B Y T

TRNG I HC DC H NI

TRN QUC LONG

XC NH BUFALIN TRONG MT S SN PHM H TR SC KHE LIN

QUAN N CC

KHA LUN TT NGHIP DC S

H NI - 2014

B Y T

TRNG I HC DC H NI

TRN QUC LONG

XC NH BUFALIN TRONG MT S SN PHM H TR SC KHE LIN

QUAN N CC

KHA LUN TT NGHIP DC S

Ngi hng dn:

1. TS. L nh Chi

2. TS. L Th Hng Ho

Ni thc hin:

Vin Kim Nghim An Ton V Sinh Thc Phm Quc Gia

H NI - 2014

LI CM N

Li u tin cho em gi li chn thnh cm n ti thy TS. L nh Chi tn

tnh hng dn, ng gp kin qu bu cho em trong sut qu trnh thc hin ti

v vit kha lun.

Em xin by t lng bit n ti ban lnh o Vin kim nghim An ton v sinh

thc phm Quc gia, c bit l TS. L Th Hng Ho to mi iu kin cho em

c thc hin ti ti y.

Em xin chn thnh cm n Ths. Trn Cao Sn, CN. Th Thu Hng cng ton

th cc anh ch trong labo Ha nhit tnh ch bo, hng dn, ng vin em trong

sut qu trnh thc hin ti ny.

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

s mi kh khn cng em.

H Ni, ngy 14 thng 5 nm 2014

Sinh vin

Trn Quc Long

MC LC

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

Chng 1. TNG QUAN ................................................................................................ 2

1.1. Cc bufotoxin v bufalin. ..................................................................................... 2

1.1.1. Gii thiu chung v h cc v cc bufotoxin. .............................................. 2

1.1.2. Bufalin. ......................................................................................................... 3

1.1.3. Ng c do n cc v cc sn phm ch bin t cc.................................... 4

1.1.4. Cc ch phm v bi thuc dn gian c ngun gc t cc .......................... 4

1.2 Mt s phng php xc nh bufalin ................................................................... 5

1.2.2. Sc k phn b ngc dng tc cao (HSCCC) ....................................... 5

1.2.1. Sc k lng hiu nng cao pha o vi detector PDA (HPLC-PDA). ......... 6

1.2.3. Phng php sc k lp mng (TLC) .......................................................... 7

1.2.4. Phng php LC-MS/MS ............................................................................. 7

1.3. K thut sc k lng kt ni khi ph (LC-MS). ................................................. 9

1.3.1. Sc k lng. .................................................................................................. 9

1.3.2. Khi ph (Mass Spectrometry). .................................................................. 11

2.1. i tng nghin cu. ........................................................................................ 15

2.2. Nguyn vt liu- trang thit b. ........................................................................... 15

2.2.1. Nguyn vt liu ........................................................................................... 15

2.2.2. Thit b, dng c ......................................................................................... 16

2.3. Ni dung, phng php nghin cu ................................................................... 17

2.3.1. Ni dung nghin cu .................................................................................. 17

2.3.2. Phng php nghin cu ............................................................................ 17

3.1. Thit lp iu kin phn tch bufalin bng LC-MS/MS ..................................... 22

3.1.1. Ti u cc iu kin ca detector khi ph (MS) ...................................... 22

3.1.2. Ti u ha cc iu kin chy sc k lng hiu nng cao (HPLC) ............ 24

3.1.2.1. Pha tnh. ..................................................................................... 24

3.1.2.2. Pha ng ..................................................................................... 25

3.2. Xy dng quy trnh x l mu ........................................................................... 30

3.2.1. Chn quy trnh x l mu. .......................................................................... 30

3.2.3. Kho st th tch chit ................................................................................ 32

3.2.4. Kho st ct chit........................................................................................ 33

3.2.5. Kho st dung dch ra gii. ....................................................................... 33

3.2.6. Kho st th tch dung dch ra gii. .......................................................... 34

3.3. Thm nh phng php. ................................................................................... 36

3.3.1. Tnh c hiu/chn lc. .............................................................................. 36

3.3.2. Gii hn pht hin (LOD), gii hn nh lng (LOQ). ............................ 38

3.3.3. Xc nh khong tuyn tnh v ng chun. ............................................ 38

3.3.4. lp li ..................................................................................................... 39

3.3.5. ng ...................................................................................................... 40

3.3.6. Bn lun ...................................................................................................... 41

3.4. Phn tch mu thc t ......................................................................................... 42

KT LUN V KIN NGH........................................................................................ 43

Kt lun ..................................................................................................................... 43

Kin ngh ................................................................................................................... 44

DANH MC CC K HIU, CC CH VIT TT

K hiu Ting anh Ting vit

AOAC Association of Official Analytical

Community

Hip hi cng ng phn tch chnh thc

APCI Atmospheric pressure chemical

ionization

Ch ion ha p sut kh quyn

ESI Electrospray ionization Ion ha phun in t

GC-MS Gas chromatography mass spectrometry Sc k kh khi ph

HPLC High performance liquid

chromatography

Sc k lng hiu nng cao

HSCCC High speed countercurrent

chromatography

Sc k phn b ngc dng tc cao.

LC-MS/MS Liquid chromatography tandem mass

spectrometry

Sc k lng ghp khi ph hai ln

LOD Limit of detection Gii hn pht hin

LOQ Limit of quantification Gii hn nh lng

MS Mass spectrometry Khi ph

PDA Photodiode Array Dy diod quang

TLC Thin layer chromatography Sc k lp mng

SPE Dispersive solid phase extraction Chit pha rn

R(%) Recovery Hiu sut thu hi

DANH MC CC BNG

Bng 1.1: Cc nghin cu dng phng php HPLC-PDA phn tch bufalin. .......... 6

Bng 1.2: Cc nghin cu dng phng php sc k lng khi ph phn tch

bufalin............................................................................................................................... 8

Bng 2.1: Sn phm c thnh phn lm t cc c nghin cu trong ti .............. 15

Bng 3.1: Cc thng s ti u ha iu kin phn mnh ............................................. 22

Bng 3.2: Cc thng s ti u MS/MS ........................................................................... 24

Bng 3.3: nh hng ca thnh phn pha ng n thi gian lu v din tch pic

bufalin............................................................................................................................. 26

Bng 3.4: Kho st cc chng trnh chy gradient ..................................................... 27

Bng 3.5: Kt qu kho st chng trnh chy gradient 28

Bng 3.6: Kho st tc dng pha ng ..................................................................... 29

Bng 3.7: Chng trnh gradient ti u ........................................................................ 29

Bng 3.8: Kho st quy trnh x l mu......................................................................... 31

Bng 3.9: Kho st dung mi chit ................................................................................ 32

Bng 3.10: Kho st th tch dung mi chit ................................................................. 32

Bng 3.11: Kho st ct chit pha rn ........................................................................... 33

Bng 3.12: Kho st dung dch ra gii ........................................................................ 34

Bng 3.13: Kt qu kho st th tch dung dch ra gii .............................................. 35

Bng 3.14: Ion m v 2 ion con ca bufalin .................................................................. 37

Bng 3.15: S ph thuc ca din tch pic vo nng ca bufalin ............................ 39

Bng 3.16: Kt qu nh gi lp li ......................................................................... 40

Bng 3.17: Kt qu nh gi ng ........................................................................... 41

Bng 3.18: Kt qu phn tch bufalin trong mu sn phm dinh dng c thnh phn

t cc .............................................................................................................................. 42

DANH MC CC HNH V, TH

Hnh 1.1: Sc k lp mng pht hin bufalin................................................................... 7

Hnh 1.2: M hnh n gin h thng LC-MS ................................................................. 9

Hnh 1.3: S cu trc ca my khi ph MS ............................................................ 11

Hnh 1.4: B ngun ion ha ........................................................................................... 12

Hnh 1.5: M hnh b phn tch t cc chp ba ............................................................ 14

Hnh 2.1:Cc bc ca qu trnh chit pha rn ............................................................ 18

Hnh 3.1: Sc k ca dung dch bufalin chun vi pha ng ACN - HCOOH 0,1% 26

Hnh 3.2: Quy trnh x l mu d kin .......................................................................... 31

Hnh 3.3: Quy trnh x l mu ti u ............................................................................. 36

Hnh 3.4: Sc mu trng ........................................................................................... 37

Hnh 3.5: Sc mu trng thm chun 50 ng/ml ........................................................ 37

Hnh 3.6: Sc mu bt m cc pht hin c bufalin ............................................... 38

Hnh 3.7: Sc bufalin ti LOD .................................................................................. 38

Hnh 3.8: ng hi quy tuyn tnh gia nng v din tch pic chun bufalin ....... 39

1

T VN

Cc nh (Bufo melanostictus) thuc h Cc-Bufonidae, c dng trong ng y

v dn gian nh mt loi thc phm b dng cho ngi gi, mt loi thuc qu h

tr, tng cng dinh dng sau m dy, dng cha bnh tr em suy dinh dng,

chn n, chm ln, gy cm, ci xng, cam tch, l nga tr em [2]. Trong tht cc, c

rt nhiu acid amin c gi tr (asparagin, histidin, tyrosin, methionin, leucin, isoleucin,

phenylamin, tryptophan, cystein), hm lng mt s nguyn t vi lng c ch nh

km, mangan cao hn rt nhiu so vi cc loi ng vt khc [2]. Nh vy gi tr dinh

dng ca tht cc rt cao.

Bn cnh gi tr dinh dng c chng minh ca tht cc, c nhiu

trng hp t vong do n tht cc c nhim c t cc, tnh ti ngy 8/9/2010, ton

quc ghi nhn 06 v ng c do c t trong cc (bufotoxin) xy ra trn a bn 6

tnh/thnh ph lm 17 ngi mc, 13 ngi i vin v 05 ngi cht [1]. Cc nghin

cu cng b xc nh trong nha cc Vit Nam Bufo melanostictus c bufalin,

resibufogenin, bufotalin v nhiu bufadienolides khc, trong bufalin l thnh phn

chnh. y l im khc bit so vi cc Trung Quc - Bufo gargarizans (trong nha c

cha cinobufagin l thnh phn chnh) [21].

Hin nay Vit Nam cha c mt phng php chnh thc phn tch pht

hin c t cc trong cc sn phm dinh dng c thnh phn t cc. V vy, gp

phn kim sot cc nguy c tim n ng c c t cc, chng ti thc hin ti:

Xc nh bufalin trong mt s sn phm h tr sc khe lin quan n cc vi

mc tiu:

- Xy dng phng php xc nh bufalin trong cc sn phm dinh dng ch

bin t cc bng k thut LC MS/MS.

- ng dng phng php xy dng c phn tch bufalin trong mt s loi

sn phm dinh dng ch bin t cc.

2

Chng 1. TNG QUAN

1.1. Cc bufotoxin v bufalin.

1.1.1. Gii thiu chung v h cc v cc bufotoxin.

H Cc (Bufonidae) c 529 loi [11], trong 16 loi c cc cht c nhm

bufotoxin (trong ni tng (gan, trng) v nha da, tuyn mang tai), bao gm loi cc

nh Vit Nam B. melanostictus. Trong tht cc khng c cht c. Nha cc cn gi

l thim t (secretio bufonis) l nha tit tuyn sau mang tai, tuyn trn mt v cc

tuyn trn da ca cc. Cht c trong nha cc, trong gan v bung trng cc c c

tnh rt cao [7].

Hp cht bufotoxin l nhm cc cht c thng thy tuyn nha sau tai, da

v ni tng ca nhiu loi cc, mt s loi lng c khc v mt s loi cy v nm

[12]. y l mt hn hp gm hn 36 hp cht c hot tnh sinh hc khc nhau: 5-

MeO-DMT, bufalin, resibufogenin, cinobufagin, bufagin, bufotalin, bufogenin,

bufothionin, epinephrin, norepinephrin v serotonin trong c nhng cht c

xp vo nhm khng gy c bao gm cholesterol, provitamin D, ergosterol v gamma

sitosteral. Nhng cht gy c c xp thnh 3 nhm:

- Nhm 1: Cc glycosis c tc dng ln tim (hay bufadienolides) ch yu gm

bufalin, cinobufagin v resibufogenin c tc ng trn h tim mch ca ng vt v

con ngi.

- Nhm 2: Phenethylamines v dn xut. Bao gm cc catecholamines nh:

dopamine, norepinephrine v epinephrine.

- Nhm 3: Tryptamines v dn xut: serotonin, bufotenin, 5-methoxy-N,N-

dimethyltryptamin (5-MeO-DMT) chng c tc dng tng huyt p v gy o gic

v/hoc hng thn (psychoactive).

3

1.1.2. Bufalin [24].

Tn thng thng: Bufalin.

Danh php quc t: (3,5)-3,14-Dihydroxybufa-20,22-dienolide,

5,20(22)-Bufadienolide-3,14-diol.

Cng thc phn t: C24H34O4.

Cu trc phn t:

Khi lng phn t: 386,52 g/mol.

Tnh cht vt l:

Tan trong chloroform (5 mg/ml), DMSO (25 mg/ml), ethanol (25

mg/ml).

Nhit nng chy 235-2360C.

T trng 1,23 g/cm3.

Tc dng v c tnh:

Bufalin c tc dng trn tim ging digoxin [13]: c ch knh Na+-K+-ATPase

(gip trao i gia K+ ngoi t bo v Na+ trong t bo), khi knh b c ch nng

Na+ trong t bo tng dn n gim hot tnh ca bm Na+/Ca2+ (bm ny trao i Na+

ngoi t bo v Ca2+ trong t bo). Hu qu l tng nng Ca2+ ni bo dn ti co c,

tng in th mng lc ngh dn ti tng tc kh cc t nhin v tng tnh t ng,

tng trng lc i giao cm v gim hot tnh giao cm trong m c tim. Ngoi ra,

bufalin c ch knh Na+-K+-ATPase lm gim s trao i Na+ v K+ dn ti tng K+

mu.

4

Gn y bufalin c nghin cu c tc dng c ch s pht trin ca khi u.

LD50 ca bufalin trn chut l 2.35 mg/kg, rt c i vi tim gy sung huyt t

bo c tim [25].

Tc gi Yasuharu H. [29] nghin cu so snh hot lc ca bufalin tc dng

trn knh Na+-K

+-ATPase ti t bo c tim ca chut vi cc glycosis tim khc, kt

qu nh sau: bufalin > digoxin > digitoxin > telocinobufagin > gamabufotalin >

cinobufotalin > cinobufagin > g-strophanthin > digitoxingenin > resibufogenin [29].

1.1.3. Ng c do n cc v cc sn phm ch bin t cc.

Trong thi gian qua trong thc t c rt nhiu v ng c do c t cc gy ra.

Triu chng ng c c t cc biu hin cp tnh, xut hin t 1 - 2 gi sau khi n (c

th sm hn nu ung ru, bia) vi cc biu hin sau:

+ Ri lon tiu ho: Bnh nhn b chng bng, au bng trn rn; km theo nn ma

d di; c th b tiu chy.

+Ri lon tim mch: Bnh nhn hi hp, nh trng ngc, tim p nhanh; sau lon

nhp tim (ngoi tm thu tht, cn nhp nhanh tht), rung tht, block nh - tht dn n

tru tim mch; huyt p lc u cao sau tt.

+Ri lon tm thn kinh: Bnh nhn ri lon cm gic (au nh kim chch u ngn

tay, ngn chn, t mi...), chng mt, o gic, v m hi lnh, tng tit nc bt; c th

kh th, ngng th, ngng tim.

+Ri lon tiu tin: Bnh nhn xut hin b i, thiu niu, v niu v nng dn n

suy thn cp.

+Mt s triu chng khc: Bnh nhn xut hin bng rt, ph n nim mc, mt nu b

nha cc bn dnh vo trc tip nim mc.

1.1.4. Cc ch phm v bi thuc dn gian c ngun gc t cc

- Dn gian [2]: Nha cc l 1 trong 6 v ca n thuc Lc thn hon. Trong

nhn dn dng nha cc cha gim au, tn thu, dng cha pht bi, inh c, yt

5

hu sng au, au rng. Tht cc kh dng vi liu 2 3 g tn bt ung hoc lm thnh

thuc vin, cha gy cm, chm ln, km n, suy dinh dng,..Hin nay c kh nhiu

sn phm h tr sc khe trn th trng c ngun gc lm t cc nh: bt m cc

(Vin Dinh Dng), bt cc baby (Cng ty CP Dc Hi Phng), bt cc royal baby

(Cng ty CP Cng ngh v Ha sinh H Ni),

- Ti Trung Quc thuc Chansu (c bit n vi tn Senso ti Nht Bn, tn

Somsa ti Hn Quc) ch t cht tit trn da v tuyn mang tai cc hin c s dng

kh rng ri [12]. Thuc c hot tnh gy t cc b, tng huyt p, cng h hp,

khng ung th v nh hng trn tim mch tuy nhin gii hn iu tr hp nn kh thn

trng khi s dng [11].

1.2 Mt s phng php xc nh bufalin

1.2.2. Sc k phn b ngc dng tc cao (HSCCC)

Sc k phn b ngc dng l phng php sc k trong c pha tnh v pha

ng trng thi lng. Do khng s dng gi mang rn nn sc k phn b ngc

dng loi tr c nhng kh khn thng gp trong cc phng php sc k trn pha

tnh rn nh mt mu hay phn hy mu.

Tc gi Li J. v cng s [14] s dng phng php HSCCC kt hp vi sc

k iu ch HPLC phn lp v tinh ch thnh cng tm bufadienolides (trong c

bufalin) t ChanSu. Tc gi s dng ch ra gii tng bc ca HSCCC vi h

thng dung mi gm petroleum ether-ethyl acetate-nc-methanol (4:6:4:6, 4:6:5:5, v /

v). Sau s dng sc k iu ch HPLC tch cc bufadienolides, cu trc ca

chng c xc nhn bi ESI-MS v ph 1H-NMR.

6

1.2.1. Sc k lng hiu nng cao pha o vi detector PDA (HPLC-PDA).

Bng 1.1: Cc nghin cu dng phng php HPLC-PDA phn tch bufalin.

TLTK Nn mu v iu kin phn tch nh gi phng php

[9]

- Nn mu: Venenum Bufonis (mt loi thuc tng

t Chansu).

- Ct: Diamonsil C18 (250 mm x 4.6 mm, 5m).

- Pha ng: Acid acetic 0,3% - acetonitril theo

chng trnh gradient.

- Tc dng: 0,8 ml/pht.

- Bc sng pht hin 296 nm.

- thu hi t

100,6%.

- LOD=0,48 ng.

- LOQ=1,55 ng.

[16]

- Nn mu: Nha cc.

- Ct: Alltech Alltima C18 (250 mm x 4.6 mm, 5

m).

- Pha ng: ng dng, acetonitrile - m acetate

(pH 3,2) (50:50, v/v).

- Tc dng 0,8 ml/pht.

- Bc sng pht hin 299 nm.

- tuyn tnh tt

trong phm vi t 0,15-

0,525 g (r = 0,9990).

- thu hi trung bnh

ca bufalin l 98,0%.

[22]

- Nn mu: Gan ngi.

- Cht ni chun: Ethinyl estradiol (10 g/ml)

- Dung mi chit: Hn hp acid photphoric m c

v chloroform.

- Mu c lm sch bng ct OasisHLB.

- Ct: Symmetry C18 (250 mm x 4.6 mm, 5 m).

- Pha ng: ng dng, acetonitril-nc (48:52)

- Tc dng: 1 ml/pht.

- Bc sng pht hin: 299 nm.

- Thi gian lu ca

bufalin: 9.7 pht.

- ng chun (n=5)

c h s tng quan l

0.9994.

- LOD = 0,4 ng.

- thu hi > 70%.

7

1.2.3.Phng php sc k lp mng (TLC)

Tc gi Hyo-Jae Lee v cng s [13] s dng phng php sc k lp mng

phn bit v pht hin bufadienolides (bufalin, cinobufagin, resibufogenin,

cinobufotalin) chit t nc c cc. iu kin phn tch: bn mng silica GF254, pha

ng: chloroform aceton (10 : 3), hin mu bng dung dch acid sulfuric 10%.

Hnh 1.1: Sc k lp mng pht hin bufalin

1.2.4 Phng php LC-MS/MS

C nhiu nghin cu v phng php sc k lng khi ph phn tch bufalin.

Mt s nghin cu c th hin bng sau:

8

Bng 1.2: Cc nghin cu dng phng php sc k lng khi ph phn tch bufalin.

Ti

liu

tham

kho

Nn mu Dung mi

chit

Pha ng/ Tc

dng SPE K thut

thu

hi LOD

[27]

Huyt

tng

chut

Ethyl

acetate

diethyl

ether (4:1)

Acetonitrile - H2O

(0,1% HCOOH),

ch gradient/ 0,2

ml/pht

UPLC-ESI-

MS/MS 86.30.7

1

ng/ml

[26] Chansu Ethyl

acetat

Acetonitrile - H2O

(0,3 %

CH3COOH), ch

gradient/ 0,7

ml/pht

Silicagel HPLC/APCI-

MS/MS 99.9

1

ng/g

[24]

Huyt

tng

chut

Ethyl

acetate

Acetonitrile - H2O

(0,1% HCOOH),

ch gradient/ 0,3

ml/pht

LCMS/MS 85 1

ng/ml

[10] Venenum

Bufonis Methanol

Acetonitrile - H2O

(0,1% HCOOH),

ch gradient

LC/ESI-

MS/MS

84-

106%

0.25

ng/ml

[6]

Huyt

tng

ch

Methanol

Acetonitrile - H2O

(0,2% HCOOH),

ch gradient/ 0,9

ml/pht

OasisHLB

HPLC/TOF-

MS 85,28

0.15

ng/ml

phn tch hm lng cc c t ca cc, hin ti phng php chnh c s

dng l sc k lng hai ln khi ph (LC-MS/MS). V c bn, y l phng php sc

k lng s dng b phn pht hin l thit b khi ph. Phng php c nhiu u im

9

nh: gii hn pht hin thp, thi gian phn tch nhanh, c th nh lng ng thi

cc cht c thi gian lu ging nhau, c tnh chn lc cao, vi kh nng xc nh cc

cht da vo khi lng v cu to ca cht nn rt c trng cho tng cht [18]Vi

nhng u im trn, chng ti chn phng php sc k lng khi ph nghin

cu thit lp phng php phn tch bufalin.

1.3. K thut sc k lng kt ni khi ph (LC-MS).

V c bn, sc k lng khi ph l phng php sc k lng s dng b phn

pht hin l detector khi ph. M hnh n gin ca h thng LC-MS nh hnh 1.2

Hnh 1.2: M hnh n gin h thng LC-MS

1.3.1. Sc k lng [4].

Sc k l qu trnh tch xy ra trn ct tch vi pha tnh l cht rn v pha ng

l cht lng. Mu phn tch c chuyn ln ct tch di dng dung dch. Khi tin

hnh chy sc k, cc cht phn tch c phn b lin tc gia pha ng v pha tnh.

Trong hn hp cc cht phn tch, do cu trc phn t v tnh cht l ho ca cc cht

khc nhau, nn kh nng tng tc ca chng vi pha tnh v pha ng khc nhau. Do

vy, chng di chuyn vi tc khc nhau v tch ra khi nhau.

Trong HPLC, pha tnh chnh l cht nhi ct lm nhim v tch hn hp cht

phn tch, l nhng cht rn, xp v kch thc ht rt nh, t 2 5 m. Trong phn

tng quan ny, chng ti ch i su trnh by v k thut sc k phn b l loi k thut

ph bin nht. Tu theo bn cht ca pha tnh, pha ng s dng, sc k lng phn b

c chia lm 2 loi: sc k pha thun (NP-HPLC) v sc k pha o (RP-HPLC)

Sc k lng Ion ha B phn

tch khi

Detector/Lu

gi s liu

Chn khng

10

Sc k pha thun: pha tnh c b mt l cc cht phn cc ( l cc silica khng x

l ha hc mang nhm silanol hoc cc silica c gn cc nhm chc phn cc: -

NH2, -CN...), pha ng l cc dung mi hu c khng phn cc nh: n-hexan,

toluene...H ny c th tch a dng cc cht khng phn cc hay t phn cc.

Sc k pha o: pha tnh thng l cc silica c ankyl ho, khng phn cc,

loi thng dng nht l C18H37, cn pha ng phn cc: nc, methanol,

acetonitril...Trong rt nhiu trng hp th thnh phn chnh ca pha ng li l nc

nn rt kinh t. H ny c s dng tch cc cht c phn cc rt a dng: t rt

phn cc, t phn cc ti khng phn cc.

Pha ng trong HPLC ng gp mt phn rt quan trng trong vic tch cc

cht phn tch trong qu trnh sc k nht nh. Mi loi sc k u c pha ng ra

gii ring cho n c c hiu qu tch tt.

C th chia pha ng lm hai loi:

Pha ng c phn cc cao: c thnh phn ch yu l nc, tuy nhin phn

tch cc cht hu c, cn thm cc dung mi khc gim phn cc nh MeOH,

ACN. Pha ng loi ny c dng trong sc k pha lin kt ngc.

Pha ng c phn cc thp: bao gm cc dung mi t phn cc nh

cyclopentan, n-pentan, n-heptan, n-hexan, 2-chloropropan, cacbondisulfua (CS2),

chlorobutan, triclomethan, toluen...

Tuy nhin pha ng mt thnh phn i khi khng p ng c kh nng ra

gii, ngi ta thng phi hp 2 hay 3 dung mi c c dung mi c phn cc

t thp n cao ph hp vi php phn tch. S thay i thnh phn pha ng theo thi

gian gi l ra gii gradient nng .

Pha ng trong LC-MS/MS khng ch ng vai tr tch cc cht m cn gp

phn vo kh nng ion ho ca cht. Chng cng c nhng yu cu cao hn v tinh

khit, hm lng cc tp cht. C nhng loi pha ng c sn xut ring cho LC-

MS/MS.

11

Detector l b phn quan trng quyt nh nhy ca phng php. Tu thuc

bn cht l ho ca cht phn tch m la chn detector cho ph hp. Mt s loi

detector dng trong HPLC nh: Detector quang ph hp th phn t (UV-VIS),

detector hunh quang (RF), detector dn, detector mng diot (PDA), detector khi

ph (MS).

1.3.2. Khi ph (Mass Spectrometry) [18].

Khi ph l thit b phn tch da trn c s xc nh khi lng phn t ca

cc hp cht ha hc bng vic phn tch cc ion phn t theo t s gia khi lng v

in tch (m/z) ca chng. Cc ion c th to ra bng cch thm hay bt in tch ca

chng nh loi b electron, proton ha...Cc ion to thnh ny c tch theo t s m/z

v pht hin, t c th cho thng tin v khi lng hoc cu trc phn t ca hp

cht.

Hnh 1.3: S cu trc ca my khi ph MS

Cu to ca mt thit b khi ph bao gm 3 phn chnh: ngun ion, thit b

phn tch v b phn pht hin (hnh 1.3). Trc ht, cc mu c ion ha trong

ngun ion, sau a vo b phn phn tch khi tch cc ion theo t s m/z. Sau

Ngun ion Phn tch

khi

Detector

X l v

lu gi liu Ph khi

Chn khng cao Trnh ion phn tch va chm

vi ion trong khng kh

12

cc ion i vo b phn pht hin (detector), s c khuch i v chuyn thnh tn

hiu. Cc tn hiu thu c s chuyn vo my tnh x l v lu tr.

Ngun ion

Cht phn tch sau khi ra khi ct tch s c dn ti ngun ion chuyn

thnh dng hi v c ion ha nguyn t. Mt s k thut ion ha thng c s

dng trong sc k lng khi ph nh: ion ha u phun in t (ESI), ion ha ha hc

p sut kh quyn (APCI). Trong nghin cu ny chng ti s dng k thut ion ho

phun in t (ESI).

K thut ion ha phun in t bao gm ba qu trnh c bn sau:

+ To thnh cc git mang in tch.

+ Lm gim kch thc ca cc ht, v phn nh cc ht.

+ Qu trnh hnh thnh pha hi cc ion.

Hnh 1.4: B ngun ion ha

Khi dung dch mu ra khi ct sc k, c a vo ng mao qun bng kim

loi. u mao qun ny c p in th cao (4-6 kV). Khi in tch d trn u mao

qun vt qua sc cng b mt ca dch mu th s to thnh cc git mang in tch.

Tip cc git mang in ny c lm gim kch thc nh hai qu trnh lin tc

xy ra, l s ha hi dung mi nh dng kh N2 c lin tc thi vo v s bn ph

ca cc git tch in cng du. Cui cng dn n s hnh thnh pha hi ca cc ion.

13

Nh lc ht tnh in m cc ion ny c dn vo b phn tch khi ph qua mt ca

s rt nh. Dung mi v kh tr N2 c ht ra ngoi do mt dng kh (Curtain Gas).

C 2 ch bn ph: bn ph vi ch ion dng v ion m.

Loi hnh thnh ion dng.

o Ph hp nht cho phn tch cc loi cht phn tch c tnh baz.

o Thng hnh thnh nn ion [M+H]+.

o Cng c th hnh thnh ion [M+nH]n+ , [M+Na+]+

Loi hnh thnh ion m.

o Thch hp nht cho s phn tch cc loi cht phn tch c tnh acid.

o Hnh thnh nn ion [M-H]-, [M-nH]n-

y l k thut ion ha mm, c nhy cao. K thut ny ng dng phn tch

cc cht khng phn cc nh: protein, peptit, cacbonhydrat, nucleotit, polyetilen

glycocol,... v cc cht phn cc c khi lng phn t nh.

B phn phn tch khi: Sau khi c ion ho, cc ion c a n b phn

tch khi nhm loi b nhng ion khng cn thit, la chn cc ion phn t, thc hin

bn pha thm thu c cc ion con. Cc k thut phn tch khi c s dng ph

bin l b phn tch t cc, b phn tch by ion, b phn tch thi gian bay. B phn

tch khi ba t cc c s dng ph bin trong k thut LC-MS/MS v y chnh l

k thut c chng ti s dng trong nghin cu ny.

T cc c cu to bi 4 thanh in cc song song to thnh mt khong trng

cc ion bay qua. Mt trng in t c to ra bng s kt hp gia dng mt

chiu (DC) v in th tn s radio (RF). Cc t cc c ng vai tr nh mt b lc

khi. Khi mt trng in t c p vo, cc ion chuyn ng trong n s dao ng

ph thuc vo t s gia m/z v trng RF. Ch nhng ion c t s m/z ph hp mi c

th i qua c b lc ny.

B phn tch khi ba t cc gm ba t cc ghp ni vi nhau (hnh 1.5). Trong

, t cc th nht (Q1) c nhim v tch cc ion, la chn ion m vi m/z nht nh

14

t ngun ion chuyn n chuyn n Q2. t cc th hai (Q2) cc ion phn t b

phn li do va chm vi kh tr c mt nh kh N2, Ar, He. B Q2 to ra phn ly do cc

ion m b phn mnh tip theo to ra cc ion nh hn, ion con (daughter ions). Q2

khng ng vai tr l b lc ion m n chp nhn tt c cc ion do Q1 chuyn n. Sau

tt c cc ion con c chuyn qua b tch Q3. B t cc th ba (Q3) lm nhim v

tch cc ion c chuyn t Q2 i ti b phn pht hin.

.

Hnh 1.5: M hnh b phn tch t cc chp ba

B phn pht hin: Sau khi i ra khi thit b phn tch khi, cc ion c a ti

phn cui ca thit b khi ph l b phn pht hin ion. B phn pht hin cho php

khi ph to ra mt tn hiu ca cc ion tng ng t cc electron th cp c

khuch i hoc to ra mt dng do in tch di chuyn. C hai loi b phn pht hin

ph bin: b phn pht hin nhn electron v b phn pht hin nhn quang.

B phn pht hin nhn electron l mt trong nhng detector ph bin nht, c

nhy cao. Cc ion p vo b mt diod lm bt ra cc electron. Cc electron th cp

sau c dn ti cc diod tip theo v s to ra electron th cp nhiu hn na, to

thnh dng cc electron. B phn pht hin nhn quang cng ging nh thit b nhn

electron, cc ion ban u p vo mt diod to ra dng cc electron. Khc vi detector

nhn electron, cc electron sau s va p vo mt mn chn phospho v gii phng

ra cc photon. Cc photon ny c pht hin bi mt b nhn quang hot ng nh

thit b nhn electron. u im ca phng php ny l cc ng nhn quang c t

trong chn khng nn loi b c cc kh nng nhim bn.

Q1 Q2

Q3 Q0

15

Chng 2. I TNG V PHNG PHP NGHIN CU

2.1. i tng nghin cu.

Bufalin trong mt s sn phm h tr sc khe lin quan n cc hin c trn

th trng:

Bng 2.1: Sn phm c thnh phn lm t cc c nghin cu trong ti

Tn mu S kim sot Thnh phn Ngy sn xut Hn dng

Bt cc royal

baby-Cng ty

CP Dc v

CN Ha sinh

H Ni

4777/2012/ATTP-

XNCB

Bt cc, lyzin,

vitamin B1,

km gluconat,

canxi lactat,

bt u.

15/07/2013 15/7/2016

Bt m cc-

Vin Dinh

Dng

CBCL

3095/2011/YT-

CNTC

Cc sy kh,

u tng, u

xanh, natri

sorbat, hng

vani

02/08/2013 02/08/2014

2.2. Nguyn vt liu- trang thit b.

2.2.1. Nguyn vt liu

a. Cht chun

- Cht chun Bufalin: Hng Wako Pure, Ltd. tinh kit > 98%.

- Dung dch chun gc bufalin nng 1000 g/ml: Cn 10 mg cht chun pha

vo 10 ml methanol, bo qun trong bnh ti trnh nh sng nhit 2-80C.

- Dung dch chun trung gian 100 g/ml: Ht 0,5 ml mi dung dch chun gc

vo bnh nh mc 5 mL v nh mc va bng. Bo qun 2-80C.

16

- Dung dch chun lm vic c nng 10 g/ml, 5 g/ml, 2 g/ml, 1 g/ml.

Nng dung dch chun trung gian (g/ml) 100 100 100 100

Th tch ht chun trung gian (l) 500 250 100 50

nh mc (ml) 5 5 5 5

Nng chun lm vic (g/ml) 10 5 2 1

b.Ha cht, dung mi

- Dung mi tinh khit cho HPLC: methanol, acetonitril (Merck).

- Ha cht tinh khit phn tch: acid formic, acid acetic bng, amoni acetat

(Merck).

2.2.2. Thit b, dng c

Thit b

- H thng sc k lng khi ph khi ph LC/MS/MS bao gm: HPLC 20 AXL

ca Shimadzu v khi ph ABI 5500 QQQ ca Aplied Biosystem: b phn bm dung

mi, b loi kh, b phn iu nhit v detector MS.

- Ct sc k Symmetry C18 (250 mm x 4,6 mm, c ht 5 m) v tin ct C18 (4

mm x 4,6 mm, c ht 5 m).

- My lc vortex IKA.

- My li tm (Heraus)

- Cn phn tch Metter Toledo (c thang c ti 0,1 mg v 0,01 mg).

- My ct quay chn khng (Eyala).

- B chit pha rn (Sulpelco) v my ht chn khng (New Askir 30).

- B thi kh (OA-Sys).

Dng c

- ng li tm 50 ml.

- Bnh c quay 100 ml.

- Vial loi 1,8 ml.

17

- ng nghim thy tinh.

- ng ong, phu, giy lc.

- Autopipet loi 100 l ,200 l, 1000 l, 5000 l v u cn tng ng.

2.3. Ni dung, phng php nghin cu

2.3.1. Ni dung nghin cu

Ni dung nghin cu bao gm:

Ti u ha iu kin xc nh bufalin bng phng php sc k lng khi ph

(LC-MS/MS).

Kho st quy trnh chit bufalin t nn mu.

Thm nh phng php:

c hiu ca phng php.

Khong tuyn tnh v ng chun.

lp li ca phng php.

thu hi ca phng php.

Gii hn pht hin, gii hn nh lng

ng dng phng php: p dng phng php mi xy dng xc nh

bufalin trong mt s sn phm h tr sc khe c cha thnh phn lm t cc hin

ang lu hnh.

2.3.2. Phng php nghin cu

a. Phng php tch chit mu.

i tng mu phn tch trong lun vn ny l sn phm dinh dng c nn

mu phc tp. Do cn c phng php tch chit mu thch hp gim nh hng

ca nn mu, trnh lm bn detector, tng kh nng pht hin.

Trong ti ny, chng ti s dng phng php chit pha rn (SPE) tch

chit mu [6][15][22].

18

Chit pha rn l mt phng php chun b lm giu v lm sch mu phn

tch t dung dch bng cch hp ph ln ct chit pha rn. Sau cht phn tch c

ra gii bng mt lng dung mi thch hp, cc cht nh hng c loi b.

Cc bc ca qu trnh chit pha rn c m t nh sau [3]:

Hnh 2.1:Cc bc ca qu trnh chit pha rn

1. Hot ha cht hp ph v pha rn.

Lm t vt liu nhi, solvat ha cc nhm chc ca cht hp ph.

Loi khng kh trong cc khong trng trong lp hp ph.

Khng c cht hp ph b kh.

2. Mu v cht phn tch c chy qua ct

Cht phn tch c lm giu trn cht hp ph.

Mt vi thnh phn nh hng khc cng c th b gi li.

Cc thnh phn khng b hp ph b loi ra.

3. Loi b cc cht gy nh hng ra khi ct.

Loi b cht gy nh hng v nn mu ch gi li cht phn tch.

19

Nu mu l dung dch nc, s dng h m hoc hn hp nc-dung mi hu c

ra tp.

Nu mu b ha tan trong dung mi hu c th khi ra ct c th s dng chnh

dung mi .

4. Ra gii

Dung mi c chn phi ph v d dng tng tc gia cht phn tch v cht

hp ph.

Th tch dung mi ra gii cng t cng tt nhng phi m bo ra sch cht phn

tch ra khi vt liu hp ph.

Cht hp ph chit pha rn thng s dng gm cc loi:

Cht hp ph pha thng: Cc silica trung tnh, oxit nhm.

Cht hp ph pha ngc: Cc silica c ankyl ha nhm OH.

Cht c kh nng trao i ion.

Cht ry hay sng lc phn t theo ln, kch thc.

Cht hp ph pha kh-rn.

Hin nay, phng php chit pha rn ngy cng c s dng rng ri v ph

bin do nhng u im vt tri sau:

Hiu sut thu hi cao.

Kh nng lm giu v lm sch cht phn tch d dng.

Gim lng dung mi s dng.

C kh nng kt hp cc phng php phn tch.

An ton, n gin v c th tin hnh hng lot.

b. Phng php sc k lng khi ph.

Chng ti la chn phng php phn tch l sc k lng khi ph (LC-

MS/MS) xc nh bufalin trong sn phm dinh dng c thnh phn lm t cc. C

s l thuyt c nu trong phn tng quan.

c. Phng php thm nh [5].

20

Gii hn pht hin, gii hn nh lng

Gii hn pht hin LOD: l nng thp nht ca cht phn tch trong mu c

th pht hin c nhng cha th nh lng c.

Gii hn nh lng LOQ: l nng ti thiu ca mt cht c trong mu th

m ta c th nh lng v cho kt qu c chm mong mun.

Xc nh LOD, LOQ da trn t l tn hiu nhiu ng (S/N): Phn tch mu

thm chun nng thp cn c th xut hin tn hiu ca cht phn tch (n=4). Xc

nh t l tn hiu chia cho nhiu (S/N = Signal to noise ratio).

LOD l nng m ti S/N = 3.

LOQ l nng m ti S/N = 10.

Trong : S l chiu cao tn hiu ca cht phn tch

N l nhiu ng nn

Khong tuyn tnh v ng chun

Khong tuyn tnh ca mt phng php: l khong nng c s ph

thuc tuyn tnh gia i lng c o v nng cc cht phn tch.

ng chun: l ng biu din s ph thuc tuyn tnh gia i lng c

o v nng cc cht phn tch.

xc nh khong tuyn tnh ngi ta thc hin o cc dung dch chun c

nng thay i v kho st s ph thuc ca tn hiu vo nng . Sau v ng

cong s ph thuc gia din tch pic thu c vo nng , quan st s ph thuc cho

n khi khng cn tuyn tnh.

C th xy dng ng chun trn nn mu thc, nhm mc ch loi tr nh

hng ca nn mu n kt qu phn tch.

lp li v thu hi.

lp li (nh gi chm) l mc gn nhau ca cc gi tr ring l ca

cc php o lp li v c biu din bng lch chun S hay h s bin thin

CV(%):

21

1

1

2

N

xx

S

N

i

i

Trong :

xi: Nng tnh c ca ln th nghim th i

x : Nng trung bnh tnh c ca N ln th nghim.

N: S ln th nghim.

ng l mc gn nhau ca gi tr phn tch vi gi tr thc hoc gi tr

c chp nhn. ng l khi nim nh tnh v c biu din nh lng di

dng thu hi (recovery). thu hi (nh gi ng) l t l phn trm gia gi

tr thu c so vi gi tr l thuyt.

R(%) = C

Cc x100%

Trong :

R: thu hi (%)

C (Ctt) : Nng cht phn tch trong mu trng thm chun (ng/ml).

Cc (Clt) : Nng chun thm (l thuyt) (ng/ml).

d. Phng php x l s liu.

Cc kt qu c tnh ton t ng theo phn mm phn tch ca thit b (phn

mm Analyst 1.5.1, ABSciex). X l kt qu bng phn mm Microsotf Excel.

22

Chng 3: KT QU V THO LUN

3.1. Thit lp iu kin phn tch bufalin bng LC-MS/MS

3.1.1. Ti u cc iu kin ca detector khi ph (MS)

Qua tham kho mt s ti liu [7][10][24][27], chng ti tin hnh kho st xc

nh bufalin bng k thut ion ha phun in t ESI vi ch bn ph ion dng.

a.Ti u ha iu kin phn mnh ca bufalin (Compound Optimization)

Trong k thut ion ha phun in t vi ch bn ph ion dng, cc ion m

c dng (M+H)+ . Detector c s dng trong nghin cu ny l khi ph hai ln, do

pht hin ng cht phn tch th vic chn c ion con l rt quan trng. Vi

h thng Tripquad 5500, iu kin phn mnh ca tng cht c ti u ha t ng.

Dng xilanh 500 L, bm chun bufalin 100 ng/ml trc tip vo detector khi

ph, la chn 2 ion con c cng tn hiu cao nht nh tnh v nh lng

bufalin.

Bng 3.1: Cc thng s ti u ha iu kin phn mnh

Ion m

(m/z)

Ion con

(m/z)

DP

(V)

CE

(V)

CXP

(V)

387 351

150 30 12

255 30 12

Trong :

+ DP (declustering potential): Th u vo, th p vo mn chn ca b phn

tch khi. N c tc dng b gy cm ion [M+H3O]+ v kh nhiu ha hc to thnh,

lm tng nhy ca php phn tch.

+ CXP (collision cell exit potential): Th p gia b t cc Q2 v Q3.

+ CE (collision energy): L nng lng va chm c to ra do th p vo b

t cc Q2, to ra nng lng phn mnh ion m.

23

Mnh ion con m/z=351 c cng ln nht dng nh lng, mnh con th

hai m/z=255 c cng thp hn dng nh tnh bufalin.

b.Ti u ha cc iu kin ca ngun v kh (Source and gas Optimization)

ti u ha iu kin MS cho tng cht, chng ti s dng k thut FIA,

ngha l hn hp chun c bm trc tip vo my MS m khng qua b HPLC.

Chn ch kho st t ng i vi tng ion con nh lng v nh tnh ca

bufalin.

Cc thng s cho b phn to ngun ion.

+ IS (IonSpray Voltage): Th ion ha, th ny c p ln u phun v mn

chn ca b phn phn tch ion. Th ny s quyt nh loi ion chuyn n b phn

phn tch khi. i vi loi ion dng 4000 5500V, ion m (-)3000 (-)4000V.

+ GS1 (Ion Source Gas 1): To p sut kh hai bn u phun, c tc dng lm

cho s hnh thnh nn cc git c d dng hn. Tc kh ca Gas 1 thng cao

hn so vi Gas 2.

+ TEM ( Temperature): Nhit ca ngun kh nng thi vo (Gas2). N thc

y qu trnh ha hi cc git cht phn tch khi i ra khi u phun.

+ GS2 (Ion Source Gas 2): To p sut ca lung kh nng, h tr qu trnh

lm bay hi dung mi, tng hiu qu ca qu trnh ion ha.

+ CUR (Curtain Gas): Lung kh N2 tinh khit c thi vo khe gia 2 mn

chn ca b phn ion ha v b phn phn tch ph. N c tc dng y cc git dung

mi v cc phn t trung ha, gi cho Q0 (ngun ion m ) sch hn.

Cc thng s ca b phn phn phn tch khi:

+ DP, CE, CXP nh gii thch trn.

+ EP (Entrance Potential): Th p vo ngun ion m Q0.

+ CAD (Collisionally activated dissociation): Kim sot p sut kh N2 trong

b t cc Q2, thc y qu trnh phn mnh th cp, ngoi ra cn c tc dng lm mt

cc ion con v hng chng n b t cc Q3.

24

T ng kho st cc thng s cho tng cht trn vi cc gi tr nh sau:

IS (V): 4000,0; 4500,0; 4500,0; 5000,0; 5500,0.

TEM (oC) : 300,0; 350,0; 400,0; 450,0.

GS1 (psi): 10,0; 20,0; 30,0; 40,0

GS2 (psi): 10,0; 15,0; 20,0; 25,0.

CUR (psi): 20,0; 25,0; 30,0.

DP (eV) : 140,0; 150,0; 160,0; 170,0.

EP (eV) : 8,0; 9,0; 10,0; 11,0; 12,0.

CAD (psi): 6,0; 7,0; 8,0; 9,0; 10,0;

CXP (eV): 10,0; 12,0; 14,0; 16,0; 18,0; 20,0.

Qua kho st ta thu c gi tr ti u ca tng thng s trn cho c cc cht

nghin cu. Gi tr c lit k trong bng:

Bng 3.2: Cc thng s ti u MS/MS

Thng

s

IS

(V)

TEM

(oC)

GS1

(psi)

GS2

(psi)

CUR

(psi)

DP

(V)

EP

(V)

CAD

(psi)

CXP

(V)

CE

(V)

Gi tr

ti u 5500 450 40 25 20 150 12 9 12 30

Kt lun: Chng ti tin hnh kho st cc iu kin MS v a ra c cc

iu kin ti u nh trong bng trn.

3.1.2. Ti u ha cc iu kin chy sc k lng hiu nng cao (HPLC)

3.1.2.1. Pha tnh.

Trong nghin cu ny, qua tham kho cc ti liu [16][24] [26], chng ti chn

ct pha o C18 phn tch Bufalin. y l loi pha tnh ph bin, ph hp cc iu

kin ca phng th nghim. bo v ct, chng ti s dng thm tin ct. Thng s

ct tch v tin ct:

Ct: Symmetry C18 (250 mm x 4,6 mm x 5 m).

25

Tin ct C18 (4 mm x 4,6 mm x 5 m).

3.1.2.2. Pha ng

a. La chn thnh phn pha ng

Trong phng php sc k lng khi ph pha ng khng ch nh hng ti qu

trnh tch cc cht m n cn nh hng ti qu trnh ion ha v tn hiu ca cht phn

tch. Vi k thut ion ha phun in t bn ph ch ion dng, qu trnh ion ha

tng khi c thm cc cht nh acid acetic, acid formic Dng dung dch chun bufalin

500 ng/ml kho st dung mi pha ng. Hn na c th va tch c bufalin,

va tit kim c thi gian phn tch v dung mi, chng ti tin hnh chy pha ng

vi ch gradient. Cc iu kin chy my c c nh nh sau:

Ct: Symmetry water C18 (250 mm x 4,6 mm, 5m).

Tin ct C18 (4 mm x 4,6 mm, 5 m).

Detector khi ph vi cc thng s bng 3.2.

Pha ng: ln lt s dng cc hn hp acetonitrile acid formic 0,1 %;

acetonitril acid acetic 0,1% v acetonitril amoni acetat 0,1 % theo chng trnh

gradient di y:

Thi gian (pht)

T l (%)

Acetonitril Acid formic (hoc acid acetic,

amoni acetat) 0,1%

1,00 20 80

4,00 80 20

7,00 80 20

7,01 20 80

10,00 20 80

Tc dng: 1 ml/pht.

26

S dng dung dch chun nng 500 ng/ml.

Kt qu thi gian lu v din tch pic ca bufalin thu c khi thay i thnh phn pha

ng c tp hp bng 3.3 (sc k ph lc 3.1).

Bng 3.3: nh hng ca thnh phn pha ng n thi gian lu v din tch pic bufalin

Thnh phn pha ng TR (min) Spic

ACN - CH3COOH 0,1% 7.45 58500

ACN - HCOOH 0,1% 7.43 85400

ACN -CH3COONH4 0,1% 7.44 44100

Hnh 3.1: Sc k ca dung dch bufalin chun vi pha ng ACN - HCOOH 0,1%

Nhn xt: S dng pha ng l acetonitril - acid formic 0,1% cho din tch pic

cao nht, tng nhy ca php o khi phn tch bufalin, pic bufalin cn i, khng b

ch, dong chn pic. Do chng ti chn pha ng acetonitril - dung dch acid formic

0,1 % cho nhng kho st tip theo.

27

b. La chn chng trnh chy gradient

c th va tch cc cht va tit kim c thi gian phn tch v dung mi,

chng ti tin hnh chy pha ng vi ch gradient. Tin hnh kho st cc

chng trnh chy gradient khc nhau vi tc dng 1 ml/pht (bng 3.4). Kt qu

th hin bng 3.5 v sc k ph lc 3.2.

Bng 3.4: Kho st cc chng trnh chy gradient

STT Thi gian

(pht)

T l (%) thnh phn pha ng

Acid formic 0,1% Acetonitril

1

1,00 80 20

4,00 20 80

7,00 20 80

7,01 80 20

10,00 80 20

2

1,00 40 60

4,00 60 40

10,00 60 40

10,01 40 60

13,00 40 60

3

1,00 20 80

4,00 80 20

10,00 80 20

10,01 20 80

16,00 20 80

4

1,00 90 10

4,00 10 90

10,00 10 90

28

Gradient

Kt qu

10,01 90 10

14,00 90 10

5

1,00 60 40

4,00 40 60

7,00 40 60

7,01 60 40

10,00 60 40

Bng 3.5: Kt qu kho st gradient

1 2 3 4 5

Din tch pic 529 000 517 000 219 000 345 000 271 000

Chiu cao pic 100 000 20 300 40 900 99 800 58 500

Nhn xt: Sau qu trnh kho st, chng ti nhn thy chng trnh gradient 1

cho p ng pic ca bufalin c cng ln nht, pic cn xng, khng b ch pic. V

chng trnh gradient ny c la chn thit lp phng php phn tch bufalin.

c. Kho st tc dng pha ng.

Thi gian lu v hnh dng ca pic sc k ph thuc nhiu vo tc dng pha

ng. Nu tc dng pha ng ln, cng c ngha th tch pha ng ln, kh nng

phn b cc cht trong pha ng ln. Nu l hn hp nhiu cht s ra nhanh v gy

chng lp cc pic. Nhng nu tc qu chm th cht phn tch b gi li trong ct

lu, thi gian phn tch ko di, nh hng n hnh dng ca pic (pic b ko ui, t),

phn tch km chnh xc. V vy chng ti tin hnh kho st tc dng pha ng 3

tc : 0,8 ml/pht, 1,0 ml/pht, 1,2 ml/pht. Kt qu c th hin bng 3.6 v sc

k ph lc 3.3.

29

Bng 3.6: Kho st tc dng pha ng

Tc dng (ml/pht) 0,8 1,0 1,2

Din tch pic 94700 271000 106000

Chiu cao pic 17100 58500 24300

Thi gian lu (pht) 6,78 7,45 8,42

Nhn xt: kt qu bng trn cho thy tc dng pha ng 1 ml/pht cho

din tch pic v chiu cao pic ln nht, do chng ti la chn tc dng pha ng

1 ml/pht.

Nh vy, qua qu trnh kho st thc nghim, chng ti thit lp quy trnh phn

tch bufalin vi cc iu kin sc k nh sau:

Ct: Symmetry water C18 (250 mm x 4,6 mm, 5m).

Tin ct C18 (4 mm x 4,6 mm, 5 m).

Pha ng: Dung dch acid formic 0,1% - acetonitril.

Chng trnh gradient dung mi (bng 3.7).

Bng 3.7: Chng trnh gradient ti u

Thi gian (pht) Tc dng

(ml/pht)

T l (%) thnh phn pha ng

Acid formic 0,1 % Acetonitril

1,00 1 80 20

4,00 1 20 80

7,00 1 20 80

7,01 1 80 20

10,00 1 80 20

Detector MS/MS vi cc thng s bng 3.1 v 3.2.

30

3.2. Xy dng quy trnh x l mu

3.2.1. Chn quy trnh x l mu.

Hai quy trnh x l mu di y c nh gi s b la chn:

Quy trnh 1 [22]:

Cn chnh xc khong 1g mu ng nht vo ng ly tm, thm 10 ml

methanol lc votex 1 pht, ng nht. Sau ly tm 5 pht tc 6000 vng/pht.

Gn ly phn dch, c quay 400C, ha cn bng 1ml MeOH, pha long bng 10 ml

nc, chuyn sang ng ly tm, ly tm 5 pht tc 6000 vng/pht. Lc qua giy

lc, ri cho qua ct HLB. Ct c hot ha bng 1 ml MeOH, 1ml H2O; mu c

np sau khi hot ha. Ct c ra bng 1ml dung dch CH3COOH 2% trong MeOH -

H2O (50 : 50) v 1 ml dung dch NH4OH trong MeOH - H2O (50:50). Ra gii bufalin

bng 1 ml CH3COOH 0,5% trong MeOH. Ly dch ra gii em phn tch.

Quy trnh 2 [27]:

Cn chnh xc khong 1g mu ng nht vo ng ly tm, thm 10 ml MeOH

lc votex 1 pht, ng nht, sau ly tm 5 pht tc 6000 vng/pht. Gn ly

phn dch, c quay 400C, ha cn bng 1ml MeOH, lc qua mng 0,22 m ly dch

em phn tch.

Nhm mc ch tm ra quy trnh x l mu ti u nht. Chng ti tin hnh

kho st trn 2 quy trnh x l mu nu trn. Cn chnh xc khong 1g mu bt

m cc ng nht vo ng ly tm 50 ml, thm 100 l dung dch chun nng 5

g/ml vo mi ng mu nh vy dch cui thu c s c nng 500 ng/ml, tin

hnh x l mu theo 2 quy trnh trn. Mi quy trnh tin hnh lm li 2 ln ly kt qu

trung bnh. Kt qu c ch ra bng sau:

31

+1 ml MeOH

+1ml H2O

+ 10 ml H2O

Hot ha

Bng 3.8: Kho st quy trnh x l mu

Kt qu Quy trnh 1 Quy trnh 2

Clt (ng/ml) 500 500

Ctt (ng/ml) 480 170

R(%) 96 34

Nhn xt: T kt qu thu c ta nhn thy quy trnh 1 cho hiu sut thu hi tt

nht. Do trong kha lun ny chng ti s tp trung kho st cc iu kin ca quy

trnh 1 a ra mt quy trnh x l mu ti u nht.

Hnh 3.2: Quy trnh x l mu d kin

Ra tp

+ 10 ml MeOH

Gn dch vo bnh c, c quay 400C

Ha cn bng 1ml MeOH

Lc qua giy lc, ly dch lc

Ct HLB hot ha Ct

Ra gii 1 ml CH3COOH 0,5% trong MeOH

Lc votex 1 pht; Ly tm 5 pht, 6000 vng/pht

Cn 1g mu/ng ly tm 50 ml

Chuyn sang ng ly tm, ly tm 5 pht,

tc 6000 vng/pht

Vial, LC-MS/MS

+1 ml CH3COOH 2% trong

MeOH-H2O (50:50,v/v)

+1 ml NH4OH 2% trong

MeOH-H2O (50:50, v/v)

32

3.2.2. Kho st dung mi chit Dung mi chit l mt yu t v cng quan trng quyt nh hiu sut ca qu

trnh x l mu. Cn chnh xc khong 1g bt m cc (khng cha bufalin) vo cc

ng ly tm 50 ml, thm chun bufalin nng 250 ng/ml, tin hnh lm theo quy trnh

1. Qua tham kho mt s ti liu [20][25] , chng ti tin hnh kho st 5 loi

dung mi chit l methanol, ethanol, ethyl acetate, cloroform, acetonitril. Kt qu th

hin bng 3.9:

Bng 3.9: Kho st dung mi chit

Dung mi Methanol Ethanol Ethyl

acetate Cloroform Acetonitril

Din tch

pic 45000 40800 33800 214 36300

Nhn xt: T kt qu thu c, chng ti chn methanol lm dung mi chit.

3.2.3. Kho st th tch chit

Chit mu khng cha bufalin c thm chun nng 250 ng/ml bng

dung mi methanol 1 ln, 2 ln, mi ln bng 5 ml methanol v 10 ml methanol. Cc

bc c tin hnh theo quy trnh 1. Ta thu c kt qu bng 3.10 v sc k ph

lc 3.5.

Bng 3.10: Kho st th tch dung mi chit

Ln chit Clt (ng/ml) Kt qu V=5 ml V=10 ml

1 250 Ctt (ng/ml) 85,8 111,3

R (%) 34,3 44,5

2 250 Ctt (ng/ml) 102,8 200,0

R (%) 41.1 80,0

33

Nhn xt: Qua qu trnh kho st, chng ti nhn thy chit 1 ln th cht phn

tch vn cha hon ton c chit ln dung mi. Kt qu chit 2 ln, mi ln bng 10

ml methanol cho hiu sut thu hi cao nht l 80% l chp nhn c do chng ti

chn chit 2 ln mi ln bng 10 ml methanol.

3.2.4. Kho st ct chit

V bufalin c tnh phn cc trung bnh [13] nn chng ti chn 2 loi ct

kho st l ct C18 [15] v ct polydivinylbenzen-N-vinylpyrolidon HLB Oasis

(hydrophile lyophile balance) [6][22] . kho st ta tin hnh nh sau: i vi ct

HLB lm nh quy trnh 1, chit mu 2 ln mi ln 10 ml methanol. i vi ct C18

giai on chit mu lm tng t nh vi ct HLB. Giai on qua ct, hot ha ct

bng 3 ml MeOH v 3 ml nc; ra tp bng 3 ml dung dch MeOH-H2O (20:80); ra

gii bng 3ml dung dch MeOH-H2O (80:20); Thi kh dch ra gii, ha cn bng 1

ml MeOH em phn tch. Kt qu thu c th hin bng 3.11 v sc k ph lc

3.6.

Bng 3.11: Kho st ct chit pha rn

Ct Clt (ng/ml) Ctt (ng/ml) R (%)

C18 250 100 40

HLB 250 208 83.1

Nhn xt: Kt qu ch ra rng i vi bufalin th vic s dng ct chit pha rn

HLB

Oasis tt hn so vi ct C18.

3.2.5. Kho st dung dch ra gii.

Trong qu trnh ra gii, nng acid acetic trong dung dch ra gii nh hng

ti lc y cht phn tch ra khi ct. V bufalin c tnh base yu nn dung dch ra

gii phi c tnh acid y cht phn tch, ng thi dung mi MeOH c tch dng

li ko cht phn tch ra khi ct. Tuy nhin dung dch cng khng c qu acid,

34

v s gy phn hy cht phn tch. Do cn phi tm c nng acid acetic ph

hp vi loi ct v cht phn tch. Tin hnh x l mu theo quy trnh 1, sau ra

gii bng cc dung dch methanol, acid acetic 0,1%, acid acetic 0,5%, acid acetic 1%

trong methanol. Kt qu thu c ch ra bng 3.12:

Bng 3.12: Kho st dung dch ra gii

Dung dch ra gii Clt (ng) Ctt (ng) R (%)

Methanol 250 96,8 38,7

0,1% CH3COOH trong methanol 250 216,8 86,7

0,5% CH3COOH trong methanol 250 156,3 62,5

1% CH3COOH trong methanol 250 82,8 33,1

Nhn xt: Kt qu cho thy dung dch ra gii acid acetic 0,1% cho hiu sut

thu hi cao nht do chng ti chn dung dch acid acetic 0,1% trong methanol lm

dung dch ra gii.

3.2.6. Kho st th tch dung dch ra gii.

Th tch dung dch ra gii nh hng ln n kt qu chit mu ca quy trnh

x l mu. Lng dung dch ra gii phi ln cht phn tch c th c ra gii

ra khi ct hon ton. Nhng nu lng qu ln cng nh hng khng tt n kt

qu v nu th tch ln s dn n hm lng nc cha trn ct qu ln, gy nh

hng khng tt n kt qu v th tch ln hm lng dung mi cha trn ct ln, gy

nh hng qu trnh trao i ion ca ct.

Cn chnh xc khong 1g mu bt m cc (khng cha bufalin) ng nht

vo ng ly tm 50 ml, thm chun bufalin nng 250 ng/ml, tin hnh x l mu

theo quy trnh, kho st ti cc th tch ra gii khc nhau l: 1, 2, 3 ml dung dch acid

acetic 0,1% trong methanol. Ta thu c kt qu (bng 3.13):

35

Bng 3.13: Kt qu kho st th tch dung dch ra gii

Th tch ra gii (ml) 1 2 3

Clt (ng/ml) 250 250 250

Ctt (ng/ml) 209,8 153 154

thu hi R (%) 83.9 61,2 61,7

Nhn xt: Theo kt qu bng trn chng ti chn th tch dung dch ra gii l 1

ml dung dch acid acetic 0,1% trong methanol cho cc th nghim tip theo.

36

+1 ml MeOH

+1ml H2O

Hnh 3.3: Quy trnh x l mu ti u

3.3. Thm nh phng php.

3.3.1. Tnh c hiu/chn lc.

xc nh tnh c hiu/chn lc i vi sc k lng khi ph chng ti s

dng cc phng php xc nhn (confirmation method). Theo hi ng Chu u im

+ 10 ml H2O

+1 ml CH3COOH 2% trong MeOH-H2O (50:50)

+1 ml NH4OH 2% trong MeOH-H2O (50:50)

+ 10 ml methanol

Ly tm 5 pht, tc 6000 vng/pht

Gn dch vo bnh c, c quay 400C

Ha cn bng 1ml MeOH

Lc qua giy lc, ly dch lc

Ct HLB hot ha Ct HLB

Ra gii 1 ml CH3COOH 0,1% trong MeOH

Vial, LC-MS/MS

Lc votex 1 pht

Cn 1g mu/ng ly tm 50 ml

Chuyn sang ng ly tm, ly tm 5 pht, tc

6000 vng/pht

B

+10 ml methanol

37

IP (im nhn dng- identification point) i vi k thut sc k lng khi ph 2 ln

(LC-MS/MS) l 4, tc l cn 1 ion m bn ph ra 2 ion con.

Bng 3.14: Ion m v 2 ion con ca bufalin

Ion m Ion con S im IP

387 351

4 255

Nh vy phng php p ng c yu cu. Hn na xc nh chc chn

phng php nghin cu c tnh c hiu/chn lc ph hp, chng ti tin hnh

phn tch cc mu trng. Kt qu cho thy mu trng khng cho cc mnh m/z ca cht

phn tch. S c mt ca cc mnh cho php nh tnh, nh lng bufalin mt cch

c hiu.

Hnh 3.4: Sc mu trng

Hnh 3.5: Sc mu trng thm chun 50 ng/ml

38

Hnh 3.6: Sc mu bt m cc pht hin c bufalin

3.3.2. Gii hn pht hin (LOD), gii hn nh lng (LOQ).

xc nh LOD, LOQ chng ti pha long dung dch chun 100 ng/ml ca

bufalin v phn tch cho n khi khng cn p ng. Chng ti xc nh c nng

1,25 ng/ml p ng pic ca bufalin gp 3 ln nhiu ng nn do chng ti

xc nh c LOD=1,25 ng/ml. Theo l thuyt thng k trong ha phn tch th

LOQ=3,33LOD. Vy LOQ=4,16 ng/ml.

Hnh 3.7: Sc bufalin ti LOD

Nhn xt: Chng ti s dng phng php xc nh trc tip LOD, LOQ da

trn t s tn hiu/nhiu (S/N) [5]. Gii hn pht hin ca bufalin l 1,25 ng/ml.

3.3.3. Xc nh khong tuyn tnh v ng chun.

Chng ti kho st quan h gia din tch pic v nng bufalin trong

khong nng t 10 1000 ng/ml. Kt qu phn tch thu c c tp hp trong

bng 3.15.

39

Bng 3.15: S ph thuc ca din tch pic vo nng ca bufalin

Nng

(ng/ml) 10 20,0 50,0 100,0 200,0 500,0 1000,0

Din tch

pic 26732 51500 78300 111000 214000 462000 899000

Hnh 3.8: ng hi quy tuyn tnh gia nng v din tch pic chun bufalin

Nhn xt: Trong khong nng 10,0-1000,0 ng/ml c quan h cht ch gia

nng bufalin v p ng pic trn sc k .

3.3.4. lp li

nh gi lp li ca phng php, chng ti tin hnh phn tch c lp 6

ln trn mu bt Royal Baby ( xc nh s b c bufalin). lp li c th hin

qua gi tr lch chun tng i RSD (%) ca hm lng bufalin tnh c t 6 ln

phn tch k trn (bng 3.16).

40

Bng 3.16: Kt qu nh gi lp li

Ln phn tch Lng cn mu

(g)

Din tch pic bufalin

Nng bufalin trong

dung dch th (ng/ml)

Hm lng bufalin trong

mu (ng/g)

1 1,1503 43878 16,4 14,3

2 1,0226 43443 15,9 15,5

3 1,0207 42051 14,3 14,0

4 1,1652 44226 16,8 14,4

5 1,0671 42225 14,5 13,6

6 1,1255 43617 16,1 14,3

TB 14,3

RSD (%) 4,6

Kt qu thc nghim cho thy, RSD (%) sau 6 ln phn tch c lp ch l 4,6 %

trong khi hm lng bufalin tm c trong mu vng thp hn 20 ng/ml. Nh vy,

phng php c lp li tt khi p dng nh lng bufalin dng vt trong mu sn

phm dinh dng c ngun gc t cc.

3.3.5. ng

nh gi ng, bufalin chun c thm vo nn dung dch mu th bt

m cc cc mc nng 10, 50, 100 ng/ml, mi mc tin hnh c lp 3 ln. Cc

mu thm chun c phn tch v tnh ton thu hi (%) ca chun bufalin so vi

nng thm vo. Kt qu phn tch c trnh by trong (bng 3.17).

thu hi ca bufalin ti 3 mc nng kho st nm trong khong 80,0

88,2%, ngha l bn trong khong thu hi 80 -110% theo quy nh ca hi ng chu

u [5]. Do phng php ny c ng tt khi phn tch bufalin trong sn phm

dinh dng t cc.

41

Bng 3.17: Kt qu nh gi ng

Nng bufalin chun

thm vo

(ng/ml)

Lng cn mu (g)

Din tch pic bufalin

Nng bufalin chun tm li c

(ng/ml)

T l tm li (%)

10,0 1,1351 50763 8,0 80,0

10,0 1,0267 49697 8,4 84,0

10,0 1,1566 51119 8,1 81,0

50,0 1,0023 79408 42,9 85,8

50,0 1,0578 79536 42,2 84,4

50,0 1,0770 78992 41,3 82,6

100,0 1,1422 119219 86,6 86,6

100,0 1,1087 119584 87,5 87,5

100,0 1,0761 119786 88,2 88,2

3.3.6. Bn lun

Kt qu thm nh v c hiu, khong tuyn tnh, ng, lp li, gii

hn nh lng v gii hn pht hin cho thy:

- V mt nh danh, phng php cho php pht hin mt cch c hiu s c mt

ca bufalin trong sn phm dinh dng t cc vi gii hn pht hin nhy (1,25 ng/ml

tnh theo nng dung dch tim sc k, ngha l tng ng vi 1,25 ng/g trong

mu phn tch khi p dng quy trnh x l mu xy dng)

- V mt nh lng, phng php cho php xc nh bufalin vi ng (t l thu

hi trn 80% khi nh gi mc nng chun thm vo 100 ng/ml), lp li

(RSD = 4,6% khi phn tch 6 ln c lp trn mu c hm lng bufalin di 20

ng/ml) tt v gii hn nh lng thp (4,16 ng/ml tnh theo nng dung dch tim

sc k, ngha l tng ng vi 4,16 ng/g trong mu phn tch khi p dng quy trnh

x l mu xy dng). Nh vy phng php c thit lp trong nghin cu ny

ph hp p dng xc nh bufalin trong cc sn phm dinh dng lm t cc.

42

3.4. Phn tch mu thc t

Do iu kin thi gian c hn, trong khun kh nghin cu ny, phng php

phn tch bufalin c ng dng nh gi 2 sn phm dinh dng c ngun gc

t cc sn xut trong nc (bng 2.1).

Kt qu phn tch cho thy trong c hai mu u pht hin c mt bufalin hm

lng ln lt l 14,3 v 19,0 ng/g (bng 3.18).

Bng 3.18: Kt qu phn tch bufalin trong mu sn phm dinh dng c thnh phn t

cc

Mu Hm lng bufalin (ng/g)

Bt cc Royal baby 14,3

Bt m cc 19,0

Vi LD50=2,35 mg/kg th hm lng bufalin c trong 2 mu trn rt nh, kh

nng gy c rt thp.

Nh vy, ch mi qua kim tra trn 2 mu sn phm dinh dng c thnh phn

nguyn liu t cc, c th thy nguy c nhim bufalin trong cc sn phm ny l rt

thc t. Tuy nhin, vi s mu phn tch cn hn ch, khun kh nghin cu ny cha

th a ra c mt nh gi xc thc v nguy c tng th nhim bufalin trong sn

phm dinh dng. Chnh v vy, mt nhu cu cp thit mang tnh thc t cao l cn m

rng kim tra bufalin trn nhiu i tng sn phm dinh dng c nguyn liu t cc

nh gi chnh xc nguy c, mc nhim bufalin trong nhm sn phm ny, t

a ra cc quy nh qun l ph hp m bo an ton cho ngi s dng.

43

KT LUN V KIN NGH

Kt lun

Trn c s cc kt qu thc nghim nghin cu xc nh bufalin trong mt

s thc phm chc nng bng phng php sc k lng khi ph, chng ti thu

c cc kt qu sau.

1. ti u ha cc iu kin khi ph xc nh bufalin.

Kho st tm c ion m ca bufalin m/z=387, ion con nh tnh m/z=255, ion

con nh lng m/z=351.

Tm cc iu kin ti u khi ph.

Thng s IS

(V)

TEM

(oC)

GS1

(psi)

GS2

(psi)

CUR

(psi)

DP

(V)

EP

(V)

CAD

(psi)

CXP

(V)

Gi tr

ti u 5500 450 40 25 25 150 12 9 12

2. ti u ha iu kin sc k lng hiu nng cao (HPLC).

Pha tnh:

Ct: Symmetry water C18 (250 mm x 4,6 mm, 5 m).

Tin ct C18 (4 mm x 4,6 mm, 5 m).

Pha ng:

L h dung mi gm 2 knh, knh A: acid formic 0,1% v knh B

acetonitril, tm c chng trnh chy gradient ti u.

Tc dng 1ml/pht.

3. kho st quy trnh x l mu.

Chng ti kho st v a ra quy trnh x l mu ti u.

44

4. Thm nh phng php: Phng php c nhy tt vi LOD l 1,25 ng/ml,

thu hi t yu cu trong khong 80-110% v lp li t yu cu CV% < 15%.

5. ng dng phn tch trn mt s mu thc t: p dng phng php phn tch 2

mu thc phm chc nng bt m cc (Vin Dinh Dng), bt cc royal baby (Cng

ty CP Cng ngh v Ha sinh H Ni) cho thy c 2 mu u c bufalin v theo tm

hiu ca chng ti, hin nay cha c quy nh no v mc gii hn hm lng bufalin

trong thc phm.

Kin ngh

1. Tin hnh p dng phng php thu c i vi cc c s ch bin, sn xut cc

sn phm h tr sc khe lin quan n cc nhm trnh cc trng hp ng c do

bufalin.

2. Tip tc nghin cu xc nh thm cc cht khc trong nhm bufadienolide trong

sn phm h tr sc khe lin quan n cc.

45

TI LIU THAM KHO

TING VIT

[1] Trn Minh Hong (2010), Lm g ngn chn nhng ci cht thng tm do Cc,

Thng Tin - Truyn Thng ATTP, S y t Bnh Dng.

[2] Tt Li (2004), Nhng cy thuc v v thuc Vit Nam, Nh xut bn Y Hc.

[3] Nguyn nh Luyn, Nguyn Thanh Bnh (2012), Tiu lun i cng chit pha

rn v ng dng chit pha rn.

[4] Nguyn Vn Ri (2004), Gio trnh cc phng php tch, H Quc Gia H Ni.

[5] Vin kim nghim an ton v sinh thc phm quc gia (2010), Thm nh phng

php trong phn tch ha hc & vi sinh vt, NXB Khoa hc v K thut.

TING ANH

[6] Cao Y., Zhao L., Liang Q., Bi K., Wang Y., Luo G. (2007), "Study of the

determination and pharmacokinetics of bufadienolides in dogs plasma after

administration of Liu-Shen-Wan by high performance liquid chromatography time-of-

flight mass spectrometry", Journal of chromatography. B, Analytical technologies in

the biomedical and life sciences, 853, 227233.

[7] Gao H., Zehl M., Leitner A., Wu X., Wang Z., Kopp B. (2010), "Comparison of

toad venoms from different Bufo species by HPLC and LC-DAD-MS/MS", J

Ethnopharmacol, 131, 368376.

[8] Gao F. v cng s (2014), "Recent developments in the field of the determination

of constituents of TCMs in body fluids of animals and human", Journal of

Pharmaceutical and Biomedical Analysis, 87, 241260.

46

[9] Huang H., Liang M., Luo H., Zhang W., (2009), "Simultaneous Determination of

Bufadienolides and Qualitative Evaluation for Venenum Bufonis by High Performance

Liquid Chromatography", Chemical Research in Chinese Universities, 25, 801806.

[10] Hu Y., Yu Z., Yang ZJ., Zhu G., Fong W., (2011), "Comprehensive chemical

analysis of Venenum Bufonis by using liquid chromatography/electrospray ionization

tandem mass spectrometry", Journal of Pharmaceutical and Biomedical Analysis, 56,

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PH LC

Ph lc 3.1: sc k kho st pha ng.

Pha ng: acetonitril-acid acetic 0,1%.

Pha ng: acetonitril-amoni acetat 0,1%

Pha ng: acetonitril-acid formic 0,1%

Ph lc 3.2: sc k kho st gradient

Gradient 1

Gradient 2

Gradient 3

Gradient 4

Ph lc 3.3: Sc k kho st tc dng pha ng.

Tc dng pha ng 0,8 ml/pht

Tc dng pha ng 1ml/pht

Tc dng pha ng 1,2 ml/pht

Ph lc 3.4: Sc k kho st dung mi chit.

Dung mi acetonitril

Dung mi chloroform

Dung mi methanol

Dung mi ethyl acetat

Dung mi ethanol

Ph lc 3.5: Sc k kho st th tch dung mi chit.

Chit 1 ln 5 ml methanol

Chit 2 ln mi ln 5 ml methanol

Chit 1 ln 10 ml methanol

Chit lp li 2 ln mi ln 10 ml methanol

Ph lc 3.6: Sc k kho st ct chit

Ct HLB

Ct C18

Ph lc 3.7: Sc k kho st dung dch ra gii

methanol

0,1% CH3COOH/MeOH

0,5% CH3COOH/MeOH

1% CH3COOH/MeOH

Ph lc 3.8: Sc mt s im chun trn nn mu

im chun 20 ng/ml

im chun 50 ng/ml

im chun 100 ng/ml

Ph lc 3.9: Sc phn tch mu thc t

Mu bt m cc pht hin c bufalin

Mu bt cc baby pht hin c bufalin

Bia.docKha lun_Trn Quc Long.pdfPH LC.docx