I HC QUC GIA TP.

HCM
TRNG I HC BCH KHOA

NGUYN PHC MINH

NGHIN CU NH LNG KHNG SINH

ERYTHROMYCIN TRONG TM, C BNG K
THUT SNG VUNG QUT NHANH TRN CC
GIT CHM V KH NNG O THI

Chuyn ngnh: Ch Bin Thc Phm v Ung

M s chuyn ngnh: 62.54.02.01

TM TT LUN N TIN S K THUT

Tp. H Ch Minh nm 2012

Cng trnh c hon thnh ti: Trng i hc Bch Khoa HQG-HCM

Ngi hng dn khoa hc 1: TS. Trn Bch Lam

Ngi hng dn khoa hc 2: TS. Nguyn Trng Giao

Phn bin c lp 1:
Phn bin c lp 2:
Phn bin 1:.
Phn bin 2:.
Phn bin 3:.

Lun n s c bo v trc Hi ng chm lun n hp ti:

.....................
.................
vo lc

gi

ngy

thng

nm

C th tm hiu lun n ti th vin:

- Th vin Khoa hc tng hp Tp. HCM
- Th vin Trng i Hc Bch Khoa - HQG-HCM

DANH MC CNG TRNH CNG B

1. Nguyen Phuoc Minh et al., Simultaneous determination of
erythromycin A in giant prawn and tilapia in Mekong region by
stripping square wave voltammetry, European Journal of Food
Research and Review, vol. 1, no. 1, pp. 01-14, 2011.
2. Nguyen Phuoc Minh et al., Accumulation and clearance of orally
administered erythromycin in adult Nile tilapia (Oreochromis
niloticus), International Food Research Journal, vol. 18, no. 1,
pp. 95-100, 2011.
3. Nguyen Phuoc Minh et al., Tissue distribution and elimination of
erythromycin

in giant freshwater prawn (Macrobrachium

rosenbergii) depletion, African Journal of Food Science, vol. 4,

no. 9, pp. 578-584, 2010.
4. Nguyn Phc Minh v cng s. Qu trnh tch t v o thi
khng sinh erythromycin trn c r phi (Oreochromis niloticus),
K Yu Hi Ngh Khoa Hc: Pht Trin Nng Nghip Bn Vng
Thch ng Vi S Bin i Kh Hu, Cn Th, 2010, trang 430437.
5. Nguyn Phc Minh v cng s. Chuyn ha sinh hc v o
thi khng sinh erythromycin trong t bo tm cng xanh trng
thnh (Macrobrachium rosenbergii), K Yu Hi Ngh Khoa Hc:
Pht Trin Nng Nghip Bn Vng Thch ng Vi S Bin i
Kh Hu, Cn Th, 2010, trang 472-479.

TNH CP THIT CA TI
Mt trong nhng vn bc xc v v sinh an ton thc phm
nc ta hin nay l d lng khng sinh trong vt nui, d lng khng
sinh trong thc phm vt qu mc cho php nh hng ti sc khe ca
ngi tiu dng v uy tn hng xut khu ca chng ta. Do tnh cht khng
khun mnh ca erythromycin t lu n c dng phng v tr bnh
trn tm c. Do nng dn trong qu trnh nui tm cng xanh v c r phi
c s dng erythromycin v khng tun th thi gian ngng thuc trc
thu hoch nn li sn phm c d lng vt mc cho php.
Theo quy nh ca Codex, WHO/FAO, EU, M, Canada, Australia,
v.v th d lng erythromycin trong sn phm thy sn nhn chung phi
nh hn 30 ppb. Theo thng t s 15/2009/TT-BNN ngy 15/03/2009 ca
B Nng Nghip v Pht Trin Nng Thn Vit Nam, erythromycin thuc
nhm khng sinh hn ch s dng, vi mc d lng ti a cho php l
200 ppb. Hin nay c nhiu phng php xc nh d lng khng sinh
ny trong cc sn phm thy sn nh ELISA, LC-MS/MS. Mc tiu v ni
dung th nht t ra l phi tm c phng php phn tch d lng
khng sinh trong sn phm thy sn vi chi ph thp, nhanh, tin cy v
tnh chnh xc cao, c kh nng ti khng nh erythromycin.
Xt thy cha c bt k nghin cu no c cng b v qu trnh
o thi, chuyn ha, thi gian ngng khng sinh erythromycin trc thu
hoch trn i tng tm cng xanh, c r phi. Vy nn trong ti ny em
s tin hnh nghin cu thi gian o thi, chuyn ha ca erythromycin
trong c tht tm cng xanh v c r phi nhm rt ra quy lut v c lng
c thi gian ngng thuc trc thu hoch an ton.
T hai yu cu trn, em chn ti Nghin cu nh lng
khng sinh Erythromycin trong tm, c bng k thut sng vung qut
nhanh trn cc git chm v kh nng o thi.

MC TIU NGHIN CU
Mt l, trn thit b ANALYZER SQF 505 do Vit Nam sn xut
xy dng c phng php phn tch erythromycin bng k thut sng
vung qut nhanh trn cc git chm p ng tiu chun an ton thc phm
quc t ng thi t yu cu phn tch nhanh, n gin v gim chi ph
phn tch.
Hai l, nghin cu o thi, chuyn ha ca erythromycin trong c
tht thy sn (tm cng xanh, c r phi), t xc nh c quy lut v
thi gian thu hoch an ton khi s dng erythromycin.
NGHA KHOA HC V THC TIN
ngha khoa hc:
1.

Ln u tin Vit nam v trn th gii tin hnh nghin cu s dng

k thut sng vung qut nhanh trn cc git chm phn tch nh
tnh v nh lng khng sinh eythromycin trong mu thy sn.

2.

S xut hin ca sng kh vng th khong -1,4 V l do s kh ca

nhm C = O th hai trong phn t erythromycin lin kt vi nhm i
in t cha nguyn t oxi bn cnh. Qu trnh kh l khng hon ton
thun nghch vi s trao i hai in t.

3.

Pht hin trong iu kin thch hp c s hp ph ca erythromycin

trn b mt git thy ngn. C th dng hiu ng ny tin hnh
phn tch erythromycin cc nng rt thp bng k thut stripping
hp ph trn cc git chm hay cc ngi, cc treo.

4.

nghin cu v chn c dung dch nn thch hp, cc thng s

chy my ti u, quy trnh chun b mu o hp l phn tch
erythromycin trong cc mu tm cng xanh, c r phi c kt qu tin
cy. Cc s liu c so snh vi k thut phn tch truyn thng
LC-MS l c s tng ng.

5.

Chng minh rng cc dn xut erythromycin khng ch c chuyn

ho bi Sacchropolyspora erythrea bng con ng ln men m cn
chng minh c erythromycin A khi vo c th tm c cn c s
chuyn ho sinh hc sang cc dn xut erythromycin C, E, F.
ngha thc tin:

1.

nghin cu xy dng thnh cng quy trnh phn tch nh tnh v

nh lng erythromycin trong tm cng xanh v c r phi bng k
thut sng vung qut nhanh trn cc git chm vi my ANALYZER
SQF-505 do Vit Nam sn xut vi chi ph phn tch r hn, thao tc
d dng hn cc k thut khc ca nc ngoi. Cc s liu c
kim chng vi cc kt qu phn tch bng cc phng php hin i
khc.

2.

Kt qu ny c th gip cc nh sn xut kinh doanh cc a phng

c phng tin kim sot khng sinh t gc (thc n, mi trng,
ha cht, tm c trong qu trnh thu mua, ch bin,) nhm to ra cc
sn phm c uy tn quc t.

3.

S thnh cng bc u ny c th m ra mt hng nghin cu mi

s dng k thut sng vung qut nhanh n gin, khng qu tn km
phn tch nhiu khng sinh khc trong cc i tng thy sn khc
nhau nhm gip cc a phng t gim st tt vn ny.

4.

Rt ra c quy lut o thi, chuyn ha ca erythromycin trong c

tht tm cng xanh v c r phi sau khi n thc n c trn
erythromycin, t c lng thi gian ngng thuc an ton, gp
phn c c mt nn cng nghip ch bin thc phm ch ng, an
ton v bn vng.

CHNG I TNG QUAN

Erythromycin l nhm khng sinh macrolide gm erythromycin A, B,
C, D, E, F. Tt c c to ra t Sacchropolyspora erythrea bng con
ng ln men. Sn phm chnh ca qu trnh ln men ny l erythromycin
A, cn li l cc erythromycin khc ( 5%). Erythromycin gm 2 phn t
ng gm desosamin v cladinose gn vo Erythronolide. N d dng ht
m v tan t trong nc (0,2%). Erythromycin hot ha pH kim hn l
pH trung tnh v khng bn pH acid.
Erythromycin c tc dng dit khun tt cc loi vi khun Gram [+]
v hiu qu trong vic cha tr nhim khun Staphylococcus m khng
li Penicillin. Mycoplasma, Staphylococcus, Streptococcus, Neisseria,
Haemophylus, Corynebacterium, Listeria, Pasteurella multocida, Brucella,
Rickettsia, Treponema rt nhy cm vi erythromycin. Erythromycin c
s dng rng ri cha nhim khun ngi v ng vt.

Hnh 1.1: Cng thc cu to ca erythromycin

Nguyn l ca cc ph sng vung qut nhanh trn cc git

chm v stripping sng vung qut nhanh trn cc git chm: Nhng
vo dung dch nn c cha cht cn phn tch 3 in cc: Cc lm vic: l
cc git thy ngn c tc chy n nh khong 7 giy mt git. Nu p
dng k thut stripping nhanh ta dng cc git thy ngn c tc chy
trn 10 giy mt git. Cc so snh: Ag/AgCl/KCl bo ha c th khng
i. Cc h tr: dng cc Platin. t ln cc lm vic 2 thnh phn: Th
mt chiu tng dn theo thi gian dng hnh bc thang vi cc bc th 2,

4, 6, 8, 10 mV. Th xoay chiu l cc xung vung gc c gi tr bin

xung: 10, 20, 30, 40 mV v tn s vi trm Hz. trit tiu dng t in
gy nhiu cho tn hiu cn o, ta tin hnh ghi cng dng Faraday xoay
chiu u xung v cui xung. Phn mm SQF-505 cho php hin th s
bin i ca cng dng Faraday xoay chiu theo th mt chiu. ng
thu c gi l ph sng vung qut nhanh. Ph thng cha nhiu nh.
Mi nh ng vi mt cht hay mt giai on phn ng in ha ca mt
cht. Th bn sng (E1/2) vi mt dung dch nn no l mt gi tr c
trng cho mi cht v thng dng cho mc ch phn tch nh tnh.
Chiu cao ca nh (cng dng Faraday xoay chiu) hay din tch nh
(cng sut to sng) t l vi nng cht tham gia phn ng in ha trn
cc lm vic nn c dng cho mc ch nh lng.
Stripping sng vung qut nhanh trn cc git chm: Dng mt
cc git thy ngn chy rt chm, khong 10 giy mt git, lm cc lm
vic. Phn mm PSA-F cho php t mt gi tr th mt chiu ty tch
gp cht cn phn tch ln b mt ca mt git thy ngn ang ln dn ln.
Sau qu trnh ny, my s t ng tin hnh qut ph theo ch sng
vung qut nhanh.
Cc nghin cu nh lng erythromycin: Nghin cu trong
nc, cha thy c nghin cu no v qu trnh nh lng erythromycin,
ngay c cc nghin cu nc ngoi cng c rt t nghin cu v phng
php Von-ampe nh lng erythromycin m ch yu l cc phng
php sc k lng ghp khi ph. c bit cha thy mt cng trnh nghin
cu no dng k thut sng vung qut nhanh trn cc git chm dng
nh lng erythromycin trn nn mu tm cng xanh v c r phi.
Mt s cng trnh nghin cu v kh nng o thi, chuyn ha
erythromycin trn mt s loi thy sn nh: Esposito cng cng s
(2006) nghin cu qu trnh o thi erythromycin trn c hi vn dng
Oncorhynchus mykiss. Moffitt cng cng s (1988) nghin cu s tch

ly v o thi ca erythromycin thiocyanate trn c hi Oncorhynchus

tshawytscha. Moffitt cng cng s (1999) tin hnh nghin cu c tnh,
kh nng gy t bin v hiu qu ca vic tim 10, 20, hoc 40 mg
erythromycin/kg th trng c hi Oncorhynchus tshawytscha, cch 20 ngy
tim 1 ln v tim 6 ln, trc khi c hi pht dc. Moffitt v cng s
(2001) kim tra tnh c cp tnh v c mn tnh ca erythromycin trn c
hi Oncorhynchus tshawytscha 2 nhit nc khc nhau. Haukenes v
cng s (2002) tim erythromycin phosphate vo c hi ci Oncorhynchus
tshawytscha trc khi trng. Fairgrieve v cng s (2005) cho c hi
Oncorhynchus tshawytscha n thc n c cha azithromycin (30 mg/kg c
trong 14 ngy) hoc erythromycin (100 mg/kg c trong 28 ngy).
Tm li, nhng nghin cu trc v bin i ca erythromycin trn
c th thy sn ch yu mi tp trung trn i tng c hi, l c nc lnh
v cng ch mi dng li vic theo di s o thi erythromycin c.
Cha c bo co v erythromycin trn thy sn nhit i v cng cha c
bo co no cho bit trong c th thy sn erythromycin s chuyn ha th
no, v vy y s l nhng ni dung m ti ny nghin cu.
V sao chn i tng thy sn trong nghin cu ny l tm
cng xanh v c r phi: y l hai loi thy sn c nui rt ph bin
Vit Nam v c s dng erythromycin. Rt nhiu vi sinh vt gy bnh trn
tm cng xanh bao gm protozoans nh Epistylis, Zoothamnium, v
Vorticella; cc vi khun gy bnh nh Vibrio, Aeromonas, Pseudomonas,
Edwardsiella. Trong khi cc bnh ch yu trn c r phi khi nui
thng l Streptococcus iniae, Aeromonas hydrophila, Trichodina,
Flexibacter

Columnaris,

Edwardsiella.

Ph

bin

nht

do

Streptococcosis, vi khun gram dng, gy thit nng n m khng c

vacccine no phng c, thm ch trn c trng thnh v khe mnh. V
vy, erythromycin cng l khng sinh t lu c dng phng v tr
bnh trn c r phi do Streptococcus.

CHNG II. NGUYN VT LIU, PHNG PHP NGHIN CU

2.1 NGUYN VT LIU, THIT B DNG C, HA CHT
2.1.1 Nguyn vt liu
- Tm cng xanh ( 50 g/con), c r phi (500550 g/con).
- Erythromycin base 96,5% t cty C Phn Dc Hu Giang.
2.1.2 Thit b, dng c
- Thit b Analyzer SQF-505 ca Trung Tm Nhit i Vit Nga.
- B chit pha rn SPE ca Phenomenex, ct HLB ca Waters.
- Cc dng c phn tch thng thng khc.
2.1.3 Ha cht
-

Erythromycin

A,

chloramphenicol,

florfenicol,

furazolidone,

ciprofloxacin, enrofloxacin, danofloxacin chun c tinh khit > 98% ca

hng Sigma Aldrich, Fluka Chemical.
- Cc dung mi, ha cht phn tch khc.
2.2 PHNG PHP NGHIN CU
2.2.1 Thuyt minh nghin cu
Ni dung th I: Nghin cu cc iu kin c bn ca k thut
stripping sng vung qut nhanh trn cc git chm nh lng
erythromycin. Sau tin hnh nh gi thm nh hiu nng phng
php. Cui cng l ly mu tm cng xanh, c r phi thc t ti 10 tnh
BSCL nh gi bc tranh thc trng nhim erythromycin.
Ni dung th II: Theo di qu trnh o thi v chuyn ho
erythromycin theo thi gian khi khng sinh ny i vo c th tm cng
xanh v c r phi bng con ng thc n. T rt ra quy lut thi gian
ngng thuc an ton trc thu hoch.
2.2.2 Phng php thc nghim
2.2.2.1 Kho st cc iu kin ti u cho vic nh lng erythromycin
trn thit b Analyzer SQF - 505

Kho st v la chn cc loi dung dch nn: Amoni axetat, Natri

axetat, Borax, Tris, Citrat - phosphat
Kho st nng dung dch nn: 0,05 0,25 M.
Kho st dung mi ha tan erythromycin: Methanol, Ethyl axetat,
Acetonitril.
Nghin cu k thut o Stripping SWV: chiu qut, Vstart, Vstep, Vpulse,
Tdrop, Velectrolise, Telectrolise
Kho st nh hng ca cc yu t gy nhiu: cc ion v c.
Xy dng ng chun trong khong 50 900 ppb.
La chn quy trnh trch ly mu: s dng ct chit pha rn tch loi
tp v lm giu erythromycin.
chnh xc, ng, hiu sut thu hi
Kh nng nhn danh erythromycin A vi cc khng sinh khc: Kho
st s hin din ca ln lt cc khng sinh chloramphenicol, florfenicol,
furazolidone, ciprofloxacin, enrofloxacin, danofloxacin xem c tch bit v
nhn danh r so vi erythromycin A.
So snh i chng kt qu Stripping SWV vi LC-MS/MS: Tm cng
xanh (c r phi) c nui n trng lng thng phm, c ly 3 mu
v phn tch ti trung tm kim nh Intertek Vit Nam dng lm mu
trng blank. Sau tm (c) nui c gy nhim erythromycin bng
con ng thc n; liu gy nhim 100 mg erythromycin/kg th trng/
ngy; thi gian gy nhim 7 ngy lin tip v tin hnh ly mu sau ngy
th 7, 8 v 9 k t thi im ngng thuc. Lc ny ta s c 3 nhm mu
nhim erythromycin (cao, trung bnh, thp). mi nhm nng gy
nhim (6 mu), chia lm 2 phn bng nhau:
- Phn I: Phn tch bng Stripping SWV (3 mu nng thp, 3 mu nng
trung bnh, 3 mu nng cao).

- Phn II: Gi phn tch i chng bng LC-MS/MS ti trung tm kim

nh Intertek Vit Nam (3 mu nng thp, 3 mu nng trung bnh, 3
mu nng cao).
ng dng phn tch erythromycin trn cc mu thc t tm, c ti cc
a phng: Ly cc mu tm cng xanh, mu c r phi i din 10 tnh
(mi tnh ly mu 3 huyn) ng bng sng Cu Long v phn tch v
x l thng k nh gi thc trng nhim erythromycin. Mi mu c
phn tch 5 ln (n=5) trn my Analyzer SQF - 505. Kt qu th hin: trung
bnh SD, % RSD.
2.2.2.2 Xc nh quy lut chuyn ha sinh hc, o thi ca
erythromycin trong c th thy sn khi nui
Kim sot t giai on th ging n khi t kch c thng phm
nh: mt th ging, kch c th ging nhit , pH, kim, hm lng
oxy, kh c, lng thc n cho n, kch c tm khi x l khng sinh,
khng sinh nguyn liu, sng lc cc dng erythromycin B, C, D, E, F c
trong nguyn liu erythromycin base ny. Thi gian x l khng sinh, con
ng a khng sinh vo c th, liu khng sinh x l, s ln cho n thc
n trong ngy cng c gim st ht sc cht ch. Mu c phn tch
khng nh d lng erythromycin A, phn tch sng lc cc dng chuyn
ha ca erythromycin nh erythromycin B, C, D, E, F theo phng php
LC-MS/MS thng qua Cty kim nghim TUV Rheinland Aimex Vietnam.
2.2.3 X l s liu
Tt c s liu phn tch u c x l thng k bng phn mm
Statgraphics v WT14.

10

CHNG III. KT QU V BN LUN

3.1 NGHIN CU CC IU KIN TI U CHO VIC NH
LNG ERYTHROMYCIN
3.1.1 Nghin cu thm d nh hng, chn vng qut th v chiu
qut th
Erythromycin kh d tan trong nc nn cc dung dch nn dng
cho ngi cu ch yu l dung dch nc vi cc cht in ly khc nhau.
Trong cng thc cu to ca erythromycin c nhm C=O lin kt vi mt
nguyn t oxy khc, ngoi ra c mt s d vng cha oxy c lin kt mt
nguyn t oxy na nn c th d on s xut hin nhng sng kh trong
khong th t -1,0 V n -1,7 V. V th em s tp trung nghin cu vng
th ny.
Qua thm d s b em thy c mt vi sng trong khong th ny,
nhng sng th vng khong -1,4 V l tt nht i vi mc ch phn tch
nh lng, nn cc kho st tip theo em tp trung tm iu kin ti u
cho sng ny.
3.1.2 Nghin cu tm dung dch nn v pH ph hp
Cng dng ca erythromycin b nh hng bi loi dung dch
nn. Trong s cc dung dch nn kho st nh Natri axetat, Amoni axetat,
Citrat-Phosphat, Borax, m Tris th dung dch nn amoni axetat cho gi tr
cng dng erythromycin cao nht v hnh dng nh peak p nht.
Khong pH ca dung dch amoni axetat c kho st trong
khong 7,0 n 10,0. Erythromycin c cng dng cao nht pH 8,0
(E1/2 = -1.430 mV, I = 351,7 5,7 nA). V th dung dch nn amoni axetat
(pH 8,0) c chn kho st tip theo.
3.1.3 Nghin cu nh hng ca nng dung dch nn
Nng dung dch nn amoni axetat pH 8,0 c kho st trong
khong 0,05 0,25 M. Erythromycin c cng dng cao nht amoni
axetat 0,1 M (E1/2 = -1.430 mV, I = 254,8 10,2 nA).

Cng dng erythromycin A

(nA)

11
270,0
250,0
230,0
210,0
190,0
170,0
150,0
0,00

0,05

0,10

0,15

0,20

0,25

0,30

Nng dung dch nn ammonium acetate (M)

Hnh 3.9: nh hng ca nng dung dch nn amoni axetat n cng dng
ca erythromycin A

3.1.4 Nghin cu nh hng ca dung mi ha tan erythromycin

Tin hnh ghi ph ca erythromycin trong dung dch nn amoni
axetate 0,1 M pH 8,0 vi s c mt ca ba dung mi methanol, acetonitril,
ethyl axetate ha tan chun erythromycin. Kt qu thu c thy khi c
mt ca cc dung mi trn vn khng nh hng nhiu ti tnh cht ca
ph eythromycin, chng ta vn ghi c cc ph r rng vng th nh c.
Qua , ta thy trong qu trnh chun b mu o c th dng cc dung mi
trn.
3.1.5 Nghin cu cc iu kin chy my thch hp
3.1.5.1 Nghin cu chiu qut
Chiu qut xui (0 n -1.800 mV) v chiu qut ngc (-1.800
mV n 0) ln tn hiu ca cng dng c kho st. Chiu qut
xui (0 n -1.800 mV) cho nh peak cao v p. Trong khi , chiu qut
ngc (-1.800 mV n 0) khng th hin nh peak. Chiu qut xui (0 n
-1.800 mV) c chn kho st cc thng s tip theo. iu ny ni ln
rng tnh cht khng thun nghch ca erythromycin khi qut.
3.1.5.2 Nghin cu chn th bt u ghi ph Vstart
Tin hnh ghi ph erythromycin nng 100 ppb trong dung dch
nn amoni axetat 0,1 M; pH 8,0 vi cc gi tr th bt u ghi ph V start
khc nhau v thu c cc kt qu sau:

12
Cng dng erythromycin A
(nA)

120,0
100,0

y = 0,1332x + 162,8

80,0
60,0
40,0
20,0

0,0
-200 -300 -400 -500 -600 -700 -800 -900 -1000 -1100 -1200
Vstart (mV)

Hnh 3.13: nh hng ca Vstart n cng dng ca erythromycin A

3.1.5.3 Nghin cu chn bc th 1 chiu Vstep

Tin hnh ghi ph dung dch erythromycin nng 100 ppb trong
dung dch nn amoni axetat 0,1 M; pH 8,0 mt s tc qut th mt chiu
Cng dng erythromycin
A (nA)

tng ng vi th bc Vstep = 6 , 8 , 10 mV/ bc th v thu c:

220
210
200
y = -12,395x + 284,64

190
180
170
160
150
4

10

12

Vstep (mV)

Hnh 3.15: nh hng ca Vstep n cng dng ca erythromycin A

3.1.5.4 Nghin cu chn bin xung vung Vpulse

Tin hnh ghi ph erythromycin nng 100 ppb trong nn amoni
Cng dng erythromycin
A (nA)

axetat 0,1 M; pH 8,0 vi cc bin khc nhau 10, 20, 30, 40 mV

800
700
600

y = 15,258x + 78,47

500
400
300
200
100
0

10

20

30

40

50

Vpulse (mV)

Hnh 3.17: nh hng ca Vpulse n cng dng ca erythromycin A

13

3.1.5.5 Nghin cu chn thi im qut th trong i sng 1 git thy

ngn Tdrop
Gi tr Tdrop ny th hin thi im bt u ghi sng k t thi im
hnh thnh mt git thy ngn mi. ghi ton ph ta nn chn gi tr
1.000 5.000 ms trong qu trnh ghi git khng b ri. Gi tr Tdrop cng
ln sng cng ln v c ghi vng git ln, cng dng qua cc lm

Cng dng erythromycin

A (nA)

vic cng cao, nhy cng cao.

450
400
350

y = 0,0631x + 70,59

300
250
200
150
100
0

1.000

2.000

3.000

4.000

5.000

6.000

Tdrop (ms)

Hnh 3.19: nh hng ca Tdrop n cng dng ca erythromycin A

3.1.5.6 Nghin cu chn thi gian in phn tch gp Telectrolise

Tin hnh ghi ph erythromycin sau cc khong thi gian tch gp
3 - 4 - 5 giy v thu c:
Cng dng erythromycin
A (nA)

1800
1750
1700
1650

y = 181,7x + 815,2

1600
1550
1500
1450
1400
1350
1300
2

Telectrolise (s)

Hnh 3.21: nh hng ca Telectrolise n cng dng ca erythromycin A

3.1.5.7 Nghin cu chn th in phn tch gp Velectrolise

Tin hnh th nghim vi cc th tch gp: -400, -900, -1100 mV
v thu c:

14

Cng dng erythromycin

A (nA)

1900
1850

y = -0,2187x + 1620,8

1800
1750
1700
1650
-1200 -1100 -1000

-900

-800

-700

-600

-500

-400

-300

Velectrolise (mV)

Hnh 3.23: nh hng ca Velectrolise n cng dng ca erythromycin A

3.1.6 Nghin cu nh hng ca cc ion v c Na+, K+, Ca++, Mg++, Fe3+,

Cl- , SO4-2 , HPO42- ti ph erythromycin
nng 05 ppm th cc ion K+, Na+, Ca2+, Mg2+, Mn2+, Fe3+,
Cu2+, Cl-, I-, SO42-, HPO42- nh hng khng ng k (<5%) n cng
dng ca erythromycin.
3.1.7 Nghin cu xy dng ng chun
ng chun tuyn tnh trong khong 50 400 g/kg vi h s
tng quan iu chnh radjusted = 1,0; LoD = 0,52 g/kg.

Hnh 3.24: ng chun erythromycin A trn dung dch nn amoni axetat 0,1 M; pH 8,0

3.1.8 Nghin cu la chn quy trnh trch ly mu

Kt qu kho st ny cho thy trn nn mu tm cng xanh v c r
phi th quy trnh trch ly III cho hiu sut thu hi erythromycin cao nht
(90,59%, 83,57%) nn quy trnh trch ly III c chn kho st cc
thng s tip theo.

15

90.59

92.00
90.00
88.00
86.00
84.00
Hiu sut trch 82.00
ly (%)
80.00
78.00
76.00
74.00
72.00
70.00

88.62

87.90

81.63
77.27

II

III

IV

Quy trnh trch ly

Hnh 3.25: Hiu sut trch ly erythromycin trn tm cng xanh

90.00
80.00

70.69

75.25

83.57
69.33

72.34

70.00
60.00
Hiu sut 50.00
trch ly (%) 40.00
30.00
20.00
10.00
0.00

II

III

IV

Quy trnh trch ly

Hnh 3.26: Hiu sut trch ly erythromycin trn c r phi

3.2 THM NH PHNG PHP

3.2.1 Khong tuyn tnh
ng chun erythromycin tuyn tnh trong khong nng 50
400 g/kg vi h s tng quan iu chnh R2adjust = 1,0 v lch chun
tng i %R.S.D trong khong 0,4 3,7 %. Phng trnh ng chun
Y=1,08*X + 87.
3.2.2 Gii hn pht hin LOD
- Gii hn pht hin trn nn dung dch m 0,52 g/kg
- Gii hn pht hin trn nn mu tm cng xanh 0,57 g/kg
- Gii hn pht hin trn nn mu c r phi 0,80 g/kg
3.2.3 chnh xc, ng v hiu sut thu hi

16
Bng 1.16 (ph lc): chnh xc (RSD %), ng (RE %) v hiu sut thu hi
erythromycin A trn mu tm cng xanh

Ngy Nng thm Nng phn tch c RSD RE

(g*kg-1)
(TB SD, g*kg-1)
(%) (%)
1
100
91,45 1,44a
1,58 91,45
2
100
90,40 1,25a
1,38 90,40
3
100
90,92 1,43a
1,57 90,92
1
200
187,23 2,79b
1,49 93,61
b
2
200
184,76 1,97
1,07 92,38
3
200
185,18 2,43b
1,31 92,59
c
1
300
286,03 4,01
1,40 95,34
2
300
288,27 2,71cd
0,94 96,09
3
300
289,50 2,65d
0,91 96,50
* Mi gi tr l trung bnh ca 6 mu o

Bng 1.17 (ph lc): chnh xc (RSD %), ng (RE %) v hiu sut
thu hi erythromycin A trn mu c r phi
Ngy Nng thm Nng phn tch c RSD
(g*kg-1)
(TB SD, g*kg-1)
(%)
1
100
85,07 1.26a
1,48
2
100
85,34 1.79a
2,10
3
100
85,31 1.24a
1,45
b
1
200
173,25 2.34
1,35
2
200
173,03 2.09b
1,21
3
200
172,82 1.39b
0,80
c
1
300
261,62 4.53
1,73
2
300
262,57 3.42cd
1,30
d
3
300
265,68 5.38
2,02

RE
(%)
85,07
85,34
85,31
86,63
86,51
86,41
87,21
87,52
88,56

* Mi gi tr l trung bnh ca 6 mu o

3.2.4 nh gi kh nng nhn danh erythromycin A vi cc khng sinh

khc

17

Bng 3.3: Th bn sng c trng ca mt s khng sinh c nh danh

bng phng php sng vung qut nhanh trn cc git chm
Stt
Khng sinh
Th bn sng E1/2
1 Erythromycin
-1.430 mV
2 Chloramphenicol
-168 mV
3 Florfenicol
-78 mV
4 Furazolidone
-1.152 mV
5 Ciprofloxacin
-1.336 mV
6 Enrofoxacin
-214 mV
7 Danofoxacin
-1.120 mV
S tch bit th bn sng ny chng t c phng php stripping
sng vung qut nhanh trn cc git chm ngoi kh nng sng lc m cn
c kh nng nhn danh v tnh khng nh.
Gii thch c ch xut hin sng ca erythromycin:
Trong cng thc cu to ca eythromycin (hnh 3.1) c hai nhm
C=O, l c s hy vng c th bt c tn hiu kh c trng ca
eythromycin. Cc nhm C=O, nh trong cc aldehyde v xeton, thng cho
sng kh trc tip theo c ch sau:

Trong giai on th nht xy ra phn ng kh thun nghch vi s

tham gia mt in t to ra gc anion:
RRC=O + e- (+ H+) RRC` O-

(hay l RRC` OH)

Cc gc anion tip tc kh vi s tham gia thm mt in t na

v to mt sng kh c s tham gia hai in t.
Tuy nhin ty nguyn t cacbon ca nhm C=O lin kt vi cc

nhm i nhn hay i in t m vic kh nhm C=O d hay kh. Nu n

lin kt vi cc nhm i in t cng mnh th vic kh nhm C=O cng
d, sng cng chuyn dch v pha dng.
Ta hy xt th bn sng ca mt s cht hu c c nhm C=O sau:
Formaldehyde HCH=O

m pH 8

E1/2 = -1,50 V

m pH 10

E1/2 = -1,63 V

m pH 12

E1/2 = -1,74 V

18

Axetaldehyde CH3CH=O

m pH 7

E1/2 = -1,93 V

m pH 9

E1/2 = -2,08 V

Propionaldehyde CH3CH2CH=O LiOH 0,1M E1/2 = -1,96 V

Qua cc v d trn ta thy khi nguyn t cacbon ca nhm C=O
lin kt vi cc mch cacbon cng di, mt in t trong nhm C=O
cng cao, qu trnh kh cng kh, th bn sng cng tng v pha m.
Ngc li khi cc nhm i in t cng gn nguyn t cacbon ca nhm
C=O, th nhm C=O cng d kh nh cc v d sau:
Methylglyoxal CH3COCH=O m pH 7 E1/2 = -1,36 V
Glycoaldehyde HOCH2CH=O m pH 7 E1/2 = -1,54 V
Nhm C=O th nht ca erythromycin lin kt vi mt lot nhm
CH v CH3 l cc nhm i nhn nn mt in t kh cao nn rt kh
kh. Th bn sng ca nhm ny phi nm ln cn vng -2,0 volt.
Nhm C=O th hai lin kt trc tip vi mt nguyn t oxy l mt
tc nhn i in t, cho nn c th kt lun sng ca erythromycin c th
nh vng -1,4 volt l sng kh ca nhm C=O th hai.
Khi qut ho kh nng phn tch mt s h khng sinh:
a) H Phenicol:
Trong cng thc cu to ca h khng sinh phenicol c 3 nhm
chc in ho:
- Nhm -Cl2: CH2=CCl2 c E1/2 > -0,2 V; CH2=CH-CH2Cl c E1/2: -1,95 V
- Nhm C=O cng c th tham gia phn ng kh.
- Nhm nitro c tr s E1/2 dao ng trong khong -0,2 V n -1,0 V ph
thuc vo gi tr pH, cc nhm lin kt vi nhm NO2 v dung mi s
dng. Tu thuc pH dung dch nn, nhm ny cho 1 sng hay 2 sng kh.
b) H Nitrofuran:
Trong h khng sinh nitrofuran c 3 nhm chc in ho:
- Nhm NO2 c E1/2 trong khong - 0,2 V n - 1,0 V.
- Nhm C=N c E1/2 trong khong -1,0 V n - 1,8 V.

19

- Ngoi ra cn c nhn d vng 2-oxo-1,3-oxolidin cng c th tham gia

phn ng kh.
c) H Fluoroquinolon:
Nhm fluoroquinolon trong cng thc cu to c khung quinolon,
nhn quinon, nhm ny c phn ng kh trn in cc git thu ngn.
3.2.5 So snh i chng kt qu SSWV vi LC-MS/MS
3.2.5.1 Trn mu tm cng xanh
Hiu sut thu hi: > 90,92 %, MDL: 0,57 g.kg-1; R_adjust:
0,99999; RSD: 0,91 1,58 %. (Hnh 3.29)

Hnh 3.29: ng chun erythromycin trn nn mu tm cng xanh

3.2.5.2 Trn mu c r phi

Hiu sut thu hi: > 85,07%; MDL: 0,80 g.kg-1; R_adjust: 1,0;
RSD: 0,80 2,10 % (Hnh 3.30).

Hnh 3.30: ng chun erythromycin trn nn mu c r phi

3.2.5.3 So snh kt qu phn tch bi SSWV v LC-MS/MS

a) Tm cng xanh

20
Bng 3.4: So snh kt qu phn tch mu tm cng xanh bi 2 phng php
S nhn din
GP Blank
GP Blank
GP Blank
GP I
GP I
GP I
GP II
GP II
GP II
GP III
GP III
GP III

SSWV (LOD=0,57 g*kg-1)

D lng
TB SD
RSD
(g*kg-1)
(g*kg-1)
(%)
KPH
KPH
KPH
51,37
50,19
1,17
50,75 0,59a
50,68
65,45
64,97
65,48 0,53b 0,80
66,02
80,61
79,16
80,00 0,75c 0,94
80,22

LC-MS/MS (LOD=10 g*kg-1)

D lng
TB SD
RSD
(g*kg-1)
(g*kg-1)
(%)
KPH
KPH
KPH
50,73
56,89
52,61 3,72a 7,06
50,21
72,82
68,00
70,49 2,41b 3,42
70,65
74,60
85,12
80,23 5,30c 6,61
80,98

* LOD: Gii hn pht hin, ** KPH: Khng pht hin

b) C r phi
Bng 3.5: So snh kt qu phn tch mu c r phi bi 2 phng php
S nhn din
TL Blank
TL Blank
TL Blank
TL I
TL I
TL I
TL II
TL II
TL II
TL III
TL III
TL III

SSWV (LOD=0,80 g*kg-1)

D lng
TB SD
RSD
(mg*kg-1)
(mg*kg-1)
(%)
KPH
KPH
KPH
1,31
1,27
1,29 0,02a 1,61
1,30
1,95
2,09
2,02 0,07b 3,47
2,02
2,80
2,80
2,78 0,03c 1,25
2,74

LC-MS/MS (LOD=10 g*kg-1)

D lng
TB SD
RSD
(mg*kg-1)
(mg*kg-1)
(%)
KPH
KPH
KPH
1,23
1,28
1,26 0,03a 2,10
1,27
3,14
2,72
2,72 0,43a 15,64
2,29
2,80
2,80
2,80 0,01b 0,21
2,81

* LOD: Gii hn pht hin, ** KPH: Khng pht hin

Qua bng 3.4 v 3.5, cng nh kt qu phn tch ANOVA bng

3.28-3.31 (phn ph lc) ta thy c c s tng ng kh cht ch v kt
qu phn tch gia 2 phng php SSWV v LC-MS/MS. Xt v lp li
ca phng php th phng php sng vung qut nhanh c lp li tt

21

hn phng php LC-MS/MS da trn ch s % R.S.D trn c 2 i tng

mu l tm cng xanh v c r phi.
3.3 NG DNG PHN TCH ERYTHROMYCIN TRN CC MU THC
T TM, C TI CC A PHNG
700
600
D lng
erythromycin A
(ppb)

500
400
300
200

Cao Lanh
Tam Nong
Thap Muoi
Go Quao
Giong Rieng
Vinh Thuan
Thoai Son
Long Xuyen
Phu Tan
Tam Binh
Binh Minh
Long Ho
Mo Cay
Chau Thanh
Giong Trom
Duyen Hai
Tra Cu
Cang Long
Phung Hiep
Long My
Vi Thuy
My Tu
My Xuyen
Ke Sach
Vinh Thanh
O Mon
Co Do
Phuoc Long
Hong Dan
Gia Rai

100

Dong Kien An Vinh Ben

thap Giang Giang Long Tre

Tra Hau Soc Can

Vinh Giang Trang Tho

Bac
Lieu

a phng

Hnh 3.31: Tnh hnh nhim erythromycin tm cng xanh ti cc tnh BSCL
800
700
600
500
D lng
erythromycin A 400
(ppb)
300
200

Cao Lanh
Tam Nong
Thap Muoi
Go Quao
Giong Rieng
Vinh Thuan
Thoai Son
Long Xuyen
Phu Tan
Tam Binh
Binh Minh
Long Ho
Mo Cay
Chau Thanh
Giong Trom
Duyen Hai
Tra Cu
Cang Long
Phung Hiep
Long My
Vi Thuy
My Tu
My Xuyen
Ke Sach
Vinh Thanh
O Mon
Co Do
Phuoc Long
Hong Dan
Gia Rai

100

Dong Kien
An
Vinh
thap Giang Giang Long

Ben
Tre

Tra Hau Soc Can

Vinh Giang Trang Tho

Bac
Lieu

a phng

Hnh 3.32: Tnh hnh nhim erythromycin c r phi ti cc tnh BSCL

T l nhim trn tm cng xanh (45/150 mu) cao hn so vi trn

c r phi (30/150 mu). Ngi nng dn bt u s dng loi khng sinh
ny, thm ch nng cao iu tr bnh cho thy sn nui v khng
tun th thi gian ngng thuc cn thit (hnh 3.31 & 3.32).
3.4 XC NH QUY LUT CHUYN HA SINH HC, O THI
KHNG SINH ERYTHROMYCIN TRN C TH THY SN KHI NUI

22

3.4.1 o thi erythromycin trong c tht

Erythromycin A (g/kg)

700.0
600.0
500.0
400.0
300.0
200.0
100.0
0.0
0

100

200

300

400

500

600

700

Thi gian (oC - ngy)

50 mg erythromycin/kg th trng/ngy

100 mg erythromycin/kg th trng/ngy

Hnh 3.33: Kh nng o thi erythromycin A trong c tht tm cng xanh

Ey rthro m y cin A ( g /kg )

c cho n 50 v 100 mg.kg-1 th trng.ngy-1 trong 7 ngy

50,000
45,000
40,000
35,000
30,000
25,000
20,000
15,000
10,000
5,000
0
0

100

200

50 mg ery t hromy cin/kg t h t rng/ngy

300

400

500

600

700

Thi g ian ( o C - ng y)

100 mg ery t hromy cin/kg t h t rng/ngy

Hnh 3.34: Kh nng o thi erythromycin A trong c tht c r phi c cho

n 50 v 100 mg.kg-1 th trng.ngy-1 trong 7 ngy

Bng phng php thng k, rt ra c thi gian ngng thuc 976

C_ngy (tng ng 34,86 ngy; nhit 28 oC) i vi tm cng

xanh c cho n erythromycin 100 mg.kg-1 th trng.ngy-1 trong thi
gian 7 ngy. Ring vi mc nng 50 mg.kg-1 th trng.ngy-1 trong thi
gian 7 ngy th d lng li l khng ng k v mc an ton.

23

Cng bng phng php thng k ny, rt ra c thi gian ngng

thuc 908 C-ngy (tng ng 32,42 ngy, 28 oC) i vi c r phi

c cho n erythromycin 50 mg.kg-1 th trng.ngy-1 trong thi gian 7
ngy. Trong khi th cn n 1.150 C-ngy ngng thuc (tng ng
41,07 ngy, 28 oC) nu c r phi c c cho n 100 mg.kg-1 th
trng.ngy-1 trong thi gian 7 ngy.
3.4.2 Chuyn ho sinh hc ca khng sinh gc
3.4.2.1 Tm cng xanh
Khi cho n liu 100 mg.kg-1 th trng.ngy-1 trong 7 ngy bng
con ng thc n, erythromycin A dn chuyn ha sang erythromycin
E (2,09 g/kg) ngy th nht sau ngng thuc. ngy th 23 sau ngng
thuc, erythromycin E (5,81 g/kg) v erythromycin F (3,52 g/kg) c
pht hin mc nng khng ng k.
3.4.2.2 C r phi
Khi c r phi c cho n erythromycin base liu thp (nhm A),
ngy th nht sau ngng thuc th khng pht hin c bt k dn xut
chuyn ha no ca erythromycin. Tuy nhin n ngy th 23 th pht hin
c cc dn xut erythromycin E (2,30 g/kg) v erythromycin F (2,37
g/kg) trong c tht c. Trong trng hp c r phi c cho n
erythromycin base liu cao (nhm B) th ngay ngy th nht sau ngng
thuc, pht hin thy dn xut chuyn ha erythromycin C (131,5 g/kg) v
erythromycin E (258,3 g/kg). ngy th 23 sau ngng thuc,
erythromycin E (6,94 g/kg) v erythromycin F (5,90 g/kg) c pht
hin mc nng khng ng k. Khi phn tch c ch chuyn ha, thy
rng: C s oxy ha ti v tr R2 v R3 trong phn t erythromycin A lm
bin i erythromycin A thnh erythromycin E c tm cng xanh v c r
phi v c s kh methyl ca R4 lm bin i erythromycin A thnh
erythromycin C c r phi. Nh vy cc enzyme oxy ha kh c trong gan
v trong m c ca tm v c xc tc phn gii erythromycin.

24

KT LUN
1.

ti xy dng c phng php sng vung qut nhanh trn

cc git chm trong vic phn tch khng sinh erythromycin trong tm, c
t c gii hn pht hin, p ng yu cu gii hn d lng cho php
theo tiu chun quc t. Phng php cho thy hiu sut thu hi cao (85,05
96,50 %), nhy cao (gii hn pht hin 0,52 g/kg), chnh xc cao
( lch chun tng i, RSD t 0,80 2,10 %), ng (sai bit tng
i, RE t 3,50 4,95 %) cng nh mc tuyn tnh rt cao (r2
0,99999). C s tng ng gia kt qu phn tch bi phng php ny
vi phng php sc k lng ghp khi ph.
2.

a ra c c ch gii thch xut hin sng erythromycin. T

khi qut ho v a ra c gi thuyt kh nng phn tch tng h khng

sinh nh phenicol, nitrofuran, fluoroquinolon (thng gp trong nui trng
thu sn) bng k thut sng vung qut nhanh trn cc git chm.
3.

Kt qu ti lm cho thy rng cc dn xut erythromycin

khng ch c chuyn ho t Sacchropolyspora erythrea bng con ng

ln men, m cn chng minh rng erythromycin A khi vo c th tm c,
di tc dng ca cc enzyme oxy ha kh v enzyme chuyn methyl ha,
bin thnh erythromycin C, E, F, sau b o thi. ti cng a ra
c gi thuyt gii thch c ch chuyn ho erythromycin trong tm cng
xanh, c r phi khi nui.
4.

nh gi v rt ra c quy lut thi gian ngng khng sinh

erythromycin trc thu hoch trn i tng tm cng xanh v c r phi.