TI:

XC NH CLENBUTEROL TRONG M LN BNG PHNG PHP CHIT

PHA RN KT HP VI CHIT VI LNG PHN TN LNG LNG V
PHT HIN BNG HPLC-UV

HC VIN: L TH NHN MSHV: 1670671

HC VIN: NGUYN CH THANH MSHV:
NHM:
A. TNG QUAN
I.CLENBUTEROL
II. CC PHNG PHP XC NH CLENBUTEROL
III. CC PHNG PHP TIN X L MU
IV. KT LUN
B. THC NGHIM
I HA CHT
II. THIT B
III. PHA DD CHUN V DD MU
C. KT QU V THO LUN
I. LA CHN DM CHIT
II. TI U HA QUY TRNH SPE
III. TI U HA QUY TRNH DLLME
IV. GI TR O CA PHNG PHP
V. PHN TCH MU
D. KT LUN
 A. TNG QUAN

I.Clenbuterol

(4-amino-3,5-dichloro-a-tert-butylaminomethylbenzyl alcohol hydrochloride, CLB)

 A. TNG QUAN I.Clenbuterol

Clenbuterol
Cu to phn t: C12H9OCL3
Trng lng phn t: 313.65
Nhit nng chy: 174 175.5C
Hnh dng: bt tinh th nh khng
mu
Tnh tan: tan nhiu trong nc v
ru, t tan trong Chloroform, khng
tan trong Benzen.
 A. TNG QUAN I.Clenbuterol
CLENBUTEROL C S DNG

Thuc tr su Thuc gin ph qun Thuc b tim

Cht kch thch tng trng

Cht kch thch pht trin c Gm lng m trong thc n cho ng vt
A. TNG QUAN I.Clenbuterol
 A. TNG QUAN II. CC PHNG PHP XC NH CLENBUTERON

CC PHNG PHP CHNH C S DNG

XC NH D LNG CLENBUTEROL TRONG M THT LN

XT NGHIM MIN DCH

IN DI MAO QUN (CZE)

SC K LNG U D UV (LC-UV)

SC K KH U D KHI PH (GC-MS)

SC K LNG U D KHI PH (LC-MS)

SC K LNG U D IN HA (LC-ECD)

SC K LNG U D HUNH QUANG (LC-FD)

A. TNG QUAN III. CC PHNG PHP TIN X L MU

Do s phc tp ca cc nn mu
v cc lng vt ca CLB trong
cc sn phm tht, k thut tin x
l mu thch hp l cn thit
lm sch nn mu v lm giu
cht phn tch trc khi a vo
thit b phn tch
 CHIT
Chit pha rn l qu trnh phn b
LNG-
LNGca
(LLE)cc cht gia 2 pha, trong lc
u cht VImu
CHIT
dng lng (pha
CHIT PHA nc, hayPHAhuRNc), cn cht chit
RN (SPE)
dng rn, dng ht nh v xp
PHNG
ng knh 25 - 70 m. V th nn
PHPcX
tn l chit pha rn (Solid Phase
Extraction ), hay chit rn-lng.
L MU
VI CHIT
DIPHASIC LNG-
SPE da trnDIALYSIS
cht hp th khc nhau nh C, Alumina, v phi
LNG
trn cht hp ph l phng php s dng rng ri
NNMU
PHN TN
PHA RN
A. TNG QUAN III. CC PHNG PHP TIN X L MU

Tuy nhin, tt c cc phng php trn u b chn lc

thp i vi vic chit xut cht phn tch t nn mu phc tp.
Mc d phn t polymer c p dng cho ci thin tnh chn
lc, thu hi thp ca CLB khng ph hp vi tiu chun
C.D.2002/657/EC. S chn lc cng c ci thin bng cch
s dng cc cht hp th t tin, nhng tn nhiu thi gian v
cc iu kin c th hn ch ng dng rng ri
 A. TNG QUAN III. CC PHNG PHP TIN X L MU

Gn y, SPE kt hp vi mt quy trnh lm sch cho thy mt cch tim

nng lm tinh khit v lm tng nng lng vt cht phn tch t
cc mu thc

Chit vi lng phn tn

lng lng
(DLLME)

SPE
 A. TNG QUAN III. CC PHNG PHP TIN X L MU

Chit vi lng phn tn lng lng

(DLLME)

L mt k thut vi chit mi
c pht trin bi Assadi et al,
n c da trn mt h thng dung mi th ba nh LLE ng nht v
che cht cn tch. Trong phng php ny, hn hp dung mi chit v
dung mi phn tn thch hp c thm nhanh vo mt dung dch nc,
kt qu l mt cht trng thi che bao gm cc git tinh khit ca dung
mi chit xut c phn tn vo pha nc, n lm tng b mt tip xc
gia cc pha v gim thi gian chit cng nh yu t lm giu.
u im ca DLLME l s n gin, nhanh chng, chi ph thp, thu
hi cao v cc yu t lm giu cht phn tch
 A. TNG QUAN IV. KT LUN

Mc tiu ca nghin cu ny l trnh by n

lc u tin kt hp vi nhng u im ca
SPE v DLLME pht trin mt phng php
tin x l trit CLB t m m ln. Cc mu
c chit trc tip bng quy trnh SPE cht ra
ca SPE c s dng lm dung mi phn tn
ca theo quy trnh DLLME lm sch thm v
lm giu thm CLB trc khi phn tch HPLC.
Cc yu t lm giu (EF) ca phng php SPE-
DLLME-HPLC c th c ci thin nhiu hn
62 ln. So vi quy trnh SPE thng thng,
phng php SPE-DLLME-HPLC cung cp kh
nng lm chn lc v tinh khit cao hn.
 B. THC NGHIM I HA CHT

Chloroform (CHCl3), dichloromethan

(CH2Cl2), 1,2-dichloroethane (C2H4Cl2),
1,1,2,2 tetrachloroethane (C2H2Cl4),
tetrachloroethylene (C2Cl4), chlorobenzene
(C6H5Cl) v tetrachloromethane (CCl4).
Acetone, methanol, ethanol, acetonitrile,
isopropanol, ammonia v hydrochloricacid.
Hp PCX v hp C18 (3.0mL, 60mg). Hp
OASIS HLB (3.0mL, 60mg). Tt c cc cht
th khc c s dng trong th nghim l
loi cao cp nht c mt trn th trng
thng mi. Nc kh ion c lc qua mt
mng si 0,45 m trc khi s dng.
B. THC NGHIM II. THIT B

Thit b o c Phn tch HPLC c thc

hin bng h thng HPLC Shimadzu c trang
b hai b cung cp dung mi LC-20AT, mt b
trn SUS20A, v mt u d SPD-20A. Mt
phn mm ghi nhn tn hiu N-2000. Ct phn
tch (150 mm 4.6 mm ID, C 18, 5,0m). Pha
ng l nc-methanol (64:36, v/v, cha 0,2
trifluoracetic Acid, pH 2,8) v tc dng chy
ca n c t mc 1.0mL / pht. u d
c ci bc sng pht hin l 210nm. Thit b
HSE-12D SPE v b dao ng siu m
KQ3200E c ci t 40kHz v 25 C. Hai
my ly tch pha.
 B. THC NGHIM III. PHA dd CHUN V dd MU
Chun b mu chun gc CLB bng cch ha tan mt
lng chun trong methanol nng 0.2mg/mL v lu tr
trong bng ti -4 C v dung dch chun lm vic CLB
(0.00096 1.92 mg/L) c chun b bng cch pha
long dn dn vi methanol. Cc mu tht v gan ti mua
t ch. Sau c ng nht bng my nghin, mu 1,0g
c a vo mt ng hnh nn 5,0ml v chit bng dao
ng siu m trong 10 pht vi 2,0 ml anhydrous ancohol
nh dung mi chit. Quy trnh chit c lp li trong ba
ln v cc dung dch ni trn b mt c thu bi ly tm
16.000 vng/pht trong 5 pht c dn vi nhau v
c kh m bng 1.0 mL hexane. Sau khi ly tm 4000
rpm cho 5,0 pht v loi b giai on ty nhn, dung dch
c c c n 1,3 ml tip tc SPE-DLLME.
 B. THC NGHIM III. PHA dd CHUN V dd MU

1g mu tht v gan ti
C c n 1,3 ml
nghin

a vo mt ng hnh nn 5,0ml
Ty nhn

chit bng dao ng siu m

Trong 10 pht vi Ly tm 4000 rpm cho 5 pht
Lp li trong ba ln
2,0 ml anhydrous ancohol

Dung dch ni trn b mt c

kh m bng 1.0 mL hexane
thu bi ly tm 16.000 vng/pht
 B. THC NGHIM IV. QUY TRNH SPE - DLLME

Hp PCX c ti tp hp bng methanol 5,0 ml v axit clohiric (30

mmol/L) tc dng chy 1,0 mL/pht. Sau khi mu c tch bng lc
hp dn, hp c ra sch vi 5,0ml nc v methanol vi tc dng
chy 1,0 mL/pht loi b cc nhiu nn. Cui cng, cht phn tch c
ra vo ng hnh nn 10ml bng 5.0mL methanol- dung dch amoniac
(95:5, v/v) v c c n 0,5 ml 35C. Cui cng, 5.0mL nc, 50 L
amoniac v 150L C2H2Cl 4 c thm vo ra gii v sau siu m
trong 2,0 min che tt. Vic tch pha c thc hin bng ly tm ti
4000 rpm trong 5 pht. Pha lng ng c bc hi n kh v ti to
trong 20L pha ng phn tch vi HPLC. Hiu sut tch c tnh
bng cch ly t l phn trm ca tng s cht phn tch (n0) v cht phn
tch (na) c chit t cht lng phn tch bng HPLC.
 C. KT QU V THO LUN I. LA CHN DM CHIT

Hiu qu chit CLB lin quan trc tip n dung

mi chit c s dng cho mu m. Xem xt cc tnh
cht ha hc ca CLB v cc bo co trc , 0,1
mol/L Axit clohiric, 0,1 mol/l axit perchloric, v cn
khan nh mt dung mi ph bin chit xut CLB t
cc m ng vt. Cc kt qu cho thy rng chit hiu
qu (>96%) vi nhiu nn t thu c t h chit
ru khan. Ngoi ra, ru khan c c tnh thp hn
v c th c s dng nh cht lng protein ng
thi khng kt t protein. V vy, khan Ru c s
dng lm dung mi chit trong trng hp ny.
C. KT QU V THO LUN II. TI U HA QUY TRNH SPE

Loi cht hp th SPE l mt trong nhng yu t chnh cho

s c lp v s tinh khit cc cht mc tiu t cc mu phc
tp. PCX, C18 v cht hp th HLB c nghin cu v thu
hi tt nht CLB c gi t cht hp ph PCX. Cht hp
ph PCX cho thy mt cht duy nht phn cc theo bin i
b mt, n l nguyn nhn protein v phopholipid trong mu
khng th vo trong trung tm cc l k nc v b mt a
nc, do n c hiu qu lm gim nhiu nn ca cc mu
m. i vi hp PCX, CLB hp ph mnh ln cht hp
ph trong iu kin axit, trong do n chuyn i thnh
mui ammonium bc bn tng ng (NH 2 RR +) . V vy,
cn gi iu kin axit chc chn rng CLB c nh
mnh vo cht hp ph bng cch kt ni vi nhm SO3-
ca PCX theo hnh thc mui ammonium
C. KT QU V THO LUN II. TI U HA QUY TRNH SPE

Da trn cc cng trnh trc, nc v methanol

c la chn lm dung mi ra v th tch trong phm
vi 1,0-10,0 mL i vi cc mu c thm chun.
thu hi ca CLB hu nh khng thay i vi dung dch
ra gii c quan st khi tng th tch dung mi ra t
1,0 n 5,0 ml v sau thu hi ca CLB gim dn
cng vi s gia tng dung mi ra t 5,0 n 10,0 ml.
Xem xt hiu qu chit v nh hng ca nn, 5,0 ml
dung mi ra c chn cho cng vic tip theo. Do ra
gii ca SPE cng ng vai tr mi trng phn tn
theo quy trnh DLLME, methanol, ethanol,
Acetonitrile, aceton, v isopropanol cha nhiu lng
khc nhau Amoniac (1,0-9,0%) nh l dung mi ra
gii c nghin cu
 C. KT QU V THO LUN II. TI U HA QUY TRNH SPE

Cc kt qu trong hnh cho thy rng s phc

hi tt nht ca CLB thu c khi s dng
methanol-amoniac (95: 5, v/v) lm dung mi ra,
trong l do ammonia c th trung ha cc CLB
b tch ra v ph v lin kt cng ha tr gia
CLB v cht hp ph PCX. S khc bit lng
dung mi ra gii trong khong 1,0 9,0mL c
nghin cu v kt qu cho thy rng thu hi r
rng tng vi lng tng t 1,0 n 4,0 mL, v sau
n gi khng i thm ch cn tng ln n 9,0
mL. Xem xt thi gian tch, thu hi v mc
ca CLB trong cc mu, 5,0 mL c s dng
nh lng ti u lm dung mi ty ra.
 C. KT QU V THO LUN III. TI U HA QUY TRNH DLLME

Hiu sut ca DLLME ch yu c xc

nh bi cc loi v th tch ca cht chit, n
c khi lng ring cao hn nc, kh nng
chit cao ca cc cht phn tch, v tan thp
trong nc. Xem xt cc nguyn tc ca
DLLME v cc cng trnh trc [36,38] ,
lng khc nhau ca CHCl3, CH2Cl2, C2H2Cl4,
C2H4Cl2, C2Cl4, C6H5Cl, CCl4 trong mt phm
vi 50-200L c nghin cu v cc kt qu
trong hnh. 3 cho thy 150L C2H2Cl4 cht chit
t c s hi phc tt nht cho CLB. Lng
cht phn tn l mt yu t quan trng khc cn
c xem xt trong DLLME.
 C. KT QU V THO LUN III. TI U HA QUY TRNH DLLME

V th, Cc th tch khc nhau ca cht phn tn methanol -

ammonia (0,3, 0,5, 0,8, 1,0, 1,2 v 1,5 mL) c nghin cu
v kt qu cho thy EF ban u tng ln v sau r rng l
gim xung khi th tch cht phn tn trn 0.5 mL. Hn na,
thu hi ca chit CLB cng gim vi s gia tng th tch phn
tn trn 0.5 mL, nguyn nhn bi cht phn tn c th lm tng
tan ca cht phn tch trong nc v y nhanh cc git cht
chit xut vo trong dung dch nc. Do 150L C2H2Cl4 nh
chit v 0,5 mL methanol-amoniac lm cht phn tn c
s dng.
 C. KT QU V THO LUN IV. GI TR O CA PHNG PHP

Trong cc iu kin c ti u ha trn, phng php SPE-

DLLME-HPLC c gi tr bi kh nng tuyn tnh, chnh xc, gii
hn pht hin, thu hi, lch chun. ng chun c xy
dng bng cch s dng din tch ca cc nh sc k bi 9 ln im
tng dn, trong khong 0,19 192 g/kg. tuyn tnh tt thu c
cho CLB trong phm vi nng v phng trnh ng chun l
y = 7.11105 x 7,13103 vi h s tng quan (r 2) ca 0,9995.
chm th hin bng lch chun tng i (RSD) tnh t cc mu
kim sot cng ngy v nm ngy khc nhau t hn 4,0% v 5,9%.
Trong iu kin ti u ha chit, EF ca CLB l 62 ln.
 C. KT QU V THO LUN IV. GI TR O CA PHNG PHP

Da trn t l tn hiu nhiu nn (S/N) l 3, gii

hn pht hin (LOD) l 0,07 g/kg, thp hn gii
hn d lng ti a FDA v WHO xy dng. Ngoi
ra, nm mu trng c chit xut v c phn
tch theo quy trnh SPE-DLLME-HPLC nh gi
cc nhiu tim n. Khng c cc nh ni gy nhiu
t nn mu c tm thy trng vi thi gian lu
CLB, n chng t kh nng thc hnh tt ca
phng php SPE DLLME HPLC. Hn na, sc
k r hn c quan st khi dng quy trnh SPE-
DLLME (so vi SPE), n ch ra phng php
c pht trin r rng ci thin tnh chn lc ca
qu trnh tin x l mu
C. KT QU V THO LUN IV. GI TR O CA PHNG PHP

9 mu m (tht v gan) ly t cc
mu ch khc nhau ca thnh ph a
phng c p dng xc nhn d
kin phng php. Tt c cc mu thc
t c tin x l trc theo mc 2.3 .
Mt mu gan c quan st thy s
lng vt ca CLB mc 0,5 g/kg,
di mc d lng ti a ca n
0,6g/kg) c xy dng bi FDA v
WHO ( Fig. 5 )
 C. KT QU V THO LUN V. PHN TCH MU

Nghin cu nh hng ca nn mu v chnh xc ca

phng php SPE-DLLME-HPLC, cc th nghim thu hi c
thc hin bng cch thm chun ba nng CLB trong mu m
(Bng 1). thu hi trung bnh cho CLB c 3 mc thm chun
trong khong 87,9 103,6% vi SRD t hn 3,9% (n = 3), cho
thy phng php ny l c lp li c th c s dng xc
nh lng vt CLB trong mu m.
 D. KT LUN

SPE-DLLME-HPLC mi c pht trin xc nh CLB trong

cc mu m. Cc mu u tin c chit bi SPE v ra gii n
phn tn theo DLLME lm sch v lm giu thm cc cht phn
tch trc khi phn tch HPLC. tuyn tnh tt c quan st thy
trong mt 0,19-192 g/kg vi LOD 0,07 g/kg (S/N = 3). thu hi
ti ba mc thm chun dao ng t 87,9% n 103,6% vi RSD t
hn hn 3,9% v c th t c EF 62 ln. xut phng php
SPE-DLLME-HPLC c tnh chn lc cao hn v kt hp u im
ca SPE v DLLME, n c th c s dng trong vic xc nh cc
cht phn tch dng vt trong cc mu sinh hc.