Nghiên cứu định lượng đồng thời Strychnin và Brucin trong huyết tương bằng phương pháp HPLC.pdf

B Y T

TRNG I HC DC H NI

LNG TH PHN

NGHIN CU NH LNG NG

THI STRYCHNIN V BRUCIN TRONG

HUYT TNG BNG PHNG PHP

HPLC

KHA LUN TT NGHIP DC S

H NI 2014

B Y T

TRNG I HC DC H NI

LNG TH PHN

NGHIN CU NH LNG NG

THI STRYCHNIN V BRUCIN TRONG

HUYT TNG BNG PHNG PHP

HPLC

KHA LUN TT NGHIP DC S

Ngi hng dn:

1. PGS.TS.Nguyn Tin Vng

2. TS.Nguyn Th Thun

Ni thc hin:

1. B mn Ha Dc- Trng H Dc H Ni

2. Khoa Y Dc, HQGHN

H NI 2014

LI CM N

Trc ht, ti xin chn thnh by t lng knh trng v bit n su sc ti

PGS.TS. Nguyn Tin Vng - Vin Php y Quc gia, TS. Nguyn Th Thun -

ging vin b mn Ha dc- Trng H Dc H Ni v ThS. V c Li-

khoa Y Dc, HQGHN, hng dn tn tnh, ng vin v gip ti trong

sut qu trnh thc hin kha lun.

Ti cng xin gi li cm n ti cc thy c, cc k thut vin b mn ha

dc Trng i hc Dc H Ni v khoa Dc Trng i hc Quc gia

H Ni gip ti trong sut qu trnh lm thc nghim ti trng.

Xin cm n Ban gim hiu, Phng o to, cc phng ban to mi iu

kin gip ti hon thnh kha lun. Cm n cc thy c trng i hc Dc

H Ni quan tm du dt v truyn kin thc cho ti trong 5 nm hc va

qua.

Cui cng, ti xin gi li cm n ti gia nh v bn b lun ng h,

ng vin v khch l ti trong qu trnh hc tp v lm kha lun.

H Ni, thng 5 nm 2014

Sinh vin

Lng Th Phn

MC LC

LI CM N

MC LC

DANH MC CC K HIU, CH VIT TT

DANH MC CC BNG

DANH MC CC HNH V, TH

Trang

T VN ..... 1

Chng 1. TNG QUAN.. 3

1.1. Hp cht strychnin... 3

1.1.1. Cng thc cu to.................... 3

1.1.2. Tnh cht.. 3

1.1.3. Dc ng hc. 3

1.1.4. Tc dng.. 4

1.2. Hp cht brucin 4

1.2.1. Cng thc cu to.................... 4

1.2.2. Tnh cht.. 4

1.2.3. Dc ng hc. 4

1.2.4. Tc dng.. 5

1.3. Ngun gc ca strychnin v brucin 5

1.3.1. Tn khoa hc................... 5

1.3.2. Thnh phn ha hc................... 5

1.3.3. Cng dng ca m tin................... 5

1.4. Phng php nh lng strychnin v brucin...................... 6

1.5. Phng php HPLC. 9

1.5.1. Khi nim v sc k lng hiu nng cao. 9

1.5.2. Nguyn tc ca qu trnh sc k. 10

1.5.3. Cc thng s c trng cho qu trnh sc k. 10

1.5.4. C s l thuyt ca vic la chn iu kin sc k 10

1.5.5. ng dng ca phng php HPLC................... 11

1.6. Cc phng php x l mu huyt tng. 12

1.6.1. Ta protein.. 12

1.6.2. Chit lng- lng................... 12

1.6.3. Chit pha rn................... 13

1.7. Thm nh phng php nh lng. 13

1.7.1. Tnh thch hp h thng.. 13

1.7.2. chn lc.. 13

1.7.3. ng chun v khong tuyn tnh... 13

1.7.4. Gii hn nh lng di... 14

1.7.5. ng.................... 14

1.7.6. chnh xc 14

1.7.7. Hiu sut chit. 14

1.7.8. n nh.................... 14

Chng 2: I TNG V PHNG PHP NGHIN CU 15

2.1. Nguyn vt liu v thit b... 15

2.1.1. Nguyn vt liu 15

2.1.2. Trang thit b , dng c................... 15

2.2. Phng php nghin cu. 15

2.2.1. Chun b cc mu huyt tng... 15

2.2.2. Xc nh iu kin sc k.................... 17

2.2.3. Xy dng quy trnh x l mu huyt tng................... 18

2.2.4. Thm nh phng php phn tch 19

2.2.5. Phng php x l kt qu. 22

Chng 3: THC NGHIM, KT QU V BN LUN 23

3.1. Xy dng phng php nh lng strychnin v brucin trong huyt

tng bng HPLC...........................................

23

3.1.1. Kho st iu kin HPLC v chun ni ca phng php................... 23

3.1.2. Kt qu kho st quy trnh x l mu huyt tng.................... 26

3.2. Thm nh phng php phn tch 28

3.2.1. thch hp ca h thng.. 28

3.2.2. c hiu- chn lc.. 29

3.2.3. ng chun- khong tuyn tnh.. 31

3.2.4. Gii hn nh lng di (LLOQ). 33

3.2.5. ng- chnh xc trong ngy v khc ngy.. 34

3.2.6. Hiu sut chit. 37

3.2.7. n nh ca mu huyt tng................... 39

KT LUN V KIN NGH 42

1. Kt lun 42

2. Kin ngh...... 42

TI LIU THAM KHO..... 43

DANH MC CC K HIU, CC CH VIT TT

ACN : Acetonitril

BR : Brucin

ESI : Ion ha electrospray (Electrospray ionization).

GC/MS : Sc k kh/ detector khi ph (Gas Chromatography/Mass

Spectrometry).

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

Chromatography).

HPLC/DAD : Sc k lng hiu nng cao/detector diod array (High Performance

Liquid Chromatography/Diode Array Detection).

HPLC/MS : Sc k lng hiu nng cao/detector khi ph (High Performance

Liquid Chromatography/Mass Spectrometry).

HPLC/UV : Sc k lng hiu nng cao/detector UV ((High Performance

Liquid Chromatography/Ultraviolet).

HQC : Mu kim chng nng cao (High quality control).

HT : Huyt tng

IS : Cht chun ni (Internal standard).

LC/MS : Sc k lng/ detector khi ph (Liquid Chromatography/Mass

Spectrometry).

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

LQC : Mu kim chng nng thp (Low Quality Control).

MeOH : Methanol

MQC : Mu kim chng nng trung bnh (Middle Quality Control).

QC : Mu kim chng (Quality Control).

r : H s tng quan.

RP-HPLC : Ngc pha sc k lng hiu nng cao (Reversed Phase- High

Performance Liquid Chromatography).

RSD : lch chun tng i (Relative Standard deviation).

SK : Sc k

ST : Strychnin

ULOQ : Gii hn nh lng trn (Upper Limit of Quantification).

UPLC : Sc k lng siu hiu nng (Ultra Performance Liquid

Chromatography).

DANH MC CC BNG

Tn Bng Trang

Bng 1.1. Danh mc cc phng php nh lng strychnin v brucin 6

Bng 3.1: Kt qu kho st thch hp ca h thng sc k 28

Bng 3.2a: S tng quan gia nng strychnin trong HT v t l

p ng pic strychnin/IS

31

Bng 3.2b. S tng quan gia nng brucin trong HT v t l p

ng pic brucin/IS

32

Bng 3.3a. Kt qu xc nh gii hn nh lng di ca strychnin

(LLOQ)

33

Bng 3.3b. Kt qu xc nh gii hn nh lng di ca brucin

(LLOQ)

33

Bng 3.4a: Kt qu kho st ng, chnh xc trong ngy ca

Strychnin

34

Bng 3.4b. Kt qu kho st ng, chnh xc trong ngy ca

brucin

35

Bng 3.5a: Kt qu kho st ng, chnh xc khc ngy ca

strychnin

36

Bng 3.5b: Kt qu kho st ng, chnh xc khc ngy ca

brucin

36

Bng 3.6: Kt qu kho st hiu sut chit IS (900ng/ml) 37

Bng 3.7a: Kt qu kho st hiu sut chit ST 38

Bng 3.7b: Kt qu kho st hiu sut chit BR 39

Bng 3.8: Kt qu nghin cu n nh ca mu HT sau 3 chu k

ng-r

39

Bng 3.9: Kt qu nghin cu n nh mu HT thi gian ngn 40

Bng 3.10: Kt qu n nh mu huyt tng thi gian di 41

DANH MC CC HNH V, TH

Tn hnh v, th Trang

Hnh 3.1: Ph hp th t ngoi ca dung dch ST v BR 1000 ng/ml

trong dung mi ACN:m pH9 (38:62).

23

Hnh 3.2: SK vi t l pha ng ACN: m pH9 (38:62). 24

Hnh 3.3: SK dung dch chun ni ibuprofen trong pha ng 25

Hnh 3.4: SK dug dch chun ni cloramphenicol trong pha ng 25

Hnh 3.5: SK dung dch chun ni cafein trong pha ng 25

Hnh 3.6: SK ca ST, BR v chun ni ta protein bng MeOH 26

Hnh 3.7: SK ca ST, BR v chun ni ta protein bng ACN 26

Hnh 3.8: SK ca ST, BR v chun ni ta protein bng acid

percloric

27

Hnh 3.9: SK ca ST, BR v chun ni c chit lng- lng bng

chloroform

27

Hnh 3.10: SK ca ST, BR v chun ni chit lng lng bng

ethylacetat

27

Hnh 3.11a: SK mu HT trng 29

Hnh 3.11b: SK mu HT c IS 29

Hnh 3.11c: SK mu HT c ST 30

Hnh 3.11d: SK mu HT c BR 30

Hnh 3.11e: SK mu HT c ST, BR v IS 30

Hnh 3.12a: th ng chun biu th mi tng quan gia nng

ST trong HT v t l din tch pic ST/IS.

31

Hnh 3.12b: th ng chun biu th mi tng quan gia nng

BR trong HT v t l din tch pic BR/IS.

32

1

T VN

M tin (Semen Strychni) l mt v thuc qu c s dng t lu trong c ng

y v ty y. V thuc ny cng c a vo mt s Dc in ca cc nc nh: Dc

in Trung Quc 1997, Dc in Nht 1996, Dc in Anh 1980.. M tin c

ch t ht chn phi hay sy kh ca cy M tin (Strychnos nux-vomica).

V thuc ny c y hc dn gian Trung Quc s dng hiu qu trong gim

bt tnh trng vim, au khp, triu chng d ng v iu tr cc bnh thn kinh. Tuy

nhin do c tnh ca ht rt cao nn khng c php s dng trc tip m phi qua

ch bin. Alcaloid c bit n l thnh phn th hin hot tnh sinh hc ca loi cy

ny, trong strychnin v brucin l hai alcaloid c nhiu nht trong ht m tin v c

nh hng quan trng n tc dng cng nh c tnh ca ht. Mt mt, Strychnin v

brucin c tc dng gim au, chng vim, chng ung th, nhng bn cnh chng c

th dn ti cc ri loi vn ng, tng trng lc c, tng hot ng cm gic, liu

cao c th gy co git h thng thn kinh trung ng v dn n t vong do lit h hp,

lit ct sng hoc ngng tim. Ngoi ra, strychnin cn c s dng lm thuc dit

chut v mt s loi ng vt gm nhm. N cng c bit n l mt trong nhng

cht u tin c s dng nng cao hiu sut trong th thao. Strychnin tc dng

chn lc v i khng cnh tranh vi glycin trn receptor glycin ty sng. Liu cao

tc ng c ln receptor glycin no. Ng c strychnin c th gy t vong cho ngi

v ng vt. [12][30][23]

Brucin cng l mt alcaloid m t lu c s dng nh mt loi thuc truyn thng

iu tr ung th biu m t bo gan. N cn c chng minh c hiu qu gim au

v chng vim. c ca brucin thp hn strychnin nhng cng c th gy co git h

thng thn kinh ngoi bin. Do c c tnh mnh nh vy nn phm vi s dng hai

cht ny b gii hn ng k v c kim sot cht ch bi php lut nhiu nc

2

trn th gii. Mc d vy vn c nhng trng hp ng c strychnin c pht hin,

nguyn nhn c th do b nhm ln, u c hoc t st. [41] [35]

y l hai thnh phn c c tnh rt mnh, ch vi mt liu lng nh cng c

th gy nguy him n tnh mng con ngi. Do vy gip cho vic chn on, pht

hin nhanh nguyn nhn gy c v c bin php cp cu kp thi, nng cao hiu qu

cu cha nn nhn cng nh phc v cho cng tc php y th i hi phi c phng

php phn tch nhanh cc mu bnh phm. V vy vic xc nh hai alcaloid ny l v

cng quan trng trong gim nh c cht.

Trong nhng nm gn y, trn th gii c nhiu phng php c xut

xc nh strychnin v brucin trong dch sinh hc nh: HPLC/DAD[14], HPLC/MS[32],

LS-ESI-MS[42], HPLC-UV[15], UPLC-MS/MS[41] Tuy nhin nhng phng php

ny cha ph hp vi tnh hnh thc t ti Vit Nam. Xut pht t yu cu thc t trn,

chng ti tin hnh thc hin ti:

Nghin cu nh lng ng thi strychnin v brucin trong huyt tng bng

phng php HPLC vi cc mc tiu sau:

1. Xy dng phng php nh lng ng thi strychnin v brucin trong huyt

tng chut bng HPLC.

2. Thm nh phng php nh lng strychnin v brucin va xy dng.

3

CHNG 1.TNG QUAN

1.1. Hp cht strychnin

1.1.1 Cng thc cu to

Cng thc phn t: C21H22N2O2

Phn t lng: 334,41

Tn khoa hc: (4aR,5aS,8aR,13aS,15aS,15bR)-4a,5,5a,7,8,13a,15,15a,15b,16-decahydro-2H-4,6-methanoindolo[3,2,1-ij]oxepino[2,3,4-de]pyrrolo[2,3-h]quinoline-14-one.

1.1.2. Tnh cht

L tinh th khng mu hoc mu trng, khng mi, v rt ng. Nhit nng

chy: 270C. Khi lng ring 1,36 g/cm3. PH ca dung dch bo ha: 9,5.

tan trong: nc l 1/6400 (g/ml), nc si l 1/3100 (g/ml), ru l 1/150

(g/ml), chloroform l 1/35 (g/ml), benzen l 1/180 (g/ml), toluen l 1/200 (g/ml),

methanol l 1/260 (g/ml), glycerol l 1/320 (g/ml), rt t tan trong ether. [33][39][28]

1.1.3. Dc ng hc

Strychnin hp thu nhanh chng t ng tiu ha, ng h hp, ng tim v

da nguyn vn. [28][25]

Strychnin d dng c chuyn ha trong c th, ch yu bi cytochrome P450 2B

gan, v hu nh khng c nh hng tch ly. Thi gian bn thi l 10-12 gi.[28]

4

Mt vi pht sau khi ung, strychnin c bi tit khng i trong nc tiu, v

chim khong 5% mt liu gy cht trong vng hn 6 gi. Thuc c thi tr ch yu

qua thn v gn nh hon ton trong 48 n 72 gi. [13][26][28]

1.1.4. Tc dng

i vi thn kinh trung ng v ngoi vi c tc dng kch thch vi liu nh, v

tc dng co git vi liu cao. Lm tng bin v tn s h hp, tng tun hon khi cc

trung tm ny b c ch. Liu c kch thch mnh ty sng lm tng phn x v gy

cn co git ging nh co git do un vn. [10][12]

1.2. Hp cht brucin

1.2.1. Cng thc cu to

Cng thc phn t: C23H26N2O4

Phn t lng: 394,46

Tn khoa hc: (4aR,5aS,8aR,13aS,15aS,15bR)-10,11-dimethoxy-4a,5,5a,7,8,13a,15, 15a,15b,16-decahydro-2H-4,6-methanoindolo[3,2,1-ij]oxepino[2,3,4-de]pyrrolo[2,3-h]quinoline-14-one.

1.2.2. Tnh cht

Tinh th nh mu trng, khng mi, v cay ng. pH ca dung dch nc bo

ha 9,5. Nhit nng chy: Dng khan: 178C; dng hydrat: 105C.

tan: Trong methanol 1/0,8 (g/ml); trong chloroform 1/5 (g/ml), trong ethyl

acetate 1/25 (g/ml); trong glycerol 1/36 (g/ml); trong ether 1/187 (g/ml); trong nc

1/1320 (g/ml); trong nc si 1/750 (g/ml).[29]

1.2.3 Dc ng hc

5

Th nghim trn chut cho thy: sau khi ung brucin c hp thu nhanh (Tmax

< 0,5h). S gia tng AUC cho thy t l thun vi s gia tng liu. Sinh kh dng

ng ung (F) khng thay i theo liu. Gi tr Cmax c tnh ton l 0,730,23

g/ml.[16][17][27]

1.2.4. Tc dng

Brucin c tc dng tng t strychnin nhng yu hn. N gy lit dy thn

kinh ngoi bin v to ra cn co git d di. Brucin c th gy bun nn, nn, phn

khch, co git v co git vi liu lng ln. Ngoi ra brucin cn c tc dng gim au,

chng vim v chng ung th. [29][40]

1.3. Ngun gc ca strychnin v brucin

Strychnin v brucin l hai thnh phn alcaloid trong ht M tin c c tnh v

tc dng sinh hc mnh.

* Mt s c im ca cy M tin

1.3.1. Tn khoa hc

Strychnos nux-vomica L.

Thuc h M tin Loganiaceae.

1.3.2. Thnh phn ha hc

Trong ht m tin c cha rt nhiu alcaloid trong ch yu l strychnin, brucin, kt

hp vi acid igasuric (acid clorogenic). Nhng alcaloid khc thng gp l vomixin,

struxin, colubrine v . T l alcaloid ton phn trong m tin thay i t 2,5 n

5,5% , trong strychnin chim 43-45%. [10]

1.3.3. Cng dng ca m tin

Tr phong thp, hot lc, thng kinh, gim au, dng trong cc bnh phong thp

hoc mn tnh. Mnh gn ct, dng trong cc trng hp gn v c t au, c th suy

nhc, au nhc thn kinh ngoi bin. Kh phong ch kinh, dng trong cc bnh kinh

gin, co qup, chn tay b quyt lnh. Tn , tiu thng, dng trong cc bnh ung c

hoc chn thng c nhc sng ty. [11]

6

1.4. Phng php nh lng strychnin v brucin

Hin nay trn th gii c mt s cng trnh nghin cu nh lng

strychnin v brucin trong dch sinh hc cng nh trong dc liu. Qua tham kho mt

s ti liu, chng ti thy c cc phng php nh lng nh sau:

Bng 1.4.1: Danh mc cc phng php nh lng strychnin v brucin

STT Ti

liu

Mu th Phng

php

Ct sc k iu kin sc k

1 14 Mu HPLC/

DAD

ThermoHyper-

sil C18 ( 250

mm 4,6 mm

i.d, 5m)

-Pha ng: acetonitril trong m

phosphat 20 mM pH 3,8

- Tc dng: 1,0 ml/pht

- Nhit ct: 358C

- Bc sng pht hin: 254 nm

- Th tch tim: 100 l

-Cht chun ni: Chloroquin

-Phng php x l mu: chit

lng-lng

2 32 Dch

chit t

cn trng

HPLC/

MS

Phenomenex

Luna C18 (150

mm2,0 mm,

5m)

-Pha ng: MeOH 10% : acid

acetic 90%

-Tc dng: 0,25 ml/pht

-Nhit ct: nhit phng

- Bc sng pht hin: 254 nm

-Th tch tim: 10l

3 43 Bt dc

liu

HPLC-

ESI/MS

Zorbax Bonus

RP (150

mm2,1 mm,

5m)

-Pha ng: acetonitril- amoni acetat

20 mM thay i t 30-70% n 70-

30 % trong 30 pht

-Tc dng: 0,2 ml/pht

-Nhit ct: 40C

-Bc sng pht hin: 254 nm

7

-Th tch tim: 5l

4 42 Huyt

tng

chut

LC-ESI-

MS

Waters

SymmetryTM

C18 (100

mm4,6 mm,

5m)

-Pha ng: methanol-amoni format

20mM- acid formic (32:68:0,68,

v:v:v).

-Tc dng: 0,4 ml/pht

- Nhit ct: 30C

-Th tch tim: 10l

-Cht chun ni: tacrin


lng-lng.

5 15 M

chut

HPLC-

UV

Kromasil C18

(250 mm4,6

mm, 5m)

-Pha ng: 24% acetonitril- 76%

m (hn hp ng cc ca 0,01

mol/l natri sulfonat heptan v 0,02

mol/l kali dihydrogen phosphat gi

tr pH 2,8 bng acid phosphoric 10

%)

-Tc dng: 1,0 ml/pht

-Nhit ct: 35C


-Chut chun ni: hyperzin A


lng-lng.

-Th tch tim: 20l

6 41 Huyt

tng

chut

UPLC-

MS/MS

BEH C18 (50

mm2,1 mm

i.d, 1,7 m)

-Pha ng: methanol: acid formic

0,1 % (25:75, v/v)


-Nhit ct: 40C

-Th tch tim: 2 l

- Cht chun ni: moclobemide

8

7 31 Nc

tiu

LC-MS Cyanopropyl

(100 mm4,6

mm, 3m,

Chrompack,

Antwerp,

Belgium)

-Pha ng: acetonitril v acid acetic

1 % trong nc.

-Tc dng: 1,0 ml/pht

- Th tch tim: 50 l

-Cht chun ni: nalorphin


lng-lng.

8 22 M GC/MS Silica

(12,5m0,2m

m i.d, 0,33m

film thickness)

GC

-Phun: chia tim (1:10)

-Kh mang: Helium (1ml/pht)

-Nhit phun: 250C

-Nhit giao din MS: 280C

-Nhit l: 170C

MSD

-Ch ion ha: EI (70ev)

-Nhn in t: 2000 V

9 30 Mu v

nc tiu

HPLC-

ESI-MRM

XBridgeTM

Shield RP 18

(250 mm3,0

mm i.d, 5m)

-Pha ng: acetonitril v amoni

bicarbonat 10mmol/l (dung dch

nc ammoniac c dng iu

chnh pH 10,5).

-Tc dng: 0,5 ml/pht

-Nhit : 35C


-Th tch tim: 10l

-Phng php x l mu: chit pha

rn.

10 38 Mu RP- HPLC XTerra TM RP

18

(150mm4,6m

-Pha ng: 10mM amoni

bicarbonat (iu chnh pH 10 vi

amoni hydroxid)/acetonitril (70:30,

9

m, 5m) v:v)

-Tc dng: 1 ml/pht

-Nhit : 30C

-Th tch tim: 10l



rn

-Cht chun ni: gelsemine

11 35 Mu,

nc

tiu, gan,

no

LC-

MS/MS

Phenomenex

Luna

(50mm4,6

mm, 5m)

-Pha ng: amoni acetat 10 mM v

acid acetic 0,1% (pH 3,2) v hn

hp ca nc/methanol (95:5, v:v).

-Tc dng: 0,3 ml/pht


rn.

12 18 Mu,

nc tiu

HPLC-

ESI-

MS/MS

BDS

HYPERSIL

C18

(150mm2,1m

m , 2,4 m)

-Pha ng: 10 mmol/l amoni format

trong nc vi acid formic 0,1%.


-Nhit : 40C

Nhn xt: Phng php nh lng strychnin v brucin trong huyt tng

bng GC/MS, UPLC-MS, HPLC-ESI, LC-ESI-MS, kh phc tp, tn km v cha ph

hp vi a s phng th nghim Vit Nam. V vy, chng ti t vn nghin cu

xy dng phng php nh lng strychnin v brucin trong huyt tng bng

HPLC/UV n gin, c ng, chnh xc cao v kinh t, ph hp vi iu kin cc

phng th nghim ca Vit Nam hin nay.

1.5. Phng php HPLC

1.5.1. Khi nim v sc k lng hiu nng cao

10

Phng php HPLC l mt phng php phn tch sc k da trn s phn chia

khc nhau ca cc cht gia hai pha khng trn ln vi nhau. Trong , pha ng l

cht lng c y qua pha tnh trong ct di p lc cao ca mt bm cao p. Sc k

lng da trn c s l c ch hp ph, phn b khi lng, trao i ion, loi c hay

tng tc ha hc b mt. [1] [2]

1.5.2. Nguyn tc ca qu trnh sc k

Pha tnh c nhi vo ct tch theo mt k thut nht nh. Pha tnh l yu t

quyt nh bn cht ca ca qu trnh sc k. Nu pha tnh l cht hp ph th ta c sc

k hp ph pha thng hay pha o. Nu pha tnh l cht lng th ta c sc k phn b.

Nu pha tnh l cht trao i ion th ta c sc k trao i ion.

Khi t cht phn tch ln pha tnh u ct ri cho pha ng lin tc i qua

chng ta thc hin qu trnh sc k. Yu t quyt nh hiu qu s tch sc k y

l tng cc tng tc:

- Tng tc gia cht phn tch v pha tnh (F1).

- Tng tc gia cht phn tch v pha ng (F2).

- Tng tc gia pha tnh v pha ng (F3).

Trong tng tc F1 v F2 ng vai tr quyt nh [1], [2].

1.5.3. Cc thng s c trng cho qu trnh sc k

- Thi gian lu tR v th tch lu VR

- H s phn b K

- H s dung lng k

- H s chn lc

- phn gii Rs

- H s bt i ca pc (AF)

- S a l thuyt N. [2]

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

1.5.4.1. La chn ct (pha tnh)

11

Pha tnh trong HPLC l cht nhi ct c nhim v tch mt hn hp c nhiu

thnh phn. Pha tnh c bn cht l cht rn, xp, kch thc ht rt nh v ng knh

c ht t 3-10 m.

Yu cu ca pha tnh: phi tr v bn vng vi mi trng sc k, c kh nng

tch trong iu kin nht nh, tnh cht b mt n nh, cn bng ng hc ca s tch

phi xy ra nhanh v chnh xc tt, c ht phi ng nht.

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

nn hp cht cao phn t (cellulose) .... Pha tnh trn nn silica c nhiu u im v

c s dng nhiu nht.

1.5.4.2. La chn pha ng cho HPLC

Pha ng l dung mi dng ra gii cht cn phn tch ra khi ct tch

thc hin mt qu trnh sc k. Pha ng l yu t th hai quyt nh hiu sut tch sc

k ca mt hn hp. Pha ng c th l nc, dung mi hu c hay hn hp dung mi

theo t l nht nh.

Yu cu: phi tr i vi pha tnh, phi ha tan c cht mu, n nh theo

thi gian, c tinh khit cao, nhanh t cn bng trong qu trnh sc k.

1.5.4.3. Chn h m

Trong sc k to cp ion, sc k trao i ion, sc k hp ph m cht tan c tnh

acid hay base thng phi cho thm m vo pha ng n nh pH cho qu trnh sc

k v lm tng hiu lc tch. Thng thng vi cc cht tan l acid hu c th phi

dng m pH acid nhng cng c nhng trng hp ngoi l, cn sc k to cp ion

th pH ty tng trng hp.

1.5.4.4. Tc dng

Sau khi c pha ng, pha tnh, pH thch hp th cn phi la chn tc dng

cho pha ng qu trnh tch tt hn. [1][2][6]

1.5.5. ng dng phng php HPLC

- Phn tch nh tnh: thng da vo thi gian lu.

12

- Phn tch nh lng: thng da vo chiu cao hay din tch pc v da trn

c s ca phng php chun ngoi hoc chun ni, hoc thm chun.

Phng php chun ngoi: c hai mu th v chun u c tin hnh sc k

trong cng mt iu kin. Sau so snh trc tip chiu cao hay din tch pc ca mu

th vi mu chun.

Phng php chun ni: l phng php cho thm nhng lng ging nhau

ca cht chun th hai c thi gian lu v p ng gn ging mu th vo c mu

chun v mu th ri tin hnh sc k. Cht chun th hai gi l chun ni.

Phng php thm chun: Mu th c thm mt lng chnh xc cht

chun. Nng cha bit ca mu th c tnh da trn s chnh lch nng v

tng ca din tch. Vi phng php thm nhiu ln ta c phng php thm chun.

[1][2][6]

1.6. Cc phng php x l mu huyt tng

1.6.1. Ta protein

Ta protein c s dng rng ri trong x l cc mu sinh hc, loi b cc tp

protein trong huyt tng khi cc thnh phn cht tan khc. C ch ca ta protein l

thay i s solvate ha ca dung mi lm gim kh nng tan trong huyt tng ca cc

protein. C th gy ta protein cho huyt tng bng nhiu cch:

Ta protein bng mui: thng l mui trung tnh nh amoni sulfat.

Ta protein ti im ng in (pI).

Ta protein vi dung mi hu c: nh methanol hoc ethanol.

Ngoi ra cn c th ta protein bng cc cch khc nh: dng cc polymer a

nc khng ion ha nh dextran, polyethylene glycol, ta bng cc ion kim loi a ha

tr nh Fe2+[3].

1.6.2. Chit lng-lng (liquid-liquid extraction)

Chit lng-lng l phng php tch cc cht da vo s phn b khc nhau ca

cht tan trong hai cht lng khng th ha trn vo nhau, thng l nc v mt dung

13

mi hu c. nh gi hiu qu ca phng php chit lng-lng, c th s dng cc

thng s sau:

H s phn b

H s tch

H s kh tp [3]

1.6.3. Chit pha rn (solid phase extraction)

Chit pha rn l phng php tch cc cht da vo i lc ca cht tan vi pha

ng khi cho pha ng chy qua pha tnh. Pha tnh trong chit pha rn thng gm

silica gn vi mt nhm chc c bit. [3]

x l mu huyt tng, thng s dng 1 trong 3 phng php chit nh

trn.Tuy nhin phng php chit pha rn thng phc tp, tn thi gian v khng

kinh t nn chng ti kho st vi 2 phng php l kt ta protein v chit lng-lng

x l mu huyt tng.

1.7. Thm nh phng php nh lng

1.7.1. Tnh thch hp h thng

nh gi tnh thch hp h thng l nhng php th nhm nh gi tnh tng

thch ca ton h thng phn tch c cu thnh bi cc yu t nh my mc, thit b,

h thng in, cch tin hnh phn tch v mu th. [36][21]

1.7.2. chn lc

chn lc th hin kh nng phng php phn tch c th xc nh (nh

tnh-nh lng) cht cn phn tch, khng b nhm ln bi cc thnh phn khc c

trong mu.[8]

1.7.3. ng chun v khong tuyn tnh

ng chun biu din mi quan h gia p ng ca pic (din tch hay chiu

cao) v nng thuc trong dch sinh hc.

Khong tuyn tnh l khong nng t thp nht n cao nht c p ng

tuyn tnh.[8]

14

1.7.4. Gii hn nh lng di (LLOQ)

Gii hn nh lng di ca mt quy trnh phn tch l lng nh nht ca

cht phn tch trong mu th c th nh lng c vi ng v chnh xc

thch hp. [36][21]

1.7.5. ng

ng (accuracy): Gi tr phn nh st gn ca kt qu phn tch vi gi tr

thc ca mu bit. [8]

1.7.6. chnh xc (precision)

chnh xc (precision): Mc chm gia cc kt qu ring bit khi lp li

quy trnh phn tch nhiu ln trn cng mt mu th ng nht, c biu th bng gi

tr RSD (%). chnh xc bao gm lp li trong ngy v lp li khc ngy. [8]

1.7.7. Hiu sut chit

L t l thu hi hot cht sau khi mu c chit tch theo quy trnh chn so vi

mu c cng nng khng c x l qua chit tch. [36][21]

1.7.8. n nh

Cc nghin cu n nh bt buc phi c i vi mt qu trnh, phng php

phn tch thuc trong dch sinh hc bao gm:

- n nh 3 chu k ng-r ng.

- n nh thi gian ngn nhit phng.

- n nh sau khi x l mu, chit tch hot cht.

- n nh di ngy.[8]

15

CHNG 2. I TNG V PHNG PHP NGHIN CU

2.1 Nguyn vt liu v thit b

2.1.1. Nguyn vt liu

2.1.1.1. i tng nghin cu

- Cht chun strychnin hm lng 99,2%.

- Chut chun brucin hm lng 98,9%.

- Huyt tng chut nht trng, B mn Dc l- i hc Y H Ni cung cp.

2.1.1.2. Ha cht

- Methanol, acetonitril dng cho HPLC, acid percloric, ethylacetat, cloroform (hng

grade- Merck), acid H3PO4, KOH, K2HPO4, ibuprofen, cafein, cloramphenicol t tiu

chun PA (Merck).

- Nc ct 2 ln.

2.1.2. Trang thit b, dng c

- My sc k lng hiu nng cao: DIONEX Ultimate 3000- detector Diode Array.

- My quang ph UV- VIS Cary 60.

- My siu m Utrasonic cleaner- MRC.

- My o pH AL 20 pH- Aqualitic.

- My ly tm HSCEN- 204- MRC.

- My lc xoay in Velp- Zx3.

- Cn phn tch AUW 220.

- My khuy t: RO15 POWER- KiKa- Werke.

- T lnh m su Biomedical Freezer MDF- U334.

- Bnh nh mc, cc c m, pipet, micro pipet, a thy tinh, mng lc 0,45 m, bm

tim, l ng mu.

2.2. Phng php nghin cu

2.2.1. Chun b cc mu huyt tng

16

- Mu huyt tng trng: Huyt tng khng cha ST v BR, bo qun trong t

lnh su (-20). r ng nhit phng trc khi s dng.

- Dung dch gc strychnin (SST): Cn chnh xc khong 20mg ST, ha tan trong MeOH

sau cho vo bnh nh mc 100 ml v thm MeOH n vch, c dung dch ST

trong MeOH c nng khong 0,2mg/ml, bo qun trong t lnh su (-20C).

r ng nhit phng trc khi s dng.

- Dung dch gc brucin (SBR): Cn chnh xc khong 20mg BR, ha tan trong MeOH

sau cho vo bnh nh mc 100 ml v thm MeOH n vch, c dung dch BR

trong MeOH c nng khong 0,2mg/ml, bo qun trong t lnh su (-20C).

r ng nhit phng trc khi s dng.

T 2 dung dch gc ny ta pha thnh cc dung dch sau:

- Mu ST v BR chun trong huyt tng c nng ST v BR ln lt l 3600ng/ml

v 3600ng/ml, bo qun trong t lnh su (-20C). r ng nhit phng

trc khi s dng.

- Mu huyt tng t to cha ST v BR: T dung dch huyt tng chun ST v BR

trn, thm huyt tng trng, lc u c cc mu ST v BR trong huyt tng c

nng khong t 40- 1800ng/ml (tng ng vi khong nng ST v BR t 1/30-

1/20 Cmax n 2-3 Cmax).

- Dung dch chun ni: cn chnh xc khong 20mg cht chun ni cho vo bnh nh

mc 100ml, thm MeOH n vch, lc u c dung dch nng 0,2mg/ml, bo

qun trong t lnh su (-20C). r ng nhit phng trc khi s dng. T

dung dch ny pha thnh dung dch chun ni gc nng 45g/ml.

- Mu kim chng (QC): Chun b tng t mu chun nhng t dung dch chun gc

c lp vi dung dch chun gc pha mu chun. Mu QC gm 3 loi:

+ Mu LQC c nng 3 ln nng LLOQ.

+ Mu MQC c nng gn im gia ca ng chun.

+ Mu HQC c nng bng 75- 80% ULOQ.

17

2.2.2. Xc nh iu kin sc k

Kho st phng php phn tch trn h thng sc k DIONEX s dng ct C18

vi cc pha ng c thnh phn c t l khc nhau. T kt qu kho st la chn

nhng iu kin sc k ph hp c th tin hnh nh lng strychnin v brucin

trong huyt tng chut bng HPLC vi detector Diode Array.

Bc sng

xc nh bc sng thch hp pht hin strychnin v brucin, chng ti tin

hnh qut ph UV trn my quang ph UV-VIS Cary 60 dung dch strychnin v brucin

chun c nng khong 1000 ng/ml. Qut ph hp th t ngoi ca dung dch ny

vi di sng t 200-500 nm, cuvet thch anh dy 1cm, mu trng l dung mi pha

ng.

Pha ng:

Trong phng php HPLC pha ng hay dng nht l methanol, acetonitril,

nc hay hn hp ca chng. Chng ti tin hnh sc k mu strychnin v brucin

chun vi mt s h dung mi nh sau:

MeOH: ACN: H2O (50: 20: 30, v/v/v).

MeOH: ACN: H2O: m K2HPO4 1M pH 9 (50:20:20:10, v/v/v/v).

ACN: H2O: m K2HPO4 1M pH9 (30:30:40, v/v/v/).

ACN : m K2HPO4 1M pH9 vi cc t l: 40:60; 30:70; 38: 62; 35:65 (v/v).

Tc dng: 0,6 ml/pht; 0,8 ml/pht; 1,0 ml/pht;

Th tch tim mu: 20 l.

Chun ni:

Do c im ca nn mu huyt tng c cha nhiu thnh phn khc nhau nn

phng php nh lng thng phi c giai on x l chit tch hot cht, loi b

cc yu t nh hng ca nn mu. gim thiu sai s trong qu trnh chit tch, x

l mu, ngi ta thng thm cht ni vo mu chun v mu th. Da trn yu cu

chung ca cht chun ni v tham kho cc ti liu cng kinh nghim thc tin, chng

18

ti tin hnh kho st chn chun ni vi cc cht l: ibuprofen, cafein,

cloramphenicol.

2.2.3. Xy dng quy trnh x l mu huyt tng

2.2.3.1. Phng php ta protein

- Ta trong MeOH (khng kim ha): Ly chnh xc 2,0ml HT cha ST v BR

+ 50l dung dch chun ni gc 45g/ml + 3ml MeOH. Lc xoy trong 15 giy. Ly

tm vi tc 5000 vng/pht trong 10 pht. Lc ton b dch, bay hi, ha tan cn

trong 1,5ml pha ng, lc qua mng 0,45 m, em phn tch HPLC.

- Ta trong MeOH (kim ha): Ly chnh xc 2,0ml HT cha ST v BR + 50l

dung dch chun ni gc 45g/ml + 50l dung dch NaOH 1M (kim ha) + 3ml

MeOH. Lc xoy trong 15 giy. Ly tm vi tc 5000 vng/pht trong 10 pht. Lc

ton b dch, bay hi, ha tan cn trong 1,5ml pha ng, lc qua mng 0,45 m, em

phn tch HPLC.

- Ta trong ACN: Ly chnh xc 2,0ml HT cha ST v BR + 50l dung dch

chun ni gc 45g/ml + 50l dung dch NaOH 1M (kim ha) + 3ml ACN. Lc xoy

trong 15 giy. Ly tm vi tc 5000 vng/pht trong 10 pht. Lc ton b dch, bay

hi, ha tan cn trong 1,5ml pha ng, lc qua mng 0,45 m, em phn tch HPLC.

- Ta trong acid percloric: Ly chnh xc 2,0ml HT + 50l dung dch chun ni

gc 45g/ml + 400l acid percloric 1N + 50l dung dch NaOH 1M (kim ha). Lc

xoy trong 15 giy. Ly tm vi tc 5000 vng/pht trong 10 pht. Lc ton b dch

qua mng 0,45 m, em phn tch HPLC.

2.2.3.2. Phng php chit lng-lng

- Chit trong chloroform: Ly chnh xc 2,0ml HT cha ST v BR + 50l dung

dch chun ni gc 45g/ml + 50l dung dch NaOH 1M (kim ha), lc xoy 5 pht.

Sau thm 2 ml CHCl3, lc xoy 10 giy. Ly tm ly lp dung dch pha di. Bay

hi dung mi hu c. Ha tan cn bng 1,5ml pha ng, lc qua mng 0,45 m, em

phn tch HPLC.

19

- Chit trong ethyl acetat: Ly chnh xc 2,0ml HT cha ST v BR + 50l dung

dch chun ni gc 45g/ml + 50l dung dch NaOH 1M (kim ha), lc xoy 5 pht.

Sau thm 2 ml ethyl acetat, lc xoy 10 giy. Ly tm ly lp dung dch pha trn.

Bay hi dung mi hu c. Ha tan cn bng 1,5ml pha ng, lc qua mng 0,45 m,

em phn tch HPLC.

2.2.4. Thm nh phng php phn tch

Thm nh phng php phn tch theo hng dn ca Vin Kim nghim

thuc Trung ng, tin hnh trn cc mu: mu trng, mu chun, mu th v mu QC,

nh gi cc ch tiu sau:

2.2.4.1. thch hp ca h thng

Nguyn tc: Tin hnh tim chnh xc 6 ln cng mt dung dch chun ST v

BR vo h thng sc k theo chng trnh chn. thch hp ca h thng HPLC

c biu th qua lch chun tng i RSD (%) ca thi gian lu v din tch pic.

Tin hnh: Tim 6 ln mu chun vo h thng HPLC, tin hnh sc k theo

iu kin chn. Ghi kt qu: thi gian lu, din tch v h s bt i ca pic to bi

ST v BR.

2.2.4.2. chn lc

X l mu HT theo quy trnh la chn, tin hnh sc k theo iu kin

xc nh cc mu: mu HT trng, (mu HT ST, BR gn gii hn nh lng) mu HT

cha IS, mu HT cha ST, mu HT cha BR v mu HT cha ST, BR v IS. Ghi li

cc sc k v p ng pic ti cc v tr tng ng vi thi gian lu ca hot cht.

Phng php c coi l c tnh chn lc khi:

Pic ca ST v BR phi m bo c nhn din r rng, tch hon ton khi cc

pic tp, cc pic ca cc dc cht dng ng thi v cht chuyn ha.

p ng ca mu trng ti thi im trng vi thi gian lu ca ST v BR

khng c vt qu 20% p ng ca ST v BR nng LLOQ.

20

p ng ca mu trng ti thi im trng vi thi gian lu ca IS khng c

vt qu 5% p ng ca IS.

2.2.4.3. ng chun v khong tuyn tnh

Chun b dy nng chun gm t nht 6 nng ca ST v BR trong huyt

tng trng vi cc nng trong khong 40-1800ng/ml. Tin hnh phn tch cc mu.

T p ng pic ca ST v BR ti cc nng tng ng, xc nh s tng quan gia

p ng pic v nng pha. Xy dng phng trnh hi quy v xc nh h s

tng quan r. Tnh li nng theo phng trnh hi quy xy dng, xc nh

ng vi tng nng . ng chun phi tha mn cc yu cu sau:

- ng so vi gi tr thc ca cc nng phi t t 85% n 115%.

ng ca im c nng thp nht ca ng chun c php t 80-120%.

- C t nht 75% s im trong dy ng chun/khong tuyn tnh (bao gm c

2 im c nng thp nht v cao nht) t c tiu chun trn.

- Khong tuyn tnh: C h s r 0,98.

2.2.4.4. Gii hn nh lng di (LLOQ)

Tin hnh sc k 6 mu huyt tng chun c nng trong khong 1/30-1/20

Cmax. Ghi li p ng pic ca mu huyt tng chun. Tnh li nng ca cc mu

kho st da vo ng chun xy dng trong cng iu kin. Xc nh chnh

xc bng cch tnh lch RSD% gia kt qu 6 ln phn tch.

Mu chun c nng thp nht trn ng chun (1/30-1/20 Cmax) c chp

nhn l LLOQ nu tha mn:

- Pic ca cht cn phn tch c th nhn bit, nm tch ring v c p ng t nht

bng 5 ln p ng ca mu trng.

- ng phi t t 80-120% so vi nng thc.

- chnh xc vi gi tr RSD phi nh hn 20%.

2.2.4.5. ng

21

Tin hnh chy sc k cc l mu HT bao gm LQC (ca ST l 139,2ng/ml v

BR l 140,0ng/ml), MQC (ca ST l 905,0ng/ml v BR l 907,0ng/ml), HQC (ca ST

l 1448,0ng/ml v BR l 1451,2ng/ml). Mi l gm 6 mu c lp c cng nng .

Xc nh kt qu nh lng cc mu QC theo ng chun xy dng trong cng

iu kin. So snh gi tr trung bnh cc ln nh lng ti mi nng vi gi tr thc.

2.2.4.6. chnh xc

- chnh xc trong ngy: lch chun tng i (RSD) gia cc ln nh

lng ca ca mi nng c phn tch trong cng 1 ngy.

- chnh xc khc ngy: tin hnh tng t nh xc nh chnh xc trong

ngy, sc k cc mu QC. Tnh gi tr RSD ca kt qu nh lng cho mi nng

trong t nht 3 ngy phn tch khc nhau.

Yu cu vi mi nng :

ng phi nm trong khong t 85-115% nng thc pha.

lp li trong ngy: Gi tr RSD phi 15%.

lp li gia cc ngy: Gi tr RSD phi 15%.

2.2.4.7. Hiu sut chit

Tin hnh sc k cc l mu HT QC bao gm LQC, MQC, HQC theo quy trnh

xy dng, mi l mu HT gm 6 mu c lp c cng nng . Song song tin hnh

nh lng cc mu chun pha trong pha ng khng qua giai on chit tch, c nng

tng ng vi cc mu QC. So snh kt qu p ng ca ST v BR trong mu QC

pha trong huyt tng (c qua chit tch) vi p ng ca ST v BR trong mu tng

ng pha trong dung mi (khng qua chit tch).

2.2.4.8. n nh ca mu th trong huyt tng

- n nh sau 3 chu k ng-r: Kho st n nh sau 3 chu k ng-r,

thc hin trn mu HT 2 nng LQC v HQC. Bo qun mu nhit -20C v

r ng nhit phng. Sau 3 chu k ng-r, tin hnh phn tch xc nh nng

ST v BR trong mu huyt tng. So snh vi kt qu xc nh nng ST v BR

22

c trong cc mu tin hnh phn tch ngay sau khi pha (nng ban u). Nng ST

v BR trong mu sau 3 chu k ng-r phi sai khc vi nng ban u khng qu

15% v gi tr RSD 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 (4 gi), so snh vi nng mu c x l ngay sau

khi r ng. Nng ST v BR trong HT c x l sau khi bo qun mt thi gian

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

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

bng 15%.

- n nh ca huyt tng trong thi gian di: Khong thi gian th nghim

n nh di ngy ti thiu phi tin hnh ly mu huyt tng v phn tch ht

s mu huyt tng. Bo qun mu huyt tng 2 nng LQC v HQC nhit -

20C, phn tch mu ti thi im ban u v sau tng khong thi gian bo qun nht

nh (7, 14, 21 ngy). So snh nng ca mu HT sau khi bo qun vi nng

ca mu ti thi im ban u. Nng ST v BR trong mu sau khi bo qun mt

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

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

2.2.5. Phng php x l kt qu

- Gi tr trung bnh, lch chun tng i, phng trnh hi quy, h s tng

quan hi quy xc nh bng phn mm Microsolf Excel 2013.

- Nng ST v BR trong huyt tng c xc nh da vo phng trnh hi

quy biu din mi tng quan gia in tch pic v nng ST v BR trong mu

chun c xy dng trong mi ngy phn tch.

23

CHNG 3. THC NGHIM, KT QU V BN LUN

3.1. Xy dng phng php nh lng strychnin v brucin trong huyt tng

bng HPLC

3.1.1. Kho st iu kin HPLC v chun ni ca phng php

Qua nghin cu tnh cht ha l ca strychnin v brucin, kt hp vi cc ti liu tham

kho v iu kin sn c ca phng th nghim, chng ti la chn iu kin sc k

HPLC pha o, s dng ct sc k Acclaim C18 (4,6x150mm; 5m). Trong qu trnh

thc nghim, chng ti tin hnh kho st cc iu kin sc k nh: la chn bc

sng, pha ng, pH ca h m, tc dng, cht chun ni.

3.1.1.1. La chn bc sng thch hp

Sau khi qut ph hp th t ngoi ca dung dch vi di sng t 200-500 nm, kt qu

thu c ghi hnh 3.1.

Hnh 3.1: Ph hp th t ngoi ca dung dch ST v BR 1000 ng/ml trong dung mi

ACN:m pH9 (38:62).

Nhn xt: Ph hp th ca dung dch ST v BR chun 1000ng/ml cho cc i hp th

bc sng 260 nm trong h dung mi pha ng ACN: m K2HPO4 1M pH9 (38:62).

V vy chng ti chn bc sng 260nm kho st nh lng 2 cht ny.

3.1.1.2. Kho st chn thnh phn pha ng

Sau khi chy sc k vi cc h pha ng nh trn, kt qu hnh 3.2 cho thy h dung

mi ACN: m pH9 (38:62) l h dung mi cho thi gian lu thch hp, pic cn xng

24

nht, p ng pic tt v thi gian lu ca ST v BR ln lt l 5,56 v 4,23 pht. Do

chng ti chn h dung mi ny trong cc nghin cu tip theo.

c c s tt hn khi la chn pH ca m, chng ti chy sc k mu ST v BR

chun trong pha ng trn vi cc pH m l: pH5, pH9, pH10 cho kt qu vi m

pH9 cho p ng pic tt nht v pic cn i (AF= 1,4), thi gian lu ph hp, vi m

pH5 p ng pic km hn v pic dong hn (h s bt i AF=2,1), vi h m pH 10

pic b dong (AF= 1,9). Vy chng ti chn h m pH9.

Hnh 3.2: SK vi t l pha ng ACN: m pH9 (38:62).

3.1.1.3. La chn tc dng

Qua kt qu thc nghim, chng ti tin hnh chy sc k vi tc dng 0,6 ml/pht,

0,8 ml/pht, 1,0 ml/pht v nhn thy: Vi tc dng 0,6 ml/pht pic b ko ui.

Vi tc dng 1,0 ml/pht pic xut hin sm, d b trng vi cc tp trong huyt

tng. Vi tc dng 0,8 ml/pht cho pic gn, kh cn i, thi gian lu thch hp

nht. V vy, chng ti quyt nh chn tc dng l 0,8 ml/pht tin hnh sc k.

3.1.1.4. Kho st cht chun ni (IS)

Sau khi tin hnh kho st chn chun ni vi cc cht l: Ibuprofen, cafein,

cloramphenicol, thu c kt qu nh sau:

BR ST

25

Hnh 3.3: SK dung dch chun ni ibuprofen trong pha ng

Hnh 3.4: SK dung dch chun ni cloramphenicol trong pha ng

Hnh 3.5: SK dung dch chun ni cafein trong pha ng

Qua kt qu trn cho thy thi gian lu ca ibuprofen l 3,12 pht, ca

cloramphenicol l 2,20 pht, ca cafein l 2,61 pht. Mt khc pic ca c 3 cht u

cn xng, p ng pic tt. Tuy nhin vi mu huyt tng thng c pic tp cht ra

khong 0- 3 pht, ng thi ibuprofen p ng cc yu cu chung ca chun ni. Do

vy chng ti quyt nh chn ibuprofen lm chun ni kho st tip.

T kt qu kho st, chng ti s b la chn iu kin sc k nh sau tip tc

nghin cu xy dng phng php x l, chit tch ST v BR trong mu huyt tng:

- Ct sc k C18: Acclaim C18 (4,6150mm; 5m).

- Detector Diode Array: = 260 nm.

26

- Pha ng: ACN: m kali hydrophosphat 1M( iu chnh v pH= 9) t l 38:62 v/v.

- Th tch tim: 20 l.

- Tc dng: 0,8 ml/pht.

- Nhit phn tch: nhit phng th nghim.

- Cht chun ni: ibuprofen.

3.1.2. Kt qu kho st quy trnh x l mu huyt tng

Tin hnh kho st mu huyt tng theo phng php nu trong mc 2.3.3,

kt qu thu c nh sau:

So snh gia mu huyt tng c kim ha bng NaOH 1M v mu khng c

kim ha th mu huyt tng khng kim ha cho p ng pic ca strychnin v brucin

khng cn xng, din tch pic nh.

Trong khi vi mu huyt tng c kim ha th p ng pic cn xng, din tch,

chiu cao pic ln hn. Do vy quy trnh x l mu huyt tng cn phi kim ha.

Hnh 3.6: SK ca ST, BR v chun ni ta protein bng MeOH

Hnh 3.7: SK ca ST, BR v chun ni ta protein bng ACN

IS

IS

BR

BR

ST

ST

27

Hnh 3.8: SK ca ST, BR v chun ni ta protein bng acid percloric

Hnh 3.9: SK ca ST, BR v chun ni c chit lng- lng bng chloroform

Hnh 3.10: SK ca ST, BR v chun ni chit lng lng bng ethylacetat

Kt qu kho st cc quy trnh x l mu huyt tng trn mu huyt tng t

to cho thy: Tc nhn gy ta l MeOH hoc ACN phi cn mt lng dung mi gp

1,5 ln th tch huyt tng mi c th ta c lng protein c trong 2ml mu nn

lm gim gi tr pht hin v nh lng ca phng php. Tc nhn gy ta acid

percloric do ch cn mt lng nh (400l) gy ta lng protein c trong huyt

tng nn mu khng b pha long, qu trnh c tin hnh nhanh, n gin, kt qu

sc k vn m bo cc cht tch hon ton, hnh dng pic cn xng, chiu cao,

din tch pic v thi gian lu ca ST, BR, IS ph hp. Khi s dng dung mi chit l

chloroform v ethylacetat cho kt qu: pic cn ln nhiu tp, din tch pic nh, ng

IS

IS

IS

BR

BR

BR ST

ST

ST

28

nn nhiu, tin hnh phc tp. Do chng ti la chn phng php ta protein bng

cid percloric x l mu.

Nh vy, chng ti la chn c quy trnh x l mu v iu kin sc k c

kh nng tch ring pic ca ST, BR v IS khi cc pic tp c trong mu huyt tng

nh sau:

- Quy trnh x l mu:

Ly chnh xc 2,0ml huyt tng + 50l dung dch chun ni gc 45g/ml + 400l

acid percloric 1N + 50l dung dch NaOH 1M (kim ha). Lc xoy trong 15 giy. Ly

tm vi tc 5000 vng/pht trong 10 pht. Lc ton b dch qua mng lc 0,45 m,

em phn tch HPLC.

- iu kin sc k:

- Ct sc k Acclaim C18 (4,6150mm; 5m)

- Detector Diode Array: = 260 nm

- Pha ng: ACN: m kali hydrophosphat 1M (iu chnh v pH = 9,0 bng

KOH 2M hoc H3PO4 1M) t l 38:62 v/v.

- Th tch tim: 100 l.

- Tc dng: 0,8 ml/pht.

- Nhit phn tch: nhit phng.

- Ni chun: ibuprofen nng 900ng/ml.

3.2. Thm nh phng php phn tch

3.2.1. thch hp ca h thng

Tim 6 ln mu chun pha trong pha ng vo h thng HPLC, tin hnh sc k theo

iu kin chn. Kt qu kho st c trnh by bng 3.1.

Bng 3.1: Kt qu kho st thch hp ca h thng sc k

Ln tim tr ST tr BR tr IS SST/IS SBR/IS

1 5,56 4,23 3,12 1,786 0,902

2 5,52 4,21 3,06 1,779 0,908

3 5,57 4,20 3,08 1,810 0,911

29

4 5,53 4,19 3,14 1,790 0,906

5 5,56 4,25 3,15 1,802 0,923

6 5,55 4,18 3,11 1,768 0,915

TB 5,548 4,21 3,11 1,789 0,911

RSD% 0,35 0,62 1,11 0,85 0,81

Nhn xt: Kt qu kho st cho thy gi tr RSD (%) ca thi gian lu v din

tch pic ST v BR u nh hn 2%, cc gi tr ca h s bt i xng nm trong

khong cho php. iu ny chng t h thng HPLC ny ph hp cho php phn tch

nh lng strychnin v brucin.

3.2.2. c hiu- chn lc

Tin hnh phn tch cc mu HT trng, (mu HT ST, BR gn gii hn nh

lng), mu HT cha IS nng 900ng/ml, mu HT cha ST, BR v IS theo phng

php xy dng. SK mu HT trng, mu HT cha IS, mu HT cha ST, mu HT

cha BR v IS c trnh by hnh 3.11a, 3.11b, 3.11c v 3.11d, 3.11 e.

Hnh 3.11a: SK mu HT trng

Hnh 3.11b: SK mu HT c IS

IS

30

Hnh 3.11c: SK mu HT c ST

Hnh 3.11d: SK mu HT c BR

Hnh 3.11 e: SK mu HT c ST, BR v IS

Nhn xt: Kt qu thc nghim cho thy mu HT c IS xut hin pic ti thi

gian lu l 3,1 pht; mu HT c ST c pic xut hin ti thi gian lu l 5,5 pht; mu

HT c BR xut hin pic ti thi gian lu l 4,2 pht; trong khi th mu HT trng ti

cc thi gian lu trn khng thy xut hin pic. Mt khc trn sc k mu HT cha

ng thi IS, BR v ST th thy xut hin 3 pic tch khi cc thnh phn tp trong mu

v c thi gian lu l 3,1; 4,2 v 5,5 pht trng vi thi gian lu ca IS, BR v ST

trong cc mu HT cha ring tng thnh phn ny. Do phng php c tnh c

hiu- chn lc i vi ST v BR.

ST

BR

IS BR ST

31

3.2.3. ng chun- Khong tuyn tnh (40-1800ng/ml)

Tin hnh phn tch cc mu HT chun cha ST v BR c nng t 40-1800

ng/ml theo phng php xy dng. Thit lp mi quan h tng quan gia nng

ST vi t l p ng pic ST/IS, gia nng BR vi t l p ng pic BR/IS, xc nh

phng trnh hi quy, h s tng quan r. Tnh li cc nng theo ng chun xy

dng c xc nh ng ca nng tm li. Kt qu phn tch c trnh by

bng 3.2a v 3.2b.

Bng 3.2a: S tng quan gia nng strychnin trong HT v t l p ng pic

strychnin/IS (Nng HT ST chun 3620ng/ml pha thnh cc dd HT t to; BR

3628 ng/ml)

C ng/ml

S (mAU.min) C xc nh t ng chun

ST IS ST/IS ng/ml So vi gi tr

thc (%)

45,2 0,623 6,863 0,091 46,188 102,2

90,5 1,203 6,924 0,174 87,672 96,9

181,0 2,385 6,903 0,346 173,55 95,9

452,5 6,301 6,805 0,926 463,77 102,5

905,0 12,389 6,910 1,793 897,25 99,1

1810,0 24,863 6,897 3,605 1803,3 99,6

.

Hnh 3.12a: th ng chun biu th mi tng quan gia nng ST trong

HT v t l din tch pic ST/IS

32

Nhn xt: Trong khong nng ST t 45,2 n 1810,0ng/ml, c s tng quan

tuyn tnh gia nng ST v t l din tch pic ST/IS (phng trnh hi quy tuyn

tnh c h s tng quan gn bng 1). ng ca cc nng tm li nm trong

khong 85-115%. Nh vy, ng chun v khong tuyn tnh thch hp nh lng

ST trong mu huyt tng.

Bng 3.2b. S tng quan gia nng brucin trong HT v t l p ng pic

brucin/IS

C ng/ml

S (mAU.min) C xc nh t ng chun

BR IS BR/IS ng/ml So vi gi tr

thc (%)

45,3 0,238 6,863 0,035 53,9 118,9

90,7 0,574 6,924 0,083 102,1 112,6

181,4 1,249 6,903 0,181 200,1 110,3

453,5 3,018 6,805 0,444 462,7 102,2

907,0 6,228 6,910 0,901 920,5 101,5

1814,0 13,056 6,897 1,893 1912,2 105,4

Hnh 3.12b: th ng chun biu th mi tng quan gia nng BR trong

HT v t l din tch pic BR/IS.

Nhn xt: Trong khong nng ST t 45,3 n 1814,0ng/ml, c s tng quan

tuyn tnh gia nng BR v t l din tch pic BR/IS (phng trnh hi quy c h s

tng quan gn bng 1). ng ca cc nng tm li nm trong khong t 85-

33

115%. ng ca im c nng thp nht ca ng chun nm trong khong 80-

120%. Nh vy ng chun v khong tuyn tnh thch hp nh lng BR trong

mu huyt tng.

3.2.4. Gii hn nh lng di (LLOQ)

Phn tch cc mu HT trng v cc mu HT c nng ST v BR thp nht

trong khong tuyn tnh (ST: 45,2ng/ml, BR: 45,3ng/ml). Kt qu c trnh by trong

bng 3.3a v 3.3b.

Bng 3.3a. Kt qu xc nh gii hn nh lng di ca strychnin (LLOQ)

STT

HT trng

Din tch

(mAU*min)

Mu HT chun

ST

(mAU*min) IS (mAC*min)

Nng tm

thy (ng/ml) ng (%)

t/

Khng t

1 0,002 0,623 6,863 46,2 102,2 t

2 0,003 0,605 6,858 44,9 99,4 t

3 0,005 0,614 6,814 45,9 101,4 t

4 0,004 0,621 6,806 46,4 102,7 t

5 0,007 0,617 6,836 45,9 101,6 t

6 0,002 0,609 6,843 45,3 100,2 t

TB 101,3 t

RSD (%) 1,2 t

Bng 3.3b. Kt qu xc nh gii hn nh lng di ca brucin (LLOQ)

STT

HT trng

Din tch

(mAU*min)

Mu HT chun

BR

(mAU*min) IS (mAC*min)

Nng tm

thy (ng/ml)

ng

(%)

t/

Khng

t

1 0,002 0,210 6,863 49,8 109,9 t

2 0,003 0,207 6,854 49,4 109,1 t

3 0,005 0,201 6,839 48,6 107,3 t

4 0,004 0,203 6,805 49,0 108,2 t

5 0,007 0,195 6,826 47,8 105,5 t

6 0,002 0,190 6,835 47,0 103,8 t

TB 107,3 t

RSD (%) 2,3 t

34

Nhn xt: p ng ca mu trng ti thi im trng vi thi gian lu ca ST,

BR v IS nh hn 5% p ng ca cc pic ny trong mu LLOQ (bng 3.3a v bng

3.3b) v ng khi tnh li theo ng chun phn tch trong ngy u nm trong

khong t 80-120% vi RSD < 20%. Do vy gii hn nh lng di ca phng

php i vi ST l 45,2ng/ml v BR l 45,3ng/ml.

3.2.5. ng chnh xc trong ngy v khc ngy

* Thm nh ng, chnh xc trong ngy:

Phn tch cc mu huyt tng LQC, MQC, HQC theo phng php c

xc nh. Ghi li kt qu p ng ti mi nng (mi mu QC tin hnh o 6 ln

xc nh ng v chnh xc). Da vo p ng pic ca strychnin, brucin v IS,

tnh t l p ng ST/IS, BR/IS, t xc nh nng thc ca ST v BR theo ng

chun xy dng trn. T nng tnh c, xc nh ng, chnh xc tng

ng. Kt qu v ng, chnh xc trong ngy c trnh by trong bng 3.4a v

3.4b.

Bng 3.4a: Kt qu kho st ng, chnh xc trong ngy ca Strychnin

Mu

LQC (139,2ng/ml) MQC (905,0ng/ml) HQC (1448,0ng/ml)

T s

din tch

pic

ST/IS

Nng (a)

(ng/ml)

ng(b)

(%)

T s

din tch

pic

ST/IS

Nng (a)

(ng/ml)

ng(b)

(%)

T s

din tch

pic

ST/IS

Nng (a)

(ng/ml)

ng(b)

(%)

1 0,284 142,8 102,6 1,805 903,3 99,8 2,895 1448,3 100,0

2 0,272 136,8 98,3 1,810 905,8 100,1 2,887 1444,3 99,7

3 0,279 140,3 100,8 1,779 890,3 98,4 2,845 1423,3 98,3

4 0,285 143,3 102,9 1,792 896,8 99,1 2,869 1435,3 99,1

5 0,279 140,3 100,8 1,814 907,8 100,3 2,894 1447,8 100,0

6 0,279 140,3 100,8 1,798 899,8 99,4 2,872 1436,8 99,2

TB 101,0 99,5 99,4

RSD (%) 1,6 0,7 0,7

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

Kt qu bng 3.4a cho thy:

- Mu HT LQC c ng t 98,3% n 102,9 % vi RSD = 1,6%.

35

- Mu HT MQC c ng t 98,4 % n 100,3% vi RSD = 0,7%.

- Mu HT HQC c ng t 98,3% n 100,0% vi RSD = 0,7%.

Nh vy cc mu HT QC u c ng nm trong khong 85-115% vi

lch chun tng i RSD 15%.

Bng 3.4b. Kt qu kho st ng, chnh xc trong ngy ca brucin

Mu


T s

din tch

pic

BR/IS

Nng (a)

(ng/ml)

ng(b)

(%)

T s

din tch

pic

BR/IS

Nng (a)

(ng/ml)

ng(b)

(%)

T s

din tch

pic

BR/IS

Nng (a)

(ng/ml)

ng(b)

(%)

1 0,124 143,2 102,3 0,891 910,2 100,4 1,413 1432,2 98,7

2 0,131 150,2 107,3 0,898 917,2 101,1 1,409 1428,2 98,4

3 0,128 147,2 105,1 0,920 939,2 103,6 1,417 1436,2 99,0

4 0,136 145,2 103,7 0,915 934,2 103,0 1,459 1478,2 101,9

5 0,139 158,2 113,0 0,922 941,2 103,8 1,435 1454,2 100,2

6 0,125 144,2 103,0 0,916 935,2 103,1 1,452 1471,2 101,4

TB 105,7 102,5 99,9

RSD (%) 3,8 1,4 1,5

(a): tnh t phng trnh hi quy

(b): % so vi nng thc

Kt qu bng 3.4b cho thy:

- Mu HT LOQ c ng t 102,3% n 113,0% vi RSD = 3,8%.

- Mu HT MOQ c ng t 100,4% n 103,8% vi RSD = 1,4%.

- Mu HT HOQ c ng t 98,4 % n 101,9 % vi RSD = 1,5%.

Nh vy cc mu HT QC u c ng nm trong khong 85-115% vi

lch chun tng i RSD 15%.

*Kho st ng, chnh xc khc ngy:

Tip tc kho st ng v chnh xc khc ngy qua vic kho st cc mu

huyt tng QC, mi mu tin hnh trong 3 ngy- mi ngy o 6 mu ng vi mi

mu huyt tng QC. Kt qu xc nh c trnh by bng 3.5a v 3.5b. Cch thc

tnh ton vn tin hnh tng t nh i vi ng, chnh xc trong ngy.

36

Bng 3.5a: Kt qu kho st ng, chnh xc khc ngy ca strychnin

Ngy


Nng (a)

(ng/mg) Nng (b) (%)

Nng (a)

(ng/mg) Nng (b) (%)

Nng (a)

(ng/mg)

Nng (b)

(%)

138,0 99,1 910,1 100,6 1419,1 98,0

137,6 98,9 908,7 100,4 1442,4 99,6

141,3 101,5 902,6 99,7 1425,6 98,5

135,2 97,1 901,5 99,6 1429,7 98,7

143,8 103,3 903,8 99,9 1471,8 101,6

134,9 96,9 904,3 99,9 1452,9 100,3

143,6 95,3 911,2 100,6 1429,3 99,3

137,4 94,7 912,1 100,3 1432,7 97,5

135,8 102,9 909,4 99,7 1416,5 99,9

134,7 103,8 903,7 100,0 1463,1 100,6

142,9 97,6 902,8 100,1 1472,0 102,2

144,5 99,1 904,7 99,5 1421,5 98,2

144,8 95,8 901,8 99,9 1440,2 99,4

143,4 93,3 902,6 100,9 1414,6 99,0

140,8 98,1 908,4 100,4 1429,8 100,1

138,6 104,6 911,0 100,0 1470,6 97,6

136,4 102,6 911,5 100,8 1452,9 100,3

135,3 101,8 900,3 100,6 1460,2 102,9

TB (%) 99,2 100,2 99,7

RSD (%) 3,5 0,4 1,5

Kt qu bng 3.5a cho thy, ng khc ngy ca cc mu HT QC nm trong

khong 85-115%, vi RSD 15%.

Bng 3.5b: Kt qu kho st ng, chnh xc khc ngy ca brucin

Ngy

LQC (140ng/ml) MQC (907,0ng/ml) HQC (1451,2ng/ml)

Nng (a)

(ng/mg) Nng (b) (%)

Nng (a)

(ng/mg) Nng (b) (%)

Nng (a)

(ng/mg)

Nng (b)

(%)

145,2 103,7 901,5 99,4 1423,4 98,1

143,8 102,7 905,2 99,8 1412,8 97,4

136,9 97,8 910,8 100,4 1464,3 100,9

137,8 98,4 909,7 100,3 1473,8 101,6

135,6 96,9 912,1 100,6 1437,6 99,06

138,1 98,6 913,2 100,7 1441,0 99,3

37

132,7 94,8 910,5 100,4 1437,6 99,1

131,8 94,1 907,3 100,0 1411,2 97,2

143,2 102,3 902,5 99,5 1446,5 99,7

144,5 103,2 904,6 99,7 1456,8 100,4

135,8 97,0 905,8 99,9 1479,8 102,0

137,9 98,5 900,3 99,3 1422,3 98,0

133,4 95,3 904,2 99,7 1439,4 99,2

129,8 92,7 913,0 100,7 1432,8 98,7

136,5 97,5 908,9 100,2 1449,2 99,9

145,6 104,0 904,7 99,8 1413,6 97,4

142,8 102,0 911,8 100,5 1452,9 100,1

141,7 101,2 910,2 100,4 1489,8 102,7

TB (%) 98,9 100,1 99,5

RSD (%) 3,6 0,5 1,6

Kt qu bng 3.5b cho thy, ng khc ngy ca cc mu HT QC nm

trong khong 85-115% vi RSD 15%.

3.2.6. Hiu sut chit

Kho st hiu sut chit IS:

Xc nh hiu sut chit ca IS trn cc mu huyt tng bng cch so snh

din tch pic ca cc mu huyt tng qua x l chit tch v cc mu tng ng

khng chit tch (pha trong pha ng). Tin hnh sc k 6 mu c lp, ghi li p ng

pic ca cc mu trong pha ng v cc mu huyt tng qua x l, t cc p ng

tnh c hiu sut chit ca IS.

Bng 3.6: Kt qu kho st hiu sut chit IS (900ng/ml)

STT SIS trong huyt tng (mAU*min) SIS trong dung mi pha ng (mAU*min) Hiu sut chit (%)

1 6,863 8,716 78,7

2 6,854 8,692 78,9

3 6,839 8,708 78,5

4 6,805 8,669 78,5

5 6,826 8,725 78,2

6 6,835 8,689 78,7

TB 78,6

RSD (%) 0,3

38

Nhn xt: Kt qu thc nghim cho thy hiu sut chit ca ni chun l 78,6%,

chnh xc ca phng php chit tt (RSD= 0,3%). Do vy quy trnh x l mu huyt

tng c nghin cu l thch hp tch IS.

Kho st hiu sut chit ST v BR:

Vi hiu sut chit ST v BR, tin hnh o ti 3 nng ca 3 mu huyt tng

QC. Chun b cc l mu huyt tng LQC, MQC, HQC mi l gm 6 mu c lp v

cc mu chun pha trong pha ng c nng tng ng. Tin hnh nh lng trong

cng iu kin sc k. So snh din tch pic ST v BR thu c t cc mu huyt

tng QC (qua x l chit tch) vi din tch pic ST v BR thu c t cc mu tng

ng pha trong pha ng (khng qua chit tch). Kt qu xc nh hiu sut c trnh

by trong bng 3.7a v 3.7b:

Bng 3.7a: Kt qu kho st hiu sut chit ST

STT


Huyt

tng

Dung

mi pha

ng

Hiu

sut (%)

Huyt

tng

Dung

mi pha

ng

Hiu

sut (%)

Huyt

tng

Dung

mi pha

ng

Hiu

sut

(%)

1 1,917 2,338 82,0 12,389 14,926 83,0 19,802 23,526 84,2

2 1,958 2,390 81,9 12,406 14,910 83,2 19,837 23,598 84,1

3 1,906 2,317 82,3 12,367 14,958 82,7 19,846 23,573 84,2

4 1,942 2,308 84,1 12,328 14,983 82,3 19,858 23,535 84,4

5 1,932 2,385 81,0 12,354 15,006 82,3 19,892 23,603 84,3

6 1,936 2,371 81,7 12,410 14,967 82,9 19,817 23,610 83,9

TB 82,2 82,7 84,2

RSD (%) 1,3 0,5 0,2

Nhn xt: Kt qu thc nghim cho thy c 3 khong nng 139,2ng/ml;

905,0ng/ml; 1448,0ng/ml, phng php u cho hiu sut chit cao (82,2- 84,2%),

chnh xc tt (RSD < 2%). Do phng php x l mu c xy dng l ph hp

chit tch ST t huyt tng chut.

39

Bng 3.7b: Kt qu kho st hiu sut chit BR

STT


Huyt

tng

Dung

mi pha

ng

Hiu

sut (%)

Huyt

tng

Dung

mi pha

ng

Hiu

sut (%)

Huyt

tng

Dung

mi pha

ng

Hiu

sut

(%)

1 1,012 1,220 83,0 6,257 7,448 84,0 10,014 11,923 84,0

2 1,140 1,241 91,9 6,310 7,419 85,1 10,146 11,869 85,5

3 1,096 1,159 94,6 6,218 7,436 83,6 10,072 12,036 83,7

4 1,182 1,314 90,0 6,325 7,452 84,9 10,039 12,051 83,3

5 1,085 1,205 90,0 6,218 7,423 83,8 10,124 11,893 85,1

6 1,108 1,209 91,6 6,242 7,456 83,7 10,052 11,968 84,0

TB 90,2 84,2 84,3

RSD (%) 4,3 0,7 1,0

Nhn xt: Kt qu thc nghim cho thy c 3 khong nng 140,0ng/ml;

907,0ng/ml; 1451,2 ng/ml phng php u cho hiu sut chit cao (84,2- 90,2%),

chnh xc tt (RSD < 5%). Do phng php x l mu c xy dng l ph

hp chit tch BR t huyt tng chut.

3.2.7. n nh ca mu huyt tng

nh sau 3 chu k ng-r

Pha cc l mu HT LQC, HQC mi l gm 6 mu. Phn tch mu theo phng

php c xy dng. Xc nh nng ST v BR trong cc mu HT ban u (phn

tch ngay sau khi pha mu) v cc mu HT qua 3 chu k ng-r t ng chun

xy dng c.

Kt qu so snh nng ST v BR trong cc mu HT ban u v cc mu HT

n nh qua 3 chu k ng-r c trnh by bng 3.8.

Bng 3.8: Kt qu nghin cu n nh ca mu HT sau 3 chu k ng-r.

Mu

Nng ST (ng/ml) Nng BR (ng/ml)

LOQ (139,2ng/ml) HQC (1448,0ng/ml) LQC (140,0ng/ml) HQC (1451,2ng/ml)

Ban u

Sau 3 chu

k ng-

r

Ban u

Sau 3 chu

k ng-

r

Ban u

Sau 3

chu k

ng-r

Ban u

Sau 3

chu k

ng-r

1 136,0 135,2 1429,3 1413,7 129,8 126,4 1423,4 1413,5

40

2 143,2 137,4 1432,7 1420,6 132,6 130,2 1412,8 1424,6

3 135,7 137,4 1416,5 1425,6 148,7 145,7 1464,3 1458,3

4 138,0 132,1 1463,1 1456,7 146,3 143,5 1473,8 1464,8

5 131,8 134,5 1472,0 1467,8 142,9 140,8 1437,6 1425,0

6 143,8 142,7 1421,5 1415,5 127,8 124,8 1441,0 1437,8

TB 138,1 136,6 1439,2 1433,3 138,0 135,2 1442,2 1437,3

RSD (%) 3,4 2,6 1,6 1,6 6,5 6,8 1,6 1,4

T l so vi

ban u (%) 98,9 99,6 98,0 99,7

Nhn xt: Kt qu c 2 khong nng thp v cao ca ST v BR u cho

thy: nng ST v BR mu tri qua 3 chu k ng- r chnh lch khng qu 15%

so vi mu ban u, RSD ca mi mu u < 15% nn mu t v n nh sau 3

chu k ng- r.

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

Tin hnh pha cc l mu HT LQC v HQC mi l gm 6 mu. Phn tch mu

HT theo phng php c xy dng. Xc nh nng ST v BR trong cc mu

HT ban u (phn tch ngay sau khi pha mu) v cc mu HT n nh qua 4 gi sau

khi pha c trnh by bng 3.9.

Bng 3.9: Kt qu nghin cu n nh mu HT thi gian ngn

Mu


LQC (139,2ng/ml) HQC (1448,0ng/ml) LQC (140,0ng/ml) HQC (1451,2ng/ml)

X l

ngay Sau 4 gi

X l

ngay Sau 4 gi

X l

ngay Sau 4 gi X l ngay

Sau 4

gi

1 135,6 136,7 1440,2 1442,8 143,5 142,8 1437,6 1435,8

2 143,8 145,0 1414,6 1415,6 141,0 136,7 1411,2 1425,4

3 136,5 137,1 1429,8 1424,7 140,5 142,9 1446,5 1448,1

4 137,2 136,4 1470,6 1469,8 144,2 136,9 1456,8 1454,2

5 142,9 139,8 1452,9 1449,2 136,5 132,8 1479,8 1466,0

6 144,0 142,7 1460,2 1452,9 131,0 135,7 1422,3 1420,5

TB 140,0 139,6 1444,7 1442,5 139,5 138,0 1442,4 1441,7

RSD(%) 2,8 2,6 1,4 1,4 3,6 2,9 1,7 1,2

T l so vi

ban u (%) 99,7 99,8 98,9 100,0

41

Nhn xt: Kt qu thm nh cho thy, cc mu c RSD < 15%, nng ST v

BR ca mu HT o ngay sau khi r ng v mu HT sau 4 gi chnh lch nhau

khng qu 15% nn mu t v n nh trong thi gian ngn.

n nh ca mu huyt tng thi gian di

Bng 3.9: Kt qu n nh mu huyt tng thi gian di

Mu


LQC (139,2ng/ml) HQC (1448,0ng/ml) LQC (140,0ng/ml) HQC (1451,2ng/ml)

Ban

u

Sau 7

ngy

Sau

21

ngy

Ban u Sau 7

ngy

Sau 21

ngy

Ban

u

Sau 7

ngy

Sau

21

ngy

Ban

u

Sau 7

ngy

Sau 21

ngy

1 137,3 134,9 135,4 1419,1 1412,4 1418,9 135,2 136,0 134,8 1439,4 1436,0 1432,6

2 142,6 137,8 136,8 1442,4 1440,6 1440,8 138,1 139,1 140,9 1432,8 1430,2 1428,5

3 138,0 141,2 143,1 1425,6 1427,5 1425,4 142,9 143,2 144,5 1449,2 1437,4 1435,7

4 144,2 143,5 142,8 1429,7 1430,1 1423,8 144,2 143,5 142,8 1413,6 1411,0 1415,9

5 140,9 138,0 136,4 1471,8 1468,8 1463,2 134,6 141,0 136,7 1452,9 1446,8 1448,2

6 136,9 137,4 135,3 1452,9 1451,5 1450,9 135,7 136,3 137,8 1489,8 1486,8 1474,2

TB 140,0 138,8 138,3 1440,3 1438,5 1437,2 138,5 139,9 139,6 1446,3 1441,4 1439,2

RSD

(%) 2,2 2,2 2,6 1,4 1,4 1,2 3,0 2,3 2,7 1,8 1,8 1,4

T l

so vi

ban

u

(%)

99,2 98,8 99,9 99,8 101,0 100,8 99,7 99,5

Nhn xt: Kt qu thm nh cho thy, cc mu c RSD < 15%, nng ST v

BR ca mu HT sau 7 ngy v 21 ngy so vi mu HT ca ngy u tin chnh lch

nhau khng qu 15% nn mu t v ch tiu n nh di ngy.

42

KT LUN V KIN NGH

KT LUN

Qua qu trnh kho st, thc hin nghin cu, kha lun hon thnh c cc

mc tiu sau:

1. xy dng phng php nh lng ng thi strychnin v brucin trong huyt

tng chut bng HPLC vi:

Quy trnh x l mu: Ly chnh xc 2,0ml HT + 50l dung dch chun ni gc

45g/ml + 400l acid percloric 1N + 50l dung dch NaOH 1M (kim ha). Lc

xoy trong 15 giy. Ly tm vi tc 5000 vng/pht trong 10 pht. Lc ton

b dch qua mng 0,45 m, em phn tch HPLC.

iu kin sc k:

- Ct sc k Acclaim C18 (4,6150mm; 5m) vi Detector Diode Array: = 260 nm.

- Pha ng: ACN: m kali hydrophosphat 1M (iu chnh v pH = 9,0 bng KOH

2M hoc H3PO4 1M) t l 38:62 v/v.

- Th tch tim: 100 l; tc dng 0,8 ml/pht;

- Nhit phn tch: nhit phng; Ni chun: ibuprofen nng 900ng/ml.

2. thm nh phng php nh lng strychnin v brucin va xy dng t cc tiu

ch ca mt phng php nh lng thuc trong dch sinh hc vi ng t yu

cu (98,4-113,0%), chnh xc, n nh cao (RSD = 1,4- 3,8).

KIN NGH

Do thi gian cn hn ch, chng ti ch mi kho st trn cc mu huyt tng

v vy cn tip tc nghin cu:

- Vi dch ra d dy c th a ra phng php nh gi nhanh v chnh xc

nguyn nhn ng c do m tin hay khng.

- Trn cc dch sinh hc ngi xc nh nhanh nguyn nhn gy c, c bin php

cu cha kp thi hoc chng minh nguyn nhn t vong trong gim nh php y.

43

TI LIU THAM KHO

TING VIT

1. Trn T An(2007), Ha phn tch II, Nh xut bn Y hc, H Ni, tr. 125-143, 168-

200.

2. Trn T An (2007), Kim nghim dc phm, Nh xut bn Y hc, H Ni, tr. 84-

110.

3. Trn T An (2002), Phng php chit ng dng trong kim nghim thuc v c

cht, Nh xut bn Y hc, H Ni, tr. 33-39, 76-83.

4. B Y t (2009), Dc in Vit Nam IV, Nh xut bn Y hc, H Ni, tr. 819- 820.

5. L Trn c (1997), Cy thuc Vit Nam, nh xut bn nng nghip, H Ni, tr.

1327- 1329.

6. Nguyn Minh c (2006), Sc k lng hiu nng cao v mt s ng dng vo

nghin cu, kim nghim dc phm, dc liu v hp cht t nhin.

7. Phm Hong H (2000), Cy C Vit Nam, nh xut bn tr, tr. 668- 670.

8. T Mnh Hng, L Th Thu Huyn, Trn Hong, Nguyn Th Thanh Tho, Phng

Th Vinh(2013),Hng dn thm nh phng php sc k lng hiu nng cao phn

tich thuc trong huyt tng ngi tnh nguyn tham gia nghin cu tng ng sinh

hc, Tp ch kim nghim thuc, tp 11(40), tr 1-4.

9. Phm Thanh K (2007), Dc liu hc tp 2, Nh xut bn Y hc, H Ni, tr. 119-

125.

10. Tt Li (1999), Nhng cy thuc v v thuc Vit Nam, Nh xut bn Y hc, tr.

529- 530.

11. Phm Xun Sinh (2006), Dc hc c truyn, Nh xut bn Y hc, H Ni, tr.

260- 261.

12. Mai Tt T, V Th Trm (2007), Dc l hc tp 1, Nh xut bn Y hc, H Ni,

tr 130-131.

44

TING ANH

13. Boyd RE, Brennan PT, Deng JF, Rochester DF, Spyker DA (1983), Strychnine

poisoning, Recovery from profound lactic acidosis, hyperthermia and rhabdomyolysis.

Am J Med, pages 507-512.

14. Charlotte Duverneuil, Geoffroy Lorin de la Grandmaison, Philippe de Mazancourt,

Jean-Claude Alvarez (2004), Liquid chromatography/photodiode array detection for

determination of strychnine in blood: a fatal case report, Forensic Science

International, pages 17-21.

15. Chen J, Hou T, Fang Y, Chen ZP, Liu X, Cai H, Lu TL, Yan GJ, Cai BC (2011),

HPLC determination of strychnine and brucine in rat tissues and the distribution study

of processed semen strychni, Yakugaku Zashi: Journal of the Pharmaceutical Society

of Japan, pages 721-729.

16. Chen J, Li L, Qi Y, Chen ZP, Cai BC (2012), Preparation and pharmacokinetics of

brucine hydrogel patch, Zhong Yao Cai, pages 1295-1298.

17. Chen J, Xiao HL, Hu RR, Hu W, Chen ZP, Cai H, Liu X, Lu TL, Fang Y, Cai BC

(2011), Pharmacokinetics of brucine after intravenous and oral administration to rats,

Fitoterapia, pages 1302-1308.

18. Chunhua Zhang, Huiqin Wu, Xiaolan Huang, Zhixin Zhu, Huitai Luo, Fang Huang,

Xiaoshan Lin (2012), Simultaneous Determination of Toxic Alkaloid in Blood and

Urine by HPLC-ESI-MS/MS, Chromatographia, pages 499-511.

19. Cooper P (1974), Poisoning by drugs and chemicals. London, Alchemist

Publications, pages 193-194.

20. Edmunds M, Sheehan TMT and Vant Hoff W (1986), Strychnine poisoning:

clinical and toxicological observations on a non-fatal case, Journal of Toxicology-

Clinical Toxicology, 24 (3): 245-255.

21. European Medicines Agency (2009), Guidance on Validation Of Bioanalytical

Methods, London.

45

22. E.P. Marquens, F. Gil, P. Proenca, P. Monsanto, M.F. Oliveira, A. Castanheira,

D.N. Vieira (2000), Analytical method for the determination of strychnine in tissues by

gas chromatography/mass spectrometry: two case reports, Forensic Science

International, pages 145-152.

23. Genevive Philippe, Laurent Nguyen, Luc Angenot, Michel Frdrich, Gustave

Moonen, Monique Tits, Jean-Michel Rigo (2006), Study of the interaction of

antiplasmodial strychnine derivatives with the glycine receptor, European Journal of

Pharmacology, pages 15-22.

24. Goodman LS, Gilman AG and Gilman A (1985), The pharmacological basis of

therapeutics, New York, Macmillan publishing Co., Inc., pages 582-584.

25. Greene R, Meatherall R (2001), Dermal exposure to strychnine, J Anal Toxicol,

pages 344-347.

26. Jiri Patocka (2009), CHAPTER 14- strychnine, Handbook of Toxicology of

Chemical Warfare Agents, pages 199-205.

27. Li J, Miao F, Lin L, Zhu D, Lin C, Liu J (2012), Determination of brucine and

strychnine in rat after cutaneous administration of semen strychni noisome gel by LC-

MS/MS, Article in Chinese, pages 853-857.

28. Makarovsky I, Markel G, Hoffman A, Schein O, Brosh-Nissimov T, Tashma Z,

Dushnitsky T, Eisenkraft A (2008), Strychnine- a killer from the past, Isr Med Assoc J,

pages 142-145.

29. Merck Index (1989), Rahway, 11th edition, New Jersey, USA, Merck and Co., Inc.,

201.

30. Peihong Qiu, Xiaohong Chen, Xiang Chen, Li Lin, Changchun Aj (2008),

Simultaneous determination of five toxic alkaloids in body fluids by high-performance

liquid chromatography coupled with electrospray ionization tandem mass

spectrometry, Journal of Chromatography B, pages 471-477.

46

31. P. Van Eenoo, K. Deventer, K. Roels, F.T. Delbeke (2006), Quantitative LC-MS

determination of strychnine in urine after ingestion of a Strychnos nux-vomica

preparation and its consequences in doping control, Forensic Science international,

pages 159-163.

32. Randal S. Stahl, Wendy M rjo, Kim K. Wagner, Carol Furcolow, Dale L. Nolte,

John J. Johnston(2004), Development of a high performance liquid

chromatography/mass spectroscopy method for determination of strychnine

concentrations in insects used to assess potential risks to insective, Journal of

Chromatography, pages 257-262.

33. Sax NI (1988), Strychnine, Dangerour properties of industrial material, Report,

8(1): 78-83.

34. Sharma R.K (2008), Consice textbook of forensic medicine and toxicology,

Elsevier.

35. Teske J, Weller JP, Albrecht UV, Fieguth A (2011), Fatal intoxication due to

brucine, J Anal Toxicol, pages 248-253.

36. US.Department of Health and Human Services, CDER, FDA, CVM (2001),

Guidance for Industry Bioanalytical Method Validation, BP.

37. Wang X, He C, Chen Y, Chen J, Cai B (2011), Determination and comparison of

plasma protein binding rate of alkaloids from seed of Strychnou nux-vomica,

Zhongguo Zhong Yao Za Zhi, pages 185-188.

38. Wang Z, Zhao J, Xing J, He Y, Guo D (2004), Analysis of strychnine and brucine

in postmortem specimens by RP-HPLC: a case report of fatal intoxication, J Anal

Toxicol, pages 141-144.

39. Windholz (1983), The Merck Index Rahway, New Jersey, USA, Merck and Co.

Inc., 1268.

47

40. Wu Yin, Tian-Shan Wang, Fang-Zhou Yin, Bao-Chang Cai (2003), Analgesic and

anti-infammatory properties of brucine and brucine N-oxide extracted from seeds of

Strychnos nus-vomica, Journal of Ethnopharmacology, pages 205-214.

41. Yanwen Liu, Ronghua Zhu, Huande Li, Miao Yan, Yanqing Lei (2011), Ultra-

peformance liquid chromatography-tandem mass spectrometric method for the

determination of strychnine and brucine in mice plasma, Journal of Chromatography

B, pages 2714-2719.

42. Yanyan Xu, Duanyun Si, Changxiao Liu (2009), Determination of strychnine and

brucine in rat plasma using liquid chromatography electrospray ionization mass

spectrometry, Journal of Pharmaceutical and Biomedical Analysis, pages 487-491.

43. Young Hae Choi, You-Min Sohn, Chul Young Kim, Kwan Yul Oh, Jinwoong Kim

(2004),Analysis of strychnine from detoxified Strychno nux-vomica seeds using liquid

chromatography-electrospray mass spectrometry, Journal of Ethnopharmacology,

pages 109-112.

1.5.3. Cc thng s c trng cho qu trnh sc k1.5.4. C s l thuyt ca vic la chn iu kin sc k1.5.5. ng dng phng php HPLC1.6. Cc phng php x l mu huyt tng1.6.1. Ta protein1.6.2. Chit lng-lng (liquid-liquid extraction)1.7.2. chn lc1.7.4. Gii hn nh lng di (LLOQ)

Documents

Nghiên cứu định lượng đồng thời Strychnin và Brucin trong huyết tương bằng phương pháp HPLC.pdf