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I HC QUC GIA H NI

TRNG I HC KHOA HC T NHIN


____________________

inh ng Huy

NGHIN CU NH LNG C T SINH HC BIN ASP


TRONG THY SN V SN PHM THY SN BNG PHNG PHP
SC K LNG GHP KHI PH TANDEM LC-MS/MS

LUN VN THC S KHOA HC

H Ni - Nm 2009

I HC QUC GIA H NI
TRNG I HC KHOA HC T NHIN
_____________________

inh ng Huy

NGHIN CU NH LNG C T SINH HC BIN ASP


TRONG THY SN V SN PHM THY SN BNG PHNG PHP
SC K LNG GHP KHI PH TANDEM LC-MS/MS

Chuyn ngnh

: Ho phn tch

M s

: 60 44 29

LUN VN THC S KHOA HC

NGI HNG DN KHOA HC


GS.TS. PHM HNG VIT

H Ni - Nm 2009

LI CM N
Bn lun vn ny c thc hin v hon thnh ti Trung tm Cht
lng Nng lm thy sn vng 4, 30 Hm Nghi Qun 1, Thnh ph HCM
vi s hng dn ca GS.TS. Phm Hng Vit.
Vi lng bit n su sc, ti xin trn trng cm n GS.TS. Phm Hng
Vit hng dn v gip ti hon thnh lun vn ny.
Ti xin chn thnh cm n TS. Dng Hng Anh hng dn, gip
ti trong qu trnh nghin cu.
Xin chn thnh cm n Ban lnh o Trung tm Cht lng Nng lm
thy sn vng 4, Tp th phng kim nghim Trung tm vng 4, bn b v
ng nghip quan tm gip ti trong thi gian va qua.
Xin chn thnh cm n Ban lnh o Trng, Khoa Ha hc, cng cc
thy c Trng i hc Khoa hc T nhin tn tnh ch dy v hng dn
trong sut qu trnh hc v lm lun vn.

H Ni, thng 12 nm 2009


Hc vin

inh ng Huy

MC LC
M U..........................................................................................................................1
Chng 1 - TNG QUAN...............................................................................................4
1.1. Hin tng thy triu ...................................................................................4
1.2. c t sinh hc bin trong thy sn..................................................................6
1.3. i cng v axt domoic (DA)........................................................................8
1.3.1. Tnh cht ha l. .......................................................................................8
1.3.2. Ngun tch t DA trong nhuyn th: ........................................................8
1.3.3. c tnh ca DA .......................................................................................9
1.4. Mt s phng php phn tch DA ...................................................................9
1.4.1. Phng php sinh ha trn chut............................................................10
1.4.2. Phng php sc k lng (LC-UV, LC-DAD, LC-FLD, LC-MS/MS)..11
1.5. u v nhc im ca cc phng php dn n vic s dng phng
php LC-MS/MS trong phn tch DA.............................................................12
1.5.1. Phng php sinh ha trn chut............................................................12
1.5.2. Phng php sc k lng ........................................................................12
1.6. i cng v sc k lng hiu nng cao ghp khi ph [2], [5] ........................12
1.6.1. Mt s nh ngha v phng trnh c bn .............................................13
1.6.2. Nhng thnh phn c bn ca h thng LC-MS/MS (Waters) ..............15
1.6.3. Loi hp cht ph hp phn tch bng sc k lng ................................23
1.6.4. Cc yu t nh hng n qu trnh tch ca cc cht trong ct............23
1.7. K thut chun b mu cho phn tch sc k...................................................26
1.7.1. Chit lng - lng: ....................................................................................27
1.7.2. Chit pha rn SPE: ..................................................................................27
Chng 2 NI DUNG V PHNG PHP NGHIN CU ..................................29
2.1. Ni dung nghin cu ca ti.......................................................................29
2.2. M hnh thc nghim ......................................................................................29
2.3. Thit b, dng c, ho cht: .............................................................................29
2.3.1. Thit b, dng c: ....................................................................................29
2.3.2. Thuc th, ha cht: ...............................................................................30
2.4. Thng tin v mu nghin cu:.........................................................................30
2.5. Xc nh cc thng s ti u:..........................................................................31
2.5.1. Xc nh cc thng s ti u cho MS .....................................................31
2.5.2. Ct:..........................................................................................................31
2.5.3. Pha ng v ch gradient: ..................................................................32
2.5.4. Dung mi chit:.......................................................................................32
2.5.5. Thit lp bng mu: ................................................................................32
2.5.6. Tnh ton : ...............................................................................................33
2.5.7. Kho st khong tuyn tnh: ...................................................................33
2.5.8. Gii hn pht hin ca phng php: .....................................................33
2.5.9. lp li ca phng php: ...................................................................34
2.5.10. thu hi ca phng php:................................................................34
2.5.11. Thc nghim xc nh DA trn mu nhuyn th..................................35
Chng 3- KT QU V BN LUN........................................................................36
3.1. Xc nh cc thng s ti u:..........................................................................36
3.1.1. Xc nh cc thng s ti u ca MS/MS ..............................................36
i

3.1.2. Pha ng v chng trnh chy gradient. ...............................................40


3.1.3. Dung mi chit:.......................................................................................45
3.2. Khong tuyn tnh: ..........................................................................................47
3.3. Gii hn pht hin ca phng php: .............................................................49
3.4. lp li v thu hi ca phng php: .....................................................49
3.5. Thc nghim xc nh DA trn nhuyn th. ...................................................51
3.6. nh gi tin cy ca phng php:............................................................52
3.7. Qui trnh phn tch Axt domoic. ....................................................................54
3.7.1. Phm vi p dng: ....................................................................................54
3.7.2. Nguyn tc: .............................................................................................54
3.7.3. Thit b, dng c, ha cht, dung dch:...................................................54
3.7.4. Chun b mu: .........................................................................................57
3.7.5. Tin hnh th nghim: ............................................................................58
3.7.6. m bo cht lng ................................................................................60
3.7.7. Tnh ton kt qu: ...................................................................................60
KT LUN ....................................................................................................................61
TI LIU THAM KHO..............................................................................................62
PH LC 1: CNG THC CU TO C T SINH HC BIN...................1
PH LC 2: MT S SC K TIU BIU KHI TI U ...........................4
PH LC 3. NG BIU DIN TUYN TNH ......................................13
PH LC 4. SC K CHY MU NHUYN TH 2 MNH V ...........19
PH LC 5. SC K PHN TCH MU NHUYN TH 2 MNH V
.BNG HPLC UV ........................................................................29

ii

DANH MC CC CH VIT TT
Ch vit tt
DA
DAD
EU
FA
FLD

Ting Anh
Axt domoic
Diot Array Detector
European
Formic acid
Fluorescence Detector

Ting Vit
Axt Domoic
u d Diot Array
Lin minh Chu u
Axt Formic
u d hunh quang

HPLC

High Performance Liquid


Chromatography
Liquid Chromatograph
Tandem Mass Spectrometer
Limit of Detection
Limit of Quantitative
Normal Phase Liquid
Chromatography
Not detected
Reversed Phase Liquid
Chromatography
Solid Phase Extraction
Trifluoroacetic acid
Ultra Violet
Visible

Sc k lng hiu nng cao

LC-MS/MS
LOD
LOQ
NPLC
ND
RPLC
SPE
TFA
UV
VIS

iii

Sc k lng ghp 2 ln
khi ph
Gii hn pht hin
Gii hn nh lng
Sc k lng pha thun
Khng pht hin
Sc k lng pha o
Chit pha rn
Axt Trifluoro axetic
Cc tm
Nhn thy

DANH MC CC BNG
Bng

Din gii

Trang

Bng 01. Mt s dung mi s dng trong HPLC .....................................................26


Bng 02. Chi tit mu thc nghim ..........................................................................30
Bng 03. Kt qu kho st gi tr hiu in th mao qun........................................36
Bng 04. Kt qu kho st gi tr hiu in th cone................................................37
Bng 05. Kt qu kho st nng lng va chm.......................................................38
Bng 06. Cc Ion th cp v cc iu kin ti u ca nng lng va chm ............39
Bng 07. iu kin gradient 1 pha ng 1.............................................................40
Bng 08. Bng gradient 2 pha ng 1....................................................................41
Bng 09. Bng gradient 1 pha ng 2....................................................................42
Bng 10. Bng gradient 2 pha ng 2....................................................................43
Bng 11. Bng gradient 3 pha ng 2....................................................................44
Bng 12. So snh cng tn hiu ion 266,25 nng 2 ppm ...........................47
Bng 13. Bng tng hp thng s v tuyn tnh theo cc ion: Error! Bookmark
not defined.
Bng 14. Kt qu xc nh gii hn pht hin ca phng php ............................49
Bng 15. Kt qu phn tch lp li v thu hi ca phng php. ...................49
Bng 16. Kt qu phn tch mu nhuyn th (tnh trn Ion 266,25) ........................51
Bng 17. So snh kt qu phng php HPLC-UV v LC-MS/MS.........................53

iv

DANH MC CC HNH V, TH
Hnh

Din gii

Trang

Hnh 01. S n gin ca h thng sc k lng...................................................16


Hnh 02. S van cao p v tr np......................................................................17
Hnh 03. S van cao p v tr tim ....................................................................17
Hnh 04. Pha tnh ca ct sck pha o. .................................................................18
Hnh 05. Pha tnh ca ct sc k pha thun. .............................................................19
Hnh 06. B kt ni phun in t..............................................................................20
Hnh 07. B ion ha ha hc.....................................................................................20
Hnh 08. H thng u d t cc ..............................................................................22
Hnh 09. Nguyn l hot ng ca u d MS/MS ..................................................23
Hnh 10. kh nng tch ca ct C8 v C18...............................................................24
Hnh 11. nh hng ca pH dung mi n kh nng tch ca cht.........................24
Hnh 12. nh hng ca phn cc dung mi i vi qu trnh sc k. ..............25
Hnh 14. M hnh thc nghim .................................................................................29
Hnh 15. Sc k ng vi gi tr ti u Capillary = 2 KV.....................................37
Hnh 16. Sc k ti u Ion m ng vi gi tr ti u cone volt = 30 V ...............38
Hnh 17. Cch phn mnh ion ca DA .....................................................................39
Hnh 18. Sc k iu kin Collision energy 15 eV...........................................40
Hnh 19. Sc k chy chun DA gradient 1 pha ng 1 ....................................41
Hnh 20. Sc k chy chun DA gradient 2 pha ng 1 ....................................42
Hnh 21. Sc k chy chun DA gradient 1 pha ng 2 ....................................43
Hnh 22. Sc k chy chun DA gradient 2 pha ng 2 ....................................44
Hnh 23. Sc k chy chun DA gradient 3 pha ng 2 ....................................45
Hnh 24. Sc k chy mu chit bng dung mi MeOH:H20: 1:1 ......................46
Hnh 25. Sc k chy mu chit bng dung mi Formic: metanol:H20: 2:5:93..46
Hnh 26. Sc k chy mu chit bng dung mi MeOH: H20: 2:1 .....................47
Hnh 27. th biu din ng tuyn tnh .............................................................48
Hnh 28. Kt qu chy mu nghu ti Nam nh trn LC-MS/MS v HPLC-UV...53
Hnh 29. Kt qu chy mu CRM trn LC-MS/MS v HPLC-UV ..........................54

M U
Theo c tnh ca T chc nng lng (FAO), tng kim ngch xut
nhp khu cc sn phm thy sn trong nm 2008 ca th gii ln u tin
trong lch s vt 100 t USD. Mt na xut khu thy sn trn th gii
bt ngun t cc nc ang pht trin trong khi 80% nhp khu thuc v cc
nc pht trin. Xut khu rng t cc nc ang pht trin t mc 25,4 t
USD trong nm 2008. Cc sn phm t thy sn l mt ngun thu ngoi t
quan trng ti cc nc ang pht trin. Vit Nam, thy sn ngy cng
ng vai tr thit yu vo kim ngch xut khu ca Vit Nam. Sau hn 1 nm
gia nhp WTO, ngnh thy sn Vit Nam c mt bc tin nhy vt trong
cng tc xut khu thy sn, ch trong nm 2007 sn lng thy sn c nc
c t 3,9 triu tn vi kim ngch xut khu 3,75 t USD, trong sn
phm nhuyn th hai mnh v cng chim mt t trng ng k. n nm
2008, kim ngch xut khu thy sn nc ta vt ngng 4 t USD.
Mt trong cc th trng nhp khu ln ca ngnh thy sn Vit Nam
l Lin minh Chu u (EU). Theo quy nh ca y ban lin minh Chu u,
mt nc ngoi khi EU c php xut khu thy sn vo EU phi m
bo cc yu t: (i) H thng vn bn quy pham php lut v nng lc c quan
qun l an cht lng v sinh ton thc phm ca nc xut khu v EU l
tng ng. (ii) H thng phng kim nghim tham gia vo cng tc kim
tra, chng nhn cht lng m bo v sinh an ton thc phm ca nc xut
khu v EU tng ng nhau. (iii) Bt buc phi thc hin cc chng trnh
gim st d lng c hi trong thy sn nui v gim st iu kin m bo
v sinh vng thu hoch nhuyn th 2 mnh v. (iv) ng thi thy sn phi
c phn tch cc ch tiu theo quy nh ca EU trc khi xut khu cng
vi cc i hi nghim ngt v k thut phn tch.
Nh vy, thc hin chng trnh gim st iu kin m bo v sinh
vng thu hoch nhuyn th 2 mnh v l mt iu kin tin quyt gip Vit
Nam c php xut khu nhuyn th hai mnh v vo EU. Hai ni dung lin
1

quan n k thut ng vai tr chnh trong vic thc hin chng trnh ny l
nh danh, phn loi to c (cc loi to c c kh nng sinh c t) v
phn tch c t sinh hc bin (ASP- c t gy mt tr nh, DSP c t gy
tiu chy, PSP c t gy lit c).
Dng tn ti chnh ca c t gy mt tr nh (ASP) l axit Domoic c
cng thc cu to:

Ngnh thy sn hin nay ang rt cn c nhng qui trnh phn tch ph
hp theo yu cu ca cc th trng nhp khu gip c quan chc nng kim
sot cc ha cht c hi ni chung v c bit l cc c t c mi nguy gn
lin vi loi (c t sinh hc bin vi loi nhuyn th hai mnh v, Histamine
i vi h c thu ng). V vy, cn thit phi xy dng qui trnh phn tch
xc nh Axt domoic trong nhuyn nh 2 mnh v. Ngoi mt s nghin
cu ca mt s t chc khoa hc hoc tiu chun nc ngoi vi phng
php c s dng xc nh hm lng Axt domoic bng k thut HPLCUV v LC/MSn, phng php sinh ha trn chut th hin nay Vit nam cha
c tiu chun ring v phng php th cho loi c t ny. V vy vn
nghin cu, ci tin phng php c nghin cu trn th gii c th
p dng xc nh hm lng DA trong nhuyn th hai mnh v, ph hp vi
iu kin ca Vit nam (nn mu phn tch, thit b, ha cht mi trng )
l rt cn thit. N gip cc phng th nghim ng dng thc t gip c quan
chc nng kim sot cht ch d lng c t ny p ng yu cu xut
khu.
Vi khun kh lun vn thc s chuyn ngnh ha phn tch, chng ti
tp trung tm ra iu kin phn tch ti u trn thit b LC-MS/MS hin c ti
2

phng th nghim Nghin cu nh lng c t sinh hc bin ASP


(Axt domoic) trong thy sn v sn phm thy sn bng phng php sc
k lng ghp 2 ln khi ph v thc hin phn tch trn mt s mu nhuyn
th ti cc vng thu hoch ti Vit Nam.

Chng 1 - TNG QUAN


1.1. Hin tng thy triu

Trong cc h sinh thi thy vc, cc loi vi to l nhng sinh vt sn


xut s cp ng thi cn l ngun dinh dng ch yu ca nhiu loi ng
vt ph du, u trng tm cua, mt s loi thn mm n lc v c, v.v. Phn
ln cc loi vi to l c li cho cc sinh vt thy sinh. Tuy vy, mt phn nh
trong chng, vi khong 100 trong tng s trn 5.000 loi to ph du c
pht hin trn th gii thuc to khu (Bacillariophyta), to gip
(Dinoflagellata), cc to c roi khc v vi khun lam (Cyanobacteria) c cha
cc c t gy hi cho sinh vt khc.
Cc loi to v vi khun c cha cc sc t mu khc nhau, sng tri
ni, di nhng iu kin mi trng nht nh c th sinh trng thnh cc
qun th to ln vng ven b gy nn s i mu nc. S i mu nc t
nhin thnh mu , nu, vng nu nht (mu h phch) hoc xanh vng cc
vng nc rng ln din ra l kt qu ca s n hoa (Algal blooms) ca cc
loi vi to v vi khun lam trong thy vc. V d: mu c trng ca Bin
gy nn bi s n hoa ca vi khun lam Oscillatoria erythraeum c cha cc
sc t phycoerythrin ...
Thy triu l s sinh trng mnh m ca cc loi ph du no
gy ra lm i mu nc. Cc loi ny c th sn sinh ra c t gy cht tm,
c, thn mm v con ngi n phi cng b ng c v c th b t vong.
Hin tng n hoa ca to c hi (Harmful Algal Blooms) l cc s
kin m ti s tng mt ca mt hoc mt s loi to c hi t ti
mc c th gy nguy hi ti cc sinh vt khc.
Ngi ta chia hin tng n hoa ca to c hi thnh mt s loi sau:
a. Cc loi khng cha c t nhng khi n hoa lm thay i mu
nc; di nhng iu kin c bit chng hn nh trong cc vnh kn, to n
hoa c th tng n mt rt cao lm cht c v cc ng vt khng xng
sng c trong thy vc do cn kit oxy. Tiu biu trong nhm ny l cc
4

loi: Gonyaulax polygramma, Noctiluca scintillans (to gip), Trichodesmium


erythraeum (to lam).
b. Cc loi sn sinh ra cc c t mnh m ta c th pht hin c
thng qua chui thc n ti con ngi, gy nn mt lot cc chng bnh v
thn kinh v tiu ha, chng hn nh:
c t PSP (Paralytic Shellfish Poisoning) do cc loi to gip:
Alexandrium acatenella, A. catenella, A. cohorticula, ... sn sinh ra.
c t DSP (Diarrhetic Shellfish Poisoning) do cc loi to gip
Dinophysis acuta, D. acuminata, D. fortii, D. norvergica, ... sn sinh ra.
c t ASP (Amnesic Shellfish Poisoning) do cc loi to khu
Pseudonitzschia multiseries, P. pseudodelicatissima, P. australis, ... sn sinh
ra.
c t CFP (Ciguatera Fish Poisoning) do cc loi to gip ch yu
sng y Gambierdiscus toxicus, Ostreopsis spp, Prorocentrum spp, ... sn
sinh ra.
c t

NSP

(Neurotoxic

Shellfish

Poisoning)

do

cc

loi

Gymnodinium breve, Gymnodinium G. cf.breve (New Zealand), ... sn sinh


ra.
c t Cc c t to lam do cc loi to lam Anabaena circinalis,
Mycrocystis aeruginosa, Nodularia spumigena, ... sn sinh ra.
c. Cc loi khng c vi ngi nhng li c vi c v cc ng vt
khng xng sng (c bit trong cc h thng nui thm canh) do ph hy
hoc lm tc cc mang ca chng; bao gm cc loi to khu Chaetoceros
convolutus, to gip Gymnodinium mikimotoi,... gy nn.
Mt vi loi to c th gy c ngay mt thp cha lm thay i
mu nc, chng hn nh loi Alexandrium tamarense, cc c t PSP c
pht hin trong thn mm khi mt ca loi ny thp hn 103 t bo/lt,
trong khi cc to khc thng gy hi khi chng xut hin cc mt cao
hn, lm thay i mu nc v kt qu l gy nn thy triu . Loi

Gyrodinium aureolum gy cht c v cc ng vt y mt cao hn 107


t bo/lt (Andersen, 1996).
Nhng tc hi do to c hi n hoa gy nn l rt ln. Ngi ta
thng k c t thng 9/1988 n thng 3/1989 ti cc vnh Villareal,
Carigara v vng Samar (Philippin) c 45 ngi b ng c, trong c 6
ngi cht. vnh Manila t nm 1988 n nay c 672 trng hp b ng
c, trong c 101 ngi cht. Nm 1989 vnh False (Nam Phi) loi
Gymnodinium sp. n hoa lm cht khong 40 tn bo ng. Monte
Hermosa (Achentina) t ngy 11 n ngy 17/11/1995 to c n hoa lm
cht khong 45 triu con ngao (Mesoderma macroides).
Theo thng k ca Fukuyo (1992), cc loi to c hi n hoa bin
Seto Inland (Nht Bn) gy nn nhng thit hi v kinh t rt ln; c th l
t nm 1987 n nm 1991 khu vc ny xut hin 745 ln to n hoa
trong c 46 ln gy cht c hng lot vi tng s thit hi l 4.452 triu
yn, v.v.
Vit Nam, mt s ln to c hi n hoa lm thit hi v kinh t
c ghi nhn: vo thng 5, 6/1995 to Noctiluca scintillans n hoa khu vc
vnh Vn Phong Bn Gi thuc vng bin Khnh Ho lm cht khong
20 tn tm hm vi thit hi c tnh khong 6 t ng (Nguyen Ngoc Lam et
al., 1996).
1.2. c t sinh hc bin trong thy sn

a. c t sinh hc bin trong nhuyn th c ngun gc t sinh vt ph


du. Thng qua ng thc n ca nhuyn th, c t c tch t hoc
chuyn ha trong c th nhuyn th, bao gm:
c t gy lit c (paralytic shellfish poisonings - PSP): c khong 20
loi l cc dn xut ca saxitoxin. Liu lng gy cht cho ngi l t 0,1 ti
1 mg. Cc c t PSP bn vi nhit v c th tan trong nc.

c t gy tiu chy (Diarrheic shellfish poisoning DSP) l nhm c


t c khi lng phn t ln, bao gm axit okadaic, dinophysis,
pectenotoxins v yessotoxin.
c t gy mt tr nh (Amnesic shellfish poisoning - ASP) gy ln bi
domoic axit. Domoic axit thng c pht hin ti hm lng thp trong
rt nhiu loi to (Chondria armata, Alsidium corallinum v Digenea
simplex).
c t thn kinh (Neurotoxin Shellfish Poisoning - NSP) bao gm cc
ng phn ca brevetoxin. Cc phn t ny c th tan trong m.
Tt c cc loi nhuyn th (loi thn mm n thc n bng phng thc
mng lc) u c nguy c b nhim c t. Tuy nhin, chng thng thy
mt s loi sau: PSP thng c trong vm, nghu, s, ip; DSP thng c
trong vm, trai, ip; ASP thng ch c trong vm, NSP thng c trong
trai, vm.
b. Khi con ngi n nhuyn th 2 mnh v b tch t c t c th gy
ra nhiu triu chng nhng cc triu chng ny ty thuc vo loi c t, hm
lng c t trong nhuyn th v lng nhuyn th m chng ta n vo c
th. Cc triu chng:
Gy ra bi PSP: phn ln s nh hng n h thn kinh, bao gm: t lit
c, ni ngng, kh th, mm nga, st, mt mi.
Gy ra bi DSP: gy ra cc triu chng ri lon tiu ha, v d: nn ma,
tiu chy, au bng, i vi tr em th km theo cc du hiu nh st, au
u.
Gy ra bi ASP: tng t nh DSP c t ASP cng gy ra cc triu
chng nn ma, tiu chy, au bng ng thi km theo cc triu chng i
vi h thn kinh (ln, mt tr nh, tai bin mch mu, hn m)
Gy ra bi NSP: Gii phng Na+ trong qu trnh vn chuyn ion vo
trong t bo dn ti vic khng iu chnh c Na+ vo trong t bo lm
thay i c tnh ca t bo, gy ra s gii phng thn kinh ph hu
Acetylcholine gy co c.
7

1.3. i cng v axt domoic (DA)

1.3.1. Tnh cht ha l.


DA l cht bt mu trng, nng chy nhit 223-224oC, tan tt trong
nc (8mg/ml), tan t trong metanol (0.6mg/ml).
Tn y : ([2S-[2,3,4 (1Z,3E,5R)]]-2-Carboxy-4-(5-carboxy-1methyl-1,3-hexadienyl)-3- pyrrolidineacetic acid)
Cng thc phn t: C15H21NO6:
Cng thc cu to:

Trng lng phn t: 311.34 g/mol.


tan trong nc l 8 mg/ml 25oC, trong Metanol l 0.6 mg/ml.
Nhit nng chy t 223-224oC.
1.3.2. Ngun tch t DA trong nhuyn th:
DA c pht hin u tin t loi to ln Chondria pha nam
Nht Bn (Take moto and Daigo, 1958). Tuy nhin n nm 1987 n li bng
pht ti Canada do loi Pseudo-nitzschia sinh ra (Perl et all.., 1990).
Pseudo-nitzschia phn b rng khp trong nc bin trn th gii, c
kh hu m v kh hu lnh. To n hoa tng ln khi thi tit chuyn t ma
xun sang ma thu khi c ma ro v di do cht gip cho qu trnh tng
trng [B. Jeffery*, T. Barlow, K. Moizer, S. Paul, C. Boyle1]. Ngoi ra cng
vi vic tc gi v dng nc thp gip cho vic tng s tch t to trn
vng nc mt m.
DA c tm thy trong cc loi nhuyn th nh s (Cerastoderma
edule), cua (Cancer magister), nghu (Scrobicularia plana), trai (Mytilus

edulis), razor clams (Siliqua patula) and ip (Pecten maximus) [Wekell et al.,
1994; Rhodes et al., 1998; Vale and Sampayo, 2001].
DA b tch t trong nhuyn th do c ch n kiu mng lc nn c th
trc tip cc loi to c hoc cc vi sinh vt c hm lng DA b tch t
trong NT2MV, hm lng c t h thng tiu ha ca nhuyn th cao hn
so vi c tht, tc tch t DA trong nhuyn th cng rt khc nhau gia
cc loi nhuyn th [Vale and Sampayo, 2001; Hay et al., 2000], Kh nng
chuyn ha DA trong nhuyn th l rt thp.
Khi nhuyn th b tch t c t sinh hc bin, ta c th nui lu
(nui trong mi trng nc bin sch) th c th lm gim hm lng c
t. V d, theo nghin cu ca (Novaczek, 1992): 50% hm lng DA trong
loi vm xanh c ra gii trong vng 24 h nui lu, trong khi phi mt
86 ngy ra gii 50% DA trong razor clams (Siliqua patula) (Horner et al.,
1993)
1.3.3. c tnh ca DA
LD50 ca DA khong 2,3 mg/kg khi lng c th theo nghin cu ca
Iverson v cc cng s (1989).
Quy nh ca cc th trng nhp khu (Canada, M, Chu u, c...) v
Vit Nam quy nh mc gii hn ti a cho php trong nhuyn th l 20 g
DA/g tht nhuyn th (20ppm), khi kt qu phn tch vt mc trn s b nh
ch vng thu hoch v ly mu tng cng kim tra ch tiu ny.
1.4. Mt s phng php phn tch DA

Da vo tnh cht v cu trc ca Axt domoic (DA), hin nay trn th


gii c mt s phng php xc nh trong cc nn mu khc nhau (nc
bin, to, nhuyn th hai mnh v...) nh: sinh ha trn chut (mouse
bioassay), sc k lng vi u d UV-VIS, hunh quang (HPLC-UV/FLD)
hoc LC-MS/MS.

Tuy nhin ti Vit Nam, hin cha c mt tiu chun hoc phng php
no cp n vic phn tch DA ngoi tr mt s qui trnh t xy dng ca
mt s phng kim nghim.
Sau y l mt s phng php in hnh, ph hp p ng c phn
tch d lng DA trong thy sn v sn phm thy sn:
1.4.1. Phng php sinh ha trn chut
Pha long dung dch chun ASP mt s nng tng dn bng nc
v trng v tim vo mng bng mt s con chut, tim mi con 1 ml cho n
khi xc nh c nng gy cht chut trong khong thi gian t 5 n 7
pht. Lu : kim tra pH ca cc dung dch trc khi tim. pH ca dung dch
phi nm trong khong t 2-4, khng c ln hn 4,5.
T nng c chn, pha long thm hai nng mi bng cch
thm hay bt 1 ml nc v trng so vi th tch ban u. Th d nu 10ml
chun c pha long vi 25ml nc v trng th khi pha thm hai dung dch
mi s dng th tch nc v trng l 24ml v 26ml.
Tim mi nng chun pha long thu c bc trn vo mt
nhm gm 10 chut (10 chut/ mt nng ), mi con chut 1ml dung dch.
Xc nh thi gian gy cht chut tnh t thi im tim xong n thi im
tim chut p ln cui cng.
Lp li qui trnh trn 1 hoc 2 ngy sau vi dung dch chun lm
vic mi c chun b.
Tnh thi gian gy cht trung bnh ca mi nhm 10 chut ng vi mi
pha long. Loi b cc pha long c thi gian gy cht chut trung bnh
nh hn 5 pht hoc ln hn 7 pht. T thi gian gy cht chut thu c, tra
bng Sommers, xc nh hm lng c t tnh theo n v MU trong
1ml dung dch (MU/ml) ng vi mi pha long. Tnh h s chuyn i CF
(h s chuyn i gia lng c t tnh bng g v tnh bng MU) cho mi
pha long bng cch chia nng ca dung dch (g/ml) vi s n v c
t tnh theo MU va tra (MU/ml). n v ca CF s l g/MU
10

Tnh gi tr trung bnh ca cc h s CF thu c trn. S dng h s


CF ny trong tnh kt qu.
H s chuyn i CF cn c kim tra thng xuyn, vic kim xot
li h s chuyn i CF c thc hin bng cch chch vo 5 con chut dung
dch chun pha long sao cho thi gian gy chut cht nm trong khong
5-7 pht. Xc nh h s CF v so snh vi CF tham chiu xc nh c
trc . S sai lch phi nm trong khong 20 %. Nu s sai lch vt
khong ny, tim thm 5 con chut v b sung vo 5 con chut chch trc
c nhm chut gm 10 con, chn v chch thm nhm chut th hai
gm 10 con vi cng nng dung dch chun nh nhm 1. Tnh h s CF
ca 2 nhm chut v tnh CF trung bnh. H s CF thu c ny c xem
nh gi tr CF mi s dng trong tnh kt qu, v lch nm ngoi khong
20 % c kh nng do k thut hoc do s thay i p ng ca ging chut
i vi c t. S thay i ny i hi phi thay i h s CF [S tay phng
php ca Trung tm Cht lng Nng lm thy sn vng 4, 2009].
1.4.2. Phng php sc k lng (LC-UV, LC-DAD, LC-FLD, LCMS/MS)
Thng mu c ly trch bng mt dung mi hu c v c lm
sch bng cch cho qua ct chit pha rn (nu cn). Sau , dch chit c
lm sch v c ha tan trong dch pha ng tim vo h thng HPLC vi
cc u d UV, PDA, FLD hoc MS/MS. i vi u d hunh quang FLD,
sau khi qua ct phn tch DA c cho phn ng vi mt cht ph hp to
thnh hp cht c tnh cht hunh quang trc khi vo u d.
Da vo i lc ca cc cu t gia pha tnh (pha o hoc pha thun)
thng l cht rn v pha ng (lng) m mi cu t s c ra gii ra khi
ct theo th t khc nhau. Cu t ra khi ct c a vo u d UV,
PDA/DAD (Photo Diot Array detecter) hoc u d khi ph MS/MS. i
vi u d UV hay DAD bn cnh vic pht hin bi tn hiu b dung mi
cha cu t ra gii hp th mt bc sng nht nh nh lng. i
11

vi u d khi ph cu t ra khi ct c a vo u d MS v y cu
t c ion ha, phn mnh v c pht hin da vo thng s m/z. Trn c
s ny nn u d MS c th nh danh v nh lng mt cch r rng,
khng nh v mt hp cht no .
1.5. u v nhc im ca cc phng php dn n vic s dng
phng php LC-MS/MS trong phn tch DA

1.5.1. Phng php sinh ha trn chut


Phng php sinh ha trn chut c u im l thc hin nhanh, n
gin, d lm, c th thc hin vi s lng mu ln, u t ban u t (v
nhn lc, thit b, ha cht, dng c), i vi phn tch c t th phng
php sinh ha trn chut ang c chn l mt trong cc phng php kim
khng nh. Tuy nhin i vi cc loi c t c nhiu ng phn th khng
th phn bit c hm lng ca tng loi c t (v d DSP, PSP c nhiu
hp cht cng nhm). Ngoi ra, ti mt s nc lut php khng cho php
lm cc th nghim trn ng vt.
1.5.2. Phng php sc k lng
Phng php sc k lng c u im l mt phng php kh nhy v
c tnh chn lc rt cao, c th nh lng v khng nh ngay m chi ph
phn tch mc trung bnh. Tuy nhin phng php li kh c th pht trin
phn tch thm cc nhm cht c t sinh hc bin khc nh DSP, PSP vi
mc gii hn cho php thp (ppb).
Vi nhng tn ti ca cc phng php trn i vi DA th vic cn c
mt phng php khc nh phng php LC-MS/MS ring cho DA v c th
pht trin nh lng DSP, PSP cng vi vic d dng p dng cho hu ht
cc phng kim nghim v an ton thc phm v p ng yu cu ra
ca cc tiu chun quc t cng nh yu cu trong nc l v cng cn thit.
1.6. i cng v sc k lng hiu nng cao ghp khi ph [2], [5]

12

Sc k lng l mt trong nhng k thut phn tch dng c lin quan


n qu trnh tch cc cht khc nhau trong cng mt mu ra khi nhau. K
thut ny cho php ngi phn tch nhn danh v nh lng tng cu t
trong mu thng qua cc dung dch chun v ph tng ng ca chng.
1.6.1. Mt s nh ngha v phng trnh c bn
1.6.1.1. Chiu rng pic
Chiu rng pic c o bng cch m rng hai bn pic xung ng
nn v o chiu rng ng nn. Tuy nhin, ngi ta thng o ti bn
chiu cao picv thc hin d dng v c tin cy cao hn.
1.6.1.2. Thi gian lu (tr) v thi gian lu hiu chnh (tr)
Thi gian lu l tng thi gian hp cht trong pha ng v pha tnh, c
n v o thng dng l pht. Ti mt nhit ct v tc dng c nh,
mi hp cht tn cng mt thi gian trong pha ng. Thi gian trong pha
ng c xc nh bng cch tim mt hp cht khng b lu li vo h
thng HPLC v o thi gian lu ca n. Thi gian lu ny c gi l thi
gian cht ca ct v biu th l tm hoc to.
Bi v mi hp cht tiu tn cng thi gian trong pha ng, s khc
nhau trong thi gian lu l do bi s khc nhau thi gian tiu tn trong pha
tnh. Nu ly thi gian lu ca mt hp cht tr thi gian cht ca ct (tm) ta
s c c thi gian tiu tn tht s ca mt hp cht trong pha tnh, gi tr
ny c gi l thi gian lu hiu chnh (tr) v c tnh bi:
tr = tr - tm (0.1)
Trong :
tr : thi gian lu
tm : thi gian ca pic khng b gi li hay thi gian cht ca ct
1.6.1.3. T s phn b
T s phn b k hay h s cha (k) cho bit thi gian mt hp cht
nm trong pha tnh bao lu so vi hp cht khc. Nu k = 0 th ta ni l
hp cht khng b lu gi.
13

k=

tr tm
(0.2)
tm

T s phn b phn nh ln tht s lu gi hp cht hn thi


gian lu.
1.6.1.4. Hng s phn b (KD)
Hng s phn b c nh ngha l t s nng ca hp cht
trong pha tnh v pha ng.
KD =

Cs
(0.3)
Cm

Cs: Nng hp cht trong pha tnh


Cm: Nng hp cht trong pha ng
Hng s phn b rt tin li m t v d on nhng thay i
kh nng lu gi da trn thay i kch c v nhit ct.
1.6.1.5. Hiu nng ct
Hiu nng tch ca ct c biu din bi s a l thuyt (n).
2

t
t
n = 5,545 r = 16 r
Wb
Wh

(0.4)

Wb = 1,699Wh: chiu rng pic ti y


tr : thi gian lu ca pic
Wh: Chiu rng pic ti na chiu cao (cng n v vi tr)
Ngoi ra cn dng s a l thuyt hiu dng N
2

t r'
t r'
N = 5,545 = 16 (0.5)
Wb
Wh

Mt i lng khc dng o hiu nng ca ct l tng ng


chiu cao ca mt a l thuyt (h hoc HETP).
h=

n : tng s a l thuyt
L : chiu di ca ct (mm)

14

L
(0.6)
n

1.6.1.6. H s tch ()
Cn c gi l chn lc, cho bit khong cch gia 2 pic.
=

k2
(0.7)
k1

k1: t s phn b ca pic gii hp trc


k2: t s phn b ca pic gii hp sau
1.6.1.7. phn gii (R)
phn gii dng nh gi kh nng tch gia 2 cht
t t
R = 1,18 r 2 r1
Wh1 Wh 2

(0.8)

tr1: thi gian lu ca pic 1


tr2: thi gian lu ca pic 2
Wh1: chiu rng bn chiu cao ca pic 1 (cng n v vi tr)
Wh2: chiu rng bn chiu cao ca pic 2 (cng n v vi tr)
Khi phn gii R 1,5 th 2 cht tch hon ton khi nhau.
1.6.2. Nhng thnh phn c bn ca h thng LC-MS/MS (Waters)
H thng gm 7 phn chnh:
H thng bm (Pump system)
H thng tim mu (Injection system)
Bung ct (Column ovent - iu chnh nhit ct)
Ct sc k (pha tnh stationary phase)
Pha ng (mobile phase (hn hp) dung mi)
u d khi ph (Detector)
H thng x l d liu

15

Hnh 01. S n gin ca h thng sc k lng


1.6.2.1. H thng bm
c im v yu cu ca mt h thng bm cao p:
- p sut c th t n 1000 bar (15.000 Psi)
- C th iu chnh tc dng trong khong 0,01 10 ml/min
- Th tch cht nh
- Tr vi dung mi ca pha ng
- C th trn 2 hay nhiu dung mi vi nhau
1.6.2.2. H thng tim mu:
a. c im v yu cu ca mt h thng tim mu:
- C tc dng tim mu
- T ng tt khi p sut cao
- C th bm tun hon mu
b. Cu to v s hot ng ca van cao p:
- V tr np: mu c np vo ng cha mu Hnh 02
- V tr tim: van cao p xoay 1 gc 60o v dung mi s y mu
sample loop theo ng ng v i vo ct (Hnh 03)

16

Hnh 02. S van cao p v tr np

Hnh 03. S van cao p v tr tim


1.6.2.3 Bung ct
Dng n nh nhit ct v dung mi qua ct trong qu trnh phn
tch
1.6.2.4. Ct sc k
L mt loi ct c bit c nhi y bng cc ht nhi (pha tnh), kch
thc ct: Di (L): 5-30cm, ng knh trong: 2-5mm, kch c ht: 1.7, 3, 5,
17

10m. C 2 loi ct thng dng trong sc k lng lag ct pha o v ct


pha thun.
a. Ct sc k lng pha o (Reversed Phase Liquid Chromatography RPLC):
Pha tnh gm cc phn t silic dioxyt (silica) c gn cc nhm chc
khng phn cc v cc nhm silanol (Hnh 04).
Cc cht phn tch c i lc khc nhau vi nhm chc khng phn cc:
- Cht khng phn cc: phn ln dng ct C18
- Cht phn cc: mt s dng ct C18
- Cht phn cc: a s dng ct C8

Hnh 04. Pha tnh ca ct sck pha o.


b. Ct sc k lng pha thun (Normal Phase Liquid Chromatography NPLC)
Pha tnh gm cc phn t silic dioxyt (silica) c gn cc nhm chc
phn cc bi s thay th mch (CH2)nCH3 bng cc gc phn cc nh gc
Cyano, Amino... (Hnh 05)

18

Hnh 05. Pha tnh ca ct sc k pha thun.


1.6.2.5. Pha ng
L cc dung mi hoc hn hp dung mi hu c hoc dung dch m,
yu cu:
- tinh khit cao (v d: nc/m vi MeOH/ACN)
- Khng phn ng vi cht phn tch v pha tnh
- Khng c bt kh, khng c bi (nh siu m, lc).
1.6.2.6. u d khi ph MS
u d MS c ni vi u ra ca ct sc k. Vic phn tch khi ph
(phn tch t l khi lng theo in tch (m/z) ca ion) da trn s bn ph
cc phn t hp cht hu c trung ha thnh cc ion phn t mang in tch
(+) hoc ph v thnh cc mnh ion, cc gc bng cc phn t mang nng
lng cao (nh va chm electron, ion ha ha hc, ion ha proton, bn ph
ion, ion ha trng...). Ion phn t v cc mnh ion l cc phn t c khi
lng t ngi ta tnh c s khi z=m/e (khi lng ion/in tch).
1.6.2.6.1. B kt ni phun in ( Electrospray Ionizaton : ESI)

19

Mao qun

Bung ha hi

Hnh 06. B kt ni phun in t


Nguyn tc ca phng php ion ha phun in l dng chy t my
sc k lng cao p (HPLC) ra vi tc 1-40ml/pht i qua 1 ng capillary c
ng knh c mm v in trng cao 1-6kV. Cht lng c phun ra di
dng ht rt nh mang in p sut thng. Cc ht ny i n in cc ca
ngun ion ha p sut kh quyn (API). Dng kh N2 thi vo lm mu bay hi
nhanh bin cc ht rt nh tr thnh cc nh trc khi a n bung ion ha.
1.6.2.6.2. Ion ho ho hc p sut kh quyn (Atmospheric Pressure
Chemical Ionization: APCI)
in cc phng in
B lc
p sut kh quyn

Chn khng cao

Cht lng
B phn phn tch
Bm chn khng

Gia nhit
Khng kh
kh

Kh nebulizer

Hnh 07. B ion ha ha hc

20

Hn hp kh ny n mt vng c p sut kh quyn, v xy ra s ion


ho ho hc khi va chm vi 1 dng ion dng (H2, CH4, H2O...) hoc m
bin cc phn t trung ha thnh cc ion phn t hay cc mnh ion qua s bn
ph bi dng electron mang nng lng cao do que Corona to ra. Mi phn
t kh c th to ra cc ion dng khc nhau lm tc nhn cho ion ha ha
hc.
Cht phn tch (M)
v hi dung mi
u kim dn mu
V ng dn kh
B phn phn
tch

Vng va chm
Kh nebulizer

Block lm bay hi
bng gia nhit

Vng phun in
(plasma)

Din gii vng phun


in (plasma)

u kim dn mu
(hiu in th cao)

Thit b m chng ti s dng phn tch y s dng khi ph t


cc (quadrupole mass spectrometer). Khi ph k t cc s dng 4 thanh trn
ngn t song song nhau thnh 1 b, hai u t trn 2 gi :

21

u d
Ion cng hng
Ion khng cng hng

Ngun

Hiu in th mt chiu
v xoay chiu

Hnh 08. H thng u d t cc


Tng cp i din, tch in m hay dng ca ngun in DC. Chng
lm cho cc phn t chuyn ng quanh trc trung tm. Do ch cc ion
nht nh mi ti c b gp, s thay i tn s v in th cp lm cho
cc ion c s khi khc nhau ln lt ti b phn thu gp.
K thut MS mt ln c mt s nhc im nh : khng nghin cu
c c ch phn mnh, s khc bit gia cc ng phn, xc nh thm chi
tit cu trc ho hc, do k thut ion ho nh nn khi ph ch cho thy
ion phn t K thut MS/MS (2 ln) khc phc c nhng im ny ng
thi tng thm nhy, chnh xc.
u d khi ph MS/MS hay my o khi ph hai ln lin tip gm hai
h my khi ph ring bit c lp nhau c ni lin vi nhau cch nhau bi
mt bung va chm ( collision cell ). MS u tin ( t cc Q1 ) c s dng
c lp ion m ( precursor ion ), ion ny lin ngay sau s b phn mnh
ti bung va chm collision cell cho ra nhng ion con ( product ion ), tip
theo MS th hai (t cc Q2 ) s phn tch nhng ion ny. Nhng ion mong
mun s i ti detector v chuyn thnh tn hiu.

22

B mt photon
va chm

Ion s cp

Ion th cp

Hnh 09. Nguyn l hot ng ca u d MS/MS


1.6.2.7. H thng thu nhn v x l d liu
L mt h thng bao gm h thng kt ni v thu nhn d liu th v
phn mm kim sot, iu khin thit b, qu trnh phn tch v tnh ton, x
l, bo co, kt xut d liu th va thu c.
1.6.3. Loi hp cht ph hp phn tch bng sc k lng
Cc hp cht phn cc, khng phn cc, nhng hp cht sinh hc,
dc liu d b phn hy nhit sc k kh v nhng hp cht c th phn
ng vi vt liu dng trong GC v ct tch ca GC.
1.6.4. Cc yu t nh hng n qu trnh tch ca cc cht trong ct
C 3 yu t chnh nh hng n qu trnh tch trong ct l:
- Tnh cht ha hc ca ct.
- Tnh cht ca dung mi pha ng.
- pH ca pha ng.
1.6.4.1. Tnh cht ha hc ca ct
Ct Silica C18 tch tt hn ct Silica C8 nhng thi gian lu ca C18
di hn v kch thc l xp ca ht ln hn ct C8 do c u k nc di
hn (18C so vi 8C) dn n s tng tc gia cht phn tch v pha tnh
trong loi ct ny cng ln hn so vi C8 (Hnh 1010).

23

ng knh ct cng nh, lng tiu tn dung mi cng t, nhng kh


ch to. Ct cng di, c ht cng nh th kh nng tch cng tt nhng thi
gian chy cng lu v p sut cng phi cao.

Hnh 10. kh nng tch ca ct C8 v C18.


1.6.4.2. Tnh cht ca dung mi pha ng
- pH ca pha ng:
pH ca pha ng cng nh hng n hiu qu tch gia cc cht. Tuy
nhin ty thuc vo bn cht ca cc cht phn tch m nh hng ca pH c
th lm tng hoc gim kh nng tch ca ct (Hnh 11).

Hnh 11. nh hng ca pH dung mi n kh nng tch ca cht


- phn cc ca dung mi
phn cc ca dung mi nh hng mnh n qu trnh sc k (Hnh
)
24

Hnh 12. nh hng ca phn cc dung mi i vi qu trnh sc k.

25

Bng 01. Mt s dung mi s dng trong HPLC


phn cc

Dung mi

Kh nng tan
trong nc

Khng phn cc

Hexan

Khng

Iso-octan

Khng

Petroleum ete

Khng

Cyclo-hexan

Khng

Cacbon tetra-hydrochlorit

Khng

Cloroform

Khng

Dicloro metan

Khng

Tetra-hydrofuran

Phn cc

Dietyl ete

Khng

Etyl axetat

Axeton

Axetonitril

Isopropanol

Metanol

Nc

Axt axetic

1.7. K thut chun b mu cho phn tch sc k

Trong cc mu sinh hc, mu mi trng thng c thnh phn rt phc


tp, hm lng cht cn phn tch kh thp nn khng tng thch cho vic
phn tch trc tip trn h sc k. Do vy, trc khi phn tch mu chng ta
cn phi tch, lm giu cc hp phn ca mu cn phn tch. C rt nhiu
phng php x l mu nh: lc, bay hi, lm kh, ly tm, chit lng-lng,
sc k ct, chit Soxhlet, SPE, xung siu m, vi sng.... nhng tt c cc
phng php ny u phi p ng c cc tiu ch:
- Lm sch mu mt cch chn lc.
26

- Tng nng ln nhiu ln.


- Lng mu khng cn nhiu.
- Lng dung mi cn t.
- thu hi cao, khng gy nhiu.
- n gin, nhanh, gi thnh thp.
Da trn cc mi lin h gia chn lc, c tnh dung mi, gi thnh,
thi gian... m chng ta chn phng php chit mu sao cho hiu qu nht.
1.7.1. Chit lng - lng:
Phng php chit lng - lng l phng php lm sch mu thng
dng, dng c thit b kh n gin nhng hiu qu chit kh cao.
Qu trnh chit lng - lng da trn nh lut phn b Nernst: bt c mt hp
phn trung tnh no cng s phn b gia hai dung mi khng trn ln vi t
s nng trong hai pha l mt hng s.
KA : hng s phn b

[A]hc
KA=

(0.9)
[A]n

[Ahc]: nng ca cht A trong pha hu c.


[An]: nng ca cht A trong pha nc.

1.7.2. Chit pha rn SPE:


Ngy nay, lm sch mu trong k thut sc k ngi ta thng s
dng phng php chit pha rn SPE (chim hn 50% cc phng php lm
sch mu).
Phng php chit pha rn ng dng cho nhiu nn mu (matrix) nh
cc loi mu mi trng, cc loi mu dc phm, mu sinh ha, mu hu c
v mu thc phm.
C 6 bc chnh trong qu trnh chit pha rn:
Bc 1. Chun b dch mu: Mu phi dng dung dch v tng tc c
vi cht hp ph. Dch mu khi cn thit phi c lc hoc ly tm trc khi
cho vo ct SPE trnh lm tc ct.

27

Bc 2. Hot ha ct: lm t pha rn, to mi trng thch hp cho vic


hp thu cht phn tch. Th tch dung mi cn s dng khong 1mL/100mg
cht hp ph. Nu th tch dung mi s dng t hn th tch quy nh ny s
lm tng nguy c pha rn khng c solvat ha hon ton, kt qu thu hi
ca mu thp.
Bc 3. Cn bng ct: Trc khi cho mu vo, ct phi c iu kin tng
ng vi iu kin chy ca mu (v d: pH) bng cch cho thm dung mi
c iu kin tng ng dung mi cha mu.
Bc 4. Np mu: mu c cho qua ct SPE. Tc dng chy ca mu
qua ct khong 3ml/pht.
Bc 5. Ra pha rn: dng dung mi thch hp loi cc tp cht ra khi
ct nhng vn gi li c cht cn phn tch.
Bc 6. Ra gii: s dng dung mi thch hp tch cht cn phn
tch ra khi ct, tc dng chy khi ra gii khng c qu nhanh. Tc
ny ph thuc vo ng knh ct v khi lng cht hp ph, ngi ta
thng ra vi tc khong 1ml/pht.
Qua cc phn tch nu trn, ta thy vic kim sot c t gy mt tr
nh ASP (axt domoic) trong thy sn ni chung v nhuyn th hai mnh v
ni ring m bo an ton v sinh thc phm l cn thit. V vy vic
nghin cu xy dng mt quy trnh phn tch axt domoic trong thy sn bng
phng php LC-MS/MS l cn thit p ng c yu cu kim sot an ton
thc phm ca Vit Nam cng nh cc th trng nhp khu trn th gii.

28

Chng 2 NI DUNG V PHNG PHP NGHIN CU


2.1. Ni dung nghin cu ca ti

- Kho st, ti u ha cc iu kin xy dng quy trnh phn tch


axt domoic trong thy sn trn thit b LC-MS/MS .
- Kho st cc thng s xc nh gi tr s dng ca phng php (gii
hn pht hin, thu hi, lp li, v so snh kt qu phn tch trn LCMS/MS vi phng php HPLC-UV).
- Thu thp v phn tch mt s mu nhuyn th 2 mnh v (NT2MV)
ti mt s vng thu hoch NT2MV 12 vng thu hoch NT2MV ti Vit
Nam.
2.2. M hnh thc nghim

Tin hnh thc nghim theo m hnh di y (Hnh 1):


Chn cc thng s
LC, MS/MS

Xy dng qui
trnh chy mu

Chn thnh phn


pha ng, gradient
Chn phng
php chit

Xy dng
phng php
Xy dng qui
trnh chit mu

Chn dung mi
chit

Kim tra
lp li

Kim tra
thu hi

Tm gii hn
pht hin

Xc nh
c hiu

Phn tch mt
s mu tht

Hnh 14. M hnh thc nghim


2.3. Thit b, dng c, ho cht:

2.3.1. Thit b, dng c:


2.3.1.1. Ct sc k lng C18, kch thc ct 250 x 4.6 mm, kch thc
ht 5 m (Merck- Licrocart 250-4 RP-18), c.
29

2.3.1.2. Ct sc k lng C8 kch thc ct 4,6x150mm kch thc ht


5m (Merck- Licrocart 150-4 RP-8), c.
2.3.1.3. My ng ha mu (KCH-1000), Trung Quc.
2.3.1.4. My ly tm (Sigma 4 K 15), M .
2.3.1.5. B nh siu m (Branson 2210), M.
2.3.1.6. Cn k thut (Sartorious, d= 0,1g), c.
2.3.1.7. Cn phn tch (Sartorious, d= 0,1mg), c.
2.3.1.8. My lc mu (IKA KS-260), c.
2.3.1.9. Transferpette 1ml (Eppendorf).
2.3.1.10. ng ly tm 50ml.
2.3.1.11. Mng lc mu 0.45m, : 25mm.
2.3.1.12. ng tim nha loi 5ml.
2.3.1.13. Giy lc.
2.3.1.14. Cc dng c thy tinh: bnh nh mc, ng ong, l cha mu;
cc c m,...
2.3.1.15. H thng LC-MS/MS, model Micromass API, hng Waters M
2.3.2. Thuc th, ha cht:
Tt c cc thuc th phi t tiu chun dng cho phn tch
2.3.2.1. Chun axt domoic (Sigma). Bo qun chun rn trong ngn ng
t lnh.
2.3.2.2. Nc tinh khit dng cho HPLC (nc HPLC), Merck c.
2.3.2.3. Dung mi: axetonitrile v metanol loi dng cho HPLC, Merck
c.
2.3.2.4. Axt Trifluoroacetic TFA) tinh khit phn tch, Merck c.
2.3.2.5. Axt Formic(FA) tinh khit phn tch, Merck c.
Cch thc pha ha cht, cht chun c thc hin theo mc 3.7
2.4. Thng tin v mu nghin cu:

Tin hnh thu thp 36 mu nhuyn th hai mnh v ti 12 vng thu


hoch trong c nc, chi tit theo ti Bng 02:
Bng 02. Chi tit mu thc nghim
TT

Tn mu

1.

Nghu Bn Tre

2.
3.

S mu
03

Nghu Bn Tre

03

Tu hi

03

Ni ly mu
Giao Thy Nam nh
Tin Hi Thi Bnh
Vn n - Qung Ninh
30

Thi gian ly
mu
20/7; 18/8;
09/11
20/7; 18/8;
09/11
20/7; 17/8;
09/11

Thi gian ly
mu
ip
03
28/7; 18/8;
4.
Phan Thit Bnh Thun
10/11
S anti
03
28/7; 18/8;
5.
Tuy Phong Bnh Thun
10/11
Nghu Bn Tre
03
28/7; 18/8;
6.
Bnh i Bn Tre
10/11
Nghu Bn Tre
03
28/7; 18/8;
7.
Ba Tri Bn Tre
10/11
S huyt
03
28/7; 18/8;
8.
Thnh Ph Bn Tre
10/11
Nghu Bn Tre
03
28/7; 18/8;
9.
Cn Gi - H Ch Minh
10/11
Nghu Bn Tre
03
28/7; 18/8;
10.
Tn Thnh Tin Giang
10/11
S Lng
03
20/7; 17/8;
11.
B La Kin Giang
09/11
Nghu la
03
20/7; 17/8;
12.
Hip Thnh Tr Vinh
09/11
o
Tt c cc mu c bo qun ng t nht -20 C cho n khi x l.
TT

Tn mu

S mu

Ni ly mu

2.5. Xc nh cc thng s ti u:

2.5.1. Xc nh cc thng s ti u cho MS


Chng ta cn ti u mt s thng s sau: Capillary, Cone volte,
Collision energy: thay i ln lt tng thng s trn, nguyn tc chn iu
kin ti u cho tng thng s nh sau:
- Capillary (in th mao qun) v Cone Volte (in th cone): ti gi
tr m cng pic ca ion s cp (ion m) l ln nht.
- Collision energy (nng lng va chm): ti gi tr ng vi cng
Pic ca mi mnh l ln nht. Mi mt Ion con s c mt gi tr Collision
energy ti u tng ng.
2.5.2. Ct:
la chn ct s dng, chng ti tin hnh nh sau: s dng dng
dch chun nng 5ppm v tin hnh kim tra kh nng tch v thi gian
lu. Trong khun kh lun vn ny chng ti ch thc hin kho st v kh
nng tch, thi gian lu trn ct C8 4,6x150mm 5m v C18 4,6x150mm
31

5m. Ct c chn phi l ct c kh nng phi m bo pic cn phn tch


trong sc k khng b chp vi cc pic nhiu khc, kh nng tch r rng,
pic cn i, t b dong pic, thi gian lu khng c qu di.
2.5.3. Pha ng v ch gradient:
Thay i v ti u thnh phn pha ng vi mu chun v mu thm
chun bng cch thay i thnh phn v t l pha ng: thc hin phn tch
mu thm chun ln lt vi pha ng. Chng ti la chn, nghin cu 02
pha ng sau:
- Pha ng 1: 0,1% FA trong H20 (A), 0.1%FA trong ACN
- Pha ng 2: 0.05% TFA trong H2O (A), 0,05% TFA trong ACN (B).
Pha ng c chn phi m bo pic cn phn tch trong sc k
khng b chp vi cc pic nhiu khc, kh nng tch r rng, t b dong pic,
thi gian lu khng c qu di.
2.5.4. Dung mi chit:
Sau khi chn c ct tch v thnh phn pha ng ph hp, s dng
phng php chit l chit lng lng vi 03 loi dung mi:
- Dung mi 1: MeOH:H20: 1:1
- Dung mi 2: Axt Formic: metanol:H2O: 2:5:93
- Dung mi 3: MeOH: H2O: 2:1.
Quy trnh chit: Cn chnh xc 2,0 g (m) mu ng ha trn cn
k thut vo ng ly tm. Thm chnh xc 8,0 ml dung mi chit mu. Lc
mu trong vng 20 pht trn my lc mu. Ly tm 4500 vng/ pht trong
vng 10 pht trn my ly tm. Lc dch trong qua mng lc mu 0,45m vo
l thy tinh vial 1,5 ml.
Sau khi c kt qu, phng php c chn phi l phng php loi
ng k nhiu nn c th gy nh hng n pic DA ng thi thu hi tt.
Nu c hai tiu ch trn u t th chn la phng php n gin, gi thnh
thp v ph hp vi iu kin thc t.
2.5.5. Thit lp bng mu:

32

Thit lp bng mu vi th t sau:


- Mu chy th (pre-test).
- 5 mu chun sp xp t nh n ln.
- Mu trng v mu kim sot.
- Cc mu th nghim.
- Mu chun
Chng trnh chy ra ct (50% MeOH:50%H2O, 1mL/pht, 50 pht)
2.5.6. Tnh ton :
Vic tnh ton kt qu bng phn mm trn h thng x l d liu
Masslynx 4.0. Da vo thi gian lu ca mu chun, lp ng cong chun
tuyn tnh da trn din tch pic v nng ca cc mu chun sau mu
c tnh theo ng hi quy y=ax+b (xi = (yi-b)/a) vi y l din tch pic ca
mu cn x l nng .
Mu trng, mu kim sot c dng tnh ton thu hi ca mi
t kim v xy dng gin kim sot.
Sau khi xc nh c cc thng s ti u, tin hnh xc nh khong
tuyn tnh, gii hn pht hin ca phng php, lp li, thu hi.
2.5.7. Kho st khong tuyn tnh:
xc nh khong tuyn tnh ca phng php, thc hin chy dy
chun vi 5 nng pha t 0,5 ppm n 30 ppm trong 3 ngy lin tc. Nu
th tuyn tnh trong khong nng ny (R2 >= 0,99) th chp nhn dy
nng ny, nu khng phi tip tc thu hp di nng cho n khi no R2
0,99.
2.5.8. Gii hn pht hin ca phng php:
- Trong trng hp khi tin hnh phn tch mu trng m c pic ti thi
gian lu hoc trong vng ln cn thi gian lu ca pic DA th tin hnh xc
nh nhy ca phng php bng cch dng dy mu thm chun (spike)
gim dn vi nmg phn tch trong 3 ngy. Gii hn pht hin (LOD)
l nng nh nht trong dy chun c din tch pic trung bnh cao t nht
gp 3 ln dn tch pic ca mu trng. Gii hn nh lng (LOQ) l nng
33

nh nht trong dy chun c din tch pic trung bnh cao gp 10 ln dn tch
pic ca mu trng.
- Trong trng hp mu trng khng c pic ti vng ln cn thi gian
lu ca pic DA th tin hnh xc nh nhy ca phng php bng cch
dng dy mu thm chun (spike) gim dn vi nmg phn tch trong 3
ngy v kim tra nhy ca pic thng qua tnh nng signal-to-noise ratio
ca chng trnh. Gii hn pht hin (LOD) l nng nh nht trong dy
chun c t l pic:nhiu trung bnh ca pic DA t nht gp 3 ln so vi nhiu
nn. Gii hn nh lng (LOQ) l nng nh nht trong dy chun c t l
pic:nhiu trung bnh ca pic gp 10 ln so vi nhiu nn.
2.5.9. lp li ca phng php:
th nghim lp li ca phng php, tin hnh phn tch trong 3
ngy, mi ngy 7 mu nhuyn th thm chun DA 2ppm. Tnh ton lp li
ca kt qu kt qu thu c thng qua lch chun Sr :
Sr =

Trong :

(X

X) 2

n 1

n 1

(0.1)

Sr = lp li
Std: lch chun
xi : kt qu thu c trn mu th i

2.5.10. thu hi ca phng php:


th nghim thu hi ca phng php, chng ti tin hnh phn
tch trong 3 ngy, mi ngy 7 mu trng l nhuyn th v 7 mu thm chun
DA 2ppm. Tnh ton kt qu thu c nh sau:

Rm =
Trong :

C C uns
C spike

(0.2)

Rm : thu hi trung bnh


C : gi tr trung bnh ca cc kt qu kim nghim,

c
tnh nh sau: C = xi/n
34

XSpike : nng ca dung dch mu thm chun.


2.5.11. Thc nghim xc nh DA trn mu nhuyn th.
Tin hnh kim nghim 36 mu th cp Bng 01. Quy trnh x
l mu thc hin ging nh trong mc 2.5.4. Sau khi c kt qu, tin hnh ly
ngu nhin mt s mu em i phn tch v so snh kt qu vi phng php
phn tch DA bng HPLC-UV nh gi tin cy ca phng php i
vi mu tht cng nh cc thng s lin quan khc.

35

Chng 3- KT QU V BN LUN
3.1. Xc nh cc thng s ti u:

ti u ha mt quy trnh phn tch DA trn thit b LC-MS/MS, chng


ta cn ti u 03 cng on: (i) cc thng s cho u d MS/MS, (ii) cc thng
s cho sc k lng, (iii) iu kin tch chit.
3.1.1. Xc nh cc thng s ti u ca MS/MS
Chng ta cn la chn c cc thng s ti u ion m bao gm hiu
in th mao qun (capillary) v hiu in th cone (cone volt); ti u ion con
bng cch ti u gi tr nng lng va chm (collision energy). Di y
chng ti ln lt ti u tn thng s:
3.1.1.1. Capillary (hiu in th mao qun): tim chun DA thng vo u
d MS/MS v thay i cc thng s v Capillary t nh ti ln (c nh gi
tr ca cc thng s khc) v chn gi tr ti u capillary ng vi kt qu
cng tn hiu ca ion [M+H]+ thu c l ln nht.
Kt qu kho st c trnh by Bng 03 (chi tit kho st c th tham
kho ti mc 1 Ph lc 2). Qua bng 03 ta thy gi tr hiu in th mao
qun gi 2 kV l ti u nht do thu c cng tn hiu ln nht:
Bng 03. Kt qu kho st gi tr hiu in th mao qun
STT

Capillary

Cng tn hiu

1.

1 kV

2,93 e5

2.

2 kV

1,00 e6

3.

3 kV

8,13 e5

4.

4 kV

7,1 e5

36

Hnh 15. Sc k ng vi gi tr ti u Capillary = 2 KV


3.1.1.2. Hiu in th cone: tng t nh phn ti u thng s Capillary,
iu chnh cone volt sao cho cng tn hiu ca ion m 312 ln nht. Kt
qu kho st in th cone v gi tr ti u hiu in th cone c trnh by
Bng 04 (chi tit kho st c th tham kho ti mc 2 Ph lc 2). Qua
Bng 04 ta thy ti gi tr cone volt 30 eV l gi tr ti u do thu c cng
tn hiu l ln nht.
Bng 04. Kt qu kho st gi tr hiu in th cone
STT

Cone volt

Cng tn hiu

1.

10 V

9,74 e4

2.

20 V

4,78 e5

3.

30 V

4,89 e5

4.

40 V

1,36 e5

5.

50 V

5,81 e5

37

Hnh 16. Sc k ti u Ion m ng vi gi tr ti u cone volt = 30 V


3.1.1.3. Nng lng va chm
Chy ch Full-scan ca ch ESI (+) i vi ion phn t [M+H+],
i vi DA c m/z = 312: Thay i cc gi tr nng lng va chm, chn
mnh ion con v gi tr nng lng va chm tng ng vi gi tr m cng
ca Ion con l ln nht.
Kt qu kho st c trnh by Bng 05 (chi tit kho st c th tham
kho ti mc 3 Ph lc 2):
Bng 05. Kt qu kho st nng lng va chm
STT
1.
2.
3.
4.
5.
6.
7.

Nng lng
va chm
10 eV
15 eV
20 eV
25 eV
30 eV
35 eV
40 eV

184,51
0
0
52348
0
21304
0
0

Cng tn hiu cc ion


219,31
247,64 266,25
0
0
0
0
19793
80749
22530
0
0
0
0
0
0
0
0
0
0
0
0
0
0

38

293,78
0
21573
0
0
0
0
0

- H2O

184
Hnh 17. Cch phn mnh ion ca DA
Kt qu kho st: chng ti chn c cc mnh Ion th cp v cc
iu kin ti u ca Collision energy tng ng nh sau:
Bng 06. Cc Ion th cp v cc iu kin ti u ca nng lng va chm
STT

Ion

Collision energy (eV)

1.

184,51

20 eV

2.

219,31

20 eV

3.

247,64

15 eV

4.

266, 25

15 eV

5.

293,78

15 eV

39

Hnh 18. Sc k iu kin Collision energy 15 eV


Chng ti chn mnh Ion 266 dng nh lng do mnh 266,25 cho
cng ln nht so vi cc mnh khc.
3.1.2. Pha ng v chng trnh chy gradient.
Chng ti s dng 02 pha ng tham kho theo mt s bi bo trn tp
ch ScienDirect nh sau:
1. Cp pha ng 1: 0,05% TFA trong ACN (A) v 0,05% TFA trong
H20 (B)
Bng 07. iu kin gradient 1 pha ng 1
Thi gian
(Pht)
0,00
4,90
5,00
6,00
10,00
10,01

A%

B%

10,0
10,0
50,0
90,0
90,0
10,0

90,0
90,0
50,0
10,0
10,0
90,0

40

Tc dng
(mL/pht)
0,400
0,400
0,400
0,400
0,400
0,400

Curve
1
1
1
1
1
1

Hnh 19. Sc k chy chun DA gradient 1 pha ng 1


Bng 08. Bng gradient 2 pha ng 1
Thi gian (Pht)

A%

B%

Tc dng

Curve

(mL/pht)
0,00

50,0

50,0

0.400

5,00

50,0

50,0

0.400

6,00

90,0

10,0

0.400

10,00

90,0

10,0

0.400

10,01

50,0

50,0

0.400

41

Hnh 20. Sc k chy chun DA gradient 2 pha ng 1


Nhn xt: ta thy pha ng B c phn cc mnh hn pha A. Nn ch
gradient 1, ti thi im xut hin pic DA t l A:B = 10:90, phn cc
ca pha ng mi mnh ko DA ra khi ct. Do vy rt ngn thi
gian chy chng ti thay i tng phn cc ca pha ng ngay t u vi
chng trnh gradient 2 vi t l A:B = 50:50. Kt qu pic ca DA c ra sm
hn tuy nhin pic cha c tch tt.
2. Cp pha ng 2: 0,1% FA trong H2O (A) v 0,1%FA trong ACN (B)
Thi gian (Pht)
0,00
4,90
5,00
6,00
10,00
10,01

Bng 09. Bng gradient 1 pha ng 2


A%
B%
Tc dng
(mL/pht)
10,0
90,0
0,400
10,0
90,0
0,400
50,0
50,0
0,400
90,0
10,0
0,400
90,0
10,0
0,400
10,0
90,0
0,400

42

Curve
1
1
1
1
1
1

Hnh 21. Sc k chy chun DA gradient 1 pha ng 2


Bng 10. Bng gradient 2 pha ng 2
Thi gian (Pht)

A%

B%

Tc dng

Curve

(mL/pht)
0,00

50,0

50,0

0,400

5,00

50,0

50,0

0,400

6,00

90,0

10,0

0,400

10,00

90,0

10,0

0,400

10,01

50,0

50,0

0,400

43

Hnh 22. Sc k chy chun DA gradient 2 pha ng 2


Bng 11. Bng gradient 3 pha ng 2
Thi gian (Pht)

A%

B%

Tc dng

Curve

(mL/pht)
0,00

50,0

50,0

0,400

5,00

50,0

50,0

0,400

6,00

90,0

10,0

0,400

10,00

90,0

10,0

0,400

10,01

50,0

50,0

0,400

44

Hnh 23. Sc k chy chun DA gradient 3 pha ng 2


Nhn xt: Vi pha ng s 2, phn cc ca pha ng tng khi tng
thnh phn pha A (H2O). Theo kt qu cc ln chy ta thy iu kin
gradient 3 cho pic ra vi thi gian ngn v pic sc nhn, cn i nht.
3.1.3. Dung mi chit:
La chn dung mi chit: Chng ti chn 03 loi dung mi
a. Dung mi 1: MeOH:H20: 1:1 (mu spiked 2 ppm)

45

Hnh 24. Sc k chy mu chit bng dung mi MeOH:H20: 1:1


b. Dung mi 2: Formic: metanol:H20: 2:5:93 (mu spiked 2 ppm)

Hnh 25. Sc k chy mu chit bng dung mi Formic: metanol:H20: 2:5:93

c. Dung mi 3: MeOH: H20: 2:1

46

Hnh 26. Sc k chy mu chit bng dung mi MeOH: H20: 2:1


Nhn xt: C 03 dung mi trn u cho kt qu kh tt v u c th s dng
c v chng ti xut chn dung mi 1 vi l do cng tn hiu ca
ion nh lng 266,25 cho tn hiu tt nht v thnh phn dung mi n gin.
Bng 12. So snh cng tn hiu ion 266,25 nng 2 ppm
Dung mi 1
Cng tn hiu ion

2,07 e5

266,25 nng 2,0 pmm


3.2. Khong tuyn tnh:

Kho st khong tuyn tnh ca DA

47

Dung mi 2
2,03 e5

Dung mi 3
1,74 e5

Hnh 27. th biu din ng tuyn tnh


Tng hp tuyn tnh theo cc Ion c th hin Bng 13.
Bng 13. Bng tng hp thng s v tuyn tnh theo cc ion
Ion

H s hi quy

H s gc a

H s gc b

tuyn tnh R2
219,31

0,9984

346,635

-133,465

184,51

0,9846

314,745

-196,319

293,78

0,9972

1733,17

-784,847

266,25

0,9995

16764

-5244,29

247,64

0,9969

1812,02

-902,768

Kt lun: T kt qu kho st khong tuyn tnh ng chun ca DA


trn ta thy trong khong nng t 0,5 ppm n 10ppm th th c
tuyn tnh tt vi R2> 0,98 (chi tit v R2 ti Bng 13).

48

3.3. Gii hn pht hin ca phng php:

Bng 14. Kt qu xc nh gii hn pht hin ca phng php


nhy (signal-to-noise ratio) theo nng

Ion

Blank

0.5 ppm

2 ppm

4 ppm

7 ppm

10 ppm

184,51

2,4

7,9

53

144

259

439

266,25

20,7

373

2372

5368

8850

9189

T kt qu thu c Bng 13. ta c ta thy nng 0,5 ppm t s S/N


ca ion nh lng 266,25 18 (S/N = 373/20,7) tuy nhin ion nh danh
184,51 c t l S/N 3,2 (S/N = 7,9/2,4) nn ta chn LOD = 0.5 ppm v gii
hn nh lng LOQ = 1,5 ppm (LOQ = 3 x LOD). Tuy nhy ny l khng
cao i vi phng php phn tich trn LC/MS/MS v iu quan trng nht l
nhy ca phng php hon ton p ng vi yu cu ra trong vic
kim sot c t ASP (mc gii hn cho php trong thc phm l 20 ppm).
3.4. lp li v thu hi ca phng php:

Bng 15. Kt qu phn tch lp li v thu hi ca phng php.


TT

Hm lng DA trong mu spike ( 2.5 ppm)


Ngy 1

Ngy 2

Ngy 3

2,63

2,61

2.40

2,43

2,93

2,58

2,45

2,58

2,53

2,46

2,25

2,36

2,72

2,73

2,20

2,60

2,42

2,74

2,43

2,61

2,60

Trung bnh (1 ngy)

2,53

2,59

2,49

SD (1 ngy)

0,12

0,22

0,18

Trung bnh (3 ngy)

2,53

49

TT

Hm lng DA trong mu spike ( 2.5 ppm)


Ngy 1

SD (3 ngy)

Ngy 2
0,17

CV%

6,72 %

thu hi (3 ngy)

101,2 %

T Bng 11. ta c kt lun nh sau:


- lp li ca phng php SD = 0,17 hay CV = 6,72%
- thu hi ca phng php: R = 101,2 %

50

Ngy 3

3.5. Thc nghim xc nh DA trn nhuyn th.

p dng qui trnh chy mu v x l mu chng ti phn tch trn mu


tht, kt qu phn tch c a ra trn Bng12
Bng 16. Kt qu phn tch mu nhuyn th (tnh trn Ion 266,25).
Thi gian lu

Cng

Nng

(pht)

pic

(ppm)

1.

5,07

1016

0,37

2.

5,10

1018

0,37

3.

5,10

997

0,37

4.

5,13

933

0,37

5.

4,93

775

0,36

6.

5,10

854

0,36

7.

5,07

852

0,36

8.

4,97

260

0,33

9.

4,70

20

0,31

10.

5,17

811

0,36

11.

5,13

894

0,37

12.

5,13

762

0,36

13.

4,77

39

0,32

14.

5,13

559

0,35

15.

4,80

52

0,32

16.

5,10

808

0,36

17.

5,07

580

0,35

18.

5,03

734

0,36

19.

5,07

739

0,36

STT

51

20.

4,73

28

0,31

21.

5,03

515

0,34

22.

5,03

429

0,34

23.

5,03

376

0,34

24.

5,03

485

0,34

25.

5,07

494

0,34

26.

5,07

482

0,34

27.

4,93

90

0,32

28.

5,03

474

0,34

29.

5,00

444

0,34

30.

5,00

462

0,34

31.

5,07

484

0,34

32.

5,00

560

0,35

33.

5,07

355

0,33

34.

5,13

486

0,34

35.

5,10

277

0,33

36.

5,00

346

0,33

Nhn xt: Tt c cc mu pht hin nng rt thp, nh hn LOD


nn c th kt lun khng pht hin DA trong mu phn tch.
3.6. nh gi tin cy ca phng php:

nh gi tin cy ca phng php, ng thi kim chng li kt


qu va thc hin trn 50 mu thu thp c, chng ti thc hin kim khng
nh ngu nhin 1 s mu m tnh v do khng c mu pht hin mc
>0,5ppm nn chng ti s dng mu spiked nng 2 ppm so snh, kt
qu nh sau:
52

Bng 17. So snh kt qu phng php HPLC-UV v LC-MS/MS


TT

Tn mu

Kt qu
(HPLC-UV)

Kt qu
(LC-MS/MS)

1.

Mu nghu Nam nh

ND

ND

2.

Mu Tu hi Vn n

ND

ND

3.

Mu Nghu Tin Giang

ND

ND

4.

Mu S lng B La

ND

ND

5.

S huyt Thnh Ph

ND

ND

6.

Mu Spiked 2 ppm

2.12 ppm

2.06 ppm

7.

Mu nghu Spiked 2.5 ppm

2.64 ppm

2.53 ppm

8.

Mu CRM
DA: 41 2 ppm
C5 Epiaxt domoic:
3.2 0.3

42.5 ppm

48.12 ppm

DA

Hnh 28. Kt qu chy mu nghu ti Nam nh trn LC-MS/MS v HPLC-UV

53

Hnh 29. Kt qu chy mu CRM trn LC-MS/MS v HPLC-UV

Chi tit v kt qu phn tch mu trn HPLC-UV v sc k c th


tham kho ti Ph lc 5.
T kt qu Bng 13 ta thy:
Nh vy, chng ti c th khng nh: Phng php chng ti xy
dng t tin cy cao v c th p dng kim nghim DA trong thy sn
v sn phm thy sn.
Vi khng nh ny, chng ti xin c a ra qui trnh kim nghim
c t sinh hc bin ASP (DA) nh sau:
3.7. Qui trnh phn tch Axt domoic.

3.7.1. Phm vi p dng:


Hng dn ny qui nh phng php xc nh hm lng ASP trong
mu thy sn bng LC-MS/MS.
Gii hn pht hin ca phng php l 0.5 mg/kg.
3.7.2. Nguyn tc:
ASP c chit tch t mu nhuyn th bng hn hp metanol-nc
(1:1). Hm lng ASP trong dch chit mu c xc nh trn sc k ghp
hai ln khi ph (LC-MS/MS).
3.7.3. Thit b, dng c, ha cht, dung dch:
3.7.3.1. Thit b- dng c:

54

3.7.3.1.1. My sc k lng ghp khi ph LC-MS/MS, gm cc b phn


sau: Bm pha ng, b tim mu t ng, b iu nhit ct, u d MS/MS
(Micro mass), b iu khin h thng (Waters) v phn mm x l.
3.7.3.1.2. Ct sc k lng C18, kch thc ct 250 x 4.6 mm, kch thc
ht 5 m (Merck- Licrocart 250-4 RP-18), c hoc tng ng.
3.7.3.1.3. My ng ha mu (KCH-1000), Trung Quc hoc tng
ng.
3.7.3.1.4. My ly tm (Sigma 4 K 15), M hoc tng ng.
3.7.3.1.5. B nh siu m (Branson 2210), M hoc tng ng.
3.7.3.1.6. Cn k thut (Sartorious, d= 0,1g), c hoc tng ng.
3.7.3.1.7. Cn phn tch (Sartorious, d= 0,1mg), c hoc tng ng.
3.7.3.1.8. My lc mu (IKA KS-260), c hoc tng ng.
3.7.3.1.9. Transferpette 1ml (Eppendorf).
3.7.3.1.10. ng ly tm 50ml.
3.7.3.1.11. Mng lc mu 0.45m, : 25mm.
3.7.3.1.12. ng tim nha loi 5ml.
3.7.3.1.13. Giy lc.
3.7.3.1.14. Cc dng c thy tinh: bnh nh mc, ng ong, l cha mu;
cc c m,...

3.7.3.2. Ha cht, dung dch:


3.7.3.2.1. Chun axt domoic (Sigma) hoc tng ng. Bo qun chun
rn trong ngn ng t lnh.
3.7.3.2.2. Nc tinh khit dng cho HPLC (nc HPLC).
3.7.3.2.3. Dung mi: axetonitrile v metanol loi dng cho HPLC.
3.7.3.2.4. Axt Formic (FA) tinh khit phn tch
3.7.3.2.5. Dung dch pha ng cho my HPLC: Trn u 100ml ACN v
khong 900ml nc HPLC trong bnh nh mc 1000ml, thm 1.0 ml FA, v
nh mc n 1L vi nc HPLC. ui kh bng cch nh siu m 15 n
20 pht trn b siu m. Bo qun dung dch nhit phng, s dng trong
1 tun.
55

3.7.3.2.6. Dung dch chit mu : Trn u metanol v nc HPLC theo t


l th tch (1:1). Chun b dung dch trc mi ln s dng.
3.7.3.2.7. Dung mi pha chun: Trn u axetonitril v nc HPLC theo
t l th tch (1:9). Chun b dung dch trc mi ln s dng.
3.7.3.2.8. Dung dch chun gc 1000g/ml: ht chnh xc 950l Metanol
bng micropipette 1ml vo chai ng cht chun axt domoic, lc u. Bo
qun trong ngn ng t lnh, Dung dch c dng trong 1 nm.
Lu : Th tch ht metanol trn phi c iu chnh hp l sau khi
tnh ton v hiu chnh khi lng theo giy chng nhn ca nh sn xut
sau tnh ton li nng thc t pha.
3.7.3.2.9. Dung dch ASP 25g/ml: Ht chnh xc 250l dung dch
chun gc vo bnh nh mc 10ml. Thm dung dch axetonitrile 10 % n
vch, lc u. Bo qun trong t lnh (2 - 8oC), a v nhit phng khi s
dng, dung dch bn trong 6 thng.
3.7.3.2.10. Dung dch ASP 10 g/ml: Ht chnh xc 100l dung dch
chun gc vo bnh nh mc 10ml. Thm dung dch axetonitrile 10 % n
vch, lc u. Bo qun trong t lnh (2 - 8oC), a v nhit phng khi s
dng, dung dch bn trong 6 thng.
3.7.3.2.11. Dung dch ASP 7 g/ml: Ht chnh xc 70l dung dch
chun gc vo bnh nh mc 10ml. Thm dung dch axetonitrile 10 % n
vch, lc u. Bo qun trong t lnh (2 - 8oC), a v nhit phng khi s
dng, dung dch bn trong 6 thng.
3.7.3.2.12. Dung dch ASP 4 g/ml: Ht chnh xc 40l dung dch
chun gc vo bnh nh mc 10ml. Thm dung dch axetonitrile 10 % n
vch, lc u. Bo qun trong t lnh (2 - 8oC), a v nhit phng khi s
dng, dung dch bn trong 6 thng.
3.7.3.2.13. Dung dch ASP 2.5 g/ml: Ht chnh xc 1000l dung dch
chun ASP 25g/ml vo bnh nh mc 10ml. Thm axetonitrile 10 % n

56

vch, lc u. Bo qun trong t lnh (2 - 8oC), a v nhit phng khi s


dng, dung dch bn trong 6 thng.
3.7.3.2.14. Dung dch ASP 2.0 g/ml: Ht chnh xc 800l dung dch
chun domoic 25g/ml v nh mc 10ml. Thm axetonitrile 10 % n
vch, lc u. Bo qun trong t lnh (2 - 8oC), a v nhit phng khi s
dng, dung dch bn trong 6 thng.
3.7.3.2.15. Dung dch ASP 1.0 g/ml : Ht chnh xc 400l dung dch
chun 25 g/ml solution vo bnh nh mc 10ml. Thm axetonitrile 10 %
n vch, lc u. Bo qun trong t lnh (2 - 8oC) khi khng s dng, a v
nhit phng khi s dng, dung dch bn trong 6 thng.
3.7.3.2.16. Dung dch ASP 0.5g/ml: Ht chnh xc 200l dung dch
chun 25g/ml solution vo bnh nh mc 10ml. Thm axetonitrile 10 %
n vch, lc u. Bo qun trong t lnh (2 - 8oC) khi khng s dng, a v
nhit phng khi s dng, dung dch bn trong 6 thng.
3.7.4. Chun b mu:
3.7.4.1. Chun b mu th
Tch v nhuyn th, ra sch nc mui. ng ha t nht 100g tht
nhuyn th trn my ng ho mu. Bo qun t nht -100C nu mu khng
c phn tch ngay khong 1 tun.
3.7.4.2. Chun b mu th nghim:
Cn chnh xc 2.0 g (m) mu ng ha trn cn k thut vo ng ly
tm. Th nghim 1 ln/mu
3.7.4.3. Chun b mu trng:
Mu trng l mu nhuyn th xc nh khng c ASP. Chun b
mu trng ging nh i vi mu th nghim.
3.7.4.4. Chun b mu thm chun:
3.7.4.4.1. Mu kim tra gii hn pht hin: uc chun b bng cch
thm 40 L dung dch chun 25 g/mL vo 2.0 g mu trng c hm
lng ASP l 0.5 mg/Kg. Tin hnh phn tch nh i vi mu th nghim.
57

3.7.4.4.2. Mu kim sot 4.0mg/kg: uc chun b bng cch thm


16L dung dch chun 500g/mL vo 2.0g mu trng. Tin hnh phn tch
nh i vi mu th nghim.
3.7.5. Tin hnh th nghim:
3.7.5.1. Chit mu v lm sch mu:
- Thm chnh xc 8.0 ml dung dch chit mu (3.7.3.2.6) vo cc ng ly
tm cha mu chun b cc bc (3.7.4.2), (3.7.4.3), (3.7.4.4). Lc mu
trong 20 pht trn my lc mu.
- Ly tm 4500 vng/ pht trong 10 pht trn my ly tm Lc dch trong
qua mng lc mu 0.45m vo l thy tinh 1.5 ml.
Xc nh hm lng ASP trong dch chit ny trn my LC-MS/MS.
3.7.5.2. Phn tch mu trn HPLC:
3.7.5.2.1. Thng s ci t cho HPLC:
- Pha ng A: 0.1% FA trong H20
- Pha ng B: 0.1%FA trong ACN
- Thnh phn pha ng thay i theo thi gian (ch gradient) c
m t bng sau:
Thi gian

A%

B%

(Pht)

Tc dng

Curve

(mL/pht)

0,00

50,0

50,0

0,400

5,00

50,0

50,0

0,400

6,00

90,0

10,0

0,400

10,00

90,0

10,0

0,400

10,01

50,0

50,0

0,400

- Th tch tim: 20l


- Tc dng: 0,4 mL/pht
- Thi gian phn tch: 10 pht
- Nhit ct: 35oC
58

3.7.5.2.2. Thng s ci t cho u d MS


- Ch vn hnh: ESI+
- Ngun ESI (+)
- in th mao qun: 1kV
- in th extractor: 2,0 V
- in th RF: 0,0 V
- Nhit ngun: 120oC
- Nhit kh dung mi:400oC
- Tc kh cone: 40 l/gi
- Tc kh kh dung mi: 460 l/gi
- B phn tch
phn gii vng khi lng thp LM1: 15,0
phn gii vng khi lng cao HM1: 15,0
Nng lng ion ho 1: 0,5
in th ca vo: -1
in th ca ra: 1
phn gii vng khi lng thp LM2: 15,0
phn gii vng khi lng cao HM2: 15,0
Nng lng ion ho 2: 3
in th b khuych i: 650 V
p sut chn khng: 4-5e-3 mbar
Ch vn hnh : chun
- Cc iu kin phn ly MS/MS:
Tn cht

Axt Domoic

ion s

ion th

Thi gian

in th

Nng

cp

cp

Dwell (s)

cone (V)

lng

(m/z)

(m/z)

312

cone (eV)

184,51

0,1

30

20

266,25

0,1

30

15

59

Th t tim mu:
-Tim cc dch mu xy dng ng chun.
-Tim dch mu trng.
-Tim cc dch mu th nghim.Tnh Hm lng DA trong mu th
nghim.
3.7.6. m bo cht lng
3.7.6.1. nh tnh: Cht cn phn tch c khng nh l c mt khi
cc yu cu v thi gian lu, mc dung sai cng ion tng i p ng
theo ch th 2002/657/EC ca Cng ng Chu u.
3.7.6.2. nh lng:
- T l tn hiu/ nhiu ca cc ion th cp trong mu phi 3:1
- H s hi qui tuyn tnh ca ng chun R2 phi 0.99
3.7.7. Tnh ton kt qu:
3.7.7.1. Tnh t s ion theo phng trnh:

R=

100 A1
(%)
A2

Trong :
R: t s ion (%)
A1: din tch ca cc pic ion th cp c cng thp.
A2: din tch ca cc pic ion th cp c cng cao
3.7.7.2. Dng th h s tn hiu ca dch mu xy dng ng chun
vi nng cht chun b sung theo phng php hi qui tuyn tnh:
Y = ax + b
Trong :
b: tung gc ca ng chun
a: h s gc ca ng chun
3.7.7.4. Tnh hm lng cht cn phn tch trong mu da theo h s tn hiu
ca tng mu phn tch v ng chun.

60

KT LUN
Qua qu trnh nghin cu thc nghim, chng ti hon thnh cc
nhim v ca lun vn ra vi kt qu nh sau:
- Kho st, ti u ha cc iu kin xy dng quy trnh phn tch axt
domoic trong thy sn trn thit b LC-MS/MS: (i) Phng php chit: la
chn phng php chit lng-lng vi dung mi chit l MeOH:H2O t l 1:1.
(ii) Ti u cc thng s lin quan n HPLC: s dng ct C18, kch thc ct
250 x 4.6 mm, kch thc ht 5 m (Merck- Licrocart 250-4 RP-18); la chn
v s dng pha ng l 0.1% FA trong H20 (A) v 0.1%FA trong ACN (B);
tc dng 0,4 ml/pht. (iii) Ti u ha cc thng s ca u d MS/MS:
hiu in th mao qun (capillary = 2 kV), in th cone (cone volt = 30 V),
nng lng va chm (collission energy = 20 eV v 15 eV tng ng vi 02
ion con l 184,51 v 266,25).
- Kho st gi tr s dng ca phng php trn mu tht v mu chun: (i)
Khong tuyn tnh: khong tuyn tnh c nng t 0.5 ppm n 10 ppm (R2
> 0,98). (ii) nhy ca phng php: Gii hn pht hin LOD: 0,5 ppm.
Gii hn nh lng LOQ: 1,5 ppm. (iii) lp li ca phng php: thu
hi ca phng php: 101.2%. lp li ca phng php SD = 0.17 hay CV
= 6.72%.
- phn tch 36 mu nhuyn th ti 12 vng thu hoch nhuyn th ca
Vit Nam.
- Kim i chng: ly xc sut 1 s mu nhuyn th, mu thm chun
(spiked) v mu vt liu chun (CRM).
- Trong khun kh lun vn, vi thi gian v iu kin hn hp nn
chng ti ch mi ch nghin cu trong phm vi l phn tch DA trong nhuyn
th hai mnh v. V vy mong mun ca chng ti l trong thi gian sp n
cn phi m rng nghin cu phng php ny thnh phng php chung c
th p dng phn tch ng thi nhiu cht nhm c t sinh hc bin khc
nh DSP, PSP.
61

TI LIU THAM KHO


Ting Vit
1. Nguyn Hu nh, Trn Th (1999), ng dng mt s phng php
ph nghin cu cu trc phn t, Nh xut bn gio dc.
2. Phm Lun (2000), C s l thuyt ca HPLC trng i hc Khoa
hc T nhin, H Ni.
3. Bi Th Nh Thun, Nguyn Phng Tin, Bi Minh c (1991), Kim
nghim cht lng v thanh tra v sinh an ton thc phm, Nh xut
bn y hc, tr. 236-242.
4. T Th Tho, Bi ging chuyn Thng k trong ha phn tch,
Trng i hc Khoa hc t nhin, 2005
5. Phm Hng Vit (2003), Sc k kh, NXB Khoa hc K thut.
Ting Anh
6. H. Dizer, B. Fischer, A.S.A. Harabawy, M.-C. Hennion, P.-D. Hansen
(2001), Toxicity of axt domoic in the marine mussel Mytilus edulis,
Aquatic Toxicology, Vol.55, pp.194-156.
7. Intergovernmental Oceanographic Commission (1995), Manuals and
Guides No. 31, Volume 1.
8. Jean-Philippe Antignac, Bruno Le Bizec, Fabrice Montau, Francois
Andre (2003), Validation of Analytical Mehtods Based on Mass
Spectrometric Detection according to 2002/657/EC European
Decision: Guideline and Application, Analytica Chimica Acta, Vol.
483, pp. 325-334.
9. Jonathan Clayden,* Benjamin Read and Katherine R. Hebditch (2005),
Chemistry of axt domoic, isoaxt domoics, and their analogues,
Tetrahedron report number 720.

62

10. Marios Maroulis, Ioannis Monemvasiosa, Elisabeth Vardakaa, Pantelis


Rigasa (2008) Determination of axt domoic in mussels by HPLC with
post-column derivatization using 4-chloro-7-nitrobenzo-2-oxa-1,3diazole

(NBD-Cl)

and

fluorescence

detection,

Journal

of

Chromatography B, 876, pp. 245251.


11. Lefebvre KA, Powell CL, Busman M, Doucette GJ, Moeller PD, Silver
JB, Miller PE, Hughes MP, Singaram S, Silver MW, Tjeerdema RS
(1999), Detection of axt domoic in northern anchovis and California
sea lions associated with an unusual mortality event, Natural toxins ,
7(3), pp.85-92.
12. Philipp Hess, Steven Morris, Lesley A. Stobo, Nigel A. Brown, John
D.G. McEvoy, Glenn Kennedy, Paul B. Young, Deirdre Slattery, Evin
McGovern, Terry McMahon, Susan Gallacher (2005), LC-UV and LCMS methods for the determination of axt domoic, Trends in Analytical
Chemistry, Vol. 24, No. 4, 2005.
13. Quilliam MA (1995), Seafood toxins, Journal of AOAC International,
78(1), pp. 144-148.
14. Yasumoto T; Fukui M; Sasaki K; Sugiyama K (1995), Determinations
of marine toxins in foods, Journal of AOAC International, 78(2), pp.
574-582.
15. Whitney PL, Baden DG (1996), Complex association and dissociation
kinetics of brevetoxin binding to voltage-sensitive rat brain sodium
channels, Natural toxins, 4, pp. 261-270.

63

PH LC 1: CNG THC CU TO C T SINH HC BIN

1. Nhm ASP Axt domoic

2. Nhm DSP
2.1 Dinophysis Toxin

2.2 Okadaic Acid

2.3 Pectenotoxin

2.4 Yessotoxin

3. Nhm PSP

4. Nhm NSP

PH LC 2: MT S SC K TIU BIU KHI TI U

1. Kho st capillary

Hnh 32. Sc k ca Ion s cp ti iu kin Capillary 1 kV

Hnh 33. Sc k ca Ion s cp ti iu kin Capillary 2 kV

Hnh 34. Sc k ca Ion s cp ti iu kin Capillary 3 kV

Hnh 35. Sc k ca Ion s cp ti iu kin Capillary 4 kV


Nhn xt: ti iu kin capillary = 2 kV l ti u nht.

2. Kho st cone volt

Hnh 36. Sc k ca Ion s cp ti iu kin cone volt 10 V

Hnh 37. Sc k ca Ion s cp ti iu kin cone volt 20 V

Hnh 38. Sc k ca Ion s cp ti iu kin cone volt 30 V

Hnh 39. Sc k ca Ion s cp ti iu kin cone volt 40 V

Hnh 40. Sc k ca Ion s cp ti iu kin cone volt 50 V


Nhn xt: Ti iu kin 30 V l ti u nht v ion m l 312
3. Kho st nng lng va chm (Collission energy)

Hnh 41. Sc k ca Ion th cp ti iu kin Collision energy 5 eV

Hnh 42. Sc k ca Ion th cp ti iu kin Collision energy 10 eV

Hnh 43. Sc k ca Ion th cp ti iu kin Collision energy 15 eV

Hnh 44. Sc k ca Ion th cp ti iu kin Collision energy 20 eV

Hnh 45. Sc k ca Ion th cp ti iu kin Collision energy 25 eV

Hnh 46. Sc k ca Ion th cp ti iu kin Collision energy 30 eV

Hnh 47. Sc k ca Ion th cp ti iu kin Collision energy 35 eV

Hnh 48. Sc k ca Ion th cp ti iu kin Collision energy 40 eV


Nhn xt: Tng hp tn hiu ca cc mnh ion con (5 mnh) ta c bng
tm tt nh sau:
Mass
10
184.51
184.92
219.31
219.92
247.64
247.84
248.04
265.65
265.85
266.05
266.25
293.58
293.78

15

Collision Energy
20
25
30
52348
21304
16651
22530
17764

35

40
184
219

19793
13518
11555

247

40379
17217
25038
266

80749
14042
21573
Bng 14. Bng tng hp tn hiu ca cc mnh Ion s cp

294

PH LC 3. NG BIU DIN TUYN TNH

Hnh 49. Sc k ca chun DA nng : 0.5 ppm

Hnh 50. Sc k ca chun DA nng : 1 ppm

Hnh 51. Sc k ca chun DA nng : 2ppm

Hnh 52. Sc k ca chun DA nng : 3 ppm

Hnh 53. Sc k ca chun DA nng : 5 ppm

Hnh 54. Sc k ca chun DA nng : 7ppm

Hnh 55. Sc k ca chun DA nng 10 ppm

1. ng tuyn tnh ca Ion 219

2. ng tuyn tnh ca Ion 184

3.

ng tuyn tnh ca Ion 293

4. ng tuyn tnh ca Ion 266

5. ng tuyn tnh ca Ion 247

PH LC 4. SC K CHY MU NHUYN TH 2 MNH V

Hnh 56. Sc k chy mu nghu s 1 ti Giao Thy Nam nh

Hnh 57. Sc k chy mu nghu s 2 ti Giao Thy Nam nh

Hnh 58. Sc k chy mu nghu s 3 ti Giao Thy Nam nh

Hnh 59. Sc k chy mu nghu s 1 ti Tin Hi Thi Bnh

Hnh 60. Sc k chy mu nghu s 2 ti Tin Hi Thi Bnh

Hnh 61. Sc k chy mu nghu s 3 ti Tin Hi Thi Bnh

Hnh 62. Sc k chy mu Tu Hi ti Vn n Qung Ninh

Hnh 63. Sc k chy mu ip s 1 ti Phan Thit

Hnh 64. Sc k chy mu ip s 2 ti Phan Thit

Hnh 65. Sc k chy mu ip s 3 ti Phan Thit

Hnh 66. Sc k chy mu S s 1 ti Tuy Phong

Hnh 67. Sc k chy mu S s 2 ti Tuy Phong

Hnh 68. Sc k chy mu S s 3 ti Tuy Phong

Hnh 69. Sc k chy mu nghu s 1 ti Bnh i

Hnh 70. Sc k chy mu nghu s 2 ti Bnh i

Hnh 71. Sc k chy mu nghu s 3 ti Bnh i

Hnh 72. Sc k chy mu nghu s 1 ti Ba Tri

Hnh 73. Sc k chy mu nghu s 2 ti Ba Tri

Hnh 74. Sc k chy mu nghu s 3 ti Ba Tri

Hnh 75. Sc k chy mu s huyt s 1 ti Thnh Ph

PH LC 5. SC K PHN TCH MU NHUYN TH 2 MNH


V BNG HPLC UV

Hnh 76. ng chun chy DA bng HPLC-UV

DA

Hnh 77. Kt qu chy mu Spiked 2 ppm

DA

Hnh 78. Kt qu chy mu Spiked 2.5 ppm

Hnh 79. Kt qu chy mu nghu ti Giao Thy Nam nh

Hnh 80. Kt qu chy mu Tu hi ti Vn n

Hnh 81. Kt qu chy mu nghu ti Tin Giang

Hnh 82. Kt qu chy mu s lng ti B La

Hnh 83. Kt qu chy mu s huyt ti B La

DA

Hnh 84. Kt qu chy mu CRM trn HPLC

Hnh 85. Kt qu chy mu CRM trn LC-MS/MS

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