Xác định Ochratoxin trong thực phẩm bằng phương pháp sắc ký lỏng khối phổ (LC-MS/MS)

I HC QUC GIA H NI

TRNG I HC KHOA HC T NHIN

---------------------

Nguyn Th H Bnh

XC NH OCHRATOXIN

TRONG THC PHM BNG PHNG PHP

SC K LNG KHI PH (LC-MS/MS)

LUN VN THC S KHOA HC

H Ni - 2013

I HC QUC GIA H NI

TRNG I HC KHOA HC T NHIN

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Nguyn Th H Bnh

XC NH OCHRATOXIN

TRONG THC PHM BNG PHNG PHP

SC K LNG KHI PH (LC-MS/MS)

Chuyn ngnh: Ha phn tch

M s: 60440118

LUN VN THC S KHOA HC

NGI HNG DN KHOA HC:

HDC: TS. L TH HNG HO

HDP: PGS.TS NGUYN XUN TRUNG

H Ni - 2013

Lun vn thc s Nguyn Th H Bnh

1

LI CM N

Li u tin cho em gi li cm n su sc ti thy PGS.TS. Nguyn Xun

Trung, Trng i hc Khoa hc t nhin HQGHN v c L Th Hng Ho,

Vin Kim nghim an ton v sinh thc phm Quc gia B Y t tn tnh hng

dn, ng gp nhng kin qu bu, to mi iu kin gip em trong sut qu

trnh thc hin ti v vit lun vn.

Em xin by t lng bit n ti cc thy c gio ging dy ti khoa Ho hc,

c bit l cc thy c trong b mn Ho Phn Tch, cho em nhng kin thc

qu gi, to iu kin cho em c hc tp v nghin cu trong mi trng hin

i.

Ti xin c gi li cm n chn thnh ti ban lnh o Vin kim nghim

An ton v sinh thc phm Quc gia to mi iu kin thun li ti c hc

tp v hon thnh ti ny.

Ti cng xin gi li cm n ti cc ng nghip ti labo Ha Vin kim

nghim An ton v sinh thc phm Quc gia gip ti rt nhiu trong qu trnh

lm thc nghim.

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

chia s mi kh khn cng ti.

H Ni, nm 2013

Hc vin

Nguyn Th H Bnh


2

MC LC

M U1

Chng 1: TNG QUAN....4

1.1 Gii thiu chung [14-15].4

1.1.1 nh ngha..4

1.1.2 Tnh cht ha l ca Ochratoxins..5

1.1.3 Gii hn tn d ti a cho php (MRL)7

1.2 Cc phng php xc nh..8

1.2.1 Phng php ELISA (enzyme-linked immunosorbent assay).8

1.2.2 Phng php sc k kh (Gas chromatography-GC) 9

1.2.3 Phng php sc k lng hiu nng cao (HPLC) v ct i lc min

dch (IAC).9

1.2.4 Phng php sc k lng khi ph10

Chng 2: I TNG V PHNG PHP NGHIN CU. 12

2.1 i tng v ni dung nghin cu.12

2.1.1 i tng nghin cu12

2.1.2 Ni dung nghin cu12

2.2 Phng php nghin cu..12

2.2.1 Phng php tch chit mu.12

2.2.2 Phng php sc k lng khi ph.13

2.2.2.1 H thng sc k lng hiu nng cao..13

2.2.2.2 Khi ph (Mass Spectrometry) .15

2.3 Phng tin nghin cu..20

2.3.1 Thit b, dng c.20

2.3.1.1 Thit b20

2.3.1.2 Dng c..21

2.3.2 Dung mi, ha cht.21

2.3.3 Ly mu.22

Chng 3: KT QU V THO LUN.23


3

3.1 Ti u cc iu kin chy my LC-MS/MS..23

3.1.1 Ti u cc iu kin chy ca my khi ph MS/MS..23

3.1.1.1 Kho st ion m v ion con..23

3.1.1.2 Ti u cc iu kin MS..24

3.1.2 Ti u cc iu kin chy sc k lng hiu nng cao (HPLC).26

3.1.2.1 Pha tnh26

3.1.2.2 Kho st thnh phn pha ng..27

3.1.2.3 Kho st tc pha ng...............................................................29

3.1.2.4 Chn chng trnh chy gradient.30

3.2 Ti u quy trnh x l mu33

3.2.1 Chn quy trnh x l mu33

3.2.2 Kho st th tch (s ln chit) dung mi chit.. 36

3.2.3 Chn ct chit pha rn.36

3.2.4 Kho st tc qua ct chit pha rn.......................................................37

3.2.5 Kho st th tch dung mi ra gii.38

3.2.6 Kho st nh hng pH trong qu trnh chit mu..................................38

3.3 Thm nh phng php phn tch.42

3.3.1 Tnh c hiu / chn lc42

3.3.2 Gii hn pht hin (LOD), gii hn nh lng (LOQ)44

3.3.3 Xc nh khong tuyn tnh45

3.3.4 Xy dng ng chun47

3.3.5 chnh xc (accuracy) ca phng php phn tch ( ng v

chm) [9]48

3.4 Phn tch mu thc t.52

KT LUN V KIN NGH...57

TI LIU THAM KHO......................................................................................59

PH LC.........63


4


5

DANH MC T VIT TT K hiu Ting Anh Ting Vit

CE Collision energy Nng lng va chm

EI Electron Ionization Ion ha bng dng electron

ESI Eelectrospray ionization Ch ion ha phun in t

EU European Union Chu u

HPLC High performance liquid

chromatography

Sc k lng hiu nng cao

IUPAC International Union of Pure and

Applied Chemistry

Lin minh quc t v ha hc

c bn v ng dng

LOD Limit of detection Gii hn pht hin

LOQ Limit of quantity Gii hn nh lng

MeOH Methanol metanol

MRL Maximum Residue Limit Gii hn d lng ti a

PSA Primary and secondary amine Cc amin bc 1, bc 2

RSD Relative standard deviation lch chun tng i

SPE solid phase extraction Chit pha rn

IARC International Agency for Research on

cancer

T chc nghin cu ung th

quc t

UPLC-

MS/MS

Ultral performance liquid

chromatography tandem mass

spectrometry

Sc k lng siu hiu nng kt

ni khi ph

RF Fluorescence detector Hunh quang


6

DANH MC CC BNG

Trang

Bng 1.1: Mc d lng ti a cho php ca ochratoxin A Vit Nam 7

Bng 2.1: Bng pha dung dich chun chy my 22

Bng 3.1: Ion m ca tng cht 23

Bng 3.2: Cc thng s ti u cho ESI-MS/MS 23

Bng 3.3: Cc thng s ti u MS/MS 26

Bng 3.4: Chng trnh chy gradient 28

Bng 3.5: Kt qu kho st thnh phn pha ng 28

Bng 3.6: Kho st cc chng trnh gradient 31

Bng 3.7: Chng trnh gradient ti u 32

Bng 3.8: Kho st quy trnh x l mu 34

Bng 3.9 : Kho st th tch dung mi chit 36

Bng 3.10: Kho st ct chit pha rn 37

Bng 3.11: nh hng ca tc ra gii ti hiu sut ca qu trnh chit

mu 37

Bng 3.12: Kt qu kho st th tch dung dch ra gii 38

Bng 3.13: Ion m v hai ion con ca cc cht trong nhm ochratoxins 42

Bng 3.14: Kt qu xc nh LOD, LOQ ca ochratoxin A, B 45

Bng 3.15: S ph thuc ca din tch pc vo nng 46

Bng 3.16: Kt qu nh gi chnh xc ca phng php 50


7

DANH MC HNH V

Trang

Hnh 1.1: Cu trc cc ochratoxins 4

Hnh 2.1: M hnh h thng LC-MS 11

Hnh 2.2: S n gin ca h thng sc k lng 11

Hnh 2.3: S cu trc ca my khi ph MS 13

Hnh 2.4: B ngun ion ho 14

Hnh 2.5: B phn tch t cc 16

Hnh 2.6: M hnh b phn phn tch r cc chp ba 17

Hnh 3.1: Sc t l cc ion ca cc ochratoxins Hnh 3.2: Sc ca ochratoxin A, B nng 10 ppb khi dung dung mi pha ng l CH3COONH4 10mM

24

29

Hnh 3.3: Sc ra gii cc cht ti tc dng 0,8 ml/pht 29

Hnh 3.4: Sc ra gii cc cht ti tc dng 1,0 ml/pht 30

Hnh 3.5: Sc ra gii cc cht ti tc dng 1.2 ml/pht 30

Hnh 3.6: Sc khi chy sc k bng chng trnh gradient 1 32

Hnh 3.7: Quy trnh x l mu d kin 35

Hnh 3.8: th s ph thuc ca din tch pc vo gi tr pH chit mu 40

Hnh 3.9: Quy trnh x l mu ti u 41

Hnh 3.10: Sc mu trng 43

Hnh 3.11: Sc mu trng thm chun 2.5ppb 43

Hnh 3.12: Sc ochratoxins ti LOD 44

Hnh 3.13: th biu din s ph thuc ca din tch pc vo nng OTA 46

Hnh 3.14: th s ph thuc ca din tch pc vo nng OTB 47

Hnh 3.15: ng chun ca OTA 47

Hnh 3.16: ng chun ca OTB 48

Hnh 3.17: Sc OTs ti mc thm chun 2,5 ppb 52

Hnh 3.18: Sc mu ru khng c ochratoxin A,B 54

Hnh 3.19: Sc mu ng khng c ochratoxin A,B 54

Hnh 3.20: Sc mu lc khng c ochratoxin A,B 54

Hnh 3.21: Sc mu lc c ochratoxin A 55

Hnh 3.22: Sc mu ng c ochratoxin A,B 55


8

M U

Vn an ton v sinh thc phm ngy cng c quan tm bi n lin quan

trc tip ti sc khe ca tt c ngi tiu dng. Thi gian va qua trong nc lin

tip pht hin cc v ng c thc phm trong c cc v ng c mycotoxin c

bit nghim trng gy ra tm l lo ngi cho ngi tiu dng khi la chn thc

phm cng nh s dng thc phm th no l ng cch. Theo thng k ca Cc

An ton thc phm v cc v ng c thc phm trong nhng nm gn y trn c

nc:

- Nm 2009: Ton quc xy ra 147 v ng c thc phm, c 5026 ngi mc;

3958 ngi nhp vin; 33 ngi t vong.

- Nm 2010: Tnh hnh NTP trong nm 2010 phc tp. Ton quc xy ra

175 v ng c trong c 34 v ng c trn 30 ngi lm 5664 ngi mc 42

trng hp t vong, so snh vi s liu trung bnh trn nm ca giai on 2006-

2009, s v ng c gim 9,1%; s mc gim 17,6% v s t vong gim 19,2%.

- Nm 2011: 148 v, 4700 ngi mc, 27 ngi t vong.

- Nm 2012: 168 v, 5541 ngi mc, 34 ngi t vong.

Theo TS Trn Quang Trung, Cc trng Cc An ton thc phm - B Y t,

trong 9 thng ca nm 2013, c nc c 108 v ng c thc phm lm hn

2.800 ngi mc, trong c 18 ca t vong. Trong 40 v ng c thc phm c

thng k trong qu III ny th nguyn nhn do vi sinh vt l 23 v, do c t t

nhin 4 v, do ha cht 2 v v 11 v cha xc nh c nguyn nhn. Cc v ng

c xy ra khp ni, t gia nh ring n tp th.

Ochratoxin l mt loi c t c sinh ra bi cc chng nm mc thuc cc

ging Aspergilus ochraceus v Penicillium verrucusum v l loi c t c tim

nng gy ung th v vim thn ngi v ng vt. c t ny c pht hin

trn nhiu nng sn khc nhau bao gm ng cc v cc sn phm ca chng. iu

ng lo ngi l khi chng ta n phi thc n b nhim ochratoxin n khng gy ng


9

c cp tnh m tch ly dn trong c th - l nguy c tim n e da sc khe cho

con ngi.

Trong s 10.000 loi nm mc khc nhau c bit n th c khong 50 loi

l c hi i vi gia sc gia cm v con ngi. Cc loi nm ny sn sinh ra cc c

t c gi chung l Mycotoxin... Mycotoxin la cht c sinh ra t nm mc , c

hnh thnh khi nm chuyn ha cc cht dinh dng c trong thc n v nguyn

liu. Theo t chc lng thc v nng nghip Lin Hip Quc (FAO), khong 25%

s ng cc th gii c cha mt hm lng mycotoxin mt mc no . Ty

vo a l, kh nng nhim mycotoxin li khc nhau . iu kin nhit i v cn

nhit i , nguy c nhim mycotoxin cng cao. c th kh hu v nn sn xut

nng nghip Vit Nam tinh trang nhim c t nm mc la kh ph bin. S hnh

thnh nm mc v c t ca chng c th bt u t khi cy cn trn ng, lc

thu hoch, trong khi bo qun hoc ngay c trong qu trnh ch bin thc n cho vt

nui. Nh vy, khng ni no trn th gii c th thot khi nm mc v c t t

chng, v tc hi ca chng l v cng to ln i vi nng sut vt nui v sc khe

con ngi.

kim sot mc nhim ochratoxin trong thc phm, Vit Nam cng

nh trn th gii c nhiu phng php phn tch c nghin cu v ng dng.

Nhng vi nhng c im u vit v chnh xc cao nn cc phng php phn

tch sc k lng khi ph c coi l phng php phn tch c gi tr php l

pht hin v nh lng nng ochratoxin lng vt hay siu vt.

Xut pht t tnh cp thit ca x hi v tnh u vit ca phng php phn

tch, chng ti xy dng phng php nghin cu:

"Xc nh hm lng Ochratoxin trong thc phm bng phng php sc k

lng hai ln khi ph (LC-MS/MS).

Mc tiu ca thc hin ti nghin cu l:

1. Xy dng phng php xc nh hm lng ca Ochratoxin trong thc

phm bao gm:

- Kho st cc iu kin trn my sc k lng hai ln khi ph


10

- Kho st cc iu kin x l mu.

- Thm nh phng php xy dng.

2. p dng phng php xc nh ochratoxin trong ng cc, sn phm t ng cc,

ru ln men trn th trng trong nc.


11

CHNG 1: TNG QUAN

1.1.Gii thiu chung [14-15]

1.1.1.nh ngha

c t ochratoxin, l mt sn phm chuyn ho th cp ca mt s loi nm

mc. Ochratoxin c mt trong khp cc loi nng sn thc phm: ng cc, tho

dc, bia, c ph... trong cc sn phm c ngun gc ng vt do b ly nhim

trc. Ochratoxins c bit n l sn phm ca cc loi nm Aspergillus v

Penicillium v thng c tm thy trong nhiu loi sn phm lng thc v thc

n chn nui.

Ochratoxin A ln u tin c tm thy nm mc A. ochraceus vng Nam

Phi bi Scott (1965) trn ht la b nhim A.ochraceus. c tm thy Ochratoxin

thng xuyn trong tht. Anh, chng c tm thy trong u nnh, bp bt, ca

cao. M.Nakajima nm 1997 ghi nhn t l chim 30% hm lng OTA t 0.1

17,4 g/kg 47 mu caf c nhp vo Nht Bn t cc nc Nam Phi , v mt

s nc ASIAN. Ti Vit Nam, nghin cu tin hnh trn 123 mu ng ca 2 x

Cn T v Lng Tm huyn Qun B tnh H Giang. Kt qu cho thy: trong 123

mu ng c phn tch c ti 50 mu (40,7 %) pht hin c ochratoxin A, trong s

c 2 mu (1,6 %) vt mc d lng theo quy nh ca B Y t.

Cho ti nay pht hin c 3 loi ochratoxin khc nhau. Trong khun kh

lun vn chng ti quan tm nghin cu n ochratoxin A v B

Ochratoxin A

Ochratoxin B

Hnh 1.1: Cu trc cc Ochratoxins


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1.1.2.Tnh cht ha l ca Ochratoxins

Ochratoxins l c t tinh th khng mu, bn vi nhit, tan trong dung mi

hu c phn cc nh chloroform, methanol, , t tan trong nc v tan trong m

carbonat long). Ochratoxins rt bn vng vi cc x l nhit v ha cht. c t

c sn sinh nhiu nht nhit t 20-250C. S sn sinh ochratoxins ph thuc

chng nm mc, hot tnh ca nc trong ht, c cht, nhit . Ochratoxins d b

phn hy bi nh sng, trong mi trng kim hoc cht ty ra.

Ochratoxin A pht hunh quang v hp th UV cc i ti 365nm. Ochratoxin

A c im nng chy 1690C. Ph hng ngoi trong cloroform cho cc pc c

di 3380, 1723,1678, 1655 cm-1 OTA c tnh axit yu pKa1 = 4,2-4,4 v pKa2 = 7,0-

7,3. Ochratoxin A pht hunh quang xanh khi dng thit b sc k lp mng (TLC)

chiu tia UV 366nm.

Ochratoxin B c trng lng phn t 369,37. Ochratoxin B c th pht hunh

quang mu xanh khi chiu tia UV bc sng 318 nm. Nhit nng chy khong

2210C.

C ch tc ng ca ochratoxins: ochratoxins gy c ch s vn chuyn ca

ribonucleic axit (tARN) v cc axitamin. Ochratoxins cn c ch vi khun, nm

men v phenylalanine - tARN gan. Tc ng lm c ch s tng hp protein trong

t bo v c th. S c ch min dch ca ochratoxin c biu hin lm gim thc

bo v c ch t bo lympho. c ch tng t nh trn cc amino axit synlaza

tARN tng ng ochratoxin A gy c ch hydroxylase phenylalanine, mt na

phenylalanine ca ochratoxin A l mt phn hydroxyl ha tyrosin gy bnh cc

t bo gan trong c th. Ochratoxin c ch s tng hp ARN lm nh hng n

cc protein trong vng tun hon. Tc ng n cc t bo mng ti th v gy ra cc

hiu ng khc nhau trn ti th. Kch thch s hnh thnh ADN trong thn, gan v l

lch. Cc ADN ny l cc si n b ph v.

Cc nguyn liu d nhim c t ny nh cm go, la m, bt m, bp, u

nnh, c ph. D lng ochratoxin cng c tm thy trong tht heo v tht gia


13

cm. c t ny gy hi n gan v thn ng vt. Vi nng ln hn 1 ppm c

th lm gim sn lng trng g , nng ln hn 5 ppm c th gy nn nhng

tn thng gan v rut. Tng t nh Aflatoxin, c t ny cng gy nn s gim

sc khng v l tc nhn gy ung th ngi.

- Gy tn thng t bo gan: tt c cc trng hp xc nh s ng c

Ochratoxin u c bnh tch ging nhau ch gan b h hi nng. Ty theo mc

nhim t hay nhiu, lu hay mau m tnh trng bnh trn gan khc nhau. Biu hin

chung l ban u gan ng vt (in hnh l g) bin thnh mu vng ti, mt sng

sau gan sng phng v bt u ni cc mn nh trn b mt lm cho n g gh

i khi c nhng nt hoi t mu trng, sau cng do nhim khun m gan tr nn

b v d v

- Thn sng to lm cho vic o thi cht c ra khi c th tr nn ht sc

kh khn, t lm cho triu chng ng c tr nn trm trng

- Lm gim kh nng khng ca ng vt, c ch h thng sinh khng th,

c th gy t vong cho ng vt. Khi nhim c ochratoxin c th rt mn cm vi

cc loi bnh thng thng

- Bo mn nim mc ca ng tiu ha do lp t bo nim mc b cht bong

ra v b kh li hnh thnh nn mt lp mng bc lm cn tr s chuyn thc n i

trong ng tiu ha

- Lm thay i hot ng sinh l bnh thng gy ri lon sinh sn. th

mang thai c th gy cht thai. i vi gia cm c th gy t l cht phi giai

on u rt cao, t l n thp

- Lm gim tnh ngon ming i vi thc n do s pht trin ca nm mc

lm mt mi thc n

- Lm h hi cc vitamin trong thc n do s ln men phn gii ca nm

mc

- Ngoi cc tc hi trn nm mc c trong thc n cn ln men phn gii cc

ngun dng cht (glucid, protein, acid amin, vitamin...) lm cho thc n b gim


14

gi tr nghim trng, lm mt mi t nhin, chuyn sang mi hi mc, vt nui

khng thch n.

Nh vy c th ni rng c t nm gy ra nhng tc hi rt ln v hu qu

v cng nghim trng cho c th ca ngi v ng vt.

Ty theo tng loi m c t nm mc c th gy nhi m c cp tnh v mn

tnh. c t nm mc t khi gy ra ng c cp tnh, n gy hi c th t t do

lm cho chng ta khng h hay bit. Nhng khi pht sinh triu chng th nhng c

quan b phn chng tn cng h hi nghim trng kh cha tr. Tuy nhin, cc

c t nm mc trong thc n s gy nn nhng hu hoi thm lng i vi h

thng min dch ca gia sc, lm cho chng mn cm hn i vi bnh.

Khc vi bnh nhim trng l khng sinh khng iu tr c nhim c t

nm mc. Cch tt nht l ngn nga khng cho c t nm mc nhim vo trong

thc n.

Trn heo: Liu gy cht : LD50 1-6 mg/kg, g: LD50 3,6 mg/kg i vi g

con 10 ngy tui

1.1.3. Gii hn tn d ti a cho php (MRL)

- Mc d lng ti a cho php l gii hn d lng ca mt loi thuc,

c php tn ti v mt php l hoc xem nh c th chp nhn c trong nng

sn, thc n m khng gy hi cho ngi s dng v vt nui khi dng.

Bng 1.1: Mc d lng ti a cho php ca Ochratoxin A Vit Nam

Loi g/g

Ng cc cha qua ch bin 5

Ht c ph rang, c ph bt 5

C ph ung lin 10

Ru vang, nc nho p 2

Thc phm dnh cho tr di 36 thng tui 0,5

Gia v 30

Sn phm chit xut t cam tho 80

Ng cc v bt ng cc 3


15

Hin cha c quy nh c th no v mc MRL cho ochratoxin B k c trong

nc v tiu chun Chu u (EN).

1.2 Cc phng php xc nh

1.2.1 Phng php ELISA (enzyme-linked immunosorbent assay).

Nguyn tc da trn s kt hp c hiu gia khng nguyn v khng th.

Ochratoxin chun c cng hp vi enzyme, to thnh phc hp Ochratoxin-

enzyme v c s dng nh mt khng nguyn nng nht nh, cnh tranh

vi ochratoxin c trong mu. Khng th c hiu vi ochratoxin c gn ln b

mt ging nha polystryren. Dch chit mu trong methanol 70% c trn cng

hp vi ochratoxin enzyme v c cho vo ging c ph khng th, cc

ochratoxin c trong mu (nu c) s cnh tranh vi phc hp ochratoxin- enzyme

gn vo khng th c nh trn polystryren. Sau ra sch cc phc hp tha,

c cht phn ng vi enzyme c a vo to mu. mt thi gian sau thm

vo dung dch ngng pht mu to iu kin ng u v thi gian cho mi

ging. Tin hnh o hp th ca mu v dy chun bng my o mu, t tnh

c nng ca ochratoxin c trong mu. Mu cng m chng t cng hp

ochratoxin enzyme c gi trong ging cng nhiu, ng ngha vi nng

ochratoxin trong mu cng thp. Ngc li, mu cng nht th nng Ochratoxin

cng cao.

S dng phng php ELISA xc nh ochratoxin c th k n mt s

nghin cu sau:

Nhm tc gi Aihua Zhang, Yanna Ma, Lulu Feng, Ying Wang, Chenghua

He, Xichun Wang, Haibin Zhang [14] xc nh Ochratoxin trn i tng mu ng

cc nguyn liu ti Nanjing, Trung Quc. Phng php phn tch c gii hn pht

hin 0,15 ng/ml. thu hi ca phng php t t 87-101% ti mc thm chun

2,5-10 ppb. Nhm tin hnh phn tch trn 65 mu ng, go, la m. Kt qu cho

thy 8 trong s 22 mu la m cha Ochratoxin A trong khong 2- 9 ppb (chim

36% tng s mu), 6 trong s 23 mu ng (chim khong 26% tng s mu) dng


16

tnh vi Ochratoxin A trong khong 3-23 ppb, 3 trong s 20 mu go (chim

khong 15% tng s mu) dng tnh vi Ochratoxin A trong khong 2-3 ppb.

Nhm tc gi Pedro Novoa, Graud Moulasa, Duarte Miguel Franc

Prazeresb, Virginia Chua, Joo Pedro Conde [32] xc nh Ochratoxin trong mu

ru vi gii hn pht hin l 0,85 ng/ml. Ochratoxin c pht hin trong dung

dch m PBS vi vic s dng cu hnh mt knh thng.

K thut c u im: nhanh, n gin,tit kim dung mi, c hiu v kh

nhy nhng so vi phng php HPLC th km hiu qu hn do d cho kt qu

dng tnh gi hoc m tnh gi

1.2.2 Phng php sc k kh (Gas chromatography-GC)

Phng php sc k kh c mt s tc gi ng dng xc nh cc

loi c t Ochratoxin. Tuy nhin phng php ch phn tch c nhng cht d

bay hi, cn cc cht kh bay hi th phi to dn xut nn tn thi gian v ha

cht. Hn na, phn tch c ng thi cc cht th cn thi gian phn tch di.

Do vy, phng php t c ng dng phn tch cc loi c t Ochratoxin ny

Tc gi Yuying Jiao v cc cng s [31] tin hnh xc nh Ochratoxin

trong thc phm bng phng php sc k kh. Ochratoxin A c chuyn

thnh dng dn xut ester 0-methyl ochratoxin A v c xc nh bng sc k kh

khi ph vi ch ESI m.

1.2.3. Phng php sc k lng hiu nng cao (HPLC) v ct i lc min dch

(IAC)

Ging nh ELISA, ct i lc min dch cng da trn s kt hp c hiu

gia khng nguyn v khng th. Khng th c hiu vi ochratoxin c gn ln

gi rn ca ct sc k (thng dng Sepharose 4B), to cho IAC c tnh va tinh

sch va c c ochratoxin. Mu c chit vi acetonitril/H2O, sau c pha

long v cho qua ct i lc min dch. Ct c ra sch nhng tp cht khng gn

ln khng th v c gii hp bng methanol. Tin hnh nh lng bng HPLC

vi detector hunh quang . Bn thn Ochratoxin l cht pht hunh quang ti bc


17

sng hp th 333 nm, bc sng pht x 460 nm nn c rt nhiu nghin cu s

dng phng php sc k lng hiu nng cao vi detector hunh quang xc nh.

Tc gi Catherine Tessini v cng s [18] tin hnh thc hin phn tch

154 mu ru xut x Chi L, kho st trn ct i lc min dch vi cc h dung

mi khc nhau, gii hn pht hin ca phng php t 1-9 ppb, hiu sut thu hi

nm trong khong 60-90%.

Tc gi R. Ghali v cng s [27] tin hnh thc hin trn 180 mu thc

phm xut x Tunisia. Mu c chit bng ACN/H2O (80/20) v lm sch bng

ct i lc min dch. thu hi mc nng 0,5 v 2 ng/g trong khong 84 n

94 %. Phng php c gii hn pht hin 0,1 ng/g. Loi mu thng b nhim l la

mch, la m.Tuy nhin, khi s dng detector hunh quang phng php c

nhy tng i tt, nhng ch c th nhn bit cht phn tch thng qua thi gian

lu. i vi nhng nn mu phc tp, cc cht phn tch rt d b nh hng bi

nn mu, nu ch da vo thi gian lu s rt kh c th khng nh ng n

cht cn phn tch.

1.2.4. Phng php sc k lng khi ph

Phng php sc k lng khi ph, c bit l phng php sc k lng hai

ln khi ph l mt k thut mi c pht trin trong nhng nm gn y. V c

bn, n l phng php sc k lng s dng b phn pht hin l detector khi ph.

Phng php c nhiu u im nh chn lc cao, gii hn pht hin thp, thi

gian phn tch nhanh, c th nh lng ng thi cc cht c thi gian lu ging

nhau m phng php sc k lng thng khng lm c.

C s l thuyt ca phng php c tm tt di y: Cu trc ca h

thng LC-MS/MS


18

Hnh 2.1: M hnh h thng LC-MS/MS

1.2.4.1. H thng sc k lng hiu nng cao

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.

Hnh 2.2: S n gin ca h thng sc k lng.

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 3 7 m. Tu

theo bn cht ca pha tnh, trong phng php sc k lng thng chia lm 2 loi:

sc k pha thng (NP-HPLC) v sc k pha ngc (RP-HPLC)

Sc k lng Ion ha B phn tch khi

Detector/ Lu gi s liu


19

Sc k pha thng: pha tnh c b mt l cc cht phn cc ( l cc silica

trn hoc cc silica c gn cc nhm ankyl c t cacbon mang 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 ngc: 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, axetonitril....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, CCl4, toluene...

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 .

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 (DAD), detector

khi ph (MS).


20

1.2.4.2 Khi ph (Mass Spectrometry)

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 2.3: 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. 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 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 (electrospray

Ngun ion Phn tch khi

Detector

X l v lu gi

liu

Ph khi

Trnh ion phn tch va chm

vi ion trong khng kh Chn khng cao


21

ionization ESI), ion ha ha hc p sut kh quyn (atmospheric pressure

chemical ionization APCI).

a) Ch ion ha u 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 2.4: 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. 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 thuc c tnh bazo.

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


22

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 thuc c tnh axit.

o [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) Ch ion ho ho hc p sut kh quyn (APCI).

Cht phn tch v dung mi dng lng c chuyn thnh cc git nh ri ha

hi nhit cao khong 500C. Mt in th cao t 3 5kV to ra cc electrron

v ion ha cht phn tch.

y cng l mt k thut ion ha mm. APCI c s dng phn tch cc

cht c khi lng phn t trung bnh. K thut ny c th bn ph 2 ch : ion

m v ion dng.

B phn phn tch khi

L tri tim ca my khi ph, c nhim v phn tch cc ion c tr s m/z

khc nhau thnh tng phn ring bit, c nhiu b phn tch khi khc nhau nh:

B phn tch t cc, phn tch t cc chp ba, phn tch by ion, phn tch thi gian

bay... Hin nay, c ba loi phn tch khi thng c s dng nht, l:

B phn tch t cc (Quadrupole Analyser)- Phn gii bng t hp in.

My t cc da trn nguyn tc cc ion c khi lng khc nhau s dao ng

khc nhau theo in p tng hp mt chiu v xoay chiu t vo mi trng di

chuyn ca n.

My gm 4 thanh cc ghp song song ni vi nhau tng i mt i din

nhau, to thnh hai cp, sau chng ni vi in p mt chiu to thnh 2 cp

dng v m. Ngoi in p mt chiu, hai cp in cc cn c ni vi in p

xoay chiu. T hp in p ny c bit l in p xoay chiu s thay i theo chu

k qut khi lng cc ion:

+ Ion cng hng


23

+ Ion khng cng hng.

Hnh 2.5: B phn tch t cc

Mt s ion c t s m/z xc nh cng hng vi th xoay chiu xc nh c

th i thng qua khong khng n detector. Trong khi cc ion khc khng s c

qu o khng n nh va chm vi cc cc v b gi li . Tuy nhin thu

c tt c cc ion ta qut in p theo chu k t zero n mt in p nht nh

tng dn sau li tr li zero, ln lt cc ion s vt qua c t cc cng c

khi lng t nh n ln n detector

B phn tch t cc by ion (Quadrupole Ion-Trap Mass Analyser)

By t cc hot ng theo nguyn l b phn tch khi t cc, ch c mt

im khc l cc ion c lu gi v a dn ra khi by. Bng cch thay i th

xoay chiu p vo cc cc cc ion c t s m/z khc nhau c th vt qua khong

khng n detector. Cc ion ny cng c th b bn ph trong by thu c

cc ion con. V nguyn tc loi phn tch khi ph by ion c th lm n MS nhiu

ln.

Loi thit b ny bao gm mt in cc vng (ring electrode) vi nhiu in

cc bao xung quanh, in cc u ct (end-cap electrode) trn v di. Tri vi

li thit b t cc trn, cc ion sau khi i vo by ion theo mt ng cong n

nh c by li cho n khi mt in p RF c t trn in cc vng. Cc ion

khc nhau m/z sau tr nn khng n nh v s c hng i v pha detector. Do

in p RF khc nhau trong h thng ny m thu c mt ph khi lng y .


24

B phn tch t cc chp ba (triple Quadrupole Analyser).

Gm ba t cc ghp ni vi nhau.

.

Hnh 2.6: M hnh b phn tch t cc chp ba

Trong :

Q0: Ngun ion m.

Q1: B t cc th nht, c nhim v tch cc ion. La chn ion m vi m/z nht

nh t ngun ion chuyn n chuyn n Q2.

Q2: B t cc th hai, iu kin p sut cao, cc ion m 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.

Q3: B t cc th ba lm nhim v tch cc ion c chuyn t Q2 i ti b

phn pht hin.

Thit b khi ph ba t cc thng c gi l my khi ph hai ln (LC-

MS/MS).

B phn pht hin.

Sau khi i ra khi thit b 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 multipler) v b phn pht

hin nhn quang (photo multipler).

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

c nhy cao. Mt ion p vo b mt diot lm bt ra cc electron. Cc electron

Q1 Q2

Q3 Q0


25

th cp sau c dn ti cc diot tip theo v s to ra electron th cp nhiu

hn na, to thnh dng cc electron (1-106)

B phn pht hin nhn quang cng ging nh thit b nhn electron, cc ion

ban u p vo mt diot to ra dng cc electron. Khc vi detector nhn electron,

cc electron sau s va p vo mt mn chn photpho v gii phng ra cc

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

b nhn electron. S lng cc photon t l vi cng tn hiu.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.

R. Vatinnoa, D. Vuckovica, C.G. Zamboninb, J. Pawliszyna [28] tin

hnh xc nh Ochratoxin trong 96 mu nc tiu bng phng php vi chit pha

rn kt hp vi sc k lng khi ph. Mu c chnh pH v 3 bng m

photphatsalin 0,5 M trc khi vi chit pha rn. Chng trnh sc k lng c

gradient khong 8 pht c th cho ra cht phn tch vi tn hiu tt. thu hi,

chm c tin hnh o 3 mc nng 1, 10, 50 ng/ml. Gii hn pht hin v

gii hn nh lng trong nc tiu tng ng l 0,3 v 0,7 ng/ml. Ct sc k s

dng l ct C18 Waters, tc dng 0,5 ml/pht. Pha ng: 90% knh A (nu,

acetonitril, aceticacid 90:10:0,1) trong 1 pht, tng t l 60% A/40%B (acetonitril

v acetic acid 100:0,1) trong 6 pht, a v 90% B giy tip theo v gi trong

vng 1 pht. Tng thi gian chy sc k l 8 pht. Detector khi ph thc hin

ch ion m, IS=-4V, nhit ngun 4000C, vi ochratoxin A c cc thng s

tng ng: DP=-20V, FP=-74V, EP=-8,6, CE=-25V, CXP=-9,5V, vi ochratoxin B

c cc thng s tng ng: DP=-20V, FP=-86, EP=-8,6, CE=-44,41V, CXP=-

15,11V. Ion nh dng: OTA: m/z 402.1->357.9, OTB: m/z 368.0->133.1. trong

nghin cu ny tc gi coi ochratoxin B nh l mt ni chun, c thm vo t

u kim sot hiu sut thu hi. u vi chit pha rn c nhng vo 1 ml mu

tc khuy 850 rpm, thi gian hp th v gii hp th l 1 gi 15 pht. Phng

php c u im tnh t ng ha cao, t c hi cho ngi phn tch.


26

Ti Vin nghin cu thuc tr su v nc, i hc Jaume, Ty Ban Nha,

nhm tc gi Eduardo Beltrn [20] nghin cu xc nh mt s mycotoxin

trong thc phm v sa bng sc k lng khi ph. Mu c chit bi acetonitril:

nc (80:20), lm sch mu bng ct i lc min dch (Aaochra HPLCTM) c

cung cp bi Vicam (Tecasa, Madrid, Ty Ban Nha). Phng php cho thu hi

80-110% ti mc thm chun 0,025 v 0,1 ng/g, RSD nh hn 15%.

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

ph nghin cu xc nh cc loi c t ochratoxins c trong sn phm ru

v cc loi ng cc v cc sn phm lm t ng cc.


27

CHNG 2: I TNG V PHNG PHP NGHIN CU

2.1. i tng v ni dung nghin cu

2.1.1. i tng nghin cu

- Ochratoxin l c t gy tc hi vo cc c quan quan trng ca c th nh:

thn kinh, gan, thn v h min dch. Ochratoxin A gy hu qu nghim trng i

vi ngi v ng vt nui nng cc thp (ppb). Ochratoxin B c tnh c t

hn ochratoxin A , tuy nhin chng u l cht c thuc nhm IIB theo T chc

nghin cu ung th Quc t IARC.

- Nn mu phn tch: ng cc v cc sn phm t ng cc, cc loi ru ln

men.

2.1.2. Ni dung nghin cu

Ni dung nghin cu bao gm:

Ti u ha iu kin chy my xc nh c t Ochratoxin c trong sn

phm ru ln men, ng cc v sn phm t cc loi ng cc bng phng php sc

k lng khi ph (LC-MS/MS).

Xy dng quy trnh tch chit cht phn tch.

Thm nh phng php:

c hiu/chn lc ca phng php.

Gii hn pht hin LOD, gii hn nh lng LOQ

Khong tuyn tnh.

chnh xc ca phng php ( ng v chm)

p dng phng php xc nh Ochratoxins trong cc loi mu thc

phm, ru ln men.

2.2. Phng php nghin cu

2.2.1.Phng php tch chit mu

Chn dung mi d ho tan cht phn tch chit cht phn tch ra khi nn

mu. Dung mi c th l: chloroform, methanol, dung dch cacbonate

long,V loi dung mi ny thng ko theo rt nhiu cht tp khc c tnh cht


28

tng t cht phn tch b chit vo nn rt cn loi bt cc cht tp ny. Chit pha

rn l mt phng php chun b mu l 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 nh dung mi thch hp.

2.3. Phng tin nghin cu

2.3.1. Thit b, dng c

2.3.1.1. Thit b

H thng sc k lng khi ph khi ph LC-MS/MS ca Shimadzu bao gm:

+ My sc k lng ca Shimadzu. Model 20 AD-UFLC.

+ My khi ph.

Nc sn xut: M

Model: AB sciex Triplequard 5500

Ct sc k: Water- ct C18 (250mm 2,1mm 5m) v tin ct C18 (4mm

2,1mm 3m)

My lc Vortex.

Cn phn tch (c chnh xc 0,1mg v 0,01mg) (Metter Toledo).

Cn k thut (c chnh xc 0,01g) (Metter Toledo).

My ct quay chn khng (Eyela).

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

B thi kh (OA-Sys).

2.3.1.2. Dng c

Bnh nh mc: 5, 10, 50, 100 ml.

ng ly tm 50 ml

Bnh c quay 50 ml, 100 ml

Vial loi 1,8 ml.

ng ong, phu, giy lc.

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

2.3.2. Dung mi, ha cht

Cc loi ho cht s dng u thuc loi tinh khit phn tch.


29

o Cht chun ca Supelco

o Methanol (Merk)

o n-hexan (Merk)

o Amoniacetat CH3COONH4 (Merk)

o Axit acetic (Merk), acetonitril (Merk)

o Nc ct 2 ln

* Chun b dung dch chun.

- Dung dch chun trung gian 500 g/l: Ly chnh xc khong 10l cht chun

ln lt ca tng chun gc c nng 50000 g/l vo mt vial mu nu np kn,

thi kh, ho tan cn trong 1 ml MeOH. Chun gc c bo qun nhit t 0

5C, trong 6 thng.

- Dung dch chun hn hp lm vic: c pha ty vo mc ch xy dng

khong ng chun, thng thng nng chun c pha trong khong nng

t 2,5 g/l - 50 g/l

Bng 2.1: Bng pha dung dch chun chy my

2.3.3 Ly mu

- i tng mu: cc loi ng cc: ng, lac, , go,, cc loi ru

- Phng php ly mu: ngu nhin

Nng hn hp

chun lm vic

(g/l)

500 50 50 50 25

Th tch ly chun

lm vic (l) 100 100 500 200 100

nh mc (ml) 1 1 1 1 1

Nng chun

(g/l) 50 5 25 10 2,5


30

- a im: mt s ch trn a bn ni thnh H Ni, Bc Giang, Thanh

Ho, Ngh An.

- Khi lng mu ly: 100 500 g/mu

- Bo qun: nhit thng


31

CHNG 3: KT QU V THO LUN

3.1 Ti u cc iu kin chy my LC-MS/MS

3.1.1 Ti u cc iu kin chy ca my khi ph MS/MS

3.1.1.1 Kho st ion m v ion con

V ochratoxin A, B l nhng cht c khi lng phn t khng ln v

phn cc trung bnh. Qua tham kho ti liu chng ti tin hnh kho st xc nh

cc ochratoxin bng k thut ion ha phun in t ESI vi ch bn ph ion m.

ti u ha iu kin khi ph, dng xilanh bm hn hp 2 cht chun 50 ng/ml

tim trc tip vo detector khi ph kho st. Chn ch kho st t ng i

vi tng cht chn c ion m, ion con dng nh lng v nh tnh i vi

tng cht.

Ion m ca tng cht c ch ra bng 3.1.

Bng 3.1: Ion m ca tng cht

Cht Khi lng phn t (g/mol) Ion m (m/z)

Ochratoxin A 403 402 [M-H]-

Ochratoxin B 369 368[M-H]-

Bng 3.2 Cc thng s ti u cho ESI-MS/MS.

Cht Ion m Ion con DP (V) CE (V) CXP (V)

Ochratoxin A 402 358

-100

-24 -25

166,9 -35 -25

Ochratoxin B 368 324 -24 -21

220 -32 -15


32

Trong :

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

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. Tuy nhin, th ny cng khng

c qu cao, v n c th b gy lun c cu trc ca phn t ion m.

+ 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.

Mnh ion con m/z c cng ln nht dng nh lng, mnh con th 2

c cng thp hn dng xc nhn cht phn tch.

XIC of -MRM (4 pairs): 368.000/324.000 Da ID: OTB1 from Sample 3 (test) of DataSET1.wiff (Turbo Spray) Max. 2.2e5 cps.

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 9.5Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.2e5

In

te

ns

it

y,

c

ps

6.38

-MRM (4 pairs): 6.655 min from Sample 3 (test) of DataSET1.wiff (Turbo Spray) Max. 2.0e4 cps.

368.000/324.000 368.000/220.000 402.000/358.000 402.000/166.900Q1/Q3 Masses, Da

0.0

5000.0

1.0e4

1.5e4

2.0e4

In

te

ns

it

y,

c

ps

-MRM (4 pairs): 6.413 min from Sample 3 (test) of DataSET1.wiff (Turbo Spray) Max. 7.3e4 cps.

368.000/324.000 368.000/220.000 402.000/358.000 402.000/166.900Q1/Q3 Masses, Da

0.0

2.0e4

4.0e4

6.0e4

7.3e4

In

te

ns

it

y,

c

ps

Hnh 3.1: Sc t l cc ion ca cc ochratoxins

Kt lun: Sau khi tin hnh kho st, chng ti chn c ion m v ion con

thch hp cho cc ochratoxins ti cc iu kin MS nh bng trn.

3.1.1.2 Ti u cc iu kin MS

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

vi iu kin nh sau: Pha ng l amoni acetate 10 mM : methanol (95/5), hn hp

chun c nng 50 ppb, th tch bm 10 l, thi gian 1 pht. Chn ch kho

st t ng i vi tng ion con nh lng v nh tnh ca tng cht.


33

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 (Collision Gas Pressure): 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.

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

IS (V): -4000,0; 4500,0; -3500,0; -3000,0;

TEM (oC) : 250,0; 300,0; 350,0; 400,0;450,0

GS1 (psi): 25,0; 30,0; 10,0;15,0

GS2 (psi): 8,0; 9,0; 10,0; 5,0; 2,0;

CUR (psi): 15,0;20,0; 5,0; 10,0;

DP (V) :-110,0; -120,0; -100,0; -90,0; -80,0;

EP (V) : -7,0; -8,0; -9,0; -10,0; -11,0;

CAD (psi): 7,0; 8,0; 9,0; 5,0; 6,0;


34

CXP (V): -18,0; -21,0; -22,0; -24,0; -26,0; -15,0; -12,0;

CE (V): -20,0; -24,0; -28,0; -32,0; -35,0; -40,0; -44,0;

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 3.3

Bng 3.3: Cc thng s ti u MS/MS.

Thng s Ochratoxin A Ochratoxin B

OTA1 OTA2 OTB1 OTB2

EP (V) -5 -5 -12 -11

CE (V) -24 -35 -24 -32

CXP (V) -25 -25 -21 -15

IS (V) -3500 -3500

TEM (oC) 400 400

GS1 (psi) 10 10

GS2 (psi) 5 5

CUR (psi) 10 10

DP (V) -100 -100

CAD (psi) 7 7

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 cc iu kin chy sc k lng hiu nng cao (HPLC)

3.1.2.1 Pha tnh

Ct tch l b phn quan trng ca h thng sc k, n ng gp mt phn

quan trng trong vic quyt nh qu trnh tch. Ochratoxin l cht km phn cc,

do ct tch c s dng l ct pha o. Hn na, cht nhi pha o s dng h

dung mi phn cc c tnh kinh t cao, ph hp vi iu kin ca phng th nghim.

Trong nghin cu ny, chng ti chn ct pha o C18 tch cc cht. bo v

ct, chng ti s dng thm tin ct. Thng s ct tch v tin ct:


35

Ct: Waters- ct C18 (250 mm 4,6 mm 5 m).

Tin ct: C18 (4 mm 2,1 mm 5 m).

3.1.2.2 Kho st 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 m, qu trnh

ion ha tng khi c thm cc cht nh acid acetic, acid formic, amoni acetate...

Trong phng php nghin cu, pha tnh s dng l ct C18, t phn cc nn pha

ng phi l h dung mi phn cc. Cht tan t phn cc s b lu gi trong ct lu

hn cht tan phn cc. Do tnh cht phn cc trung bnh ca Ochratoxin nn chng

ti chn pha ng trn 2 knh:

Knh A: Dung dch amoniacetate 10 mM.

Knh B: Methanol.

Chng ti tin hnh dng dung dch chun hn hp 50 ng/ml kho st

dung mi pha ng. Hn na c th va tch c cc cht, 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:


Tin ct: C18 (4 mm 2,1 mm 5 m).

Detector: khi ph vi cc thng s nh bng 2.2; 2.3.

Pha ng: knh B c nh l Methanol, knh A ln lt l CH3COOH 0,1%,

CH3COONH4 10 mM, CH3COONH4 20 mM, CH3COONH4 5 mM.

Chng trnh gradient:

- Tc dng 1,0 ml/pht.


36

Bng 3.4: Chng trnh gradient pha ng

Thi gian (pht) T l (%) knh B

0,01 5

4 100

6 100

6,01 5

10 5

Kt qu kho st dung mi pha ng n thi gian lu v din tch pic c ch

ra bng sau:

Bng 3.5: Kt qu kho st thnh phn pha ng

Dung mi Kt qu Ochratoxin A Ochratoxin B

CH3COOH 0,1% t (pht) 6,21 6,23

S pic 1,23*104

4,62*104

CH3COONH4

10mM

t (pht) 6.90 6.70

S pic 1,44*104

8,46*104

CH3COONH4

20mM

t (pht) 7,05 7,01

S pic 1,52*104

3,25*104

CH3COONH4 5mM t (pht) 6,92 7,02

S pic 1,2*104

3,2*104

Nhn xt: Khi nng amoniacetate cao, s lng ion i vo bung ion ca

detector MS tng, gy cnh tranh vi cc ion ca cht phn tch, lm gim tn hiu.

ng thi nng mui cao s gy hi n hiu lc v tui th ca ct. Ti nng

amoniacetate 10 mM cc cht u cho tn hiu cao nht, hnh dng pic p,

khng b ch pic, dong chn pic. Ngoi ra amoniacetate cn khng c hi, d s

dng Chng ti quyt nh chn nng amoniacetate 10 mM. Nng ny cng

ph hp vi cc h LC/MS/MS.


37

XIC of -MRM (4 pairs): 368.000/324.000 Da ID: OTB1 from Sample 7 (Std mix 10ppb) of DataSET1.wiff (Turbo Spray) Max. 2.2e4 cps.

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 9.5Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

In

te

ns

it

y,

c

ps

6.70


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 9.5Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

In

te

ns

it

y,

c

ps

6.70

XIC of -MRM (4 pairs): 402.000/358.000 Da ID: OTA1 from Sample 7 (Std mix 10ppb) of DataSET1.wiff (Turbo Spray) Max. 1.4e4 cps.

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 9.5Time, min

0.0

5000.0

1.0e4

1.4e4

In

te

ns

it

y,

c

ps

6.90

Hnh 3.2: Sc ca ochratoxin A, B nng 10 ppb khi dng dung mi pha

ng l CH3COONH4 10 mM

3.1.2.3 Kho st tc pha ng

Tc pha ng nh hng n thi gian ra gii v lng dung mi tiu

tn. c bit, vi h UFLC, tc dng nh hng rt nhiu n s n nh ca

chng trnh gradient v lp li ca qu trnh sc k. Nu tc qu nh,

chng trnh gradient s km n nh. Tc dng cao, nh hng n p sut ca

ton h thng. C nh cc iu kin:

- Ct C18 (250mm 2,1mm 5m)

- Pha ng: knh B (MeOH), knh A: amoniacetat

- Mu phn tch: hn hp chun Ochratoxin A, B nng : 10 ng/ml

- Tc pha ng kho st ln lt l: 0,8 ml/pht; 1,0 ml/pht; 1,2 ml/pht.

XIC of -MRM (4 pairs): 368.000/324.000 Da ID: OTB1 from Sample 2 (test) of DataSET1.wiff (Turbo Spray) Max. 7707.0 cps.

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 9.5Time, min

0

2000

4000

6000

7707

In

te

ns

it

y,

c

ps

6.65

XIC of -MRM (4 pairs): 368.000/324.000 Da ID: OTB1 from Sample 2 (test) of DataSET1.wiff (Turbo Spray) Max. 7707.0 cps.

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 9.5Time, min

0

2000

4000

6000

7707

In

te

ns

it

y,

c

ps

6.65

XIC of -MRM (4 pairs): 402.000/358.000 Da ID: OTA1 from Sample 2 (test) of DataSET1.wiff (Turbo Spray) Max. 3773.0 cps.

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 9.5Time, min

0

1000

2000

3000

3773

In

te

ns

it

y,

c

ps

6.88

Hnh 3.3: Sc ra gii cc cht ti tc dng 0,8 ml/pht


38


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 9.5Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

In

te

ns

it

y,

c

ps

6.70


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 9.5Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

In

te

ns

it

y,

c

ps

6.70

XIC of -MRM (4 pairs): 402.000/358.000 Da ID: OTA1 from Sample 7 (Std mix 10ppb) of DataSET1.wiff (Turbo Spray) Max. 1.4e4 cps.

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 9.5Time, min

0.0

5000.0

1.0e4

1.4e4

In

te

ns

it

y,

c

ps

6.90


XIC of -MRM (4 pairs): 368.000/324.000 Da ID: OTB1 from Sample 3 (TEST03-TC) of DataSET1.wiff (Turbo Spray) Max. 1.0e5 cps.

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 9.5Time, min

0.00

2.00e4

4.00e4

6.00e4

8.00e4

1.00e5

1.20e5

In

te

ns

it

y,

c

ps

6.78

XIC of -MRM (4 pairs): 368.000/324.000 Da ID: OTB1 from Sample 3 (TEST03-TC) of DataSET1.wiff (Turbo Spray) Max. 1.0e5 cps.

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 9.5Time, min

0.00

2.00e4

4.00e4

6.00e4

8.00e4

1.00e5

In

te

ns

it

y,

c

ps

6.78

XIC of -MRM (4 pairs): 402.000/358.000 Da ID: OTA1 from Sample 3 (TEST03-TC) of DataSET1.wiff (Turbo Spray) Max. 1.2e5 cps.

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 9.5Time, min

0.00

2.00e4

4.00e4

6.00e4

8.00e4

1.00e5

1.20e5

In

te

ns

it

y,

c

ps

6.98


Nhn xt: Khi tng tc dng ca pha ng, cc pic sc k c xu hng

nhn v r nt. Ti tc dng 1,2 ml/pht cc pic rt sc v nhn. Tuy nhin, ti

tc ny, p sut ca h cao, nh hng rt ln n p sut u vo ca ct v lm

gim tui th ca ct. Do , chng ti quyt nh la chn tc dng 1,0

ml/pht. Ti tc ny, cc cht bt u c ra gii trong khong t 6-8 pht,

cc pc tch tt, hnh dng pc sc nhn.

3.1.2.4 Chn chng trnh chy gradient

Thc hin tch v xc nh ng thi 10 hp cht trong cng mt nhm, c

cu trc gn ging nhau, s dng ch ra gii ng dng (isocractic) l khng

ph hp. c th va tch c cc cht, va tit kim c thi gian phn tch


39

v dung mi, chng ti tin hnh chy pha ng vi ch gradient. Tin hnh

kho st cc chng trnh chy gradient khc nhau

Bng 3.6: Kho st cc chng trnh gradient

Chng trnh

gradient

Thi gian

(pht)

Tc dng

(ml/pht)

Knh A:

CH3COONH4 10 mM

Knh

B:MeOH

1

0,01 1,0 95 5

4 1,0 0 100

6 1,0 0 100

6,01 1,0 95 5

10 1,0 95 5

2

0,01 1,0 95 5

4 1,0 0 100

5 1,0 0 100

5,01 1,0 95 5

10 1,0 95 5

3

0,01 1,0 95 5

4 1,0 0 100

8 1,0 0 100

8,01 1,0 95 5

10 1,0 95 5

4

0,01 1,0 95 5

4 1,0 20 80

6 1,0 20 80

6,01 1,0 5 5

10 1,0 5 5


40


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 9.5Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.2e5

In

te

ns

it

y,

c

ps

6.38


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 9.5Time, min

0.0

5.0e4

1.0e5

1.5e5

2.0e5

2.2e5

In

te

ns

it

y,

c

ps

6.38

XIC of -MRM (4 pairs): 402.000/358.000 Da ID: OTA1 from Sample 3 (test) of DataSET1.wiff (Turbo Spray) Max. 7.2e4 cps.

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 9.5Time, min

0.0

2.0e4

4.0e4

6.0e4

7.2e4

In

te

ns

it

y,

c

ps

6.62

Hnh 3.6: Sc khi chy sc k bng chng trnh gradient 1.

Nhn xt: Khi gi t l MeOH 100% qu t hoc qu lu u lm pc b ch

v t, lm gim nhy ca phng php.

Qua qu trnh kho st chng ti chn c chng trnh chy gradient 1 cho

tn hiu pic p nht, cc cht c tch ra khi nhau, pic thu c khng b t,

khng b ch pc, v din tch pic cng cao nht i vi tt c cc cht nghin cu.

Tm li, cc thng s ti u cho qu trnh chy sc k nh sau:


Tin ct: C18 (4 mm 2,1 mm 5 m).

Thnh phn pha ng: Knh A l dung dch CH3COONH4 10 mM, knh B l

MeOH

Chng trnh chy gradient

Bng 3.7: Chng trnh gradient ti u.

Thi gian

(pht)

Tc dng

(ml/pht)

Knh A:

CH3COONH4

10mM

Knh B:MeOH

0,01 1,0 95 5

4 1,0 0 100

6 1,0 0 100

6,01 1,0 95 5

10 1,0 95 5


41

Detector MS/MS vi cc thng s bng 3.2 v 3.3.

3.2 Ti u quy trnh x l mu.

3.2.1 Chn quy trnh x l mu.

Ta kho st s b trn 3 quy trnh x l mu sau:

Quy trnh 1.

Cn chnh xc khong 5g mu ng ng nht vo ng ly tm 50 ml.

Thm 100 l chun hn hp 100 ng/ml, thm 30 ml CHCl3, lc xoy. Ly lp hu

c sang ng ly tm khc, thm 10ml NaHCO3 1%, lc xoy, ly tm 6000v/p trong 3

pht. Ly lp nc em qua ct chit pha rn C18. Ct chit pha rn c hot ha

bi 5ml MeOH, 5 ml H2O. Sau , ct chit c ra tp bng 2 ml H2O, 2 ml

MeOH:H2O (6:4). Cht phn tch c ra gii bng 2 ml MeOH:CH3COOH

(99,5:0,5, v/v). Dch ra gii c chuyn vo vial ng mu ri bm vo h thng

LC-MS/MS (dch c th c lc trc khi em o trn my bng mng lc mu c

0,2 m, nu dch b c).

Quy trnh 2 [3].

Cn 10 g mu ng ng nht vo bnh tam gic 250 ml. Thm 100 l

chun hn hp 100 ng/ml, thm 200ml NaHCO3 1%, lc 30 pht. Lc ly 10 ml

dch chit, thm 10 ml m phosphat saline (PBS) pH=7-8. Hn hp dch chit

c qua ct chit pha rn C18. Ct c hot ha bng 10 ml PBS. Sau , ct

chit c ra tp bng 5 ml nc ct. Cht phn tch c ra gii bng 3 ml

MeOH. Dch ra gii c em thi kh ri ha cn li bng 1 ml MeOH, em i

o my.

Quy trnh 3 [21].

Cn chnh xc khong 5 g mu ng ng nht vo ng ly tm 50 ml.

Thm 100 l chun hn hp 100 ng/ml, thm 10 ml ethyl acetate, em lc xoy. Ly

5 ml dch chit em bay hi n kh di dng kh N2 40 oC. Ha cn bng 5 ml

acetonitril:H2O (84:16), sau cho qua ct chit pha rn C18. Cc bc tip theo

ging quy trnh 1.


42

Nhm mc ch tm c ra quy trnh x l mu ti u nht, chng ti

tin hnh kho trn 3 quy trnh x l mu nu trn. Mi quy trnh tin hnh

lm lp li 2 ln v ly kt qu trung bnh. Kt qu thu c ch ra trong bng sau:

Bng 3.8: Kho st quy trnh x l mu

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

l tt nht trong 3 quy trnh kho st. Nu ch chit mu bng NaHCO3 th dch

chit thu c rt c lm nh hng n qu trnh qua ct, d gy tc ct. iu

ny va lm mt nhiu thi gian chit va lm gim hiu sut chit ca ct chit.

Trong khi , phn cc ca chloroform l 4,1, ca ethylacetate l 4,4 trong khi

ochratoxins l cc cht c phn cc yu. Do dng ethylacetat chit mu

cng khng cho hiu sut cao .

Do , chng ti s tp trung kho st cc iu kin ca quy trnh 1 a ra

c mt quy trnh x l mu ti u nht .

Quy

trnh

C

thm chun

(ppb)

Kt qu

Ochratoxin A

OchratoxinB

1 5 C(ppb) 4,2 3,9

R (%) 84 78

2 5 C(ppb) 3,4 2,8

R (%) 68 56

3 5 C(ppb) 2,6 2,8

R (%) 52 56


43

Quy trnh x l mu d kin c m t theo s sau:

Hnh 3.7:Quy trnh x l mu d kin.

5g mu / ng ly tm 50ml

+ 30 ml CHCl3

Lc xoy, ly tm

Ly lp hu vo ng ly tm khc

+10 ml NaHCO3 1%

Lc xoy, ly tm

Ly lp nc

Ct C18 hot ha

Ct C18

+5ml MeOH

+5ml H2O

+2 ml H2O

+2 ml MeOH:H2O(6/4, v/v)

Ra gii 2 ml MeOH-acid acetic (99,5:0,5 v/v)

Vial

LC-MS/MS


44

3.2.2 Kho st th tch (s ln chit) dung mi chit

Th tch dung mi chit nh hng n hiu sut chit cht cn phn tch.

Nu dung mi chit qu t s khng ho tan trit cht cn phn tch, nhng nu

qu nhiu s gy lng ph v nh hng n mi trng.

Chng ti s dng nn mu ng khng pht hin ochratoxins tin hnh

kho st tip. Cn chnh xc khong 5 g bt ng , thm 100l chun hn hp c

nng 100 ng/ml. Tin hnh chit lp 1, 2, 3 ln cc mu bng dung mi CHCl3

vi th tch mi ln chit ln lt l 5, 10, 15 ml. Cc bc c tin hnh theo quy

trnh 1. Ta thu c kt qu nh sau:

Bng 3.9: Kt qu s ph thuc ca th tch dung mi chit v hieuj sut chit

S ln chit

Ochratoxin A Ochratoxin B

Kt qu

V5 V10 V15 Kt qu

V5 V10 V15

1 C(ng/g) 2,9 3,1 3,5 C(ng/g) 2,7 3,1 3,2

R% 58 62 70 R% 54 62 64

2 C(ng/g) 3,1 3,5 4,2 C(ng/g) 2,9 3,4 4,1

R% 62 70 84 R% 58 68 82

3 C(ng/g) 3,3 4,1 4,1 C(ng/g) 3,1 4 4,1

R% 66 82 82 R% 62 80 82

Nhn xt: Qua qu trnh kho st, chng ti nhn thy chit cloroform mt

ln 15 ml th cht phn tch vn cha chit c hon ton. Kt qu chit 2 ln v 3

ln khng khc nhau ng k. Hiu sut chit lp ba ln mi ln 10 ml cho kt qu

tng t khi chit lp 2 ln mi ln 15 ml. Do tit kim dung mi, chng ti

chn chit lp 2 ln mi ln 15 ml CHCl3.

3.2.3 Chn ct chit pha rn

V Ochratoxin c tnh axit yu nn chng ti chn 2 loi ct kho st l

ct C18 v ct Polydivinylbezen-N-vinylpyrolidon HLB Oasis ( hydrophile

lyophile balance).

S dng nn mu ng khng pht hin OTs. Qu trnh chit mu c thc

hin theo quy trnh 1. Kt qu thu c nh sau:


45

Bng 3.10: Kho st ct chit pha rn.

Ct chit

C

thm chun

(ng/ml)

Kt qu Ochratoxin A Ochratoxin B

C18 10 C(ng/ml) 8,6 7,4

R (%) 86 74

HLB 10 C(ng/ml) 6,7 5,8

R (%) 67 58

Nhn xt: Kt qu ch ra rng chit Ochratonin A, B vic s dng ct

chit pha rn C18 th tt hn so vi ct oasis HLB. Ct HLB nhi cc phn t c

hai u mt u a nc, mt u k nc nn c phn cc ln hn so vi ct

chit C18. Trong khi Ochratoxin l cht t phn cc nn n b lu gi trn ct C18

tt hn ct HLB trong sut qu trnh np mu ln ct v qu trnh ra gii. Do ,

chng ti chn ct chit C18 lm sch dch chit.

3.2.4 Kho st tc qua ct chit pha rn

Tc ra gii nh hng kh ln n hiu sut chit. Nu tc ra gii

qu ln s phi s dng mt lng ln dung mi, d ko i mt cht phn tchlm

hiu sut chit gim. Cn nu tc ra gii thp s ko di thi gian phn tch

mu. Do , chng ti cn kho st tm tc ra gii ph hp.

Chng ti kho st tc ra gii ti 0,5 ml/pht, 1 ml/pht, 2 ml/pht

tm tc ti u.

Kt qu phn tch nh sau:

Bng 3.11: nh hng ca tc ra gii ti hiu sut ca qu trnh chit mu

C (ng/g)

thm chun Tc ra gii

C(ng/g) R%

OTA OTB OTA OTB

5

0,5 4,1 3,8 82 76

1 4,2 4,1 84 82

2 2,5 2,3 50 46


46

T cc kt qu ch ra bng chng ti nhn thy ti tc 0,5 ml/pht v

1,0 ml/pht cho hiu sut chit khng khc nhau nhiu, nhng tit kim thi gian

chng ti chn tc ra gii 1 ml/pht .

3.2.5 Kho st th tch dung mi ra gii

Lng dung dch ra gii phi ln cht phn tch c th c ra gii

ra khi ct hon ton. Qua tham kho ti liu chng ti chn dung mi ra gii l

dung dch MeOH-acid acetic (99,5:0,5, v/v).

S dng nn mu ng khng pht hin ochratoxins. Cn chnh xc 5 g mu

trng bt ng ng nht vo ng ly tm 50 ml, thm 200 l chun hn hp

nng 100 ng/ml, tin hnh x l mu theo quy trnh , kho st ti cc th tch ra

gii khc nhau l: 1, 2, 6 ml dung dch MeOH-acid acetic (99,5:0,5, v/v). Dch ra

gii 6 ml s c em thi kh di dng kh N2, ri ha cn 1 ml pha ng em o

my. Ta thu c kt qu kho st nh sau:

Bng 3.12: Kt qu kho st th tch dung dch ra gii

V ra gii

(ml)

C

ng/g Din tch OTA Din tch OTB

R(%)

OTA OTB

1

10

45630 4620 45 51

2 8820 8520 70 68

6 8930 8540 72 70

Nhn xt: Kt qu cho thy khi ra gii vi th tch 2 ml dung dch MeOH-

acid acetic (99,5:0,5) hiu sut thu hi cc cht phn tch l cao nht. V vy chng

ti chn th tch dung dch cn ra gii l 2 ml dung dch cho cc th nghim tip

theo.

3.2.6 Kho st nh hng pH trong qu trnh chit mu

Khong pH chit mu l kh quan trng lin quan n hiu sut chit. Mi

cht nghin cu c nhng c tnh ring trong khi nn mu thc phm l rt phc


47

tp. Do vy chng ti tip tc kho st so snh gia cc quy trnh chit sau mi qui

trnh lm lp 3 mu v tnh hiu sut thu hi theo cng thc sau:

100lt

tt

C

CH (1)

Trong :

H: hiu sut thu hi (%)

Ctt: Nng thc t ca mi OTs thu c (tnh theo ng chun) (ng/ml)

Clt: Nng l thuyt ca mi OTs tnh ton t lng chun thm vo (10

ng/ml)

S dng phn mm nh lng ca my, cc mu c nh dng dng

mu quality control vi nng bit trc l 10 ng/ml. Phn mm s t ng lp

ng chun, tnh ton nng thc trong mu v a ra hiu sut thu hi theo

cng thc trn.

Cn chnh xc khong 5g mu ng nht vo ng ly tm 50 ml. Thm

100l chun hn hp 100 ng/ml, axit ha bng HCl 2% n cc pH khc nhau

trongkhong 1-9 (dng giy ch th mu nhn bit). Thm 15 ml CHCl3, lc

Vortex, ly tm 6000 v/p trong 3 pht. Ly lp hu c sang ng ly tm khc. Phn

b tip tc c chit vi 15 ml cloroform. Gp dch chit vo ng ly tm, thm 10

ml NaHCO3 1%, lc Vortex, ly tm 6000 v/p trong 3 pht. Ly lp hu c em qua

ct chit pha rn C18. Ct chit pha rn c hot ha bi 5ml MeOH, 5 ml H2O.

Sau ct chit c ra tp bng 2 ml H2O, 2 ml MeOH:H2O (6:4). Cht phn

tch c ra gii bng 2 ml MeOH:CH3COOH (99,5:0,5,v/v). Dch ra gii c

chuyn vo vial ng mu ri bm vo h thng LC-MS/MS (dch c th c lc

trc khi em o trn my bng mng lc mu c 0,2 m, nu dch b c).


48

Kt qu thu c nh sau:

Bng 3.13: S ph thuc gia pH chit mu v din tch pc sc k

Khong pH Gi tr pH S OTA SOTB

1 1-2 4400 4620

2 3-4 3850 4521

3 5-6 3694 4312

4 7-8 3375 3821

5 9-10 3256 3622

Hnh 3.8 th s ph thuc ca din tch pc vo gi tr pH chit mu

Kt lun: Ochratoxin ch c chit tt vo cloroform trong mi trng axit.

iu ny c th gii thch: trong mi trng pH=1-2 ochratoxins trng thi ng

in, dng cation v anion ngang bng, dn n kh nng phn cc gim. Do

gip n d tan vo trong cc loi dung mi khng phn cc nh cloroform. Mt

khc vic axit ha cn gip lm m c cht, gip chit mu vo mi trng hu c

trit hn


49

QUY TRNH X L MU TI U CHO MU THC PHM

Hnh 3.9: Quy trnh x l mu ti u cho mu thc phm.

5 g mu / ng ly tm 50ml

+ Thm 100 l chun hn hp 100ng/ml + Thm 5ml HCl 2% + 15ml cloroform

Lc xoy, ly tm

Gp dch vo ng ly tm 50ml

Lc xoy, ly tm

+ 10 ml NaHCO3 1%

Ly lp nc

Ct C18 hot ha Ct C18

+5ml MeOH

+5ml H2O +2ml H2O

+2ml MeOH:H2O(6/4)

Ra gii 2ml MeOH-acid acetic (99,5:0,5 v/v)

Vial

LC-MS/MS

Phn b

+ 15 ml cloroform


50

QUY TRNH X L MU TI U CHO MU RU

Hnh 3.10: Quy trnh x l mu ti u cho mu ru

5 ml mu / ng ly tm 50ml

+ Thm 100 l chun hn hp 100ng/ml + Axit ha n pH=1-2 bng HCl 6N + 15ml cloroform

Lc xoy, ly tm

Gp dch vo ng ly tm 50ml

Lc xoy, ly tm

+ 10 ml NaHCO3 1%

Ly lp nc

Ct C18 hot ha Ct C18

+5ml MeOH

+5ml H2O +2ml H2O

+2ml MeOH:H2O(6/4)

Ra gii 2ml MeOH-acid acetic (99,5:0,5 v/v)

Vial

LC-MS/MS

Phn b

+ 15 ml cloroform


51

3.3 Thm nh phng php phn tch

3.3.1 Tnh c hiu / chn lc

Tnh c hiu l kh nng pht hin cc cht phn tch khi c mt cc tp

cht khc nh cc tin cht, cc cht chuyn ha, cc cht tng t, tp cht...

Trong php phn tch nh tnh phi chng minh c kt qu l dng tnh khi

c mt cht phn tch, m tnh khi khng c mt n, ng thi kt qu l phi m

tnh khi c mt cc cht c cu trc gn ging vi cht phn tch. Trong php phn

tch nh lng, l kh nng pht hin chnh xc cht phn tch trong mu khi b nh

hng ca tt c cc yu t, nhm hng n kt qu chnh xc [9].

Tnh chn lc: L khi nim rng hn tnh c hiu, lin quan n vic phn

tch mt s hoc nhiu cht trong mt quy trnh. Nu cht cn phn tch phn bit r

vi cc cht khc th phng php phn tch c tnh chn lc [9].

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 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.

Bng3.13: Ion m v 2 ion con ca cc cht trong nhm ochratoxins

Chun Ion m Ion con S im IP

Ochratoxin A 402 358

4 166,9

Ochratoxin B 368 324

4 220

Nh vy phng php nghin cu c tnh c hiu p ng c yu cu.

Hn na xc nh chc chn phng php nghin cu c tnh c hiu / chn lc

cao, chng ti tin hnh phn tch cc mu trng. Kt qu cho thy mu trng

khng cho tn hiu phn tch.


52

XIC of -MRM (4 pairs): 368.000/324.000 Da ID: OTB1 from Sample 6 (1138-1-5.26) of DataSET1.wiff (Turbo Spray) Max. 125.0 cps.

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 9.5Time, min

0

50

100

150

180I

nt

en

si

ty

,

cp

s

7.37

6.668.47 9.048.798.10 9.40

XIC of -MRM (4 pairs): 368.000/324.000 Da ID: OTB1 from Sample 6 (1138-1-5.26) of DataSET1.wiff (Turbo Spray) Max. 125.0 cps.

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 9.5Time, min

0

50

100

125

In

te

ns

it

y,

c

ps

7.37

6.66

8.47 9.048.798.10 9.40

XIC of -MRM (4 pairs): 402.000/358.000 Da ID: OTA1 from Sample 6 (1138-1-5.26) of DataSET1.wiff (Turbo Spray) Max. 180.0 cps.

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 9.5Time, min

0

50

100

150

180

In

te

ns

it

y,

c

ps

6.94

7.55 8.518.157.91 9.208.72

Hnh 3.10: Sc mu trng

XIC of -MRM (4 pairs): 368.000/324.000 Da ID: OTB1 from Sample 12 (Spike 2.5) of DataSET1.wiff (Turbo Spray) Max. 1265.0 cps.

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 9.5Time, min

0

1000

2000

3000

4000

5000

5827

In

te

ns

it

y,

c

ps

7.26

6.428.39 8.486.87

XIC of -MRM (4 pairs): 368.000/324.000 Da ID: OTB1 from Sample 12 (Spike 2.5) of DataSET1.wiff (Turbo Spray) Max. 1265.0 cps.

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 9.5Time, min

0

200

400

600

800

1000

1200

In

te

ns

it

y,

c

ps

7.26

6.42

8.398.486.87

XIC of -MRM (4 pairs): 402.000/358.000 Da ID: OTA1 from Sample 12 (Spike 2.5) of DataSET1.wiff (Turbo Spray) Max. 5827.0 cps.

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 9.5Time, min

0

1000

2000

3000

4000

5000

5827

In

te

ns

it

y,

c

ps

6.66

Hnh 3.11: Sc mu trng thm chun 2,5 ng/ml


53

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

Gii hn pht hin LOD c xem l nng thp nht ca cht phn tch

m h thng phn tch cn cho tn hiu phn tch khc c ngha vi tn hiu ca mu

trng hay tn hiu nn.

xc nh gii hn pht hin da trn lch chun, chng ti tin

hnh lm trn nn mu th. Lm 10 ln song song, cn chnh xc khong 5 g trng

bt ng cho vo ng ly tm 50 ml, thm chun cc cht nng 0,5 ng/ml (gp 5

ln LOD c lng, LOD c lng l: 0,1 ng/ml). Thc hin tin hnh x l mu

theo quy trnh ti u ha trn.

Tnh LOD: Tnh gi tr nng trung bnh xtb, lch chun SD (S)

LOD = 3.S

1

1

2

N

xx

S

N

i

i

nh gi LOD tnh c ta kim tra qua gi tr R = xtb

LOD

Nu 4 < R < 10 th nng dung dch em th l ph hp v gi tr LOD

tnh c l ng tin cy.

Gii hn nh lng LOQ c xem l nng thp nht m h thng phn

tch nh lng c vi tn hiu phn tch khc c ngha nh lng vi tn hiu

ca mu trng hay tn hiu nn. Theo l thuyt thng k trong ha phn tch th LOQ

= 3,33 LOD.

XIC of -MRM (4 pairs): 368.000/324.000 Da ID: OTB1 from Sample 4 (ruou-01) of DataSET1.wiff (Turbo Spray) Max. 620.0 cps.

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 9.5Time, min

0

200

400

600

In

te

ns

it

y,

c

ps

6.38

6.98

7.86

XIC of -MRM (4 pairs): 368.000/324.000 Da ID: OTB1 from Sample 4 (ruou-01) of DataSET1.wiff (Turbo Spray) Max. 620.0 cps.

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 9.5Time, min

0

200

400

600

In

te

ns

it

y,

c

ps

6.38

6.98

7.86

XIC of -MRM (4 pairs): 402.000/358.000 Da ID: OTA1 from Sample 4 (ruou-01) of DataSET1.wiff (Turbo Spray) Max. 247.0 cps.

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 9.5Time, min

0

50

100

150

200

247

In

te

ns

it

y,

c

ps

6.59

7.67

8.766.44 9.02

Hnh 3.12: Sc ochratoxins ti LOD


54

Bng 3.14: Kt qu xc nh LOD, LOQ ca Ochratoxin A, B

KQ OTA OTB

0,42 0,42

0,48 0,38

0,39 0,47

0,41 0,39

xi(ng/ml) 0,46 0,38

0,42 0,36

0,46 0,39

0,41 0,37

0,39 0,41

0,47 0,46

xtb(ng/ml) 0,431 0,45

SD 0,03 0,03

LOD (ng/ml) 0,1 0,1

LOQ (ng/ml) 0,3 0,3

R 4,2 4,0

RSD(%) 7,7 8,2

Nhn xt: T bng kt qu thu c nhn thy cc cht u c gi tr R tha

mn yu cu, hn na gi tr RSD (%) thu c cng tha mn theo AOAC nn

phng php p dng cng c chm t yu cu. V vy cc gi tr LOD, LOQ

thu c bng trn l ng tin cy.

3.3.3 Xc nh khong tuyn tnh

xc nh khong tuyn tnh chng ti thc hin o cc dung dch chun

c nng thay i t 1 ng/ml n 1000 ng/ml v kho st s ph thuc ca tn

hiu vo nng . Sau v ng cong ph thuc gia din tch pic thu c vo


55

nng , quan st s ph thuc cho n khi khng cn tuyn tnh. Kt qu thu c

nh sau:

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

Nng (ng/ml) 1 10 50 100 200 300 500 1000

Din tch OTA 894 5640 27600 55400 113000 173000 278000 335000

Din tch OTB 860 9050 48600 95500 185000 286000 474000 691000

Hnh 3.13: th biu din s ph thuc ca din tch pic vo nng OTA.


56

Hnh 3.14: th biu din s ph thuc ca din tch pic vo nng OTB.

T cc th ta thy gii hn tuyn tnh (LOL) ca OTA, OTB l 500 ng/ml

Nh vy t cc kt qu trn chng ti c th ch ra khong tuyn tnh ca

cc cht l 1 ng/ml-500 ng/ml.

3.3.4 Xy dng ng chun.

Lp dy chun c nng t 1ng/ml n 500 ng/ml

Hnh 3.15: ng chun ca Ochratoxin A


57

Hnh 3.16: ng chun ca Ochratoxin B

Nhn xt: Nhn thy cc h s hi quy tuyn tnh r ca cc ng chun trn

u nm trong khong: 0,995 r 1. Do vy cc ng chun dng trn u

chp nhn c.

3.3.5 chnh xc (accuracy) ca phng php phn tch ( ng v

chm) [11]

chm hay lp li l mc gn nhau ca cc gi tr ring l ca cc

php o lp li. chm l mt khi nim nh tnh v c biu din nh lng

bng lch chun S hay h s bin thin CV(%).:

CV(%) = 100.x

S

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.


58

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 chch (Bias) hoc hiu sut thu hi (recovery)

R(%) = C

Cc .100

Trong :

R: thu hi (%)

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

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

xc nh chm v ng ca phng php phn tch, chng ti tin

hnh th nghim nh sau: Tin hnh th nghim lp li trn nn mu trng ( mu

ng c phn tch khng cha cht cn phn tch) thm chun 3 mc nng

khc nhau (mi mc lm lp li 6 ln). C th thm chun hn hp 3 mc l 2,5

ng/ml , 5 ng/ml , 10 ng/ml. Kt qu thu c nh sau:


59

Bng 3.16: Kt qu nh gi chnh xc ca phng php

Cspike Ln

OTA OTB

xi x SD RSD% R% xi x SD RSD% R%

2,5

1 2,3

2,1 0,2 9,1 85

1,9

1,9 0,2 11 77

2 1,8 2,1

3 2 2,2

4 2,2 2

5 2,1 1,8

6 2,3 1,6

5

1 3,9

4,2 0,3 7,6 84

3,8

4 0,3 7,2 79

2 4,5 3,6

3 4,2 3,7

4 3,8 4,2

5 4,6 4,1

6 4,1 4,3

10

1 8,6

8 0,7 8,9 80

7,4

7,6 0,7 9,0 76

2 7,4 6,9

3 6,9 8,5

4 8,5 8,2

5 8,1 7,6

6 8,6 6,8

Nhn xt: Theo qui nh ca Chu u 2002/657/EC [27], ng v

chm ph thuc nng cht nh sau:


60

Bng 3.16: Qui nh ng v chm ca cc phng php nh lng ph

thuc nng cht theo 2002/657/EC

Theo 2 bng trn, ti cp t 10 100 g/kg, h s bin thin CV 20%;

hiu sut thu hi ti cp t 1 10 g/kg phi nm trong khong 70 110%. Nh

vy, phng php c ng v chm t yu cu ca Chu u.


61

Sample Name Sample ID Sample Type File NameAnalyte Peak Area (counts)

Analyte Peak Height (cps)

Analyte Concentration

(ng/mL)

Standard Query Status

Use RecordRecord Modified

Calculated Concentration

(ng/mL)

Accuracy (%)

1 Std 0.5 Standard 2013\T7\11\DataSE 1.34e+003 3.50e+002 0.500 N/A 0.493 98.6

2 Spike 2.5 Quality Control 2013\T7\11\DataSE 2.80e+003 7.38e+002 2.50 N/A 2.34 93.4

3 Std 5 Standard 2013\T7\11\DataSE 5.02e+003 1.19e+003 5.00 N/A 5.14 103.

4 Std 10 Standard 2013\T7\11\DataSE 8.77e+003 1.81e+003 10.0 N/A 9.87 98.7

5 M1-5.98 Unknown 2013\T7\11\DataSE 0.00e+000 0.00e+000 N/A N/A No Peak N/A

Sample Name: "Std 0.5" Sample ID: "" File: "DataSET1.wiff"Peak Name: "OTA1" Mass(es): "402.000/358.000 Da"Comment: "" Annotation: ""

Sample Index: 1

Sample Type: Standard

Concentration: 0.500 ng/mL

Calculated Conc: 0.493 ng/mL

Acq. Date: 7/11/2013

Acq. Time: 1:30:06 PM

Modified: Yes

Proc. Algorithm: Specify Parameters - MQ III

Noise Percentage: 50

Base. Sub. Window: 1.00 min

Peak-Split. Factor: 2

Report Largest Peak: Yes

Min. Peak Height: 0.00 cps

Min. Peak Width: 0.00 sec

Smoothing Width: 0 points

RT Window: 30.0 sec

Expected RT: 6.93 min 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0Time, min

0

100

200

300

400

In

te

ns

it

y,

c

ps

7.15

6.52

5.48

9.397.905.204.80 8.24

Sample Name: "Spike 2.5" Sample ID: "" File: "DataSET1.wiff"Peak Name: "OTA1" Mass(es): "402.000/358.000 Da"Comment: "" Annotation: ""

Sample Index: 2

Sample Type: QC



Acq. Date: 7/11/2013


Modified: Yes









RT Window: 30.0 sec


0

100

200

300

400

500

600

700

800

In

te

ns

it

y,

c

ps

7.06

7.37

6.709.53

8.065.65 5.853.43 5.37 8.35

Sample Name: "Std 5" Sample ID: "" File: "DataSET1.wiff"Peak Name: "OTA1" Mass(es): "402.000/358.000 Da"Comment: "" Annotation: ""

Sample Index: 3




Acq. Date: 7/11/2013


Modified: Yes









RT Window: 30.0 sec


0

200

400

600

800

1000

1200

In

te

ns

it

y,

c

ps

6.90

7.21

6.539.406.025.77

Sample Name: "Std 10" Sample ID: "" File: "DataSET1.wiff"Peak Name: "OTA1" Mass(es): "402.000/358.000 Da"Comment: "" Annotation: ""

Sample Index: 4




Acq. Date: 7/11/2013


Modified: Yes









RT Window: 30.0 sec


0

500

1000

1500

In

te

ns

it

y,

c

ps

6.97

6.596.41

Untitled 4 (OTA1): "Linear" Regression ("1 / x" weighting): y = 792 x + 951 (r = 0.9998)

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 10.0Concentration, ng/mL

0

5000

9000

Ar

ea

,

co

un

ts

Hinh3.17: Sc OTs tai mc thm chun 2,5 ng/ml

Nh vy phng php nghin cu trn c th p dng vo thc t xc

nh Ochratoxin A, B trong i tng mu thc t.

3.4. Phn tch mu thc t

Sau khi nghin cu cc iu kin tch chit, chng ti tin hnh phn tch

trn cc mu thc khc nhau. Cc mu chng ti tin hnh kho st bao gm: ng

cc v cc sn phm ch bin t ng cc v cc loi ru ln men. Cc mu c

ng nht, sau c em cn v tin hnh phn tch nh qui trnh ti u ha

trn. Mi mu tin hnh lm lp li 3 ln , ly kt qu trung bnh v tnh lch

chun.

Chng ti tin hnh phn tch trn 20 mu ru nhp ngoi ln men t

hoa qu, xut x Chi L, Php, Colombia v cc mu ng, lc ly ngu nhin trn

a bn cc tnh Bc Giang, Thanh Ha, Ngh An. Kt qu pht hin mt s mu c

cha ochratoxin A, B, tuy nhin u di ngng cho php. Hiu sut thu hi cc

mu thm chun u nm trong khong 70-110%.


62

Kt qu phn tch cc mu nh sau:

Bng 3.17: Kt qu phn tch mu thc t.

STT i tng m (g)

/V(ml)

Din tch

OTA

Din tch OTB

C-OTA

(ng/g.

ng/ml)

C-OTB

(ng/g.

ng/ml)

1 Ru L'estaminet

- Php 5 KPH KPH KPH KPH

2 Ru Raiz - Chi L 5 KPH KPH KPH KPH

3 Ru Mirabeau-

Php 5 KPH KPH KPH KPH

4 Ru Piraat - B 5 KPH KPH KPH KPH

5 Ru Bordeaux

-Php 5 KPH KPH KPH KPH

6 LN-BG-01 5,12 KPH KPH KPH KPH


8 LN-BG-03 5,23 3500 KPH 3,9 KPH

9 LC-BG-04 6,12 KPH KPH KPH KPH


Documents

Xác định Ochratoxin trong thực phẩm bằng phương pháp sắc ký lỏng khối phổ (LC-MS/MS)