VIEN CHAN NUOI - Tgp chi Khoa hgc Cdng nghe Chdn nudi - S6 12-Thang 6-2008
ITNG DUNG KY THUAT ELISA DE XAC DINH CLENBUTEROL vA SALBUTAMOL TRONG THLTC A N C H A N NUOI
Doan Thi Khang, Phan Thanh Dgm*, Duang Thi Tim Anh vd Dao Dure Hao.
Phong Phan tich Thirc an va san pham chan nuoi -Vien Chan nuoi Thuy Phuong - Tir Liem - Ha Noi
Tac gia lien he: Phan Thanh Dam
Tel: (04) 8.385.941 / 0913.084.222; Fax: (04) 8.389.775; Email: vcnvilas053(gyahoo.com
ABSTRACT
Appplying the ELISA method for determination of Clenbuterol, Salbutamol in animal feeds The occurrence of hormonal residues in animal tissue, meat and urine samples was investigated by semi- quantitative enzyme-linked immunoassay technique.
The Beta-agonist ELISA test kit of Biooscientific were used for determining Clenbuterol, Salbutamol in animal feeds, RSD of calibration was acceptable 2.25 -3.89%. The tests were able to detect Clenbuterol, Salbutamol in animal feed through recovery rates of Clenbuterol, Salbutamol (90.05 - 97.0%).
The results of Clenbuterol, Salbutamol in the same sample were determined by ELISA method are lower than GC/MS method. It means that ELISA method can be used for qualitative determination of Clenbuterol, Salbutamol in animal feeds.
Key words : Beta-Agonist, ELISA method, Clenbuterol, Salbutamol. ', j ,;.•,
DAT V A N D E
Clenbuterol va salbutamol la nhiing chat thudc nhdm Beta-Agonits cd tac dung lam gian phe quan. Day la mdt dugc pham sir dung de dieu tri hen suyen tren ngudi, cac trudng hgp viem phdi di img tren ngua va viem dudng hd hap tren bd. Khi udng lieu cao cac chat Beta-Agonits cd the lam tang khdi lugng co the va lam giam mo.
Do vay, thudc dugc bd sung vao thiic an gia siic, gia cam nham tang thjt nac va tang trgng nhanh hon (Eshaq va cs, 2003). Tuy nhien, su tdn du ciia Clenbuterol va Salbutamol trong thit va phu tang, dac biet la gan cua nhiing con vat dugc nudi bang thirc an cd chiia cac chat nay da cd anh hudng xau den sire khoe ngudi tieu diing (Yu va Yang, 2004). Hien nay My, Canada, va cac nude trong lien minh Chau Au da cam sir dung cac chat thudc nhdm Beta- Agonits lam thirc an chan nudi. d Viet Nam, Bd Ndng nghiep va PTNT ciing da ra quyet dinh sd 54/2002/QD-BNN ngay 20 thang 06 nam 2002: Cam sir dung cac chat thudc nhdm Beta- Agonist trong thirc an chan nudi. Tuy nhien, viec sir dung bat hgp phap cac chat cam nhu Clenbuterol va Salbutamol van ed tren thi trudng thiic an chan nudi nudrc ta.
Tren the gidi Clenbuterol va Salbutamol Uong thiirc an chan nudi da dugc dinh lugng bang sac ky khi khdi phd (Hassnoot va cs, 1994), (He va cs, 2005). Phuang phap nay ddi hdi thiet bi dat tien nen chi phi qua cao.
Phuang phap ELISA(ENZYM-LINK IMUNOSORRBENT ASSAY) da dugc su dung rgng rai de xac djnh cac chat du lugng thudc trir sau, ddc td nam mdc (Skeritt,1998). Nguyen ly ciia phuong phap ELISA xac dinh Clenbuterol va Salbutamol la dua tren nguyen ly cua phan ung khang the - khang nguyen.
DOAN THI KHANG - (fng dung ky thudt ELISA di xdc dinh ...
Khang nguyen dugc cd djnh tren be mat ciia gieng phan tich. Khang nguyen tu do (chit cin phan tich) va khang nguyen cd djnh se canh tranh de bam vao khang the. Lugng khang nguyen tu do cang nhieu thi lugng khang the bi khang nguyen cd dinh giir lai cang it. Sau khi nia trdi cac chat cdn du, dung mdt lugng khang the thir 2 ed danh dau enzyme de phat hien lugng khang the ban dau bi giii lai. Bang phan iing tao mau va phep do cudng do mau tai budc sdng 450 run va cudng do mau nay ty le nghich vdi ndng do chat can phan tich.
Tir dd suy ra, lugng khang nguyen tu do(chat can phan tich) la bao nhieu. d Viet Nam, viec kiem tra cac ddc td trong thirc an chan nudi gap rat nhieu khd khan do chua cd thiet bi hien dai de cd the xac dinh chinh xac cac chat nay va chua cd phdng thi nghiem nao phan tich Clenbuterol va Salbutamol trong thiic an chan nudi.
Chiing tdi trien khai cac thi nghiem phan tich Salbutamol va Clenbuterol bang ky thuat ELISA tai phdng phan tich thirc an va san pham chan nudi - Vien Chan nudi vdi muc dich khao nghiem phuong phap xac dinh Clenbuterol va Salbutamol trong thiic an chan nudi.
VAT LIEU vA PHUONG P H A P NGHIEN ClTU.
Vat lieu nghien cuu
Mau thirc an chan nudi, nhirng san pham de bd sung vao trong thirc an chan nudi.
Sir dung 2 loai bd kit chuin Salbutamol va Clenbuterol cua hang BlOO SCIENTIFIC - M y.
Npi dung va phuong phap nghien cuu
Xay dimg dudng chuan vdi 6 loai ndng do chat chuan (Salbutamol hoac Clenbuterol) khac nhau: 0 ppb; 0.05ppb; 0.15ppb; O.Sppb; 1.5ppb; 4.5ppb.
Moi loai ndng do thuc hien lap lai 8 lin. Dgc do hap thu cua cac ndng do chuan khac nhau.
Dd xac dinh miic thu hdi chit chuin Clenbuterol va Salbutamol dugc them vao mau trang d cac ndng do khac nhau: Ippb; 2ppb; 3ppb; 4ppb. Moi mirc ndng do them vao mau trang dugc laplailOlin.
So sanh kdt qua phan tich ciia 3 mau thiic an chan nudi dugc phan tich bang phuong phap sac ky khi khoi phd(GC-MS) tai phdng thi nghiem - Bd Ndng nghiep ciia Malaysia va phuong phap ELISA tai phdng phan tich Vien Chan nudi dd xac dinh do chinh xac ciia phuang phap.
Cac budc tien hanh ciia ky thuat ELISA dugc thuc hien theo diing hudng dan cua hang BIOO SCINTIFIC - My.
Cac phan tich dugc tidn hanh lap lai va kdt qua phan tich dugc xir ly Thong ke.
Xir ly so lieu
Cac so lieu thu thap dugc xir ly theo phuong phap thong ke sinh vat hgc bing phan mem Minitab version 14.0
VIEN C H A N N U O I - Tgp chi Khoa hgc Cdng nghi Chdn nudi - So 12-Thing 6-2008
KET QUA VA THAO LUAN Ket qua xay dung duong chuan Clenbuterol va Salbutamol
De dinh lugng dugc Clebuterol va Salbutamol can cd Dd thi chuan cua cac chat nay.
Vdi moi ndng do khac nhau cua moi chat Uong bd Kit chuan cua hang BIOO SCIENTIFIC- My da dung dugc hai dudng chuan Clenbuterol va Salbutamol. Bieu thi d Dd thi I va Dd thi 2.
(ppb)
Do thj 1, Duong chuan Clenbuterol
(ppb)
Do thj 2. Duong chuan Salbutamol
Xay dung dudng chuan cua Clenbuterol hoac Salbutamol dua tren % hap thu tdi da ciia chat chuan Clenbuterol hoac Salbutamol va ndng do tuong ung cua chiing la mdt dudng khdng tuyen tinh. Do vay, cac ket qua duong tinh dugc dgc tir dudng chuan chi cd gia tri tuong ddi chinh xac trong gidi han ciia dudng chuan.
Chieu hudng ciia ca hai dd thi deu cd % do hap thu giam dan theo miic tang cua ndng dd chat chuan. Dieu nay la phii hgp vdi nguyen ly ciia phuang phap ELISA da neu tren. Hai dd thi
DO AN TH! KHANG - lyng dung ky thudt ELISA di xdc dinh ...
nay dugc diing de xac dinh ham lugng cua Clenbuterol va Salbutamol trong thirc an chan nudi.
Ket qua thu hoi cua cac chat chuan bo sung
Dua vao mau trang cac chit Clenbuterol hoac Salbutamol cac nong do xac dinh (1,2,3,4) ppb . Tien hanh ky thuat ELISA da dinh lugng dugc cac chit Clenbuterol hoac Salbutamol ciia moi gieng tuong img vdi cac ndng do bd sung (Bangl va Bang 2)
Bang 1. Kdt qua thu hdi chit chuin Clenbuterol dugc them vao mau tring Ndng do Ket qua thu dugc Thu hdi cua chuin
Clenbuteri n (ppb) (%) R.S.D (%) (ppb) Mean SE Mean SE
1 2 3 4
Bang 2.
Ndng do Salbutamol (ppb)
1 . 2
3 4
10 10 10 10 Ket qua
n 10 10 10 10
0,973 1,81 2,79 3,75
0,0067 0,0155 0,0644 0,041
97,3 90,05 93,00 93,75
0,67 0,78 1,16 1,03
thu hdi chat chuan Salbutamol dugc them vao mau Ket qua thu dugc
Mean 0,92
1,87 2,75 3,71
(ppb) SE 0,0092
0,015 0,027 0,046
Thu hdi ciia (%) Mean
92,3 93,4 91,67 92,75
Ichuan SE 0,92 0,75 0,85 1,14
2,25 2,72 4„57 3,46 trang
R.S.D (%) 3,15 2,53 2,94 3,89
Cac ket qua Bang 1 va Bang 2 cho thay, hieu suit thu hoi ciia chit Clenbuterol va Salbutamol d cac mirc them vao mau tring tir 1-4 ppb da dat kdt qua tren 90% (90,05 - 97,3%)va do lech chuan (R.S.D) cung nhd hon 5%. Nhu vay, do chinh xac cua phuong phap la kha cao va cho phep cac phan tich tiep theo.
Ket qua djnh lugng Clenbuterol va Salbutamol trong thirc an chan nuoi bang phuong ph^p ELISA tai Vien Chan nuoi.
Bang 3. Ham lugng Clenbuterol va Salbutamol trong mau phan tich (ppb)
TT Ten mau n Clenbuterol Salbutamol 1 Thirc an hdn hgp cho lgn 1
2 Thirc an hdn hgp cho Ign 2 3 Thirc an hdn hgp cho ga 3 4 Thirc an hdn hgp cho Ign 3 5 Thirc an ngau nhien(l 16 mau )
10 10 10 10 10
Mean 56,35 24,42 3.074
- 0
SE 0,69 0,56 0.042
- -
Mean - - - 4,004
0
SE - - - 0,049
-
VIEN CHAN NUOI - Tgp chi Khoa hgc Cdng nghi Chdn nudi - S6 12-Thang 6-2008
Trong tdng sd 120 mau thiic an chan nudi dugc lay ngau nhien de phan tich ELISA da dinh lugng dugc Clenbuterol va Salbutamol (Bang 3) nhung chi tim thay Salbutamol Uong mdt mau thirc an chan nudi cho lgn 3 la 4,004 ppb. Cac tri sd thu dugc ciia Clenbuterol trong thiic an cho lgn d mau 1 va miu 2 tuong iing la 56,35 ppb va 24,42 ppb va Uong thiic an chan nudi cho ga 3 la 3,074 ppb. • .. , . .
So sanh ket qua phan tich giua 2 phugng phap ELISA va GC/MS.
De khang dinh do chinh xac cua phuang phap phan tich cac chat Beta-Agonist, chiing tdi fien hanh phan tich dinh lugng cac chit nay bang ky thuat ELISA tai Vien Chan nudi ddng thai ciing nhiing mau thirc an da xir ly dugc giri phan tich bang phuong phap sac ky khdi phd (GC- MS) tai Malaysia. Kdt qua ghi d Bang 4.
Bang 4: Ham lugng Clenbterol va Salbutamol phan tich bdi ELISA hoac GC- MS.
Ten mau
TAHH lgn 4 TAHH ga 3 TAHH lgn 3
n
10 10 10
ELISA Vien Chan Clenbuterol Mean SE
0,00
3,074 0,042 (ppb)
nudi VN Salbutamol Mean SE
0,00
4,004 0,049
GC-MS (ppb) Malaysia Clenbuterol
Mean SE 0,00
5,978 0,059
Salbutamol Mean SE
0,00
6,524 0,064 Bang 4 chi rd, vdi mau TAHH cho lgn 4, ca hai phuang phap deu khdng phat hien thay Clenbuterol va Salbutamol, trong khi dd d mau TAHH cho ga 3, Clenbuterol dugc phan tich va cd kdt qua dinh lugng la 3,074 ppb ddi vdi ELISA va 5,978 ddi vdi GC- MS.
Mau TAHH lgn 3 dugc dinh lugng Salbutamol vdi cac tri sd 4,004 ppb (ELISA) va 6,2524 ppb (GC-MS). So sanh ket qua dinh lugng cac chat nghien ciiu bang hai phuong phap khac nhau thay rang: Phuang phap GC-MS tai Malaysia cho ket qua gan gap 2Ian vdi Clenbuterol (5,978 so vdi 3,074 ppb) va gap 1,6 lan cho Salbutamol (6,524 so vdi 4,004 ppb) so vdi phuong phap ELISA trien khai tai Vien Chan nudi - Viet Nam.
Ket qua nay ggi len mdt suy nghi ve sir dung ELISA de phan tich cac chat Beta-Agonits.
Nen chang chi diing phuong phap ELISA de dinh tinh cac chat Clenbuterol va Salbutamol trong thiic an chan nudi.
KET L U A N VA DE NGHI Ket luan
Vdi bd Kit chuan ciia hang BIOO SCIENTIFIC- My da dung dugc hai dudng chuan cho hai chat Clenbuterol va Salbutamol dap iing dugc cac thdng sd ky thuat cua hang.
Phan tich bang ky thuat ELISA cho thay hieu suat thu hdi Clenbuterol va Salbutamol bo sung vao mau tring la 90,05-97,3% vdi do lech chuin la 2,25 - 3,89 %.
Ham lugng Clenbuterol trong thiic an hdn hgp cho lgn chgn phan tich bing ky thuat ELISA tuong img la 24,42± 0,56 va 56,35±0,69 ppb va trong thiic an hdn hgp cho ga la 3,074 ppb.
Ham lugng Salbutamol trong thiic an hdn hgp lgn 3 phan tich bang ELISA la 4,004 ppb.
Vdi ciing mau va ciing each xir ly ban dau, hai phuang phap ELISA (Phan tich tai Vien Chan
DOAN THI KHANG - &ng dung ky thudt ELISA dixdc dinh
ve dinh tinh va khac nhau ve dinh lugng Clenbuterol va Salbutamol. Phuong phap GC-MS cho ket qua gap 1,6-1,9 lan so vdi ELISA.
De nghj
Sir dung ky thuat ELISA de dinh tinh Clenbuterol va Salbtamol trong thiic an chan nudi d Viet Nam.
TAI LIEU THAM KHAO
Eshq. S.C., Chai.S., Jamokha., G.Aznar and M.K. Hoffman, (2003). Determinationof ractopamin in animal tissue by liquid chromatography-fluorescence and liquid chromatography/tandem mass spectrometry, Anal.Chim, Acta 483 (2003), pp. 137-145,
Hassnoot,W„ P.Stouten and A,M,Lommen, (1994), Determination of fenoterol and ractopamin in urine by enzyme immunoassay. Analyst 119 (1994), pp. 2675-2680.
He L.M, Y.J.Su and H.J.Zhang, (2005). Detection of clenbuterol in animal tissues and feeds by cappilary gas chromatography- mass spectrometry, J.Chin.Mass Spectrom.Sos.26 (2005), pp, 168-172,
Yu H,X and S.M. Yang, (2004) Advance in detremination of ractopamin residues in animal tissues end excrements, Chin.J.Vet.Drug 38 (2004), pp. 44- 47.
Skeritt.,(1998). Approate analytical Technologies for Monitoring Agrochemical Residues.In Seeking Agricultural Produce Fee of Pesticide Residues. Intematioanal Workshop held in Yogyakarta, Indonesia, 17-19 February 1998. ACIAR Proceeding No.85. ISBN 1 86320 2277, Canberra. Australia.
*Ngu-6i ph^n bifn: GS, TS. Le Van Lien; TS. Trin Xuan Hoan
