh by bng 3.8

Bng 3.8: Kt qu thm nh t l thu hi RIF

p ng RI F (mAU.s)

LQC (0,6 g/mL) MQC (9,0 g/mL) HQC (18,0 g/mL) STT

Huyt

tng

Dung

mi

Huyt

tng

Dung

mi

Huyt

tng

Dung

mi

1 13614 12661 198897 191051 395893 388350

2 13280 12541 201631 191605 399660 385584

3 13188 12540 201283 189818 397386 377560

4 13084 12588 203755 190974 400252 382496

5 12864 12653 196816 188198 405840 384346

6 13259 12938 192219 188601 397722 382187

TB 13215 12654 199100 190041 399459 383421

CV (%) 1,9 1,2 2,1 0,7 0,9 1,0

T l thu hi

(%) 104,4 104,8 104,2

Kt qu thc nghim cho thy c 3 khong nng (0,6; 9,0; 18,0

g/mL), phng php u cho hiu sut chit nm trong khong 30 110% (

104,4 ; 104,8 ; 104,2%), gi tr CV% mi nng ca RIF u 15% (t 0,7

2,1%). Do phng php x l mu c xy dng l ph hp chit tch

RIF t huyt tng.

42

3.2.6. n nh ca mu huyt tng

3.2.6.1. n nh ca mu huyt tng sau 3 chu k ng r ng

Pha cc mu LQC v HQC nh mc 2.3.1 v tin hnh nh mc 2.3.3.7. Kt

qu xc nh nng RIF trong mu LQC, HQC sau 3 chu k ng r v cc mu

tin hnh phn tch ngay sau khi pha (nng ban u) c trnh by trong bng 3.9:

Bng 3.9: Kt qu n nh ca mu huyt tng sau 3 chu k ngr

Nng (g/mL)

Mu LQC (0,6 g/mL) Mu HQC (18,0 g/mL) Mu

Ban u Sau 3 chu k ng- r Ban u Sau 3 chu k ng- r

1 0,612 0,561 17,487 18,159

2 0,549 0,599 18,520 17,884

3 0,574 0,635 18,281 17,530

4 0,582 0,502 17,832 17,642

5 0,551 0,499 18,310 17,282

6 0,567 0,502 18,605 17,765

TB 0,573 0,550 18,172 17,710

CV (%) 4,1 10,6 2,4 1,7

lch (%) -4,0 -2,5

Kt qu cho thy, nng RIF trong mu sau 3 chu k ng r ng u

sai khc so vi nng ban u khng qu 15% (LQC: -4,0% v HQC: -2,5%) v

gi tr CV% gia cc kt qu nh lng u 15%. Nh vy mu huyt tng n

nh sau 3 chu k ng r ng.

3.2.6.2. n nh mu huyt tng nhit phng trong thi gian ngn

Pha cc mu LQC v HQC nh mc 2.3.1 v tin hnh nh mc 2.3.3.7. Kt

qu xc nh nng RIF trong mu LQC, HQC nhit phng sau 5 gi v

nng RIF c x l ngay sau khi r ng c trnh by trong bng 3.10

43

Bng 3.10: Kt qu n nh ca mu huyt tng nhit phng trong thi

gian ngn:

Nng (g/mL)

Mu LQC (0,6 g/mL) Mu HQC (18,0 g/mL) Mu

X l ngay Sau 5 gi X l ngay Sau 5 gi

1 0,586 0,551 16,583 16,380

2 0,578 0,568 16,649 16,108

3 0,576 0,566 15,933 15,986

4 0,570 0,575 16,558 15,903

5 0,587 0,577 16,167 16,363

6 0,562 0,573 16,541 16,440

TB 0,577 0,568 16,405 16,197

CV (%) 1,7 1,6 1,8 1,4

lch (%) -1,4 -1,3

Kt qu cho thy, nng RIF trong mu c x l sau khi 5 gi nhit

phng u sai khc so vi nng mu x l ngay khng qu 15% (LQC: -1,4%

v HQC: -1,3%) v gi tr CV% gia cc kt qu nh lng u 15%. Nh vy

mu huyt tng n nh nhit phng sau 5 gi.

3.2.6.3. n nh mu huyt tng nhit -800C trong thi gian di Pha cc mu LQC v HQC nh mc 2.3.1 v tin hnh nh mc 2.3.3.7.

Trong khun kh ca phm vi lm kha lun nn chng ti mi ch tin hnh th

n nh ca mu huyt tng sau 21 ngy. Kt qu xc nh nng RIF trong mu

LQC, HQC sau 21 ngy v cc mu tin hnh phn tch ngay sau khi pha (nng

ban u) c trnh by trong bng 3.11

44

Bng 3.11: Kt qu n nh ca mu huyt tng trong thi gian di

Nng (g/mL)

Mu LQC (0,6 g/mL) Mu HQC (18,0 g/mL) Mu

Ban u Sau 21 ngy Ban u Sau 21 ngy

1 0,612 0,580 17,487 16,504

2 0,549 0,603 18,520 18,009

3 0,574 0,576 18,281 17,559

4 0,582 0,579 17,832 18,006

5 0,551 0,614 18,310 17,627

6 0,567 0,597 18,605 18,320

TB 0,573 0,591 18,172 17,671

CV (%) 4,1 2,6 2,4 3,6

lch (%) 3,3 -2,8

Kt qu cho thy, nng RIF trong mu sau 21 ngy u sai khc so vi

nng ban u khng qu 15% (LQC: 3,3 % v HQC: -2,8%) v gi tr CV% gia

cc kt qu nh lng u 15%. Nh vy mu huyt tng n nh sau nhit

-800C trong khong thi gian 21 ngy.

Nhn xt chung:

T nhng kt qu thm nh c hiu chn lc, khong tuyn tnh, gii

hn nh lng di, ng - chnh xc, t l thu hi, nghin cu n nh

thu c trn cho thy phng php c xy dng p ng cc yu cu ca

mt phng php phn tch dng trong sinh hc. Chng ti cho rng phng php

ny hon ton ph hp nh lng rifampicin trong huyt tng v c th c

p dng nh lng rifampicin huyt tng ngi tnh nguyn trong cc nghin

cu sinh kh dng v nh gi tng ng sinh hc cc ch phm cha rifampicin

cng nh trong iu tr.

45

3.3. BN LUN 3.3.1. Phng php x l mu huyt tng c Rifampicin

X l mu gip loi b mt hoc nhiu thnh phn tp cht c trong mu, to

iu kin thun li cho qu trnh phn tch, nh lng rifampicin bng phng

php HPLC. Da vo c tnh ca rifampicin, tham kho mt s ti liu v kho st

thc t chng ti xy dng c mt phng php x l mu dng nh lng

rifampicin trong huyt tng.

So vi phng php SPE, phng php x l mu s dng trong kha lun

ta protein vi dung mi MeOH n gin, d dng thc hin trong iu kin

phng th nghim do khng i hi trang thit b c bit, d dng trin khai v tit

kim v mt chi ph, ng thi hiu sut chit cao nn to iu kin ti u cho nh

lng RIF trong huyt tng.

Phng php x l mu ca chng ti cng tng ng vi cc nghin cu

ca tc gi R.Panchagnula [18] v AK.Hemanth Kumar [11].

3.3.2. Phng php nh lng rifampicin trong huyt tng

Phng php HPLC nh lng rifampicin trong huyt tng s dng trong

nghin cu ny c thm nh theo cc ch tiu ca US-FDA bao gm: c

hiu chn lc, khong tuyn tnh, gii hn nh lng di, ng - chnh

xc, t l thu hi, nghin cu n nh.

Vi mt iu kin sc k khng qu phc tp, d dng trin khai ti cc

phng th nghim ca Vit Nam, cht phn tch rifampicin tch hon ton khi

cc thnh phn tp c trong huyt tng vi thi gian trin khai sc k l 9 pht.

Thi gian trin khai sc k khng qu di cho php nh lng c mt s lng

mu ln trong mt ngy, gip tit kim c thi gian v chi ph, c bit l trong

iu kin hn hp v thit b hin i v ti chnh.

So vi nghin cu ca Co, B G, Wagan, E G, Lagdameo, E E, Molinar,

Adelaida M [14] c s dng cng cht ni chun l dipyridamol th phng php s

dng trong ti c khong nng tuyn tnh rng hn (t 0,20 n 20,0 g/mL

so vi 0,50 n 20 g/mL). Khong nng tuyn tnh ca phng php cha bng

46

kt qu ca Lau YY, Hanson GD, Carel BJ (t 0,50 n 35 g/mL) vi phng

php x l mu SPE [15] nhng im c nng cao nht ca ng chun (20

g/mL) vn nm trong khong t 2 3 ln gi tr Cmax (7 9 g/mL) nn khong

nng tuyn tnh p ng c yu cu ca nghin cu ny.

Gi tr gii hn nh lng di (LLOQ) ca phng php s dng trong

ti l kh thp 0,20 g/mL thp hn so vi cc nghin cu ca Co, B G, Wagan, E

G, Lagdameo, E E, Molinar, Adelaida M [14]; Lau YY, Hanson GD, Carel BJ [15]

nn phng php c nhy cao hn v chnh xc hn.

n nh ca phng php: chng ti tin hnh nh gi c ch tiu

cn bn v n nh ca phng php, cho thy s sai khc rt nh gia nng

RIF sau 3 chu k ng r, sau thi gian 5 gi nhit phng v sau thi gian 21

ngy so vi dung dch ngay sau khi pha, p ng yu cu phng php phn tch

dch sinh hc. Tuy nhin, trong khun kh lun vn tt nghip dc s i hc

nn chng ti cha thm nh c y cc ch tiu nh: n nh ca dung

dch chun gc, dung dch chun ni gc v n nh ca mu huyt tng vi

thi gian di hn phc v cc nghin cu nh gi tng ng sinh hc.

47

CHNG 4: KT LUN V KIN NGH 4.1.KT LUN

Qua qu trnh thc hin ti, chng ti t c 2 mc tiu ra nh sau:

1. Xy dng c quy trnh phn tch RIF trong huyt tng mt cch tng

i hon thin bao gm x l mu v iu kin sc k. C th l:

Quy trnh x l mu:

-Mu huyt tng r ng nhit phng

-Ngay sau khi r ng, ly 0,5 mL huyt tng thm Vitamin C

(nng khong 1,0 mg/mL), thm 50 L dung dch ni chun v 1,0

mL MeOH

-Lc xoy 15 giy, ly tm 10000v/pht x 5 pht, ht lp dung mi, tim

sc k.

iu kin sc k:

-Ct sc k: Rp18, 250x4,6 mm, 5 m. Bo v ct Rp18, 4 x 3 mm, 5m.

-Pha ng: MeOH MeCN m phosphat 0,02M pH 4,5 t l

48,8 : 16,2 : 35

-Tc dng: 0,01 6,50 pht: 1,5 mL/pht

6,51 9,51 pht: 2,0 mL/pht

-Detector: UV, = 337 nm

-Th tch tim: 50 L

-Autosampler: 40C

Ton b qu trnh x l mu v phn tch sc k c tin hnh trong iu

kin trnh nh sng.

2. Tin hnh thm nh phng php nh lng RIF vi cc ch tiu: c

hiu chn lc, khong tuyn tnh, gii hn nh lng di, ng - lp li,

t l thu hi, nghin cu n nh. Kt qu cho thy:

- Phng php c c hiu chn lc vi RIF.

- C s tng quan tuyn tnh cht ch gia p ng phn tch v nng

RIF trong khong nng kho st vi r > 0,999.

48

- Phng php c ng tt (88,0 107,8 %), lp li vi gi tr

CV% 15%.

p ng yu cu ca phng php phn tch cc cht trong dch sinh hc ca

US FDA.

4.2.KIN NGH

ng dng phng php ny nh lng RIF trong huyt tng ngi tnh

nguyn trong cc nghin cu sinh kh dng v nh gi tng ng sinh hc ca

cc ch phm c cha RIF trn th trng.

Tip tc nghin cu nh lng RIF trong huyt tng ca bnh nhn s

dng cc thuc chng lao phi hp.

TI LIU THAM KHO Ting Vit:

1. B Y t (2009), Dc th quc gia Vit Nam, Nh xut bn Y hc H Ni tr.

1008 1010.

2. B Y t (2010), Dc in Vit Nam IV, tr. 546 550.

3. B Y t (2007), Dc l hc, trng i hc Dc H Ni, tp 2, Nh xut

bn Y hc, tr.185 186, 188 190.

4. B Y t (2007), Ha dc, Trng i hc Dc H Ni, tp 2, Nh xut

bn Y hc, tr 175 176.

5. B Y t (2007), Ha phn tch, Trng i hc Dc H Ni, tp 2, Nh

xut bn Y hc, tr. 123 138, 142 143, 168 188.

6. B Y t (2007), Kim nghim dc phm, Nh xut bn Y hc, tr. 84 95,

104 110.

7. B Y t (2010), Hng dn bo co s liu nghin cu sinh kh dng/ tng

ng sinh hc trong ng k thuc, s 08/2010/TT-BYT

8. T Mnh Hng (2006), Nghin cu nh gi tng ng sinh hc vin

nang Cefaclor Stada 250 sn xut ti Vit Nam, Lun vn Thc s Dc hc,

Trng i hc Dc H Ni, H Ni.

9. L Th Luyn (2005), ng dng phng php sc k lng hiu nng cao

nh lng Rifampicin trong huyt tng ngi ung ng thi rifampicin

pyrazinamid isoniazid, tp ch dc hc, s 347, tr.32 35.

10. Nguyn Th Nhung (2011), Xy dng phng php nh lng acid

fenofibric trong huyt tng bng sc k lng hiu nng cao, Kha lun tt

nghip dc s i hc kha 2005 2010, Trng i hc Dc H Ni, H Ni.

Ting Anh:

11. AK Hemanth Kumar, I Chandra, R Geetha, KS Chelvi, V Lalitha, G Prema

(2004), A validated high-performance liquid chromatography method for the

determination of rifampicin and desacetyl rifampicin in plasma and urine, The

Indian Journal of Pharmacology, Volume 36, Issue 4, p. 231 233.

12. A.L. Allanson, M.M. Cotton, J.N.A. Tettey, A.C. Boyter (2007),

Detemination of rifampicin in human plasma and blood spots by high

performance liquid chromatography with UV detection: A potential method for

therapeutic drug monitoring, Journal of Pharmaceutical anh Biomedical

Analysis, Volume 44, Issue 4, p. 963 969.

13. British Pharmacopoeia 2009, pp.5206 5208.

14. Co, B G, Wagan, E G, Lagdameo, E E, Molinar, Adelaida M (2003), The

quantification of rifampicin in human plasma using high performance liquid

chromatography (HPLC) with ultraviolet, Science and Technology Research

Colloquium.

15. Lau YY, Hanson GD, Carel BJ (1996), Determination of rifampin in human

plasma by high-performance liquid chromatography with ultraviolet detection,

Journal of Chromatography, Vol 676(1):147-152.

16. Michael W.Dong and Ahuja Satinder (2005), Handbook of pharmaceutical

analysis by HPLC, United Kingdom, p.19 45.

17. Pharmacopoeia of the peoples republic of China (2005), Volume II, pp. 294

296.

18. R Panchagnula, A Sood, N Sharda, K Kaur, C.L Kaul (1999),

Determination of rifampicin and its main metabolite in plasma and urine in

presence of pyrazinamide and isoniazid by HPLC method, Journal of

Pharmaceutical anh Biomedical Analysis, Volume 18, Issue 6, p. 1013 1020.

19. The United Stated Phamcopenia 32 (2009), Volume 1 &2, pp. 3501 3502.

20. WHO (2009), Global tuberculosis control: epidemilogy, strategy, financing:

WHO report 2009.

21. WHO (2009), The Global TuberculosisEpidemic WHO factsheet.

22. Yuri Karakevich & Rosario Lobrutto (2007), HPLC for Pharmaceutical

Scientists, p.139 p.188, p.75 p.132.

Trang Web:

23. www.thuocbietduoc.com.vn.