DANH GIA MU'C DO

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DANH GIA MU'C DO 6 NHIEM CAC CHAT GAY UNG THU' NHOM POLYCYdic AROMATIC HYDROCARBON (PAH)

TRONG THU'C PHAM 6 HA NOI VA TP HO CHI MINH

Le Hdng Dung', Nguyen Vdn ST ^ vd CS.

• Muc tieu: Chuan hoa ky thuat sac ky long hieu nang cao de phan dch 15 hgp chat 6 nhiem nhom hydrocarbon thom da nhan (PAH) va budc dau xac dinh mirc dp 6 nhiem cac PAH trong 15 loai thuc pham che bien bang nirdng, ran, quay thupc 3 nhom thit, thiiy san va nguon goc thuc vat.

Phucmg phdp: Mau thuc pham dugc thiiy phan bang KOH trong ethanol, cac PAH dugc chiet bang hexane va lam sach qua cpt chiet pha ran loai Si. Cac PAH dugc dinh dnh va dinh lugng bang ky thuat sac ky long hieu nang cao sir dung cpt tach LC-PAH, phat hien bang detector PDA va detector huynh quang. Ket qud: Phuong phap phan tich thu dugc cd dp nhay cao doi vdi hau het cac PAH, vdi gidi han phat hien tir 0,002 ppb den 0,06 ppb. Phuong phap eo dp lap lai tot, vdi he so bien sai nam trong khoang 6,6% den 20,7% khi phan tich lap lai mau chuan cd nong dp lOppb. He so thu hoi nam trong khoang cho phep la tir 55,5% den 83,6%. Ket qua phan tich mau cho thay hau het cac mau thyc pham che bien bang nudng, ran, quay deu bi d nhiem cac chat PAH, trong do cd benzo(a)pyrene. Cac mau thyc pham c6 ham lugng benzo(a)pyrene >5 ng/g (gidi ban quy dinh ciia EU) gom cha ca, banh ran man, banh chung ran, dau phu ran, thit xien nudng, nem ran, chan ga nudng, thit nudng cha, ga nudng va banh quay, trong d6 thit nudng cha va banh quay chiia nhieu B(a)P nhat, vdi ham lugng tuong img la 14,0 ng/g va 9,4 ng/g. Ket lugn: Cac sd lieu phan dch budc dau se gop phan danh gia mirc dp 6 nhiem cac chat dpc hai PAH cho cac nghien ciiu phoi nhiem va danh gia nguy co tiep theo, gop phan bao ve sire khoe cpng dong.

Til- khoa: PAH: thirc phdm che bien: HPLC

L D A T V A N D E , hiiu ca. O nhiet do cao, cac hgp chat hdu Cac hydrocarbon da nhan t h a m c a bj nhiet phan thanh cac phan tir nhd (Polycyclic aromaric hydrocarbon - PAH) h a n (chii yeu la cac gde ty do), sau dd la mgt nhdm gdm nhieu chat hda hgc cd chiing tai kSt hgp vdi nhau thanh cac hgp cau tnie vdng thom, dugc sinh ra do sy chat t u a n g ddi ben la cac PAH. Day la ddt chay khdng hoan toan cac hgp chSt mpt nhdm chat dugc c a quan bao ve mdi

' Ths, KS - Vien Dinh ducmg - KS - Vien Dinh duong

81

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TC.DD &TP5 (3+4) - 2009

tradng My (EPA) va Uy ban chau Au (EC) xSp vao nhdm cac chat d nhiSm uu rien do cd kha nang gay dot biSn gen va gay ung thu dugc thS gidi biSt dSn hr rat lau. Khi phan tich khdi va tan thudc la, ngudi ta phat hien nhieu hgp chat PAH, trong dd benzo(a)pyrene dugc xera la hgp chat nguy hiem nhat gay benh ung thu.

Ngoai thudc la, nhung ngudn d nhiSm cac hgp chat PAH chinh trong mdi tradng la khi thai cua cac phuang rien giao thdng, ngudn nudc thai va khi thai cdng nghiep (khu vyc luyen kim, nhiet dien sir dung than lam nguyen lieu...), chay rimg, boat ddng ciia nui lira, d nhiSm dau md trong dat va nudc [1].

Do sy d nhiSm mdi trudng rii- rat nhieu ngudn nhu vay, nguy ca d nhiem cac hgp chat PAH vao thyc pham la rat cao trong timg chang ciia day chuySn thyc pham, dd la sy d nhiSm tir dat, nudc va khong khi. Tuy nhiSn, cac hgp chat PAH cdn cd thS hinh thanh tryc tiep trong thyc pham do qua trinh chS bien d nhiet dp cao nhu nudng, quay, ran, hoac d nhiet do thap ban nhu bun khdi... Mac dii chua rd rang nhung mdt sd ca chS hinh thanh PAH trong thyc pham da dugc xac djnh, dd la d nhiet do cao, md ndng chay rai xudng tiSp xtic vdi lua va bj chay khdng hoan toan, hoac PAH dugc tao thanh khi cac loai thjt bj nhiet phan d nhiet do cao.

Mpt sd nghiSn ctiu vS mirc dp d nhiSm PAH trong thyc pham do qua trinh ehS biSn cho thay hara lugng mgt sd PAH cao tdi 200 |ag/kg trong thjt va ca bun khdi [I]. Trong thjt quay, rairc d nhiSm trung

binh cua cac PAH la 130 yg/kg so vdi ham lugng trung binh trong thyc pham chua chS biSn la rii 0,1-1 yg/kg. Cac san pham dau thyc vat ciing la mdt ngudn dS bj d nhiSm cac PAH do mdt sd cdng doan trong qua trinh che bien nhu say tryc riSp bang lira, khdi va san pham dot chay khac cd thS tiSp xiie vdi dau hoac hat chiia dau.

Cac PAH cdn cd thS cd mat trong cac san pham che va ca phS do qua trinh say va rang san phara. Mdt nghiSn cim d Phan Lan cho thay ca phS xay va la che kho chiia PAH tdi 100-200 jiig/kg and 480-

1400 yg/kg tuong iing [1].

Ddi vdi cac chat gay ung thu nhdm PAH, d Viet Nam hien nay chi cd mgt sd nghien cim trong ITnh vyc mdi tradng [2, 3, 4]. Viec sir dyng cac thyc pham cd ngudn gde ddng vat bang phucmg phap nudng, quay, ran, bun khdi ... la rat phd bien d khu vyc thanh thj. Nhiing mdn an til- cac san pham nhu vay la ngudn d nhiSm quan trgng cac hgp chat PAH qua dudng an udng va can phai dugc danh gia. Vi vay, nghiSn ciru nay dugc thyc hien nham danh gia mire do d nhiSm 15 chat thudc nhdm cac hydrocarbon da nhan thom (PAHs) trong thyc pham thudng gap d Ha Ndi va TP Hd Chi Minh.

II. VAT LIEU VA PHI OnVG PHAP NGHIEN ClTU .-' ••-••-- • Hda chat, dung cu

Cac hda chat su dyng trong nghien ciiu deu thudc loai tmh khiet phan tich. 15 chat chuan PAH gdm Naphtha- lene, Acenapthene, Fluorene, Phenan- threne, Anthracene, Fluoranthene, Pyrene,

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TC.DD & TP5(3+4) - 2009

Benzo(a)anthracene, Chrysene, Benzo(j )fluoranthene, Benzo(k)fluoran-

thene, Benzo(a)Pyrene, Dibenzo(a,h)anthracene, Benzo(ghi)Pyrene va lndeno( 1,2,3cd)pyrene dugc mua tir hang Dr Ehrenstorfer (My). Cdt chiSt pha ran (SPE) silica (lOOmg, 3ral) la cua hang Alltech (my). De tai sir dyng he thdng HPLC Alliance (Waters, My). Cdt sic ky la loai LC-PAH (250mm x 4,6mm x 5mm) (Supelco, My). Pha ddng cho HPLC gdm acetonitril (A) va nudc cat (B), vdi chuang trinh dung mdi nhu sau:

0-5 phut (40%A), chuySn 100%oA trong 25 phut, 30-46 phut (100%oA), 46-50 phut (40%)A). Tdc do ddng pha ddng la Iml/phiit. Nhiet do budng cdt la 30°C. De- tector PDA dat d 254nm, detector huynh quang dugc lap chuang trinh budc sdng dS cd thS xac djnh 15 chat PAH.

Quy trinh phan tich mau

De tai ap dyng quy trinh phan tich cac hgp chat nhdm PAH theo Takatsuki K va cdng sy [5], vdi mdt sd thay ddi nhd va dugc tdm tat nhu sau: Can 50 g mau ddng nhat vao binh cau day trdn 500 ml, thSm 200 ml ethanol, 35 ml KOH 50%, 2 g Na2S.9H20, dun hdi luu nhe trong 2 gid. De nguoi, thSm 150 ml hexane, chuySn vao binh gan. Trang binh cau bang 20 ml ethanol va chuyen vao binh gan.

ChiSt Idng-ldng va chuyen Idp hexan vao mdt binh gan thir 2. Chiet lai ldp nudc 2 lan bang 150 ml va 100 ml hexane, gdp Idp hexane, rii-a bang 100 ml nudc cat.

Lgc qua bdng thuy tinh cd chiia mdt ldp Na2S04 khan. Djch chiet dugc Cd quay

chan khdng dSn khd (< 40"C). Hda tan phan cdn lai bang 2ml n-hexane va lam sach bang cdt chiSt pha ran (SPE) loai silica. Hoat hda cdt SPE lan lugt bang 2ml n-hexane, 2ml dichlormathane, 2 ml n- hexane. Cho 2ml mau da hda tan trong n- hexane vao cdt SPE, rira cdt bang 2ml n-hexane. Rira giai PAH bang 2ml hdn hgp n-hexane/dichlormathane, ty IS 70/30 vao dng nghiSm. Thdi khd mau bang khi nita, hda tan lai bang 2ral acetonitrile.

Lgc dung djch acetonitrile chiia PAH qua mang lgc 0.45 ym va bam vao HPLC.

III. KET QUA NGHIEN CUU

3.1. Tham d|nh cac thong so phan tfch Xac dinh gioi han phat hien cac hop chat PAH

Gidi ban phat hien (Limit of detection, LOD) ddi vdi cac chat PAH dugc xac dinh tren nguyen tac tin hieu pic/nhieu dudng nSn (S/N) ~3. Phuang phap sir dyng detector huynh quang cd kha nang phat hien cac chat PAH d ndng do rat thap, khoang tir 0.002ppb vdi B(j)fluoranthene den O.Odppb ddi vdi naphthalene.

Khoang tuyen tinh cua phuong phap Day chuan cac PAH dugc chuan bj d cac ndng do 0.1 ppb, I ppb, lOppb, 50ppb,

lOOppb, 200ppb va 400ppb. KSt qua cho thay phuang phap cd mdi tuang quan tuySn tinh bac nhat giira dien tich pic va ndng do trong khoang 0.1 ppb den 400ppb, vdi he sd tuang quan R > 0.9970.

Sac dd mau chuan hdn hgp ndng do lOOppb dugc the hien d hinh la.

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Hinh 1. Sac do hon hop chuan PAH ndng do lOOppb (la) va cac PAH trong mau quay ran (lb) tren detector huynh quang. Dieu kien phan tich nhu- muc 2. Naph = naphthalene, ace = acenaphthene, ilur

= flurene, phe = phenanthrene, anth = anthracene, fluo = fluoranthene, pyr = pyrene, B(k)F = benzo(k)fluoranthene, B(-a)P = benzo(a)pyrene, D(a,h)A = dibenzo(a,h)anthracene, B(ghi)P = benzo(ghi)pyrene

Do lap lai va do ciia phuo'ng phap Them 0.8mi dung djch chuan hdn hop PAH cd ndng do 0.4 pg/ml vao 25g mau thit (mau nem ran), tiic la ndng do mdi chuan thSm vao la 12.8 ng/g mlu.

Phan tich lap lai 5 lan va he sd bien sai (RSD) ddi vdi cac PAH nam tning khoang 6,6% dSn 20,7%, nam trong gidi han cho phep cua tiSu chuan AOAC. Do thu hdi

ddi vdi cac chat PAH trong khoang tir 55.5% (indeno(l,2,3-cd)pyrene) den 83.6% (benz(a)anthracene), dat yeu eau eho phep cua tiSu chuan AOAC [6, 7].

3.2. Kit qua phan tich cac chat PAH trong mot so mau thuc pham

DS tai da thu thap 180 mau thuc pham tu 15 loai thit, thuy san va thirc pham ngudn gde thue vat ehS bien bf^ng

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TC.DD &TP5 (3+4) - 2009

cac each nudng, ran, quay tai hai thanh pho Ha Ndi va TP Hd Chi Minh va phan tich ham lugng 15 chat 6 nhiem PAH. Ket qua phan dch chung cho thay cac mau thuc pham deu bi nhiSm cac hgp chat PAH va a mirc do khac nhau. Ham lugng cao nhat tim thay ddi vdi cac chat cd dgc tinh thap hoac chua rd rang nhu naphthalene, fluorene, phenanthrene, anthracene, fluoranthene va pyrene, thS hien d cac hinh 2a va 2b. Ba loai PAH cd kha nang gay ung thu thudc nhdm 2A la benzo(a)pyrene, benz(a)anthracene va dibenzo(a,h)anthracene dugc tim thay d nong do thap ban. Ham lugng trung binh

Clia B(a)P va B(a)anthracene (n=6) trong 15 loai thuc pham thu thap tai Ha Ndi dugc tdng hgp d hinh 3. Hai chat nay cd nhiSu nhat trong thit nudng cha vdi muc 14,0 va 28,4 ng/g mau. Mdt nguySn nhan cd thS la do thit nudng cha chua nhiSu md, dugc dot true tiSp trSn than va md chay xudng bi chay khdng hoan toan, ddng thdi khdi va thit khi chay se bam trd lai vao san pham cudi ciing gay nSn su d nhiSm cao cac PAH ndi chung va ca B(a)P, B(a)anthracene. Mdt sd thuc pham khac bi nhiSm hai chat nay vdi ham lugng ciing kha cao la thit xien nudng, nem ran, va chan ga nudng.

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Hinh 2. Phan bo cac loai PAH trong cac loai thu-c pham khac nhau tai Ha noi (hinh 2a) va TP Ho Chi IVlinh (hinh 2b). Ten cac PAH phia ben phai tu-ong irng voi mij-c 6 nhiem trong moi cot

theo thu-tu-tir tren xuong duoi.

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TC.DD &TP5 (3+4) - lutty

Trong cac mau thu thap tai TP Hd Chi Minh, kSt qua tdng hgp d hinh 4 cho thay benzo(a)pyrene va benz(a)anthracene cd ham lugng cao nhat trong mau quay ran (n=6) va mau xiic xieh nudng, tu'ong ung la 9,4 ng/g vdi B(a)P va 25,8 ng/g ddi vdi B(a)anthracene. Ddi chieu vdi cac quy dinh qudc tS hien nay, mdi chi co mgt sd quy dinh chinh thuc cua LiSn minh chau Au doi vdi gidi ban B(a)P trong mdt sd san pham nhu dau/md, thit va ea bun khdi, sua cdng thiic tre em.

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Hinh 4: Mirc do nhiem B(a)P va B(a)anthracene trong 15 loai thyc pham of TP HCM I V . K E T L U A N .

Phuang phap phan tich cac hgp ch4t PAH bang ky thuat sac ky long hieu nang cao ket hgp detector huynh quang va detector PDA da dugc chuan hoa de xac dinh ham lugng cac chat 6 nhiem trong trong thuc pham che bien dang nudng, ran, quay.

Phuang phap thu dugc c6 dp nhay eao ddi vdi cac chat PAH, vdi gidi ban phat hien tren detector huynh quang tu ().005pg/kg dfin 0.06 pg/kg doi vdi 13 ch4t PAH va hai chat tren detector PDA co gidi ban phat hien la 1.2 pg/kg va 2.7 pg/kg. Do lap lai va do thu hdi

Clia phuang phap deu nam trong gidi ban cho phep Clia tieu chuan AOAC, dam bao do tin cay Clia ket qua phan tich mau thuc

Ket qua phan tich 180 mau thu thap d Ha Noi va TP Ho Chi Minh cho thay hau het cae thuc pham che bien bang nudng, ran hoac quay deu bi 6 nhicm cac PAH, trong do co benzo(a)pyrene la chat cd nguy ca gay ung thu nhom 2A. Day la nhiing ket qua budc dau gop phan xay dung bo sd lieu ve cac chat 6 nhiein chi diem quan trong nhom PAH, ddng thdi gop phan irdc lugng muc dp phai nhicm cac chat dpc hai PAH va phuc vu cho cac

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TC.DD &TP5 (3+4) - 2009

nghien ci'm danh gia nguy ca gay ung thu tiep theo. Ket qua nghien ciiu con cung cap thong tin cho ngudi tieu diing va cac nha quan ly ve mpt trong nhung nhom chat 6 nhiem quan trpng trong thirc pham dc gop phan bao ve siic khoe cpng ddng.

T A I L I E U THAM KHAO

1. EUROPEAN COMMISSION.

HEALTH and CONSUMER PRO- , „ TECTION DIRECTORATE-GEN-

ERAL. Polycychc Aromatic Hydrocarbons - Occurrence in foods, dietary exposure and health effects.

SCF/CS/CNTM/PAH/29 ADDl Final,

"'•' 4 December 2002

2. Kim Oanh, N.T, Nghiem, L.H., Phyu, Y.L., (2002). Emission of Polycyclic Aromatic Hydrocarbons, Toxicity and Mutagenicity from Domestic Cooking Using Sawdust Briquettes, Wood, and Kerosene. Environmental Science and

" ' Technology 36, 833-839.

3. Kim Oanh, N . T , Reutergardh L.B., Dung, N.T, (1999), Emission of Poly-

6,

cyclic Aromatic Hydrocarbons and Particulate MaUer from Domestic Combustion of Selected Fuels, Envi- ronmental Science and Technology 33, 2703-2709.

N.T. Kim Oanh, D.O. Albina, Li Ping and Xiaoke Wang (2005). Emission of particulate matter and polycyclic aromatic hydrocarbons from select cook- stove-fuel systems in Asia. Biomass and Bioenergy, Volume 28: 579-590 Takatsuki K, Suzuki S, Sato N, Ushizawa I (1985). Liquid chromato- graphic determinadon of polycyclic aromatic hydrocarbons in fish and shellfish. / Assoc Off Anal Chem:

68(5):945-949

AOAC. Peer-Verified Methods Pro- gram: Manual on Policies and Proce- dures, Arlington, VA. 1998.

http://www.aoac.org/vmeth/PVM.pdf Ludwig Huber (2007). Validadon and Qualification in Analydcal Laborato- ries, Second Edition,. Informa Health- care USA, Inc

Summary

DETERMINATION OF CONTAMINATION LEVEL OF POLYCYCLIC ARO- MATIC HYDROCARBON (PAH) IN FOODS IN HANOI AND HCM CITY

Objective: The aims of this study are to validate a HPLC method for the determination of PAH compounds in food, and to provide inidal data on PAH contamination levels in common foods from meat, fish and starch sources, which were processed by direct contact with headng source like grill, barbecue, or being fried in oil.

Method: Food samples were saponified by KOH in ethanol, and the PAHs were extracted by hexane, followed by cleaning-up by solid phase extraction (Si SPE). PAH compounds were iden- tified and quantified by HPLC technique using PDA and fluorescent detectors.

Results: The validated HPLC method can determine PAH compounds at very low level, with the range of detection from 0.()02ppb to 0.06ppb. The repeatability ofthe method is good with RSD ranges from 6.6% to 20.7% when analyzing PAH at lOppb level. The method provides acceptable recovery data, which range from 55.5% to 83,6% when analyzing at 10 ppb level.

Sample analysis data showed that most of the food samples were contaminated with PAH compounds, including benzo(a)pyrenc which is the most toxic PAH. Numbers of food items contaminated B(a)P with > 5 ng/g level (the limit set by EU), among that the grilled pork contained highest level of 14.0 ng/g.

Conclusion: This initial data on PAH contamination in common processed food can be used for fur- ther studies on PAH exposure as well as risk assessment studies for carcinogen PAH aiming to pro- tect health ofthe consumers.

Keywords: PAH: proccessed foods; HPLC