xác định ATX A bằng HPLC

8/2/2019 xc nh ATX A bng HPLC

1/24

TR NG I H C CNG NGHI P TP.HCM TR NG I H C CNG NGHI P TP.HCM KHOA CNG NGH HO H C KHOA CNG NGH HO H C

TIU LUN MN PHN TCH TH C PH M TIU LUN MN PHN TCH TH C PH M

ti: ti: XAC INH OCHRATOXIN A TRONG NGU XAC INH OCHRATOXIN A TRONG NGU CC B NG PH NG PHAP SC KY LONG CC B NG PH NG PHAP SC KY LONG

HIU NNG CAO HIU NNG CAO GVHD: Ths Nguyn c V ng GVHD: Ths Nguyn c V ng

SVTH : Vo Thanh Danh SVTH : Vo Thanh Danh Huynh Th Thu Ha Huynh Th Thu Ha Nguyn Ngoc T ng Vy Nguyn Ngoc T ng Vy

Thanh Ph H Chi Minh thang 5 - 2011 Thanh Ph H Chi Minh thang 5 - 2011


2/24

NI DUNG BAO CAO NI DUNG BAO CAO

XAC INH OCHRATOXIN A XAC INH OCHRATOXIN A ..

T NG QUAN V OCHRATOXIN T NG QUAN V OCHRATOXIN ..


3/24

T NG QUAN V OCHRATOXIN T NG QUAN V OCHRATOXIN

Ochratoxin l c t vi n m, c t o ra t m t s loi n m thu c gi ng Aspergillusochraceus v Penicillium viridicatum. ct n m m c thu c nhm ny g i l Ochratoxin

Trong s cc lo i ochratoxin, ochratoxin A (OTA) c tnh c cao nh t. Ochratoxin A c m t trong kh p cc lo i nng s n th c ph m: ng c c, th o d c, bia, c ph...


4/24

Ochratoxin A gy c n gan, th n v c kh nng gy ung th cho con ng i. Ochratoxin A th hi n c y u t gy t bi n.

T NG QUAN V OCHRATOXIN A T NG QUAN V OCHRATOXIN A

N m thu c gi ng N m thu c gi ng AspergillusAspergillus ochraceusochraceus


5/24


Tnh ch t ha l c a Ochratoxin A

- Tr ng l ng phn t : 403.8

- i m nng ch y: 169 oC

- Ph h p th UV: 2 nh; 216 333 nm

- Cng th c phn t : C 20H18ClNO6

- L tinh th khng mu, b n v i nhi t, tan trong dung mi h u c phn c c nh : chloroform, methanol, ethanol t tan trong n c v tan trong pha m carbonat long. D b phn h y b i nh sng, trong mi tr ng ki m ho t ch t t y r a.


6/24


Cng th c phn t Ochratoxin A Cng th c phn t Ochratoxin A


7/24

XAC INH OCHRATOXIN A XAC INH OCHRATOXIN A

PH NG PHAP SC KY LONG HIU PH NG PHAP SC KY LONG HIU NNG CAO (HPLC) LAM SACH BNG NNG CAO (HPLC) LAM SACH BNG

SILICAGEL TCVN 7595-1:2007SILICAGEL TCVN 7595-1:2007

PH NG PHAP SC KY LONG HIU PH NG PHAP SC KY LONG HIU NNG CAO (HPLC) LAM SACH BNG NNG CAO (HPLC) LAM SACH BNG BICABONAT TCVN 7595-2:2007BICABONAT TCVN 7595-2:2007

1

2


8/24

PH NG PHAP HPLC LAM SACH PH NG PHAP HPLC LAM SACH BNG SILICAGEL BNG SILICAGEL

Pham vi ap dung .

Nguyn tc .

Quy trinh phn tich .

Tinh toan .

i u ki n xc nh phn tich .


9/24

Pham vi ap dung Pham vi ap dung

Tiu chun nay qui inh ph ng phap xac inh Tiu chun nay qui inh ph ng phap xac inh

ochratoxin A v i cac ham l ng l n h n 0.4 ochratoxin A v i cac ham l ng l n h n 0.4 g/kg trongg/kg trong

cac mu ng cc, qua kh, cac hat co du, u , ca ph cac mu ng cc, qua kh, cac hat co du, u , ca ph nguyn liu nguyn liu

Ph ng php ny th nghi m trn b t m c ch a hm Ph ng php ny th nghi m trn b t m c ch a hm l ng ochratoxin A t 0.4 l ng ochratoxin A t 0.4 g/kg n 1.2g/kgg/kg n 1.2g/kg


10/24

Nguyn tcNguyn tc

Sau khi axit hoa bng axit HCl v b sung Sau khi axit hoa bng axit HCl v b sung

MgClMgCl22 dung toluene chit ochratoxin A (OTA). dung toluene chit ochratoxin A (OTA). Lam sach dich chit trn ct silicagel v xac inh Lam sach dich chit trn ct silicagel v xac inh OTA bng sc k long hiu nng cao trn ct pha OTA bng sc k long hiu nng cao trn ct pha ao, nhn dang v s a i b ng detect huynh ao, nhn dang v s a i b ng detect huynh quang.quang.


11/24

i u ki n v n hnh HPLC i u ki n v n hnh HPLC Pha ng: acetonitril (HPLC) : N c : acid acetic Pha ng: acetonitril (HPLC) : N c : acid acetic

bng = 49:49:2 (thay i t l n u c n). bng = 49:49:2 (thay i t l n u c n). T c dng: 1ml/pht T c dng: 1ml/pht

C t pha tch pha o: 250mm x 4.6mm x C18 (5m). C t pha tch pha o: 250mm x 4.6mm x C18 (5m). Detector : hunh quang 420nmDetector : hunh quang 420nm

V b m: 20lV b m: 20l


12/24

i u ki n ng chu n i u ki n ng chu n

Dung d ch chu n g c A: cn 20mg vo BM 200ml Dung d ch chu n g c A: cn 20mg vo BM 200ml thm 25 ml dd h n h p (toluen : acid acetic acetic thm 25 ml dd h n h p (toluen : acid acetic acetic bng = 99 :1 (V+V)), ha tan m u, sau thm h n bng = 99 :1 (V+V)), ha tan m u, sau thm h n

h p trn t i v ch, pha long thch h p thu c h p trn t i v ch, pha long thch h p thu c dung d ch 20g/ml n 30 g/ml (dd1) dung d ch 20g/ml n 30 g/ml (dd1) Dung d ch lm vi c: pha long dd1 v i dung mi Dung d ch lm vi c: pha long dd1 v i dung mi

trn c n ng 4g/ml. Sau l y trn c n ng 4g/ml. Sau l y 5,10,25,50,100l cho vo l nh , c kh d i dng 5,10,25,50,100l cho vo l nh , c kh d i dng nit sau thm pha ng c n ng achratoxin nit sau thm pha ng c n ng achratoxin cu i cng 0.5;1;2.5;5;10 ng/25l.cu i cng 0.5;1;2.5;5;10 ng/25l.


13/24

Quy trinh phn tich Quy trinh phn tich

B loc mang

20g Mu (m0)

ng ly tm250ml Ly tm

+ 30 ml HCl 2 mol/L

+ 50 MgCl2 0.4 mol/L

Khuy u

+ 100 ml toluen (V1)

50ml dd(V2)

Ct chit pha rn

Hut 1 ml

(V3)

HPLCV b m = 20l (V 4)

K t qu


14/24

Tinh toan Tinh toan

WOTA

: tinh bng g/ Kg

m0 : khi l ng mu th , tinh bng gam

mt : khi l ng OTA t ng ng v i di n tch pic c c t ng chu n, tinh bng nanogam

F : h s pha long


15/24

PH NG PHAP HPLC LAM SACH PH NG PHAP HPLC LAM SACH BNG BICACBONAT BNG BICACBONAT

Pham vi ap dung .

Nguyn Tc .

Quy trinh phn tich .

Tinh toan .


16/24

Pham vi ap dung Pham vi ap dung

Tiu chun nay qui inh ph ng phap xac inh Tiu chun nay qui inh ph ng phap xac inh ochratoxin A v i cac ham l ng l n h n 3 ochratoxin A v i cac ham l ng l n h n 3 g/kgg/kgtrong cac mu ngu cc nh ng hat, cam, lua mi trong cac mu ngu cc nh ng hat, cam, lua mi Ph ng php ny th nghi m trn m u ng c c Ph ng php ny th nghi m trn m u ng c c ch a hm l ng ochratoxin A t 3.0g/kg n ch a hm l ng ochratoxin A t 3.0g/kg n

16.3g/kg16.3g/kg


17/24

Nguyn tcNguyn tc

Ochratoxin A (OTA) c chit ra khoi ng cc Ochratoxin A (OTA) c chit ra khoi ng cc bng axit phosphoric pha loang trong clorofoc v bng axit phosphoric pha loang trong clorofoc v

c tach phn oan bng dung dich bicacbonat c tach phn oan bng dung dich bicacbonat loang. R a giai bng axit etyl axetat-metanol-axetat. loang. R a giai bng axit etyl axetat-metanol-axetat. OTA c tach bng HPLC pha ao, ri nhn bit OTA c tach bng HPLC pha ao, ri nhn bit v inh l ng bng detect huynh quang v inh l ng bng detect huynh quang ..


18/24

i u ki n v n hnh HPLC i u ki n v n hnh HPLC Pha ng: axetonitril : N c : acid acetic t l Pha ng: axetonitril : N c : acid acetic t l

49:49:2 (thay i t l n u c n). 49:49:2 (thay i t l n u c n). T c dng: 1ml/pht T c dng: 1ml/pht

C t pha tch pha o: 250mm x 4.6mm x C18 (5m). C t pha tch pha o: 250mm x 4.6mm x C18 (5m). Detector : hunh quang 420nmDetector : hunh quang 420nm

V b m: 20lV b m: 20l


19/24

i u ki n ng chu n i u ki n ng chu n Dung d ch chu n g c A: cn 20mg vo BM 200ml Dung d ch chu n g c A: cn 20mg vo BM 200ml

thm 25 ml dd h n h p (toluen : acid acetic acetic thm 25 ml dd h n h p (toluen : acid acetic acetic bng = 99 :1 (V+V)), ha tan m u, sau thm h n bng = 99 :1 (V+V)), ha tan m u, sau thm h n

h p trn t i v ch, pha long thch h p thu c h p trn t i v ch, pha long thch h p thu c dung d ch 20g/ml n 30 g/ml (dd1) dung d ch 20g/ml n 30 g/ml (dd1) Dung d ch lm vi c: pha long dd1 v i dung mi Dung d ch lm vi c: pha long dd1 v i dung mi

trn c n ng 4g/ml. Sau l y trn c n ng 4g/ml. Sau l y 5,10,25,50,100l cho vo l nh , c kh d i dng 5,10,25,50,100l cho vo l nh , c kh d i dng nit sau thm pha ng c n ng ochratoxin nit sau thm pha ng c n ng ochratoxin cu i cng 0.5; 1; 2.5; 5; 10 ng/25l.cu i cng 0.5; 1; 2.5; 5; 10 ng/25l.


20/24

Quy trinh phn tich Quy trinh phn tich

+ 10 mlNaHCO3 30g/l

50g Mu (m0)

My tr n t c cao

+ 250 ml clorofom

+ 25 ml H3PO4 0.1mol/l

Tr n 3 phut

+10 g diatomit

B loc s i thuy tinh ph 10g diatomit

50 ml dich loc

Phu chit

5 mlCt chit HPLC

Nghin

M u thM u th

K tK tququ


21/24

Tinh toan Tinh toan

WOTA

: tinh bng g/ Kg

m0 : khi l ng mu th , tinh bng gam

mt : khi l ng OTA t ng ng v i di n tch pic c c t ng chu n, tinh bng nanogam

F : h s pha long


22/24


23/24

Tai liu tham khao Tai liu tham khao

- TCVN 7595-1:2007

- TCVN 7595-2:2007- Ths Ph m Lu n, C s l thuy t phn tch s c k l ng hi u nng cao, HQGHN, 1999


24/24

Cm n Th y & Cc B n L ng Nghe

Documents

xác định ATX A bằng HPLC