Xc nh ng thi d lng khng sinh nhm

nitrofuran trong mt s loi thc phm ti

sng trn a bn H Ni bng phng php
sc k lng khi ph LC/MS/MS


Trn Th Hng


Trng i hc Khoa hc T nhin
Lun vn ThS. ngnh: Ha phn tch; M s: 60 44 29
Ngi hng dn: TS. L Th Hng Ho
Nm bo v: 2012


Abstract. Gii thiu v khng sinh nhm nitrofuran; tc dng ca cc cht khng
sinh nhm nitrofuran; d lng khng sinh nhm nitrofuran trong thc phm. Xy
dng phng php xc nh ng thi 4 cht chuyn ha ca nitrofuran trong thc
phm ti sng bng LC-MS/MS. ng dng phng php xc nh cc cht
chuyn ha ca nitrofuran trong thc phm ti sng cu th trong thi t l n , tht bo,
tht g, gan. Tm hiu kt qu t c.

Keywords. Ha phn tch; D lng khng sinh; Thc phm


Content
M U

An ton v sinh thc phm l vn ang c c x hi quan tm, c bit l
th v cc khu cng nghip, khi ngy cng c nhiu tc nhn c hi b pht hin trong thc
phm khin d lun lo ngi. Tuy nhin trong lnh vc sn xut kinh doanh ni chung v trong
chn nui gia sc, gia cm ni ring con nhiu vn ng lo ngi, nh vic qun l s dng
thuc khng sinh con lng lo, tnh trng s dng cc cht b tr trong chn nui kh ty
tin. T li tn d cc ha cht, khng sinh trong sn phm chn nui, gy nguy hi
nghim trng n sc khe ngi tiu dng.
Nitrofuran l mt nhm khng sinh tng hp thng c s dng trong thc n gia
sc nh kch thch tng trng v l phng php iu tr d phong, iu tr cc bnh nhim
trng ng tiu ha gy ra bi Escherichia v Salmonella trong chn nui gia sc, gia cm.
Chng cng c s dng iu tr nhim trng do vi khun v sinh vt n bo trong
nui trng thy sn...Tuy nhin chng gy tc hi cho sc khe con ngi, c bit l gy
ung th v t bin ngi. Nm 2002 2003 pht hin d lng cht chuyn ha ca
nitrofuran trong s lng ln cc mu t gia cm v nui trng thy sn cc sn phm nhp
khu vo Chu u t mt s khu vc ng Nam v cc nc Nam M. T dn n
lnh cm s dng trong sn xut thc phm ng vt ti nhiu quc gia bao gm M, Canada
v EU... Cc nc ny t lnh cm trn tt c cc loi thc phm nhp khu c cha d
lng nitrofuran. Thng 3 nm 2003 EU quy nh yu cu gii hn nh nht cn thc hin
phng php (MRPL) i vi cc cht chuyn ha nhm nitrofuran l 1 g/kg [29]. Nm
2002, Vit Nam, B nng nghip v pht trin nng thn quyt nh cm sn xut, nhp
khu, lu thng v s dng mt s loi khng sinh ha cht trong sn xut v kinh doanh
TACN trong c nitrofuran [9].
Hin nay vic s dng d lng khng sinh sai mc ch ang mc bo ng nh
hng ti sc khe con ngi v ng vt. V vy vi nhu cu bc thit v vn m bo
VSATTP hin nay, chng ti tin hnh nghin cu pht trin phng php Xc nh
ng thi d lng khng sinh nhm nitrofuran trong mt s loi thc phm ti sng
trn a bn H Ni bng phng php sc k lng khi ph LC/MS/MS.


CHNG 1: TNG QUAN

1.1. Gii thiu v khng sinh nhm nitrofuran
Nitrofuran l mt nhm khng sinh tng hp co

cha nhm 5-nitro, thng c s

dng lm thc n gia sc kch thch s tng trng, ch yu i vi gia sc (nh gia cm,
ln), nui trng thy sn (c, tm) v nui ong iu tr cc vi khun v sinh vt n bo
nhim trng nh vim rut tiu ha gy ra bi Escherichia coli v Salmonella spp, gia cm
bnh t v bnh cu trng mu en u [12].
Cc cht nhm n itrofuran bao g m Furazolidone, furaltadone, furazolidone,
nitrofurazone, khi i va

o c th sinh v t n to tha

nh ca

c ch t chuy n ho

a tng

ng (AOZ,
AMOZ, AHD, SEM) lin k t trong ca

c m t n ta i trong nhi u tu n sau khi s

du ng [12].
1.2. Tc dng ca cc cht khng sinh nhm nitrofuran
McCracken va

ca

c c ng s 1995, Nouws v Laurensen 1990 ch ra rng cc hp cht

nhm nitrofuran sau khi vo c th to thnh cc cht chuyn ha tng ng lin kt trong
cc m.
V mt c ch tc dng, cc cht chuyn ha t

nitrofuran km hm hoc ph hy
cc h thng men iu hoa trao i cht vi khun. Do vi khun khng pht trin va

khng sinh sn c na. Tc dng tt vi vi khun gram m v gram dng. ng thi con
tc dng c vi nguyn sinh ng vt, mt s cht c tc dng t t trong vi c gi t tr

giun a
[7].
V

i nng thp, nitrofuran c tc dng khng sinh, nng cao c tc dng dit
khun.
Theo nhiu bo co, nitrofuran tc dng rt tt trong vi c di t khu n salmonellosis,
colisepticeamia. Nitrofuran con c tc dng kch thch dinh dng. Trn nitrofuran vi thc
n, theo t l thch hp se

gip cho g v ln con tng trng nhanh.

Nitrofuran nh hng n sc khe con ngi nh gy ung th v t bin [7], khi
th c ph m co

t n d nitrofuran va

ca

c d n xu t cu

a no

.
Do h u h t ca

c n

c trn th gi

i a

c m s

du ng kha

ng sinh nho

m nitrofuran
trong chn nui . Vit Nam, b Nng nghi p va

pha

t tri n nng thn a

quy t i nh c m s

dng nitrofuran cho va

o trong th

c n chn nui [9]. EU quy nh yu cu gii hn nh

nht cn thc hin phng php (MRPL) i vi cc cht chuyn ha nhm nitrofuran l 1
g/kg [29].
1.3. D lng kha

ng sinh nho

m nitrofuran trong thc phm

T nm 2002 2003 nitrofuran thng xuyn c pht hin th y trong tht gia cm
v cc sn phm nui trng thy sn nhp khu vo cc nc EU t Thi Lan, Trung Quc,
i Loan, n , Vit Nam, Ecuador v Brazil [25].
Hn na, d lng nitrofuran cng c tm thy trong sn phm gia sc v gia cm
Chu u nh: B o Nha, , Hy Lp, Romania v Bulgari. Sau EU kim tra v cho
thy nitrofuran nhim trong cc sn phm c ngun gc t hn 9 quc gia trong nm 2007,
t l mc cao nht l t n (37%), Trung Quc (37%), Bangladesh (10%) v Thi Lan
(5%) trong mt lot c sn phm bao gm tm, mt ong v tht hp [25].
T thng 10/2006 n thng 5/2007, FDA lin tc pht hin thy sn nhp khu t
Trung Quc c nhim nitrofuran. Kt qu ly trn cc sn phm tm, c tr, c ba sa..cho
thy, 22/89 mu (chim 22%) b pht hin c cht cm nitrofuran trong tm [11].
Vit Nam theo kt qu kho st ca TS . Nguy n Qu c n, ph trng phong qun
l thuc, cc th y vo nm 2007 a

pht hin 5 m u thu

y sa

n nui l y ta i ao nui nhi m

SEM (3 m u tm, 1 m u ca

va

1 m u cua l t) v

i ha

m l ng t

0 3,55 ng/g. Nm 2008 pht

hi n 1 m u tm the

chn tr ng ta i Phu

My

Bnh nh nhim AOZ vi hm lng 12,6 ng/g;

2/754 m u cua l t ta i C n Gu c Long An nhi m SEM v

i ha

m l ng t

2 2,2 ng/g [1].

1.4. Cc phng php xc nh kha

ng sinh nhm nitrofuran

Hi n nay co

nhi u phng pha

p xa

c i nh kha

ng sinh nho

m nitrofuran , bao g m:
phng pha

p s c ky

lo

ng v

i detector UV , phng pha

p vi sinh (k thut Elisa ), phng

php sc k lng vi detector MS/MS.


CHNG 2: I TNG V PHNG PHP NGHIN CU

2.1. i tng, mc tiu v ni dung nghin cu
2.1.1. i tng, mc tiu nghin cu
i tng nghin cu l cc cht chuyn ha ca nitrofuran, bao gm: AOZ, AMOZ,
SEM v AHD.
Vt liu nghin cu l sn phm thc phm ti sng bao gm tht v gan ca gia sc
gia cm.
Mc tiu chung ca ti l nghin cu xy d ng phng php xc nh ng thi 4
cht chuyn ha nhm nitrofuran: AOZ, AMOZ, AHD, SEM trong thc phm ti sng bng
k thut sc k lng khi ph LC/MS/MS. Cc mc tiu c th nh sau:
- Xy dng phng php xc nh ng thi 4 cht chuyn ha ca nitrofuran trong thc
phm ti sng bng LC-MS/MS.
- ng dng phng php xc nh cc cht chuyn ha ca nitrofuran trong thc phm
ti sng c th trong tht ln, tht bo, tht g, gan.
2.1.2. Ni dung nghin cu
2.1.2.1. Xy dng phng php
Kho st phng php bao gm:
iu kin v thng s vn hnh my LC/MS/MS,
iu kin tch chit l y ch t phn ti

ch.
Thm nh phng php:
Gii hn pht hin LOD, gii hn nh lng LOQ,
Khong tuyn tnh,
chm ( lp li),
ng ( thu hi, chch).
2.1.2.2. ng dng phng php
p dng phng php mi xy dng xc nh d lng khng sinh nhm
nitrofuran c th l cc cht chuyn ha nhm nitrofuran trong thc phm ti sng ang ba

n
trn a bn H Ni.
2.2. Phng php nghin cu
2.2.1. Phng php sc k lng khi ph
2.2.2. Phng php x l mu
Kho st phng php x l mu bng chit lng lng v chi t pha r n
2.2.3. Thm nh phng pha

p
2.2.4. Phng php x l s liu
2.3. Ha cht v dng c nghin cu
2.3.1. Dng c v thit b
2.3.1.1. Thit b
- H thng sc k lng khi ph khi ph LC/MS/MS bao gm: HPLC 20 AXL ca
Shimadzu v khi ph ABI 5500 QQQ ca Aplied Biosystem: b phn bm dung mi, b
loi kh, b phn iu nhit.
- Ct sc k Agilent C
18
(150mm x 2,1mm x 3,5m).
- Ct chit pha rn SPE (Oasis HLB).
- My lc vortex VELP.
- My ng nht mu.
- My li tm MIKRO 22R.
- Cn phn tch (c c 0,1mg v 0,01mg).
- Cn k thut (c c 0,01g).
- My thi kh N
2
lm kh c iu nhit.
- B iu nhit
2.3.1.2 Dng c
- Cc c m dung tch 50, 100, 200 ml.
- ng ong dung tch 10, 100, 200 ml.
- Pipetman, pipet nha, pipet pasteur 200 l; 1000 l; 5 mlu cn.
- ng ly tm 50 ml, mng lc 0,2 m.
- Vial loi 1,8 ml.
- Bnh nh mc loi A dung tch 10, 25, 50, 100 ml.
- ng nghim thy tinh
2.3.2 Ha cht v cht chun
Cc loi ha cht s dng u thuc loi tinh khit phn tch
2.3.2.1 Cht chun
- AOZ 10 mg/l (Dr.EhrenstorferGmbH), tinh khit 99%
- AMOZ 10 mg/l (Dr.EhrenstorferGmbH), tinh khit 99%
- AHD.HCl (Dr.EhrenstorferGmbH), tinh khit 99%
- SEM.HCl (Dr.EhrenstorferGmbH), tinh khit 99,5%
- d
4
-AOZ (Dr.EhrenstorferGmbH), tinh khit 99%
- d
5
-AMOZ (Dr.EhrenstorferGmbH), tinh khit 99%
- d
2
-AHD.HCl 10 mg/l (USA), tinh khit 99%
-
13
C
15
N
2
-SEM.HCl 10 mg/l (USA), tinh khit 98%
2.3.2.2 Ha cht
- Methanol (Merck hoc tng ng, tinh khit 99,9%)
- Amoni acetate (Merck hoc tng ng, tinh khit 98%)
- 2-NBA (Merck hoc tng ng, tinh khit 99%)
- K
2
HPO
4
.3H
2
O (Merck hoc tng ng, tinh khit 99%)
- NaOH (Merck hoc tng ng, tinh khit 99,9%)
- HCl 37% (Merck hoc tng ng, tinh khit 99,9%)
- HCl 0,2M: Ht 17 ml HCl 37% vo bnh nh mc 1000 ml, nh mc n vch bng nc
ct.
- 2-NBA 1000 mg/l: cn 100 mg 2-NBA hoa tan v nh mc vo bnh 10 ml bng methanol.
Dung dch chun b hng ngy.
- K
2
HPO
4
0,2 M: cn 45,7 mg K
2
HPO
4
.3H
2
O hoa tan v nh mc vo bnh 1000 ml bng
nc ct 2 ln.
- NaOH 2 M: cn 8 g NaOH hoa tan v nh mc vo bnh nh mc 1000 ml bng nc ct
2 ln.
- CH
3
COONH
4
10 mM: cn 0,077 g CH
3
COONH
4
hoa tan v nh mc vo bnh nh mc
1000 ml bng nc ct 2 ln.
2.3.3 Pha ch cht chun
- Chun AHD 1000 mg/l: cn 13,2 mg 0,1 mg chun AHD.HCl hoa tan v nh mc bng
methanol vo bnh nh mc 10 ml. Bo qun - 20
0
C s

du ng c trong 6 thng.
- Chun AHD 10 mg/l: ht 100 l chun AHD 1000 mg/l nh mc bng methanol vo bnh
nh mc 10 ml. Bo qun - 20
0
C s

du ng c trong 6 thng.
- Chun SEM 1000 mg/l: cn 14,9 mg 0,1 mg chun SEM.HCl hoa tan v nh mc bng
methanol vo bnh nh mc 10 ml.
- Chun SEM 10 mg/l: ht 100 l chun SEM 1000 mg/l nh mc bng methanol vo bnh
nh mc 10 ml. Bo qun - 20
0
C s

du ng c trong 6 thng.
- Hn hp chun 4 cht (AOZ, AMOZ, SEM, AHD) 1 mg/l: ht ln lt 1 ml chun AOZ 10
mg/l, AMOZ 10 mg/l, AHD 10 mg/l, SEM 10 mg/l nh mc bng methanol vo bnh nh
mc 10 ml. Bo qun - 20
0
C s

du ng c trong 3 thng.
- Hn hp chun 4 cht (AOZ, AMOZ, SEM, AHD) 20 g/l: ht 200 l hn hp chun 4
cht (AOZ, AMOZ, SEM, AHD) nh mc bng methanol vo bnh nh mc 10 ml. Bo
qun - 20
0
C s

du ng c trong 1 thng.
- d
4
-AOZ 1000 mg/l: Cn 10 mg 0,1 mg d
4
-AOZ nh mc bng methanol vo bnh nh
mc 10 ml. Bo qun - 20
0
C s

du ng c trong 6 thng.
- d
4
-AOZ 10 mg/l: ht 100 l d
4
-AOZ 1000 mg/l nh mc bng methanol vo bnh nh mc
10 ml. Bo qun - 20
0
C s

du ng c trong 6 thng.
- d
5
-AMOZ 1000 mg/l : Cn 10 mg 0,1 mg d
5
-AMOZ nh mc bng methanol vo bnh
nh mc 10 ml. Bo qun - 20
0
C s

du ng c trong 6 thng.
- d
5
-AMOZ 10 mg/l: Ht 100 l d
5
-AMOZ 1000 mg/l nh mc bng methanol vo bnh nh
mc 10 ml. Bo qun - 20
0
C s

du ng c trong 6 thng.
- Hn hp ni chun (d
4
-AOZ, d
5
-AMOZ, d
2
-AHD.HCl,
13
C
15
N
2
-SEM.HCl) 1 mg/l: ht ln
lt 1 ml chun d
4
-AOZ 10 mg/l, d
5
-AMOZ 10 mg/l, d
2
-AHD.HCl 10 mg/l v
13
C
15
N
2
-
SEM.HCl 10 mg/l nh mc bng methanol vo bnh nh mc 10 ml. Bo qun - 20
0
C s

dng c trong 3 thng.
- Hn hp ni chun (d
4
-AOZ, d
5
-AMOZ, d
2
-AHD.HCl,
13
C
15
N
2
-SEM.HCl) 20 g/l: ht ln
lt 200 l hn hp ni chun (d
4
-AOZ, d
5
-AMOZ, d
2
-AHD.HCl,
13
C
15
N
2
-SEM.HCl) 1 mg/l
vo bnh nh mc 10 ml. Bo qun - 20
0
C s

du ng c trong 1 thng.


CHNG 3: KT QU V THO LUN

3.1. Kho st iu kin xc nh cc cht nhm nitrofuran bng LC/MS/MS
Phn tch d lng khng sinh nhm nitrofuran da trn vic phn tch cc cht
chuyn ha ca nhm nitrofuran. Cc cht chuyn ha ca nitrofuran c khi lng phn t
thp gy nhiu ph nn cao, hiu qu ion ha thp v khng c hiu cho s phn mnh (ch
yu l mt nc, NH
3
, CO
2
), nhy pht hin b ng MS tng i thp. V vy s dng 2-
nitrobezaldehyte dn xut cc cht chuyn ha nhm nitrofuran c c cc hp cht
d n xu t (NPAOZ, NPAMOZ, NPAHD, NPSEM) vi nhiu c tnh thun li.
3.1.1. Kho st cc iu kin o ph kh i
3.1.1.1. Kho st ion m
Cc cht NPAOZ, NPAMOZ, NPAHD, NPSEM c khi lng phn t nh v phn
cc. Qua tham kho mt s ti liu [12, 13, 14, 16, 17, 18, 19, 21, 22, 23, 26], chng ti tin
hnh kho st xc nh NPAOZ, NPAMOZ, NPAHD, NPSEM bng k thut ion ha phun
in t ESI vi ch bn ph ion dng. ti u ha iu kin khi ph, dng xylanh
500 l bm tng chun AOZ, AMOZ, AHD, SEM 500 ng/ml c dn xut bng 2-
nitrobenzaldehyte , sau a vo detector kho st. Chn ch kho st t ng i
vi tng cht, ti u ha tng ion m, thu c iu kin chy ngun ion ha ESI bng 3.1.
Bng 3.1: iu kin chy ngun ion ha ESI

Kh mn (CUR) 20 psi
Kh va chm (CAD) 8 psi
Th ion ha (IS) 5000 V
Nhit mao qun (TEM) 400
0
C
p sut kh 2 bn u phun (GS1) 50 psi
p sut ca lung kh nng (GS2) 40 psi
Trong k thut ion ha phun in t vi ch bn ph ion dng, cc ion m c
dng (MH)
+
v

i s kh i m = (M+1) theo pha

ng:
M
0
+ H
+
(MH)
+

Tin hnh kho st bn ph t cc ion m, kt qu c ch ra bng 3.2
Bng 3.2: Kt qu bn ph cc ion m
Chuyn ha nhm nitrofuran Khi lng phn t M Ion m (M+H)
NPAOZ 235 236
NPAMOZ 334 335
NPSC 208 209
NPAHD 248 249
d
4
-AOZ 239 240
d
5
-AMOZ 339 340
13
C-
15
N
2
SEM.HCl 211 212
D
2
-AHD.HCl 250 251
3.1.1.2. Kho st iu kin bn ph ion me thu c ion con
Detector s dng trong nghin cu ny l h kh i ph 2 ln, do pht hin ng
cht phn tch th vic la chn c ion con l rt quan trng. Ion con phi c tn hiu gp t
nht 10 ln so vi ion m. thu c mnh ion con c tn hiu cao cn phi chn c mc
nng lng bn ph thch hp. Dng xylanh 500 l bm tng chun c dn xut vo
detector khi ph v la chn 2 ion con c trng c c ng tn hiu cao nht nh tnh
v nh lng. Mnh ion con m/z c cng ln nht dng nh lng, mnh ion con
th 2 c cng thp hn dng xc nhn cht phn tch. i vi cc cht ni chun, la
chn 1 ion con c trng c cng cao nht. Kt qu thu c bng 3.3
Bng 3.3: Nng lng bn ph v cc ion con ca cc cht chuyn ha nitrofuran
Chuyn ha
nhm
nitrofuran
Ion m
(M+H)
+

Ion con DP (V) CE (eV) CXP (V)
NPAOZ 236
134 80 15 10
104 80 27 14
NPAMOZ 335
291 80 15 16
262 80 21 16
NPSEM 209
192 80 11 18
166 80 13 14
NPAHD 249
134 80 15 18
104 80 27 14
d
4
-AOZ 240 134 80 17 16
d
5
-AMOZ 340 296 80 15 16
13
C-
15
N
2

SEM.HCl
212 168 80 13 12
d
2
-AHD.HCl 251 134 80 15 14
Nh n xe

t:
Theo qui i nh cu

a Chu u 2002/657/EC, s i m nh n da ng (IP) c ti

nh i v

i ky

thu t
LC/MS/M, tng

ng v

i 1 ion me va

2 ion con, s i m nh n da ng (IP) l 4. Nh v y

phng pha

p a

ng c yu c u cu

a Chu u [ph lc 4]. Ph hp vi cc nghin cu

khc ca mt s tc gi nh tc gi Leitner, tc gi D.Tyler cu

a pho

ng thi

nghi m cu

a Anh
3.1.2. La chn ct tch
Cc cht nhm nitrofuran l cc cht phn cc, ng thi theo khuyn co ca nh
sn xut thit b khi ph th h pha ng s dng an ton vi thit b l cc dung mi phn
cc v phn cc trung bnh nh methanol, acetonitrile, nc, acid formic (0 n 1%, amoni
acetate (0 ti 1%)ct tch nn s dng l ct tch pha o. Do chng ti la chn ct
tch pha o C18 v

i ca

c thng s d

i y cho ca

c nghin c

u ti p theo:
- Ct C18 ca Agilent c:
- Chi u da

i 150mm,
- ng knh 2,1mm,
- C

ha t 3,5m.
3.1.3. Kho st chng trnh gradient pha ng
Cc dn xut ca nhm nitrofuran c cu trc gn ging nhau, nn s dng ch ra
gii ng dong (isocratic) khng ph hp. Chng ti tin hnh kho st mt s chng trnh
ra gii gradient. Qua tham kho mt s ti liu [12, 16, 17, 24] chng ti s dng pha ng
knh A: Amoniacetat 10 mM, knh B l methanol. C nh cc iu kin sc k:
- Ct C18 (150 mm x 2,1 mm x 3,5 m),
- Pha ng: knh A: amoniacetat 10 mM, knh B: methanol,
- Tc dong: 0,4 ml/pht,
- Mu phn tch: hn hp d n xu t nitrofuran, nng : 20 ng/ml.
Ti n ha

nh thi

nghi m theo:
a) Chng tri

nh Gradient 1, thu c s c nh trong hi

nh 3.1 :
Thi gian (pht) 0,01 12,00 14,00 15,00 20,00
% MeOH 20 90 90 20 20


Hnh 3.1: s c h n h p chu n khi cha y ch gradient 1 nng 20 ng/ml
b) Chng tri

nh Gradient 2, thu c s c trong hi

nh 3.2:
Thi gian (pht) 0,01 5 8 9 12
% MeOH 20 90 90 20 20


Hnh 3.2: s c h n h p chu n khi cha y ch gradient 2 nng 20 ng/ml
c) Chng tri

nh Gradient 3, thu c s c trong hi

nh 3.3 :
Thi gian (pht) 0,01 8 10 13 13,01
% MeOH 20 90 90 20 20


Hnh 3.3: s c h n h p chu n khi cha y ch gradient 3 nng 20 ng/
d) Chng tri

nh Gradient 4, thu c s c trong hi

nh 3.4:
Thi gian (pht) 0,01 10 12 15 15,01
% MeOH 20 90 90 20 20


Hnh 3.4: s c h n h p chu n khi cha y ch gradient 4 nng 20 ng/ml
e) Chng tri

nh Gradient 5, thu c s c trong hi

nh 3.5:


Thi gian (pht) 0,01 12,00 13,00 13,01
16,00
% MeOH 20 90 90 20
20


Hnh 3.5: s c h n h p chu n khi cha y ch gradient 5 nng 20 ng/ml
Bn lun :
- Khi tng nng MeOH trong khong thi gian ngn (chng tri

nh gradient 1, gradient 2,
gradient 3, gradient 4) pc ca AOZ rt xu v b ch pc. Hi n t ng che

pic na

y la

do ha

m
l ng MeOH cao , AOZ t n ta i ng th

hai da ng la

amin b c nh t R -NH
2
v dng R -
NH
3
+
. Do o

, n ng MeOH c n pha

i c kh ng ch phu

h p , ng nghi

a v

i tng n ng
CH
3
COONH
4
chuyn ton b dng amin thnh dng R-NH
3
+
.
- Chng trnh gradient 5 pic nhn, cn xng va

co

ti

n hi u pi

c ro

ra

ng . Do chng ti la
chn chng trnh gradient 5 cho cc nghin cu tip theo.
3.1.4. Kho st quy trnh chit mu
Qua tham kho mt s ti liu [12, 13, 14, 16, 17, 18, 19, 21, 22, 23, 26] c mt s
quy trnh v ang c ng dng tch chit cc cht nhm nitrofuran bao gm : quy
trnh x l mu bng chit lng lng, quy trnh x l mu bng chit pha rn. Do chng
ti tin hnh kho st 2 quy trnh chit mu sau y ti n ha

nh thi

nghi m.
3.1.4.1. La chn quy trnh x l mu bng chit lng lng
D kin quy trnh x l mu nh sau:
Cn 2 g mu vo ng ly tm

Thm 10 ml HCl 0,2M v 240 l 2-NBA 10mg/ml

Thm 100 l hn hp ni chun vo mi ng

Thm dung dch chun lm vic vo mu

y ng, lc vortex, trnh nh sng qua m trong b iu nhit 40
0
C

Trung hoa axit bng 10 ml K
2
HPO
4
0,2M, sau thm 800 l NaOH 2M, lc vortex 20 giy

Ly tm 4500 rpm trong 15 pht l y dung di ch m u

Chit lp 2 ln mi ln bng 4 ml ethylactetate, ly tm ht ly lp trn thi kh bng N
2

Hoa tan cn bng 1 ml H
2
O:MeOH (60:40)

Chuyn mu vo vial v em phn tch b ng LC/MS/MS

3.1.4.2. La chn quy trnh x l mu bng chit pha rn
Th c hi n theo s sau:

Cn 2 g mu vo ng ly tm

Thm 10 ml HCl 0,2M v 240 l 2-NBA 10mg/ml

Thm 100 l hn hp ni chun vo mi ng

Thm dung dch chun lm vic vo mu

y ng, lc vortex, trnh nh sng qua m trong b iu nhit 40
0
C

Trung hoa mu bng 10 ml K
2
HPO
4
0,2M, sau thm 800 l NaOH 2M, lc vortex 20 giy

Ly tm 4500 rpm trong 15 pht l y dung di ch m u

Hot ha ct SPE (Oasis HLB): 3 ml EtAc 3 ml MeOH 2 x 2,5 ml H
2
O

Np mu vo ct, ra loi cht bn bng 2 x 2,5 ml H
2
O

Ht chn khng cho kh ct SPE

Ra gii ch t phn ti

ch bng 4 ml EtAc vo ng nghim

Thi kh bng N
2

Hoa tan cn bng 1 ml H
2
O:MeOH (60:40)

Chuyn mu vo vial v em phn tch b ng LC/MS/MS
X

ly

m u b ng 2 quy tri

nh chi t, so sa

nh thu h i cu

a 2 quy tri

nh chit c ch ra bng
3.6:
Bng 3.6: So sa

nh thu hi ca 2 quy trnh chit:

Tht ln thm chun
Chit SPE Chit lng-lng
H (%) H (%)
AOZ 20 ng/g
87,7 42,1
AMOZ 20 ng/g
92,7 45,0
AHD 20 ng/g 73,3 33,7
SEM 20 ng/g
70,5 30,6

Nh n xe

t: Nhn vo bng trn ta thy quy trnh chit SPE cho thu hi cao hn so
vi quy trnh chit lng lng v p ng c quy nh ca Chu u 2002/657EC. Do
chng ti chn quy trnh chit SPE cho cc nghin cu tip theo
3.2. Th m i nh phng pha

p
3.2.1 Tinh c hiu
Phng pha

p trn s

du ng ky

thu t s c ky

lo

ng kh i ph 2 l n, th c hi n b n pha

ion
m m/z, i nh l ng theo ion con ta o tha

nh . Theo ca

ch ti

nh i m IP thi

i 1 ion me va

2 ion
thu c 4 i m IP, nh v y ph ng pha

p co

ti

nh c hi u a

ng c yu c u cu

a chu
u 2002/657/EC [32].
3.2.1. Kho st lp ng chun
3.2.1.1. ng chun ca cc cht nhm nitrofuran s dng ni chun ( phn tch mu
thc)
Bng 3.7: Phng trnh hi quy 4 nitrofuran s dng ni chun
TT Cht Phng trnh hi qui H s R
1 AOZ y = (0,21426 0,039142) + (0,31607 0,016822)x 0,9992
2 AMOZ y = (0,01807 0,044860) + (0,45988 0,019293)x 0,9995
3 AHD y = (0,12446 0,119312) + (1,22034 0,051273)x 0,9995
4 SEM y = (0,26595 0,262915) + (1,44626 0,112983)x 0,9984

Nhn xt: t l din tch pic nitrofuran/din tch pic ni chun (IS) trong khong 0,5 5 ng/ml
c h s tng quan R
2
> 0,99.
3.2.1.2. ng chun ca cc cht nhm nitrofuran khng s dng ni chun ( nh gi
hiu sut thu hi)
Bng 3.8: Phng trnh hi quy ca 4 nitrofuran khng s dng ni chun
TT Cht Phng trnh hi qui H s R
1 AOZ y = (14569 32560) + (307751 13990)x 0,9994
2 AMOZ y = (42233 53499) + (363895 22988)x 0,9989
3 AHD y = (6879 4425) + (36695 1902)x 0,9993
4 SEM y = (592 4175) + (35485 1793)x 0,9993
Nhn xt: Din tch nitrofuran vi nng trong khong 0,5 5 ng/ml c h s tng quan
R
2
> 0,99.
3.2.2. Gii hn pht hin v gii hn nh lng ca phng php
LOD cu

a ca

c ch t kha

ng sinh nho

m nitrofuran n m trong khoa

ng t

0,100 0,378
ng/g; LOQ t

0,300 1,13 ng/g a

ng c yu c u cu

a Chu u 2002/657/EC v nha y

ca phng php. Ph hp vi cc nghin cu tng t.
3.2.3. lp li v thu hi
Thc hin thm chun vo mu tht ln khng cha nitrofuran cc mc nng
(MRPL; 1,5MRPL; 2MRPL) i vi mi cht l: 1 ng/g; 1,5 ng/g; 2 ng/g. Phn tch lp li 6
ln mi mu.
Bn lun: Ta th y thu h i trung bi

nh ca cc cht t 80,5 106%, l p la i t t (%RSD <

13,7%) a t yu c u theo quy i nh cu

a Chu u 2002/657/EC. T

ca

c i u ki n a

c t i
u

trn u

m ba

o cho phe

p phn ti

ch xa

c i nh 4 ch t nho

m nitrofuran.
3.3. ng dng phng pha

p trong phn ti

ch th c ph m
Trn c s phng php xy dng v thm nh, chng ti ng dng phn tch
mt s loi thc phm ti sng trn a bn H Ni. Cc kt qu c trnh by bng 3.16
Bng 3.16: Kt qu phn tch mu thc theo phng pha

p thm chun

STT Mu Nitrofuran
Lng thm
vo (ng)
Lng tm
thy (ng)
R%
1
Tht ln ch
Nguyn Cao
AOZ
0 KPH -
1 0,87 87
AMOZ
0 KPH -
1 0,96 96
AHD
0 KPH -
1 0,78 78
SEM
0 KPH -
1 0,72 72
2
Tht g ch Nguyn
Cao
AOZ
0 KPH -
1 1,12 112
AMOZ
0 KPH -
1 1,02 102
AHD
0 KPH -
1 0,95 95
SEM
0 KPH -
1 0,89 89
3
Tht bo ch Mai
ng
AOZ
0 KPH -
1 0,99 99
AMOZ
0 KPH -
1 1,05 105
AHD
0 KPH -
1 1,03 103
SEM
0 KPH -
1 1,09 109
4 Gan ch Mai ng
AOZ
0 KPH -
1 0,88 88
AMOZ
0 KPH -
1 0,97 97
AHD
0 KPH -
1 0,75 75
SEM
0 KPH -
1 0,69 69
5
Tht ln ch Cu
Giy
AOZ
0 KPH
1 1,02 102
AMOZ
0 KPH
1 0,79 79
AHD
0 KPH
1 1,01 101
SEM
0 KPH
1 1,03 103
6
Tht bo ch Cu
Giy
AOZ
0 KPH
1 0,92 92
AMOZ
0 KPH
1 0,88 88
AHD
0 KPH
1 0.79 79
SEM
0 KPH
1 1,06 106
7
Tht ln ch Ngc
H
AOZ
0 KPH
1 1,05 105
AMOZ
0 KPH
1 1,11 110
AHD
0 KPH
1 0,98 98
SEM
0 KPH
1 0,67 67
8
Tht g ch Ngc
H
AOZ
0 KPH
1 0,76 76
AMOZ
0 KPH
1 0,82 82
AHD
0 KPH
1 0,97 97
SEM
0 KPH
1 0,73 73
KPH: khng pht hin < LOD ca phng php,
R %: Phn trm thu hi.
Bn lun:
Qua qu trnh phn tch mt s mu thc trn a bn H Ni, khng pht hin mu
no c cha cc cht nhm nitrofuran.
thu hi hu ht cc cht t 67 112% a

ng c qui i nh cu

a Chu u
2002/657/EC. T

k t qua

thu h i na

y cho th y co

th m

r ng phng pha

p cho ca

c sa

n
ph m th c ph m ti s ng kha

c.
3.4 Hng pht trin ca ti
Trong bn lun vn ny, do iu kin con hn ch nn chng ti ch xc nh c d
lng khng sinh nhm nitrofuran bng LC/MS/MS trong mt s mu thc phm ti sng
(tht g, tht ln, tht bo, gan). Phng php con c th c m rng phn tch trong nhng
dng nn mu khc nhau nh: thc n chn nui, mu sinh hc, mu saV vy rt cn c
nhng nghin cu tip theo pht trin phng php va

p dng phu c vu phn ti

ch m u va

o
thc tin.


KT LUN

Trn c s nghin cu cc iu kin thc nghim, vi mc ch ng dng ky

thu t sc
k lng khi ph LC/MS/MS tch v xc nh cc khng sinh nhm nitrofuran trong mu
thc phm ti sng, chng ti a

thu c cc kt qu sau:
1. nghin cu c cc iu kin chy sc k lng khi ph xc nh 4 cht
chuyn ha nhm nitrofuran bao gm:
- Kho st c cc iu kin o ph kh i (th phn nhm (DP), nng lng va chm
(CE)..) thu c cc ion kh i c trng ca tng cht nitrofuran.
- Kho st cho n c cc iu kin sc k lng: ct sc C18 Agilent (150mm 2,1mm
3,5m); pha ng v

i knh A amoniacetat 10mM trong nc va

knh B l metanol;
chng trnh ra gii gradient.
2. kho st c iu kin tch v lm sch cc nitrofuran ra khi nn mu tht ln
bng chit pha rn vi ct chit oasis HLB.
3. thm nh cc thng s ca phng php bao gm:
- Khong tuyn tnh: trong khong t 0,5 5 g/kg vi h s tng quan tuyn tnh R
2

> 0,996.
- Gii hn pht hin AOZ (0,100 g/kg), AMOZ (0,119 g/kg), AHD (0,223 g/kg),
SEM (0,378 g/kg), p ng yu cu v nhy phn tch ca

c ch t nho

m
nitrofuran.
- thu hi trung bi

nh t

80,5 106% cho thy phng php c ng tt.

- lch chun tng i thp (RSD% < 13,7%) cho thy phng php c lp li
v chnh xc cao.
4. ng dng phng php phn tch cc mu thc phm ti cc ch trn a bn
H Ni, cho thy phng php c th ng dng phn tch xa

c i nh dn xut nhm
nitrofuran trong thc phm ti sng.



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