TNH HNH NHIM C T NM (AFLATOXIN B1, OCHRATOXIN A, CITRININ) TRN GO TI NHA TRANG

Nguyn Minh Tr Khoa Ch bin Tm tt Bng phng php tch chit vi acetonitrile/ nc (9/1), tinh ch lng-lng, nh lng trn HPLC, chng ti phn tch ng thi cc c t nm Aflatoxin B1 (AFB1), Citrinin (CIT), Ochratoxin A (OTA) trn go, vi gii hn nh lng theo th t l 0,15 ; 0,17 ; 0,26 ng/g. Chng ti tin hnh nh lng cc c t ny trn 64 mu go thu thp t 4 ch ln Nha Trang. Nhim AFB1 l ch yu. T l nhim AFB1 36/64 (41%), ch yu trong ma ma (84%), v loi go r tin (100%), mu nhim cao nht l 7,2 ng/g.
Abstract

Aflatoxin B1 (AFB1), Citrinin (CIT), Ochratoxin A (OTA) in the rice were simultaneously extracted by acetonitrile/ water (9/1), purified by partition method, and quantified on the HPLC. Their limits of quantification were 0,15; 0,17; 0,26 ng /g (respectively). Sixty-four samples of rice were collected in big markets in Nha Trang. Contamination of AFB1 was mainly. This ratio was 36/64 (41%), particularly in the rainy season (84%), and at the cheaper rice (100%); the highest level of AFB1 contamination was 7,2 ng/g.

1. t vn Hin nay chng ta quan tm nhiu n cht lng thc phm, bi cht lng thc phm nh hng trc tip n sc kho con ngi. C rt nhiu mi quan tm, bn cnh vn gi tr dinh dng, vn an ton thc phm c c bit ch trng, trong nhim cc cht gy hi cho ngi nh kim loi nng, thuc tr su, d lng phn bn, cc c t khc nhau Trong iu kin kh hu ca nc ta, kh hu gi ma, ch ma m cao. iu kin bo qun la go c quan tm, tuy nhin vn cn quy m ln, cc kho la go ln ca c nc, cc kho xut khu, cn i vi cc a phng, cc kho nh l cha c quan tm v hiu v n cha ng mc, hu qu s nh hng khng nh n cht lng thc phm cung cp cho ngi dn. Trong iu kin nh vy, nm mc c th pht trin sinh c t c gi l c t nm (mycotoxin), gy nh hng ng k n sc kho ngi tiu dng, c bit l gy ung th. Trong c th k n Aflatoxin B1 (AFB1), Ochratoxine A (OTA), Citrinine (CIT). nh gi mc nhim, chng ti tin hnh iu tra mc nhim c t nm go (AFB1, OTA, CIT) bn cc ch ti Nha Trang. 2. Vt liu v phng php 2.1. Thu thp mu 64 mu go c ly ngu nhin ti 4 ch ti Nha Trang : Ch m, Ch Xm Mi, Ch Vnh Hi, ch Phc Thi. Mu c ly 2 ln vo ma ma v ma kh. 2.2. Ho cht Tt c ho cht (chlorua kali, acid chlohydric, acid phosphoric, ) mc PA. Tt c dung mi (methanol, chloroform, acetonitrile, propanol-2-ol, n-hexane) mc HPLC. Nc kh ion c s dng chun b cc dung dch dng trong qu trnh tch chit, tinh ch v phn tch trn HPLC. 2.3. Chun b cc dung dch chun Cc dung dch chun c chun b 10 mg AFB1, OTA hay CIT/ml methanol. Mt lot cc dung dch chun t 0,3 n 100 ng AFB1, OTA, CIT/ml c chun b bng pha long. Nng ca dung dch chun gc c xc nh bng hp ph tia cc tm (A) theo cng thc :

( g / ml )1000ma A =
Trong : ma max nng ca c t chun trong dung dch. trng lng phn t kh nng hp thu ca phn t ti max bc sng hp thu cc i

c t Aflatoxin B1 Ochratoxin A Citrinin

ma (g/mol) 312 403 250

Dung mi Methanol Methanol Methanol

max 360 333 321

(cm2mol-1) 21800 5440 5490

2.4. nh lng AFB1, OTA v CIT trn HPLC S dng thit b HPLC ca Nht ti u ho nhy cm khi phn tch AFB1, OTA, CIT s dng cc thng s hunh quang kch thch v pht x khc nhau (AFB1 365 440 nm ; OTA 335 v 465 nm ; CIT 331 v 500 nm). Phn tch c tin hnh trn ct C18 (3m C18, 0,46 x 25 cm). H thng c phn tch vi phase ng H3PO4 (0,33 M)/ acetonitrile/ propanol-2 (650/400/50) ; tc dng 0,5 ml/ pht. Thi gian lu i vi tng c t nm : AFB1 l 14 pht, CIT l 28 pht, v OTA l 56 pht. Cc sc k c phn tch da vo phn mm Class-LC10 ca Shimadzu version 1.60. - Chit xut: Thm 10 ml dung dch KCl 4% (pH 1,5) vo 20 g mu c nghin nh. Ri c thm vo 90 ml acetonitrile, lc 20 pht. Cht chit c lc qua giy lc nh ht chn khng. - Tinh ch: 100 ml n-hexane c thm vo dch lc, lc 5 pht, sau khi lng, b lp trn, lp li bc ny 1 ln na. Thm vo lp di 50 ml nc kh ion, v 100 ml chloroform. Lp dung mi trn c chit li 2 ln vi 50 ml chloroform. Trn chung chloroform chit c. Cho bay hi trong c quay cho n khi gn kh 400C, trnh nh sng. Bnh cha chloroform c trng ra bng choloroform chuyn sang ng nghim v lm kh di kh nit. 2ml methanol c thm vo, nh siu m, lc qua giy lc c kch thc l lc nh hn 0,45 m. Lm kh di kh nit. 500 l methanol c thm vo v em phn tch trn HPLC.

Mu go nhim

Go khng nhim + c t nm

Hnh 1: Sc k mu go nhim c t v mu go khng nhim+c t chun. 3. Kt qu v bn lun 3.1. nh gi kh nng tch chit v tinh ch ca phng php. Da vo tnh cht tan trong dung mi hu c ging nhau ca cc c t nm phn tch (AFB1, OTA, CIT) chng ti a ra c phng php tch chit v tinh ch nh trn. nh gi kh nng tch chit v tinh ch da vo kh nng thu hi. Kh nng thu hi c xc nh bng cch thm c t (AFB1, OTA, CIT) vo cc mu go sch, sau nh lng cc c t ny trong cc mu go gy nhim. Xc nh t l phn trm nh lng c. Mi th nghim lp li 3 ln. Kt qu c trnh by trong bng 1. Bng 1: T l thu hi v lch chun tng i RSD ca cc c t kho st

c t AFB1 CIT OTA

Nng gy nhim 2 ng/g 2 ng/g 2 ng/g

T l thu hi (%) 96,9 84 101,9

RSD (%) 2,7 5,2 5,7

Tt c kt qu th nghim u nm trong khong gii hn cho php ca Chu u (S 472/2002 v 26/2002), nng gy nhim gia 1-10 ng/g, t l thu hi chp nhn trong khong 70-110% v RSD nh hn 20%. 3.2. Gii hn pht hin v gii hn nh lng Gii hn pht hin (LOD) v gii hn nh lng (LOQ) c xc nh bng cch ly 3,3 v 10 ln lch chun (theo th t) chia cho dc ca ng cong chun tng loi c t. Kt qu th nghim c trnh by trong bng 2. Bng 2 : Gii hn pht hin v gii hn nh lng cc c t th nghim.

LOD (ng/g) LOQ (ng/g)

AFB1 0,048 0,144

CIT 0,056 0,168

OTA 0,086 0,260

Vi kt qu ny chng ta thy phng php ny kh nhy, cho php pht hin c c t nm quan tm di 0,1 ng/g; v gii hn nh lng di 0,3 ng/g. 3.3. Kt qu phn tch Kt qu phn tch 64 mu go ti 4 ch ln Nha Trang trnh by trn bng 3, 4, 5, 6. Bng 3: Kt qu phn tch trn mu go Ch m Thi im ly mu Ma ma Ma ma Ma ma Ma ma Ma ma Ma ma Ma ma Ma ma Ma kh Ma kh Ma kh Ma kh Ma kh Ma kh Ma kh Ma kh c t (ng/g) CIT <LOD ND ND ND ND ND <LOD ND ND ND ND ND ND ND ND ND

K hiu 1(a) 2(a) 3(a) 4(a) 5(b) 6(b) 7(b) 8(b) 9(a) 10(a) 11(a) 12(a) 13(b) 14(b) 15(b) 16(b)

AFB1 <LOQ 2.33 0.45 0.47 0.52 <LOQ 0.43 0.49 0,40 ND <LOQ ND <LOQ ND ND ND

OTA <LOD ND <LOD <LOD <LOD ND ND <LOD 0.28 0.36 <LOQ <LOQ ND ND ND <LOQ

Bng 4: Kt qu phn tch trn mu go Ch Xm mi K hiu 1(a) 2(a) 3(a) 4(a) 5(b) 6(b) 7(b) 8(b) 9(a) 10(a) 11(a) 12(a) Thi im ly mu Ma ma Ma ma Ma ma Ma ma Ma ma Ma ma Ma ma Ma ma Ma kh Ma kh Ma kh Ma kh AFB1 <LOQ 7.20 <LOQ 0.50 0.52 <LOQ <LOQ ND ND ND ND 0,2 c t (ng/g) CIT ND ND ND ND ND ND ND <LOD ND ND ND ND OTA <LOD <LOD <LOD <LOD <LOD <LOD ND <LOQ ND 0,36 0,30 ND

13(b) 14(b) 15(b) 16(b)

Ma kh Ma kh Ma kh Ma kh

ND ND ND <LOD

ND ND ND ND

ND ND ND <LOQ

Bng 5: Kt qu phn tch trn mu go Ch Phc Thi K hiu 1(a) 2(a) 3(a) 4(a) 5(b) 6(b) 7(b) 8(b) 9(a) 10(a) 11(a) 12(a) 13(b) 14(b) 15(b) 16(b) Thi im ly mu Ma ma Ma ma Ma ma Ma ma Ma ma Ma ma Ma ma Ma ma Ma kh Ma kh Ma kh Ma kh Ma kh Ma kh Ma kh Ma kh AFB1 0,41 0,45 <LOQ 2,40 ND ND <LOD <LOQ ND <LOQ <LOQ ND ND ND <LOD <LOQ c t (ng/g) CIT ND ND <LOD ND ND ND ND ND ND ND ND ND ND ND ND ND OTA ND <LOD <LOD 0,48 <LOQ ND ND ND 2,78 0,32 <LOQ <LOQ <LOQ ND ND ND

Bng 6: Kt qu phn tch trn mu go Ch Vnh hi K hiu 1(a) 2(a) 3(a) 4(a) 5(b) 6(b) 7(b) 8(b) 9(a) 10(a) 11(a) 12(a) 13(b) 14(b) 15(b) 16(b) Thi im ly mu Ma ma Ma ma Ma ma Ma ma Ma ma Ma ma Ma ma Ma ma Ma kh Ma kh Ma kh Ma kh Ma kh Ma kh Ma kh Ma kh AFB1 <LOQ <LOQ 0,50 3,31 ND 0,43 ND ND ND ND <LOQ 0,25 ND ND <LOD ND c t (ng/g) CIT ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND OTA <LOD <LOD <LOD 0,42 <LOQ <LOQ ND ND ND 0.41 <LOQ ND ND <LOD ND <LOQ

(a) Ghi Ch : Gi go 4500 ng/kg go. (b) : Gi go > 4500 ng/kg go. ND : khng pht hin. LOD : Gii hn pht hin. LOQ : gii hn nh lng. Tnh hnh nhim CIT, OTA: Trn 64 mu khng pht hin c mu no nhim CIT. T l nhim OTA 22/64 mu kim tra (34%), nhng ch 9/64 (14%) nh lng c, trung bnh 0,63 ng/g. Ch c mt mu nhim 2,78 ng/g, cc mu cn li u nh hn 0,5 ng/g go. Kt qu nghin cu trn go min Nam nm 2001, cho thy nhim OTA l ch yu, c mu nhim n 26,2 ng/g (Trung T.S., 2001). So vi s liu ny, kt qu nghin cu ca chng ti thp hn nhiu.

Tnh hnh nhim AFB1: t l nhim 36/64 mu (41%), mu nhim cao nht 7,2 ng/g. Trung bnh cc mu nh lng c l 1,45 ng/g. Trong iu tra nghin cu 1998, mc nhim Aflatoxin B1 trn go ti Tp H Ch Minh cho thy t l nhim l : 33% , c mu nhim n 27 ng/g (Son C.P.N., 1998). Trong nghin cu ca chng ti, t l cc mu nhim AFB1 cao hn, nhng mc nhim thp hn. Tnh hnh nhim AFB1 khc nhau theo ma. Trong ma ma, t l nhim AFB1, 27/32 (84%) mu nhim AFB1, trong c 17 mu (53%) trn gii hn nh lng, trung bnh 1,26 ng/g, 4 mu nhim trn 2 ng/g, mu nhim cao nht l 7,2 ng/g. Trong ma kh, t l nhim AFB1: 9/32 (28,1%), ch c 3 mu nh lng c trung bnh 0,28 ng/g). Ma ma, m khng kh cao, mc nhim AFB1 cao hn nhiu so vi ma kh (p < 0,05). iu ny cho thy nh hng ln ca m ca khng kh n mc nhim c t nm iu tra trn go bn ni a, cng cho thy mc cn thit phi ch n bo qun go ti cc kho nh l v trong khi bn ti a phng nc ta. S khc nhau gia lng c t nm mc nhim vi 2 nhm go c gi bn khc nhau (nhm t 4500 ng/kg go tr xung v nhm trn 4500 ng/kg go) (p<0,05), nhm go r tin trong ma ma t l nhim l 16/16 (100%), trong c 10 mu nh lng c, trung bnh 1,8 ng/g go. iu ny cng cho thy c lin quan n vic bo qun go. Go c gi tr ln hn thng c ch gi gn hn. V cch gii thch khc l go c, km phm cht c em bn vi gi r hn. So vi gii hn cho php ca WHO th tt c u nm trong gii hn cho php. Tuy nhin cc nc pht trin, gii hn ny ngy cng c h xung, AFB1 khng c vt qu 5 ng/g ng cc, th mc nhim c t AFB1 vn cn nm trong gii hn chp nhn c (ch c 1 mu vt qu tiu chun ny). Tuy vy vi ch n nhiu go nh hin nay nc ta, nht l nhng ngi lao ng nng (trn 500 g go/ ngi/ ngy), cho thy mc nhim s ng k ; mu nhim cao, c th nhim trn 3,5 g/ ngi/ ngy. Theo Wild v cng s (1992), ch n hng ngy 1.4 g Aflatoxin v cng vi mt s yu t khc l yu t nguy c gy ung th gan. 4. Kt lun v ngh - Phng php nh lng c t nu trn c th phn tch ng thi cc c t nm AFB1, OTA, CIT. Vi gii hn nh lng theo th t l 0,15 ; 0,17 ; 0,26 ng/g. - Trong 3 c t nm kim tra, nhim AFB1 l ch yu. Khng c mu no nhim CIT, nhim OTA 34%, mc nhim thp, trung bnh cc mu nh lng l 0,63ng/g, mu nhim cao nht l 2,78 ng/g. T l cc mu nhim AFB1 trn go bn ti cc ch Nha Trang kh cao 42 %. T l nhim AFB1 khc nhau gia cc ma, ma ma t l nhim l 84% cao hn ma kh (p<0,05), trong go r tin t l nhim l 100%. Mu nhim AFB1 cao nht l 7,2 ng/g. - Cn tip tc nghin cu phn lp loi nm mc sinh c t ny trn go, xc nh iu kin bo qun go thch hp, gim nhim c t nm AFB1 trn go, nht l trong ma ma.

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