XC NH D LNG CHLORAMPHENICOL TRONG SA BNG

K THUT SC K LNG HAI LN KHI PH (LC/MS/MS)

Ch Vn Tut
(1)
, u Ngc Ho
(2)
, Trn Th Mai Tho
(1)
,
Phan Th Hu
(1)
, V Dng Minh
(1)
v Phm Th Trang
(1)

TM TT
Phng php xc nh d lng chloramphenicol (CAP) trong sa bng k thut sc k
lng hai ln khi ph (LCMSMS) c lp dng v ph duyt thnh cng. Phng php l ph
hp pht hin lng vt CAP ngng i hi ca th trng (MRPL l 0,3 g/kg).
D lng CAP trong sa c chit bng ethyl acetate, sau c lc v bm ln h
thng my LCMSMS. Pht hin CAP bng ngun ion phun in t (ESI m cc, ch chy
SIM. Khng nh CAP v CAP-d5 c kim sot bng 3 ion c trng. Phng php c
ph duyt da theo quyt nh 657/2002/EC ca cng ng chung chu u cho nhm d lng
thuc th y trong thc phm. lp li v thu hi cc nng thm chun 0,3; 0,45; 0,6
g/kg l 7,6% v nm trong khong -50 +20%. ti lp ca phng php trn cc nn mu
sa khc nhau l 16,5%. Gii hn quyt nh (CC) v kh nng pht hin (CC) tng ng l
0,10 v 0,12 g/kg. khng m bo o ca phng php l 33,1%.
T kha: Chloramphenicol, D lng, Phng php xc nh, Sa, Sc k lng hai ln
khi ph.
Determination of chloramphenicol residue in milk
by liquid chromatography tandem mass spectrometry (LCMSMS)
Chu Van Tuat, Dau Ngoc Hao

,Tran Thi Mai Thao,
Phan Thi Hue, Vu Dung Minh and Pham Thi Trang,
SUMMARY
The method for determination of chloramphenicol residue in milk by liquid
chromatography tandem mass spectrometry was successfully developed and validated. The
method is fit for purpose to detect trace amount of the CAP at Minimum Required Performance
Limit (MRPL, 0.3 g/kg).
The trace amount of CAP in milk was extracted by ethyl acetate, filtered and injected onto
the LC-MS/MS system. Detection of CAP was achieved by negative ionization electrospray (ESI)
in the selected ionization monitoring (SIM) mode. For confirmation, three characteristic mass
transitions were monitored each for the CAP and the CAP-d5 standard. The method was validated
according to the decision 657/2002/EC of European Union criteria for the analyses of veterinary
drug residues in food. The repeatability and the recovery at all three fortification levels at 0.3,
0.45, 0.6 g kg
-1
at different days of the method was 7.6% and within the ranges of -50 +20%,
respectively. The within-laboratory reproducibility on different milk matrices was 16.5%. The
decision limit (CC) and the detection capability (CC) were at 0.10 and 0.12 g kg
-1
,
respectively. The meaurement uncertainty of the method was 33.1%.
Key words: Chloramphenicol, Residue, Validation method, Milk, Liquid chromatography
tandem mass spectrometry.

I. T VN
CAP l mt cht khng khun ph rng, tng c s dng rng ri ng vt nui lm
thc phm iu tr nhiu loi bnh nhim trng v hiu qu iu tr rt tt v chi ph thp. V
CAP c th gy ra chng thiu mu do suy tu nn chng b cm s dng trong chn nui ng
vt lm thc phm Vit Nam (BNN, 2005) cng nh nhiu nc trn th gii (Reuters, 2002).
Trn th gii, nhiu tc gi nghin cu thnh cng phng php xc nh d lng
CAP trong sa bng k thut sc k khc nhau (Choon, 2005). Gn y vi s pht trin nhanh
chng ca k thut sc k, p dng k thut sc k lng khi ph phn tch CAP t ra c nhiu
-------------------------------------------------
(1) Trung tm Kim tra v sinh th y trung ng I
(2) Hi th y Vit Nam

67

u im ni tri v bng k thut ny c th pht hin v khng nh mt cch chnh xc cc cht
c hi CAP trong thc phm ngng th trng (MRPL l 0,3 g/kg).
trong nc, vic p dng k thut sc k lng khi ph pht hin d lng CAP
trong thc phm c trin khai (Nguyn Th Thu Thu v cs, 2007). Mi php th p dng p
dng ti phng th nghim phi c nghin cu thng qua qu trnh xc nh hiu lc phng
php. Khi phng php c ph duyt, kt qu phn tch ti phng th nghim s c cc t
chc khc hoc cc quc gia khc tha nhn hoc cng nhn l tng ng (Vn phng cng
nhn cht lng, 2005).
c phng php phn tch d lng CAP trong sa ph hp cho mc ch s dng,
vic lp dng v ph duyt phng php l thc s cn thit.
II. I TNG, NI DUNG V PHNG PHP NGHIN CU
2.1. i tng nghin cu
D lng CAP trong mu sa ti.
2.2. Ni dung nghin cu
- Ti u ha iu kin phn tch CAP trn my LC/MS/MS;
- Ti u ho quy trnh chun b mu th;
- Ph duyt phng php phn tch d lng CAP trong sa bng LCMSMS.
2.3. a im nghin cu
Phng th nghim ca Trung tm Kim tra v sinh th y T1 - Cc Th y.
2.4. Phng php nghin cu
2.4.1. Thit b
H thng sc k lng khi ph khi ph UPLC xevo TQMS (hng Waters).
2.4.2. Nguyn liu, vt t, ha cht
- Chun CAP v CAP-d5 (Dr. Ehrenstorfer).
- Cc loi dung mi: ethyl acetate, methanol, acetonitril, n-hexan, nc ... (loi tinh khit
dng cho LCMS).
- Mu sa trng (gm mu sa b v sa d khng nhim CAP); Mu sa trng thm chun;
Mu sa dng tnh (sa d th nghim).
2.4.3. Thit k cc th nghim
Th nghim 1. To mu sa trng, mu sa dng tnh vi CAP.
Th nghim 2. Ti u ha cc iu kin sc k v ph khi trn h thng LC/MS/MS.
Th nghim 3. Ti u ha quy trnh chun b mu th phn tch d lng CAP trong sa.
Th nghim 4. Ph duyt phng php th (theo Quyt nh 2002/675/EC ca EC).
2.4.4. Phng php x l s liu
- Bo qun d liu bng phn mn Excel.
- Phn mn Masslyns 4.1 (lp dng ng chun t ng).
- Thng k m t tnh t l %, h s phn tn (CV%)....
- Thng k phn tch dng t nghim F-test, t-test.
- nh gi hiu lc phng php theo ca quyt nh 657/2002/EC ca EC.
III. KT QU NGHIN CU
3.1. Ti u ha iu kin phn tch CAP trn my LC/MS/MS
3.1.1. iu kin bn ph ion m
CAP c ti u ha bng k thut ion ha phun in t ESI vi ch bn ph ion m.
Chn ch kho st t ng i vi CAP v CAP-d5; ti u ha tng ion m, ta c iu kin
chy ngun ion ha ESI nh sau:
Bng 1. iu kin chy ngun ion ha ESI

Ghi ch: * Cc thng s khc ty iu kin c th ca h thng my.
Thng s
*
iu kin
Tc kh phun 30 L/h
Th phun in t 3kV
Nhit mao qun 400C

68


Trong k thut ion ha phun in t vi ch bn ion m, cc ion m thng dng (M-
1). Tin hnh kho st bn ph cc ion m. Tip theo tin hnh bn ph la chn ion con. Yu cu
ion con phi c tn hiu ln gp t nht 10 ln so vi ion m. Tin hnh ti u ha mc nng lng
bn ph ion m, ion con. Kt qu thu c bng 2.
Bng 2. Kt qu bn ph cc ion m, ion con ca CAP, CAP-d5
Khi lng phn t Ion m Ion con 1 Ion con 2
CAP 323,13 326,16 257,12 152, 07
CAP-d5 226 326,15 257,12 157,07


Hnh 1. Ph khi ion con ca CAP, CAP-d5 sau khi bn ph
3.1.2. Pha tnh
CAP l cht km phn cc, do ct tch c s dng l ct pha o. Kho st cc loi
ct sc k khc nhau. Kt qu cho thy CAP c th tch ra mt cch d dng bng ct sc k pha
o (RP). Ct ph hp l ct C
18
2,1 mm x 50mm hoc tng ng.
3.1.3. Pha ng
3.1.3.1. Kho st thnh phn pha ng
Trong phng php sc k lng khi ph, vi k thut ion ha phun in t bn ph ch
ion m, qu trnh ion ha tng khi c thm cc cht nh axit focmic.
Kho st s nh hng ca thnh phn pha ng (B/A) trong pha ng 1 l acetonitrile /
nc tinh khit vi 0,1% axit focmic; pha ng 2 l acetonitrile : methanol (9:1)/ m amonium
acetate 10 mM pha trong nc tinh khit.
Tin hnh: Bm cng 1 nng cht chun CAP 0.5 ppb, chy ch ng dng, t l B/A l
85/15, tc dng l 0,3 mL/pht.
Kt qu: C 2 thnh phn pha ng ny u c th s dng lm pha ng phn tch
CAP trong mu sa (u y c CAP ra khi ct, sc ph khi gm ion m v 02 ion con
in hnh ca CAP). Tuy nhin nhc im ca thnh phn pha ng c m amonium acetate 10
mM l nhanh lm bn cone ca ngun. Do , chng ti chn pha ng l acetonitrile/ nc - axit
focmic 0,1% cho cc kho st tip theo.
3.1.3.2. Kho st ti u ha pha ng
- Da vo kt qu kho st trn, tip tc kho st ti u ha pha ng i vi dung dch
chun v mu trng thm chun khi cc iu kin th nghim khc l c nh. Kt qu tm c
chng trnh pha ng ti u phn tch CAP trong sa (bng 3).

Bng 3. Chng trnh pha ng gradient
Thi gian
(pht)
Tc dng
(mL/pht)
Knh A
(nc - axit focmic 0,1%)
Knh B
(acetonitrile)
0 0,3 30 70
0,2 0,3 50 50
1,0 0,3 50 50
1,1 0,3 80 20
2,0 0,3 30 70

Hnh nh m t qu trnh ti u ha pha ng:

69


a/ Pha ng ng dng, t l
knh B:A l 85: 15
b/ Pha ng ng dng, t l
knh B:A l 95: 5
c/ Pha ng gradient
Hnh 2: Sc ti u ha pha ng

3.2. Kt qu ti u ho quy trnh chun b mu th
3.2.1. Kho st tch b trong mu sa trc khi tin hnh chit tch mu
- Chun b 2 nhm mu, mi nhm gm 01 mu sa trng v 06 mu thm chun CAP
nng cui cng ca dch chit mu l 1 ng/mL.
- Tin hnh chit v lm sch mu nh quy trnh d kin ca AFFSSA, 2004.
- Thc t, sau khi cho ethyl acetate vo, lc, ly tm th ch ht c 3 mL lp pha trn v
c hin tng nh ha.
- Vi 3 mL lp pha trn ht oc, tin hnh cc bc chun b mu tip theo v phn tch
sc k. Trn c s so snh din tch pic (im in) ca 02 quy trnh th nghim bng th nghim
F-test. Kt qu nh bng sau:

Bng 4. Kt qu th nghim quy trnh tch chit CAP trong sa ca mu khng tch b
v mu tch b trc khi chun b mu th
Quy trnh khng tch b Quy trnh tch b
Cp mu BLK 8,347 9,650
Cp mu SPK1 35,742 54,864
Cp mu SPK1 51,470 86,122
Cp mu SPK1 32,525 60,430
Cp mu SPK1 98,372 95,814
Cp mu SPK1 117,085 272,394
Cp mu SPK1 198,685 300,657
Phng sai 4,319E
+09
1,289E
+10

F
tnh ton
0,335
F
0,05
0,233
p
0,05
0,105
Ghi ch * File kt qu lu: CAP_MILK_32-47.qld
Kt qu cho thy:
- Phng sai ca quy trnh khng tch b l 4,319E
+09
nh hn mt cht so vi phng sai ca
quy trnh c tch b l 1,289E
+10
song gi tr F
tnh ton
= 0,335 ln hn gi tr F
0,05
= 0,233, cho thy
khng c s khc bit v chnh xc v kt qu th nghim gia 2 quy trnh chun b mu th
(p>0,05).
- Khng th p dng chit ton phn;
- Khng cn phi tch b trc khi chit tch mu nhng cn phi nghin cu b sung mt
bc gim hin tng mu b nh ha v nng cao thu hi ca quy trnh.
3.2.2. Kho st ngn nga s nh ha khi chit tch mu
Chun b 2 nhm mu, mi nhm gm 01 mu sa trng v 06 mu thm chun CAP
nng cui cng ca dch chit mu l 1 ng/mL.
Tin hnh chit v lm sch mu nh quy trnh d kin nhng sau bc cho ethyl acetate
v lc, mt l mu nhit m su (-15
0
C) trong 30; l kia nhit phng th nghim.

70

L nhit m su, sau khi ly ra, lc nh cho tan dung mi v phn nh ha. C 2 l, tip
tc tin hnh cc bc tip theo nh quy trnh d kin.
Kt qu th nghim cho thy: So vi th nghim trc, lp dung mi pha trn ca l
nhit m su ht c nhiu hn (8 mL), l kia vn ch ht c khong 3 mL. Tin hnh cc
bc x l mu tip theo ri bm ln h thng my sc k. Trn c s so snh din tch (im
in) ca 02 quy trnh th nghim bng th nghim F-test; s dng th nghim t-test c phng
sai khc nhau cho 2 l mu ny tm 2 s khc bit gia 2 kt qu th nghim. Kt qu nh sau:
Bng 5. Kt qu th nghim quy trnh tch chit CAP trong sa ca
L mu nhit phng v L mu nhit m su
n v: Din tch pic (im in)
Mu L mu nhit phng
*
L mu nhit m su
*

Mu SPK1 56,658 504,284
Mu SPK2 166,647 426,315
Mu SPK3 48,535 417,863
Mu SPK4 71,870 418,250
Mu SPK5 47,907 441,825
Mu SPK6 27,456 413,700
Phng sai 2,46E
+09
1,19E
+09

F
tnh ton
2,073
F
0,05
5,050
p
0,05
0,221
t
tnh ton
14,906
t
0,05/2
2,262
p
0,05/2
0,000
Ghi ch * File kt qu lu: CAP_MILK_32-47.qld
Kt qu bng trn cho thy: Trung bnh ca L mu nhit m su (im in l
437039,5) cao hn so vi trung bnh ca L mu nhit phng (im in l 69845,5); gi
tr t
tnh ton
= 14,906 ln hn gi tr t
0,05/2
= 2,262, cho thy v kt qu th nghim ca L mu
nhit m su c thu hi tt hn r rt so vi L mu nhit phng (p<0,05).
Nh vy: bng cch b sung bc mu nhit m su (-15
0
C) khc phc c
hin tng nh ha; thu hi ca phng php tng ln r rt (p<0,05).
3.2.3. Th nghim li quy trnh chit mu th CAP trong sa theo quy trnh c iu chnh
3.2.3.1. Tm tt quy trnh chit tch CAP trong mu sa
Theo cc kt qu nghin cu, quy trnh chit tch CAP trong mu sa tm tt nh sau:
Bng 6. Tm tt cc bc chun b mu th phn tch CAP trong sa
Bc Ni dung
Bc 1. Cn 2g mu ng nht vo ng ly tm 50ml
Bc 2. Thm CAP-d5 (nng l 0,5 g/kg)
Bc 3. Voltex 30 giy tc cao nht
Bc 4. Thm vo 9ml Ethylacetate
Bc 5. Lc 10 pht tc 270 ln/pht
Bc 6. nhit m su (-15
0
C) trong 30
Bc 6. Lc nh, ly tm 4500 vng/ 5 pht/ 4
0
C
Bc 7. Chuyn 6ml b mt sang ng thu tinh
Bc 8. Bay hi bng kh N
2
n kh nhit 40
0
C
Bc 9. Ho tan bng 0,5ml n-hexan hoc iso -octan
Bc 10. Thm vo 0,4 ml H
2
O
Bc 11. Ly tm 4000 vng trong 5 pht
Bc 12. Loi b lp trn
Bc 13. Ht vo l bm trn my LCMSMS

71


3.2.3.2. Th nghim li quy trnh chit tch CAP trong mu sa
- Chun b 2 nhm mu, mi nhm gm 06 mu thm chun CAP nng cui cng
trong mu th l 0,5 g/kg. Tin hnh chun b mu th theo quy trnh tm tt ni trn.
- Dng th nghim t-test vi phng sai khc nhau ( kim tra bng th nghim F-test),
so snh trung bnh din tch (im in) ca L mu chun b theo quy trnh c iu chnh theo
bng 6 vi L nhit m su. Kt qu nh bng sau:

Bng 7. So snh kt qu quy trnh chit CAP trong sa ca L mu chun b theo quy trnh
c iu chnh v L mu chun b theo quy trnh cha c iu chnh
n v: Din tch pic (im in)
Mu L mu chun b theo
quy trnh cha c iu chnh
*
L mu chun b theo
quy trnh c iu chnh
*

Mu SPK1 504,284 855,275
Mu SPK2 426,315 830,790
Mu SPK3 417,863 594,185
Mu SPK4 418,250 705,184
Mu SPK5 441,825 748,244
Mu SPK6 413,700 669,803
Trung bnh 437039,5 733913,5
t
tnh ton
6,952
t
0,05/2
2,447
p
0,05/2
0,000
Ghi ch * File kt qu lu: CAP_MILK_32-73.qld

Kt qu bng trn cho thy: Trung bnh ca L mu chun b theo quy trnh c iu chnh (im
in l 733913,5) cao hn L mu nhit m su (im in l 437039,5); gi tr t
tnh ton
= 6,952 ln
hn gi tr t
0,05/2
= 2,447, cho thy v kt qu th nghim ca L mu chun b theo quy trnh c iu
chnh c thu hi tt hn r rt (p<0,05) so vi L mu chun b theo quy trnh cha c iu chnh (L
mu nhit m su).

3.3. Ph duyt phng php th
3.3.1. Khong tuyn tnh
3.3.1.1. Xy dng ng chun CAP tinh khit
Tin hnh lp ng chun tinh khit ca dy dung dch chun c nng 0,2; 0,5; 1,0; 2,5; 5,0;
10,0; 25 ppb trn h thng my theo iu kin ti u ca quy trnh phn tch CAP. Qu trnh ny lp li 2
ln. ng chun c thit lp t ng bng phn mm Masslyns 4.1. Kt qu cho thy khong tuyn tnh
ca ng chun CAP tinh khit l 0,2-25,0 g/mL, tng ng vi nng trong mu l 0,05-12,5 ppb.
3.3.1.2. Xy dng ng chun trn nn mu thc
Trn c s kho st khong tuyn tnh ca ng chun tinh khit, tin hnh kho st thm chun
vi nng cui cng trong mu l 0,05; 0,1; 0,25; 0,5; 1,0; 2,0; 10,0 g/kg. Tin hnh cc bc chit v
lm sch mu nh quy trnh trn.
Bng 8: Khong tuyn tnh ca ng chun CAP trn nn mu thc
n v: g/kg
Nng (g/kg) Ln (1) Ln (2)
0,1 0,15 0,13
0,25 0,13 0,17
0,5 0,42 0,44
1 1,02 1,08
2 2,30 2,15
10 9,83 9,88
R
2
0,988 0,994
Phng trnh tuyn tnh Y= 0,957X+0,127 Y = 0,942X + 0,129

72


Ghi ch: File kt qu lu: (1) CAP MILK LOD 080512.qld, (2) CAP MILK LOD 080512.qld
Nhn xt: ng chun chp nhn c (R
2
0,98). Khong tuyn tnh ca ng chun CAP
trn nn mu thc l 0,1 10,0 g/kg, tng ng vi nng trong mu l 0,1-10,0 g/kg.
Hnh 3 minh ha khong tuyn tnh ca ng chun CAP tinh khit v ng chun trn nn
mu thc:


Khong tuyn tnh ca ng chun CAP
tinh khit
Khong tuyn tnh ca ng chun CAP
trn nn mu thc
Hnh 3: Khong tuyn tnh ca ng chun CAP trn nn mu thc

3.3.2. Gii hn pht hin (LOD), gii hn nh lng (LOQ)
Tin hnh chun b 10 mu sa trng thm chun nng l 0,3 g/kg (phng php
thm chun). Kt qu phn tch c trnh by trong bng sau:

Bng 9: Kt qu th nghim v gii hn pht hin (LOD), gii hn nh lng (LOQ)
Mu* Nng (g/kg)
SPK0,3-1 0,30
SPK0,3-2 0,30
SPK0,3-3 0,32
SPK0,3-4 0,26
SPK0,3-5 0,27
SPK0,3-6 0,27
SPK0,3-7 0,30
SPK0,3-8 0,31
SPK0,3-9 0,26
SPK0,3-10 0,26
Trung binh 0,29
SD 0,023
LOD 0,07
LOQ 0,23
R
tnh ton
4,09
Ghi ch: *File kt qu lu CAP_MILK_74-133.qld
- nh gi tnh hp l: T gi tr LOD tnh c, tnh R
tnh ton
= x / LOD = 4,09 trong
x l tr s trung bnh. Do 4<R
tnh ton
<10, nn nng dung dch th ca mu sa trng thm chun
l ph hp, LOD tnh c l ng tin cy. Vy LOD l 0,07 g/kg, LOQ l 0,23 g/kg.
3.3.3. Gii hn pht hin (CC), gii hn nh lng (CC)
T kt qu cc th nghim, tin hnh tnh cc gi tr CC v CC theo tiu chun ISO11843. Kt
qu trnh by trong bng sau:

Bng 10. Tnh ton gi tr CC v CC qua cc ngy khc nhau
n v: g/kg

Nng
Tn hiu
(1) (2) (3) (4)

73

0,05 0,030 0,050 0,040 0,040
0,1 0,130 0,070 0,100 0,090
0,25 0,240 0,330 0,360 0,250
0,5 0,500 0,550 0,450 0,520
CC 0,10 0,09
CC 0,12 0,12
Ghi ch: File kt qu lu
(1): [CAP_MILK_188-216.qld] (3): [CAP_MILK_74-133.qld]
(2): [CAP_MILK_153-172.qld] (4): [CAP_MILK_173-187.qld]

Nh vy: Gi tr CC l 0,10 g/kg v gi tr v CC l 0,12 g/kg.

3.3.4. lp li ca phng php
Tin hnh chun b 3 l mu sa trng thm chun CAP, nng cui cng trong mu l
0,30; 0,45 v 0,60 g/kg, mi l gm 6 mu, lp li 3 ngy khc nhau. Kt qu trnh by trong
bng sau:
Bng 11. Kt qu xc nh chm trung gian cc nng
n v: g/kg
Tiu ch
Mc 0,30 g/kg Mc 0,45 Mc 0,60 g/kg
Tnh
chung
KQ
a
KQ
b
KQ
c
KQ
a
KQ
b
KQ
c
KQ
a
KQ
b
KQ
c

Ln th 1 0,32 0,24 0,30 0,50 0,45 0,55 0,64 0,60 0,68
Ln th 2 0,27 0,32 0,32 0,58 0,49 0,50 0,63 0,60 0,65
Ln th 3 0,37 0,28 0,30 0,46 0,45 0,47 0,62 0,65 0,63
Ln th 4 0,31 0,24 0,33 0,54 0,44 0,51 0,71 0,67 0,69
Ln th 5 0,35 0,35 0,29 0,50 0,52 0,48 0,64 0,62 0,62
Ln th 6 0,27 0,26 0,29 0,51 0,49 0,55 0,64 0,71
Trung bnh 0,32 0,28 0,31 0,52 0,47 0,51 0,65 0,63 0,66
lch chun
(SD) 0,04 0,04 0,02 0,04 0,03 0,03 0,04 0,03 0,04
H s phn tn
(CV%) 13,0 15,9 5,4 7,9 6,6 6,7 5,5 4,5 5,4
lch chun tng ng (S
R
) 0,11
Trung bnh chung (g/kg) 1,45
H s phn tn chung khc nng (CV%) 7,6
Ghi ch: KQ
a
: Kt qu o ngy 1, file lu l CAP_MILK_134-160.qld
KQ
b
: Kt qu o ngy 2, file lu l CAP_MILK_74-133.qld
KQ
c
: Kt qu o ngy 3, file lu l CAP_MILK_188-216.qld
Kt qu bng cho thy: H s phn tn chung (CV%) nng CAP trong mu sa l 0,3;
0,45 v 0,6 g/kg, gia cc ngy khc nhau l 7,6%. So vi quy nh trong quyt nh
2002/657/EC ngng <1 g/kg, kt qu ny chp nhn c.
3.3.5. thu hi ca phng php
T kt qu th nghim v lp li ca phng php tin hnh cc nng khc nhau trong
cc ngy khc nhau, tin hnh tnh thu hi ca tng mu mi nng v thu hi chung
mi nng . Kt qu trnh by trong bng sau:

Bng 12. thu hi ca phng php
thu hi (%)
Nng 0,3 g/kg Nng 0,45 g/kg Nng 0,6 g/kg

74

KQ
a
KQ
b
KQ
c
KQ
a
KQ
b
KQ
c
KQ
a
KQ
b
KQ
c

Ln th 1 106,7 80,0 100,0 111,1 100,0 122,2 106,7 100,0 113,3
Ln th 2 90,0 106,7 106,7 128,9 108,9 111,1 105,0 100,0 108,3
Ln th 3 123,3 93,3 100,0 102,2 100,0 104,4 103,3 108,3 105,0
Ln th 4 103,3 80,0 110,0 120,0 97,8 113,3 118,3 111,7 115,0
Ln th 5 116,7 116,7 96,7 111,1 115,6 106,7 106,7 103,3 103,3
Ln th 6 90,0 86,7 96,7 113,3 108,9 122,2 0,0 106,7 118,3
thu hi
chung (%)
100,2 111,0 107,8
Ghi ch: KQ
a
: Kt qu o ngy 1, file lu l CAP_MILK_134-160.qld
KQ
b
: Kt qu o ngy 2, file lu l CAP_MILK_74-133.qld
KQ
c
: Kt qu o ngy 3, file lu l CAP_MILK_188-216.qld
Kt qu bng cho thy: thu hi (%) ca php th CAP trong mu sa thm chun:
- nng 0,6 g/kg bin ng t 100,0-118,3%.
- nng 0,3 g/kg bin ng t 80,0-123,3% trong 1 php th ngy th nht nm
ngoi khong chp nhn (vt ra ngoi khong quy nh mt cht) nhng thu hi chung trong
cc ngy l 100,2%.
- nng 0,45 g/kg bin ng t 97,8-128,9% trong 1 php th ngy th nht v 1
php th ngy th 2 nm ngoi khong chp nhn (vt ra ngoi khong quy nh mt cht)
nhng thu hi chung trong cc ngy l 111,0%.
Kt qu thu hi chung trong cc ngy cc nng l chp nhn c theo Quyt nh
2002/657/EC ngng <1 g/kg.
3.3.6. ti lp v tnh khng m bo o ca phng php
ti lp v khng m bo o ca phng php c nghin cu trn nn mu sa b
v sa d. Kt qu nh sau:
Bng 13. ti lp v tnh khng m bo o ca phng php
Thng s Sa d
*
Sa b
**
Tnh chung
Nng spike (g/kg) 0,30 0,45 0,60 0,30 0,45 0,60
Trung bnh (g/kg) 0,30 0,50 0,61 0,29 0,38 0,65
t do - 1 17 17 16 5 5 5
lch chun (SD) 0,037 0,039 0,156 0,061 0,024 0,107
H s phn tn (CV%) 12,3 7,7 25,5 20,8 6,3 16,5
Phng sai 0,258 0,101 1,044 0,215 0,020 0,137 1,775
Tng t do - 1 65
Phng sai chung

0,03
H s phn tn chung (CV%)

16,5
H s ph

2,0
khng m bo o (%)


33,1
Ghi ch: Sa d
*
: file lu kt qu l CAP_MILK_134-160.qld; CAP_MILK_74-133.qld;
CAP_MILK_188-216.qld; Sa b
**
: file lu l CAP MILK LOD 080512.qld
Kt lun:
- ti lp ca phng php trn nn mu sa khc nhau l 16,5%.
- khng m bo o ca phng php l 33,1%.
3.3.7. Nghin cu m bo cht lng kt qu th nghim
3.3.7.1. Kim tra nhim ca h thng my
Trc mi ngy chy lot mu, bm dung mi ACN kim tra s tp nhim ca my.

75

3.3.7.2. Kim tra s nhim bn sau khi bm lot mu
C sau khi bm mt lot 10 mu, bm 01 mu ACN. Kt qu my khng b nhim bn t
nhng ln chy my trc th mi c bm mu tip.
3.3.7.3. Kim tra mu m
Th nghim b tr ngi phn tch mu khng c bit thng tin v mu. S dng mu sa d
th nghim (mu sa trng v mu dng tnh vi CAP) c ng nht, chia lm 02 phn; mt
phn phn tch ti PTN ca Trung tm, phn kia gi ti PTN khc. Kt qu nh sau:
Bng 14. Kt qu phn tch CAP gia phng th nghim ca Trung tm (VSTY1)
vi phng th nghim khc
Mu
Phn tch nh tnh Phn tch nh lng (g/kg) p
2

(=0,05) VSTY1 PTN khc VSTY1 PTN khc
Mu (-) - - 0,00 0,00
0,469
Mu (+) + + 0,69 0,76
Ghi ch : (-) : m tnh ; (+) : Dng tnh.
Kt qu bng trn cho thy:
+ Kt qu phn tch nh tnh: C 02 PTN u cho kt qu m tnh i vi mu m tnh,
ph hp vi th nghim c b tr.
+ Kt qu phn tch nh lng: Dng th nghim F-test nh gi mc chnh xc ca
kt qu th nghim gia 2 phng th nghim. Kt qu bng trn cho thy: Phng sai phng th
nghim Trung tm (VSTY1) = 0,24 nh hn mt cht so vi phng sai phng th nghim gi
mu i phn tch = 0,29 song gi tr F
tnh ton
= 0,824 ln hn gi tr F
0,05
= 0,006 cho thy khng c
s khc bit v chnh xc ca phng php m phng th nghim p dng so vi phng php
ca phng th nghim kia.
IV. KT LUN V KHUYN NGH
-. nghin cu v lp dng thnh cng c quy trnh phn tch d lng CAP trong sa bng
k thut sc k lng khi ph LCMSMS trong hiu lc ca phng php c xc nh da
theo hng dn Quyt nh 2002/675/EC ca EC ngy 20/8/2002.
-. Phng php l ph hp xc nh d long CAP trong sa ngng i hi ca th trng
(0,3 g/kg).
TI LIU THAM KHO
1. AFSSA (2004): Chloramphenicol identification by liquid chromatography tandem mass
spectrometry. AFSSA - France.
2. Balizs, G. & Arnold, D. (1989) Reference standards for residue analysis of
chloramphenicol in milk and meat: a critical study. Chromatographia, 27, 489494.
3. BNN (2005): Quyt nh s 25/2005/Q-BNN
4. Choon K. N. v cs (2005): A rapid and sensitive analysis method of Sudan I, II, III & IV in
tomato sause using ultra performance LC MS/MS. Waters agency.
5. EU (2002): EU regulation, European commission decision 2002/657/EC
6. Reuters (2002): Chloramphenicol antibiotic is banned in foods - FAO.
7. Nguyn Th Thu Thu, Lm Vn X, Phm Th nh, Trn Kim Tnh, Chu Phm Ngc
Sn, Kefei Wang (2007): Phn tch clenbuterol trong tht heo v thc n chn nui bng k
thut sc k lng khi ph.
8. Tyler, D. (2006): Determination of chloramphenicol at residue levels in food by solid
phase extraction and LC/MS/MS. DEFRA Central Science Laboratory, Britain.
9. Vn phng cng nhn cht lng (2005): Hng dn nh gi khng m bo o trong
phn tch ho hc nh lng. Vn phng cng nhn cht lng.
Nhn: 10-6-2012 Phn bin 15-6-2012