Nghiên cứu định lượng Tetrodotoxin trong phủ tạng cá Nóc bằng sắc ký lỏng...

B Y T

TRNG I HC DC H NI

---------

HONG THANH H

NGHIN CU NH LNG

TETRODOTOXIN TRONG PH TNG

C NC BNG SC K LNG KHI

PH (LC-MS)

KHA LUN TT NGHIP DC S

H NI-2014

B Y T

TRNG I HC DC H NI

HONG THANH H

NGHIN CU NH LNG

TETRODOTOXIN TRONG PH TNG

C NC BNG SC K LNG KHI

PH (LC-MS)

KHA LUN TT NGHIP DC S

Ngi hng dn:

1. ThS. Phng Minh Dng

2. CN. V Tng Lm

Ni thc hin:

1. B mn Ha Phn Tch

2. Vin kim nghim thuc Trung ng

3. Cng ty CP Dc Phm Mediplantex

H NI-2014

H NI-2014

LI CM N

Vi long kinh trong va bit n su sc ti xin c bay to li cam n chn

thanh ti:

ThS. Phng Minh Dng, CN.V Tng Lm va cc thy c gio trong b mn

Ha Phn Tich, nhng ngi nhit tnh giang dy ti v ca l thuyt va thc hanh,

trc tip cung cp, gp cho ti nhng thng tin, kin thc rt hu ich ti c th

hoan thanh c kha lun nay.

TS. Trn Vit Hng, Ph vin trng Vin kim nghim thuc Trung ng

to iu kin gip cho ti trong sut qu trnh nghin cu va thi nghim thc t trn

c Nc.

TS. Bi Hng Cng, Giang vin B mn Dc C Truyn, mt ngi thy ng

kinh trong cng vic cung nh trong cuc sng. Thy nh hng va cho ti nhng

li khuyn b ich ti hoan thanh kha lun nay.

Dc s Dng Minh Tn trc tip hng dn, chi bao va to moi iu kin

thun li cho ti trong qu trnh lam vic, nghin cu va thu thp s liu thc t trn

c Nc ti c th hoan thanh c kha lun.

Xin chn thanh cam n cc thy c trong Ban gim hiu, Phong ao to Trng

i hoc Dc Ha Ni to moi iu kin thun li cho ti trong qu trnh hoc tp va

hoan thanh kha lun.

Xin chn thanh cam n gia nh, ngi thn va bn b lun bn cnh ng

vin va gip ti hoc tp, lam vic va hoan thanh kha lun tt nghip.

Ha Ni, ngay 07 thng 5 nm 2014

SINH VIN

Hong Thanh H

MUC LUC

TRANG PHU BA

LI CM N

MUC LUC

DANH MUC CC CH VIT TT

DANH MUC CC BNG

DANH MUC CC HNH

Trang

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

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

1.1 Tng quan v c Nc v Tetrodotoxin ................................................................... 2

1.1.1 C Nc ............................................................................................................ 2

1.1.2. Tetrodotoxin ................................................................................................... 3

1.1.3. Phng php x l mu trong cc b phn khc nhau ca c Nc nh

lng Tetrodotoxin: K thut chit v lm giu mu bng chit pha rn (SPE) ....... 7

1.1.4. Cc phng php nh lng Tetrodotoxin ................................................... 9

1.2. Tng quan v sc kh lng khi ph ................................................................... 10

1.2.1. Mt s nt s lc v sc k lng khi ph ................................................. 10

1.2.2. Thit b sc k lng khi ph ....................................................................... 12

1.2.3. Mt s k thut LC-MS ................................................................................ 17

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

2.1. Nguyn vt liu, thit b ..................................................................................... 19

2.1.1 Nguyn vt liu ............................................................................................. 19

2.1.2 Thit b, dng c, ha cht ........................................................................... 19

2.2. Ni dung nghin cu .......................................................................................... 21

2.3. Phng php nghin cu ..................................................................................... 21

2.3.1. Chun b mu ................................................................................................ 21

2.3.2. Ti u ha iu kin khi ph i vi TTX ................................................. 23

2.3.3. Xy dng chng trnh sc k lng khi ph (LC-MS) nh lng TTX

................................................................................................................................ 23

2.3.4. nh gi phng php. ................................................................................. 24

Chng 3. THC NGHIM, KT QU V BN LUN .......................................... 26

3.1. Chun b mu ....................................................................................................... 27

3.1.1. Mu chun .................................................................................................... 27

3.1.2. Mu th ......................................................................................................... 27

3.2. Ti u ho iu kin khi ph i vi TTX ....................................................... 28

3.3. Xy dng chng trnh sc k lng khi ph (LC-MS) nh lng .............. 29

3.3.1 Kho st iu kin sc k ............................................................................. 29

3.4. nh gi s ph hp ca h thng LC-MS....................................................................35

3.5. p dng phng php nh lng mt s mu c Nc bng sc k lng khi

ph LC-MS 41

3.6. Bn lun ............................................................................................................... 41

3.6.1. V quy trnh chit tch v x l mu ............................................................ 41

3.6.2. V phng php phn tch LC/MS ............................................................... 41

3.6.3. V kt qu nh lng mt s mu c Nc .................................................. 43

KT LUN V KIN NGH ....................................................................................... 44

KT LUN ................................................................................................................ 44

KIN NGH ............................................................................................................... 45

TI LIU THAM KHO

PH LC

DANH MUC CC CH VIT TT

APCI Atmospheric Pressure Chemical Ionization (K thut ion ha ha

hc p sut kh quyn)

APPI Atmospheric Pressure Photoionization (K thut ion ha nh sng

p sut kh quyn)

ELISA Enzyme Linked Immunosorbent Assay (Xt nghim hp th min

dch lin kt vi enzyme)

ESI Electro Spray ionization (K thut ion ha bng phun in t)

FLD Fluorescence Detector (u d hunh quang)

GC-MS Gas Chromatography-Mass Spectrometry (Sc k kh ghp khi

ph)

HPLC High Performance Liquid Chromatography (Sc k lng hiu nng

cao)

LC-MS Liquid Chromatography- Mass Spectrometry (Sc k lng ghp

khi ph)

LOD Limit of detection (Gii hn pht hin)

LOQ Limit of quantitation (Gii hn nh lng)

MLD Mean Lethal Dose (Liu gy cht trung bnh)

SIM Selected Ion Monitoring (K thut phn tch chn lc ion)

SPE Solid Phase Extraction (Chit pha rn)

SRM Selected Reaction Monitoring (Chn lc ion con sau phn ng)

TIC Total Ion Chromatogram (Sc ton ion)

TTX, UV Tetrodotoxin, Ultra Violet (Tia cc tm)

AG, SG Auxiliary Gas (kh b tr), Sheath Gas (kh thi)

RSD Relative Standard Deviation ( lch chun tng i)

DANH MUC CC BNG

Bng 3.1 Kt qu kho st tnh thch hp ca h thng Tr.35

Bng 3.2 Kt qu kho st tuyn tnh ca phng php Tr.35

Bng 3.3 Kt qu kho st lp li ca phng php Tr 39

Bng 3.4 Kt qu kho st ng ca phng php Tr.40

Bng 3.5 Kt qu kho st LOD v LOQ ca phng php Tr.40

Bng 3.6 Kt qu nh lng mt s mu c Nc Tr.41

DANH MUC CC HNH

Hnh 1.1 nh hai trong s cc loi Nc c Tr.3

Hnh 1.2 Cu trc phn t Tetrodotoxin Tr.4

Hnh 1.3 C ch epimer ha nhm OH C-4 ca Tetrodotoxin Tr.5

Hnh 1.4 Cu to h thng sc k lng hiu nng cao Tr.10

Hnh 1.5 Th nghim xc nh ng v Neon ca F.W Aston nm 1913 Tr.11

Hnh 1.6 S khi cu to thit b LC/MS-MS (ngun ion ho kiu ESI

kt ni phun sng) Tr.14

Hnh 1.7 K thut Electrospray Ionization Tr.16

Hnh 2.1 Ni tng c Nc c m ly ra em i x l Tr.19

Hnh 2.2 S quy trnh x l mu, chit v lm giu Tr.22

Hnh 3.1 Kt qu ti u ha iu kin khi ph i vi TTX Tr.29

Hnh 3.2 Sc k vi iu kin sc k A Tr.30

Hnh 3.3 Sc k vi iu kin sc k B Tr.31

Hnh 3.4 Sc k vi iu kin sc k C Tr.32

Hnh 3.5 Sc k vi iu kin sc k D Tr.33

Hnh 3.6 Sc k vi iu kin sc k E Tr.34

Hnh 3.7 th biu din mi tng quan nng TTX v din tch peak Tr.36

Hnh 3.8 Sc k dung dch mu trng Tr.37

Hnh 3.9 Sc k dung dch chun TTX nng 10g/mL Tr.37

Hnh 3.10 Sc k dung dch th Tr.38

Hnh 3.11 Hai loi Lagocephalus lunaris v Arothran hispidus Tr.43

1

T VN

Mc d cc phng tin thng tin i chng ni rt nhiu v mi nguy him

ca c Nc ti tnh mng con ngi nhng s nn nhn t vong do n c Nc nc ta

vn khng h gim, c bit l cc tnh duyn hi min Trung.

c t trong c Nc c thnh phn ch yu l Tetrodotoxin (TTX), thuc nhm

c t thn kinh cc k nguy him, kh nng gy t vong cao [15]. Tuy nhin khng

phi loi c Nc no cng c, hm lng c t khc nhau cc loi c Nc khc

nhau v cc b phn khc nhau ca c. Hm lng c t trong c th cn thay i

theo ma, vng a l v giai on pht trin ca c th [6]. c t trong c Nc hin

c nhiu nc trn th gii nghin cu, c bit l Nhn Bn, Hn Quc v Trung

Quc. Chng c dng trong y dc, nghin cu khoa hc v lnh vc khc. Do vy,

chng ti xut hng nghin cu pht hin v xy dng quy trnh nh lng c t

ca mt s loi c Nc bin Vit Nam c kh nng ng dng trong y hc (lm thuc),

trong c bit quan tm n tetrodotoxin.

Sc k lng khi ph hin ang l phng php ph bin, vi nhiu u im

nh nhy cao, chnh xc,... v vy, ng dng phng php ny nh lng c t

TTX vi mt hm lng nh l ht sc ph hp v cn thit.

T thc t trn chng ti thc hin ti Nghin cu nh lng

Tetrodotoxin trong ph tng c Nc bng sc k lng khi ph (LC/MS) vi cc

mc tiu:

- Xy dng phng php nh lng Tetrodotoxin bng sc k lng khi ph.

- p dng phng php xy dng c nh lng Tetrodotoxin trong ph tng c

Nc.

2

Chng 1: TNG QUAN

1.1 TNG QUAN V CA NOC VA TETRODOTOXIN

1.1.1 C nc [3], [7], [8], [9], [17], [28], [29], [30].

B C nc (tn khoa hc l Tetraodontiformes) cha 10 h cn sinh tn vi

khong 360 loi v khong 9 h tuyt chng [17]. Phn ln cc loi l c nc mn

v sinh sng trong hay xung quanh cc bi san h ngm vng nhit i, nhng c

vi loi l cc nc ngt, sinh sng trong sng sui hay ca sng.

Cc hnh dng k d c thy trong b c ny: c th gn nh l hnh vung hay tam

gic (cc loi c nc hm), hnh cu (cc loi c nc) cho ti dp bn (cc loi c u)

[3].

C nc phng th bng cch hy sinh tc : loi ny lp vy bin i thnh cc

tm hay cc gai cng. Cc gai ny i khi c th tht vo v c th kha ti ch (nh

cc loi c nc gai), hay vi lp da dai nh da th (cc loi c u v c b giy). Mt

c im phng ng ng ch khc c thy cc loi c nc v c nc nhm l

kh nng phnh to c th, tng cc kch thc so vi hnh dng thng thng. Nhiu

loi ca cc h Tetraodontidae (c nc bn rng), Triodontidae (c nc ba rng) v

Diodontidae (c nc nhm) cn c kh nng t bo v (thm) chng cc k n tht nh

tetraodotoxin (TTX), mt cht c thn kinh cc mnh hin cha c thuc gii, tp

trung ch yu trong cc c quan ni tng [9].

Ti Vit Nam, c Nc phn b dc b bin t Bc vo Nam, tp trung nhiu ven bin

min Trung. Theo kt qu iu tra s b ca Vin Nghin cu Hi sn, c khong 46

loi trong 4 h (Diodontidae, Ostraciidae, Tetraodontidae, Triodontidae) sng bin,

trong h C nc (Tetraodontidae) l ch yu, chim khong 85%.

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c t ch yu, nguy him nht i vi ngi trong c nc l TTX. Ti Vit Nam,

ngi ta tin hnh phn tch c t ca 35 loi [7], [8], trong :

- 10 loi c c tnh rt mnh.

- 7 loi c c tnh mnh.

- 4 loi c c tnh nh.

- 14 loi cha pht hin thy c t.

Hnh 1.1. nh hai trong s cc loai Nc c

1.1.2. Tetrodotoxin [5], [10], [13], [20], [22], [24].

Tetrodotoxin (TTX) l mt c t thn kinh cc mnh, c t tn theo loi c

nc u tin pht hin thy c cha c t ny [20]. TTX c tng hp t mt s loi

vi khun: Vibrio species, Pseudomonas species, Photobacterium phosphoreum. Ngoi

c Nc c th tm thy cht c ny ni tng con Sa ging (Newt), k nhng

(Salamander), tuyn nc bt bch tuc vng xanh, ch Harlequin, cua Chn nga,

cua Philippine... cho ti nay khng c cht gii c.Tuy nhin, c Nc vn l nguyn

liu chnh tch, chit v nghin cu v TTX [5].

TTX l (4R,4aR,5R,6S,7S,8S,8aR,10S,12S)-2-azaniumyliden-4,6,8,12-tetra

4

hydroxy-6-(hydroxymethyl)-2,3,4,4a,5,6,7,8-octahydro-1H-8a,10-methano-5,7-

(epoxymethanooxy)quinazolin-10-olat, c cu trc ha hc, cng thc phn t v phn

t lng nh sau: ( hnh nh xc nh cu trc TTX bng chy cng hng t ht nhn

ti ph lc)

Hnh 1.2. Cu trc phn t TTX

Cng thc phn t: C12H17N3O8

Phn t lng: 319,3

TTX tinh khit l bt tinh th khng mu; TTX sm mu khong 220oC khng km

phn hy [13] Trong phn t TTX c mt vi nhm hydroxyl thn nc, khin n

khng tan trong cc dung mi hu c. Khung phn t ca TTX tng t nh cu trc

lng ca , khin rt kh hydrat ha, do vy n t tan trong nc. Do trong phn t c

nhm guanidin perhydroquinazolin (guanidin c tnh kim mnh), nn TTX tan trong

cc dung dch acid trong nc. TTX cng c cu trc ni este, nn d b cc dung dch

acid mnh phn hy, do cch duy nht gi TTX bn vng trong dung dch l ha

tan trong acid hu c yu [24]. c tnh C-4 c th d dng nhn thy t cu trc phn

t ca TTX. C-4 l v tr ortho ca nguyn t nit vi nhm OH v tr xch o v

nguyn t H v tr trc. Bi vy, cc hot tnh ha hc v sinh hc ca nhm

hydroxyl C-4 l rt ng k. Nu H+ c mt trong dung dch, nguyn t xy t nhm

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hydroxyl ca C-4 s kt hp vi n, to ra cu trc B ha tr dng t cu trc A. Cu

trc B mt phn t H2O to thnh cu trc C vi C-4 ha tr dng [22].

Cu trc C c th tng tc vi H2O trong dung dch. H2O c th tn cng v tr

ni phn t H2O gc b loi b v to thnh cu trc E, hoc tn cng v tr i din ni

phn t H2O gc b loi b v to thnh cu trc D. Nu phn t H2O b loi khi cu

trc E, cu trc A gc ca TTX c to thnh. Cu trc D chuyn thnh cu trc F

sau khi H2O b loi b. S khc nhau gia cu trc F v cu trc A l v tr ca H v

OH hon i cho nhau. H trong C-4 ca cu trc A l trc v OH l xch o, trong khi

cu trc F nguyn t H trong C-4 l xch o v OH l trc.

Hnh 1.3. C ch epimer ha nhm OH C-4 ca Tetrodotoxin [22].

Tetrodotoxin cu trc A c gi tetrodotoxin, l nng ch yu ca TTX

thu c t c nc t nhin. Tetrodotoxin cu trc F thng c gi 4-epi

tetrodotoxin. Do nhm hydroxyl C-4 gn vi nhm hydroxyl C-9 trong 4-epi

tetrodotoxin, phn t H2O d dng b loi bi tng tc vi H+, to ra mt analog ca

TTX cha lin kt ether, c gi l 4-epi anhydrotetrodotoxin. Cc c tnh ha

hc ca ba phn t tetrodotoxin ny khc nhau khng ng k. Nhng chng khc

nhau ng k v hot tnh sinh hc. V d, c t ca TTX l 4500 n v chut/mg;

6

ca 4-epi TTX l 710 n v chut/mg v ca 4-epi anhydrotetrodoxin ch l 92 n v

chut/mg [22].

S quan trng ca nhm hydroxyl C-4 cn th hin ch: c tnh ca n gim

ng k khi n c thay th bng nhng nhm khc, nh H, CH3 hay CH3CO-. Do ,

v l thuyt, cn gi nhm hydroxyl trong C-4 v tr xch o trong qu trnh chit

TTX. Bi vy, iu quan trng l la chn ng cc vt liu v thit b chit, pH v

nhit ca dung dch, thi gian chit. c cht chit t c nc l hn hp ca hn 10

analog, ch yu l TTX, (chim ti 70% - 80% khi lng chit). Ba analog chnh

khc l acid tetrodonic, 4-epi TTX v 4-epi anhydrotetrodotoxin. So vi TTX, ba cht

ny khng khc nhiu v c tnh ha hc, nhng khc nhau ng k v c tnh sinh

hc (c t tnh) [22].

TTX tc dng c ln h thn kinh trung ng ln thn kinh ngoi vi, vi cc biu hin

sau :

- Tim mch: Mch nhanh, huyt p h, ri lon nhp tim. [5], [20].

- H hp: Kh th, do lit c h hp v lit trung khu h hp, da v nim mc

xanh tm [10], [20].

- Thn kinh:

+ Thn kinh trung ng: Chong vng, au u, co git. Khng c ri lon thc.

+ Thn kinh ngoi vi: Lit a dy thn kinh, ri lon cm gic (d cm) li, mi,

mt, ngn tay, ngn chn, ri lon li ni, kh nut [20].

+ Thn kinh c vn v c trn: Lit, rung git cc c, c ng hn n, yu c v lit

chi di, lit vn ng nhn cu, c bit nguy him l lit c lin sn, c ngc v c

honh.

7

+ Mt phn x ty v phn x gn xng ...[20]

1.1.3. Phng php x l mu trong cc b phn khc nhau ca c Nc nh

lng Tetrodotoxin: K thut chit v lm giu mu bng chit pha rn (SPE)

[11], [16], [22].

Nh vo nhng c tnh u vit ca phng php, chit pha rn (SPE) hin

ang c s dng rt rng ri, nht l trong cc trng hp chit hot cht t cc dch

c ngun gc sinh hc, c chng ti la chn thc hin trong kha lun ny

Chit pha rn (SPE) l phng php chun b mu, trong chit v lm giu cht cn

phn tch t dung dch bng cch hp ph chng vo pha rn, ri ra gii bng dung

mi thch hp. Thng thng, th tch ca dung mi dung ra gii nh hn th tch

ca dung dch mu ban u rt nhiu, v th mu c lm giu.

C bn, c ch ca SPE ging nh c ch phn tch ca HPLC, vi ba c ch chnh l:

hp ph pha thun, hp ph pha o v trao i ion.

- C ch hp ph pha thun:

L s hp ph cht cn phn tch t dung mi khng phn cc ln b mt phn cc ca

pha rn. C ch ca qu trnh phn tch da trn lc tng tc phn cc. Trong SPE

pha thun, pha tnh thng c s dng l: silica, alumina, magnesi silica, nhng

thng dng nht vn l silica.

- C ch hp ph pha o:

Ngc vi c ch hp ph pha thun, pha tnh y l cc cht khng phn cc (nh

C18), cn pha ng l phn cc. C ch hp ph pha o l tng tc khng phn cc.

Cc cht hp ph trong pha o thng l C8, C18 v mt s loi khc.

- C ch trao i ion:

Da vo s trao i ion ca cht tan (mang in tch) trong cc dung mi phn cc hay

khng phn cc vi cht hp th trao i ion Qu trnh ny ph thuc vo chn lc

ca ion hay s lng ion cnh tranh cc v tr. Nu l cc cht trao i ion mnh th

8

dng silica vi nhm sulfonic acid, cn vi cc cht trao i ion yu thng lin kt

vi nhm COOH.

Ra gii cc cht cn phn tch trn pha tnh c th tin hnh theo nhiu cch: nu trao

i cation c th dng H+ ca cc acid mnh, hoc dung cation mnh hn (v d, ra

gii Na+ th dung dung dch K+), cn ra gii anion th dng OH- hoc cc nhm anion

mnh hn.

*Qui trnh ca SPE [11], [16].

SPE c bn bc:

- Bc 1: iu kin ha pha rn. n gin l cho dung mi chy qua ct thm t

pha rn v kch hot cc nhm chc nng ca cht hp ph. ng thi loi b khng

kh (nu c) trong ct v cc khong trng tn ti gia cc ht ca pha rn. Thng

thng, u tin ngi ta cho methanol chay qua ct, tip theo l nc hay m pha

trong nc. S dng nc hay m pha trong nc tip theo methanol l c ch hp

ph hot ng tt i vi mu l dung dch trong nc. Cn trnh, khng pha rn b

kh: ch cn cht hp ph kh trong vi pht iu kin chn khng, c ch hp ph

s hot ng km hiu qu, t l chit s rt thp.

Nu cn, c th b sung mt bc lm sch cht hp ph na trong qu trnh iu kin

ha: sau khi lm m vi methanol, cho dung mi ra gii chy qua ct loi b cc

tp cht c th c trong pha rn. Sao , ra li vi methanol, ri vi m pha trong

nc.

- Bc 2: Cho mu chy qua ct. Ph thuc vo th tch ca mu, t 1 ml n 1 lt, m

p dng ht chn khng, bm ht hay cc h thng thch hp khc. Trong bc ny,

cht cn phn tch c lu gi v c c trong pha rn.

- Bc 3: Ra loi cht nn ca mu v lu gi cht cn phn tch. Nu nh cht nn

ca mu l nc, s dng m pha trong nc hay hn hp nc v dung mi hu c

ra. Nu mu c pha trong dung mi hu c, th dung chnh dung mi .

9

- Bc 4: Ra gii cht cn phn tch ra khi pha rn bng dung mi thch hp. Dung

mi ra gii c chn la c bit ph v s tng tc gia cht cn phn tch v

cht hp ph trong qu trnh ra gii. Dung mi c chn ch ra gii t nht c th

cc cht khc cng c hp ph trong pha rn.

1.1.4. Cc phng php nh lng TTX [20], [21], [27].

Mouse bioassay: phng php sinh ha trn chut.

Phng php ELISA (Enzyme Linked Immunosorbent Assay- xt nghim

hp th min dch lin kt vi enzyme).

Phng php sc k kh khi ph (Gas-Chromatography-Mass Spectrometry)

Phng php sc k lng vi cc detector UV, FLD, MS [20],[27].

Sc k lng hiu nng cao l phng php dng tch v nh lng cc thnh

phn trong hn hp da vo i lc khc nhau ca cc cht khc nhau gia hai pha lun

tip xc v khng ng tan vi nhau. Pha ng l cht lng chy qua ct vi mt tc

nht nh di p sut cao, cn pha tnh l cht lng c bao trn b mt ca cc

ht cht mang hoc c gn ha hc vi cht mang. Pha ng cng vi mu phn tch

c bm qua ct vi tc ph hp ty theo i lc ca chng vi hai pha v dn ti

s tch cc cht.

Cc cht sau khi ra khi ct s c pht hin bi b phn pht hin gi l Detector v

c truyn qua b x l kt qu. Kt qu cui cng c hin th trn mn hnh hoc

a ra my in

Qu trnh sc k lng da trn c ch hp ph, phn b, trao i ion ...

o Pha tnh

C rt nhiu loi pha tnh c th c s dng trong sc k lng, loi ph bin

nht c ch to t silic dioxyd (silica). Cc nhm chc hu c lin kt vi b mt

ca cc tiu phn silica qua cc nhm silanol. Tnh phn cc ca pha tnh ph thuc

vo tnh phn cc ca cc nhm chc lin kt.

10

H bao gm pha tnh phn cc v pha ng khng phn cc c gi l sc k pha

thun. Ngc li, pha ng phn cc, pha tnh khng phn cc c gi l sc k pha

o.

o Pha ng

Pha ng c th l dung mi n hay hn hp ca 2, 3, 4 thnh phn. phn

cc ca dung mi c xc nh bng h s P, P cng cao th dung mi cng phn

cc. Ngi ta c th thay i phn cc ca pha ng bng cch thay i thnh phn

pha ng, t l ca cc thnh phn dung mi trong hn hp. K thut ny c gi l

ra gii gradient hay chng trnh ha dung mi.

Hnh 1.4. Cu to h thng sc k long hiu nng cao

Phng php sc k lng khi ph (Liquid chromatography mass

spectrometry)

1.2. TNG QUAN V SC K LNG KHI PH

1.2.1. Mt s nt s lc v sc k lng khi ph [4], [15], [20], [23], 25], [26].

*Phng php ph khi lng (Mass Spectrometry - MS) l mt phng

php phn tch dng c quan trng trong phn tch thnh phn v cu trc cc cht. Bt

u t cui th k XIX, Goldstein (1886) v Wein (1898) thy rng mt chm tia ion

dng c th tch ra khi nhau di tc dng ca mt in trng v t trng. Nm

1913, F.W Aston (nh bc hc t gii Nobel nm 1922 cho cc nghin cu ng v)

11

nghin cu thy kh neon t nhin gm 2 loi c khi lng nguyn t khc nhau

(isotope) l 20 v 22 (g/mol). Hng lot cc nghin cu v phng php ph khi

lng nh c ch v k thut ion ho, thit b phn tch ph khi v cc ng dng ca

phng php ph khi lng trong cc lnh vc ho hc, vt l, sinh hc, c

thc hin nh my GC/MS ra i nhng nm 1950, my HPLC/MS c pht minh

nhng nm 1970

Hnh 1.5. Thi nghim xc nh ng v Neon ca F.W Aston nm 1913

Ngy nay, phng php phn tch ph khi lng c ng dng rng ri trong nhiu

ngnh vi cc ng dng chnh nh: Xc nh khi lng, cu trc phn t; Nhn dng,

nh danh v cu trc chui peptip, protein; Nghin cu ng v; nh tnh, nh lng

cc cht nng vt v vi lng trong cc mu sinh hc, thc phm, dc phm, nng

thu sn, mi trng...

* Sc k lng khi ph (LC-MS) l s kt ni gia sc k lng hiu nng cao

(HPLC) v detector khi ph (MS).

Trong k thut LC-MS , hn hp cc cht trong pha ng sau khi c tch qua

ct sc k s c pht hin bng detector khi ph.

Trong phn tch dc phm, ngi ta thng s dng phng php LC-MS

cho:

- Cc hp cht tng i phn cc n phn cc nhiu, kh bay hi

- Phn tch thuc trong dch sinh hc

- Phn tch cc hp cht t nhin trong cy thuc

12

-Trng hp khng s dng c cc detector khc: Khng pht hin c

bng detector khc hoc trong phn tch khng nh

- Nt ni bt ca phn tch khi ph l tnh chn lc v nhy, c hiu

cao. Gii hn pht hin c th n 10-14 gam. Do vy thng dng k thut ny

phn tch hm lng siu vt trong mu thnh phn phc tp.

1.2.2. Thit b sc k lng khi ph [1], [2], [4], [6], [12], [19], [26].

a. Pha ng

Pha ng trong sc k lng l mt trong nhng yu t nh hng n s tch.

Pha ng thng l hai dung mi (hu c hoc dung dch nc) ho tan vo nhau

c kh nng tch vi phn gii ph hp.

C hai cch dng pha ng ra gii:

ng dng: Thnh phn pha ng khng thay i trong sut qu trnh sc k.

Gradient: Pha ng l hn hp ca nhiu dung mi, thng l 2 n 4 loi

c ng trong cc bnh khc nhau. T l cc thnh phn thay i trong qu trnh sc

k theo chng trnh nh (chng trnh dung mi).

b. H thng bm

Bm HPLC c chc nng to p sut cao y pha ng t bnh dung mi qua

h thng sc k. H thng bm trong sc k lng cn p ng cc yu cu sau:

Phi y c dung mi ra gii thnh dng qua ct tch, dng khng lin tc

s gy nhiu ng nn.

Khng c xung p sut cao

C th chu c tc ng ca nhiu loi dung mi (khng b n mn).

c. Ct tch v pha tnh

Ct HPLC thng c ch to bng thp khng g, thu tinh hoc cht do c

chiu di 10-30 cm, ng knh trong 4-10 mm. Kch thc ca ht nhi trong ct

13

thng l 5-10 m. Cc cht nhi thng c s dng l Silica gel, nhm oxyd,

polyme xp hoc cc loi pha o C2, C8, C18...

Ct tch sc k lng yu cu phi tr, c thnh phng, ng nht trn b mt v

c th chu c p sut cao.

d. H tim mu

Mu lng hoc dung dch c tim thng vo pha ng cao p ngay u ct

m khng cn dng dng bng mt van tim c vng cha mu. Vng cha mu c

dung tch khc nhau: thng dng loi 0,50 20 L

e. Detector khi ph [4], [12], [19], [26].

Nguyn tc hot ng

- Phng php ph khi lng l mt k thut o trc tip t s khi lng v

in tch ca ion (m/z) c to thnh trong pha kh t phn t hoc nguyn t ca

mu. T s ny c biu th bng n v khi lng nguyn t (Atomic mass unit)

hoc bng Dalton. 1 amu = 1 Da v bng khi lng ca nguyn t hydro.

- Cc ion c to thnh trong bung ion ha, c gia tc v tch ring nh b

phn phn tch khi trc khi n b phn pht hin (detector). Qu trnh phn tch

khi v pht hin c thc hin trong mi trng chn khng (p sut khong 10-5

n 10-8 Torr).

- Tn hiu tng ng vi cc ion s c th hin bng mt s vch (pic) c

cng khc nhau tp hp li thnh mt khi ph hoc ph khi. N cung cp

thng tin nh tnh (khi lng phn t, nhn dng cc cht) xc nh cu trc v nh

lng cc cht.

Nhim v ca detector khi ph

- Trong phn tch nh tnh: Khi ghp vi thit b sc k lng, cht phn tch

c xc nhn bng:

+ Sc k thi gian lu xc nh tR c trng cho cht phn tch

14

+ Ph khi tng ng ca ho cht phn tch hoc ph khi biu din cng

cc ion sinh ra t mt ion xc nh ca cht phn tch.

Nh vy vic nhn danh thng qua va thi gian lu, va ph khi s chc chn hn l

ch da vo thi gian lu.

- Trong phn tch nh lng: Do c th ca detector nh nu, gip cho nh

lng ng i tng v c nhy cao.

Cc b phn ca detector khi ph [1], [4], [12], [19], [26].

- B phn nap mu: a mu vo my. Trong my sc k lng khi ph, b

np mu l u ra ca my sc k lng c kt ni vi khi ph. Vi LC/MS, u ra

ca LC l dng dung mi pha ng nu a trc tip v ton b dng dung mi vo

khi ph k s lm gim chn khng nh hng n nhy v vn hnh ca thit b.

Mt khc, khi cc ion c trong thnh phn dng dung mi d dng va chm vi cc

phn t, nguyn t trung ha lm lch hng di chuyn, v b bm chn khng ca

khi ph k ht thi ra ngoi. Do vy, qu trnh np mu t LC vo MS thng phi c

cc giao din ghp ni ph hp (ai chuyn, chia dng, phun sng,). Mt ghp ni

LC/MS cn c cc c tnh: Cho php chuyn mu hiu qu v chnh xc t LC sang

MS m khng lm ph hy hay mt cht phn tch, loi bt dung mi ra gii, gim

pha long mu v lm mt cht phn tch...

Hnh 1.6. S khi cu to thit b LC/MS-MS (ngun ion ho kiu

ESI kt ni phun sng)

15

- B phn ion ho (ion source):

+ Nhim v:

Ion ho cht cn phn tch

Chuyn ion t pha dung dch vo pha hi.

Kh dung mi a tip ion vo b phn tch khi.

Cch ly phn to ion p sut kh quyn vi b phn nm trong chn khng

su.

Rt ra ngoi cc phn t trung ho v ion khc du c th nh hng n

php o.

+ K thut ion ho: C nhiu k thut ion ha nh:

- Ky thut ion ha va chm in t (Electron Impact ionization)

- Ky thut ion ha ha hoc (Chemical ionization)

- Ky thut ion ha in trng (Field ionization)

- Ky thut bn ph nhanh nguyn t (Fast Atom Bombardment)

- Ky thut phan hp th va ion ho bng tia laser

- Ky thut ion ha phun sng in (Electron Spray Ionization)

- Ky thut ion ha ha hoc p sut khi quyn (APCI)

- K thut ion ha nh sng p sut khi quyn (APPI)

Hin nay ch yu s dng hai k thut phun in t (ESI) v ion ho bng ho hc

p sut thng (APCI) hoc kt hp 2 k thut ny.

K thut s dng trong kho lun ny l k thut ESI [18], k thut c s dng ph

bin nht trong LC-MS. K thut ny c m t nh sau:

16

Hnh 1.7. Ky thut Electrospray Ionization

ESI l mt k thut ion ha c ng dng cho nhng hp cht khng bn

nhit, phn cc, c khi lng phn t ln. ESI c kh nng to thnh nhng ion a

in tch (dng hoc m, ty thuc vo p cc in th), c xem l k thut ion ha

m du hn APCI, thch hp cho phn tch cc hp cht sinh hc nh protein, peptide,

nucleotide... Trong ESI, ti u ng dn mao qun, di nh hng ca in th cao v

s h tr ca kh mang, mu c phun thnh nhng ht sng nh mang tch in ti

b mt. Kh xung quanh cc git ny to nhit nng lm bay hi dung mi ra khi

git sng, khi , mt in tch ti b mt ht sng gia tng. Mt in tch ny

tng n mt im gii hn (gii hn n nh Rayleigh) t ht sng phn chia

thnh nhng ht nh hn v lc y lc ny ln hn sc cng b mt. Qu trnh ny

c lp li nhiu ln hnh thnh nhng ht rt nh. T nhng ht rt nh mang

in tch cao ny, cc ion phn tch c chuyn thnh th kh bi lc y tnh in

sau ri sau i vo b phn tch khi. Trong k thut ESI, phn t nht thit phi

c bin thnh cht in ly, tan trong dung dch dng phun sng. iu ny ph

thuc vo: dung mi s dng, pKa ca cht in ly v pH ca dung dch.

- B phn phn tch khi (mass analyzer):

Cc ion hnh thnh ngun ion ho c khi lng m v in tch z (t s m/z c

gi l s khi) s i vo b phn phn tch khi. B phn phn tch khi c coi l

qu tim ca my khi ph, c nhim v tch cc ion c s khi m/z khc nhau thnh

17

tng phn ring bit nh tc dng ca t trng, in trng trc khi n n b phn

pht hin v x l s liu.

- B phn pht hin (detector)

Sau khi i ra khi b phn phn tch khi lng, 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 (electron multiplier) v b phn pht hin nhn quang

(photomultiplier). B phn pht hin nhn electron l mt trong nhng detector ph

bin nht, c nhy cao. Cc ion p vo b mt dinot lm bt ra cc electron. Cc

electron th cp sau c dn ti cc dinot 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 dinot 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.

1.2.3. Mt s k thut LC-MS [4].

a. K thut phn tch ton thang (Full scan)

Vi mt hn hp nhiu cht, sau khi c tch ra qua ct sc k lng v a

vo detector khi ph, khi ph k ghi nhn tng cng cc ion sinh ra t mi cht.

Ta c mt sc ton ion TIC (Total Ion Chromatogram) v ph khi c ly ton b

cc ion (FULL SCAN).

FULL SCAN cho y thng tin v cht phn tch hn, tuy nhin phng

php ny c nhy khng cao, nhiu ng nn c th ln.

b. K thut phn tch chn lc ion (SIM, Selected Ion Monitoring)

18

K thut ny ch cho khi ph k nhn din mt ion v ghi sc theo thi gian.

K thut SIM c tc dng lm gim bt nhiu ng nn v do tng nhy, tc

tng t l tn hiu S/nhiu ng nn N.

K thut SIM nhy hn FULL SCAN

c. K thut MS/MS

i vi k thut MS/MS, mt ion chn lc ch MS ln 1 c phn tch tip bng

cch s dng nng lng b gy to ra mt hoc vi ion con c trng ch phn

tch MS ln 2.

K thut MS/MS c s dng tng chn lc v tng nhy do lm gim

nhiu ng nn rt ng k.

d. K thut SRM (Selected Reaction Monitoring)

Nu khi ly khi ph 2 ln MS/MS (MS2) m khi ph k ch ghi nhn 1 mnh

con thi th ta ni s dng k thut SRM (Chn lc ion con sau phn ng). y l mt

k thut nh lng kh chnh xc c chng ti p dng trong kha lun ny.

19

Chng 2. I TNG V PHNG PHP NGHIN CU

2.1. NGUYN VT LIU, THIT B

2.1.1 Nguyn vt liu

Ni tng c Nc (trng, gan, rut) thu mua Nha Trang Khnh Ha c bo

qun ng lnh.

Hnh 2.1. Ni tng c Nc c m ly ra em i x li

2.1.2 Thit b, dng c, ha cht

a. Thit b

- My xay GL-1903

- B iu nhit BCHI 461 water bath

- My ct quay Buchi RE111 Rotavapor Evaporator

- Bm ht chn khng model DOA-P504-BN

- Cn phn tch (Sartorius, d = 0,1 mg, c)

- Cn k thut

- My sc k lng khi ph Thermo Scientific TSQ quantum Ultra

b. Dng c

X l, chit v lm

giu mu bng

chit pha rn (SPE)

c th nh

lng hm lng

TTX trong mu c

Nc

20

- Bnh nh mc

- Mng lc Satorius RC 0,45 m

- My o pH (Cyber Scan 500, Singapore)

- Bm tim cho HPLC

- Ct: Alltech Apollo C8 (25 cm x 4,6mm; 5m)

- Ct Zorbax 300SB C3 (25 cm x 4,6mm; 5m)

- B chit pha rn, ct chit pha rn C18.

c. Dung mi, ha cht

- Cht chun TTX (Sigma Aldrich, 1mg/l, HL: 99%, SKS: 038K1804)

- Nc kh ion

- Acid acetic khan (Merck)

- Dung dch acid acetic 10% (tt/tt)

o (Pha long 100 ml acid acetic khan vi nc kh ion thanh 1000 ml, lc u).

- Dung dch acid acetic 5% (tt/tt)

o (Pha long 50 ml acid acetic khan vi nc kh ion thanh 1000 ml, lc u).

- Amoniac m c (Merck)

- Dung dch amoniac 10% (kl/tt) trong nc

o (Pha long 100 ml ammoniac m c vi nc kh ion thanh 1000 ml, lc

u).

- Nha trao i ion cation amoniac acid yu (nha LD152 NanKai,

TianJing, Trung quc)

- Ethanol 20% (tt/tt)

- Dung dch acid acetic 0,2% (tt/tt) trong ethanol 20%

o (Pha long 2 ml acid acetic khan vi ethanol 20% thanh 1000 ml, lc u).

- m acetat: Dung dch trong nc ct cha 15 mM amoni acetate v 15

mM acid acetic.

- Methanol dng cho HPLC (Merck)

21

2.2. NI DUNG NGHIN CU

- Xy dng quy trnh x l mu.

- Ti u ha iu kin khi ph, kho st iu kin sc k nh lng

Tetrodotoxin bng sc l lng khi ph (LC-MS).

- nh gi phng php.

- p dng phng php nh lng mt s mu ph tng c Nc.

2.3. PHNG PHP NGHIN CU

2.3.1. Chun b mu

Bao gm mu chun v mu th:

* Mu chun: Pha cc dung dch chun t cht chun gc.

* Mu th: bao gm x l mu v pha dung dch mu th.

- Mu ni tng c Nc c em x l theo quy trnh la chn di hnh 2.2( theo

tham kho ti liu [11], [16], [22], v trong gii hn ca kha lun tt nghip dc s

nn chng ti khng trnh by c th cc bc xy dng quy trnh x l mu) thu c

dch chit TTX.

- Dung dch th: dch chit thu c t quy trnh x l, em c cn qua my ct quay,

thu c cn. Ha tan cn trong 20mL hn hp m acetat Methanol (20 80), lc

qua mng lc 0,45m.

22

10g ph

tang c nc

H2O kh ion &

acid acetic

khan

Dung dch

ngm chit

B

un ti 80oC,

lm lnh &

Lc

Dch lc

iu chnh ti pH 7,5 vi d2

amoniac 10%, lc.

Tin hnh qua ct trao i ion

amino (NH2).

Ra gii vi d2 acid acetic 10%

Phn c TTX

Phn khng

c TTX

iu chnh ti pH 8,5 bng d2

amoniac 10%

Tin hnh SPE vi ct C-18

Dung dch

trong

khng c

TTX

Ra gii vi

d2 acid

acetic 0,2%

trong ethanol

20%

D2 TTX

Hnh 2.2. S quy trnh x li mu, chit va lam giau

23

2.3.2. Ti u ha iu kin khi ph i vi TTX

xc nh iu kin khi ph, trc tin chng ti pha dung dch chun ca

tng cht trong pha ng, sau bm trc tip bng syringe v chy chng trnh

Tune iu chnh cc thng s khi ph.

a. Ti u ho iu kin bt ion phn tch i vi TTX, mnh m/z = 320

+ Pha dung dch TTX 10ppm trong pha ng bm trc tip vo my khi ph qua

syringe chun (Unimetrics 500 L) vi tc 5L/pht.

+ Gi phng php Tune Plus xc nh iu kin khi ph:

Bc 1: Bt my khi ph, khi ng phn mm v gi phng php bng

cch: trn ca s Instrument Setup, chn biu tng my khi ph LCQ Advantage

Max MS s xut hin hnh my khi ph.

Bc 2: Nhp chut vo Tune Plus, xut hin ca s Tune Plus, chn ch

Positive ESI (+ESI).

Bc 3: Bm dung dch TTX vo h thng khi ph vi tc dng

5 L/pht, sau mt thi gian s xut hin mnh ion c m/z = 320.

Bc 4: Nhp vo biu tng Tune trn mn hnh s xut hin ca s chy ch

Automatic Tune.

Bc 5: Trong Mass (m/z), nh s 320 Tune cho mnh 320, v chn la

bn ph mnh m m/z = 320 thnh 4 mnh con. My s t ng iu chnh v ti u

ho cc thng s sao cho bt mnh ion m m/z = 320 cng vi cc mnh con tng

ng tt v nhy nht. Qu trnh ny mt khong 5 pht. Sau khi my t ng iu

chnh, a ra iu kin ph hp tn hiu ca mnh ion m m/z 320 tt hn v iu

kin c cc mnh con ti u. Lu file Tune li t tn l Tune_TTX 10ppm_320-

4_product_ion. LCQTune

b. Thit lp phng php LC-MS cho TTX

phn Instrument Setup, gi file Tune_TTX10ppm_320-4_product_ion.

LCQTune.

24

Chn phn tch MS 2 ln (MS/MS), ch SRM, la chn mnh m/z = 320

162.

Thit lp cc iu kin khi ph: Ngun ion ha (+ESI), kh thi (SG), kh b

tr (AG), nhit ha hi, nhit mao qun, th ion ha.

2.3.3. Xy dng chng trnh sc k lng khi ph (LC-MS) nh lng TTX

Kho st tm iu kin sc k nh ct, pha ng, tc dng, iu kin khi

ph cho chng trnh sc k LC-MS nh lng TTX.

2.3.4. nh gi phng php.

- tuyn tinh:

tuyn tnh biu th s tng quan hi qui gia din tch pic v nng cht

phn tch. Chun b dy cc dung dch chun c nng trong khong nng phn

tch kho st, phn tch mu xc nh phng trnh hi qui (y=ax+b) v h s tng

quan r.

- lp li:

+ lp li ca h thng: Xc nh bng lch chun tng i (RSD) din

tch pic cng mu chun tim 6 ln.

+ lp li kt qu phn tch: nh gi thng qua sai s tng i ca kt qu

phn tch.

- ng:

Bng phng php thm cht chun v nh lng xc nh kh nng tm li.

- Gii hn pht hin LOD va gii hn nh lng LOQ.

+ Gii hn pht hin (LOD) l nng thp nht ca cht phn tch c th xc

nh c nhng khng cn thit phi nh lng c trong iu kin th nghim c

th. LOD c coi l nng cht phn tch gy nn s tng tn hiu p ng 3 ln so

vi lch chun p ng ca mu trng. LOD l mt thng s ca php th gii hn.

25

Trong : S : l lch chun p ng ca mu trng

b : nhy

+ Gii hn nh lng (LOQ) l nng thp nht trong mu th c th nh

lng c vi tnh ng v tnh chnh xc chp nhn c. LOQ chp nhn c nu

t cc iu kin 1) p ng ca cht phn tch phi t nht gp 10 ln p ng ca mu

trng; 2) pic ca cht cn phn tch phi thy r, ring bit v gi tr p ng lp li vi

chnh xc 20%. Gii hn nh lng (LOQ) c tnh bng 3,3 ln gii hn pht

hin.

Ngoi ra, gii hn nh lng cn c xc nh bng nng cht cho p ng

c t s S/N = 10.

e. Phng php x l s liu

Cc c trng thng k c tnh da vo cc cng thc hoc da vo cc hm

s trong Microsoft Excel.

Tin hnh th nghim n ln, thu c cc kt qu xi l x1, x2, x3xn. Khi :

Trung bnh cc kt qu:

n

i

ixn

x1

_ 1

lch chun : S = 1

)(1

2

n

XXin

i

lch chun tng i: RSD (%) = 100X

S

Tng quan hi qui tuyn tnh:

26

- Phng trnh hi qui tuyn tnh bc nht th hin quan h gia din tch

pic sc k v nng cht phn tch:

Y = ax + b

Trong :

+ H s gc a c tnh da vo hm Slope trong Microsoft Excel.

+ H s b c tnh da vo hm Intercept trong Microsoft Excel.

- H s tng quan r c tnh da vo hm Correl trong Microsoft

Excel.

(Vo phn mm ny bng cch chn biu tng ca n trn Desktop, chn

Insert/Function).

27

Chng 3. THC NGHIM, KT QU V BN LUN

3.1. CHUN B MU

3.1.1. Mu chun

- Cht chun gc TTX (Sigma Aldrich, 1mg/l, HL: 99%, SKS: 038K1804)

- Pha dy cc dung dch chun c nng ln lt l: 20,00ppm; 10,00ppm; 5,00ppm;

1,00ppm; 0,50ppm; 0,25ppm t cht chun gc.

3.1.2. Mu th

* X l mu nh quy trnh trn hnh 2.2 c th hn nh sau:

- X l, ngm chit v lc

(a). Ct nh khong 10g ni tng (trng, gan, rut) c nc, cho vo cc c m 250mL.

(b). Thm 30 mL nc kh ion v 30 L acid acetic khan.

(c). Khi ng thit b khuy trn v khuy hn hp trong 30 pht nhit phng.

(d). Lc v p, thu ly dch lc trong mu vng (dch lc ngm chit u).

(e). Thm 30 mL nc kh ion vo cc c m v lp li qu trnh nh m t bc (a),

(b), (c) va (d), thu dch lc ngm chit th hai.

(f). Lp li tng t bc (e), thu c dch lc ngm chit th ba.

(g). Gp cc dch lc ngm chit, un nng ti 80oC v duy tr nhit ny trong 5

pht. Lm lnh, lc, loi b ta, thu ly dch lc.

- Qu trnh lm giu bng trao i ion v SPE

(a). iu chnh dch lc thu c bc x l trn (bc (g)) ti pH 7,5 bng dung

dch amoniac 10% (kl/tt).

(b). Lc, cho dch lc chy qua ct trao i ion, vi tc 200 ml/h.

28

(d). Sau khi ton b dch lc chy qua ct trao i ion, ra ct vi nc kh ion cho

n khi khng cn protein trong nc ra.

(e). Ra gii ct trao i ion bng dung dch acid acetic 10% (tt/tt), vi tc 100 ml/h,

thu dch TTX.

(f). Gp dch TTX thu c.

(g). iu chnh ti pH 8,5 bng dung dch amoniac 10% (kl/tt).

(h). Tin hnh SPE vi cc iu kin nh sau:

- Cht hp ph: Cht hp ph C-18, ct 1 ml. Hot ha ct vi 2 x 1 ml methanol, tip

theo l 2 x 1 ml dung dch amoniac pH 8,5.

- Ra giai: Sau khi cho dch TTX, ra ct vi 2 x 1 ml dung dch amoniac pH 8,5, ri

vi 2 x 1 ml nc ct. ct kh trong khng kh 2 pht v ra gii vi 2 x 1 ml dung

dch acid acetic 0,2% trong ethanol 20%.

(i). Gp dch TTX thu c.

* Dch chit thu c t quy trnh x l, em c cn qua my ct quay, thu c cn.

Ha tan cn trong 20mL hn hp m acetat Methanol (20 80), lc qua mng lc

0,45m, tim sc k.

3.2. TI U HA IU KIN KHI PH I VI TTX

Tin hnh cc bc nh mc 2.3.2

Mnh ion m m/z = 320

Mnh ion con m/z = 162

SRM: 320 162 vi mc nng lng CE = 35V

29

Hnh 3.1. Kt qua ti u ha iu kin khi ph i vi TTX

3.3. XY DNG CHNG TRNH SC K LNG KHI PH (LS-MS)

NH TNH V NH LNG

3.3.1 Kho st iu kin sc k

Tin hnh kho st cc iu kin sc k, s dng dung dch chun TTX 10ppm

a. iu kin (A)

- Ct: Zorbax 300SB C3; 5m; 250 x 4,6mm

- Pha ng: m acetat MeOH = 35 65

(m acetat: dung dch amoni acetat 15mM, acid acetic 15mM)

- Tc dng: 400L/pht

- Th tch tim: 25L

- iu kin khi ph

30

+ Ngun: ESI+

+ Kh: SG: 30, AG: 20

+ Th ion ha: 3200V

+ Nhit ha hi: 200oC

+ Nhit mao qun: 360oC

+ SRM: 320 162 vi mc nng lng CE = 35V

Kt qu: C:\Xcalibur\...\Data\Vat ly\TTX\TTX10ppm

RT: 0.00 - 10.00

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Relat

ive

Abun

danc

e

7.69

0.23 0.70 1.701.18 1.35 2.00 2.14 2.47 5.914.01 5.414.43 6.592.88 3.53 5.213.25 4.74 6.19

NL:

1.09E6

TIC M S

TTX10ppm

TTX10ppm #900 RT: 7.69 AV: 1 NL: 1.09E6

T: + c ESI SRM ms2 320.000 [161.500-162.500]

161.5 161.6 161.7 161.8 161.9 162.0 162.1 162.2 162.3 162.4 162.5

m/z

0

10

20

30

40

50

60

70

80

90

100

Relat

ive

Abun

danc

e

162.26

161.64

Hnh 3.2. Sc k vi iu kin sc k A

Peak trn sc k thu c c thi gian lu 7,69 pht; tuy nhin, peak dong =>

khng ph hp

b. iu kin sc k (B)




- Tc dng: 500L/pht

- Th tch tim: 10L

- iu kin khi ph

31

+ Ngun: ESI+

+ Kh: SG: 20, AG: 10

+ Th ion ha: 3200V

+ Nhit ha hi: 200oC



Kt qu: C:\Xcalibur\...\Vat ly\TTX\TTX10ppm1

RT: 0.00 - 10.00

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Abu

ndan

ce

7.87

0.04 0.860.64 1.04 2.332.141.891.39 2.64 2.78 3.36 4.07 4.313.79 5.074.52 5.98 6.145.705.35 7.236.48 7.07

NL:

5.37E5

TIC M S

TTX10ppm1

TTX10ppm1 #918 RT: 7.84 AV: 1 NL: 5.34E5

T: + c ESI SRM ms2 320.000 [161.500-162.500]

161.5 161.6 161.7 161.8 161.9 162.0 162.1 162.2 162.3 162.4 162.5

m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Abu

ndan

ce

162.24

Hnh 3.3. Sc k vi iu kin sc k B

Peak trn sc k thu c c thi gian lu 7,87 pht; tuy nhin, peak rt dong =>

khng ph hp

c. iu kin sc k (C)




- Tc dng: 500L/pht

32

- Th tch tim: 10L

- iu kin khi ph

+ Ngun: ESI+

+ Kh: SG: 20, AG: 10

+ Th ion ha: 3200V

+ Nhit ha hi: 200oC




RT: 0.00 - 8.76

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Abu

ndan

ce

6.30

8.620.40 0.80 1.321.01 1.95 2.09 2.761.58 2.99 5.872.51 3.75 4.673.35 4.794.09 4.50 5.24 5.46

NL:

6.76E4

TIC M S

TTX10ppm3

TTX10ppm3 #739 RT: 6.31 AV: 1 NL: 6.76E4

T: + c ESI SRM ms2 320.000 [161.500-162.500]

161.5 161.6 161.7 161.8 161.9 162.0 162.1 162.2 162.3 162.4 162.5

m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Abu

ndan

ce

162.21

Hnh 3.4. Sc k vi iu kin sc k C

Peak trn sc k thu c c thi gian lu 6,30 pht; tuy nhin, peak vn rt dong

=> khng ph hp

d. iu kin sc k (D)



33


- Tc dng: 500L/pht

- Th tch tim: 10L

- iu kin khi ph

+ Ngun: ESI+

+ Kh: SG: 25, AG: 10

+ Th ion ha: 3200V

+ Nhit ha hi: 200oC




RT: 0.00 - 9.99

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Abu

ndan

ce

6.10

7.87 8.03 8.44 8.80 9.311.00 4.590.54 3.04 4.780.18 5.595.152.702.44 4.323.54 4.131.53 2.081.77

NL:

4.59E4

TIC M S

TTX10ppm4

TTX10ppm4 #711 RT: 6.08 AV: 1 NL: 4.55E4

T: + c ESI SRM ms2 320.000 [161.500-162.500]

161.5 161.6 161.7 161.8 161.9 162.0 162.1 162.2 162.3 162.4 162.5

m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Abu

ndan

ce

162.19

Hnh 3.5. Sc k vi iu kin sc k D

Peak trn sc k thu c c thi gian lu 6,10 pht; tuy nhin, peak b t u v

ko ui => khng ph hp

e. iu kin sc k (E)

34

- Ct: Alltech Apollo C8; 5m; 250 x 4,6mm



- Tc dng: 500L/pht

- Th tch tim: 10L

- iu kin khi ph

+ Ngun: ESI+

+ Kh: SG: 20, AG: 10

+ Th ion ha: 3200V

+ Nhit ha hi: 200oC




RT: 0.00 - 14.99

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Abu

ndan

ce

RT: 6.78 NL:

1.08E5

TIC M S

Genesis

TTX10ppm7

TTX10ppm7 #794 RT: 6.78 AV: 1 NL: 1.08E5

T: + c ESI SRM ms2 320.000 [161.500-162.500]

161.5 161.6 161.7 161.8 161.9 162.0 162.1 162.2 162.3 162.4 162.5

m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Abu

ndan

ce

162.16

Hnh 3.6. Sc k vi iu kin sc k E

35

Peak trn sc k thu c c thi gian lu 6,80 pht; peak cn i, khng b ko

ui, thi gian phn tch ph hp => la chn iu kin sc k (E)

3.4. nh gi s ph hp ca h thng LC-MS

a, Kho st thch hp ca h thng:

Tim 6 ln dung dch chun Tetrodotoxin c nng 10ppm trong dung mi

pha mu, ghi din tch peak v thi gian lu. Kt qu c ghi trong bng 3.1

Bng 3.1. Kt qua khao st tinh thich hp ca h thng

TT Din tch peak Thi gian lu

Ln 1 2346060 6,80

Ln 2 2298966 6,77

Ln 3 2356975 6,61

Ln 4 2375268 6,88

Ln 5 2305867 6,92

Ln 6 2335692 6,78

Trung bnh 2336471 6.78

RSD (%) 1,26 1.67

=> H thng hot ng n nh, p ng yu cu v thch hp.

b, Xc nh tuyn tnh ca h thng:

kho st tuyn tnh ca h thng, chng ti tin hnh nh sau:

Pha cc dung dch chun 20,00ppm; 10,00ppm; 5,00ppm; 1,00ppm; 0,50ppm; 0,25ppm,

tim vo h thng sc k v ghi li din tch. Kt qu c ghi trong bng 3.2

Bng 3.2. Kt qua khao st tuyn tinh ca phng php

Nng (ppm) 0,25 0,50 1,00 5,00 10,00 20,00

Din tch peak 56469 91168 161603 1107050 2336471 5000125

Phng trnh hi quy Y = 250689x 76658

H s tng quan r = 0,999

36

Hnh 3.7. th biu din mi tng quan nng TTX va din tich peak

=> kt qu kho st cho thy c s lin quan cht ch gia nng v din tch peak,

h s tng quan r = 0,999 . tuyn tnh tt trong khong nng kh rng t 0,25-

20ppm

c. chn lc-c hiu ca phng php

S dng dung dch chun 10ppm, dung dch th chun b trn, vi iu kin sc

k (E) c kho st

Tin hnh tim ln lt dung dch trng, chun, th, ghi li sc k , din tch peak,

thi gian lu ca dung dch chun, th

Kt qua: Mu trng khng xut hin hnh nh peak v mnh m/z ca TTX, mu th v

mu chun c xut hin hnh nh peak v mnh m/z ca TTX. Kt qu cho sc k

bn di (hnh 3.8; 3.9; 3.10). Nh vy phng php p ng yu cu v chn lc-

c hiu. Ph hp nh tnh v nh lng TTX.

37

Hnh 3.8. Sc k dung dch mu trng

Hnh 3.9. Sc k dung dch chun TTX nng 10g/mL

38

Hnh 3.10. Sc k dung dch th

d. lp li ca phng php

kho st lp li ca phng php chng ti tin hnh trn 6 mu th c lp, x

l mu nh quy trnh nu trn, tin hnh chy sc k vi cc iu kin la chn,

ghi li din tch peak, p dng cng thc v din tch peak chun tnh c lng TTX

c trong mu th.

Cng thc:

Lng TTX c tnh theo cng thc

Xg = tCc

t VCS

S

Trong :

Sc, St: din tch peak trong sc k cho bi dung dch chun, th

Cc: Nng dung dch chun (10g/mL)

Vt: th tch pha mu th (mL)

Kt qu kho st lp li ca phng php c ghi bng 3.3

Din tch peak dung dch chun: Sc = 2336471 (bng 3.1)

39

Bng 3.3. Kt qua khao st lp li ca phng php

TT mt(g) St mt(g)

Ln 1 10,1 2303249 197.2

Ln 2 10,1 2301875 197.0

Ln 3 10,15 2310914 197.8

Ln 4 10,17 2317748 198.4

Ln 5 10,18 2317798 198.4

Ln 6 10,19 2341007 200.4

Trung bnh 198,2

RSD (%) 0,62

Trong 10g mu ni tng c Nc, ta xc nh c lng TTX l 198,2 g. Nh vy,

hm lng TTX trung bnh trong mu ni tng c Nc kh cao: 19,8g/g

Kt qu kho st lp li cho thy: lch chun tng i TTX trong sn

phm l 0,62 %;. Kt qu ny cho php ta p dng chng trnh nh lng TTX.

e. ng ca phng php

ng ca phng php c xc nh bng phng php thm chun. Thm

mt lng chnh xc khong 20 % cht chun TTX so vi lng TTX c trong mu

th. Sau tin hnh nh mc 3.1, tin hnh sc k nh iu kin chn la, lp li

thc nghim 6 ln. Da vo hm lng cc hot cht bit trong mu th, hm lng

ca cc cht chun v cn c vo din tch peak trn sc k tnh c hm lng

cc cht thu hi li, kt qu thu c ghi bng 3.4

40

Bng 3.4. Kt qua khao st ng ca phng php

mt(g) mTTX(g) mc thm

vo(g) St Sc

kt qu

(g)

mc tm

thy(g)

% thu

hi

10,2 202,164 3,93 2407480 2336471 206,08 3,914 99,6

10,5 208,110 4,10 2479853 2336471 212,27 4,163 101,5

10,9 216,038 4,10 2571171 2336471 220,09 4,052 98,8

11,6 229,912 4,20 2735937 2336471 234,19 4,282 101,9

11,8 233,876 4,40 2782935 2336471 238,22 4,341 98,7

11,9 235,858 4,60 2809567 2336471 240,50 4,639 100,8

Trung bnh 100,2

SD 1,41

RSD(%) 1,40

Kt qu kho st cho thy kh nng tm li c kh tt i vi hot cht nghin cu

(98,7% -101,9%) => Phng php p ng yu cu v ng.

f. Xc nh gii hn pht hin (LOD) v gii hn nh lng (LOQ)

Tin hnh tim cc dung dch long dn nng , n khi peak thu c c t s nhiu

ng nn S/N 3. Ti mc nng ny chnh l gii hn pht hin ca phng php.

Vi gii hn nh lng, mc nng cho t s nhiu ng nn S/N 10.

Bng 3.5. Kt qua khao st LOD va LOQ ca phng php

Nng (ng/mL) 250 100 50 25 10

T s S/N 185 70 30 12 4

=> nh vy, phng php c gii hn pht hin (LOD) l 10ng/mL; gii hn nh

lng (LOQ) l 25ng/mL

41

3.5 p dng phng php nh lng mt s mu c Nc bng sc k lng

khi ph LC-MS

Kt qu phn tch trn mt s mu ph tng c Nc c trnh by trong bng 3.6

Bng 3.6. Kt qua nh lng mt s mu c Nc

Mu Mu 1 Mu 2 Mu 3 Mu 4 Mu 5 Mu 6

Kt qua 19,72 g/g 19,70 g/g 19,78 g/g 19,84 g/g 19,84 g/g 20,04 g/g

3.6. Bn lun

3.6.1. V quy trnh chit tch v x l mu

Quy trnh chit tch, x l mu m chng ti s dng l phng php chit pha

rn (SPE) gip tch c TTX ra khi mu ph tng c Nc, gip loi b tp v cc

dn cht khc c trong mu, to thun li cho qu trnh nh lng bng phng php

LC/MS. Phng php chit pha rn (SPE) c s dng trong kha lun mc d kh

tn km v phc tp khi thc hin trong iu kin phng th nghim nhng t l chit

mu cao, chit kit c hot cht, ph hp nh lng TTX , trnh hin tng mt

mu nh cc phng php khc [11], [16].

3.6.2. V phng php phn tch LC/MS

LC/MS l phng php mi hin nay c s dng nh tnh v nh lng

cc cht mt cch chnh xc. Phng php ny c th s dng xc nh chnh xc

cu trc ca hp cht bng vic dng nng lng b gy phn t to ra cc mnh

ion con v vic phn tch khi ph c ghi li khi b gy chng. Do kt qu ca

phng php ny l hon ton tin cy. ng thi phng php ny cng c nhy rt

cao, pht hin tuyt i t n 10-10 - 10-14 g. Phng php ELISA cng c

nhy kh cao, c hiu tng i tt, thi gian phn tch nhanh (khong 30 pht)

42

nhng gi ca cc khng th n dng rt t, nn chi ph phn tch cao, kh nng ng

dng s b hn ch. Phng php Mouse Bioassay (phng php sinh ha trn chut)

c nhc im l khng c hiu, bi c t Tetrodotoxin c nhiu dn cht cng

mang c tnh, hn na nhy li khng cao. [21]

Bn thn TTX ch tan c trong cc acid hu c yu, nn khng th phn tch

trc tip bng phng php sc k kh khi ph (GC/MS) [20]. TTX phi c to dn

xut vi 2-amino-6-hydroxymethyl-8-hydroxyquinazoline [24].

Phng php sc k lng hiu nng cao vi c trng s phn tch gia pha

ng v pha tnh, c th ng dng phn tch TTX. Tuy nhin, vi cc detector khc

nhau th phng php chun b, phn tch khc nhau, qua nhy cng khc nhau,

gii hn pht hin, gii hn nh lng cng khc nhau [21]

Vi Detector UV (DAD): iu kin pha ng thng phc tp, chn lc thng

khng cao bi trong c Nc c rt nhiu dn xut ca TTX

Vi Detector hunh quang (FLD): phng php ny c Yasumoto v Mitishita ng

dng phn tch TTX. Phng php ny s dng NaOH c, nng to dn xut

sau ct vi 2-amino-6-hydroxymethyl-8-hydroxyquinazoline pht hunh quang.

iu ny hn ch s phc hi TTX v gii hn nh lng ca phng php km [20].

Vi Detector khi ph (MS) m chng ti p dng, y l phng php hin i, c

kh nng ng dng cao v ang dn tr nn ph bin phn tch TTX. Trn th gii

c rt nhiu nghin cu s dng phng php ny phn tch TTX, tuy nhin,

chng ta vn phi nghin cu tm ra phng php ph hp vi iu kin mu, iu

kin c s vt cht Vit Nam [21], [20].

Phng php sc k lng khi ph (LC/MS) m chng ti xy dng c c

n nh, lp li cao, tuyn tnh tt, gii hn pht hin cng nh gii hn nh

lng nh (LOD = 10ng/mL; LOQ = 25ng/mL) p ng tt kh nng nh lng hm

lng nh TTX trong mu c Nc.

43

3.6.3. V kt qu nh lng mt s mu c Nc

V hn ch v thi gian, v kh khn trong vic thu thp, bo qun mu ( hin

nay c nhiu quy nh cm khai thc, bun bn c Nc di mi hnh thc) nn

chng ti ch nh lng c TTX trn mt s mu nht nh. Do vy cn phi c

nhiu thi gian v tin hnh trn s lng mu ln hn c c kt lun chnh xc

hn.

Hnh 3.11. Hai loai Lagocephalus lunaris va Arothran hispidus

44

KT LUN V KIN NGH

KT LUN

Sau mt thi gian thc hin kha lun chng ti thc hin c mc tiu ra

vi kt qu c th nh sau:

1. xy dng v thm nh c quy trnh nh lng Tetrodotoxin trong mt s

mu ph tng c Nc.

Quy trnh x li mu: Chit pha rn (SPE) nh nu trn

iu kin sc k nh sau:

- Ct: Alltech Apollo C8; 5m; 250 x 4,6mm


- (m acetat: dung dch amoni acetat 15mM, acid acetic 15mM)

- Tc dng: 500L/pht

- Th tch tim: 10L

- iu kin khi ph

+ Ngun: ESI+

+ Kh: SG: 20, AG: 10

+ Th ion ha: 3200V

+ Nhit ha hi: 200oC



Nh vy chng ti xy dng c quy trnh nh lng Tetrodotoxin bng sc k

lng khi ph (LC/MS) mt cch tng i ton din:

- Quy trnh x l mu c tnh kh thi cao

- Dung mi, ha cht, thit b ph bin cc trung tm kim nghim ln

45

- nh gi c phng php nh lng TTX vi cc ch tiu: tnh chn

lc, khong nng tuyn tnh, tnh thch hp, n nh, ng,

chnh xc, gii hn pht hin, gii hn nh lng,

2. p dng phng php xy dng c vo nh lng mu ph tng c Nc.

Nhng kt qu ny l ph hp v p ng c mc tiu t ra ca kha lun.

KIN NGH

ng dng phng php LC-MS nghin cu, kim nghim thm nhiu mu

dc phm, m phm, dc liu v hp cht t nhin .

p dng thm nhiu phng php nghin cu, phn tch c t c Nc c th

ng dng trong y hc v cuc sng.

TI LIU THAM KHO

TI LIU TING VIT

1. B mn ha phn tch (2006), Ha phn tich tp 2, Trng i hc Dc

H Ni, H Ni

2. B mn ha phn tch (2009), Kim nghim thuc, Trng i hc Dc H

Ni, H Ni.

3. B Thy Sn (1996), Ngun li thy san Vit Nam, NXB Nng nghip, H

Ni, 453-454.

4. Nguyn Duy Ch (2006), ng dng phng php sc k phn tich mt s

thuc tn dc nhm chng vim phi steroid c th trn ln trong thuc

ng dc , Lun vn thc s dc hc.

5. Nguyn Th D (2004), Ng c c Nc, Tp chi y hoc qun s, (4), 57-60

6. Nguyn Minh c (2006), Sc k long hiu nng cao va mt s ng dng

vao nghin cu, kim nghim dc phm, dc liu va hp cht t nhin,

Nh xut bn y hc chi nhnh Thnh ph H Ch Minh.

7. Nguyn Vn L (2005), Nghin cu c tnh c Nc v cc gii php x l

ch bin, qun l t khu khai thc n khu tiu th c Nc m bo an

ton v sinh thc phm, K yu hi thao toan quc v khai thc ch, ch

bin va dch v hu cn ngh c, 385-402.

8. Nguyn Vn L, Nguyn Hu Hong, Bi Th Thu Hin, v ctv (2006),

Kt qu phn tch c t c Nc bin Vit Nam, Tuyn tp Nghin cu

ngh c bin, (4), 256-264

9. Nguyn Hoi Nam, ng Vn Thi (2007), Mt s thng tin v c Nc bin

Vit Nam, Tp chi thy san,(1), 25-28.

10. Phng Thanh (2010), Cn cnh gic vi c Nc, Thng tin khoa hoc va

cng ngh Sc Trng, (1), 23-24.

11. Nguyn Thanh T (2012), Tch, lam giau, xc nh lng vt Cr(III) va

Cr(VI) trong nc bng ky thut chit pha rn va phng php quang ph,

Lun vn thc s, 19-24.

TI LIU TING ANH

12. Brian M. Tissue (2013), Basics of Analytical Chemistry and Chemical

Equilibria, Wiley, NewYork.

13. Chung-him Yu (2008), Detection and Biosynthesis of Puffer Fish Toxin from

Bacterial Culture for Novel Medical Application, Hong Kong Polytechnic

University, Department of Applied Biology and Chemical Technology,

Hong Kong.

14. David R. Lide (2005-2006), CRC Handbook of Chemistry and Physics 86TH

Edition, CRC Press,Taylor & Francis, Boca Raton, FL 2005, p. 3-482.

15. Donato P, Cacciola F, Tranchida PQ, Dugo P, Mondello L (2012), Mass

spectrometry detection in comprehensive liquid chromatography: basic

concepts, instrumental aspects, applications and trends, Mass spectrometry

reviews, 31(5), 523-559.

16. E.M. Thurman (1998), Solid-Phase Extraction: Principles and practice,

Wiley-interscience, NewYork.

17. Francesco Santini, James C. Tyler (2003), A phylogeny of the families of

fossil and extant tetraodontiform fishes (Acanthomorpha,

Tetraodontiformes) Upper Cretaceous to Recent, Zoological Journal of the

Linnean Society, 139(4), 565617.

18. Hofstadler SA, Sannes-Lowery KA (2006), Applications of ESI-MS in

drug discovery: interrogation of noncovalent complexes, Nature reviews

drug discovery, 5(7), 585-595.

19. Juan Zhang, Gregor McCombie, Christian Guenat; Richard Knochenmuss

(2005), FT-ICR mass spectrometry in the drug discovery process, Drug

discovery today, 10(9), 635-642.

20. Kelvin Sze-Yin Leung, Bonnie Mei-Wah Fong, Yeuk-Ki-Tsoi (2011)

Analytical Challenges: Determination of Tetrodotoxin in Human Urine and

Plasma by LC-MS/MS, Marin Drugs, 9(11), 2291-2303.

21. Manabu Asakawa, Yasuo Shida, Keisuke Miyazawa, Tamao Noguchi (2012),

Instrumental Analysis of Tetrodotoxin, Chromatography-The Most

Versatile Method of Chemical Analysis, 245-270.

22. Maoqing Zhou, Naning (CN), Frank Hay Kong Shum, North Point (2003),

Method of extracting tetrodotoxin, United States Paten, US.

23. Mike S. Lee (2005), Integrated strategies for drug discovery using mass

spectrometry, Wiley, NewYork.

24. Munetomo Nakamura, Takesi Yasumoto (1985), Tetrodotoxin derivatives

in puffer fish, Toxicon, 23(2), 271-276.

25. Robert E. Ardrey (2003), Liquid Chromatography - Mass Spectrometry: An

Introduction, Wiley, NewYork.

26. Scott E.Van Bramer (1998), An Introduction to Mass Spectrometry, Widener

University, US.

27. T. Yasumoto, T. Michishita (1985), Fluorometric determination of

tetrodotoxin by high perfomance liquid chromatography, Agric.Biol.Chem,

49, 3077-3080.

TI LIU THAM KHO NGOI

28. http://en.wikipedia.org/wiki/B_C_Nc

29. http://en.wikipedia.org/wiki/Tetradotoxin

30. http://en.wikipedia.org/wiki/Tetraodontida

PHU LUC

* Mt s hnh nh chy NMR xc nh cu trc TTX:

Thit b cng hng t ht nhn Bruker 500 Ultrashield

Vin Ha hoc, Vin Khoa Hoc Cng Ngh Vit Nam

Hnh anh chy NMR xc nh cu trc TTX

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