VIEN C H A N N U O I - Tgp chi Khoa hgc Cdng nghe Chdn nudi - 36 9 - Thang 12-2007
U N G
DUNG PHUONG
PHAP X A C DINH H A M L U O N G AFLATOXIN(B„ Bj, G„ G^) TRONG NGUYEN LIEU vA THLfC AN
C H A NNUOI BANG
M A YSAC KY LONG
CAO AP (HPLC)
Doan Thi Khang", Phan Thanh Dgm, Ddo Dice Hao vd Dicang Thu Anh
Bg mdn Phan tich thurc an va san pham chan nudi, Vien Chan nudi
*Tac gia dd lien he: TS. Doan Thi Khang, Trudng Bd mdn phan tich thiic an & san phdm chan nudi, Vien Chan nudi
DT: (04) 8 385941/0912 008346 ABSTRACT^
Using HPLC for determination of aflatoxin (B,, B,, G,, G,)
The method used is this study was AOAC (2000). After using colum Mycosep 226 aflazon for cleaning aflatoxin (B,, Bj, G,, Gj) in the samples, the samples was put into HPLC for operation.
It showed that: RSD of calibration was acceptable (< 2%) and R^ (determinant co-fficient) between standard samples and analysed sample was high (0.999). It meant that our laboratory can determine aflatoxin (B,, Bj, G,, G2) with an acceptable accuracy. Use of AOAC method, we can determine the concentration of aflatoxin in a range from 0.3 to 20 ng/g sample.
Key words: HPLC, materials, animal feeds
DAT VAN DE
Aflatoxin la san phdm tir loai nam dgc cd ten la Aspergillus flavus va Aspergillus parasiticus, nhirng loai nam mdc cd kha nang sinh dgc nay tdn tai, phat trien trong hat ngd.
dau. lac. hat gdn... Didu kien tdt nhdt cho loai ndm mdc phat trien d trong kho la 25-32°C va do dm tren 85%. Vdi ham lugng aflatoxin 100-400 pg/kg (ppb) da cd kha nang gay benh cho cac loai vat nudi nhu cdc benh vd gan. vdi cac con vat nudi nhd thi rat nhay vdi aflatoxin, dac biet la thuy cdm. Chi vdi ndng do aflatoxin >400 pg/kg (ppb) thi da cd the gay chet vat nudi (Van Egmond va cs., 1995).
O Viet Nam vdi khi hau ndng. dm la dieu kien tdt cho ndm mdc phat trien tren cac loai ndng san. thyc phdm. Nd la mgt nguyen nhan ldn gay giam siic canh tranh cua nganh chan nudi trong nude. Vi vay chiing tdi tien hanh nghien ciiu phucmg phap xdc dinh ddc td nam mdc aflatoxin B,. Bj, G,. Gj bang may sdc ky long cao ap (HPLC) dd dam bao chgn lgc dugc cac nguyen lieu sach dap ling xuat. nhap khdu ciing nhu phuc vu san xua't thitc an trong nude.
VAT LIEU vA PHUONG
P H A P NGHIEN CUUO day chiing tdi tie'n hanh xac dinh aflatoxin bdng phuong phap sdc ky long cao dp. vdi
tieu chudn AOAC (AOAC-chapter 19. P.3-5-2000) (Mary W. Truckses. 1990). Dya vao tinh
chat phan bd cua cac dgc chat trong pha dgng va pha tinh khac nhau. chiing cd sy tucmg tac
khac nhau do dd ma cac chat khac nhau se tach ra theo thdi gian khac nhau trong cot tach. Sau
dd aflatoxin dugc phat hien nhd ddu dd huynh quang. gidi ban phat hien cua phucmg phdp la
0,3 ng/g cho mdi aflatoxin B1,B2,G1.G2. Diing dudng chudn cua timg loai aflatoxin ma ta tinh
dugc ndng do cua chiing. phuong phap ddi hdi phai sach mdu de chay tren may sdc ky long.
D O A N TH] KHANG - ifng dung phuang phdp xdc dinh hdm lugng aflatoxin...
Th6ng thudng ngudi ta diing cdt sdc ky ai lye midn dich dd lam sach mdu nghia la cho mdu qua cdt lam sach, cdt cd dac tinh gitir lai aflatoxin trong cdt, sau dd diing accetontril rira phan giai chat aflatoxin ra (Leo M.E NoUet, 1992). Phuomg phap nay ddi hdi lugng hoa chat rira giai ldn tir 30 den 50ml va lam khd mdu bang khi nitg. Diing mdu cho chay tren may dd xac dinh ham lugng aflatoxin.
0 day chiing tdi sir dung cdt lam sach Mycosep # 226 columns cd tdc dung ngugc lai nghia la cdt cd tac dyng cho ddc chat (aflatoxin. zearelerone. deoxynivalenol) di qua cdn giir lai tap chat vdi kha nang thu hdi ciia cdt la 98,4% vdi aflatoxin va lugng mdu dem lam khd chi cd 1ml.
Quy trinh phdn tich
Ldc vdi 100ml accetonitril: HjO (9:1)
Lam sach mdu bdng cdt mycosep #226 can 50g mdu Ly tam, la'y dich mdu
(3000 vong/phtit, 10 phut)
Duac dich miu da lam sach Lam kh6 bing nito
Ham lugng Bl,
B2,Gi.G2 Chay uen may HPLC
Hoa tan miu vdi 0.1ml trifluoroacetic-i- 0,9ml H2O:
acetone (9: 1) Thiet bi, hod chdt
Thiet bi
May sdc ky long cao ap; Cdt tach Cj^; Cdt lam sach mycosep #226; Dau dd huynh quang;
May ly tam chan khdng.
Hod chdt
Accetonitril; Methanol; Trifluoroacetic; Acetone Nguyen ly hogt dgng
Xdc dinh ddc cha't Aflatoxin bdng phucmg phdp sdc ky long cao dp vdi tieu chudn AOAC (AOAC-chapter 19. P.3-5-2000).
Dya vao tinh chdt phan bd ciia aflatoxin trong pha dgng va pha tinh khac nhau, chiing cd sy tucmg tac khac nhau do dd ma cac cha't khac nhau se bi tach ra theo thdi gian khac nhau trong cot tach. Sau dd aflatoxin dugc phat hien nhd ddu dd huynh quang, gidi ban phat hien ciia phuong phap la 0.3 ng/g cho mdi aflatoxin. Dirng dudng chudn ciia timg loai aflatoxin ma ta tinh dugc ndng do ciia chiing. phucmg phap ddi hdi phai lam sach mdu dd chay tren may sdc ky long. 6 day chiing tdi sir dung cdt lam sach Mycosep #226 columns cd tac dung cho dgc chat (aflatoxin. zearelerone. deoxynivalenol) di qua. giir lai tap chdt vdi kha nang thu hdi cua cdt la 98.4% vdi aflatoxin va lugng mdu dem lam khd la 1ml. Hod tan mdu vdi 0.1ml trifluoroacetic + 0.9ml H^O: acetone. Phan tich tren may sdc ky long cao dp vdi
- Cdt tach C18 (250mm. 4.6mm, 5mm) - Tdc dd ddng 0.8ml/phut
- Pha ddng H^O: Methanol (3: 2)
VIEN CH AN NUOI - Tgp chi Khoa hgc Cdng nghi Chdn nudi - 3d 9 - Thang 12-2007 - Ddu dd huynh quang (1 ex = 365,1 cm =450nm)
- Lugng bgm mdu 20ml - Nhiet do cdt tach 40°C
* Ddi tugng xdc dinh ddc chat la cdc loai nguyen lieu va thiic an chan nudi - Khd ddu dau tuong, khd dau lac
- Cd khd, phu pham ndng nghiep - Cac loai thiic an cho gia siic, gia cdm
KET QUA VA THAO LUAN Sdc do Aflatoxin
R K S i S
f^ vr o a r-
Hinh 1- Sdc dd aflatoxin chudn dugc chay tren may
Qua sdc dd cua aflatoxin chudn cho tha'y kha nang tach cac aflatoxin la rat tdt. Chiing t6i tie'n hanh chay 5 ldn cho thdy do lech chudn tucmg ddi (RSD) ciia thdi gian luu < 2% va ciia dien tich peak la <5%.
A f i a G2 ; . V j ' '
' " I " " i " " i " " I " " I ' " ' I ' ' i " " i " " i " " i 1 1 1 1 1 1 1 [ I 1 1 1 1 1 I I n > 1 1 1 1 1 1 1 1 1 1 1 I n 1 1 1 n I I 11 1 1 1 1 1 1 1 1 1 1 ; I n 1 1 1 1 1 1 1 , < n t ' * * ia ( R jh • , » • . .
Hinh 2- Sdc dd aflatoxin chudn d cac ndng d6 khac nhau 5; 10; 20ppb
Tie'n hanh xay dyng dudng chudn d cac ndng do 5; 10; 20ppb dd lap dudng chudn. Qua dd tha'y rdng do tuyen tinh cac ndng do khac nhau ciia cac aflatoxin la rdt tdt R deu dat 0,999.
Ke't qua nay cho thdy rdng sy dn dinh ciia may HPLC la rdt cao.
D O A N THj KHANG - U'ng dung phuang phdp xdc dinh hdm lugng aflatoxin...
R-Squared = 0.998366 R-Squared = 0.999750
r
!
1
i
r' i
/
/ /
A m
/ /
0.0 5.0 : 0 . 0 I S O 20.0 25.0 30.c A d i ^ t W d A n ^
O.C S.O 10.D 15.C 20.0 ;?.D ID.
R-Squared =0.998036
4D0OO
3000D
20000
/ l
0.0 ; . 3 «.( 6.9 9.1 11 A d j i j t f d A m i
R-Squared = 0.998965
y
,y
y'
0-0 ; , 3 4 ft • » * . 1 A<juttodAn<
Hinh 3- Sdc d6 aflatoxin chuin 6 cac n6ng d6 khac nhau
Chung toi da tien hanh xir ly mSu tren cac loai nguyen lieu, thuc an chan nuoi va cho ket qua tot: Kha nang tach peak tot va hau nhu khong co peak la
H A T J K . H O D O
A
'__A
• ' T ' ' " T " " I ' ..,....,....,T....T....,....|....|..-M""I .""'"'V,'
v i n IT a O
' • " " • " J / 'J J :v
yv.
r; \'i '.^;"•|.M.M .^|. ; MVIEN C H A N NUOI - Tgp chi Khoa hgc Cdng nghe Chdn nudi - 36' 9 - Thang 12-2007
iHtrc kv nor not CHO LOF
Bot sin
13.01 12.01
ii.o;
io.o;
9.0J e.orj 7.0l B.oi
AflaB,
Afla G',
4 6 B 10 12 14 16 18
Kh6 ddu lac
13.01 12.0l 11.0J 10.01 9.0J 6.01 7.01
6.aJ
I
/ / /
] 2
AflaB,
C^,_A 1
4 6
jl
\ V
8 IJ A f l o
]i 11
I ' l l ' 1 " "
15
| | " l " " l " "
1?
Hinh 4- Sdc do aflatoxin trong cac loai nguyen Ueu thtic an chan nu6i
Chung tdi da tie'n hanh kiem tra do thu hdi ciia tat ca qua trinh phan tich (xac dinh do sai
sd he thdng ciia phuong phdp) chiing tdi tien hanh chay mdu chudn (mdu them mdt lugng
chudn da biet 2,5ppb) vdi 5 ldn lap lai. Tir dd tinh dugc do lech trung binh so vdi gia tri thyc te
la ± 10% cho ke't qua tdt nhu phuong phdp dua ra la do thu hdi tir 90,6 den 100%. Kcl qua nay
hoan toan phu hgfp vdi TCVN (TCVN 6910; 1: 2001) (2001).
O O A N THI KHANG - Ifng dung phuang phdp xdc dinh lidm lugng aflatoxin...
Bang 1: Kiem tra do thu hdi khi them 2,5ppb ciia m6i loai aflatoxin vao nguyen lieu
Ten miuMau ng6 Mdu ng6
Mau ngo + 2,5 ppb Mau ng5 -(- 2,5 ppb Miu ngo -(- 2,5 ppb Miu ngo -1- 2,5 ppb MJu ngo -t- 2,5 ppb
Bang
Ten miu Ngo mdc Ngo Ha Tay Ngo luu kho Ngo Ha Nam Ng6 hat Ha Tay NgO hat mdi thu Ngo hat Tram TN Ng6 Cty Techna Ng6 Bdc Giang Ngo TT Ha Tay Ngo Cty ADO Ngo hat Cty ADO Ngo hat Cty Techna Ngd hat Ha Nam Ng6 hat Tram TNTen mdu Kh6 d6 IV2.2 Kh6d6lV1.2 Kh6d6 11.1.2 Kh6d6lII.2.2 Khdd6V.1.2 Khd diu lac Kh6d6 1963/05 Kh6d6 111.1.2
Aflatoxin G, 0 0 2.3691 2.3731 2.3573 2.3456 2.4157
Loai doc chat aflatoxin |a.g/kg (ppb) Aflatoxin B,
5.8452 5.7845 8.1454 8.1107 8.1985 8.1756 8.0896
Aflatoxin G2 0 0 2.6879 2.7025 2.6316 2.6948 2.7152 2: Kiem tra dgc td aflatoxin trong mgt sd mdu ngd (ppb)
Aflatoxin B, 113.691
2.0857 119.6204
19.9089 0 0 0 17.5498
2.6583 23.3998
4.7017 2.3215
0 0.3887
0
Ten doc cha't (ppb) Aflatoxin Bj
11.8674 0 7.2342
1.1949 0 0 0 0.8897
0 1.7714
0 0 0 0 0
Bang 3: Thanh phdn dgc chat trong k Aflatoxin B,
2.2511 2.9477
0 0 0 71.5270
0 0
Aflatoxin B2 0 0.7843
0 0 0 12.0177
0 0
Aflatoxin G, 0 0 0 0 0 0 0 1.6903
0 0 0 0 0.2357
0 0 ho d6(ppb)
Aflatoxin G, 0 0 0 0 0 0 0 0
Aflatoxin Bj 2.7451 2.8526 5.0868 5.1577 5.0782 5.2842 5.2017
Aflatoxin G2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Aflatoxin Gj 0 0 0 0 0 0 0 0
VIEN CHAN NUOI - Tgp chi Khoa hgc Cdng nghi Chdn nudi - 86' 9 - Thang 12-2007
Bang 4: Thanh phdn ddc chat trong thiic an chan nudi {Dan vi: ppb)
Ten miuCam h6n hgp C662 Cam sieu dam dac Cam hh lgn thit
T A cho ga de HH lgn thit T A tinh 714 TA tinh 785 Cam hh 185/nc Cam hh H225
T A cho da dieu 559/nc T A cho da di^u 56Inc
T A cho ca
Thiic an gia siic 23/6 Thiic an gia siic 43/12 T A chan nuoi
T A vien Ml T A vien M2 CamTPl T A nuOi chim
T A cho da dieu 560nc
T A hh lgn 888
T A hh lgn 808 TA hh lgn 800 TAhhlgn 151S
T A D G S
Aflatoxin B, 37.3161 15.1783 13.2508 23.4333
0 11.5310
8.5409 31.3321 23.7780 16.2412 20.3268
0 3.7909
5.935 129.1855
11.9982 5.0049 5.0859
0 15.935
0 0 .0 0 I.1660
Aflatoxin Bj 2.8405 6.3836
0 1.9762
0 0 3.0480 2.6799 2.1036 3.0576
0 0 0 0 13.3154
0 0 0 0 1.941
0 0 0 0 0
Aflatoxin G, 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2.2352
0 0 0 0 0 0 0 0 0
Aflatoxin G2 0 0 0 0 0 0 0 0 0 0 0 1.2436
0 0 0 0 0 0 0 0 0 0 0 0 0
KET LUAN
Da xay dung dugc quy trinh tdi uu cho phuong phdp xac dinh ddc chat aflatoxin B,, B^, G|, Gj trong cac loai nguyen lieu va thiic an chan nudi.
Phdng Phan tich da phan tich dugc thanh phdn ddc chat aflatoxin B,, B2, G,, Gj trong cac loai nguyen lieu va thiic an chan nudi
Cac thdng sd ky thuat, do chinh xdc deu phii hgp vdi phucmg phap phan tich va hang san xudt.
T A I LIEU THAM K H A O Leo M.L Nollet. 1992. Food analysis by HPLC
Mary W. Truckses. 1990. AOAC Methods (17* Version) 2000 chapter 19,.P: 3 - 5
TCVN 6910:1; 2001 (ISO: 5725-1:1994). 2001. D6 chinh xac ciia phuang phap do va kei qua do
Van Egmond H.P, Heisterkamp S.H va Paulsh W.E.E.C. 1995. Xac dinh aflatoxin Bl trong thuc an chan nu6i, phu phdm va cac chat nhiSm ban. /.
