TIU CHUN VIT NAM TCVN 4804:1989

THC N CHN NUI PHNG PHP XC NH AFLATOXIN Animal feeding stuffs Method for determination of aflatoxin Tiu chun ny ph hp vi ST SEV 4318-83, p dng cho thc n chn nui: thc n hn hp, thc n chn nui: dng ht, cm, lc (arachis), thc n th cng nh kh du lc, kh du ci, kh du bng, kh du u tng, kh du trch ly v qui nh phng php xc nh hm lng aflatoxin (c t vi nm ) B1, B2, G1 v G2. 1. Bn cht ca phng php Phng php da trn s tch chit aflatoxin t mu th bng cloroform, ra phn chit qua ct sc k, sau xc nh hm lng cc aflatoxin (c t vi nm) ring l trn sc k bn mng (SKBM) trn c s nh gi bng mt hay bng mt hunh quang k- (Densitomet), kch thc v cng cc vt pht quang v tin hnh th nghim ho hc xc nhn trn sc k bn mng (SKBM). 2. Cc vn chung 2.1. tin hnh th nu khng c cc ch dn khc dng thuc th tinh khit phn tch (TKPT) v nc ct hay nc c sch tng ng. 2.2. Hm lng ti thiu v phng php c th pht hin l 1g/kg. 3. Thit b 3.1. Thit b cho SKBM : dng c to lp cht hp ph mng. Bung khai trin trong c t mng thu tinh, micro-pipet, dung tch 1, 2, 5 v 10mm3 hay microsranh dung tch 10mm3 cc tm thu tinh kch thc (c) 20 x 20 cm. 3.2. Ct sc k thu tinh c van mi ng knh trong 2,2 cm v di 40 cm. 3.3. Thit b bc hi chm khung quay loi rto hay loi Cudex- Danhit. 3.4. Ngun bc x t ngoi (cc tm) (OF) c bc sng 365 mm. 3.5. Mt hunh quang k c b lc sng m bo pht x t ngoi bc sng 365 mm c b lc ln hai bc sng 336 mm. 3.6. My lc. 3.7. Ph quang k m bo o trong vng ph t ngoi t 300 n 400 nm. 3.8. T sy m bo iu chnh nhit n 2000C. 3.9. Bnh kh nit hay kh tr khc. 3.10. Bp cch thu hay cch ct m bo iu chnh nhit n 1000C. 3.11. Cn phn tch vi sai s php cn khng ln hn 0,0001g. 3.12. Cn k thut vi sai s php cn khng ln hn 0,1g. 3.13. My nghin phng th nghim (my tn) m bo mn ca bt khng qu 1mm. 3.14. Bm ht bng ng nc. 3.15. Phu Bunhe (Buchner) ng knh 10 cm. 3.16. Phu thu tinh .

3.17. Bnh Eclenmaye (dung tch 500 cm3). 3.18. Bnh nn nt mi dung tch 250, 400 v 750 cm3. 3.19. Bnh nh mc dung tch 10 cm3. 3.20. ng o (hnh tr) dung tch 50, 100 v 250 cm3. 3.21. Pipt nh mc (Kor) dung tch 1, 2 v 5 cm3. 3.22. ng thu tinh nt mi dung tch 10 cm3. 3.23. Vi phun thu tinh. 3.24. Sng (li) ng knh l 1 mm. 3.25. a thu tinh. 4. Thuc th, dung dch v vt liu 4.1. Axeton 4.2. Metyl xyanua (Acetonitryl) 4.3. Benzen 4.4. St clorua 4.5. Axit focmic 85-90% 4.6. Axit triflo axetic 4.7. Axit axetic bng 4.8. Cacbonat ng - hyroxit ng CuCO3, Cu(OH)2 4.9. Natri sunfat khan 4.10. Ch axetataxit 4.11. Ru n.amylic bc 1 4.12. Ru metylic 4.13. Ru etylic 4.14. Toluen 4.15. Clorofoc 4.16. Ete dietyl mt nc khng cha peroxit 4.17. Ete petrol c nhit 30-350C hay haxan tch t du ho 4.18. Triclo etylen 4.19. Amoni sunfat dung dch 40% 4.20. Axetenitrin, dung dch 2% trong benzen 4.21. Kali hyroxit, dung dch cha (KOH) = 0,02 mol/dm3 4.22. Natri hyroxit,dung dch cha (NaOH) = 0,2 mol/dm3 4.23. Axit nitric, dung dch cha HNO3- = 2 mol/dm3 4.24. Ru metylic, dung dch 3% trong clorofoc 4.25. Axit sunfuric 0,05 v dung dch 25% 4.26. Aflatoxin (c t vi nm) dung dch 4.26.1. Dung dch gc chun b nh sau :

Dung dch I - cho 1 mg aflatoxin vo bnh nh mc dung tch 10 cm3 ho tan trong hn hp benzen - axetonitrin theo t l th tch v 98+ 2 v nh mc n vch. Nng dung dch phi l 0,1g/cm3. Dung dch II - chuyn 1 cm3 dung dch I vo bnh nh mc dung tch 10 cm3 v dng hn hp benzen axetonitrin nh mc n vch. Nng dung dch phi l 10g/cm3. i vi mi cht c aflatoxin dung dch gc cn chun b ring v bo qun trong bnh kn ch ti nhit gn 00C. 4.26.2. Dung dch hn hp cc cht aflatoxin chun b nh sau : ly 1cm3dung dch gc II ca cc aflatoxin B1v G1v 0,5cm3dung dch gc II cc aflatoxin B2v G2 vo bnh nh mc dung tch 10 cm3 v nh mc n vch bng hn hp benzen - axetonitrin. Nng cc aflatoxin trong dung dch tiu chun phi l g/ cm3 i vi B1 v G1 v 0,5 g/ cm3 i vi B2v G2. 4.26.3. Xc nh nng aflatoxin trong dung dch II bng phng php quang ph bng cch o s hp th trong ph t ngoi (UF) t 330 n 370 mm i vi hn hp benzen - axetonitrin. Nng aflatoxin (C) tnh ra microgam trn cm3, theo cng thc :

Trong : A - Hp th bc sng 350 nm mc - Phn t lng, c th i vi cc aflatoxin B1 - 312, B2 - 314, G1 - 328, G2 - 330. - H s hp th phn t c th i vi cc aflatoxin B1 - 19.800, B2 - 20.900, G1 - 17.100, G2 - 18.200. k - H s hiu chnh quang ph k s dng, nm trong khong t 0,95 n 1,05. 4.27. Hn hp cc h dung mi trin khai tch trit cho SKBM (cc pha di ng). 4.27.1. Hn hp clorofoc - triclo etylen ru n-emylic axit foemic theo t l th tch l 80 + 15 + 4 + 1. 4.27.2. Hn hp clorofoc - axeton t l th tch 90 + 10. 4.27.3. Hn hp Toluen - etyl - axetic - axit formic theo t l th tch 60 + 30 + 10. 4.28. Silicagen - H theo Stan dng cho sc k bn mng (SKBM) hay Adsorbosil - 1 hay loi khc c tnh cht tng ng. 4.29. Cc tiu chun dng cho SKBM, chun b nh sau : Ra cc tm knh trong dung dch cht kh bn, trng bng tia nc v trng k bng nc ct, sy kh v ra bng ru etylic. Cho 70g silicagen - H hay 50g Adsorbosil - 1 vo bnh nn, vo 60 cm3 nc ct v trn k; chuyn va ny vo dng c to lp cht hp ph mng v ln cc tm c dy 0,25mm (khi lng cht hp th nu chun b 10 tm c kch thc 20 x 20 cm); sy cc tm nhit phng sau cho vo t sy, tng dn nhit n 1100C v nhit ny trong 2h. Ngng sy, lm ngui nhit phng v cho vo bnh ht m. Nhng tm chun ny bo qun trong bnh ht m n 4 tun. 4.30. Silicagen vin (ht) c ng knh ht 0,05 - 0,2 mm dng cho ct sc k, c nhit 1050C trong 1 gi. Sau khi lm ngui cho nc ct theo 1% khi lng silicagen v lc trong 2 gi. Bo qun bnh y kn. 4.31. Giy lc loi nhanh .

4.32. Bng ty m bng te du ho hay bng thy tinh. 4.33. iatomit ( to ct) mc xelit - 545 hay Hyflosuper - cel hay loi khc c tnh cht tng t. 5. Chun b th 5.1. Tch cht aflatoxin t thc n chn nui Nghin mu phn tch ca thc n chn nui bng my nghin, sng qua sng v trn u. Cn 50g vi sai s cho php cn khng ln hn 0,1g v cho vo bnh nn nt mi dung tch 750 cm3, cho thm 25% diatomit, vo 25 cm3 nc, khuy k bng a thu tinh, sau cho thm vo 250 cm3 clorofoc. y kn bnh v t ln thit b, lc. Lc trong 30 pht, lc phn chit (cht chit) qua giy lc cha (5 0,1g) natri sunfat khan. Thu 50 cm3 phn lc du. 5.2. Chun b ct sc k Chun b ct ra phn lc nh sau : Cho vo y ct nt bng ty m ri cho vo 5g natri sunfat khan. clorofoc n na chiu cao ca ct, cho vo 10g silicagen chun b theo mc 4.30, v trng thnh ct bng 20 cm 3 clorofoc. Trn gel (keo) bng a thu tinh sao cho khng ph v lp sunfat. Sau khi cht ht lng (kt ta) hon ton, ph gel bng 15g natri sunfat khan v clorofoc n mc cht ht. 5.3. Lm (ra) sch phn lc trn ct sc k 50 cm3 phn lc theo a thu tinh hay theo thnh vo ct chun b nh trn, phn ny cha tng ng 10g mu phn tch thc n chn nui. M van v tho dung mi. Gii hp (ra gii) hp ph trn gel ln lt bng 150 cm3 hexan v 150 cm3 te dietylic khan. B nc gii hp (eluent).Tch aflatoxin bng 150 cm3 dung dch ru metylic trong clorofoc. Lm bc hi dung mi tch (eluent) trong thit b bc hi chn khng n khi cn c th tch 0,5 cm 3. Sau , dng 3 lng tng 2 cm3 clorofoc chuyn nh lng cn vo cc ng khc vch c nt mi v lm bc hi n khi bng dng kh nit. Ho tan phn chit c lm sch trong 0,5 cm 3 dung dch axeton nitrin trong benzen v bo qun dung dch nhn c trong t lnh n khi tin hnh th. 5.4. Chuyn dung dch v dung dch th ln cc tm sc k bn mng Co sch lp cht ht rng 0,5 cm cc cnh thng ng ca cc tm. nh du cc cnh thng ng bng cc vch rng 1 - 1,5 cm v dng ng thng ngang gii hn s di ng ca cc pha chuyn ng khong cch 13 cm t ng xut pht. Trn khong cch 2 cm t cnh di tm cho 1,2 v 5 cm3 dung dch hn hp aflatoxin chun b theo mc 4.26.2 v lm 3 ln tng 10 mm3 mi dung dch th. Trn mi vt dung dch th cho 1 v 2 mm 3 dung dch hn hp aflatoxin (chun trong). trnh nhm ln cn nh s cc vt ca dung dch chun v dung dch t pha trn tm. 5.5. Chun b bung khai trin Ngay trc khi tin hnh th mt trong nhng hn hp tch chit chun b theo mc 4.27 vo bung (bung chia bo ho) vi lng m bo phn chia sc k. 5.6. Ra ph (lm sch thm) phn chit Nu dung dch th cha cc tp cht cn tr xc nh aflatoxin cn tin hnh lm sch thm cn kh bng mt trong nhng phng php nu di y : 5.6.1. Ho tan cn kh trong 50 cm 3 axeton, cho thm 20 cm3 dung dch amonisunfat v 130 cm3 nc; trn v lng 2 -5 pht sau cho thm 10g xelit - 545, trn v lc hn hp qua giy lc cun (gp) sng. Thu 120 cm3 phn lc tng ng vi 6g thc n chn nui chuyn vo phu tch dung tch 250 cm3, cho thm 10 cm3 benzen v lc trong 2 pht. Sau khi phn lp, tch b pha nc pha di, cn trn pha benzen vi 50 cm3 nc v li lc phu. Sau li tch b pha nc, cn lc benzen qua lp natrisunfat khan c ra bng 5 cm3 benzen. Gp cc phn lc v lm bay hi bng dng kh nit v ho tan cn kh trong 0,5

cm3 dung dch axeton nitrin trong benzen. Bo qun dung dch th lm sch trong t lnh n khi tin hnh th. 5.6.2. Ho tan phn chit c lm sch trong 50 cm3 hn hp nc axetonitrin (5+45) cho thm 25 cm3 dung dch ch axetat v 2g xelit- 545. Chun b dung dch ch axetat nh sau : ho tan 200g ch axetaaxit Pb (CH3COO)2. 3H2O trong 100 cm3 nc v un nng trn bp cch thu n khi tan cho thm 3 cm3 axit axetic bng v nc n th tch 1 dm 3 ; lc phn chit c lm sch qua giy lc gp sng vo phu tch (chit) dung tch 250 cm 3 v ra phin lc bng 50 cm3 nc; chit 3 ln dung tch gp bng lng tng 50 cm3 clorofoc, gp cc pha di, sy qua lp natri sunfat trn phin lc v lm bc hi trong thit b bc hi chn khng nhit 450C. Dng 3 lung mi lng 2 cm 3 clorofoc chuyn dch lng cn kh vo ng nghim c nt mi, lm bc hi dung mi bng dng kh nit, cn cn th ho tan trong 0,5 cm3 dung dch axetonitrin trong benzen v bo qun trong t lnh n khi tin hnh th. 5.6.3. Ho tan cn kh trong 150 cm 3 axeton nc (85 +15 ). Cho vo cc dung tch 500 cm 3 ln lt 170 cm3 natri hyroxit v dung dch mi chun b gm 20g st clorua trong 300 cm3 nc. Khuy k hn hp. Cho vo dung dch phn chit trong axeton nc 3g ng cacbonat baz v chuyn vo cc cha hn hp st clorua v natri hyrxt. Cho thm vo 100g xelit- 545 v khuy k. Lc lng cha trong cc qua phu lc Bunhe (Buchner) v chuyn 150 cm3 phn lc cha 4,3g thc n chn nui vo phu tch dung tch 500 cm 3, cho thm 150 cm3 0,03% dung dch axit sunfuric v 10 cm3 cloruafoc. Lc phu trong 3 pht. Chuyn lp clorofoc bn di vo phu tch sch dung dch 250 cm3, cho thm 100 cm3 dung dch kali hyrxit v lc trong 30 giy. Sy clorofoc bng natri sunfat khan, lm bc hi trong dng kh nit v ho tan cn kh trong 0,5 cm3 dung dch axetonitrin 2% trong benzen. Bo qun dung dch trong t lnh n khi tin hnh th. 6. Tin hnh th 6.1. Tin hnh sc k bn mng Cc tm cha mu ca dung dch chun v dung dch th c t vo bung sc k v gi cho n khi tuyn (mt, tip gip) ca h tch nng cao n mc cc ng gii hn ngang, sau nhc ra khi bung v sy nhit phng trong ch ti. 6.2. nh gi nh tnh cc tm sc k Xem xt tm sc k trong nh sng t ngoi trong bung ti khong cch 20cm t ngun pht x v xc nh cc vt hunh quang mu xanh ti c i lng tng i ca yu t duy tr (khng ch)(Rf) trong h sc k 4.27.1, 4.27.2 v 4.27.3 i vi aflatoxin B 1 0,40; 0,59 v 0,35 v tng t i vi aflatoxin B2 0,40; 0,43 v 0,28. Cc aflatoxin G1 v G2 cn phi pht quang bng nh sng xanh l cy, i lng Rf ca chng trong cng h sc k trn phi l 0,24; 0,39; 0,23 i vi G1v 0,18; 0,30; 0,19 i vi G2. 6.3. S nhn bit aflatoxin Dng vi phun phun cc tm sc k cha cc hn hp tch chit ca cc dung dch chun v dung dch th, bng dung dch axit sunfuric 25% sy trong lung kh m v xem xt li trong nh sng t ngoi. Nu khng c s thay i nh hunh quang ca cc cht t xanh tm sang xanh l cy vng th c th loi tr kh nng c aflatoxin. Nu c s thay i , c th kt lun s b l c aflatoxin hay cht khc c th tham gia phn ng vi axit sunfuric. Trong trng hp ny cn tin hnh sc k li trong hai h sc k khc theo mc 4.27 v th li bng phng php ho hc. So snh hunh quang ca cc vt bng mt hoc dng mt hunh quang k. tin hnh xc nh mt hunh quang k c th dng cc ph thng cha cc cht giao thoa l trong vng tch aflatoxin. Nu bng mt c th pht hin l c cc cht , cn lm sch thm cn kh ca nc gii hp (eluent) t ct sc k dng mt trong nhng phng php nu mc 5.6.

t tm sc k trn mt hunh quang k. Ghi ph mt hunh quang ca aflatoxin theo hng s dng my. Phn tch nh lng ch tin hnh i vi cc i lng nm trong gii hn ca ch s tuyn tnh trn mt hunh quang k. Kt qu so snh l gi tr trung bnh cng ca khng t hn 3 php o. 6.4. Th nghim xc nhn bng phng php ho hc. 6.4.1. Xc nhn c aflatoxin B1 v G2. Php th da trn s to cht dn xut ca aflatoxin B 1 v G2 vi axit triflo axetat trc tip trn tm sc k. Chia tm sc k lm 2 phn bng nhau. y mt na bng tm knh sch, nh ln na kia hai vt (1 10 mm3) dung dch th cha 0,5 -5g aflatoxin B1 v G1, cng nh 2 v 5 mm3 theo mc 4.26.2. Cho thm vo mt trong cc vt th nghin cu 2 mm3 dung dch aflatoxin (chun ni b). Sau cho thm vo tt c cc vt, mi vt 2 mm3 hn hp mi chun b ca axit triflo axetic vi clorofoc (1+1). Sy tm sc k trong lung hi nng (35-40oC) trong 10 pht sau m phn hai ca tm sc k v cho ln mt khi lng nh trn dung dch aflatoxin theo mc 4.26.2 v dung dch nghin cu (th). Khng cho dung dch axit triflo axetic. Cho vo y bung sc k sch mt mng bng thu tinh hp c nc, dng pipet cho vo phn cn li ca bung gn 50 cm3 h tch chun b theo mc 4.27.2. Cho tm sc k vo sao cho mng c nc nm trc tm sc k. Tin hnh sc k n cao 13cm v xem xt trong nh sng t ngoi vi bc sng 365nm. Nhng aflatoxin khng tham gia phn ng vi axit triflo axetic u phi nm gn ng tuyn ca dung mi . Nhng aflatoxin hunh quang to thnh sau phn ng c nh xanh tm B2a v G2a phi nm pha di tm v c i lng Rf nh hn 4 ln so vi B1v G1. Phun tm knh thm bng dung dch axit sunfuric dung dch ny thay i hunh quang xanh tm ca tt c cc aflatoxin xanh l cy vng. 6.4.2. Xc nhn c aflatoxin G1 v G2. Php th ny c dng khi trn cc tm sc k xut hin cc tp cht giao thoa (nhiu) dch chuyn cng aflatoxin G. Tin hnh sc k tm sc k cha dung dch trong h tch benzen ru etylic - nc (46+35+19). H c chun b trc khi dng 24 gi bng cch lc tt c cc cht trong phu tch sau khi phn lp tch tng pha vo cc bnh ring. Nu hn hp ang chun b b c, cn un nng hn hp. 50 cm3 lp di cng vo bung sc k, cn 50 cm3 lp trn cng vo mng thu tinh ring v t mng vo bung trc tm sc k. Sau khi ly tm sc k cha cc cht tch c ra, sy tm sc k, xem xt trong nh sng t ngoi v xc nh hm lng aflatoxin. 7. X l kt qu 7.1. Khi so snh pht hunh quang ca cc vt bng mt, hm lng aflatoxin trong mu th thc n chn nui (X) tnh bng microgram trn kilogram theo cng thc:

Trong : VW - Th tch dung dch hn hp aflatoxin theo mc 4.26.2 tng ng mu th , mm3 C - Nng aflatoxin trong dung dch hn hp theo mc 4.26.2 g/cm3 Vk - Th tch cui ca dung dch th, mm3 Ve - Th tch dung dch th trn tm sc k, mm3 m - Khi lng mu cha trong th tch phn chit, ly lm sch, g.

7.2. Khi so snh pht hunh quang ca aflatoxin, bng mt k, hm lng aflatoxin trong mu th thc n chn nui tnh bng micogram trn kilogram xc nh bng cch so snh din tch cc nh (pic) theo cng thc :

Trong : Se - Din tch nh (pic) dung dch th C - Nng aflatoxin trong dung dch hn hp aflatoxin theo mc 4.26.2. Vw - Th tch dung dch hn hp aflatoxin cho ln tm sc k, mm3 VK - Th tch cui ca dung dch th, mm3 Sw - Din tch pic aflatoxin Ve - Th tch dung dch th cho ln tm sc k, mm3 m - Khi lng mu cha trong th tch phn chit , ly lm sch, g. 7.3. Kt qu xc nh cui cng l gi tr trung bnh cng ca t nht l 2 php xc nh song song. Chnh lch cho php kt qu cc php xc nh song song khng c qu 20% 8. Bin bn th Bin bn th cn ghi cc s liu sau : 1. Tn thc n chn nui v mc 2. iu kin th 3. Khi lng mu, g 4. Kt qu th 5. K hiu tiu chun SEV ny 6. Thi gian th (ngy th).