B Y T TRNG I HC DC H NI L TH HNG HO KHO ST MC NHIM MYCOTOXIN TRONG NG V LC TI TNH BC GIANG KHA LUN TT NGHIP DC S H NI -2014 B Y T TRNG I HC DC H NI L TH HNG HO KHO ST MC NHIM MYCOTOXIN TRONG NG V LC TI TNH BC GIANG KHA LUN TT NGHIP DC S Ngi hng dn TS. Phm Th Thanh H Ni thc hin: 1. B mn Ha phn tch c cht 2. Vin kim nghim an ton v sinh thc phm Quc gia H NI -2014 LI CM N Vi lng knh trng v bit n su sc, em xin gi li cm n su sc ti TS. PhmThThanhH-BmnHaphntchvccht-TrngihcDc H Ni, ngi tn tnh hng dn, ch bo, gip em trong qu trnh thc hin v hon thnh kha lun. Xin chn thnh cm n ban lnh o v cc ng nghip Vin Kim nghim an ton v sinh thc phm Quc gia, nhng ngi ht lng to iu kin thun li v trc tip gip ti trong qu trnh thc hin ti. Xinchnthnhcmnccthy,cBmnHaPhntchtrngihc Dc H Ni hng dn v gip em trong qu trnh hc tp v nghin cu. XinchnthnhcmnBangimhiunhtrng,cmnccthyc,cn b phng qun l sau i hc, cc phng ban khc ca trng i hc Dc H Ni gip emi trong sut qu trnh hc tp ti trng. Cui cng, em xin cm n gia nh, bn b lun ng vin, khch l em trong hc tp v cuc sng. H Ni, ngy 05 thng 05 nm 2014 Sinh vin L Th Hng Ho MC LC Trang DANH MC CC K HIU, CC CH VIT TT ............................................... 1 DANH MC CC BNG, HNH V V TH ................................................... 3 T VN ............................................................................................................ 1 CHNG 1: TNG QUAN ...................................................................................... 2 1.1. Khi qut v c t vi nm mycotoxin [4] ....................................................... 2 1.1.1. Gii thiu chung v aflatoxin [12] ........................................................... 2 1.1.2. Gii thiu chung v Fumonosin [20] ........................................................ 6 1.2. Hin trng v nhng cng trnh nghin cu c lin quan n ti ................. 8 1.3. Cc phng php xc nh mycotoxin ............................................................ 9 1.3.1. Phng php ELISA (enzyme-linked immunosorbent assay) .................... 9 1.3.2. Phng php sc k bn mng (TLC) ..................................................... 10 1.3.3. Phng php sc k lng hiu nng cao (HPLC) ................................... 11 1.3.4. Phng php sc k lng khi ph ......................................................... 12 1.4. Gii thiu v h thng LC-MS/MS ............................................................... 13 1.4.1. H thng sc k lng hiu nng cao [3] ................................................. 14 1.4.2Khi ph (Mass Spectrometry) [2] ......................................................... 14 1.4.3. ng dng ca sc k lng khi ph ........................................................ 14 CHNG 2:I TNG V PHNG PHP NGHIN CU ......................... 16 2.1. i tng v ni dung nghin cu ................................................................ 16 2.1.1. i tng nghin cu ............................................................................ 16 2.1.2. Ni dung nghin cu .............................................................................. 16 2.2.Trang thit b, ha cht ................................................................................ 16 2.2.1. Thit b, dng c,: .................................................................................. 16 2.2.2. Ha cht, thuc th ................................................................................ 17 2.3.Phng php nghin cu .............................................................................. 18 2.3.1. S lng mu ly: .................................................................................. 18 2.3.2. Phng php kim nghim: .................................................................... 18 CHNG 3: KT QU V THO LUN ............................................................ 26 3.1.Thm nh phng php kim nghim aflatoxin ............................................ 26 3.1.1. Tnh c hiu, tnh chn lc ................................................................... 26 3.1.2. Khong tuyn tnh v lp ng chun .................................................. 27 3.1.3.Gii hn pht hin (LOD), gii hn nh lng (LOQ) ......................... 28 3.1.4. Kho st lp li ca h thng ............................................................ 29 3.1.5. chnh xc ca phng php phn tch ( ng v chm) ........... 30 3.2. Thm nh phng php kim nghim fumonisin B1 .................................... 31 3.2.1. Tnh c hiu/chn lc: .......................................................................... 31 3.2.2. Xc nh khong tuyn tnh .................................................................... 33 3.2.3. Gii hn pht hin (LOD), gii hn nh lng (LOQ) .......................... 33 3.2.4. Kho st lp li ca h thng ............................................................ 34 3.2.5. chnh xc ca phng php phn tch ( ng v chm) ........... 35 3.3. nh gi mc nhim mycotoxin trong ng v lc .................................. 36 3.3.1. Kt qu phn tch mu lc nhn ............................................................. 36 3.3.2. Kt qu phn tch mu lc c ................................................................. 38 3.3.3. Kt qu phn tch mu ng ..................................................................... 40 CHNG IV: BN LUN ..................................................................................... 41 4.1. V Phng php phn tch aflatoxin (B1, B2, G1, G2) v fumonisinB1 bng sc k lng khi ph ............................................................................................ 41 4.2. V kt qu thm nh phng php phn tch ............................................... 42 4.3. V mc nhim cc loi aflatoxin (B1, B2, G1, G2) trong lc, fumonisinB1 trong ng ............................................................................................................. 42 4.4. nh gi cc yu t lin quan nh hng n tnh hnh nhim mycotoxin trong ng v lc ................................................................................................... 43 KT LUN ........................................................................................................ 44 5.1. Thm nh phng php ............................................................................... 44 5.2. Mc nhim Aflatoxin trong lc, fumonisinB1 trong ng: ...................... 44 5.3. xut gii php nhm gim thiu nguy c nhim mycotoxin trong ng, lc ............................................................................................................................ 45 TI LIU THAM KHO ........................................................................................ 46 PH LC I .............................................................................................................. 50 Ph lc IA: Mt s sc thm nh cc aflatoxins. ................................................. 50 Ph lc IB: Mt s sc thm nh cc fumonisinB1. ............................................ 55 Ph lc II: Quy nh ca AOAC v thu hi v lp li [22] .............................. 57 Ph lc III: Chng nhn kt qu mu lin phng cc Aflatoxins ............................... 58 Ph lc IV: CC BNG KT QU ........................................................................ 59 DANH MC CC K HIU, CC CH VIT TT AFAflatoxin AFB1Aflatoxin B1 AFB2Aflatoxin B2 AFG1Aflatoxin G1 AFG2Aflatoxin G2 BYTB Y t FAOT chc nng lng th giiFood and Agriculture Organization FB1Fumonisin B1 OAOrchatoxin ISOT chc tiu chun ho th giiInternationalStandardization Organization IECHip hi k thut in t quc tInternationalElectrotechnical CommissionKPHKhng pht hin ELISA Enzyme-linked immunosorbent assay LCSc k lngLiquid chromatography MSKhi phiMass spectrometry LODGii hn pht hinLimit of DetectionLOQGii hn nh lngLimit of Quantification QCVNQuy chun Vit Nam TCVNTiu chun Vit Nam AOAC Hip hi cc nh ha phn tch chnh thc AssosiationofOfficialAnalytical Chemists IPim nhn dngIdentification point TLCSc k lp mngThin HPLCSc k lng hiu nng cao LC-MS/MS Sc k lng hai ln khi ph UVDetector t ngoi DANH MC CC BNG, HNH V V TH Trang Hnh 1.1: Aflatoxin nhim trn c cht lc .................................................................. 3 Hnh 1.2: Cng thc ha hc ca mt s loi aflatoxin ................................................ 4 Hnh 1.3: Nm mc Fumonisin nhim trn ng ........................................................... 6 Hnh 1.4: Cu trc ho hc phn t Fumonisin ............................................................ 8 Hnh 1.5: M hnh h thng LC-MS/MS ................................................................... 13 Hnh 1.6: B ngun ion ha ....................................................................................... 14 Hnh 3.1: Sc phn mnh ca cc aflatoxins ......................................................... 26 Bng 3.1. Gi tr mnh ion m v hai ion con ca cc aflatoxins ............................... 26 Bng 3.2. Kt qu kho st khang tuyn tnh ca cc aflatoxins ............................... 27 Hnh 3.2. Kt qu kho st khang tuyn tnh ca cc aflatoxins ............................... 28 Bng 3.4. Kt qu kho st lp li ca cc aflatoxins ............................................ 30 Bng 3.5. Kt qu kho st lp li ca cc aflatoxins ............................................ 30 Bng 3.6. Kt qu tham gia th nghim lin phngca cc aflatoxins ...................... 31 Bng 3.7. Gi tr mnh ion m v hai ion con ca fumonisinB1 ................................. 32 Hnh 3.3: Sc phn mnh ca fumonisin B1 ......................................................... 32 Hnh 3.4: th kho st khong tuyn tnh ca fumonisin B1 ................................. 33 Hnh 3.5: Sc fumonisin B1 tiLOQ - 5 ng/ml .................................................... 33 Bng 3.8. Kt qu kho st lp li ca cc fumonisinB1 ....................................... 34 Bng 3.9: Kt qu nh gi chnh xc ca phng php trn nn mu ng ........... 35 Hnh 3.6. T l nhim Aflatoxin ca cc mu lc nhn ............................................... 36 Hnh 3.7. Mi quan h tnh trng mu v s nhim AF trong cc mu lc nhn ......... 38 Hnh 3.8. T l s mu lc c nhim aflatoxin ............................................................ 38 Hnh 3.9. Mi lin h gia tnh trng mu v s mu lc c nhim AF ...................... 39 Hnh 3.10: Sc mu lc c aflatoxin B2 vi mc nhim l 8,8ppb ........................ 39 Hnh 3.11: Sc mu lc c aflatoxin B1 vi mc nhim 6,6ppb ............................ 40 Hnh 3.12: Sc mu ng khng c fumonisin ........................................................ 40 Hnh 3.13: Sc mu ng nhim fumonisin 0.86 g/kg........................................... 40 1 T VN Vn antonv sinh thc phm ngy cng cquantmbinlinquan trctiptisckhecattcngitiudng.Thigianvaquatrongnclin tipphthinccvngcthcphm,trongcccvngclinquann mycotoxin gy ra tm l lo ngi cho ngi tiu dng khi la chn thc phm cng nh vic s dng thc phm th no l ng cch.Trong s 10.000 loi nmmc khc nhau cbitnthckhong50 loil c hi i vi gia sc gia cm v con ngi. Cc loi nm ny sn sinh ra cc c t cgichunglmycotoxin.Mycotoxinlchtcsinhratnmmc,chnh thnh khi nm chuyn ha cc cht dinh dng c trong thc n v nguyn liu. Theo tchc lngthcvnngnghipLinHipQuc(FAO),khong25%sngcc th gii c cha mt hm lng mycotoxin mt mc no . Ty vo v tr a l, khnngnhimmycotoxinlikhcnhau. iu kinnhitivcnnhiti, nguy cnhimmycotoxin cngcao. c th kh hu v nn sn xut nng nghip Vit Nam tnh trngnhimc t nm mc l khph bin. S hnh thnh nm mc v c t ca chng c th bt u t khi cy cn trn ng, lc thu hoch, trong khi bo qun hoc ngay c trong qu trnh ch bin thc n cho vt nui. Khng ni no trn th gii c th thot khi nm mc v c t t chng, v tc hi ca chng l v cng to ln i vi nng sut vt nui v sc khe con ngi.Vi vai tr quan trng ca cy lng thc i vi i sng con ngi cng nh nn kinh t quc dn, th vic nh gi cc yu t nguy c c th xy ra i vi vic tiu th thc phm l rt cn thit i vi Vit Nam, v vy chng ti tin hnh nghin cu nh gi nhim mycotoxin trong thc phm vi mc tiu ca ti l: 1.ThmnhphngphpxcnhAflatoxin(B1,B2,G1,G2)tronglcv FumonisinB1 trong ngbng sc k lng khi ph (LC/MS/MS). 2.p dngphngphp nhgimc nhimtng Aflatoxin(B1,B2, G1,G2) trong lc, fumonisinB1 trong ng. 2 CHNG 1: TNG QUAN 1.1. Khi qut v c t vi nm mycotoxinc t nm mc cn gi l mycotoxin l nhm hp cht c cu trc a dng, c khi lng phn t nh, c to ra bng trao i cht th cp ca cc sn phm nm mc v gy ng c i vi ng vtc v, c v gia cm. Nhngsliucgitrvccmycotoxinvccbnhvmycotoxinc thu nhn t lnh vc th y hc. Nghin cu v ng vt thc nghim cho thy tnh ccamycotoxinlrtln.Bnhnmmcngivthcvtukhngckh nng ly lan v chng do tc nhn c t ha hc gy ra. Tuy nhin, hu nh cc sn phmthcvtucthlcchtchosphttrincanmmcvsto mycotoxin tiptheo vvthn c kh nngly nhimtrctipchothcphmca conngi.Khigiascnccthcnnhimmycotoxin,chngkhngchchutc dngtrctipmcnlngunmangmycotoxinvosa,tht,vnhvytos nhim mycotoxin tip theo cho con ngi. [4] c t nm mc thu ht s quan tm ca ton cu bi chng e da n sc khe con ngi v ng vt cng nh s thit hi nghim trng vkinh t. iu ny c chng t bng nhiu hi ngh hi tho quc t, tp ch v cc bi nghin cu dnh cho vn cp thit ny. 1.1.1. Gii thiu chung v aflatoxin [12] Aflatoxin l ctvinm snsinhtnhinbimtsloi Aspergillus.Trong ngchnhtlAspergillusflavus(A.flavus)vAspergillusparasiticus (A.parasiticus)thngctrongcchtcdu (lc, utng, hthngdng, ht bng,)Aflatoxincngcthxuthintrongsacangvtcchonbng thc n nhim aflatoxin.1.1.1.1. iu kin sn sinh aflatoxin KhnngsinhctcaccchngAspergillusflavusvAspergillus parasiticusrtkhcnhau.iuphthucvoccyutnhnmmc,ccc cht, cc yu t nhit , m c cht v mi trng. Ngoi vic nh lng tng s cc aflatoxin, ngi ta cn quan tm xc nh t l ring phn ca cc aflatoxin khc nhau bit ni chung, aflatoxin B1 c to ra 3 nhiu nht c trong thin nhin ln nui cy, ri n AFG1, sau rt xa l AFB2, cn AFG2 v cc AF khc th t l kh thp. C cht v mi trng ch yu t cc ht c du c bit l ht lc v cc sn phm t lc, lng c t cha trong lc cao nht. Hnh 1.1: Aflatoxin nhim trn c cht lc Nhit thch hp sn sinh aflatoxin ca cc chng nm mc l t 25-28oC. Nu nui cy Aspergillus flavus 45oC th kh nng sinh aflatoxin s b c ch. Hm lng nc trong c cht ng vai tr quan trng cho qu trnh hnh thnh aflatoxin. lc nhn, lng nc t 15- 30% th s hnh thnh xut hin sau 2 ngy; trn go cn lng nc 24 -26 % v ng l 19-24%. Nh vy c th ni s sn sinh aflatoxindinrartnhanh.cbitsauthuhoch,cchtchmlngnckh cao, thi gian lm kh ko di l nguyn nhn dn n nhim aflatoxin. 1.1.1.2. Cu trc v tnh cht ca aflatoxin 1.1.1.2.1. Cu trc ha hc Ccaflatoxinthngnhimtrnccsnphmthcvt,hinnayngita tm thy khong 18 loi aflatoxin khc nhau, tuy nhin c 4 loi chnh thng gp nht gm 4 hp cht ca nhm bis- furanocoumarin, l sn phm trao i cht to bi nm Aspergillus flavus v Aspergillus parasiticus, c t tn l B1, B2, G1, G2. AflatoxinB1,B2trongbsacchuynhavgilaflatoxinM1v aflatoxin M2 (M l mt ch vit tt ca Milk). Afltoxin M1 c hunh quang xanh tm, afltoxin M2 c Rf thp hn v hunh quang tm. Afltoxin M1 l hidroxi- 4 aflatoxin B1, v aflatoxin M2 l hidroxi - 4 aflatoxin B2. 4 Hnh 1.2: Cng thc ha hc ca mt s loi aflatoxin Ngoi 6 loi aflatoxin ch yu trn, ngi ta cn pht hin mt s loi aflatoxin khc v c l flavatoxin hoc flavacuramin. 1.1.1.2.2. Tnh cht ha l ca aflatoxin Aflatoxin l tinh th trng, bn vi nhit, khng b phn hy khi un nu nhit thng thng ( 1200C, phi un 30 pht mi mt tc dng c). Do vy n c th tn ti trong thc phm khng cn s c mt ca nm mc tng ng; ng thi n rt bn vi cc men tiu ha. Tuy nhin n tng i khng bn khi di khng kh v di tia cc tm phin sc k bn mng, v c bit khi ha tan trong dung mi phn cccaonnvickhcthcphmsc nhiubinphphn.Ccaflatoxin trong cc dungmichloroform vbenzene bn vngtrongnhiunmnugitrongtiv lnh. Cc aflatoxin t hoc khng b phn hy di iu kin nu bnh thng v lm nng khi thanh trng. Tuy nhin, khi c m v nhit cao vn c th tiu hy aflatoxin trong mt khong thi gian nht nh. Ccaflatoxinchatantrongdungmiphnccnhnhcloroform, methanol v c bit dimethylsulfosit (dung mi thng c s dng nh phng 5 tin trong vic p dng cc aflatoxin vo cc ng vt thc nghim). Tnh tan ca cc aflatoxin trong nc dao ng t 10 - 20mg/l. Scmtcavnglactonphntaflatoxinlmchngnhycmvivic thyphntrongmitrngkim, ctnhnyquantrngtrongbtkqutrnhch binthcphm,vqutrnhxlkimlmgimhmlngaflatoxincaccsn phm, mc d s c mt ca protein, pH v thi gian x l c th thay i cc kt qu. Tuy nhin nu x l kim l nh th vic axit ha s lm phn ng ngc tr li to aflatoxin ban u. Nhitcao(khong100oC)smvngdeccarboxylationxyravcth tin xa hn, dn n s mt mt cc nhm methoxy t vng thm. Khi c cc axit v cvbsungnc,aflatoxinB1vG1chuynhathnhaflatoxinB2AvG2A.Cc sn phm cng hp tng t ca aflatoxin B1 v G1 cng c hnh thnh vi clorua axit formic thionyl, clorua axit axetic v axit thionyl trifluoacetic. 1.1.1.2.3. c tnh ca aflatoxin [29] Nu hp thu mt lng l 2,5 mg aflatoxin trong thi gian ngn (khong 3 thng) c th dn n ung th gan sau mt nm. Trong c th ng vt, aflatoxin gn ARN v AND thnh dng lin kt, cn tr vic sinhtng hpAND, ARNv protein,gyt bin dn n ung th v lm gim h suy gim min dch. S lin kt ca aflatoxin vi proteinlmvhotccenzyme,thayitnhchtcatyth,lpth,suygimqu trnh h hp ca m bo. Aflatoxintchlytrongm,chyulmgan,gynhimcchongin tht cc loi gia cm, gia sc ny, to nn mt dy chuyn sinh hc ca mm bnh. S nguy him ca aflatoxin B1 ch n c kh nng gy hi ch vi liu lng rt nh, 1 kg thc n ch cn nhim 2 miligam cng lm hng gan. 6 Bng 1.1: Cc gii hn ti a AFca B Y t Vit Nam [5] TTCc sn phm thc phm ML(g/kg) AF B1AF tng sAF M1 1.1Lcvnhnghtcdukhclmnguyn liu,hoccncxltrckhisdng lmthcnhocsdngnhmtthnh phn trong thc phm815KQ 1.5Lc,nhnghtcdukhcdngnv cc sn phm ch bin t chng Ngoi tr : du thc vt th dnh cho tinh lc v du thc vt tinh lc24KQ 1.12Ngvgo,cncxltrckhilm thc n hoc s dng nh 1 thnh phn trong thc phm510KQ 1.1.2. Gii thiu chung v Fumonosin [20] Trongccmycotoxin,miquantmvccfumonisinngycngtngcao. Fumonisin l c tmi c phthingny doFumonisinmoniliorme tnghp nn. y l nhm c tnh cao vi ng vt v con ngi. Vic nhim fumonisin trong thc n cho ngiv gia scquy m trnton thgii.RingtiM,ccboco cho thy 80 - 100% ng bo qun b nhim fumonisin. Hnh 1.3: Nm mc Fumonisin nhim trn ng 7 FumonisinchyudoccnmmcthucgingFusariumtnghpnn.Loi inhnhnhttrongsnylFusariummoniliforme.Ccchngnmmccaloi ny,doSheldonxcnhvonm1904khphbintrongmitrngvthng nhim trong lng thc, t bit l ng. Vo nm 1988, cc c tnh ca fumonisin ln u tin c xc nh v cc sn phmtraoichtcanlnutincphthintrongcanhtrngF. moniliforme. Trong s , mt hp cht c pht hin thng xuyn nht v nng caonhttrongcccanhtrngcaloinyhocthcphmmcbitlng nhim F. moniliforme. Hp cht ny sau c t tn fumonisin B1. Cc nguyn cu v sau ny cho thy rng khng ch c F. moniliforme tng hp nn fumonisin m cc chng khc thuc Fusarium cng tham gia tng hp nn c t loi nybaogmF. proliferatum, F. subglutinans,F.anthophilum, F.annulatum,F. succisae, F. beomiforme, F. dlamini, F. napiforme v F. nygamai. 1.1.2.1. Cu trc Fumonisin Fumonisinlnhmhpchtdiestercaaxittricarxylicviccrubccao khcnhau.Fumonisinchanhmaminbcnht,tantrongncvbnvngvi nhit.Trongsccfumonisin,chfumonisinB1,B2vB3cphthinvihm lngngkctrongiukintnhinviukinphngthnghim(hnh1.4 sau). 1.1.2.2. c tnh ca fumonisin Fumonisin B1 l c t c c tnh mnh nht trong s cc fumonisin. Fumonisin B1cthgycctriuchngnhngno,suygan,gym,gycctriuchngbt bnh thng cho ti t vong nga (ELEM), ung th gan chut, bnh gan g, suy tim cp kh.Ch liu lng 10 mg/kg thc n trong vng 40 - 50 ngy, fumonisin c th gyhichngELEM.FumonisinB1clinquantibnhungththcqung. Mi y, C quan Nguyn cu Quc t v ung th xp fumonisin B1 vo nhm 2B, nhm cc hp cht gy ung th cho ngi. Nguyn cu nh hng ca cc fumonisin ti ngi, ngi ta thy c s lin quan ca bnh ung th thc qun v vic s dng cc lng thc nhim cc fumonisin. 8 Hnh 1.4: Cu trc ho hc phn t Fumonisin ctnhcafumonisinB1linquanmtthitticchiunglnstraoi cht cc sphingolipid, bao gm qu trnh tng hp mi, tch lu cc sphingolipid t do, qutrnhthiloiccsphingoidtdo,tnghmlngccsnphmlipidv sphingosin. Cc hiu ng dn ti hng lot cc phn ng sinh ho gy ra cc s nhim c khc nhau. CcnhkhoahcMtinhnhthnghimchochutnthcnnhim fumonisin v pht hin rng cc c quan ch ca c t ny l gan v thn. 1.2. Hin trng v nhng cng trnh nghin cu c lin quan n ti Vit Nam c mt s nghin cu v mycotoxin trong thc phm v bin php gim nguy c nhim mycotoxin, tuy nhin cha c nghin cu no v nh gi phi nhim Aflatoxin. Trong s cc ti cng b gn y nh: Kt qu nghincu ca L Vn Giang, PhanThKimvcng s Ccan ton v sinh thc phm (2001) cho thy kt qu kho st ban u xc nh 95,4% trong s 243 mu kho st ng v lc ban u b nhim AF, trong c 23,64% vt qu gii hncho php caBY t. Sau6 thng can thipbngcchtchtpcht,sy v bo qun,ktquchothycscithinrrt:76,6%khngphthinAF;23%mu9 nhim AF vi hm lng nh hn mc quy nh [1]. NghincucaPhanThBchTrmvNguynVnBvhmlng mycotoxin trong bp tntrcho thyktqunhlngbngscklnghiunng cao cho thy xcnh c cc loi Aflatoxin Bl, B2,Gl, G2 c trong cc mu bp tn tr cc thi im 3, 5, 7, 9 v 12 tun. Trong iu kin kh thong, nhit trung bnh27,40Cvmkhngkhtrungbnh79,3%,mtrungbnhhtbpthp (11,8%) th cthtrht n 2 thngmhm lng aflatoxintngsvndimc cho php.Hm lng aflatoxin tng s cc th nghim tr bp m 72% v 89% s t nhanh n mc cao khi m ht 13,5% [4]. Virmani tin hnhnghin cu 7 vng sinh thi ca Vit Namv nhgi mi lin quan ca nguy c nhim AF ti cc vng sinh thi ti 3 giai on (1) giai on sinhtrngvphttrin,(2)giaionthuhochvlmkh,(3)giaiontngtr. NhnchungktquchothynguycnhimAFtronglctrongqutrnhtngtr mc cao trn ton Vit Nam [31]. 1.3. Cc phng php xc nh mycotoxin 1.3.1. Phng php ELISA (enzyme-linked immunosorbent assay) Reddyvcccngs(2001)[28],phntchaflatoxinB 1 (AFB 1)trongt bng vic s dng phng php ELISA. Mu ca ba loi v t c thu thp trong cc cuciutranm1998v1999,tccthtrngchnhvcccslutrlnh trong nhng khu vc trng t ln ca bang Andhra Pradesh (AP), n . Bt t c thu thp t cc siu th khc nhau ti Hyderabad, AP. trnh nh hng ca cc cht gynhiu,dngmethanolchitaflatoxintrongt. Trongchnmuidinc skthpgiaphngphpELISAvHPLCxcnhgitrcaAFB 1 vkt qu cho thy cc quy trnh p dng phng php ELISA l ng tin cy. T 182 mu tth nghim, 59% s mu b nhim vi AFB 1 v 18% c c t mc khng cho php. CcAFB1caonhtnng969mg/kgctmthytrongmtmui dincavt. Nhnchung,tltiacaqutchothyAFB 1 mccaohn30 mg/kg (mc ti achophp). Vtcboquntrongphnglnhcho thyc chatlctaflatoxinthpnht. Gn9%cabttcbntrongsiuthc cha hm lng aflatoxin vt qu mc cho php.10 Shi Chun Pei, Yuan Yuan Zhang, Sergei A. Eremin, Won Jong Lee (2009) [29] cng dng phng php cnh tranh gin tip ELISA, s dng khng th n dng o aflatoxin M1 (AFM1) trong sa v sn phm ca sa. Gii hn pht hin nng caAFM1l0,04ng/ml.McnhimAFM1cotrong12musa nguyn liu, 15 mu sa bt, 104 mu sa nc v bn mu pho mt c ly t cc siuthkhcnhaungBccaTrungQuc. 135musackimtra,c55 (41%) mu c AFM1 mc dao ng 0,32 - 0,50 ng/ml, 24 (18%) mu c 0,16 - 0,32 ng/ml, v 18 (13%) mu c 0 - 0,16 ng/ml, trong 38 (28%) mu AFM1 khng c pht hin. Kt qu cho thy cc bin php phng nga cn thit s phi c thc hin gim thiu nhim AFM1 trong sa v cc sn phm sa t vng ng Bc Trung Quc. K thut ny c u im: nhanh, n gin, tit kim dung mi, c hiu v kh nhynhngdchoktqudngtnhgihocmtnhgi.PhntchELISAthun tin cho vic xc nh ng thi cc cht nhim trong mt s lng ln cc mu vi chi ph tng i thp v thi gian ngn. Tuy nhin, n khng thch hp nh lng ccchtnhimvncthbnhhngbihiungcanhiumuvckh nng nh gi qu cao cc cht gy nhim nng rt thp. 1.3.2. Phng php sc k bn mng (TLC) Sc k lp mng l mt k thut tch cc cht c tin hnh khi cho pha ng di chuyn qua pha tnh trn t hn hp cc cht cn tch. Pha tnh l cht hp ph c chn ph hp theo tng yu cu phn tch. Pha ng l mt h dung mi n hocathnhphnctrnvinhautheotlquynhtrongtngchuynlun. Trong qu trnh di chuyn qua lp hp ph, cc cu t trong hn hp mu th c di chuyn trn lp mng, theo hng pha ng, vi nhng tc khc nhau. Kt qu, ta thu cmtsc k trnlpmng. C ch cas chiatch c thl cchhp ph,phnb,traoiion,snglcphnt haysphihpngthicanhiuc ch ty thuc vo tnh cht ca cht lm pha tnh v dung mi lm pha ng. JrgStroka,RobertvanOtterdijk,ElkeANKLAM(2000)[19]sdng phng php TLC mt chiu xc nh c t aflatoxin (B 1, B 2, G1 , G2 ) trong cc loithcphmkhcnhaubngvicsdngdungmiclo.Sdngctilcmin 11 dchlm sch sau khic chitvi methanol. Ccgiihnnhlng ctm thy l thp hn so vi gii hn quy nh hin hnh v kim sot aflatoxin bn ngoi v trong Cng ng chu u.Phng php TLC l mt phng php n gin, d thc hin, khng cn u t ln. N thay th cho phng php HPLC hin i nu khng c thit b HPLC tuy nhin khng th tch v xc nh c AF khi nng thp.1.3.3. Phng php sc k lng hiu nng cao (HPLC) TCVN [6, 7, 9] v nhiu tcgi trn th gii [17, 18, 23, 25] s dng phng php sc k lng hiu nng cao c dn xut sau ct v lm sch bng ct i lc min dchhoclmschbngchitpharnvidetectorhunhquangvdetectorUV xc nh xc nh cc mycotoxin trong cc loi ng cc khc nhau, v d nh: TCVN 7930:2008:[7] Thc phm. Xc nh aflatoxin B1 v aflatoxin tng B1, B2,G1,G2trongngcc,qucvvsnphmcachng.Phngphpsck lng hiu nng cao c dn xut sau ct v lm sch bng ct i lc min dch. TCVN8162:2009:[9]Thcphm-xcnhfumonisinB1vB2trongng phng php sc k lng hiu nng cao c lm sch bng chit pha rn. TheoZhaohui Fu, Xuexiang Huang, Shungeng Min(2008)phttrin phng php s dng UPLC vi detector UV xc nh aflatoxin B1, B2, G1 v G2 trongngvlc. Trongphngphpny,aflatoxincchitvihnhpca acetonitril trong H2O (86:14) v sau lm sch bng ct i lc min dch. thu hi trungbnhcactaflatoxintmulcvmungnng0,22-5mg/kgl 83,4% v 94,7%. Cc gii hn pht hin (S/N = 3) cho B1, B2, G1 v G2 ln lt l 0,32, 0,19, 0,32 v 0,19 mg/kg, v gii hn nh lng tng ng (S/N = 10) l 1,07, 0,63, 1,07 v 0,63 mg/kg[33]. Phng php sc k lng hiu nng cao l mt phng php thng dng xc nh cc hp cht hu c. Phng php ny c ng dng xc nh ng thi cc nhiu hp cht khc nhau. Tuy nhin, phng php c nhy khng cao khi s dng detector UV, cn khi s dng detector hunh quang, phng php c nhy tt hn nhng ch c th nhn bit cht phn tch thng qua thi gian lu. i vi nhng nn mu phc tp nh thc phm, cc cht phn tch rt d b nh hng bi nn mu, 12 nu ch da vo thi gian lu s rt kh c th khng nh cht cn phn tch. 1.3.4. Phng php sc k lng khi ph Phng php sc k lng khi ph, c bit l phng php sc k lng hai ln khi ph l mt k thut mi c pht trin trong nhng nm gn y. V c bn, n lphngphp sc k lngs dngb phn phthinldetectorkhiph.Phng phpcnhiuuimnh chnlccao,giihnphthinthp,thigianphn tchnhanh,cthnhlngngthiccchtcthigianlugingnhaum phng php sc k lng thng khng lm c. NhmtcgiL.C.Huang[21]nghincuxcnhmtnhmcccht aflatoxin M1, ochratoxin A, zearalanone v -zearalenol trong sa bng phng php UHPLC-MS/MS.MusaclmschbngctchitpharnOasisHLB.Gii hnnhlngcaccmycotoxinsnmtrongkhong0,003-0,015g/kg.Hs tng quan cao (R2 0,996) thu c trongkhong nng mycotoxin 0,01 - 1,00 g/kg vi thu hi cao (87,0 - 109%), lp li (3,4 - 9,9%) v ti lp phng th nghimtt(4,0-9,9%)mcnng0,025;0,1;0,5g/kg.Tlphthinmu dng tnh l 16,7% n 96,7% trong sa nguyn liu, sa lng v sa bt thu thp t cc trang tri v cc siu th Beijing. BaifenHuang,ZhengHan,ZengxuanCai,YongjiangWu,YipingRen(2010) [13] s dng phng php sc k lng 2 ln khi ph (UHPLC-MS/MS) xc nh ng thi cc c t aflatoxin B1, B2, G1, G2, M1 v M2 trong u phng v cc sn phmcachng. Mu cchitxutbngdungdchacetonitrile84%vlmsch bng cch qua ct chit pha rn.Cc hp cht tch ra c pht hin vi khi ph t cc 2 ln Waters MICROMASS Quattro Ultima. Phng php ny c tuyn tnh ( R 2 0,9990), thu hi trung bnh (74,7 - 86,8%) v chnh xc ( lch chun tngi10,9%). Ncchngminhlmtphngphpthchhpxc nhngthisuaflatoxintronguphngvccsnphmcachng. Phng php c p dng phn tch 73 mu c thu thp ngu nhin t cc khu vc khc nhau tnh Chit Giang. Kt qu cho thy 31 mu b uphng, 14 mu u phng ti v 5 mu ca u phng mc b nhim c t aflatoxin.EduardoBeltrn,MaraIbez,JuanVicenteSancho,MiguelngelCorts, 13 Vicent Yus, Flix Hernndez (2011) [16] cng s dng phng php sc k lng cao p(UHPLC)cngvikhiph2ln(MS/MS)xcnhccctnmmcquy nh khc nhau (aflatoxin G1,G2, B1, B2, M1,v ochratoxinA)trongccmthng thc n tr em v sa. Phng php s dng phn tch cc sn phm sa khc nhau (ng cc sa bt, sa bt cho tr s sinh, sa ng cc cho tr s sinh v sa ti). thu hi, gia 80% v 110%, vi RSD thp hn 15%, thu c trong tt c cc mu thc phm c kim tra.SouheibOueslativcngs[30]sdngphngphpUPLC-MS/MSxc nh ng thi ca cc c t aflatoxin (AFB1, AFB2, AFG1 v AFG2 ), ochratoxin A (OTA), fumonisins (FB1 v FB2), deoxynivalenol (DON), T2 v HT2 c t trong 58 mu la m th (n = 34), la mch (n = 5), bo bo ( n = 3), la m ch bin ( n = 13) v ng cc n sng ( n = 3) t cc th trng Tunisia. Mc nhim c t nm mc l 50%.Wejdan Shakir Khayoon v cng s [32] s dng phng php HPLC detector hunh quang bc sng 335 nm v 440 nm, khng nh bng LCMS/MS xc nh ccctfumonisinB1vB2trongthcphmvthcnchnnuibngcchs dngctnguynkhisilica,thnhphnphangsdngmethanolvm phosphate pH 3.35 (78:22, v/v) vi tc dng chy 1,0 ml/pht. Cc fumonisins tch trongkhong4pht,sovikhong20phtnusdngmtcthtC18.Giihn phthinl0,01-0,04mg/gfumonisinB1vB2.thuhit84,0-106,0%cho fumonisin B1 v 81,0 -103,0 % cho fumonisin B2. Vinhnguimcacaphngphpscklnghailnkhiph chngtichnphntchtngctaflatoxin(B1,B2,G1,G2)tronglcv fumonisinB1trongngkhostmcnhimtrnabnhuynLcNam, tnh Bc Giang. 1.4. Gii thiu v h thng LC-MS/MS Cu trc s b ca h thng LC-MS/MS. Hnh 1.5: M hnh h thng LC-MS/MS Sc k lng Ion ha B phn tch khi Detector/ Lu gi s liu 14 1.4.1. H thng sc k lng hiu nng cao [3] Sc k l qu trnh tch xy ra trn ct tch vi pha tnh l cht rn v pha ng lchtlng.Muphntchcchuynlncttchdidngdungdch.Khitin hnh chy sc k, cc cht phn tch c phn b lin tc gia pha ng v pha tnh. Trong hn hp cc cht phn tch, do cu trc phn t v tnh cht l ho ca cc cht khc nhau, nn kh nng tng tc ca chng vi pha tnh v pha ng khc nhau. Do vy, chng di chuyn vi tc khc nhau v tch ra khi nhau.1.4.2Khi ph (Mass Spectrometry) [2] Khi ph l thitb phn tch da trnc sxcnhkhilngphntca cc hp cht ha hc bng vic phn tch cc ion phn t hay cc ion mnh ca phn t theo t s gia khi lng v in tch ca chng (m/z). V bn cht, c th coi khi ph l mt loi detector, nhng l mt detector c bitvngoivaitrphthinkhiphcnckhnngtchccchtngragii da trn s khc nhau v khi lng ca chng.Cutocamtthitbkhiphbaogm3phnchnh:ngunion,thitb phn tch v b phn pht hin. Hnh 1.6: B ngun ion ha 1.4.3. ng dng ca sc k lng khi ph Phn tch nh tnh : -SosnhthigianlutRcachtphntchvitRcachtchunichiu trong cng iu kin sc k. - So snh sc k ca mu phn tch vi sc k ca mu phn tch thm chun i chiu. 15 - So snh ph khi ca cht phn tch vi cht chun i chiu trong cng iu kin sc k.Phn tch nh lng : - Phng php chun ha din tch: Hm lng phn trm ca mt thnh phn trong mu phn tch c xc nh bng t s (tnh theo phn trm) gia din tch pic ca thnh phn v tng din tch cc pic ca tt c cc thnh phn c mt trong mu (tr pic ca dung mi, thuc th v ca cc thnh phn c hm lng nh hn gii hn pht hin ca chng). Phng php ny thng cho kt qu khng tin cy nu khng dng h s p ng fi. - Phng phpngoichun:ngchun clpdatrnmitng quan gia cc lng cht chun i chiu khc nhau v din tch pic tng ng ca chng. Nng cc cht phn tch trong mu th c nh lng da trn ng chun ny. Phng php thm chun: Phn tch cc mu thm chun vo mu cng iu kin nh phntchmubnhthng.Datrnlngchunthmvtnhiuccmukhng thm chun v mu thm chun t tnh c nng cht c trong mu. -Phngphpnichun:Trongphngphpny,ngitachnmtcht chun ni a vo trong mu phn tch v trong dung dch chun i chiu. T s din tch ca cht phn tch v cht chun ni l thng s phn tch c dng xy dng ng chun. Phng php ny c chnh xc cao, v n c th loi b c cc yu t gy nh hng n tn hiu cht phn tch.16 CHNG 2:I TNG V PHNG PHP NGHIN CU 2.1. i tng v ni dung nghin cu 2.1.1. i tng nghin cu Mu kim tra nh gi mc nhim mycotoxin gm: lc c, lc nhn v ng mua ti cc h gia nh v h kinh doanh ti a bn huyn Lc Nam, tnh Bc Giang. Bng cch ghi li cc thng tin v tnh trng mu phn tch v mi lin quan vi cc tnh cht ca mu nh gi mi lin quan n nhim mycotoxin trong ng v lc.2.1.2. Ni dung nghin cu -Thm nh phng php: c hiu/chn lc ca phng php. Khong tuyn tnh. Gii hn pht hin LOD, gii hn nh lng LOQ chnh xc ca phng php ( ng v chm) -MuclyticchgianhtheophngphplymuTCVN-5451[8]v thc hin kho st cc yu t lin quan nh hng n mc nhim mycotoxin.-p dng phng php thm nh phn tch aflatoxin (B1, B2, G1, G2)trong lc v fumonisinB1 trong ng. 2.2.Trang thit b, ha cht 2.2.1. Thit b, dng c,: 2.2.1.1.My sc k lng khi ph LC-MS/MS vi h thng HPLC 20 AXL ca Shimadzu v khi ph ABI 5500 QQQ ca Aplied Biosystem. 2.2.1.2.Ct sc k Xbridge C18 (100 mm x 4,6 mm; 2,5 m) v tin ct. 2.2.1.3.Cn phn tch chnh xc n 0,1 mg v 0,01 mg. 2.2.1.4.Cn k thut, chnh xc 0,01 g. 2.2.1.5.My ly tm c th t c tc ti thiu 4000 rpm vi ng ly tm 15 ml. 2.2.1.6.My lc xoy. 2.2.1.7.My ng nht mu 2.2.1.8.L nung, c kh nng hot ng 500 0C 2.2.1.9.T lnh ng, c th t c nhit -20 0C. 17 2.2.1.10.Vial 1,8 ml c np kn. 2.2.1.11.Micropipet loi c th tch iu chnh c: 20-200 l v loi 100-1000 l. 2.2.1.12.Bnh nh mc cc loi: 10 ml; 50 ml v 100 ml. 2.2.1.13.ng ly tm nha 50 ml, 15ml c np kn. 2.2.1.14.Ccdng c thng thng khc ca phng th nghim. 2.2.2. Ha cht, thuc th 2.2.2.1.Ct sc k Xbridge C18 (100 mm x 4,6 mm; 2,5 m) v tin ct. 2.2.2.2.Cht chun Fumonisin B1 (FB1) c tinhkhit > 97,0 % (Sigma Aldrich hoc tng ng) 2.2.2.3.Chun aflatoxin B1, B2, G1, G2 c tinh khit 99%, Supelco hoc tng ng (c th s dng chun mix) 2.2.2.4.Methanol, loi dng HPLC (Merck hoc tng ng) 2.2.2.5.Acetonitril, loi dng HPLC (Merck hoc tng ng) 2.2.2.6.Cloroform: loi tinh khit phn tch 2.2.2.7.Diclomethan: loi dng cho HPLC (Merck hoc tng ng) 2.2.2.8.n-hexan: loi dng cho sc k lng (Merck hoc tng ng) 2.2.2.9.Magiesulfatkhan(MgSO4):dngbt,tinhkhit>98%,nung 500 0Ctrong>5giloihtnctha(Merckhoctng ng). 2.2.2.10.Natri clorua (NaCl): (Merck hoc tng ng) 2.2.2.11.Acid acetic: (Merck hoc tng ng) 2.2.2.12.Phangfumonisin:Dungdchacidacetic0,1%(v/v)trongnc:Ht 1 ml acid acetic ha vo nc ct hai ln nh mc n 1 lt. Lc qua mng lc 0.45 m, dung dch s dng trong 1 tun. 2.2.2.13.Hnhpmuichitfumonisin:Cn500mgMgSO4(6.1.4)v250 mgNaCl(6.1.5)trncn(5.1.4)chovotihocdngcchac thykn.Lctrn u.Mihnhpcsdngchomtmu phn tch. Chun b mt lot cc hn hp m bo cho cc l mu phn tch. 18 2.2.2.14.Nc ct hai ln2.2.2.15.Hn hp cloroform:acetone (90:10): Dng pipet 10 ml ht chnh xc 10 ml acetone vo bnh nh mc 100 ml v nh mc ti vch bng cloroform2.3.Phng php nghin cu 2.3.1. S lng mu ly: C mu cho nghin cu s dng cng thc tnh c mu m t: n = Z1-u22p(1 p)J2 Trong : p l t l s mu b nhim mycotoxin trong ng v lc, theo nghin cu ca L Vn Giang ti Tn K - Ngh An l 96% trong ng v 98% trong lc. d: chnh xc tuyt i: 0,05 Z=1,96 vi tin cy = 0,05 T ta tnh c s mu ng l n = 59 v s mu lc l n = 30.Ta thc hin ly 60 mu ng, 30 mu lc nhn v 30 mu lc c. 2.3.2. Phng php kim nghim: 2.3.2.1. Phng php kim nghim aflatoxin: Mu lc nhn v lc c c xc nh hm lng AF (B1, B2, G1, G2) bng sc k lng khi ph LC-MS/MS theo [6, 7]. Nguyn l: Mu c chit bng cloroform v lm sch trn ct chit pha rn Si. Aflatoxinsctchv nhlngbnghthngscklngkhiphsdngct pha o C18. Chunbmuphntch:Murncxaynh,trnu:Cnchnhxc khong 10-20 g trn cn phn tch vo bnh nn 250 ml. Mu nc v mu lng: Cn khi lng hoc ht th tch trc tip tng ng khong 10 - 20 g vo bnh nn 250 mlChitaflatoxintrongccsnphmchatchtbo:Thm100mlcloroform vomuchunbtrn.Lc30pht,tc140vng/phttrnmylcngang. Sau c lc qua giy lc vo bnh c quay 250 ml. Dch chit c c quay bng mycquaychnkhng40Cncn.Sauchacnbng10ml diclomethan19 ChitAflatoxintrongccsnphmnhiuchtbo:Thm100mlhnhp methanol: nc (85:15, .) vo mu c chun b trn, lc u. Hn hp c lc 30 pht, tc 140 vng/pht trn my lc ngang . Sau c lc quagiy lc. Ly 40 ml dch lc trn cho vo phu chit 500 ml, thm 40 ml dung dch NaCl 1%, sau thm 25 ml n-hexan vo v lc u chit loi cht bo. yn n khi phn lp ri ly lp di vo phu chit khc, lp pha trn loi b. Lp di c thm 25 ml cloroform vo v lc u chit aflatoxin, lng n khi phn lp. Ly lp di, lp trn c chit lp li ln 2 vi 25 ml cloroform. Dch ca hai ln chit c gp vo bnh ct quay 100 ml. Sau c c quay bng my c quay chn khng 40C ncn.Sauchacnbngkhong10mldiclomethanvlmschtrnct SPE-Si.Ctchothabng3mln-hexan,3mldiclomethan.Mucnpqua ct vi tc 1 - 2 ml/pht. Sau c ra tp vi 3 ml n-hexan,3 ml diclomethan. Cht phn tch c ra gii ra bng 10 ml hn hp cloroform- acetone (90 : 10) vo bnh c quay 100 ml.Dchragiiccquayncnvhacnbng1mlmethanolvc bm vo h thng LC-MS/MS.*Mukimtra(thuhi,QC):Lptngtccbcnhtrnnhngc thmchunaflatoxinsvomutrngtrckhichitmuxcnhthuhiv loibsaistrongqutrnhthchinthaotc.Muspike:thmchnhxc100l dung dch chun lm vic vo mu trng ri tin hnh phn tch ging mu th. * Mu trng: Mu trng tin hnh tng t nh mu th trn mu xc nh l khng c cc aflatoxins ni trn. iu kin chy my-Ct sck pha o C18 Waters hoc tng ng: 250 mm, 4,6 mm, 4,5m -Pha ng: chy theo chng trnh gradient nh sau: Thi gian (pht)T l% MeOHTl % Amoniacetat 10 mM 0,01595 41000 51000 20 5,01595 10595 - Th tch bm mu: 10 l. - Tc dng pha ng:1,0 ml/pht - iu kin khi ph:Bng sau mtiukinchyivithitbMS ABI 5500 QQQ. Cc iu kin khi ph c th khc nhau i vi cc thit b khi ph khc nhau, cn thc hin ti u iu kin bng cht chun aflatoxins. Ngun ion haElectrospray (ESI) Th ion ha4000 KV Ch Ion dng Nhit ngun300 0C KhCurtain gas: 25 psi Ion source gas 1 : 30 psi Ion source gas 2 : 10 psi Nng lng bn phCE = 30 eV nh tnh: xc nhn s c mt ca cht phn tch trn LC-MS/MS, ngoi s trnglpvthigianlutngngcamichtcncncvotlpngtn hiu ca ion nh lng v ion nh tnh. T l ny ca aflatoxin G2, G1, B2, B1 ln ltl:1,1-1,4;4,3-4,6;1,2-1,3;0,6-0,7.Tlnycththayitythucvo nhy ca thit b. Vimudngtnh,cncvostngquangiadintchpicvnng, cn c vo din tch pic mutnh kt qu theo cng thc sau. X = V x K x Cm/m Trong :V: Th tch dch chit cui cng chy my (ml) Cm: Nng aflatoxin trong dch chit mu tnh theo chun (ng/ml) m: Khi lng ca mu phn tch (g) X: hm lng aflatoxin trong mu phn tch (ng/g) K: H s pha long21 Mi mu lm mt ln. Thc hin bm mu theo ng quy trnh chy my. Mi l lm t nht 1 mu thu hi, mt mu trng: Mu trng phi khng c tn hiu ti thi gian lu ca cht phn tch. 2.3.2.2. Phng php kim nghim fumonisin B1: Nguynl:HmlngfumonisinB1trongngcccchitlnglngvi dungmiacetonitril(ACN),methnol(MeOH),nc(25/25/50)cbsungmui MgSO4 v NaCl gip cho qu trnh tch lp hiu qu. Dch chit c lc qua mng lc 0,2 m. Dch chit cui cng c phn tch bng LC-MS/MS. Chun b muChun b mu s b: Mu ng cc dng ht c nghin bngmy ng nht mu. Mu dng bt khng cn nghin nhng phi trn u trc khi phn tch. Muth:Cnchnhxckhong1gmutrncnphntch(2.2.1.3)vongly tm 15ml. -Thm5mldungdchchitACN:MeOH:H2O(25/25/50,v/v/v)(6.1.8)vo ng ly tm, y np kn, lc trn u bng tay vi ln.-Thmtthnhpmuichitcchunbsn(2.2.2.13),ynpkn, lc trn u bng my lc trong 1 pht.-Ly tm tc 6000 rpm trong 5 pht.-Htlptrn,thmtip5mldungdchchitACN:MeOH:H2O (25/25/50,v/v/v) v lp li bc trn.-Gp dch chit hai ln lc qua mng lc 0,2 m, thm 0,75 ml nc vo 0,5 ml dch lc v em phn tch bng LC-MS/MS. Ghi ch: Mu sau khi chit nn c phn tch ngay, nu khng th phn tch ngay th c th bo qun 2-8 0C nhng khng qu 2 ngy. Mu trng: mu trng tin hnh tng t nh mu th trn mu rau qu xc nh l khng c fumonisin B1. Mukimtra(thuhi,QC):Tinhnhtngtnhmuthnhngccho thm mt lng chun chnh xc bit nng . C th la chn cc cch thm sau y nhng ti thiu mt l mu phn tch cn c mt mu QC. 22 iu kin chy my: - iu kin LC:+ Ct sc k Xbridge C18 (100 mm x 4,6 mm; 2,5 m)v tin ct+ Tc dng 0,6 ml/min + Nhit ct: nhit phng + Th tch tim mu 10l + Pha ng: theo ch gradient nh bng di Thi gian (min)Acid acetic 0,1 % trong ncAcetonitrile (%) 0,019010 3,000100 8,000100 8,019010 12,009010 -iu kin khi ph:-Ngun ion ha ESI, ch ion dng (+), th phun 4500 kV -Nhit u phun (IS) 450 0C -Curtain Gas (CUR) 20 -Collision gas (CAD) 7 -Ion source gas 1 (GS1) 20 -Ion source gas 2 (GS1) 15 Tnh ton kt qu Cn c vo ng chun tng quan gia din tch v nng , cn c vo din tch pic muta tnh c hm lng fumonisin B1 trong dung dch chy my. Hm lng fumonisin B1 trong mu c tnh theo cng thc sau: X = C I km Trong :Xi: Hm lng fumonisin B trong mu (ng/g) Ci: Hm lng fumonisin B1 trong dch cui (ng/ml) V: Th tch dch chit (ml)23 k: H s pha long (nu c) m: Khi lng mu phn tch (g) Tnh thu hi ca mu QC theo cng thc: R% = XtCc 1uu Trong :R%: thu hi (%) Xt: Hm lng fumonisin B1 tnh c trong mu (ng/g) Cc: Hm lng fumonisin B1 thm vo ban u (ng/g) 2.3.2.3. Thm nh phng php: 2.3.2.3.1. Tnh chn lc, c hiu Tnh chn lc ni ln kh nng xc nh ng cht phn tch khi c mt cc tp cht hoc cc cht khc tin hnh nh sau.xc nhtnhchiu/chnlc iviscklngkhiphchngtis dngccphngphpxcnhn(confirmationmethod).TheoHingChuu im IP (im nhn dng- identification point) i vi k thut sc k lng khi ph 2 ln (LC-MS/MS) l 4, tc l cn 1 ion m bn ph ra 2 ion con[14].Hn na xc nh chc chn phng php nghin cu c tnh c hiu/chn lc cao, chng ti tin hnh phn tch cc mu trng khng cho tn hiu phn tch, mu trng c thm cht chun. 2.3.2.3.2. Khong tuyn tnh Khostkhongnngcachtphntchtrongcsphthuctuyn tnhgiatldintchpicchtphntch/dintchpicchtchunvinngca cht . Phng trnh hi quy tuyn tnhb x a y + =Trong : y l t l din tch pic cht phn tch/din tch cht chun x l nng cht kho st 2.3.2.3.3. Gii hn pht hin (LOD), gii hn nh lng (LOQ) Gii hn pht hin LOD (Limit of detection) l nng thp nht ca cht phn tch c th xc nh c nhng khng nht thit phi nh lng c trong iu kin th nghim c th. Trong sc k LOD c th c xc nh l nng ca cht phn tch m ti t l gia tn hiu ca cht phn tch v tn hiu nn bng 3 (S/N =3)[10]. 24 Hoc c th tnh LOD da vo lch chun nh sau: Tnh gi tr nng trung bnh xtb, lch chun SD (S) LOD = 3.S nh gi LOD tnh c ta kim tra qua gi tr R = xtb LOD

Nu 4 < R < 10 th nng dung dch em th l ph hp v gi tr LOD tnh c l ng tin cy.GiihnnhlngLOQ(Limitofquantification)lnngthpnhtca cht phn tch c trong mu th c th pht hin c v mt nh lng vi ng v chm nhn c. Trong sc k LOQ c th c xc nh l nng ca cht phn tch m ti t l gia tn hiu ca cht phn tch v tn hiu nn bng 10 (S/N =10). C th tnh LOQ theo LOD theo cng thc:LOQ = 10/3 * LOD. [10]. 2.3.2.3.4. lp li lplithhin sgnnhaucaccktquo,lmcthngnhtca cc kt qu th ring bit khi quy trnh phn tch c p dnglp li trn cngmt mu.lplicthhinbnglchchuntngiRSD.Tinhnhth nghimlpli6ln.Tnhlchchuntngi RSD%cahmlngchtphn tch. Cc cng thc tnh ton nh sau [10]: Tin hnh th nghim n ln lp li. + Gi tr trung bnh : = =n1 iixn1xTrong xl gi tr trung bnh s hc ca tp hp cc gi tr xi cn xi l gi tr kt qu ca mi ln th nghim. + lch chun : 1 n) x x (SD2n1 ii= = + lch chun tng i:( )112==Nx xSNii25100XSDRSD = 2.3.2.3.5. ng ng nh gi s ph hp ca kt qu thc nghim so vi gi tr thc (true value) hoc c chp nhn thc (accepted value) [10]. -Phn tch mu chun (vt liu chun): Mu bit trc nng ca cht phn tch. Kt qu tnh ra chch: chch = xx 100 x : kt qu phn tch c : gi tr ng ca cht phn tch Trongtrnghpthamgiaphntchccmuthnghimlinphngth nghim, c th nh gi kt qu thng qua gi tr Zscore. Zscore c tnh nh sau: Zscore = o x Trong : ol lch chun tham chiu Nu |Zscore| 2: Kt qu c chp nhn 2 < |Zscore| 3: Kt qu c nghi ng |Zscore| > 3: Kt qu c sai lch. -Khostthuhi:thuhicxcnhdatrnkthutthmchun. Lng cht chun thm vo mu phn tch phi m bo sao cho nng ca cht cn nghin cu sau khi thm chun nm trong khong kho st. thu hi (R%) c tnh nh sau: ( )100CC C% Rcm c m=+ Trong : Cm+c: Nng trong mu thm chun Cm: Nng trong mu Cc: Nng chun thm 2.3.2.4. Phng php x l s liu : -Tnh ton kt qu bng phn mm Microsoft Excel-Tnh ton thi gian lu, din tch pic, chiu cao pic v t l tn hiu trn nhiu bng phn mm ca thit b.26CHNG 3: KT QU V THO LUN 3.1.Thm nh phng php kim nghim aflatoxin 3.1.1. Tnh c hiu, tnh chn lc xcnhtnhchiu/chnlciviAFtinhnhxcnhngitrIP bng cch kim tra phn mnh ca cc AF, phn tch ccmu trng v kim tra t l p ng tn hiu ca ion nh lng v ion nh tnh bng cch thc hin trn chun v trn mu lc khng c AF. Hnh 3.1: Sc phn mnh ca cc aflatoxins Bng 3.1. Gi tr mnh ion m v hai ion con ca cc aflatoxins Tn cht phn tch Mnh m (m/z) Mnh con (m/z) Collision energy (eV) Ghi ch Aflatoxin B1 31324141nh tnh 31326937nh lng Aflatoxin B2 31525935nh tnh 31528743nh lng Aflatoxin G2 33124539nh tnh 33128533nh lng XIC of +MRM (10 pairs): 331.000/245.000 Da ID: G2 from Sample 17 (Std 5) of DataSET1.wiff (Turbo Spray) Max. 2.0e4 cps.0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5Time, min0.05.0e4Intensity, cps6.43 +MRM (10 pairs): 6.537 min from Sample 17 (Std 5) of DataSET1.wiff (Turbo Spray) Max. 5.1e4 cps.331.000/245.000 331.000/285.000 329.000/243.000 329.000/215.000 315.000/259.000 315.000/287.000 313.000/241.000 313.000/269.000 329.000/273.000 329.000/259.000Q1/Q3 Masses, Da0.05.0e4Intensity, cps +MRM (10 pairs): 6.666 min from Sample 17 (Std 5) of DataSET1.wiff (Turbo Spray) Max. 5.5e4 cps.331.000/245.000 331.000/285.000 329.000/243.000 329.000/215.000 315.000/259.000 315.000/287.000 313.000/241.000 313.000/269.000 329.000/273.000 329.000/259.000Q1/Q3 Masses, Da0.05.0e4Intensity, cps +MRM (10 pairs): 6.769 min from Sample 17 (Std 5) of DataSET1.wiff (Turbo Spray) Max. 3.3e4 cps.331.000/245.000 331.000/285.000 329.000/243.000 329.000/215.000 315.000/259.000 315.000/287.000 313.000/241.000 313.000/269.000 329.000/273.000 329.000/259.000Q1/Q3 Masses, Da0.03.3e4Intensity, cps +MRM (10 pairs): 6.433 min from Sample 17 (Std 5) of DataSET1.wiff (Turbo Spray) Max. 2.0e4 cps.331.000/245.000 331.000/285.000 329.000/243.000 329.000/215.000 315.000/259.000 315.000/287.000 313.000/241.000 313.000/269.000 329.000/273.000 329.000/259.000Q1/Q3 Masses, Da0.01.0e42.0e4Intensity, cpsB2B1 G2 G227Aflatoxin G1 32924347nh tnh 32921541nh lng Nhn xt: theo nh kt qu phn mnh ca cc AF (hnh 3.1) v (bng 3.1) mi AFmchaiAFcon,mtmnhm1,0imIP,1conc1,5imIPnhvy im IP ca tng AF l 4. Mt khc trn sc mu trng (Ph lc IA: hnh 1) khng xut hin pic ti cc thi gian lu ca cc AF nghin cu. Cn trn sc mu thm chun c xut hin cc pic ti cc thigian lu tng ng (Ph lc IA: hnh 2). Nh vy phng php c tnh c hiu v chn lc p ng yu cu. 3.1.2. Khong tuyn tnh v lp ng chun Phachunhnhpcccnngt0,3n300ppbtinhnhbmvoh thng LC-MS/MS cc sc dy chun (Ph lc IA: hnh 3). Xy dng mi quan h ph thuc tuyn tnh gia nng cht phn tch v din tch pc sc k ta c bng v hnh biu din sau: Bng 3.2. Kt qu kho st khang tuyn tnh ca cc aflatoxins

Nng AF (ppb)0,33,01575150 Din tch pic AFB1 27700353001310004550001110000 Din tch pic AFB2 79222001270073500126000 Din tchpic AFG1 152004410021800013900002330000 Din tchpic AFG2 12700262001090005070001030000 28 Hnh 3.2. Kt qu kho st khang tuyn tnh ca cc aflatoxins Nhnxt:Nhvytrongkhongnng0,3n150ppb,sphthucgia nngtngaflatoxinsvdintchpcsckltuyntnhvihstngquantuyntnhR2>0,999.VykhongtuyntnhcaccAFnghincult0,3n 150ppb. 3.1.3.Gii hn pht hin (LOD), gii hn nh lng (LOQ) xc nh gii hnpht hin da trn lch chun, chng ti tinhnh lm trn nn mu th. Lm 10 ln song song, cn chnh xc khong 10g mu lc trng y = 7278.7x + 18437R = 10500000100000015000000 50 100 150 200ng chun AFB1y = 837.44x - 424.73R = 0.99970500001000001500000 50 100 150 200ng chun AFB2y = 15578x - 9048.4R = 101000000200000030000000 20 40 60 80 100 120 140 160ng chun AFG1y = 6818.9x + 3533.9R = 0.99990500000100000015000000 50 100 150 200ng chun AFG229chovobnhnn 250ml, thmchun cc chtnng0,5ng/ml(gp 5lnLOD clng,LODclngl:0,1ng/ml).Thchintinhnhxlmutheoquy trnh v o nng tng AF (sc (Ph lc IA: hnh 4) ) c cc kt qu sau: Bng 3.3. Kt qu kho st xc nh LOD, LOQ ca cc aflatoxins Kt quAflatoxin B1Aflatoxin B2Aflatoxin G1Aflatoxin G2 Nng AF xc nh c xi(ng/ml) 0,490,450,420,45 0,480,420,460,42 0,490,440,440,39 0,480,470,480,47 0,380,390,420,42 0,470,480,380,47 0,480,440,390,43 0,490,420,450,49 0,480,410,490,41 0,480,400,420,47 xtb(ng/ml)0,470,430,440,44 SD0,030,030,040,03 LOD (ng/ml)0,100,100,100,10 LOQ (ng/ml)0,300,300,300,30 R4,74,94,04,5 RSD(%)6,407,009,106,80 Nhnxt:TbngktquthucnhnthyccchtucgitrRtha mn yu cu 450%htv,ktquchothy:11/17muphthincAflatoxin (chim64,7%).Ccyutlinquancaccmubnhimnyl:8/11muny c ghi li l ht lp, 5/11 mu ny l ht c mu khc thng, 10/11 mu khng dc v la, 3/11 mu mc mm v 2/11 mu c ht b mc. -12/30muhtlpc9/12muphthinAflatoxin,trong:4/9mucmukhc thng, 8/9 mu khng dc v la, 3/9 mu c mm v 3/9 mu c ht b mc. -8/30 mu c mu khc thng, pht hin 6 mu c aflatoxin, trong cc yu t lin quankhcnh:5/5(chim100%)muchtvvlp,4/5khngdcv,3/5c mm v 1/5 c ht b mc. -16/30mukhngdcvla,phthin12/16mucaflatoxin,trongccyut linquankhcnh:10/12muchtv,8/12muhtlp,4/12mucmukhc thng, 4/12 c mm v 2/12 mu c ht b mc. -4/30 mu c mm th c 4/4 mu (chim 100%) u pht hin c Aflatoxin. -3/30 mu c ht mc th c 3/3 mu (chim 100%) u pht hin c Aflatoxin. -c bit vi 2 mu c pht hin hm lng aflatoxin cao hn gii hn cho php c cc c im ca mu ly nh: ht v, ht lp, ht c mu khc thng, ht khng dc v la, v c ht mm. Mi quan h gia cc yu t v tnh trng mu vi s mu b nhim AF c biu din trong hnh 3.7. 38 Hnh 3.7. Mi quan h tnh trng mu v s nhim AF trong cc mu lc nhn Nhn xt: Qua hnh 3.7 ta thy s mu ht lc nhn c tnh trng khng dc v, htv, htlp, c mu khc thng,ht nymmv htmc t lthun vis mu nhim AF. S mu c htmy, ht dc v la v nguynht t l nghch vi s mu nhim AF. iu chngt lcnhnkhng dcv la(khkhngm bocho qutrnhboqun),htchtlngkm(khngnguynht,htlp,htv,htny mm) c nguy c nhim AF cao.3.3.2. Kt qu phn tch mu lc c 30 mu lc c c ly v ghi li tnh trng mu khi ly, kt qu phn tch hm lng AF trong cc mu lc c nh Bng 4.2 trong Ph lc IV. Kt qu t l mu nhim Aflatoxin trong cc mu lc c th hin trn Hnh 3.8. Hnh 3.8. T l s mu lc c nhim aflatoxin 024681012141618c nhim AF KPH nhim AFnguyn htht myc dc v lakhng dc v laht vht lpmu khc thnght ny mmht mcTng s.00%5.00%10.00%15.00%20.00%25.00%30.00%AF B1 AF B2 AF G1 AF G2 AF tng s23.333%3.333%.00% .00%26.667%39-8/30mulccbnhimAflatoxintngs(chimtl26,7%),tuynhinkhngc mu no b vt qu gii hn cho php theo quy nh trong QCVN 8-1:2011/BYT.-30/30 mu lc c kim tra (chim 100%) khng nhim Aflatoxin G1 v Aflatoxin G2. -7/30 mu lc c (chim 23,3%) b nhim Aflatoxin B1. -1/30 mu lc c (chim 3,3%) bnhim Aflatoxin B2. So snh biu hnh 3.6 v hnh 3.8 ta thy t l s mu nhim aflatoxin trong lc nhn nhiu hn lc c. iu cho thy lc c bo qun tt hn lc nhn. Mi lin h gia tnh trng mu khi ly v s mu lc c nhim AF c biu din trong hnh 3.9. Hnh 3.9. Mi lin h gia tnh trng mu v s mu lc c nhim AF Quatathylcctrongtnhtrnglntpvbmccnguycnhim Aflatoxin cao hn lc c sch. Hnh 3.10: Sc mu lc c aflatoxin B2 vi mc nhim l 8,8ppb 0510152025c nhim AF KPH nhim AFschln tpc c mcTng sSample Name: "560-2"Sample ID: ""File: "DataSET1.wiff"Peak Name: "G2"Mass(es): "331.000/245.000 Da"Comment: "" Annotation: ""0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0Time, min0102030405060708090100110120130140150160170180190200210220230240250260270280290300310320330340350360370380390400Intensity, cps6.957.538.326.499.256.245.302.17 1.60 0.80Sample Name: "560-2" Sample ID: "" File: "DataSET1.wiff"Peak Name: "G1"Mass(es): "329.000/243.000 Da"Comment: ""Annotation: ""0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0Time, min0102030405060708090100110120130140150160170180190200210220230240250260270280290300310320330340350360370380390400410420430440Intensity, cps7.457.035.928.348.429.029.179.111.14 0.98 4.65Sample Name: "560-2" Sample ID: "" File: "DataSET1.wiff"Peak Name: "B2"Mass(es): "315.000/259.000 Da"Comment: ""Annotation: ""0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0Time, min0.01000.02000.03000.04000.05000.06000.07000.08000.09000.01.0e41.1e41.2e41.3e41.4e41.5e41.6e41.7e41.8e41.9e42.0e42.1e42.2e42.3e42.4e42.5e42.6e42.7e42.8e42.9e43.0e43.1e43.2e43.3e43.4e43.5e43.6e43.7e43.8e43.9e44.0e4Intensity, cps6.707.98Sample Name: "560-2"Sample ID: ""File: "DataSET1.wiff"Peak Name: "B1"Mass(es): "313.000/241.000 Da"Comment: ""Annotation: ""0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0Time, min0102030405060708090100110120130140150160170180190200210220230240250260270280290300310320330340350360Intensity, cps7.476.778.738.199.256.465.97 5.71 1.50 3.98 0.05 2.66B2G1G2 B1 40 Hnh 3.11: Sc mu lc c aflatoxin B1 vi mc nhim 6,6ppb 3.3.3. Kt qu phn tch mu ng 60 mung ht khc ly ticchgianh vhkinhdoanhtia bn nghin cu. Kt qu phn tch v tnh trng mu ly c tm tt trong bng 4.3 trong ph lc IV. Trong 60 mu ng kim tra Fumonisin, c 35/60 mu (chim t l 58,3%) pht hin c nhim fumonisin, tuy nhin khng c mu no c hm lng Fumonisin vt qu gii hn cho php theo QCVN 8-1:2011/BYT (