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TAPCHIKHOAHOC VACONG NGHE Tap 47, so 1,2009 Tr 55-63 XAC DINH DU Ll/gNG C H A T KHANG K H U A N HO SULFOAMIT TRONG THLFC P H A M B A N G P H D ' O N G P H A P S A C KI L 6 N G HIEU N A N G C A O ( H P L C ) PHAN 2. XAC OiNH DU Ll/QNG S U L F A G U A N I D I N , S U L F A M E T H O X Y P Y R I D I N , S U L F A D O X I N VA S U L F A M E T H O X A Z O N T R O N G C A C M A U THU'C AN CHAN NUOI VA MAU T H U C PHAM CHU OlNH BlNH, NGUYEN PHLfONG THANH, NGUYEN THI A N H NGUYET, PHAM LUAN 1. G i d l THIEU Ngay nay vdi cude sdng ngay cang hien dai van de ve an toan thuc pham ludn dugc dat len hang dau, bdi nd anh hudng rat Idn den sire khoe con ngudi, nd ciing la mdt van de Idn ciia cac ca sd chan nudi, san xuat, che bien va nhat la xuat khau san pham thuc pham. Do vay, can thiet phai kiem soat, kiem djnh cac chat ddc hai, cd the gay ddc hai trong thuc pham. Sulfoamit (SAs) la mdt hg chat khang khuan phd rdng, nd la thanh phan cua cac loai thudc ho, sdt, cam ciim. Ben canh dd sulfoamit cdn dugc sir dung rdng rai trong thirc an chan nudi gia siic, gia cam va thuy hai san de phdng mdl sd benh nhiem khuan cho vat nudi. Viec tu do sir dung eac chat nay d nudc ta hien nay cd the dan den tdn du mdt lugng Idn qua gidi ban cho phep trong ca the ddng vat. Vi vay neu khdng kiem soat thdi gian dimg thudc trudc khi thu hoach lam thuc pham cd the dan den viec xuat hien cac chiing khang thudc. va khi cac vi sinh vat khang thudc xam nhap vao ca the con ngudi, cd the truyen kha nang khang thudc cho cac vi sinh vat gay benh vdi tan suat cao ban nhieu so vdi ngoai tu nhien... Cho nen yeu cau can phai cd phuang phap tdt de tach va xac djnh chinh xac ham lugng cac sulfoamit trong thirc an chan nudi va lugng tdn du ciia chung trong san pham tir ddng vat. Cd rat nhieu phuong phap xac djnh cac sulfoamit trong cac ddi tugng mau khac nhau nhu phuang phap dien di mao quan [3, 8], phuang phap sac ki Idng hieu nang cao (HPLC) [6], phuang phap sac ki Idng khdi phd (LC-MS) [7].... Nhu da de cap trong [1] phuang phap sac ki long hieu nang cao HPLC vdi detector UV - Vis la mgt phuang phap thich hgp vi nd cd do chgn Igc va do nhay kha tdt dap irng dugc yeu cau phan tich du lugng chat khang khuan trong mau thirc an chan nudi va cac mau thuc pham. 2. DUNG CU VA HOA CHAT 2.1. Hoa chat Sulfadoxin (SDO). sulfaguanidin (SGU), sulfamethoxypyridin (SMP) va sulfamethoxazon (SMX) loai chat ddi chieu hoa hgc, tinh khiet 99,9% ciia Vien Kiem nghiem Dugc, Bg Y te Hai Ba Trung, Ha Ngi. 55 Dung djch goc 1000 pg/ml dugc pha tir lugng can trong metanol tinh khiet HPLC, dung djch gdc duac bao quan trong tu lanh d 4°C. Cac dung djch chuin dugc pha tir dung djch gdc bing dung mdi gidng nhu pha ddng chay sic ki d^ tranh pic ao do sai khac dung mdi gay ra. Cac dung dich loang chi pha trong ngay, sieu am loai khi va Igc qua catridge loai 0,2 pm trudc khi cho chay HPLC.Metanol (MeOH), axetonitril (ACN), etylaxetat, n-hexan loai tinh khiet HPLC, Scharlaul, Tay Ban Nha. Axit axetic bang, muoi natri axetat, loai tinh khiet phan tich, Merck. Dung djch dem pH = 4,5. Can chinh xac mdt lugng muoi CH3COONa.H20 loai tinh khiet phan tich da tinh trudc, boa tan bing khoang 900 ml nudc cit hai lan, chinh pH ciia dung djch bing axit axetic 20% loai tinh khiet boa hgc tdi pH = 4,5. Chuyen vao binh djnh muc dung tich 1000 ml va djnh mtxc tdi vach bing nudc cat. Lgc qua catridge loc 0,45 pm, loai khi bang sieu am trudc khi chay HPLC. Cac boa chit khac: nudc cit hai lin, tricloaxetat (TCA) loai tinh khiet phan tich, Na2S04 khan loai tinh khiet phan tich, Merck. 2.2. Dung cu May quang phd hip thu phan tir UV-Vis 8453 cua hang Agilent - My (dugc dieu khien bing phin mem Chemstation cho phep quet phd tir 190 - 1100 nm). May do pH TIM 800 ciia hang Radiometer- Dan mach vdi dien cue Red - Rod dimg de do pH va bo chinh nhiet do tu ddng. May sic ki Idng hieu nang cao bao gdm: Bd loai khi cho dung mdi degassor 7100, bom cao ap 4 kenh LC 7600 Pump ciia hang Hitachi - Nhat ban, van bom mau sau chieu ciia Reodyn My, detector tir ngoai kha kien UV- 200 Linear - My, may ghi Kipp- Zonen ciia Dire. May sinh khi Nj ciia hang Chropack, Dire, may khuiy tir giai nhiet, may li tam va cac dung cu thi nghiem can thiet khac. 2.3. Mau phan tich a. Mau thuc phim (tdm ddng lanh, gan ga va gan Ign) dugc thu thap tir cac chg dau mdi, mau iiy v^ dugc phan loai va bao quan trong tii lanh d 4°C. Mau dugc nghien mjn bang cdi ma nao sau dd xir li theo quy trinh dudi day [3, 4]. b. Mau thirc an chan nudi thu thap tir cac ca sd che bien bao gdm cac mau thirc an cho tdm, thirc an cho gia cam va thirc an cho gia siic. Mau dugc xir li theo hai quy trinh. Quy trinh 1: Miu thirc an chan nudi dugc nghien nhd, trdn deu. Can mdt lugng chinh xac 0,2g cho vao binh ndn 200 ml. Them vao 10 ml dung mdi etylaxetat 20% tricloaxetat (TCA) va 5 g Na2S04 khan, lac deu tren may lac trong 30 phut. Sau dd dem li tam vdi tdc do 5000 vdng/phiit khoang 10 phut. Gan lay phan dung mdi trong, qua trinh chiet dugc lap lai 3 lan. Djch chiet dugc gom lai (khoang 30 ml) va lam bay hai djch chiet bang khi N2 d 50°C. Phan can con lai boa tan va djnh mire thanh 10ml bang pha dgng chay sac ki. Lgc dung djch qua cartridge 0,2 pm trudc khi bam vao cot HPLC. Quy trinh 2: Can mdt lugng chinh xac 0,2 g mau da dugc nghien ddng the, chuyen vao binh ndn 200 ml. Them vao 10 ml dung mdi ACN 20% tricloaxetat (TCA) lac deu tren may lac 30 phut. Rdi them 5 ml n-hexan va trdn deu trong be sieu am trong 5 phiit. Dem hdn hgp li tam vdi tdc do 5000 vdng/phiit. Gan lay phan djch trong, qua trinh chiet dugc lap lai 3 lan.Djch chiet dugc gom lai (khoang 30 ml) va lam bay hai bang khi NT d 50°C. Phan can cdn lai djnh mire thanh 10 ml bang hdn hgp pha ddng. Lgc dung djch qua cartridge 0,2 pm trudc khi bam vao cgt HPLC. 56 2,5gmau 1 Them2,5gND;SOi khan 2 Tliem 10ml ACN 3 l i e chiet trong 30 phiit 4 Li tam lOphiit (SOOOvong/phiit) 1 Can Ba con lai + NB;SOi 1 Them 1 Oml ACN 2 Sieu am 10 phiit 3 Li tam 10 phiit (5000v6ng'phut) ACN AnsT Loai bo can 1 Co can dung dich duai dong khi N2 6 50°C 2 Sieu am can thu duac v6i 3ml hon hop nuoc + axetonitril trong 5 phiit 1 Hoat hoa cot bang 10 ml MeOH Lam sach bang cot chiet SPE CI 8 2 Nap mau 3 Rira cot bang nuac 4 Riragiai 10 ml MeOH 1 Oml dung dich SAs trong MeOH 1 Co can dung dichbangd6ngkhiN2 6 50°C 2 Dinh miJC thanh 1 Oml bang pha dong chay HPLC ,, 3 LitamlOphut(2500v6ng/phiit) 4 Loc qua catridge loc 0,2/xm Phan tich bSng HPLC Hinh 2.3.1. Quy trinh xir li mau thuc pham 2.4. Cac dieu kien phan tich Tir cac dieu kien da khao sal va ket qua lhuc nghiem cac dieu kien tdi uu de tach va djnh lugng SDO, SGU, SMP va SMX nhu sau [ 1 ]: Cac dieu kien HPLC Van bam mau 6 chieu. the tich bam mau: 20 pi Cot tach Supelcosil C|8 (25 cm x 4,6 mm; 5 pm) Thanh phan pha ddng: 20% ACN 80 % dung djch dem axetat 0,01 M, pH = 4,5 chay dang ddng Tdc dg pha ddng: F = 0,8 ml/phiit Detector U V - V i s : X = 270 nm Rise time: 0,1 giay Range: 0,01 (AUFS) Nhift dg: Nhiet do phdng May ghi: Tdc do giay: 1 mm/ phut The ghi: lOmV 57 3. KET QUA VA THAO LUAN 3.1. Xac djnh hieu xuat thu hoi 3.1.1. Mau thurc an chan nuoi Vdi hai quy trinh xir li mau nhu tren da de cap, tien hanh xu li mau va phan tich trong ciing dieu kien. Ket qua thu dugc trong bang sau. Bdng 3.1.1. Ket qua xac djnh hieu suat thu hdi theo phuang phap 1 \q^ppm) SAs ^ \ 0 0,04 Lan 1 Lan 2 H Lan 3 Hpic Cx Hpic Cx tlpic Cx (mm) (ppm) (mm) (ppm) (mm) (ppm) (ppm) (%) SGU 36,0 48,0 0,0288 47,0 0,0321 47,0 0,0321 0,0310 77,50 SMP 0,0 8,0 0,0248 9,0 0,0292 9.0 0,0292 0,0280 70,00 SDO 0,0 7,0 0.0282 7,0 0,0282 7,0 0,0282 0,0292 70,50 SMX 0,0 6,5 0,0297 6,0 0,0268 7,0 0,0327 0,0297 74,33 Bang III.l .2. Ket qua xac djnh hieu suat thu hoi theo phuang phap 2 ^KI (ppm) SAs \ ^ 0 0,04 Lin 2 Lan 1 Lan 3 Cx (ppm) Cx Hpic (mm) (mm) (ppm) (mm) Cx (ppm) Hpic Hp,c //(%) (ppm) SGU 45 55,0 0,0288 57,0 0,0350 57,5 0,0360 0,0332 83,00 SMP 0 9,0 0,0292 9,0 0,0292 9,0 0,0292 0,0292 73,00 SDO 0 7,0 0,0282 7,0 0,0282 7,0 0,0282 0,0282 70,50 SMX 0 6,5 0,0297 6,5 0,0297 7,5 0,0356 0,0317 79,25 Ap dijng phuang phap thdng ke dk danh'gia su khac nhau gitra hai quy trinh xir li mau. Quy trinh xir li mau hai cho hieu suat thu hoi cac SAs cao ban nen cac mau thirc an chan nudi dugc xu Ii theo quy trinh nay. 3.1.2. Mau thu'c pham Sir dung quy trinh phan tich mau d tren d^ danh gia hieu xuit thu hoi ciia tong thS quy trinh xu li mau sir dung mau la san pham tdm ddng lanh. Mau tdm ddng lanh dugc d^ ra ddng sau dd 58 nghien nhd va can mdt lugng chinh xac va tien hanh xir li. Mdt mau khac cung tien hanh tuang tu nhung cd them cac chat phan tich. Tien hanh lam lap mdi mau ba lan va ket qua dugc chi ra lrong bang 3.1.2 va hinh 3.1.2 yy • - - -'—"-- -Ezr - ~- t— \ : :.: zr. • • .'S-' "ys - '." .'-LZiziiy —Z7. i - " " , ^-^3 ~TT-~7 1 •^.' '.'~7~ . "' i -'. — -) -1 -,—^—. -T- 1 -t ' ', : ,I i 1 .^_ j ! ' 4 q H I- — (. 1 r pMSil ^_;. t ^z zsryri'-.z^TZ'^" 1— _ E>< -_ ,_ ; -l i __^ 1 i f r "' " ' 1 ,_ i . — J — 1 ': 1 "" ^± ""m- ^fe^ _ i — r i (a) y (b) Hinh 3.1.2. SSc do ciia mai torn a. mau tom chua them, b. mlu torn da them 4 chat nong dp 0,004ppm thir tu cac pic tren s k d6 la 1. SGU, 2. SMP. 3. SDO, 4. SMX Bdng 3.1.2. Ket qua xac djnh hieu suat thu hdi cac SAs Cac SAs SGU SMP SDO SMX C,(pg/ml) Cx(pg/ml) H% 0,0400 0,0346 86,50 0,0600 0,0485 80,83 0,0800 0,0646 80,75 0,0400 0,0300 75,00 0,0600 0,0462 77,00 0,0800 0,0646 80,75 0,0400 0,0300 75,00 0,0600 0,0480 80,00 0,0800 0,0630 78,75 0,0400 0,0300 75,00 0,0600 0,0530 88,89 0,0800 0,0720 90,00 Nhu vay trong khoang ndng do 0,04 - 0,08 pg/ml, hieu suat thu hdi cac chat khang khuan SAs dat tir 75 - 90%, hi?u suat thu hdi nhu vay la dat yeu cau. Tuy nhien ket qua xac djnh hieu 59 xuit thu hoi tren cd lap lai kem vi vay cac mau phan tich dugc tien hanh bang phuang phap them chuan. 3.2. Phan tich mot sd mau thirc Phan tich mdt sd mau thirc an chan nudi 1 1 .1 1 • • i ' J h 1 ! 1^ ..__ 2 *"" •"'B i ^ ^^^^ 1 .1 (a) (b) Hinh 2.2.1. Sac do ciia mau thirc an chan nudi a. mau thirc an cho gia cam, b.mau thirc an cho gia siic Mau phar tich Thirc an gia cam Thirc an gia sue 'hire an tdm, ca SDO(ng/g) SGU(pg/g) SMP(^g/g) SMX(^g/g) Mil

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