Phan tich tetrecyline

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    10-Aug-2015

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<ol><li> 1. B GIO DC V O TO TRNG I HC CN TH KHOA KHOA HC T NHIN XC NH HM LNG NHM TETRACYCLINES TRONG THY SN V SN PHM THY SN BNG SC K LNG GHP KHI PH (LG/MS/MS) ti: CN B HNG DN TS. Nguyn Th Thu Thy SINH VIN THC HIN Dng Nht MSHV: M2014007 Nguyn Hu Tr MSHV: M2014010 Bi Thy Vy MSHV: M2014011 BO CO SEMINAR </li><li> 2. Xc nh hm lng nhm Tetracyclines trong thy sn v sn phm thy sn ti Thnh Ph Cn Th Mc tiu: </li><li> 3. Ni dung bo co Gii thiu Phng Php Phn Tch nh Gi Kt Qu Phn Tch Ti Liu Tham Kho </li><li> 4. 1.1. S lc v khng sinh nhm Tetracyclines Tetracyclines l nhm khng sinh c ph khng khun rng, c kh nng khng cc loi vi khun gy bnh trn thy sn S dng trong thi gian lu di lm cho vi khun ln thuc lm nh hng nghim trng n h min dch ca thy sn, 1. GII THIU nh hng xu n sc khe ngi tiu dng gy ra nhiu bnh v thn, d tt bm sinh v cc bnh v xng,... Tetracyclines l nhm khng sinh c ph khng khun rng, c kh nng khng cc loi vi khun gy bnh trn thy sn Tetracyclines l nhm khng sinh c ph khng khun rng, c kh nng khng cc loi vi khun gy bnh trn thy sn Tetracyclines l nhm khng sinh c ph khng khun rng, c kh nng khng cc loi vi khun gy bnh trn thy sn </li><li> 5. Khng sinh nhm tetracyclin bao gm 3 khng sinh chnh sau: tetracyclin (gi tt l TC), oxytetracyclin (gi tt l OTC) v clortetracyclin (gi tt l CTC). 1.1. S lc v khng sinh nhm Tetracyclines 1. GII THIU Tetracycline </li><li> 6. 1.1. S lc v khng sinh nhm Tetracyclines 1. GII THIU Oxytetracyclin </li><li> 7. 1.1. S lc v khng sinh nhm Tetracyclines 1. GII THIU Chlortetracyclin </li><li> 8. 1.2. Tc hi ca Tetracyclines 1. GII THIU nh Hng Ti ng Tiu Ha: + Thng gp nh n mt ngon, bun nn, chng nng rt d dy. + Bi nhim nm Candida albicans phi cha tr vi nystatin. + Vim rut do staphylocoque: thng nng ngi gi v tr em. </li><li> 9. 1.2. Tc hi ca Tetracyclines 1. GII THIU nh Hng Ti Gan: + Him gp nhng rt nng l thoi ha m gan do dng liu cao v theo ng tnh mch. + bnh nhn c thai 3 thng cui hoc sau khi sinh con. + ngi suy thn. </li><li> 10. 1.2. Tc hi ca Tetracyclines 1. GII THIU nh Hng ti thn: + Lm nng thm bnh suy thn c t trc. + Vim gan - thn trong tnh trng thoi ha m ca gan. + Gy tn thng ng thn. </li><li> 11. 1.2. Tc hi ca Tetracyclines 1. GII THIU nh Hng Ti Rng: + Lm hng men rng, hng cht xng ca rng dn n d b su rng tr em </li><li> 12. 2. Phng Php Phn Tch 11 Phm Vi p Dng 22 Nguyn Tc 33 Quy Trnh Phn Tch 44 Thng S Chy My </li><li> 13. 2.1. Phm vi p dng Phng php ny m t cch xc nh hm lng nhm tetracyclines (TCs) c trong thy sn v sn phm thy sn bng sc k lng ghp ph (LC/MS/MS). Gii hn pht hin ca phng php l 10ppb. 2. Phng Php Phn Tch </li><li> 14. 2.2. Nguyn Tc 2. Phng Php Phn Tch TCs c chit tch t mu thy sn bng m McIlvaine-EDTA, dch chit c lm sch bng cch cho qua ct chit pha rn C18. C dch chit n kh, cn kh c ha tan trong hn hp MeOH:H2O (50:50). Hm lng TCs c xc nh bng my LC/MS/MS vi gii hn pht hin l 10ppb. </li><li> 15. 2.3. Quy Trnh Phn Tch 2. Phng Php Phn Tch Chun b mu: Chun b mu th Chun b mu trng Chun b mu kim sot (QC) Dng ng chun + Nghin t nht 200g mu, ng trong ti PE bo qun trong t ng cho ti khi phn tch (i vi mu thy sn kh dng ko ct nh v trn u). + Cn 2g 0.05g mu c nghin vo ng ly tm nha 15ml. + Cn 2g 0.05g mu thy sn khng cha TCs vo ng ly tm nha 15ml. + Tin hnh chit mu trng ging nh mu th. + Cn 2g 0.05g mu thy sn khng cha TCs vo ng ly tm 15ml thm 40l dung dch chun 1ppm. + Tin hnh ging nh mu th. + Cn 2g 0.05g mu trng ln lt vo cc ng S1, S2, S3, S4, S5 c m ha trc. + Thm ln lt dung dch chun vo cc ng theo th t nh bng sau: S th t Th tch (l) TCs 1ppm Nng TCs trong mu (ppb) S1 20 10 S2 40 20 S3 100 50 S4 160 80 S5 200 100 </li><li> 16. 2.2. Quy Trnh Phn Tch 2. Phng Php Phn Tch Nghin mu Cn Mu Chit Gii Hp (10 ml dd 10mM A.Oxalic trong MeOH) Lm Sch (Bng ct Strata C18 500mg/6ml) Thi Kh (N2, 400 C) Ha Tan (1ml MeOH:H2O (50:50)) Lc (BK 0.2m)Vial LC/MS/MS Ln 1: + 8 ml m Mcllvaine-ADTA (PH = 4). + Vortex 1 pht, lc 15 pht vi tc 450 vng/pht. + Ly tm mu 4000 vng/pht trong 5 pht, 40 C. + Ht lp trn cho vo ng nghim nha 15ml khc. Ln 2: Phn cn c chit ln hai vi 7ml m McIlvaine-EDTA (pH=4) tng t nh ln mt. + Ht lp trn cho vo ng nghim nha 15ml trn. + Ly tm dch chit 4000 vng/pht trong 10 pht,40 C. HOT HA CT + 2 ml MeOH + 2ml H2O Ch : khng ct b kh+ Cho dch chit qua ct chit pha rn C18, tc chy khong 1ml/pht. + Sau khi mu chy xung ht ra ct bng 2ml nc HPLC. Ch : Phi lm cho ct kh ht nc. </li><li> 17. 2.4. Thng s ci t trn LC/MS/MS 2. Phng Php Phn Tch Ci t thng s cho LC: Pha ng A: ACN 0.3% FA. Pha ng B: H2O 0.3% FA. Th tch tim 10l. HPLC Agilent 1200. Ct: ZORBAX Eclipse XDB, 4.6 x 150mm, 5m. Ct v t l pha ng thay i nu cn. Nhit ct: 350 C. Time Flow rate %A %B Initial 0.2 10 90 0 0.2 10 90 4 0.2 50 50 8 0.2 80 20 8.5 0.2 30 70 12 0.2 10 90 20 0.2 10 90 </li><li> 18. 2.4. Thng s ci t trn LC/MS/MS 2. Phng Php Phn Tch Ci t thng s cho MS: Agilent 6410 LC/MS/MS Source ESI+ Gas temp (oC) 350 Gas flow (l/min) 9 Nebulizer (psi) 35 Capillary positive (V) 3500 Capillary negative (V 3500 MS1 Heater (o C) 100 MS2 Heater (o C) 100 </li><li> 19. 2.4. Thng s ci t trn LC/MS/MS 2. Phng Php Phn Tch iu kin phn mnh MS/MS: Component Precursor ion (m/z) Product ion (m/z) Dwell time (s) Fragmentor (V) Collision energy (eV) TC 445 445 428 410 0.2 0.2 100 100 17 15 OTC 461 461 426 444 0.2 0.2 100 100 18 25 CTC 479 479 461.8 444 0.2 0.2 100 100 17 15 </li><li> 20. 2.4. Thng s ci t trn LC/MS/MS 2. Phng Php Phn Tch Th t tim mu: Mu chun xy dng ng chun Mu trng Mu th Mu kim sot </li><li> 21. Cht cn phn tch c khng nh l hin din khi: Yu cu v thi gian lu Mc dung sai cng ion tng i, (p ng theo ch th 2002/657/EC ca cng ng Chu u.) 2. NH GI KT QU 2.1. nh Tnh Mc dung sai ti a cho php ca cc cng ion tng i: Cng tng i (% ca peak c s) LC-MS, LC-MSn ( tng i) &gt;50% 20% &gt;20 n 50% 25% &gt;10 n 20% 30% 10% 50% </li><li> 22. 2. NH GI KT QU 2.2. nh Lng H s hi qui tuyn tnh ca ng chun: R2 0.99 Khng c peak nhiu thi gian lu ca nhm TCs T l tn hiu/nhiu ca cc ion th cp trong mu phi 3:1 thu hi t 60% n 100% </li><li> 23. 2. NH GI KT QU 2.3. Tnh Ton Kt Qu Tnh t s ion theo phng trnh: 1 2 100% A R A = Trong : R : t s ion (%) A1: Din tch ca peak ion th cp c cng cao A2: Din tch ca peak ion th cp c cng thp H s pha long n: v m n = Trong : m: khi lng mu v: th tch cui cng Tnh hm lng cht cn phn tch trong mu kim da theo h s tn hiu ca tng mu phn tch v ng chun, kt qu nhn vi h s pha long tnh theo n v g/Kg vi 2 s thp phn. </li><li> 24. 4. TI LIU THAM KHO 1/ AOAC Official Method 995.09 2005 2/ Analysis of tetracyclines by HPLC of Agilent. 3/ 28TCVN 177:2002 4/ Rapid determination of residues of 4 Tetracyclines in meat by a Microbiological screening, HPLC and LC/MS/MS; 5/ High-throughput analysis of Tetracycline and Peniciclin antibiotics in animal tissues using electrospray tandem mass spectrometry with selectd reaction monitoring transition; 6/ Official Joural of the European Communities, Commission decision (2002/657/EC); 7/ Qualitative Identification of Tetracyclines, United States Department of Agriculture Food Safety and Inspection Service- Office of Public Health Science, 12/2007. </li><li> 25. XIN CHN THNH CM N </li><li> 26. 2.1. Quy Trnh Phn Tch 2. Phng Php Phn Tch Nghin mu Cn Mu Chit Gii Hp (10 ml dd 10mM A.Oxalic trong MeOH) Lm Sch (Bng ct Strata C18 500mg/6ml) Thi Kh (N2, 400 C) Ha Tan (1ml MeOH:H2O (50:50)) Lc (BK 0.2m)Vial LC/MS/MS </li></ol>