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 Likhoa 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 .....

Chng 1. TNG QUAN..

1.1. Hp cht strychnin...

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

1.1.2. Tnh cht..

1.1.3. Dc ng hc.

1.1.4. Tc dng..

1.2. Hp cht brucin

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

1.2.2. Tnh cht..

1.2.3. Dc ng hc.

1.2.4. Tc dng..

1.3. Ngun gc ca strychnin v brucin

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

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

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

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

1.5. Phng php HPLC.

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

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).

: 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

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

31

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

32

ng pic brucin/IS
Bng 3.3a. Kt qu xc nh gii hn nh lng di ca strychnin

33

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

33

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

34

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

35

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

36

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

36

brucin
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

39

ng-r
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

23

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

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

27

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

27

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

27

ethylacetat
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

31

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

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

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

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.

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,16decahydro-2H-4,6-methanoindolo[3,2,1-ij]oxepino[2,3,4-de]pyrrolo[2,3-h]quinoline14-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]

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[2,3h]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

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]

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

Mu th

liu
1

14

Phng

Ct sc k

iu kin sc k

php
Mu

HPLC/

ThermoHyper-

DAD

sil C18 ( 250

phosphat 20 mM pH 3,8

mm 4,6 mm

- Tc dng: 1,0 ml/pht

i.d, 5m)

-Pha ng: acetonitril trong m

- 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

32

Dch

HPLC/

chit t

MS

cn trng

Phenomenex

-Pha ng: MeOH 10% : acid

Luna C18 (150 acetic 90%

mm2,0 mm,
5m)

-Tc dng: 0,25 ml/pht

-Nhit ct: nhit phng
- Bc sng pht hin: 254 nm
-Th tch tim: 10l

43

Bt dc

HPLC-

Zorbax Bonus

-Pha ng: acetonitril- amoni acetat

liu

ESI/MS

RP (150

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

mm2,1 mm,
5m)

30 % trong 30 pht
-Tc dng: 0,2 ml/pht
-Nhit ct: 40C
-Bc sng pht hin: 254 nm

-Th tch tim: 5l

4

42

Huyt

LC-ESI-

Waters

tng

MS

SymmetryTM

chut

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
-Phng php x l mu: chit
lng-lng.

15

HPLC-

Kromasil C18

-Pha ng: 24% acetonitril- 76%

chut

UV

(250 mm4,6

m (hn hp ng cc ca 0,01

mm, 5m)

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
-Bc sng pht hin: 264 nm
-Chut chun ni: hyperzin A
-Phng php x l mu: chit
lng-lng.
-Th tch tim: 20l

41

Huyt

UPLC-

BEH C18 (50

-Pha ng: methanol: acid formic

tng

MS/MS

mm2,1 mm

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

i.d, 1,7 m)

-Tc dng: 0,25 ml/pht

chut

-Nhit ct: 40C

-Th tch tim: 2 l
- Cht chun ni: moclobemide

31

Nc

LC-MS

tiu

Cyanopropyl

-Pha ng: acetonitril v acid acetic

(100 mm4,6

1 % trong nc.

mm, 3m,
Chrompack,

-Tc dng: 1,0 ml/pht

- Th tch tim: 50 l

Antwerp,

-Cht chun ni: nalorphin

Belgium)

-Phng php x l mu: chit

lng-lng.

22

GC/MS

Silica

GC

(12,5m0,2m

-Phun: chia tim (1:10)

m i.d, 0,33m

-Kh mang: Helium (1ml/pht)

film thickness)

-Nhit phun: 250C

-Nhit giao din MS: 280C
-Nhit l: 170C
MSD
-Ch ion ha: EI (70ev)
-Nhn in t: 2000 V

30

Mu v

HPLC-

XBridgeTM

-Pha ng: acetonitril v amoni

nc tiu

ESI-MRM

Shield RP 18

bicarbonat 10mmol/l (dung dch

(250 mm3,0

nc ammoniac c dng iu

mm i.d, 5m)

chnh pH 10,5).
-Tc dng: 0,5 ml/pht
-Nhit : 35C
-Bc sng pht hin: 235 nm
-Th tch tim: 10l
-Phng php x l mu: chit pha
rn.

10

38

Mu

RP- HPLC XTerra TM RP -Pha ng: 10mM amoni

18
(150mm4,6m

bicarbonat (iu chnh pH 10 vi

amoni hydroxid)/acetonitril (70:30,

m, 5m)

v:v)
-Tc dng: 1 ml/pht
-Nhit : 30C
-Th tch tim: 10l
-Bc sng pht hin: 254 nm
-Phng php x l mu: chit pha
rn
-Cht chun ni: gelsemine

11

35

Mu,

LC-

Phenomenex

nc

MS/MS

Luna

-Pha ng: amoni acetat 10 mM v

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

tiu, gan,

(50mm4,6

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

no

mm, 5m)

-Tc dng: 0,3 ml/pht

-Phng php x l mu: chit pha
rn.

12

18

Mu,

HPLC-

BDS

nc tiu

ESI-

HYPERSIL

MS/MS

C18
(150mm2,1m

-Pha ng: 10 mmol/l amoni format

trong nc vi acid formic 0,1%.
-Tc dng: 0,25 ml/pht
-Nhit : 40C

m , 2,4 m)

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 1/301/20 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.

BR

ST

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:

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.
IS
BR

ST

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

IS

BR

ST

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

27

IS

ST

BR

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

IS

ST

BR

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

IS

BR

ST

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

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

5,56

4,23

3,12

1,786

0,902

5,52

4,21

3,06

1,779

0,908

5,57

4,20

3,08

1,810

0,911

29

5,53

4,19

3,14

1,790

0,906

5,56

4,25

3,15

1,802

0,923

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

IS

Hnh 3.11b: SK mu HT c IS

30

ST

Hnh 3.11c: SK mu HT c ST

BR

Hnh 3.11d: SK mu HT c BR

IS

BR

ST

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.

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)
S (mAU.min)

C xc nh t ng chun

C ng/ml

So vi gi tr

ST

IS

ST/IS

ng/ml

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

thc (%)

.
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
S (mAU.min)
C ng/ml

C xc nh t ng chun
So vi gi tr

BR

IS

BR/IS

ng/ml

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

thc (%)

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 80120%. 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)
Mu HT chun

HT trng
STT

Din tch

ST

(mAU*min)

(mAU*min)

0,002

0,623

6,863

46,2

102,2

0,003

0,605

6,858

44,9

99,4

0,005

0,614

6,814

45,9

101,4

0,004

0,621

6,806

46,4

102,7

0,007

0,617

6,836

45,9

101,6

0,002

0,609

6,843

45,3

100,2

TB

101,3

RSD (%)

1,2

IS (mAC*min)

Nng tm
thy (ng/ml)

ng (%)

t/
Khng t

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

Mu HT chun

HT trng
STT

Din tch

BR

(mAU*min)

(mAU*min)

IS (mAC*min)

Nng tm

ng

thy (ng/ml)

(%)

t/
Khng
t

0,002

0,210

6,863

49,8

109,9

0,003

0,207

6,854

49,4

109,1

0,005

0,201

6,839

48,6

107,3

0,004

0,203

6,805

49,0

108,2

0,007

0,195

6,826

47,8

105,5

0,002

0,190

6,835

47,0

103,8

TB

107,3

RSD (%)

2,3

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
LQC (139,2ng/ml)
T s
Mu

din tch
pic

Nng

(a)

(ng/ml)

ST/IS

MQC (905,0ng/ml)

ng(b)
(%)

T s
din tch
pic

Nng

(a)

(ng/ml)

ST/IS

HQC (1448,0ng/ml)

ng(b)
(%)

T s
din tch

Nng (a)

pic

(ng/ml)

ST/IS

ng(b)
(%)

0,284

142,8

102,6

1,805

903,3

99,8

2,895

1448,3

100,0

0,272

136,8

98,3

1,810

905,8

100,1

2,887

1444,3

99,7

0,279

140,3

100,8

1,779

890,3

98,4

2,845

1423,3

98,3

0,285

143,3

102,9

1,792

896,8

99,1

2,869

1435,3

99,1

0,279

140,3

100,8

1,814

907,8

100,3

2,894

1447,8

100,0

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
LQC (140,0ng/ml)
T s
Mu

din tch

Nng (a)

pic

(ng/ml)

BR/IS

MQC (907,0ng/ml)

ng(b)
(%)

T s
din tch

Nng (a)

pic

(ng/ml)

BR/IS

HQC (1451,2ng/ml)

ng(b)
(%)

T s
din tch

Nng (a)

pic

(ng/ml)

BR/IS

ng(b)
(%)

0,124

143,2

102,3

0,891

910,2

100,4

1,413

1432,2

98,7

0,131

150,2

107,3

0,898

917,2

101,1

1,409

1428,2

98,4

0,128

147,2

105,1

0,920

939,2

103,6

1,417

1436,2

99,0

0,136

145,2

103,7

0,915

934,2

103,0

1,459

1478,2

101,9

0,139

158,2

113,0

0,922

941,2

103,8

1,435

1454,2

100,2

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

LQC (139,2ng/ml)
Ngy

Nng

(a)

(ng/mg)

Nng (b) (%)

MQC (905,0ng/ml)
Nng

(a)

(ng/mg)

Nng (b) (%)

HQC (1448,0ng/ml)
Nng (a)

Nng (b)

(ng/mg)

(%)

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
LQC (140ng/ml)
Ngy

(a)

Nng

(ng/mg)

MQC (907,0ng/ml)
(a)

Nng (b) (%)

Nng

(ng/mg)

Nng (b) (%)

HQC (1451,2ng/ml)
Nng (a)

Nng (b)

(ng/mg)

(%)

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 (%)

6,863

8,716

78,7

6,854

8,692

78,9

6,839

8,708

78,5

6,805

8,669

78,5

6,826

8,725

78,2

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
LQC (139,2ng/ml)
STT

Huyt
tng

Dung
mi pha
ng

MQC (905,0ng/ml)
Hiu

Huyt

sut (%)

tng

Dung
mi pha
ng

HQC (1448,0ng/ml)
Hiu

Huyt

sut (%)

tng

Dung

Hiu

mi pha

sut

ng

(%)

1,917

2,338

82,0

12,389

14,926

83,0

19,802

23,526

84,2

1,958

2,390

81,9

12,406

14,910

83,2

19,837

23,598

84,1

1,906

2,317

82,3

12,367

14,958

82,7

19,846

23,573

84,2

1,942

2,308

84,1

12,328

14,983

82,3

19,858

23,535

84,4

1,932

2,385

81,0

12,354

15,006

82,3

19,892

23,603

84,3

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

LQC (140,0ng/ml)
STT

Dung

Huyt

mi pha

tng

ng

MQC (907,0ng/ml)
Hiu

Huyt

sut (%)

tng

Dung
mi pha
ng

HQC (1451,2ng/ml)
Hiu

Huyt

sut (%)

tng

Dung

Hiu

mi pha

sut

ng

(%)

1,012

1,220

83,0

6,257

7,448

84,0

10,014

11,923

84,0

1,140

1,241

91,9

6,310

7,419

85,1

10,146

11,869

85,5

1,096

1,159

94,6

6,218

7,436

83,6

10,072

12,036

83,7

1,182

1,314

90,0

6,325

7,452

84,9

10,039

12,051

83,3

1,085

1,205

90,0

6,218

7,423

83,8

10,124

11,893

85,1

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.
Nng ST (ng/ml)
LOQ (139,2ng/ml)
Mu

HQC (1448,0ng/ml)

Sau 3 chu
Ban u

k ng-

136,0

135,2

LQC (140,0ng/ml)

HQC (1451,2ng/ml)

Sau 3

Sau 3

Sau 3 chu
Ban u

r
1

Nng BR (ng/ml)

k ng-

Ban u

r
1429,3

1413,7

chu k

Ban u

ng-r
129,8

126,4

chu k
ng-r

1423,4

1413,5

40

143,2

137,4

1432,7

1420,6

132,6

130,2

1412,8

1424,6

135,7

137,4

1416,5

1425,6

148,7

145,7

1464,3

1458,3

138,0

132,1

1463,1

1456,7

146,3

143,5

1473,8

1464,8

131,8

134,5

1472,0

1467,8

142,9

140,8

1437,6

1425,0

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

98,9

ban u (%)

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
Nng ST (ng/ml)
Mu

Nng BR (ng/ml)

LQC (139,2ng/ml)

HQC (1448,0ng/ml)

LQC (140,0ng/ml)

X l

X l

X l

ngay

Sau 4 gi

ngay

Sau 4 gi

ngay

HQC (1451,2ng/ml)

Sau 4 gi

X l ngay

Sau 4
gi

135,6

136,7

1440,2

1442,8

143,5

142,8

1437,6

1435,8

143,8

145,0

1414,6

1415,6

141,0

136,7

1411,2

1425,4

136,5

137,1

1429,8

1424,7

140,5

142,9

1446,5

1448,1

137,2

136,4

1470,6

1469,8

144,2

136,9

1456,8

1454,2

142,9

139,8

1452,9

1449,2

136,5

132,8

1479,8

1466,0

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
Nng ST (ng/ml)
LQC (139,2ng/ml)
Mu

HQC (1448,0ng/ml)

Sau

Ban

Sau 7

ngy

137,3

134,9

135,4

142,6

137,8

138,0

Nng BR (ng/ml)
LQC (140,0ng/ml)

Sau 7

Sau 21

Ban

Sau 7

ngy

ngy

ngy

1419,1

1412,4

1418,9

135,2

136,0

136,8

1442,4

1440,6

1440,8

138,1

141,2

143,1

1425,6

1427,5

1425,4

144,2

143,5

142,8

1429,7

1430,1

140,9

138,0

136,4

1471,8

136,9

137,4

135,3

TB

140,0

138,8

2,2

RSD
(%)

Sau

HQC (1451,2ng/ml)
Ban

Sau 7

Sau 21

ngy

ngy

134,8

1439,4

1436,0

1432,6

139,1

140,9

1432,8

1430,2

1428,5

142,9

143,2

144,5

1449,2

1437,4

1435,7

1423,8

144,2

143,5

142,8

1413,6

1411,0

1415,9

1468,8

1463,2

134,6

141,0

136,7

1452,9

1446,8

1448,2

1452,9

1451,5

1450,9

135,7

136,3

137,8

1489,8

1486,8

1474,2

138,3

1440,3

1438,5

1437,2

138,5

139,9

139,6

1446,3

1441,4

1439,2

2,2

2,6

1,4

1,4

1,2

3,0

2,3

2,7

1,8

1,8

1,4

99,2

98,8

99,9

99,8

101,0

100,8

99,7

99,5

21

Ban u

ngy

21
ngy

T l
so vi
ban
u
(%)

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

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