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B GIO DC V O TO

B Y T

TRNG I HC DC H NI

BI TH LUYN

XY DNG QUY TRNH XC NH


D LNG MT S CHT NHM
QUINOLON TRONG THC PHM BNG
K THUT LC-MS/MS

LUN VN THC S DC HC

H NI 2014

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B GIO DC V O TO

B Y T

TRNG I HC DC H NI

BI TH LUYN

XY DNG QUY TRNH XC NH


D LNG MT S CHT NHM QUINOLON
TRONG THC PHM BNG K THUT LC-MS/MS

LUN VN THC S DC HC

CHUYN NGNH: KIM NGHIM THUC C CHT


M S: 60720410

Ngi hng dn khoa hc:


1. PGS.TS Nguyn Tng Vy
2. Ths. NCS Cao Cng Khnh

H NI 2014

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LI CM N
c th hon thnh lun vn, ti nhn c s hng dn v gip v mi
mt t cc thy c, ng nghip, gia nh v bn b.
Nhn dp ny, ti xin by t lng knh trng v bit n su sc ti:
PGS.TS. Nguyn Tng Vy Ph Trng Phng SH- H Dc H Ni
Th.S NCS Cao Cng Khnh Vin KN ATVSTPQG
tn tnh gip ti trong sut qu trnh thc hin v hon thnh ti ny.
ng thi ti xin cm n cc cn b Vin Kim Nghim An ton v sinh thc
phm Quc gia lun sn lng gip v to mi iu kin thun li cho ti trong
qu trnh thc nghim.
Cui cng ti xin gi li cm n ti nhng ngi thn trong gia nh v bn b
lun ng vin v nhit tnh gip ti trong qu trnh thc hin ti.
Thi Nguyn, ngy 20 thng 08 nm 2014
Tc gi

Bi Th Luyn

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MC LC
CC K HIU VIT TT
DANH MC CC BNG
DANH MC CC HNH
t vn ..................................................................................................................1
Chng 1. TNG QUAN...........................................................................................1
1.1. Vi nt v Quinolon.............................................................................................3
1.1.1. Cu to chung.....................................................................................................3
1.1.2. Phn loi ............................................................................................................3
1.1.3. C ch tc dng..................................................................................................3
1.1.4. Tc dng ph ................................................................................................... 3
1.1.5. c tnh ca cc quinolon nghin cu ................................................................4
1.2. Tng quan v sc k lng hiu nng cao v chun b mu cho phn tch sc
k ...............................................................................................................................5
1.3. Nghin cu v tn d quinolon trong thc phm ............................................12
1.3.1. Tnh hnh s dng khng sinh trong chn nui ................................................12
1.3.2. Cc nghin cu v tn d quinolon trong thc phm trong nc ..................13
1.3.3. Cc nghin cu v tn d quinolon trong thc phm ngoi nc...................14
1.3.4. Mt s quy nh v gii hn tn d quinolon trong thc phm .........................16
Chng 2. I TNG V PHNG PHP NGHIN CU
2.1. i tng nghin cu ca ti .......................................................................20
2.2. Thit b, dng c, ho cht................................................................................20
2.2.1. Thit b, dng c...............................................................................................20
2.2.2. Thuc th, ha cht ..........................................................................................20
2.3. Ni dung v phng php nghin cu .............................................................20
2.3.1. Nghin cu cc iu kin LC-MS/MS ..............................................................20
2.3.2. Nghin cu v la chn quy trnh x l mu ...................................................22
2.3.3. Thm nh li phng php xy dng..........................................................24
2.3.4. Thc nghim xc nh quinolon trn mu tht, c ............................................24
2.4. Tnh ton kt qu, x l s liu .........................................................................25

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Chng 3. KT QU NGHIN CU
3.1. Kt qu kho st v la chn cc thng s cho LC-MS ..................................27
3.1.1. Xc nh cc thng s ca detector khi ph (MS ............................................27
3.1.2. Xc nh cc thng s ca phn LC tch cc cht nhm Quinolon ...............28
3.2. Xc nh k thut x l mu.............................................................................29
3.2.1. Quy trnh tch chit cc cht cn phn tch khi nn mu .................................29
3.2.2. Quy trnh lm sch v lm giu mu qua SPE...................................................31
3.2.3. Quy trnh x l mu c la chn ...................................................................33
3.3. Thm nh phng php xy dng .............................................................34
3.3.1. Tnh thch hp ca h thng ............................................................................34
3.3.2. c hiu/ chn lc ..................................................................................35
3.3.3. Khong tuyn tnh v ng chun .................................................................35
3.3.4. Gii hn pht hin (LOD) v gii hn nh lng (LOQ) .................................39
3.3.5. lp li v thu hi ca phng php.........................................................39
3.4. Phn tch trn mu thc nghim ......................................................................44
Chng 4. BN LUN
4.1. Xy dng quy trnh k thut ............................................................................46
4.1.1. Cc iu kin phn tch trn h thng LC-MS/MS............................................46
4.1.2. iu kin x l mu .........................................................................................47
4.2. Thm nh quy trnh xy dng...................................................................47
Kt lun ...................................................................................................................50
Kin ngh .................................................................................................................52
TI LIU THAM KHO

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DANH MC CC K HIU, CH VIT TT

Ch vit tt
HPLC

Ting Anh

Ting Vit

High Performance Liquid

Sc k lng hiu nng cao

Chromatography
LC-MS/MS

Liquid Chromatograph

Sc k lng ghp 2 ln khi

Tandem Mass Spectrometer

ph

LOD

Limit of Detection

Gii hn pht hin

LOQ

Limit of Quantitative

Gii hn nh lng

SPE
CE

Solid phase extraction


Capillary electrophoresis

Chit pha rn

ESI

Electrospray ionization

EU

European Union

APCI

AOAC
IS

in di mao qun
Ion ha phun in t
Lin minh Chu u

Atmospheric pressure chemical

ion ho ho hc p sut kh

ionization

quyn

Association of Official Analytical.

Hip hi cc nh ho phn tch

Chemists
Internal Standard

Ni chun

Mu PT

Mu phn tch

TL

Tht ln

TG

Tht g

C01

C 01

RSD

Relative Standard Deviation

lch chun tng i

Ppb

Part per billion

Phn t

Ppm

Part per million

Phn triu

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DANH MC CC BNG
Bng 1.1: Gii hn tn d ti a ca cc quinolone theo quyt nh s 2377/90/EC ca
y ban Chu u....15
Bng 1.2 : Danh mc cc quinolone hn ch s dng trong sn xut kinh doanh thy
sn..........16
Bng 3.1. Thng s chung ca detector khi ph.............................27
Bng 3.2 Thng s chi tit ca 4 Quinolon...27
Bng 3.3 Gradient pha ng khi s dng ct Symmetry.....28
Bng 3.4 Gradient pha ng khi s dng ct Xbridge..........28
Bng 3.5: Kt qu th nghim dung mi chit mu khi phn tch Norfloxacin....29
Bng 3.6: Kt qu th nghim dung mi chit mu khi phn tch Ciprofloxacin.....30
Bng 3.7: Kt qu th nghim dung mi chit mu khi phn tch Enrofloxacin......30
Bng 3.8: Kt qu th nghim dung mi chit mu khi phn tch Flumequin.....30
Bng 3.9: Kt qu th nghim ct SPE khi phn tch Norfloxacin.......31
Bng 3.10: Kt qu th nghim ct SPE khi phn tch Ciprofloxacin......32
Bng 3.11: Kt qu th nghim ct SPE khi phn tch Enrofloxacin...32
Bng 3.12: Kt qu th nghim ct SPE khi phn tch Flumequin.......33
Bng 3.13. Kt qu phn tch tnh thch hp ca h thng....34
Bng 3.14: Kt qu phn tch dy chun Norfloxacin...36
Bng 3.15: Kt qu phn tch dy chun Ciprofloxacin...37
Bng 3.16: Kt qu phn tch dy chun Enrofloxacin.....37
Bng 3.17: Kt qu phn tch dy chun Flumequin.38
Bng 3.18: LOD v LOQ ca cc cht phn tch tnh trn nn mu tht...39
Bng 3.19: lp li v thu hi trn ct SPE HLB ca Norfloxacin..40
Bng 3.20: lp li v thu hi trn ct SPE HLB ca Ciprofloxacin...40
Bng 3.21: lp li v thu hi trn ct SPE HLB ca Enrofloxacin....41
Bng 3.22: lp li v thu hi trn ct SPE HLB ca Flumequin....41
Bng 3.23: lp li v thu hi trn ct SPE C18 ca Norfloxacin...42
Bng 3.24: lp li v thu hi trn ct SPE C18-E ca Ciprofloxacin42
Bng 3.25: lp li v thu hi trn ct SPE C18-E ca Enrofloxacin..41

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Bng 3.26: lp li v thu hi trn ct SPE C18-E ca Flumequin....41


Bng 3.27 : Kt qu phn tch 20 mu tht g (n v l g/kg).....44
Bng 3.28: Kt qu phn tch 20 mu tht ln (n v l g/kg).......44
Bng 3.29: Kt qu phn tch 20 mu c nc ngt (n v l g/kg).....44

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DANH MC CC HNH

Hnh 1.1 Cu trc c bn ca nhm quinolon .. .3


Hnh 1.2. Cng thc cu to ca Ciprofloxacin .....4
Hnh 1.3. Cng thc cu to ca Enrofloxacin ......5
Hnh 1.4. Cng thc cu to ca Norfloxacin ...5
Hnh 1.5. Cng thc cu to ca Flumequin .5
Hnh 3.1: Sc k th nghim dung mi chit mu khi phn tch norfloxacin..29
Hnh 3.2: Sc k th nghim ct SPE khi phn tch Norfloxacin32

Hnh 3.3: Sc k khi phn tch tnh thch hp ca h thng...35


Hnh 3.4: th ng chun ca Norfloxacin (tri) v Ciprofloxacin (phi).36
Hnh 3.5: Sc k khi xy dng ng chun ca norfloxacin.37
Hnh 3.6: th ng chun ca Enrofloxacin (tri) v Flumequin (phi)....38
Hnh 3.7: sc k ca norfloxacin khi x l mu qua ct SPE HLB .....40
Hnh 3.8: Sc k ca mu thc t c (hoc khng c) norfloxacin..45

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T VN
An ton v sinh thc phm lun c t ln hng u trong cng tc bo v
sc kho con ngi. Trong chn nui hin nay, vn s dng thuc khng sinh
(trong thc n, iu tr bnh gia sc, gia cm) l rt ph bin v c coi l mt tin
b ca cng ngh sinh hc. Tuy nhin, vic lm dng thuc khng sinh ni chung
v nhm Fluoroquinolon ni ring c th lm ngun thc phm cung cp cho con
ngi cn cha mt lng nh cht khng sinh, ngi s dng loi thc phm ny
trong thi gian di c th gy nguy hi cho sc kho.
Trong tin trnh gia nhp WTO, th trng hng ho ca Vit nam c m
rng, c bit trong lnh vc xut khu cc mt hng nng sn, thu sn. Tuy nhin,
do vic kim sot d lng ho cht khng sinh c hi n sc kho ngi tiu
dng cha c thc hin nghim tc nn mt s l hng b th trng xut nhp
khu pht hin khng sinh c hi vn cn cao. Tnh trng trn khng ch gy thit
hi ln v kinh t cho cc doanh nghip m cn nh hng nghim trng n uy tn
cht lng hng Vit nam trn th trng th gii v sc khe ngi tiu dng.
Quinolon c ph tc dng dit khun rng, hiu qu cao nn chng c s
dng rng ri cha bnh cho ngi, gia cm v thy sn trong cng nghip.
Trong qu trnh nui dng, nu khng tun th v thi gian dng thuc v thi
gian sn xut hoc ch bin th s dn n lng Quinolon cn li trong tht gia cm
hay thy sn. iu ny s gy rt nhiu mi nguy him. c bit l gy ra hin
tng khng thuc ca mt s vi khun nh: Salmonella, Campylobacter spp,
Escherichia coli,... Hu qu l gim hiu qu ca Quinolon i vi cc chng vi
khun trn, gy kh khn v tn km trong iu tr cho con ngi.
bo v quyn li ca ngi tiu dng, chu u, t chc EU thit lp
gii hn ln nht (MRLs) i vi d lng thuc trong ngun thc phm khi cung
cp cho con ngi vo nm 1990. Theo s kim tra v nh hng ca cc chuyn
gia phng th nghim, EU cng b Council directive 96/23/EC in 1996 [23] ,
trong quy nh r hm lng khng sinh theo tng loi thc phm v tng loi
thuc, v d nh vi enrofloxacin trong c, gan, thn ca b, ln, gia cm (g, vt)
l 30g/kg; trong sa b l 100 g/kg; chu M, Hoa k v cc nc Bc M

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cm s dng khng sinh h Fluoroquinolon. B Thy sn ra quyt nh s


07/2005/QD-BTS ban hnh ngy 24/02/2005 quy nh danh mc 17 loi khng sinh
cm s dng v danh mc 34 loi hn ch s dng trong c nhm Quinolon [10]
v Quyt nh s 26/2005/QD-BTS ban hnh ngy 18/8/2005 b sung nhm
Quinolon vo danh mc cm s dng trong sn xut, kinh doanh thy sn xut khu
vo th trng Bc M v Hoa K [11].
V vy chng ti thc hin ti: Xy dng quy trnh xc nh d lng mt
s cht nhm Quinolon trong thc phm bng k thut LC-MS/MS, vi 2 mc tiu
chnh nh sau:
1. Xy dng quy trnh xc nh d lng ciprofloxacin, enrofloxacin, norfloxacin ,
flumequin trong thc phm bng k thut sc k lng ghp 2 ln khi ph LC-MS/MS.
2. ng dng quy trnh xy dng s b kho st d lng Quinolon trong mt
s loi thc phm (Tht, c) trn th trng H Ni.

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Chng 1. TNG QUAN


1.1. Vi nt v Quinolon [5]
1.1.1. Cu to chung
i khi v tr 8 l N. Vy a s chng l dn cht ca acid 1,4-dihydro-4oxo-quinolein-3-carboxylic.
O
COOH

R1

6
7

1
8

R2

Hnh 1.1 Cu trc c bn ca nhm quinolon


1.1.2. Phn loi
- Quinolon th h I: Gm cc cht khng gn F (tr flumequin), hin nay hu nh
ch dng acid nalidixic.
- Quinolon th h II: c dng t nm 1985, gm cc cht c gn F (t nht l v
tr 6) hay cc fluoroquinolon c ph rng trn c vi khun gram (-) v gram (+), vi
hot tnh mnh:
+ Tc dng trn c vi khun gram (-) khng acid nalidixic, trc khun m xanh,
nhiu vi khun gram (-) kh tr khc.
+ Trn cc vi khun gram (+) th c bit l t cu vng, S.epidermitis khng
gentamycin, lin cu, cu khun rut (Enterococus) v cc vi khun k kh.
+ Trn cc vi khun pht trin trong t bo, v mt s loi vi khun c bit nguy
him (nh Legionella, Chlamydia, Ureoplasma, Mycoplasma, cc vi khun khng
alcol...)
1.1.3. C ch tc dng
Theo mt hoc hai c ch sau v u cho tc dng dit khun:
- c ch ADN-gyrase, l enzym tham gia vo qu trnh tng hp acid nhn.
- To phc vi kim loi ha tr hai ca cc protein cha cc kim loi ny, ng thi
phc ion ha thnh ion phc c hot tnh cao vi cc enzym chuyn ha
1.1.4. Tc dng ph
Tc dng ph ca thuc ch yu l ln d dy - rut, thn kinh trung ng v da.
- Tiu ha: Bun nn, nn, a chy, au bng, ri lon tiu ho

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- Da: Ni ban, nga, vim tnh mch nng.


- C xng: au cc khp, sng khp. au c, vim gn (gn gt) v m bao
quanh. C mt vi trng hp b t gn, c bit l ngi cao tui khi dng phi
hp vi corticosteroid.
- Thn kinh trung ng: Cn co git, l ln, ri lon tm thn, hoang tng, mt
ng, trm cm, lon cm ngoi vi, ri lon th gic k c o gic, ri lon thnh gic,
tai, ri lon v gic v khu gic, tng p lc ni s.
- Da: Hi chng da - nim mc, vim mch, hi chng Lyell, ban da thnh nt,
ban a dng tit dch.
- Gan: c bo co v mt vi trng hp b hoi t t bo gan, vim gan, vng
da mt.
- Tit niu - sinh dc: C tinh th niu khi nc tiu kim tnh, i ra mu, suy thn
cp, vim thn k.
- Khc: Nhy cm vi nh sng khi phi nng, ph thanh qun hoc ph phi, kh
th, co tht ph qun.
1.1.5. c tnh ca cc quinolon nghin cu
Ciprofloxacin
Tn gi: 1-Cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylic
acid
HN

Cng thc ha hc: C17H18FN3 O3

Trng lng phn t: 331.35 g/mol


Nhit nng chy: 318-3200 C

COOH
O

Hnh 1.2. Cng thc cu to ca Ciprofloxacin


Tnh cht: l bt kt tinh mu vng nht, tan mt phn trong nc, tan rt t trong ethanol,
methylen chlorid. Tan tt trong dung dch acid acetic long, c hai gi tr pKa l 6.0 v 8.8
Enrofloxacin
Enrofloxacin l khng sinh thuc nhm fluoroquinolone, loi PQ, c th chng vi
khun gram dng v gram m.

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Tn gi: acid 1-Cyclopropyl-7-(4-ethyl-1-piperazinyl)-6-fluoro-1,4-dihydro-4-oxo3-quinolinecarboxylic.


Cng thc ha hc: C19H22FN3O3

H3C

Trng lng phn t: 359.4 g/mol


Nhit nng chy: 219-2210C

COOH
O

Hnh 1.3. Cng thc cu to ca Enrofloxacin


Tnh cht: l tinh th mu vng nht, tan nh mt phn trong nc pH = 7,
c hai gi tr pKa: khong 5 v 8-9
Norfloxacin
Tn gi: acid 1-ethyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinolin-3-carboxylic
Cng thc ha hc C16H18FN3O3
Trng lng phn t: 319,3

Hnh 1.4. Cng thc cu to ca Norfloxacin


Tnh cht : Bt kt tinh mu trng hoc vng nht, d ht m, nhy cm vi nh
sng. Rt kh tan trong nc, kh tan trong aceton v ethanol 96%.
Flumequin

H
CH 3

Tn gi: acid 9-Fluoro-5-methyl-1-oxo-6,


N

7-dihydro-1H,5H-benzoquinolizin-2-carboxylic
Cng thc ha hc: C14H12FNO3

COOH
O

Trng lng phn t: 261,3

Hnh 1.5. Cng thc cu to ca Flumequin

Tnh cht: Bt trng, khng tan trong nc, t tan trong methylen chlorid, rt
t tan trong methanol, d tan trong kim
1.2. Tng quan v sc k lng hiu nng cao v chun b mu cho phn tch sc k
[2], [7]
Qu trnh tch trong k thut HPLC l do qu trnh vn chuyn v phn b
ca cc cht tan gia hai pha khc nhau (pha ng, pha tnh). Dung dch cc cht

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phn tch khi vo ct s c hp th hay lin kt vi pha tnh ty thuc vo bn


cht ca ct v ca cht cn phn tch. Khi pha ng dch chuyn vi mt tc
nht nh qua ct sc k s y cc cht tan b pha tnh lu gi ra khi ct. Ty theo
bn cht ca pha tnh, bn cht ca cht tan, bn cht ca dung mi m qu trnh
ra gii tch c cc cht ra khi nhau. Cc cht sau khi ra khi ct s c pht
hin bi detector v c chuyn qua b x l kt qu. Kt qu cui cng c a
ra my in hoc hin th trn mn hnh. Nu ghi qu trnh tch sc k ca hn hp
nhiu thnh phn, ta s c mt sc gm nhiu pic. Qu trnh tch sc k tt th
hn hp c bao nhiu thnh phn th s c by nhiu pic ring bit trn sc .

1.2.1. Kt ni sc k lng hiu nng cao (HPLC) vi ph khi lng (MS)


Trong nhiu qu trnh phn tch, cc hp cht quan tm thng nm trong
mt hn hp phc tp, v vai tr ca k thut sc k l thit lp s tch cc thnh
phn ca hn hp, cho php nh tnh hoc nh lng chng. V mt nh tnh,
nhc im chnh ca k thut sc k trong qu trnh tch l khng c kh nng
cung cp thng tin v nh tnh mt cch r rng cho cc hp phn mu, thm ch c
trong trng hp chng c tch hon ton khi nhau. Vic nh tnh c da
trn c s so snh cc c tnh lu gi, m n gin nht l thi gian lu ca mt
cht cha bit vi cc cht so snh c xc nh trong cng mt iu kin th
nghim. Tuy nhin, trn thc t rt nhiu trng hp thm ch c cng cc c tnh
lu gi ca cu t cha bit v vt liu so snh, trong gii hn sai s thc nghim,
nh tnh, ngi phn tch cng khng th kt lun mt cch tuyt i hai hp cht
ny l ging nhau. Mc d cc iu kin sc k tng i m rng cho cc nh
phn tch, nhng khng phi lc no cng c th thc hin c qu trnh tch tt
c cc cu t ca mt hn hp mu v iu ny c th nh hng n ng,
chnh xc trong qu trnh phn tch nh lng ca cc cht cn quan tm.
Kh nng ca thit b sc k - khi ph trn thc t ph thuc vo ph khi
lng ca nhiu cht m cc cht ny c y cc ph (th vin ph). iu ny
cho php vic nhn dng chng mt cch d dng vi tin cy cao, mc d nhiu
khi khng hon ton nh vy. Nu cht phn tch nm trong mt phn ca hn hp

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th ph khi lng nhn c s cha cc ion t tt c cc cht c mt. Trong


trng hp c bit, nu cht phn tch l mt hn hp ca cc thnh phn lng
nh th vic nhn dng s tr nn kh khn v dng nh khng th. S kt hp kh
nng tch ca phng php sc k cho php cc cht tinh khit c a vo my
ph khi. Kh nng nhn dng ca my ph khi rt r rng, v th c thun li v
c bit cho cc cht tng t nhau hoc ging ht nhau v cc c trng lu,
nhng c ph khi lng hon ton khc nhau v c th phn bit c cc cht
ny. Ngoi ra, ph khi lng cn cho php nh lng vi chnh xc ph thuc
vo mc phc tp ca hn hp mu v bn cht ca mi thnh phn trong hn
hp. Cng vic ny nu ch ring php sc k thi th khng th thc hin c.
Phng php sc k
- L phng php tch dnh cho hn hp mu.
- Nhn dng cc mu da trn thi gian lu.
- Cho php phn tch nh tnh v nh lng.
- Phn lp v tinh ch cht.
Phng php ph khi lng
- L phng php nhn dng cc lng mu nh.
- Cung cp ph khi lng ca cc hn hp phc tp.
S kt hp ca HPLC v ph khi lng v th cho php nhn dng r rng v xc nh
s lng ca cc hp cht m phng php sc k khng phn tch y c.
1.2.2. S thit b sc k lng - khi ph
Mt my sc k lng khi ph bao gm 3 b phn chnh (hnh 1.1).
- Thit b sc k lng (LC, Liquid Chromatography): thng thng s dng my
sc k lng hiu nng cao (HPLC, High Performance Liquid Chromatography),
hin i hn l my sc k lng siu hiu nng (UPLC Ultra Performance Liquid
Chromatography)
- B kt ni (Interface): c nhim v loi dung mi ra gii v a mu cht vo
bung ion ho ca my ph khi. y l b phn rt quan trng v l cha kho ca
phng php LC-MS.

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- Thit b ph khi lng (MS, Mass Spectrometry).


My tnh iu khin v phn tch s liu

Dung
mi

Bm chn khng

B tim
mu

Bm cao p

Ct sc k

Sc k lng

B kt
ni

Ngun
ion

B kt ni

B phn
tch khi

B pht
hin

Khi ph

Hnh 1.6. S h thng my sc k lng khi ph


B phn kt ni (interface) y ng vai tr quan trng v n l k thut
cha kho ca mt my sc k lng - khi ph. Chc nng ca b kt ni l loi
dung mi ra gii v a mu cht vo bung ion ha ca my khi ph. B kt
ni bao gm nhiu loi khc nhau v ra i cc thi im khc nhau nh:
- B kt ni bng chuyn (Moving Belt Interface): 1977.
- B kt ni np cht lng trc tip (DLI, Direct Liquid Introduction Interface):
1980
- B kt ni phun nhit (Thermospray Interface): 1983
- B kt ni bn ph nguyn t nhanh dng lin tc (CF-FBA, Continuous Flow
Fast Atom Bombardment Interface): 1985/1986.
- B kt ni ion ho ho hc p sut kh quyn (APCI, Atmospheric-Pressure
Chemical Ionization Interface): 1986.
- B kt ni ion ha phun in (ESI, Electrospray Ionization Interface): 1988.
1.2.3. Phng php ph khi lng (MS)
Phng php ph khi lng l mt trong nhng phng php phn tch
cng c quan trng. Ph khi lng cung cp cc thng tin v phn tch nh tnh,
nh lng cc nguyn t, thnh phn v cu trc ca cc hp cht v c v hu c.
a. Nguyn tc chung ca phng php
C s ca phng php ph khi lng i vi cc hp cht hu c l s
bn ph cc phn t hp cht hu c trung ho thnh ion phn t mang in tch

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dng hoc ph v thnh cc mnh ion, cc gc theo s sau bng cc phn t


mang nng lng cao:

S hnh thnh cc ion mang in tch +1 chim hn 95%, cn li cc ion


mang in tch +2 hoc ion m (-). Nng lng bn ph cc phn t thnh ion phn
t khong 10 ev. Nhng vi nng lng cao th ion phn t c th ph v thnh
mnh ion dng (+), hoc cc ion gc, cc gc hoc cc phn t trung ho nh hn:

S ph v ny ph thuc vo cu to cht, phng php bn ph v nng


lng bnph. Qu trnh ny gi l qu trnh ion ha.
Ion phn t v cc ion mnh l cc phn t c khi lng. Nu gi khi
lng ca mt ion l m v in tch ca n l e th t s z=m/e c gi l s khi.
Hin nhin cc ion c khi lng l m, 2m, 3m v in tch tng ng bng e, 2e,
3e, c s khi z bng nhau. Ion phn t c s khi k hiu l M+..
b. Thit b khi ph
Thit b ph khi u tin c ch to bi J. Thomspon (1912) v F. W.
Aston (1919). Thit b hon thin c ch to nm 1932.

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1.2.4. 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.
- 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.
a. 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.

10

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KA =

[Ahc ]
[An ]

KA: hng s phn b


[Ahc]: nng ca cht A trong pha hu c.
[An]: nng ca cht A trong pha nc.
b. 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.
Bc 2. Hot ha ct: lm t pha rn, to mi trng thch hp cho vic
hp ph 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.

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Nhiu k thut x l mu c th c s dng phn tch tn d khng


sinh trong cc nn mu khc nhau. Hai k thut ph bin c s dng trn th gii
v Vit Nam l k thut chit lng-lng v k thut chit pha rn. Cc nn mu
trong nghin cu l cc thc phm (ch yu l tht v ni tng ng vt) v thc n
chn nui, y l nn mu kh phc tp nn thng phi s dng phi hp nhiu k
thut x l mu t hiu qu cao. Cn c vo cc ti liu tham kho chng ti
cn phn tch v tm ra nhng u im ca cc nghin cu khc nhau v cc k
thut x l mu vi cc i tng khc nhau v cc k thut phn tch trn cc loi
hit b khc nhau nhm xy dng c quy trnh k thut xc nh d lng khng
sinh nhm Quinolon trong thc phm ph hp vi iu kin Vit Nam.
1.3. Nghin cu v tn d quinolon trong thc phm
1.3.1. Tnh hnh s dng khng sinh trong chn nui
Trong nhng nm gn y, khng sinh c s dng nhiu trong lnh vc nng
nghip c bit l chn nui. Theo s liu ca Ghislain Follet, trong nm 1997 tng
lng khng sinh dng trong dn y v chn nui cc nc chu u l 10500 tn (qui
theo mc 100% tinh khit ca cc thnh phn hot tnh), trong 52% s dng trong
dn y, 33% trong iu tr th y v 15% nh cht b sung trong thc n chn nui. Trong
, t l cc loi khng sinh c s dng trong chn nui: penicillin (9%); tetracycline
(66%); macrolid (12%); Aminoglycosid (4%); Fluoroquinolon (1%); Trimethomprim
/sulphamid (2%) v cc khng sinh khc (6%) [24].
Vit Nam, theo nghin cu ca mt s tc gi, khng sinh c s dng trn lan
trong thc n cho vt nui v tnh trng tn d khng sinh trong tht l ph bin. Cc
nghin cu u cho rng hu ht cc c s chn nui s dng khng sinh khng hp l
t dn n tnh trng tn d khng sinh trong sn phm cao gp hng chc ti hng
ngn ln so vi tiu chun quc t. Hin nay, vic kim sot d lng khng sinh gp rt
nhiu kh khn do thiu kinh ph, thiu trang thit b, thiu hiu bit ca ngi s dng.
Kt qu iu tra Tnh hnh s dng khng sinh v d lng khng sinh trong tht
g ti cc c s chn nui g cng nghip ca thnh ph H Ch Minh ca V Th Tr
An cho thy c 8 loi khng sinh c s dng ph bin trong chn nui g tht cng
nghip trn a bn thnh ph H Ch Minh: colistin (15,83%), enrofloxacin, diaveridin
(7,74%),

sulfadimidin

(6,72%),

trimethoprim

12

(6,38%),

norfloxacin

(5,79%),

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oxytetracyclin (4,93%), gentamycin (4,51%) v acid oxolinic (4%). C 32,61% c s s


dng khng sinh khng hp l, trong sai v liu lng chim t l cao nht (23,3%).
S c s khng ngng thuc ng qui nh chim 44,54%. Kim tra 70 mu tht g ti
cc c s s dng khng hp l pht hin 42 (60%) mu tht c tn d vi cc loi khng
sinh: enrofloxacin, norfloxacin,

tylosin, tetracyclin,

sulfadimidin, sulfadiazin,

sulfaquinoxalin. C mi lin quan gia vic ngng thuc khng ng qui nh vi tnh
trng tn d khng sinh. 35,71% mu tht g c tn d vt qu gii hn t 2 400 ln
so vi tiu chun ca Malaysia [1].
Trong cc loi khng sinh dng cho chn nui trn th trng khng t loi khng
m bo hm lng v cht lng nn khi s dng chng s c t hiu qu. Ngi chn
nui thng phi dng kt hp nhiu loi khng sinh khc nhau phng v cha bnh.
Do gy nn hin tng tn d khng sinh trong sn phm ng vt nh : tht , sa ,
trng... iu ny gy nguy hi ln cho sc khe cng ng. Con ngi s b nhim vi
khun khng thuc khi n phi tht nhim khun. c bit l s khng thuc ca nhiu
lai vi khun gy bnh nguy him.
1.3.2. Cc nghin cu v tn d quinolon trong thc phm trong nc
Phm Kim ng v ng s ng dng phng php Elisa phn tch tn d
khng sinh nhm quinolon trong Tm ti mt s ven bin khu vc pha bc cho thy:
Tnh hnh tn d quinolon trong tm c nh gi thng qua vic phn tch 90 mu
tm c ly 4 a phng i din (H Ni, Qung Ninh, Nam nh, Ngh An) bng
phng php ELISA v khng nh bng phng php Sc k lng ghp khi ph (LCMS/MS). Kt qu phn tch

nh gi tnh hnh tn d pht hin 5 quinolon

(enrofloxacin, norfloxacin, ciprofloxacin, danofloxacin v acid oxolinic) trong 9 mu


nhim (t l nhim 10%) vi nng dao ng t 0,4 n 145 ppb. Trong , c 4 mu
nhim nng cao hn gii hn tn d cho php theo Ngh nh 2377/90 CE ca U
ban Chu u [4].
Mt s kt qu kho st tnh hnh s dng thuc khng sinh trong chn nui g v
tn d khng sinh trong tht, trng g trn a bn H Ni ca L Th Ngc Dip thy
rng: Kim tra tn d khng sinh theo phng php vi sinh vt (theo Dc in Vit
Nam 3, tp II, 1994). Mu dng tnh l mu c ng knh vng v khun > 8mm.
Khng sinh c s dng trong chn nui g ti cc huyn ngoi thnh H Ni l cc
nhm beta- lactamin (46,58%), aminoglycosid (50,96%), quinolon (78,14%), macrolid

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(86,89%), tetracycline (46,58%), sulfamid (54,92%) v polypeptide (22,27%). Cc


khng sinh khc c t l s dng thp, chim 10,11% [3].
Tc gi Nguyn Thanh Nga trong nghin cu xc nh d lng khng sinh
ciprofloxacin v enrofloxacin trong thc phm cng la chn phng php
LC/MS/MS. Ct c s dng l ct XDB-C18 (50mm x 4,6mm; 3,5m;) vi pha
ng gm acetonitril v acid formic 0,1%. Cc iu kin khi ph c ti u ha
nh sau: K thut ion phun ha in EIS vi ch bn ion dng, tc dng:
0,5 ml/pht, nhit ct: 300C, nhit MS1 (MS1 Heater) v MS2 (MS2 Heater):
100oC, nhit dng kh (gas temp): 350oC, tc dng kh (gas flow): 8,0l/min,
th p vo ng mao qun (capillary): 3840V, cng dng in ca bung
(chamber current): 1.66A, cng dng in ti ng mao qun (capillary
current): 74nA, p sut dng kh N2 cho h phun sng (nebulizer): 40.0psi. Gii
hn pht hin ca ciprofloxacin v enrofloxacin tng ng l 0.426ppb v 0,391ppb
[6].
nh gi tn d enrofloxacin trn c tra, tc gi Trn Minh Ph s
dng phng php sc k lng cao p kt hp vi u d khi ph. Phng php c
gii hn nh lng 2ppb. Cc iu kin sc k c ti u ha: s dng ct C18
(150 mm x 3mm; 3,0m), Pha ng chy sc k l acetonitril v nc c iu
chnh n pH t 2,5 bng axit formic. Th tch tim mu 20l, tc dng
500l/pht, nhit ct: 250C, nhit ngun 4500C [8].
1.3.3. Cc nghin cu v tn d quinolon trong thc phm ngoi nc
Trn th gii hin nay c rt nhiu cc nghin cu xc nh d lng ca cc
khng sinh ni chung v quinolon ni ring trn cc nn mu khc nhau. Mt s
phng php ang c s dng nh: phng php min dch enzym (ELISA) [4];
phng php vi sinh vt (test vi sinh vt hc), phng php l ha. Mi phng
php c u nhc im ring. Vi nhm quinolon, c th nhn thy, phng php
c s dng ph bin hin nay l sc k lng hiu nng cao (HPLC) do c
chnh xc cao, pht hin c mt lng nh khng sinh tn d trong thc phm.
Cc detector c s dng nhiu nht l detector UV [19], detector hunh
quang[19], [26], [34] v detector khi ph [13][22][41].

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xc nh tn d khng sinh cn phi tri qua qu trnh tch chit


ly cht cn phn tch ra khi nn mu, sau c lm sch v lm giu s dng
bng cc k thut hin i pht hin v nh lng.
Nhiu k thut x l mu c th c s dng phn tch tn d khng
sinh trong cc nn mu khc nhau. Nguyn tc chung ca qu trnh ny l kt ta
protein trong mu s dng dung mi thch hp chit c cht phn tch, sau
c lm sch v/hoc lm giu mu trc khi em i phn tch. Dung mi c s
dng ch yu l acetonitril [13], [14]...[21], [30].[41] hoc hn hp acetonitril:
acid phosphoric 0,3%, hn hp acetonitril : acid formic [13]. Mt s nghin cu
cng s dng hn hp acid acetic/ethanol hoc acid formic/ethyl acetate chit
mu cho kt qu tt. Giai on ny thng c kt hp vi k thut rung siu m,
lc Vortex cho ng nht v ly tm phn tch lp tt hn. Giai on chit c
lp li nhiu ln chit c ti a cht phn tch. Dch chit c loi cht bo
bng k thut chit lng lng (n-hexan) hoc lm sch mu s dng k thut chit
pha rn SPE, sau c cn lm giu mu, ha cn v em i phn tch [20].
Trong k thut chit pha rn, phn tch tn d khng sinh thng s dng 2
loi ct chnh l ct SPE Oasis HLB Waters v ct SPE C18 [14], [15][22],
[38].[41]. y l k thut c a s cc nghin cu s dng lm sch mu,
s dng t dung mi, cho hiu qu cao. Ngoi vai tr loi cc tp cht, k thut chit
pha rn cn c vai tr lm giu mu, tng cng kh nng phn tch mu.
Sc k lng l k thut c s dng ph bin phn tch tn d khng
sinh. K thut ny u c nhiu im thun li xy dng mt phng php phn
tch a d lng khng sinh trong thc phm ni chung..
Ngay t khi c bt u ng dng trong phn tch, sc k lng c
nghin cu pht trin xc nh rt nhiu hp cht. Vi kh nng kt hp vi
nhiu loi detector khc nhau nn phm vi ng dng ca sc k lng rt rng ri v
phong ph. xc nh tn d khng sinh, trong thi gian u, detector UV v
detector hunh quang c s dng ph bin trong cc nghin cu, c bit l
detector hunh quang (do c nhy cao). Trong nghin cu ca Verdon s dng
detector hunh quang c LOD khong 4-11 mcg/kg v LOQ khong 13-36 mcg/kg
[41]. Thi gian gn y, k thut khi ph pht trin mnh m v t c nhiu

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bc tin vt bc. Sc k lng ghp ni vi detector khi ph c nhy v tnh


c hiu rt cao, p ng c yu cu ngy cng kht khe ca th gii v phn tch
d lng.
Ngoi k thut sc k lng, mt s tc gi cng nghin cu pht trin
phn tch d lng khng sinh s dng k thut: in di mao qun (CE). Gn y,
phng php CE c s dng do tnh cht u vit v hiu qu tch cao, thi gian
tch ngn, lng mu tiu tn t. Phng php ny c D. Barrn s dng vi
detector PAD phn tch flumequin v acid oxolinic. Cc iu kin in di gm:
dung dch in di l dung dch acid phosphoric 40mM iu chnh pH 8,02 bng
NaOH, th in di 18kV, nhit mao qun duy tr 250C, bc sng pht hin l
250nm. Phng php c gii hn pht hin v gii hn nh lng cho acid oxolinic
v flumequine tng ng l 15 v 48ppb v 10 v 30ppb [20]. Tuy nhin do
nhy khng tt v tin cy khng cao nn phng php in di mao qun t c
p dng.
1.3.4. Mt s quy nh v gii hn tn d quinolon trong thc phm
EU ban hnh quyt nh s 2377/90/EC quy nh qui nh gii hn cho php
thuc th y trong sn phm ng vt, theo cc sn phm c ngun gc t ng
vt phi c kim sot d lng tun th qui trnh ca Ch th s 96/23 EC (EU,
1996). c bit cc phng php phn tch c cng nhn v p dng trong chin
lc kim sot d lng phi chun ho theo quyt nh s 2002/657/CE (CE,
2002). Trong , quy nh v gii hn tn d ti a ca cc quinolon trong nghin
cu c th nh sau.
Bng 1.1: Gii hn tn d ti a ca cc quinolone theo quyt nh s
2377/90/EC ca y ban Chu u [22]
Hot cht

Danofloxacin

nh lng
cht tn d

Loi

C quan

B, cu, d,
ln

Tht
M
Gan
Thn
Sa
Tht

Danofloxacin

16

Gii hn tn d
ti a (MRL)
(g/kg)
200
100
400
400
30
200

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Gia cm

Cc loi khc

B, cu, d

Ln

Difloxacin

Difloxacin

Gia cm

Cc loi khc

Cu, b, d

Ln, th

Enrofloxacin

Tng ca
Enrofloxacin
v
Ciprofloxacin

Gia cm

Cc loi khc

B, cu, d,
ln
Flumequin

Flumequin
Gia cm

17

Da v m
Gan
Thn
Tht
M
Gan
Thn
Tht
M
Gan
Thn
Tht
Da v m
Gan
Thn
Tht
Da v m
Gan
Thn
Tht
Da
Gan
Thn
Tht
M
Gan
Thn
Tht
M
Gan
Thn
Tht
M
Gan
Thn
Tht
M
Gan
Thn
Tht
M
Gan
Thn
Sa
Tht

100
400
400
100
50
200
200
400
100
1400
800
400
100
800
800
300
400
1900
600
300
100
800
600
100
100
300
200
100
100
200
300
100
100
200
300
100
100
200
200
200
300
500
1500
50
400

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C thu
Khc

Ln

Acid oxolinic

Acid oxolinic

C thu
G
Sarafloxacin

Sarafloxacin
C hi

M
Gan
Thn
Da v tht
Tht
M
Gan
Thn
Tht
Da v m
Gan
Thn
Tht
Da v m
Gan
Thn
Da v tht
Da v m
Gan
Da v tht

250
800
1000
600
200
250
500
1000
100
50
150
150
100
50
150
150
100
10
100
30

B Thy sn ra quyt nh 07/2005/Q-BTS ban hnh ngy 24 thng 02 nm


2005 quy nh danh mc 17 khng sinh cm s dng v danh mc 34 loi hn ch
s dng, trong c nhm flouroquinolon v quyt nh 26/2005/Q-BTS ban
hnh ngy 18/8/2005 b sung nhm khng sinh fluoroquinolon cm s dng trong
sn xut, kinh doanh thy sn xut khu vo th trng M v Bc M. Cc Quyt
nh ny c thay th bi Thng t s 15/2009/TT-BNN ban hnh ngy
17/03/2009 ca B Nng nghip v pht trin nng thn, quy nh danh mc thuc,
ha cht, khng sinh cm s dng v hn ch s dng. Fluoroquinolon vn b cm
s dng trong sn xut, kinh doanh thy sn xut khu vo th trng M v Bc
M. Cc khng sinh enrofloxacin, ciprofloxacin, ofloxacin b cm s dng trong th
y. Mt s khng sinh quinolone nm trong danh mc cc khng sinh hn ch s
dng trong sn xut kinh doanh thy sn.

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Bng 1.2 : Danh mc cc quinolone hn ch s dng trong sn xut


kinh doanh thy sn
Tn hot cht, khng sinh
D lng ti a (MRL) (ppb)
Danofloxacin
100
Difloxacin
300
Enrofloxacin + Ciprofloxacin
100
Acid oxolinic
100
Sarafloxacin
30
Flumequine
600
Mi y, ngy 16/01/2012, B Nng nghip v pht trin nng thn ban hnh
thng t s 03/2012/TT-BNNPTNT, trong quy nh a enrofloxacin ra khi
Danh mc ho cht, khng sinh hn ch s dng trong sn xut, kinh doanh thu
sn, v b sung vo enfloxacin vo Danh mc cc ha cht, khng sinh cm s
dng trong sn xut, kinh doanh thy sn ]9].

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Chng 2. I TNG V PHNG PHP NGHIN CU


2.1. i tng nghin cu ca ti
- Cc cht phn tch: Ciprofloxacin, Enrofloxacin, Norfloxacin , Flumequin
- i tng phn tch: tn d quinilon trong thc phm
- i tng nghin cu: tht g, tht ln, c
Cc mu tht, c c ly ngu nhin ti mt s ch trn th trng H Ni
2.2. Thit b, dng c, ho cht
2.2.1. Thit b, dng c
- My sc k lng khi ph ba t cc (UPLC-MS/MS): vi phn UPLC 20A ca
Shimazdu v detector MS/MS TripleQuad API 5500 ca ABSciex
- Ct sc k C18 Xbrigde (150mm x 2,1mm x 3,5 mcm) v tin ct C18 tng
ng ca Waters.
- Ct sc k C18 Symmetry (250mm x 4,6mm x 5 mcm) v tin ct C18 tng
ng ca Waters.
- My ly tm lnh Hermle (c)
- My lc xoy Vortex;
- Ct SPE Sep-Pak C18-E : (500mg, 3ml) ca Waters
- Ct SPE Oasis HLB (200mg, 6ml) ca Waters
- B ct quay chn khng Eyela
- Dng c thy tinh cc loi.
- ng ly tm nha 50ml c np kn
2.2.2. Thuc th, ha cht
Tt c cc ha cht thuc th phi t tiu chun dng cho phn tch
- Chun ciprofloxacin, norfloxacin, enrofloxacin v flumequin ca Fluka
- Nc tinh khit dng cho HPLC (nc ct 2 ln lc qua mng lc 0,45 mcm)
- Acetonitril, Methanol, Acid acetic, acid formic.. loi dng cho HPLC (Merck).
2.3. Ni dung v phng php nghin cu
2.3.1. Nghin cu cc iu kin LC-MS/MS
a. Kho st v la chn cc iu kin MS xc nh cc cht phn tch

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S dng cc dung dch chun ca cc cht nghin cu c nng thch hp


phn tch trn detector MS/MS v la chn c cc thng s ti u:
- Ch ha hi: APCI hay ESI v la chn ion (+) hay ion (-)
- Ch theo di ion: MRM, SIM, TIC,......
- Cc ion m (procusor ion) v ion con (product ion),....
- Cc thng s v nng lng bn ph theo tng cht
- Nhit ngun ion ha v th ion ha
- Nhit v tc ca kh mang
- Th phn mnh cho tng cp ion
- Mt s thng s khc: kh loi tp, well time,....
b. Kho st v la chn cc iu kin LC tch cc cht phn tch
- Chun b dng c v my mc
+ Thit b LC-MS/MS c hiu chun theo nh k m bo chnh xc
+ Ct sc k c kim tra nh gi hiu lc ct nh k.
+ Cc dng c th nghim c m bo sch s trc khi s dng.
- Chun b dung mi ng
Pha dung mi ng c pha ch chnh xc v s dng trong mt khong thi gian
nht nh. Lc dung mi sau khi pha ch (c bit l cc dung dch m) qua mng
lc 0,45 m, ui kh bng cch chy siu m.
- Chun b mu o LC-MS/MS
+ Mu th: x l theo qui trnh nghin cu, cui cng qua mng lc 0,45 m.
+ Mu chun: Pha dung dch chun ca cc cht phn tch trong cc loi dung
mi thch hp, pha long (nu cn) c c cc nng thch hp.
- Phn tch th nghim v la chn cc iu kin sc k lng (LC):
Da vo bn cht ca cc cht cn phn tch, trn c s nghin cu cc ti liu
tham kho, tin hnh th nghim v la chn cc thng s sau:
+ Pha tnh: bn cht pha tnh, cc thng s ca pha tnh.
+ Pha ng: phn tch ng thi nhiu cht nn s dng ch gradient:
Dung mi hu c: Acetonitril, methanol,... hay hn hp dung mi khc

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Dung mi phn cc: dung dch acid (formic, acetic,...) hoc dung dch m
Tc pha ng
+ Cc thng s khc: nhit bung ct, th tch tim mu, .....
2.3.2. Nghin cu v la chn quy trnh x l mu
a. Nghin cu v la chn cc iu kin chit mu
S dng cc nn mu trng c thm chun nng xc nh v cc iu
kin phn tch bng LC-MS/MS la chn c trn, tin hnh th nghim v
xc nh c quy trnh v cc iu kin chit mu ph hp vi tng i tng
mu:
- Th nghim k thut chit: lng-lng, rnlng, ....
- Th nghim v la chn dung mi chit mu, s ln chit mu,....
- Nghin cu cc yu t nh hng: nhit , m, nh sng,...
- Cc k thut ph tr: lc vortex, siu m, lc ngang,.....v cc thng s i km:
thi gian, tc , cch thc,....
Da vo bn cht ca cc cht phn tch, bn cht ca i tng mu cng
nh tham kho mt s ti liu [18], [35], [39], chng ti tin hnh nghin cu v
th nghim mt s quy trnh, iu kin chit mu nh sau:
+ Test 1: Chit vi Acetonitril, chit 02 ln, c rung siu m h tr. Cn khong 2g
mu ng nht cho vo ng ly tm 50ml c np kn. Thm chnh xc 0,2ml dung
dch chun hn hp cc Quinolon 500ppb, lc u, yn khong 1h. Thm khong
30ml ACN, lc u, em rung siu m 15 pht. Sau em ly tm 6000 vng/pht
trong 5 pht, ly ra gn phn dch trong cho vo bnh c quay 100ml. Chit li ln 2
vi 15ml ACN. Gp dch chit thu c, em c quay chn khng n cn. Ha cn
trong 2ml ACN, lc qua mng 0,45mcm v phn tch trn LC-MS/MS.
+ Test 2: Tin hnh cc giai on chit mu tng t nh Test 1 nhng thay ACN
bng hn hp ACN/TCA 4% trong nc ct (70/30).
+ Test 3: Tin hnh cc giai on chit mu tng t nh Test 1 nhng thay ACN
bng hn hp ACN/NaOH 0,1N trong nc ct (70/30).

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b. Nghin cu v la chn cc iu kin lm sch mu


S dng cc nn mu trng c thm chun nng xc nh v cc iu
kin phn tch bng LC-MS/MS la chn c trn, tin hnh th nghim v
xc nh c quy trnh v cc iu kin lm sch v lm giu mu:
- La chn c loi ct SPE: HLB, C18-E, v cc thng s ct tng ng
- La chn tng quy trnh qua tng loi ct SPE tng ng:
+ Cc giai on qua ct: hot ha, np mu, ra tp v ra gii
+ Cc loi dung mi ty theo tng giai on qua tng loi ct
+ Th tch v s ln qua ct ca cc loi dung mi
+ Mt s nhn xt trong qu trnh qua ct SPE
- Da vo bn cht ca cc cht phn tch, bn cht ca i tng mu cng
nh tham kho mt s ti liu, chng ti tin hnh nghin cu v th ghim mt
s quy trnh, iu kin lm sch v lm giu mu nh sau:
+ Ct SPE Sep-Pak C18-E : (500mg, 3ml) ca Waters:
Hot ha ct SPE: 6ml methanol, 6ml nc ct.
Np mu vi tc khng qu 2ml/pht.
Loi tp bng 3ml nc ct, 3ml MeOH 10%/nc ct
Ra gii ly cht phn tch bng 2 x 2ml acetonitril.
+ Ct SPE Oasis HLB (200mg, 6ml) ca Waters:
Hot ha ct SPE: 6ml methanol, 6ml nc ct.
Np mu vi tc khng qu 2ml/pht.
Loi tp bng 3ml nc ct, 3ml MeOH 10%/nc ct
Ra gii ly cht phn tch bng 2 x 2ml acetonitril.
+ Ct SPE Strata SCX (500mg, 3ml) ca Phenomenex
Hot ha ct SPE: 6ml methanol, 6ml dung dch HCl 0,1N.
Np mu vi tc khng qu 2ml/pht.
Loi tp bng 3ml nc ct, 3ml methanol.
Ra gii ly cht phn tch bng 2 x 2ml NH3 5%/ACN.

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2.3.3. Thm nh li phng php xy dng


a. Xc nh chn lc/ c hiu
Trn h thng LC-MS/MS, vi mi cht phn tch la chn c t nht 01
ion m v 02 ion con, mt ion con nh lng, mt ion con nh tnh.
Tin hnh phn tch mu trng, mu dung dch chun v mu trng thm
chun theo cc iu kin LC-MS/MS la chn c. Kt qu phi t nh sau:
- Trn sc mu trng khng xut hin cc pic c cng thi gian lu vi pic
ca cc cht phn tch trn sc mu dung dch chun v mu trng thm chun.
- Trn sc mu trng thm chun c cc pic c thi gian lu tng t nh
trn sc ca dung dch chun.
b. Xc nh tnh thch hp ca h thng
Tin hnh phn tch lp li t nht 06 ln dung dch chun trn h thng LCMS/MS theo cc iu kin la chn c nh trn. Tnh ton lch chun
tng i (RSD %) ca cc gi tr thi gian lu (tR) v din tch pic (t l din tch
pc nu s dng ni chun). Cc gi tr RSD phi nh hn 2%.
c. Kho st khong tuyn tnh
xc nh khong tuyn tnh ca phng php, thc hin phn tch dy
chun vi t nht 5 nng pha t 1ppb n 500ppb. Nu th tuyn tnh trong
khong nng ny (R2 0,995) th chp nhn dy nng ny, nu khng phi
tin hnh phn tch li dy chun hoc thu hp di nng cho n khi R2 0,995.
d. Gii hn pht hin LOD v gii hn nh lng LOQ
- Gii hn pht hin (LOD) l nng nh nht trong dy chun c chiu cao pic
trung bnh cao t nht gp 3 ln chiu cao pic ca mu trng. Gii hn nh lng
(LOQ) l nng nh nht trong dy chun c chiu cao pic trung bnh cao gp 10
ln chiu cao pic ca mu trng, ngha l LOQ 3,3LOD
e. lp li ca phng php
th nghim lp li ca phng php, tin hnh phn tch mu theo cc
iu kin LC-MS/MS v quy trnh x l mu la chn c trn. S dng mu

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trng thm chun nng xc nh, tin hnh phn tch ng thi t nht l 06
ln. Tnh ton lp li ca kt qu kt qu thu c thng qua lch chun SD
v lch chun tng i RSD theo cc cng thc sau:

(Xi X )

SD =

RSD (%) =

n 1

SD
100
X

Trong : SD: lch chun ; Xi: kt qu thu c trn mu th i


X

l kt qu trung bnh ca n ln

f. thu hi ca phng php


th nghim lp li ca phng php, tin hnh phn tch mu theo cc
iu kin LC-MS/MS v quy trnh x l mu la chn c trn. S dng mu
trng thm chun nng xc nh, tin hnh phn tch ng thi t nht l 06 ln
Tnh ton kt qu thu c ca thu hi theo cng thc nh sau:

R =
Trong :

X
X

Spike

100

R : thu hi trung bnh ; X

l kt qu trung bnh ca n ln

XSpike: nng ca dung dch mu thm chun.


2.3.4. Thc nghim xc nh quinolon trn mu tht, c
Tin hnh thu thp mu ngu nhin ti mt s ch trn a bn H Ni v x
l mu theo cc quy trnh c nghin cu, la chn trn. Phn tch cc mu
trn h thng LC-MS/MS vi cc iu kin xc nh.
Tnh ton kt qu phn tch c a ra cc nhn xt, nh gi s b v
phng php phn tch v tnh hnh tn d mt s cht nhm Quinolon trong thc
phm trn th trng H Ni.
2.4. Tnh ton kt qu, x l s liu
S dng phn mm Analyst 1.5.1 i km theo thit b LC-MS/MS thu c
cc sc , tnh ton din tch pic,..
S dng phn mm Microsoft Excel trong my tnh : lp ng hi quy tuyn
tnh, tnh t l thu hi, lch chun tng i,..
- Mt s cch tnh kt qu :
Da vo din tch pic

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- p dng khi: + Din tch v nng cht c s tng quan tuyn tnh.
+ phn gii m bo nhng h s dung lng k' thay i.
+ Xc nh c im u, im cui ca pic.
- Khng p dng: Khi pic qu cao hoc qu dong, khng i xng, ng nn
nhiu.
Da vo chiu cao pic
Khi pic c dng khng i th chiu cao pic l mt i lng t l vi din tch
v vy trong cc trng hp khng tnh theo din tch pic c th c th tnh kt
qu theo chiu cao pic nhng vi iu kin l h s dung lng k' hng nh.
Phng php dng ngoi chun
C th dng 1 chun nu lp li tt.
C th dng ng chun thit lp bng nhiu chun c nng khc nhau, nng
dung dch th c tnh theo t l thun gia din tch pic hoc chiu cao pic
ca dung dch th v dung dch chun.
Phng php dng ni chun
gim sai s v tng lp li, ngi ta thng dng mt chun th 2 thm
vo mu th v mu chun. Cht thm vo ny gi l ni chun. Trong cng iu
kin sc k n c thi gian lu gn vi thi gian lu ca cht phn tch nhng c
tch hon ton v c nng tng ng v cu trc ho hc tng t.
Phng php thm chun
p dng trong trng hp cht chun c nng qu nh hoc b nh hng
bi cc cht khc.
Phng php tnh theo % din tch pic
p dng trong trng hp cn bit hm lng tnh theo ch phm kh ca
nguyn liu hoc t l tp cht m ta khng c cht chun. Kt qu ch l tng i
v n i hi mi cu t trong hn hp u phi c ra gii v c pht hin
nh nhau

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Chng 3. KT QU NGHIN CU
3.1. Kt qu kho st v la chn cc thng s cho LC-MS
3.1.1. Xc nh cc thng s ca detector khi ph (MS)
- p ng yu cu ca chu u (2002/657/EC) v phn tch d lng cc
cht khng sinh trong thc phm, chng ti la chn ch MRM, vi mi cht s
c 1 ion m (precursor ion) v 2 ion con (product ion).
- Th nghim kho st c hai kiu ion ha ESI v APCI cng nh c ch
thu ion dng (M+H)+ v ion m (M-H)+. Kt qu cho thy: ch ESI (+) cho
cng tn hiu ca cc cht l tt nht. V vy chng ti la chn ch ESI (+).
- Tin hnh th nghim ln lt vi tng dung dch cht phn tch (c nng 50ppb)
v th nghim vi hn hp ca chng trn h thng LC-MS/MS. Kt qu thu c:
+ Cc thng s chung:
Bng 3.1. Thng s chung ca detector khi ph
Kh mn (Curtain gas)
Kh va chm (Collision gas )
Th phun in t (Ionspray voltage )
Nhit mao qun (TEM)
Kh ngun ion 1 (Ion source gas 1)
Kh ngun ion 2 (Ion source gas 2)
Th u vo (Entrance potential)

15 (p sut/ th tch)
7 (p sut/ th tch)
5000 (volt)
4500C
20
20
10 (volt)

+ Cc thng s chi tit cho tng cht


Bng 3.2 Thng s chi tit ca 4 Quinolon
Ion con 1
(M+H)+ DP
m/z
(volt) m/z CE (volt) CXP
Ciprofloxacin
332
91 314
27
34
Norfloxacin
320
96 276
23
26
Enrofloxacin
360
41 316
25
18
Flumequin
262
106 202
43
26
Ciprofloxacin d8
340
96 322
25
34

Cht phn tch

Ion con 2

m/z
288
302
342
244
296

CE (volt) CXP
23
22
25
14
27
20
25
28
34
38

(Trong : DP-declustering potential; CE-collistion energy; CXP-collision cell exit potential).


Nhn xt: Cc thng s thu c trong qu trnh ti u ha detector MS theo
tng cht phn tch v hn hp cc cht ny s c s dng kho st cc iu
kin khc ca phng php.

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3.1.2. Xc nh cc thng s ca phn LC tch cc cht nhm Quinolon


Da vo bn cht ca cc cht phn tch, bn cht ca i tng mu cng nh
tham kho mt s ti liu, chng ti nghin cu mt s iu kin nh sau:
- Pha tnh: tin hnh th nghim trn hai ct sc k u c bn cht C18 pha ngc
+ Ct Symmetry (250mm x 4,6mm; 5,0 m) v tin ct tng ng (Waters).
+ Ct XBridge (150mm x 2,1mm; 3,5 m) v tin ct tng ng (Waters).
- Pha ng: s dng gradient gia dung mi hu c v dung dch acid hu c
+ Dung mi hu c: th nghim Acetonitril v Methanol.
+ Dung dch acid hu c: th nghim nc ct c cha 0,1% cc acid: acid
acetic (AA) , acid formic (FA) , acid trichloacetic (TCA) v acid trifloacetic (TFA)
- Mt s thng s HPLC khc c la chn:
+ Th tch tim mu 20l, nhit bung ct 350C
+ Tc dng 0,5 ml/pht cho ct XBridge v 1,0 ml/pht cho ct
Symmetry.
- Kt qu sau khi tin hnh kho st, th nghim v la chon c nh sau:
+ Gradient pha ng khi s dng ct Symmetry:
Bng 3.3 Gradient pha ng khi s dng ct Symmetry
Thi gian (pht)
Bt u
1,0
7,0
7,5
8,5
9,0
12,0

Acetonitril (FA 0,1%)


20%
20%
50%
100%
100%
20%
20%

H2O (FA 0,1%)


80%
80%
50%
0%
0%
80%
80%

+ Gradient pha ng khi s dng ct XBridge:


Bng 3.4 Gradient pha ng khi s dng ct XBridge
Thi gian (pht)
Bt u
1,0
7,0
7,5
10,0

Acetonitril (FA 0,1%)


20%
50%
70%
20%
20%

H2O (FA 0,1%)


80%
50%
30%
80%
80%

- Nhn xt: Ct Symmetry l loi ct thng dng, c s dng nhiu trong cc


phng th nghim cn ct XBridge ang c ng dng trong cc th h my

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sc k mi. Chng ti xc nh iu kin phn tch trn c 2 loi ct ny


thun li hn cho vic trin khai phng php v sau.
3.2. Xc nh k thut x l mu
3.2.1. Quy trnh tch chit cc cht cn phn tch khi nn mu
Tin hnh kho st cc quy trnh chit mu khc nhau, mi test lm
04 ln, s dng cc mu trng cho thm mt lng chun xc nh :
Bng 3.5: Kt qu th nghim dung mi chit mu khi phn tch Norfloxacin
Dung mi chit

Acetonitril

Acetonitril/TCA

Acetonitril/NaOH

C thm vo (ng/l)
100
100
100
100
100
100
100
100
100
100
100
100

Area Std/IS

C thu c (ng/l)

1,990
1,991
1,190
2,169
2,654
2,688
2,361
2,893
2,181
1,888
2,252
2,109

69,2
69,6
66,2
75,8
92,7
93,6
82,5
99,9
76,4
65,7
78,2
73,3

TB

70,2

92,2

73,4

Hnh 3.1: Sc k th nghim dung mi chit mu khi phn tch norfloxacin

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Bng 3.6: Kt qu th nghim dung mi chit mu khi phn tch Ciprofloxacin


Dung mi chit
Acetonitril

Acetonitril/TCA

Acetonitril/NaOH

C thm vo (ng/l)
100
100
100
100
100
100
100
100
100
100
100
100

Area Std/IS
0,629
0,623
0,587
0,643
0,806
0,764
0,734
0,845
0,630
0,513
0,622
0,605

C thu c (ng/l)
73,6
73,5
69,1
75,5
95,1
89,7
86,2
99,1
74,4
60,2
73,1
71,0

TB
72,9

92,5

69,8

Bng 3.7: Kt qu th nghim dung mi chit mu khi phn tch Enrofloxacin


Dung mi chit
Acetonitril

Acetonitril/TCA

Acetonitril/NaOH

C thm vo (ng/l)
100
100
100
100
100
100
100
100
100
100
100
100

Area Std/IS
5,942
5,477
5,601
6,262
7,654
7,902
7,243
8,923
6,209
4,820
6,951
5,971

C thu c (ng/l)
67,5
62,7
63,8
71,5
91,1
93,6
87,9
104
71,2
64,9
79,1
67,9

TB
66,4

94,2

70,8

Bng 3.8: Kt qu th nghim dung mi chit mu khi phn tch Flumequin


Dung mi chit
Acetonitril

Acetonitril/TCA

Acetonitril/NaOH

Cthm vo (ng/l)

Area Std/IS

Cthu c (ng/l)

100
100
100
100
100
100
100
100
100

5,631
5,477
5,340
7,146
8,010
8,625
7,722
8,641
6,619

68,5
72,6
70,4
84,3
106
113
102
114
90,9

30

TB
74,0

108

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100
100
100

5,565
6,738
6,234

75,8
91,4
82,2

Nhn xt: t cc kt qu thu c trong cc bng trn, nhn thy: s dng


dung mi acetonitril/TCA 4% trong nc ct (70/30) cho hiu qu chit cc cht
cn phn tch cao nht. Cc dung mi nh Acetonitril v acetonitril/NaOH 0,1N
trong nc ct (70/30) cho hiu qu km hn. V vy chng ti la chn hn hp
acetonitril/TCA 4% trong nc ct (70/30) lm dung mi chit mu khi phn tch
d lng cc cht Norfloxacin, Ciprofloxacin, Enrofloxacin v Flumequin trong
thc phm.
3.2.2. Quy trnh lm sch v lm giu mu qua SPE
Tin hnh th nghim qu trnh lm sch v lm giu cht phn tch trn cc
loi ct SPE khc nhau (mi loi ct lm 04 ln song song) s dng cc trng l
mu tht, cho thm mt lng chun xc nh.
Kt qu thu c nh sau:
Bng 3.9: Kt qu th nghim ct SPE khi phn tch Norfloxacin
Ct SPE
HLB

C18-E

SCX

C thm vo (ng/l)
50
50
50
50
50
50
50
50
50
50
50
50

Area Std/IS
0,992
1,056
1,089
0,914
1,139
0,938
0,955
1,077
0,401
0,430
0,377
0,357

31

C thu c (ng/l)
35,7
36,7
37,8
36,2
38,1
34,5
35,3
37,6
14,8
15,9
13,9
12,8

R (%)
71,4
73,4
75,6
72,4
76,2
69,0
70,6
75,2
29,7
31,8
28,0
25,8

TB

73,0

72,8

28,8

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Hnh 3.2: Sc k th nghim ct SPE khi phn tch Norfloxacin

Bng 3.10: Kt qu th nghim ct SPE khi phn tch Ciprofloxacin


Ct SPE
HLB

C18-E

SCX

Ct SPE
HLB

C18-E

SCX

C thm vo (ng/l) Area Std/IS C thu c (ng/l)


R (%)
TB
50
0,274
36,1
72,2
50
0,319
39,4
78,8
74,8
50
0,301
38,5
77,0
50
0,294
35,5
71,1
50
0,327
39,6
79,2
50
0,338
40,8
81,6
75,1
50
0,255
34,1
68,2
50
0,268
35,6
71,2
50
0,163
19,9
39,8
50
0,116
17,2
34,4
36,6
50
0,113
16,9
33,8
50
0,151
19,2
38,4
Bng 3.11: Kt qu th nghim ct SPE khi phn tch Enrofloxacin
C thm vo (ng/l)
100
100
100
100
50
50
50
50
50
50
50
50

Area Std/IS
7,308
8,428
7,087
6,961
2,405
2,898
2,042
2,828
3,522
4,159
3,416
3,670

32

C thu c (ng/l)
89,5
99,4
88,3
87,1
35,9
39,9
34,3
40,6
43,8
45,1
39,9
42,9

R (%)
89,5
99,4
88,3
87,1
71,8
79,8
68,6
81,2
87,6
90,2
79,8
85,8

TB
91,1

75,4

85,8

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Bng 3.12: Kt qu th nghim ct SPE khi phn tch Flumequin


Ct SPE
HLB

C18-E

SCX

C thm vo (ng/l)

C thu c (ng/l)
90,3

R (%)
90,3

TB

100

Area Std/IS
7,457

100
100
100
100
100
100
100
100
100
100
100

7,751
7,261
7,375
6,055
5,996
4,861
4,708
2,872
3,172
2,795
2,784

91,8
88,1
90,1
77,7
76,9
75,6
72,7
41,5
46,5
39,5
39,2

91,8
88,1
90,1
77,7
76,9
75,6
72,7
41,5
46,5
39,5
39,2

91,1

75,7

41,7

Nhn xt: t cc kt qu thu c trong cc bng trn, nhn thy: s dng


ct SPE HLB v ct SPE C18-E cho hiu qu tt cho c 04 cht phn tch. Ct SPE
SCX ch hiu qu khi phn tch Enrofloxacin v Flumequin, nhng li rt km khi
phn tch Norfloxacin v Ciprofloxacin.. V vy chng ti la chn ct SPE HLB
v SPE C18-E lm sch mu khi phn tch ng thi d lng cc cht
Norfloxacin, Ciprofloxacin, Enrofloxacin v Flumequin trong thc phm.
3.2.3. Quy trnh x l mu c la chn
Cn khong 2g mu ng nht cho vo ng ly tm 50ml c np kn. Thm
khong 30ml acetonitril/TCA 4% trong nc ct (70/30), lc u, em rung siu
m 15 pht. Sau em ly tm 6000 vng/pht trong 5 pht, ly ra gn phn dch
trong cho vo bnh c quay 100ml. Chit li ln 2 vi 15ml acetonitril/TCA 4%
trong nc ct (70/30). Gp dch chit thu c, em c quay chn khng n khi
cn khong 10ml. Thm khong 20ml nc ct, lc u, lm sch qua ct SPE.
Cc giai on lm sch mu qua ct SPE HLB v ct SPE C18-E:
- Hot ha ct SPE: 6ml methanol, 6ml nc ct.
- Np mu vi tc khng qu 2ml/pht.
- Loi tp bng 3ml nc ct, 3ml MeOH 10%/nc ct
- Ra gii ly cht phn tch bng 2ln x 2ml/ln acetonitril.

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Dch ra gii c lc qua mng lc 0,45m cho vo vial 1,8ml v c em


phn tch trn h thng LC-MS/MS.
3.3. Thm nh phng php xy dng
3.3.1. Tnh thch hp ca h thng
Tin hnh phn tch lp li 09 ln dung dch chun hn hp cc cht phn
tch c nng xc nh trn h thng LC-MS/MS vi cc iu kin la chn
c trn. Xc nh RSD (%) v thi gian lu (tR) v t l din tch pc chun/din
tch pic ni chun (Std/IS) ca tng cht phn tch. Kt qu thu c nh sau:
Bng 3.13. Kt qu phn tch tnh thch hp ca h thng

Test 1
Test 2
Test 3
Test 4
Test 5
Test 6
Test 7
Test 8
Test 9
TB
SD
RSD(%)

Norfloxacin
tR
Std/IS
4,83
0,696
4,83
0,677
4,83
0,686
4,82
0,698
4,83
0,679
4,87
0,674
4,86
0,701
4,82
0,699
4,86
0,690
4,841
0,689
0,019
0,010
0,39
1,50

Ciprofloxacin
tR
Std/IS
5,03
0,185
5,01
0,189
5,03
0,183
5,00
0,184
5,03
0,181
5,04
0,185
5,04
0,187
5,01
0,186
5,03
0,181
5,024
0,185
0,014
0,003
0,28
1,46

Enrofloxacin
tR
Std/IS
5,14
1,132
5,14
1,118
5,14
1,138
5,14
1,138
5,14
1,114
5,17
1,131
5,16
1,129
5,14
1,106
5,14
1,141
5,146
1,127
0,011
0,012
0,22
1,07

Flumequin
tR
Std/IS
8,13
13,59
8,11
13,27
8,09
13,69
8,13
13,10
8,13
13,41
8,14
13,28
8,14
13,48
8,09
13,18
8,13
13,10
8,121 13,34
0,020 0,210
0,24
1,57

Nhn xt: T nhng kt qu bng trn nhn thy c 04 cht phn tch u c
gi tr RSD ca thi gian lu v t l din tch pic chun/din tch pic ni chun nh
hn 2% (trong khong t 0,24% n 1,57%). Nh vy, h thng LC-MS/MS ph
hp phn tch cc cht Norfloxacin, Ciprofloxacin, Enrofloxacin v Flumequin.

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Hnh 3.3: Sc k khi phn tch tnh thch hp ca h thng


3.3.2. c hiu/ chn lc
- Theo quy nh ca chu u (2002/657/EC) v phn tch d lng, cc phng
php phi t yu cu c t nht 4 im IP (Identification Point). Phng php ca
chng ti c 1 ion m (precursor ion = 1 IP) v 2 ion con (product ion = 1,5 IP) vi
mi cht. Nh vy, phng php xy dng t yu cu v c hiu ca chu
u.
- Tin hnh phn tch mu trng, mu dung dch chun hn hp cc cht v mu
trng thm chun trn h thng LC-MS/MS. Kt qu thu c nh sau:
+ Trn sc mu trng khng xut hin pic no c cng thi gian lu vi cc
cht phn tch trn sc mu dung dch chun v mu trng thm chun.
+ Trn sc mu dung dch chun hn hp, cc cht phn tch c cng thi
gian lu trn c 02MRM ca sc mu trng thm chun.
Nh vy phng php xy dng t yu cu v c hiu, chn lc khi
phn tch ng thi 04 cht nhm Quinolon l: Norfloxacin, Ciprofloxacin,
Enrofloxacin v Flumequin.
3.3.3. Khong tuyn tnh v ng chun
Tin hnh pha dy dung dch chun hn hp cc cht phn tch c cc nng
t 1ppb n 500ppb. Phn tch dy dung dch chun trn h thng LC-MS/MS, ti
mi im nng phn tch lp li 02 ln. Xy dng th biu th mi tng quan
tuyn tnh gia t l din tch pic chun/din tch pic ni chun vi nng cht
phn tch tng ng. Kt qu thu c nh sau:

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Bng 3.14: Kt qu phn tch dy chun Norfloxacin


C (ppb)
500
200
100
50
25
10
5
2

Std/IS
3,988
1,632
0,851
0,433
0,240
0,121
0,079
0,070

Bias (%)
-0,39
0,40
2,28
-1,07
0,80
1,70
-2,07
86,3

C (ppb)
500
200
100
50
25
10
5
2

Std/IS
4,020
1,609
0,831
0,438
0,236
0,120
0,077
0,048

Bias (%)
0,42
-1,03
-0,29
0,29
-1,39
0,60
-9,39
-52,5

Nhn xt: T cc kt qu bng trn cho thy: Norfloxacin c khong tuyn


tnh t 2ppb n 500ppb. Tuy nhin, kt qu kim tra li ng chun thng qua
nh gi gi tr bias (%) cho thy ti im nng 2ppb c cc gi tr ny ln hn
20% nn khng t yu cu. V vy ng chun ca Norfloxacin c lp trong
khong nng l 5ppb n 500ppb th phng trnh hi qui tuyn tnh c gi tr
R2 > 0,995 v cc gi tr bias < 15% t yu cu phn tch.
4.5

1.2

y = 0.0079x + 0.0406
R2 = 0.9999

3.5

y = 0.0022x + 0.0116
R2 = 0.9999

0.8

2.5

0.6

2
1.5

0.4

0.2

0.5
0

0
0

100

200

300

400

500

600

100

200

300

400

500

600

Hnh 3.4: th ng chun ca Norfloxacin (tri) v Ciprofloxacin (phi)

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Hnh 3.5: sc k khi xy dng ng chun ca norfloxacin


Bng 3.15: Kt qu phn tch dy chun Ciprofloxacin
C (ppb)
500
200
100
50
25
10
5
2

Std/IS
1,106
0,452
0,230
0,1156
0,0674
0,0321
0,0201
0,025

Bias (%)
0,14
0,77
-0,03
-4,96
1,76
-6,83
-6,13
218

C (ppb)
500
200
100
50
25
10
5
2

Std/IS
1,101
0,446
0,240
0,1187
0,0685
0,0328
0,0217
0,009

Bias (%)
-0,28
-0,57
4,30
-2,10
3,76
-3,64
-9,13
-151

Nhn xt: T cc kt qu bng trn cho thy: Ciprofloxacin c khong tuyn


tnh t 2ppb n 500ppb. Tuy nhin, kt qu kim tra li ng chun thng qua
nh gi gi tr bias (%) cho thy ti im nng 2ppb c cc gi tr ny ln hn
20% nn khng t yu cu. V vy ng chun ca Ciprofloxacin c lp trong
khong nng l 5ppb n 500ppb th phng trnh hi qui tuyn tnh c gi tr R2
> 0,995 v cc gi tr bias < 15% t yu cu phn tch.
Bng 3.16: Kt qu phn tch dy chun Enrofloxacin
C (ppb)
500
200
100
50
25
10

Std/IS
5,336
2,172
1,080
0,585
0,312
0,157

Bias (%)
0,02
0,42
-2,43
1,54
-0,55
2,04

C (ppb)
500
200
100
50
25
10

37

Std/IS
5,327
2,197
1,091
0,562
0,324
0,153

Bias (%)
-0,16
1,59
-1,43
-2,91
4,31
-1,53

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5
2

0,102
0,063

0,68
-32,3

5
2

0,105
0,064

6,64
-29,3

Nhn xt: T cc kt qu bng trn cho thy: Enrofloxacin c khong tuyn


tnh t 2ppb n 500ppb. Tuy nhin, kt qu kim tra li ng chun thng qua
nh gi gi tr bias (%) cho thy ti im nng 2ppb c cc gi tr ny ln hn
20% nn khng t yu cu. V vy ng chun ca Enrofloxacin c lp trong
khong nng l 5ppb n 500ppb th phng trnh hi qui tuyn tnh c gi tr R2
> 0,995 v cc gi tr bias < 15% t yu cu phn tch.
6

30

y = 0.0106x + 0.0487
R2 = 0.9999

25

20

15

10

y = 0.0472x + 0.2867
R2 = 0.9999

0
0

100

200

300

400

500

600

100

200

300

400

500

600

Hnh 3.6: th ng chun ca Enrofloxacin (tri) v Flumequin (phi)


Bng 3.17: Kt qu phn tch dy chun Flumequin
C (ppb)
500
200
100
50
25
10
5
2
1

Std/IS
23,77
9,676
5,027
2,700
1,479
0,775
0,521
0,371
0,232

Bias (%)
-0,50
-0,53
0,44
2,26
1,01
3,38
-0,82
-11,8
-215

C (ppb)
500
200
100
50
25
10
5
2
1

Std/IS
24,02
9,799
4,800
2,700
1,491
0,755
0,540
0,370
0,158

Bias (%)
0,56
0,76
-4,38
2,27
2,06
-0,77
7,31
-11,5
-238

Nhn xt: T cc kt qu bng trn cho thy: Flumequin c khong tuyn tnh
t 1ppb n 500ppb. Tuy nhin, kt qu kim tra li ng chun thng qua nh
gi gi tr bias (%) cho thy ti im nng 1ppb c cc gi tr ny ln hn 20%
nn khng t yu cu. V vy ng chun ca Flumequin c lp trong khong
nng l 2ppb n 500ppb th phng trnh hi qui tuyn tnh c gi tr R2 >
0,995 v cc gi tr bias < 15% t yu cu phn tch.

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3.3.4. Gii hn pht hin (LOD) v gii hn nh lng (LOQ)


T nhng kt qu thu c khi xy dng ng chun th hin mi tng quan
tuyn tnh gia nng cht phn tch v t l din tch pic chun tng ng/din
tch pic ni chun chng ti nhn thy:
- im nng thp nht trong ng chun ca Norfloxacin, Ciprofloxacin ,
Enrofloxacin l 5ppb v ca Flumequin l 2ppb. V vy, chng ti xc nh y
chnh l gii hn nh lng ca phng php phn tch cc cht ny.
- Trong khi LOQ = 3,3 x LOD nn LOD = LOQ/3,3. Nh vy, gii hn pht
hin ca phng php l khong 1,5ppb i vi Norfloxacin, Ciprofloxacin ,
Enrofloxacin v khong 0,6ppb i vi Flumequin.
- Kt qu phn tch th nghim mu trng thm chun nng thp cho thy
khi thm chun Norfloxacin, Ciprofloxacin , Enrofloxacin 1,5ppb v Flumequin 0,5
ppb cng c th xc nh c pic ca cc cht phn tch.
Gii hn pht hin v gii hn nh lng tnh trn nn mu tht s l:
Bng 3.18: LOD v LOQ ca cc cht phn tch tnh trn nn mu tht
LOD (g/kg)
LOQ (g/kg)

Norfloxacin
3,0
10

Ciprofloxacin
3,0
10

Enrofloxacin
3,0
10

Flumequine
1,2
4,0

Nhn xt: Gii hn pht hin v gii hn nh lng ca phng php thp hn
rt nhiu so vi mc gii hn tn d ti a cho php (MRL) ca cc cht phn tch
trong thc phm. V vy phng php t yu cu v nhy phn tch ng
thi d lng ca Norfloxacin, Ciprofloxacin , Enrofloxacin v Flumequin trong
thc phm.
3.3.5. lp li v thu hi ca phng php
a. Quy trnh lm sch qua ct SPE HLB
Tin hnh thm chun Norfloxacin 50ppb, Ciprofloxacin 50ppb, enrofloxacin
100ppb, Flumequin 100ppb vo mu trng v x l mu theo quy trnh s dng ct
SPE HLB la chn trn. Phn tch lp li 10 ln thu c kt qu sau:

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Bng 3.19: lp li v thu hi trn ct SPE HLB ca Norfloxacin


Test
1
2
3
4
5
6
7
8
9
10

C thu c (ng/ml)
35,3
39,5
40,8
35,9
40,1
38,9
35,7
36,7
37,8
36,2

C l thuyt (ng/ml)
50,0
50,0
50,0
50,0
50,0
50,0
50,0
50,0
50,0
50,0

R (%)
70,6
79,0
81,6
71,8
80,2
77,8
71,4
73,4
75,6
72,4

lp li

TB = 75,4%
RSD = 5,33%

Nhn xt: thu hi ca Norfloxacin khi x l mu qua ct SPE HLB trong


khong t 70,6% n 80,2% (trung bnh l 75,4%). lp li ca phng php ny
khi phn tch Norfloxacin c gi tr RSD l 5,33%.

Hnh 3.7: sc k ca norfloxacin khi x l mu qua ct SPE HLB


Bng 3.20: lp li v thu hi trn ct SPE HLB ca Ciprofloxacin
Test
1
2
3
4
5
6
7
8
9
10

C thu c (ng/ml)
37,3
39,6
36,3
38,1
42,3
41,1
36,1
39,4
38,5
35,5

C l thuyt (ng/ml)
50,0
50,0
50,0
50,0
50,0
50,0
50,0
50,0
50,0
50,0

40

R (%)
74,6
79,2
72,6
76,2
84,6
82,2
72,2
78,8
77,0
71,0

lp li

TB = 76,8%
RSD = 5,78%

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Nhn xt: thu hi ca Ciprofloxacin khi x l mu qua ct SPE HLB trong


khong t 72,2% n 84,6% (trung bnh l 76,8%). lp li ca phng php ny
khi phn tch Ciprofloxacin c gi tr RSD l 5,78%.
Bng 3.21: lp li v thu hi trn ct SPE HLB ca Enrofloxacin
Test
C thu c (ng/ml)
C l thuyt (ng/ml)
R (%)
lp li
1
88,6
100,0
88,6
2
97,8
100,0
97,8
3
98,7
100,0
98,7
4
93,2
100,0
93,2
5
89,1
100,0
89,1
TB = 93,5%
RSD = 5,07%
6
99,2
100,0
99,2
7
91,2
100,0
91,2
8
88,5
100,0
88,5
9
89,5
100,0
89,5
10
99,4
100,
99,4
Nhn xt: thu hi ca Enrofloxacin khi x l mu qua ct SPE HLB trong
khong t 88,5% n 99,4% (trung bnh l 93,5%). lp li ca phng php ny
khi phn tch Enrofloxacin c gi tr RSD l 5,07%.
Bng 3.22: lp li v thu hi trn ct SPE HLB ca Flumequin
Test
1
2
3
4
5
6
7
8
9
10

C thu c (ng/ml)
96,1
95,6
98,3
99,5
97,1
96,6
90,4
90,9
96,1
93.6

C l thuyt (ng/ml)
100,0
100,0
100,0
100,0
100,0
100,0
100,0
100,0
100,0
100.0

R (%)
96,1
95,6
98,3
99,5
97,1
96,6
90,4
90,9
96,1
93.6

lp li

TB = 95,4%
RSD = 3,11%

Nhn xt: thu hi ca Flumequin khi x l mu qua ct SPE HLB trong


khong t 90,4% n 99,5% (trung bnh l 95,4%). lp li ca phng php ny
khi phn tch Flumequin c gi tr RSD l 3,11%.
b. Quy trnh lm sch qua ct SPE C18-E
Tin hnh thm chun Norfloxacin 50ppb, Ciprofloxacin 50ppb, Enrofloxacin
50ppb, Flumequin 100ppb vo mu trng v x l mu theo quy trnh s dng ct
SPE C18-E la chn trn. Phn tch lp li 07 ln thu c kt qu sau:

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Bng 3.23: lp li v thu hi (R%) trn ct SPE C18 ca Norfloxacin


Test
1
2
3
4
5
6
7

C thu c (ng/ml)
36,0
35,5
38,6
39,5
34,9
37,1
39,2

C l thuyt (ng/ml)
50,0
50,0
50,0
50,0
50,0
50,0
50,0

R (%)
72,0
71,0
77,2
79,0
69,8
74,2
78,4

lp li

TB = 74,5%
RSD = 5,04%

Nhn xt: thu hi ca Norfloxacin khi x l mu qua ct SPE C18-E trong


khong t 69,8% n 79,0% (trung bnh l 74,5%). lp li ca phng php ny
khi phn tch Norfloxacin c gi tr RSD l 5,04%.
Bng 3.24: lp li v thu hi (R%) trn ct SPE C18-E ca Ciprofloxacin
Test
1
2
3
4
5
6
7

Cthu c (ng/ml)

Cl thuyt (ng/ml)

R (%)

lp li

36,7
37,8
40,1
39,9
38,5
39,6
40,8

50,0
50,0
50,0
50,0
50,0
50,0
50,0

73,4
75,6
80,2
79,8
77,0
79,2
81,6

TB = 78,1%
RSD = 3,71%

Nhn xt: thu hi ca Ciprofloxacin khi x l mu qua ct SPE C18-E trong


khong t 73,4% n 81,6% (trung bnh l 78,1%). lp li ca phng php ny
khi phn tch Ciprofloxacin c gi tr RSD l 3,71%.

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Bng 3.25: lp li v thu hi (R%) trn ct SPE C18-E ca Enrofloxacin


Test
1
2
3
4
5
6
7

C thu c (ng/ml)
36,3
35,4
39,5
41,9
35,9
39,9
40,6

C l thuyt (ng/ml)
50,0
50,0
50,0
50,0
50,0
50,0
50,0

R (%)
72,6
70,8
79,0
83,8
71,8
79,8
81,2

lp li

TB = 77,0%
RSD = 6,72%

Nhn xt: thu hi ca Enrofloxacin khi x l mu qua ct SPE C18-E trong


khong t 70,8% n 83,8% (trung bnh l 77,0%). lp li ca phng php ny
khi phn tch Enrofloxacin c gi tr RSD l 6,72%.
Bng 3.26: lp li v thu hi (R%) trn ct SPE C18-E ca Flumequin
Test
1
2
3
4
5
6
7

C thu c (ng/ml)
80,6
82,4
90,1
79,7
75,9
77,7
76,9

C l thuyt (ng/ml)
100,0
100,0
100,0
100,0
100,0
100,0
100,0

R (%)
80,6
82,4
90,1
79,7
75,9
77,7
76,9

lp li

TB = 80,5%
RSD = 5,97%

Nhn xt: thu hi ca Flumequin khi x l mu qua ct SPE C18-E trong


khong t 75,9% n 90,1% (trung bnh l 80,5%). lp li ca phng php ny
khi phn tch Flumequin c gi tr RSD l 5,97%.
c. Bn lun v thu hi v lp li ca phng php
Theo quy nh ca FDA [40] v AOAC [12] th khi phn tch d lng thuc th y
trong thc phm cc phng php phi c thu hi ti khong nng t 10g/kg n
100g/kg t trong khong t 70% n 110% v lp li phi c RSD < 15%.
T cc kt qu trn cho thy phng php phn tch d lng ca Norfloxacin,
Ciprofloxacin, Enrofloxacin, Flumequin trong tht c thm nh t yu cu ca
FDA, AOAC v thu hi v lp li.
Trong qu trnh x l mu, chng ti s dng c 02 loi ct SPE HLB v SPE
C18E lm sch v lm giu mu. Kt qu cho thy nu khi phn tch
Norfloxacin, Ciprofloxacin th 02 loi ct SPE ny cho hiu sut thu hi gn tng
ng nhau v s dng ct SPE C18-E cho n nh ( lp li) nhnh hn mt
cht. Tuy nhin phn tch Enrofloxacin v Flumequin th s dng ct SPE HLB
cho hiu qu v n nh cao hn hn so vi ct SPE C18-E.

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3.4. Phn tch trn mu thc nghim


Cc mu thc phm c ly ngu nhin ti cc ch trn a bn H Ni. Cc mu
nghin cu c tin hnh x l mu theo quy trnh la chn c trn v phn
tch trn h thng LC-MS/MS. Kt qu thu c nh sau:
Bng 3.27 : Kt qu phn tch 20 mu tht g (n v l g/kg)
Mu PT
Norfloxacin
Ciprofloxacin
Enrofloxacin
Flumequin
Mu PT
Norfloxacin
Ciprofloxacin
Enrofloxacin
Flumequin

TG01

TG02

TG03

TG04

TG05

TG06

TG07

TG08

TG09

TG10

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

TG11

TG12

TG13

TG14

TG15

TG16

TG17

TG18

TG19

TG20

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

Bng 3.28: Kt qu phn tch 20 mu tht ln (n v l g/kg)


TL01
Mu PT
Norfloxacin
(-)
Ciprofloxacin (-)
Enrofloxacin 49,1
Flumequin
(-)
TL11
Mu PT
Norfloxacin
(-)
Ciprofloxacin (-)
Enrofloxacin (-)
Flumequin
(-)

TL02

TL03

TL04

TL05

TL06

TL07

TL08

TL09

TL10

(-)
(-)
(-)
(-)

(-)
(-)
75,0
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

39,6
40,7
441
(-)

(-)
(-)
(-)
(-)

164
125
923
(-)

(-)
(-)
(-)
(-)

19,8
(-)
(-)
(-)

TL12

TL13

TL14

TL15

TL16

TL17

TL18

TL19

TL20

(-)
(-)
(-)
(-)

58,7
(-)
204
(-)

(-)
(-)
(-)
(-)

201
193
141
(-)

(-)
(-)
(-)
(-)

23,5
(-)
94,6
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

71,1
62,2
286
(-)

Bng 3.29: Kt qu phn tch 20 mu c nc ngt (n v l g/kg)


Mu PT

C01

C02

C03

C04

C05

C06

C07

C08

C09

C10

Norfloxacin
Ciprofloxacin
Enrofloxacin
Flumequin
Mu PT
Norfloxacin
Ciprofloxacin
Enrofloxacin
Flumequin

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

C11

C12

C13

C14

C15

C16

C17

C18

C19

C20

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-)
(-)
(-)
(-)

(-) Khng pht hin hoc di ngng pht hin ca phng php

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Hnh 3.8: Sc k ca mu thc t khng c norfloxacin


Nh vy: Sau khi phn tch 20 mu tht g, 20 mu tht ln, 20 mu c chng ti
thu c kt qu sau: Trong mu tht g v c em phn tch khng mu no c d
lng 4 loi quinolon trn; mt s mu ca tht ln c cha 3 loi quinolon l
ciprofloxacin, norfloxacin v enrofloxacin, khng c mu no cha flumequin.
So snh vi kt qu thu c vi s liu bng 1.1 (Gii hn tn d ti a ca cc
quinolon theo quyt nh s 2377/90/EC ca y ban Chu u [22] chng ti thy
rng: mt s mu tht ln c tn d norfloxacin v enrofloxacin, mt s mu tn d
c 3 loi quinolon. V norfloxacin nc ta cha c quy nh v gii hn ti a cho
php trong tht nn chng ti ch a ra kt lun l mu tht ln c cha
norfloxacin, cn trong thy sn th theo quyt nh ca B thy sn Fluoroquinolon
vn b cm s dng trong sn xut, kinh doanh thy sn xut khu vo th trng
M v Bc M. V enrofloxacin l sn phm chuyn ha ca ciprofloxacin nn
trong quy nh s tnh tn d enrofloxacin l tng ca enrofloxacin + ciprofloxacin.
Gii hn tn d ti a trong tht ca enrofloxacin + ciprofloxacin l 100 g/kg nn
chng ti c th kt lun mu TL1, TL3, TL17 u c d lng ciprofloxacin v
enrofloxacin nhng trong gii hn cho php; mu TL6,TL8,TL13,TL20, TL15 c
cha enrofloxacin v ciprofloxacin vt mc gii hn cho php.

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Chng 4. BN LUN
4.1. Xy dng quy trnh k thut
4.1.1. Cc iu kin phn tch trn h thng LC-MS/MS
Trong qu trnh nghin cu xy dng quy trnh xc nh d lng 4 loi
quinolon (ciprofloxacin, enrofloxacin, norfloxacin, flumequin trong thc phm),
thng qua cc kt qu th nghim, kt hp vi tham kho mt s ti liu [18], [35],
[39], chng ti la chn c cc thng s chy my quan trng nh sau: th
tch tim mu 20l, ct sc k Symmetry v tin ct tng ng , ct XBridge v
tin ct tng ng; nhit bung ct 350C ; tc dng 0,5 ml/pht cho ct
XBridge v 1,0 ml/pht cho ct Symmetry; gradient pha ng iu chnh ph hp
vi tng loi ct. Ct Symmetry c u im l hin nay c dng ch yu trong
cc phng th nghim, gi thnh r hn ct Xbridge nn tin trin khai s dng
trong cc phng th nghim Vit Nam; ct Xbridge c u im l tit kim dung
mi v thi gian, cho hiu qu tch tt hn nhng nhc im l t tin, phi c h
thng UPLC i km do chng ti pht trin song song c 2 loi ct ny, ty theo
iu kin phng th nghim m p dng loi ct no.
Xc nh c cc thng s chung ca detector MS cho 4 cht, xc nh c
cc thng s ti u cho tng cht phn tch; detector MS cho tn hiu tt v c
n nh cao, c chn lc, c hiu tin cy xc nh d lng 4 cht nhm
Quinolon. Nhng kt qu c la chn trn cng ging vi mt s nghin cu:
[13], [14], [19]. im khc bit y l chng ti s dng sc k lng ghp 2
ln khi ph, chng khc phc c nhc im ca ghp 1 ln khi ph (khng
nghin cu c c ch phn mnh, xc nh thm chi tit cu trc ho hc, do k
thut ion ho nh nn khi ph ch cho thy ion phn t) ng thi tng thm
nhy, chnh xc. Theo ch th ca cng ng Chu u 2002/657/EC [22],
nh danh hay khng nh cc cht b cm, cc cht c trong cc mu phn tch,
phng php kim nghim phi t c t nht l 4 im nhn danh (identification
point k hiu l IP) v ch c quyn kt hp ti a ba k thut. iu ny c ngha
l bt buc phi dng phng php khi ph phn tch khng nh cc cht b

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cm, cc ha cht bo v thc vt v tn d khng sinh, hormon trong thc phm.


Hin nay, cc phng th nghim th gii ang tp trung nghin cu pht trin hai k
thut chnh phn tch cc cht ny l LC-MS/MS v GC-MS/MS [20] do
phng php nghin cu ca chng ti ph hp vi yu cu v xu hng v k
thut ca th gii.
4.1.2. iu kin x l mu
X l mu trong phn tch thc phm l khu ht sc quan trng, mt trong
nhng yu t nh hng n chnh xc ca kt qu phn tch. Tu tng i
tng mu, tu tng ch tiu phn tch m phi c cch x l khc nhau, bo qun
mu sau x l khc nhau. Khi tin hnh phn tch d lng mt s cht nhm
Quinolon trong thc phm cn phi tin hnh x l mu qua 02 giai on chnh l
chit tch cc cht cn phn tch ra khi nn mu v tin hnh lm sch, loi tp kt
hp lm giu mu. Hm lng cc cht cn phn tch rt nh (ch khong vi chc
ppb) nn 02 giai on trn l cn thit. Nhiu k thut x l mu c th c s
dng phn tch tn d khng sinh trong cc nn mu khc nhau. Hai k thut ph
bin c s dng trn th gii v Vit Nam l k thut chit lng-lng v k thut
chit pha rn. Cc nn mu trong nghin cu l cc thc phm (ch yu l tht v
ni tng ng vt) y l nn mu kh phc tp nn thng phi s dung phi hp
nhiu k thut x l mu t hiu qu cao. Dung mi c s dng ch yu l
acetonitril [13], [14]...[22] hoc hn hp acetonitril: acid phosphoric 0,3%, hn hp
acetonitril : acid formic [13]. Mt s nghin cu cng s dng hn hp acid
acetic/ethanol hoc acid formic/ethyl acetate chit mu cho kt qu tt.. Qua
nghin cu v th nghim chng ti nhn thy chit mu bng dung mi
acetonitril/TCA 4% trong nc ct (70/30) kt hp vi qua ct chit pha rn SPE
C18-E v SPE HLB s cho hiu qu x l mu tt, t yu cu .
4.2. Thm nh quy trnh xy dng
Trong nghin cu ny, chng ti s dng h thng LC-MS/MS hin i c
trang b phn mm tnh ton tinh khit pic sc k. Kt hp vi cc thng s chy

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my la chn trn gip phn bit sc k ca 4 loi quinolon. Phng php


xc nh ng thi 4 loi quinolon trong thc phm t yu cu v chn lc.
Phng php ca chng ti c khong tuyn tnh ca ciprofloxacin,
enrofloxacin, norfloxacin l 5ppb-500ppb; flumequin l t 2ppb n 500ppb. So
snh kt qu trn vi nghin cu ca tc gi

Jonas Augusto Rizzto Paschoal v

cng s [29] cho thy nghin cu ca chng ti c khong tuyn tnh ca


ciprofloxacin v enrofloxacin rng hn (ciprofloxacin tuyn tnh trong khong
17ppb-300ppb, enrofloxacin tuyn tnh trong khng 21ppb-300ppb); so vi nghin
cu ca A. Pena v cng s [13] (c ca ciprofloxacin, enrofloxacin, norfloxacin l
6,25 ppb n 125ppb) th nghin ca chng ti c khong tuyn tnh ca 3 loi
quinolon ny rng hn rt nhiu.
Gii hn xc nh v gii hn nh lng trong nghin cu ny c xc nh
ca ciprofloxacin, enrofloxacin, norfloxacin l 3ppb v 10ppb v flumequin l 1,2
ppb v 4 ppb. So snh vi phng php khc, phng php ny c kt qu thp hn
so vi kt qu nghin cu ca Jonas Augusto Rizzato Paschoal v cng s [29]
(ciprofloxacin 4ppb v 17 ppb, enrofloxacin 6ppb v 21ppb, flumequin 5ppb v
18ppb); v c kt qu tng ng so vi kt qu nghin cu ca Cun Li v cng
s [19] (ciprofloxacin 3ppb v 10 ppb, enrofloxacin 3ppb v 10ppb) . Trong nghin
cu ca Verdon v cng s [41] s dng detector hunh quang c LOD khong 411 mcg/kg v LOQ khong 13-36 mcg/kg. Nh vy sc k lng ghp ni vi
detector khi ph c nhy v tnh c hiu rt cao, p ng c yu cu ngy
cng kht khe ca th gii v phn tch d lng.
T cc kt qu thc nghim thu c trn cho chng ta thy: khi x l mu
qua ct SPE HLB :Norfloxacin c gi tr RSD l 5,33%, Ciprofloxacin c gi tr
RSD l 5,78%, Enrofloxacin c gi tr RSD l 5,07%, Flumequin c gi tr RSD l
3,11%. Khi x l mu qua ct SPE C18-E : Norfloxacin c gi tr RSD l 5,04%,
Ciprofloxacin c gi tr RSD l 3,71%, Enrofloxacin c gi tr RSD l 6,72%,
Flumequin c gi tr RSD l 5,97%. Kt qu ny c th so snh c vi lp li
thu c trong nghin cu ca tc gi O. Ballesteros v cng s [37] gi tr RSD

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khong 4,9 %-7,5%; lp li thu c trong nghin cu ca tc gi E. Rodriguez


v cng s [21] Ciprofloxacin c gi tr RSD l 7%, Enrofloxacin c gi tr RSD l
5% v lp li thu c trong nghin cu ca tc gi A. Pena v cng s [13] l
:Norfloxacin c gi tr RSD l 7%, Ciprofloxacin c gi tr RSD l 6,5%,
Enrofloxacin c gi tr RSD l 8,7% . Nh vy phng php ca chng ti c lp
li tt hn. Ngoi ra kt qu ca nghin cu cng t theo yu cu ca AOAC [12]
Nghin cu ca chng ti cn cho thy: thu hi trung bnh khi x l mu qua ct
SPE HLB ca Norfloxacin l 75,4%, Ciprofloxacin l 76,8%, Enrofloxacin l
93,5%, Fumequin l 95,4%; thu hi trung bnh khi x l mu qua ct SPE C18-E
ca Norfloxacin l 74,5%, Ciprofloxacin

l 78,1%, Enrofloxacin l 77,0%,

Flumequin l 80,5%). Kt qu trn ca phng php gn tng ng vi kt qu


nghin cu ca tc gi Cun li v cng s [19] v thu hi ca enrofloxacin 95%
v ca tc gi A.pena v cng s [13] (c thu hi trung bnh ca norfloxacin l
76%, ciprofloxacin l 75,8%. Kt qu ca nghin cu t theo yu cu ca AOAC
[12]

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KT LUN
Sau khi hon thnh nghin cu ny, trn c s cc kt qu phn tch thc
nghim xc nh d lng 4 loi quinolon (ciprofloxacin, enrofloxacin,
norfloxacin, flumequin) trong thc phm bng k thut LC-MS/MS, chng ti thu
c cc kt qu sau:
* Xy dng c phng php nh lng ng thi 4 loi quinolon trong thc
phm tng ng vi 2 giai on:
- X l mu: Mu c chit bng hn hp acetonitril/TCA 4% trong nc ct
(70/30) ) kt hp vi qua ct chit pha rn SPE C18-E v SPE HLB s cho hiu
qu x l mu tt, t yu cu.
- Phn tch bng LC-MS/MS:
Thng s ca detector khi ph
+ Ch ESI (+) cho cng tn hiu ca cc cht l tt nht.
+ Cc thng s chung:
Kh mn (Curtain gas)
Kh va chm (Collision gas )
Th phun in t (Ionspray voltage )
Nhit mao qun (TEM)
Kh ngun ion 1 (Ion source gas 1)
Kh ngun ion 1 (Ion source gas 2)
Th u vo (Entrance potential)

15
7
5000
4500C
20
20
10

+ Cc thng s chi tit cho tng cht


Ion con 1

Ion con 2

Cht phn tch

(M+H)+
m/z

DP
(volt)

m/z

CE

CXP

m/z

CE

Ciprofloxacin
Norfloxacin
Enrofloxacin
Flumequin

332
320
360
262

91
96
41
106

314
276
316
202

27
23
25
43

34
26
18
26

288
302
342
244

23
25
27
25

CX
P
22
14
20
28

Thng s ca phn LC
+ S dng ct C18
Ct Symmetry (250mm x 4,6mm; 5,0 m) v tin ct tng ng (Waters).

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Ct XBridge (150mm x 2,1mm; 3,5 m) v tin ct tng ng (Waters).


+ Dung mi hu c: Acetonitril v H2O thay i theo gradient.
+ Dung dch acid hu c: nc ct c cha 0,1% acid formic (FA)
+ Th tch tim mu 20l, nhit bung ct 350C
+ Tc dng 0,5 ml/pht cho ct XBridge v 1,0 ml/pht cho ct Symmetry.
* Phng php xy dng c thm nh cho thy c tnh chn lc, c n
nh cao, c lp li v thu hi tt, c th p dng vo thc t.
- Khong tuyn tnh rng:
+ Ciprofloxacin, enrofloxacin, norfloxacin l 5ppb n 500ppb
+ Flumequin l t 2ppb n 500ppb
- Gii hn pht hin :
+ Ciprofloxacin, enrofloxacin, norfloxacin l 3ppb
+ Flumequin l 1,2 ppb
- Gii hn nh lng :
+ Ciprofloxacin, enrofloxacin, norfloxacin l 10ppb
+ Flumequin l 4 ppb
- thu hi v lp li ca phng php p ng theo yu cu ca AOAC
* p dng quy trnh phn tch xy dng trn, chng ti phn tch 60 mu
thc phm ti cc ch khc nhau trn a bn H ni xc nh d lng
quinolon. Kt qu thu c l mu tht g v c khng c cha d lng 4 loi
quinolon trn trn. Mt s mu tht ln c cha d lng ca 3 quinolon l
(ciprofloxacin, enrofloxacin, norfloxacin) trong c mu th t gii hn cho php v
d lng quinolon, c mu vt qu gii hn cho php v d lng quinolon.

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KIN NGH

Trong khun kh lun vn, vi thi gian v iu kin hn hp nn chng


ti mi ch nghin cu trong phm vi l xc nh d lng 4 cht nhm quinolon
(ciprofloxacin, enrofloxacin, norfloxacin, flumequin) v ng dng quy trnh xy dng
phn tch ngu nhin trn 60 mu tht v c. V vy mong mun ca chng ti l trong
thi gian sp ti phi m rng nghin cu phng php ny thnh phng php chung
c th p dng phn tch ng thi cc cht khc thuc nhm quinolon v cc nhm
khng sinh khc na; ng thi ng dng quy trnh xy dng phn tch trn nhiu
loi i tng thc phm khc vi s lng mu ln hn.

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TI LI U THAM KHO
Ting vit
1. V Th Tr An (2001): Tnh hnh s dng khng sinh v d lng khng sinh
trong tht g ti cc c s chn nui g cng nghip ca thnh ph H Ch Minh, lun
vn thc s, H Nng Lm TP HCM.
2. B y t (2009), Ha phn tch, Nh xut bn Y hc.
3. L Th Ngc Dip (2001): Mt s kt qu kho st tnh hnh s dng thuc khng
sinh trong chn nui g v tn d khng sinh trong tht, trng g trn a bn H
Ni, trng i hc Nng nghip H Ni.
4. Phm Kim ng v cng s (2008): ng dng phng php ELISA phn tch
tn d khng sinh nhm Quinolone trong tm ti mt s tnh ven bin khu vc Bc
B, Tp ch Khoa hc v pht trin, Tp VI, s 3, trng H Nng nghip HN.
5. i hc dc H Ni (2004), Ha dc tp 2, NXB Y hc H Ni.
6. Nguyn Th Thanh Nga (2011), Nghin cu quy trnh xc nh d lng
ciprofloxacin v enprofloxacin trong thc phm bng phng php HPLC-MS/MS,
lun vn thc s, i hc KHTN, Thnh ph H Ch Minh.
7. Thi Phan Qunh Nh (2005): Phng php phn tch bng sc k lng hiu nng
cao, Vin Kim nghim- B Y t.
8. Trn Minh Ph v cng s (2008): Xc nh thi gian tn lu Enrofloxacin trn
cc tra, tp ch khoa hc 215 -218, trng i hc Cn Th.
9. B Nng nghip (2009) Thng t 03/2012/TT-BNNPTNT ngy 16/01/2011 sa i,
b sung Thng t s 15/2009/TT-BNN ngy 17/3/2009 ca B Nng nghip v Pht
trin nng thn ban hnh Danh mc thuc, ho cht, khng sinh cm s dng, hn ch
s dng
10. B thu sn (2005), Quyt nh s 07/2005/QD-BTS ban hnh ngy 24/02/2005
quy nh danh mc 17 loi khng sinh cm s dng v danh mc 34 loi hn ch s
dng trong c nhm Quinolon
11. B thu sn (2005), Quyt nh s 26/2005/QD-BTS ban hnh ngy 18/8/2005 b
sung nhm Quinolon vo danh mc cm s dng trong sn xut, kinh doanh thy sn
xut khu vo th trng Bc M v Hoa K.

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Ting anh
12. AOAC International (2007): Validation and Qualification in Analytical
Laboratories, Second Edition.
13. A. Pena, L. J. G. Silva, A. Pereira & L. Meisel, C. M. Lino (2010), Determination
of fluoroquinolone residues in poultry muscle in Portugal, Analytical and
Bioanalytical Chemistry, 397, pp. 26152621.
14. Alexandra Junza, Rameshwari Amatya, Rafael Prez-burgos, Gultekin Gokce,
Edyta Grzelak, Dolores Barrn, Jos Barbosa (2010), Residues of -lactams and
quinolones in tissues and milk samples. Confirmatory analysis by liquid
chromatographymass spectrometry, Ovidius University Annals of Chemistry, 21(2),
pp. 109-122.
15. B. Delepine, D. Hurtaud-Pessel, P. Sanders (1998), Simultaneous determination
of six quinolones in pig muscle by liquid chromatography-atmospheric pressure
chemical ionisation mass spectrometry, The Analyst, 123, pp. 2743-2747.
16. Chui-Shiang Chang, Wei-Hsien Wang, Chin-En Tsai (2008), Simultaneous
determination of eleven quinolones antibacterial residues in marine products and
animal tissues by Liquid chromatography with Fluorescence detection, Journal of
Food and Drug Analysis, 16(6), pp. 87-96.
17. Chui-Shiang Chang, Wei-Hsien Wang, Chin-En Tsai (2010), Simultaneous
determination of 18 quinolone residues in marine and livestock products by liquid
chromatography/tandem mass spectrometry, Journal of Food and Drug Analysis,
18(2), pp. 87-97.
18. Chung-Wei Tsai, Chan-Shing Lin, Wei-Hsien Wang (2012), Multi-residue
determination of sulfonamide and quinolone residues in fish tissues by High
performance liquid chromatography-tandem mass spectrometry (LC-MS/MS),
Journal of Food and Drug Analysis, 20(3), pp. 674-680.
19. Cun

Li

(2008),

Simultaneous

determination

of

fluoroquinolones

and

sulfonamides in chicken muscle by LC with fluorescence and uv detection,


Chromatographia, 68, pp. 117-121.

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WWW.DAYKEMQUYNHON.UCOZ.COM

20. D. Barrn, E. Jimnez-Lozano, S. Bailac, J. Barbosa (2003), Simultaneous


determination of flumequine and oxolinic acid in chicken tissues by solid phase
extraction and capillary electrophoresis, Analytica Chimica Acta, 477, pp. 2127.
21. E. Rodriguez, M.C. Moreno-Bondi, M.D. Marazuela (2008), Development and
validation of a solid-phase extraction method coupled to liquid chromatography with
fluorescence detection for the determination of fluoroquinolone residues in powdered
infant formulae Application to the analysis of samples from the Spanish and Latin
American market, Journal of Chromatography A, 1209, pp. 136144.
22. European Commission (2002) Commission decision 2377/90/EC
23. European Commission (1996) Council directive 96/23/EC
24. Ghislain Follet, (1997), Antibiotic resistance in the EU Science, Politics and
Policy. Number 3&3. 2000, p.148-155
25. Fatima Tamtam, Fabien Mercier, Jolle Eurin, Marc Chevreuil, Barbara Le Bot
(2009), Ultra performance liquid chromatography tandem mass spectrometry
performance evaluation for analysis of antibiotics in natural waters, Analytical and
Bioanalytical Chemistry, 393, pp. 1709-1718.
26. Florentina Caada-Caada, Anunciacion Espinosa-Mans illa, Ana Jimnez Girn
and Arsenio Muoz de la Pea (2012), Simultaneous determination of the residues of
fourteen

quinolones

and

fluoroquinolones

in

fish

samples

using

Liquid

chromatography with Photometric and Fluorescence detection, Czech J. Food Sci.,


30(1), pp. 74-82.
27. Francisco J. Lara (2006), Multiresidue method for the determination of quinolone
antibiotics in bovine raw milk by capillary electrophoresis-tandem massspectrometry,
Analytical Chemistry, 78(22), pp. 7665-7673.
28. Habib Amjad, Javed Iqbal and Muhammad Naeem (2006), Estimation of selected
residual antibiotics in muscle, kidney, liver, and egg of layer chicken, Proc. Pakistan
Acad. Sci, 43(1), pp. 29-37.
29. Jonas Augusto Rizzato Paschoal, Felix Guillermo Reyes Reyes, Susanne Rath
(2009), Quantitation and identity confirmation of residues of quinolones in tilapia

WWW.FACEBOOK.COM/DAYKEM.QUYNHON

WWW.DAYKEMQUYNHON.UCOZ.COM

fillets by LC-ESI-MS-MS QToF, Analytical and Bioanalytical Chemistry, 394, pp.


2213-2221.
30. J.C. Yorke, P. Froc (2000), Quantitation of nine quinolones in chicken tissues by
high-performance liquid chromatography with fluorescence detection, Journal of
Chromatography A, 882, pp. 63-77.
31. Marni S., Mustafa A.M., Marzura M.R. (2011), Analysis of quinolones in poultry
muscles using liquid chromatographytandem mass spectrometry, Malaysian Journal
of Veterinary Research, 2(1), pp. 1-15.
32. Martin D. Rose, John Bygrave, George W. F. Stubbings (1998), Extension of
multi-residue methodology to include thedetermination of quinolones in food, The
Analyst, 123, pp. 27892796.
33. Muhammad Naeem, Khalida Khanand Samra Rafiq (2006), Determination of
Residues

of

Quinolones

in

Poultry

Products

by

High

Pressure

Liquid

Chromatography, Journal of Applied Science, 6(2), pp. 373-379.


34. M.K. Hassouan, O. Ballesteros, J. Taoufiki, J.L. Vlchez, M. Cabrera-Aguilera, A.
Navaln (2007), Multiresidue determination of quinolone antibacterials in eggs of
laying hens by liquid chromatography with fluorescence detection, Journal of
Chromatography B, 852, pp. 625-630.
35. Njla Mohamad Kassab, Anil Kumar Singh, Erika Rosa Maria Kedor-Hackmam,
Maria Ins Rocha Miritello Santoro (2005), Quantitative determination of
ciprofloxacin and norfloxacin in pharmaceutical preparations by high performance
liquid chromatography, Brazilian Journal of Pharmaceutical Sciences, 41(4), pp.
507-513.
36. N. Stoilova, M. Petkova (2010), Developing and validation of method for
detection of quinolone residues in poultry meat, Trakia Journal of Sciences, 8(1), pp.
64-69.
37. O. Ballesteros, V. Sanz-Nebot, A. Navaln, J. L. Vlchez, J. Barbosa (2004),
Determination of a series of quinolone antibiotic using Liquid chromatographyMass-spectrometry, Chromatographia,59, pp. 543-550.

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WWW.DAYKEMQUYNHON.UCOZ.COM

38. Sherri B. Turnipseed, Jos E. Roybal, Allen P. Pfenning (2003), Confirmation of


fluoroquinolone residues in salmon and shrimp tissue by LC/MS: Evaluation of single
quadrupole and ion trap instruments, Food and drug administration, USA.
39. Susan B. Clark, Joseph M. Storey, Sherri B. Turnipseed (2008), Optimization

and

Validation

of

Multi-class,

Multi-residue

LC-MS/MS

Screening

and

Confirmation Method for Drug Residues in Milk, Food and drug administration,
USA.
40. US Food and Drug Administration (May 2001), Bionalytical method validation,
Guidance for industry.
41. Verdon E, Couedor P, Roudaut B, Sanders P (2005) Multiresidue method for

simultaneous

determination

multimatrix/multispecies

of

animal

ten

quinolone

tissues

by

antibacterial

liquid

residues

chromatography

in
with

fluorescence detection: single laboratory validation study. JAOAC Int, 88:11791192.


42. Yang Xiao, Hong Chang, Ai Jia, Jianying Hu (2008), Trace analysis of quinolone
and fluoroquinolone antibiotics from wastewaters by liquid chromatography
electrospray tandem mass spectrometry, Journal of Chromatography A, 1214, pp.
100-108.

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PH LC
Cch pha dung dch chun
- Dung dch chun gc: cn chnh xc khong 10mg cc cht chun cho vo
trong cc bnh nh mc 100ml, ha tan v nh mc bng Acetonitril thu c
cc dung dch chun gc 100ppm. Bo qun -40C, trnh nh sng.
- Dung dch chun trung gian: Ly chnh xc 1ml cc dung dch chun gc
cho vo bnh nh mc 100ml, nh mc bng Acetonitril, lc u thu c
dung dch chun trung gian hn hp 1ppm (C01). Vi ni chun (IS)
Ciprofloxacin d8 pha dung dung ni chun trung gian 1ppm ring (C0IS). Bo
qun 4-80C.
- Dung dch chun hn hp 50ppb: Ly chnh xc 1ml dung dch chun
trung gian hn hp 1ppm cho vo bnh nh mc 20ml, nh mc bng
Acetonitril thu c dung dch chun hn hp 50ppb (C02).
- Pha dy chun lm vic:
Cch pha dy chun lm vic
Chun (ppb)

C01 (ml)

C02 (ml)

C0IS (ml)

500
200
100
50
25
10
5
2
1

5,0
2,0
1,0
0,5
0
0
0
0
0

0
0
0
0
5,0
2,0
1,0
0,4
0,2

1,0
1,0
1,0
1,0
1,0
1,0
1,0
1,0
1,0

Acetonitril
(ml)
4,0
7,0
8,0
8,5
4,0
7,0
8,0
8,6
8,8

Cc dung dch lm vic c pha mi mi ngy.

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Quy trnh k thut xc nh d lng 4 cht nhm quinolon

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Intensity, cps

0.0
0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

7.0e5

8.0e5

9.0e5

1.0e6

1.1e6

1.2e6

1.3e6

1.4e6

1.5e6

1.6e6

1.7e6

1.8e6

1.9e6

0.5

1.0

1.5

2.5

3.0

3.5

4.0

4.5

5.0
Time, min

5.5

6.0

6.5

6.61

7.0

Sc phn tch cc cht nhm Quinolon trn ct XBrigde

2.0

5.46

XIC of +MRM (30 pairs): 262.000/244.000 Da from Sample 44 (Std-MixQuinolon-200ppb01) of Data.wiff (Turbo Spray)

Acq. Time: 18:20


Acq. Date: Sunday, May 25, 2014

7.5

8.0

8.5

9.0

9.5

Max. 1.7e6 cps.

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Intensity, cps

0.00
0.0

5.00e4

1.00e5

1.50e5

2.00e5

2.50e5

3.00e5

3.50e5

4.00e5

4.50e5

5.00e5

5.50e5

6.00e5

6.50e5

7.00e5

7.50e5

8.00e5

8.50e5

9.00e5

9.50e5

1.00e6

1.05e6

0.5

1.0

1.5

2.0

3.0

3.5

4.0

4.5

5.0

5.5

6.0
6.5
Time, min

7.0

7.5

8.15

8.0

Sc phn tch cc cht nhm Quinolon trn ct XBrigde

2.5

6.77

XIC of +MRM (30 pairs): 262.000/244.000 Da from Sample 27 (Std-MixQuinolon-200ppb01) of Data.wiff (Turbo Spray)

Acq. Time: 02:46


Acq. Date: Monday, May 26, 2014

8.5

9.0

9.5

10.0

10.5

11.0

11.5

Max. 8.3e5 cps.

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Intensity, cps

0.0
150

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

5.5e6

6.0e6

6.3e6

170

180

176.0
190

189.0
200

202.2
210

220

217.2
230

231.0
240

250

260
m/z, Da

270

280

290

300

310

314.2
320

316.2

Ph Enrofloxacin (Ion m: 360 m/z, ion con nh lng: 316 m/z, ion con 342
m/z)

160

162.2

203.2

245.2

+MS2 (360.14) CE (47): 26 MCA scans from Sample 1 (TuneSampleName) of ENR_InitProduct_Pos.wiff (Turbo Spray)

Acq. Time: 13:31


Acq. Date: Sunday, August 05, 2012

330

340

342.2

350

360

360.2

Max. 6.3e6 cps.

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Intensity, cps

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

5.5e6

6.0e6

6.5e6

7.0e6

7.5e6

8.0e6

8.5e6

9.2e6
9.0e6

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80

90

100

100.0

110

120

119.0
130

126.2

140

150

146.0

160

170
m/z, Da

174.0

180

190

200

202.0

210

220

Ph Flumequine (Ion m: 262 m/z, ion con nh lng: 202 m/z, ion con 244 m/z)

70

75.0

98.0

99.0

+MS2 (262.10) CE (47): 26 MCA scans from Sample 1 (TuneSampleName) of FLU_InitProduct_Pos.wiff (Turbo Spray)

Acq. Time: 10:16


Acq. Date: Sunday, August 05, 2012

230

240

244.2

250

260

262.2

Max. 9.2e6 cps.

WWW.DAYKEMQUYNHON.UCOZ.COM

Intensity, cps

0.0
150

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

2.1e5

2.2e5

2.3e5

2.4e5

151.4

168.0

180

173.4 183.8

176.0

185.0

190

192.2

191.2

200

210

206.0
200.8

198.2

203.2

215.2

220

226.2

230

240
m/z, Da

238.2

231.2

233.0

228.0

225.2
217.0

219.2

250

260

256.2

270

282.0

280

276.4

Ph Norfloxacin (Ion m: 320 m/z, ion con nh lng: 276 m/z, ion con 302 m/z)

170

160.2 174.0

160

156.0

164.2

162.0

161.0

205.0

+MS2 (320.15) CE (47): 26 MCA scans from Sample 1 (TuneSampleName) of NOR_InitProduct_Pos.wiff (Turbo Spray)

Acq. Time: 12:25


Acq. Date: Sunday, August 05, 2012

290

300

302.2

310

320

320.0

Max. 2.5e5 cps.

WWW.DAYKEMQUYNHON.UCOZ.COM

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Intensity, cps

0.0
150

5.0e4

1.0e5

1.5e5

2.0e5

2.5e5

3.0e5

3.5e5

4.0e5

4.5e5

5.0e5

5.5e5

6.0e5

6.5e5

7.0e5

7.5e5

8.0e5

8.5e5

9.0e5

9.3e5

160

151.0 162.2

180

190

190.0
200

206.2
210

220

207.2 218.4

230

233.0
240

250
m/z, Da

248.4

249.2

260

270

280

290

302.2
300

296.4

310

322.2

320

Ph Ciprofloxacin-D8 (Ion m: 340 m/z, ion con nh lng: 322 m/z, ion con
296)

170

180.2

209.2

235.0

+MS2 (340.15) CE (47): 26 MCA scans from Sample 1 (TuneSampleName) of IS-D8_InitProduct_Pos.wiff (Turbo Spray)

Acq. Time: 14:21


Acq. Date: Sunday, August 05, 2012

330

340

340.2

Max. 9.3e5 cps.

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Sc k khi xy dng ng chun ca ciprofloxacin

Sc k th nghim dung mi chit mu khi phn tch


ciprofloxacin

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Sc k th nghim ct SPE khi phn tch ciprofloxacin

Sc k phn tch tnh thch hp ca h thng khi phn tch enrofloxacin

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Sc k khi x l mu qua ct SPE HLB ca flumequin

Sc k khi x l mu qua ct SPE C-18 ca enrofloxacin

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Sc k ca mu thc t khng c ciprofloxacin

Sc k ca mu thc t c enrofloxacin

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