xác định ochratoxin trong thực phẩm bằng phương pháp sắc ký lỏng khối phổ đủ


TRƢỜNG ĐẠI HỌC KHOA HỌC TỰ NHIÊN



Nguyễn Thị Hà Bình



XÁC ĐỊNH OCHRATOXIN
TRONG THỰC PHẨM BẰNG PHƢƠNG PHÁP
SẮC KÝ LỎNG KHỐI PHỔ (LC-MS/MS)













- 2013


TRƢỜNG ĐẠI HỌC KHOA HỌC TỰ NHIÊN



Nguyễn Thị Hà Bình


XÁC ĐỊNH OCHRATOXIN
TRONG THỰC PHẨM BẰNG PHƢƠNG PHÁP
SẮC KÝ LỎNG KHỐI PHỔ (LC-MS/MS)

 
 60440118






N

HDP



- 2013
Luận văn thạc sĩ Nguyễn Thị Hà Bình

1
LỜI CẢM ƠN

 
 


,
, 





 





Hà Nội, năm 2013


Nguyễn Thị Hà Bình



Luận văn thạc sĩ Nguyễn Thị Hà Bình

2
MỤC LỤC
MỞ ĐẦU
Chƣơng 1: TỔNG QUAN
1.1 Giới thiệu chung [14-15]
1.1.1 4
1.1.2 5
1.1.3 Giới hạn tồn dƣ tối đa cho phép (MRL)………………………………7
1.2 Các phƣơng pháp xác định
1.2.1 Phƣơng pháp ELISA (enzyme-linked immunosorbent assay)……….8
-GC) 9
1.2.3 Phƣơng pháp sắc ký lỏng hiệu năng cao (HPLC) và cột ái lực miễn
dịch (IAC)……………………………………………………………………….9
1.2.4 Phƣơng pháp sắc ký lỏng khối phổ……………………………………10
Chƣơng 2: ĐỐI TƢỢNG VÀ PHƢƠNG PHÁP NGHIÊN CỨU
2.1 Đối tƣợng và nội dung nghiên cứu
2.1.1 Đối tƣợng nghiên cứu…………………………………………………12
2.1.2 Nội dung nghiên cứu……………………………………………………12
2.2 Phƣơng pháp nghiên cứu2
2.2.1 Phƣơng pháp tách chiết mẫu………………………………………….12
2.2.2 Phƣơng pháp sắc ký lỏng khối phổ…………………………………….13
2.2.2.1 Hệ thống sắc ký lỏng hiệu năng cao…………………………… 13
2.2.2.2 Khối phổ (Mass Spectrometry) 
2.3 Phƣơng tiện nghiên cứu0
2.3.1 Thiết bị, dụng cụ……………………………………….……………20
2.3.1.1 Thiết bị……20
2.3.1.2 Dụng cụ 21
2.3.2 Dung môi, hóa chất…………………………………………………….21

Chƣơng 3: KẾT QUẢ VÀ THẢO LUẬN3

Luận văn thạc sĩ Nguyễn Thị Hà Bình


3
3.1 Tối ƣu các điều kiện chạy máy LC-MS/MS 23

3.1.1.1 Khảo sát ion mẹ và ion con.23
3.1.1.2 Tối ưu các điều kiện MS24
3.1.2 Tối ƣu các điều kiện chạy sắc ký lỏng hiệu năng cao (HPLC)……….26
3.1.2.1 Pha tĩnh………26
3.1.2.2 Khảo sát thành phần pha động.27
3.1.2.3 Khảo sát tốc độ pha động 29
3.1.2.4 Chọn chương trình chạy gradient…30
3.2 Tối ƣu quy trình xử lý mẫu3
3.2.1 Chọn quy trình xử lý mẫu………………………………………………33
3.2.2 Khảo sát thể tích (số lần chiết) dung môi chiết…………………… 36

 37
3.2.5 Khảo sát thể tích dung môi rửa giải………………………………….38
 38
3.3 Thẩm định phƣơng pháp phân tích2
3.3.1 Tính đặc hiệu / chọn lọc………………………………………………42
3.3.2 Giới hạn phát hiện (LOD), giới hạn định lƣợng (LOQ)………………44
3.3.3 Xác định khoảng tuyến tính……………………………………………45
3.3.4 Xây dựng đƣờng chuẩn
3.3.5 Độ chính xác (accuracy) của phƣơng pháp phân tích (độ đúng và độ
chụm) [9]………………………………………………………………………48
3.4 Phân tích mẫu thực tế
KẾT LUẬN VÀ KIẾN NGHỊ7
TÀI LIỆU THAM KHẢO 59

PHỤ LỤC63


Luận văn thạc sĩ Nguyễn Thị Hà Bình


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Luận văn thạc sĩ Nguyễn Thị Hà Bình


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DANH MỤC TỪ VIẾT TẮT
Ký hiệu
Tiếng Anh
Tiếng Việt
CE
Collision energy

EI
Electron Ionization

ESI
Eelectrospray ionization

EU
European Union

HPLC
High performance liquid

chromatography

IUPAC
International Union of Pure and
Applied Chemistry


LOD
Limit of detection

LOQ
Limit of quantity

MeOH
Methanol
metanol
MRL
Maximum Residue Limit

PSA
Primary and secondary amine

RSD
Relative standard deviation

SPE
solid phase extraction

IARC
International Agency for Research on

cancer


UPLC-
MS/MS
Ultral performance liquid
chromatography tandem mass
spectrometry


RF
Fluorescence detector
h quang

Luận văn thạc sĩ Nguyễn Thị Hà Bình


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DANH MỤC CÁC BẢNG

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2.1: 
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
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ESI-MS/MS
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

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
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
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
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
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
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3.9 : 
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
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

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
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
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
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
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6: 
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Luận văn thạc sĩ Nguyễn Thị Hà Bình


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DANH MỤC HÌNH VẼ

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-MS
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
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
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
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
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 
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

CH
3
COONH
4
10mM
24

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
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
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
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
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
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
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
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
43

43

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
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
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
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
48


52

54
n A,B
54

54

55

55
Luận văn thạc sĩ Nguyễn Thị Hà Bình


8
MỞ ĐẦU



trong 

 . 


- 

- 

-
 

- 
- 
- 
      





            

 

Luận văn thạc sĩ Nguyễn Thị Hà Bình


9
- 



 Mycoto

 


m . 
,  .   
,    cao. 

















. 


 


och, 


  
ochratoxin 
 
:
"Xác định hàm lượng Ochratoxin trong thực phẩm bằng phương pháp sắc ký
lỏng hai lần khối phổ (LC-MS/MS)”.


1.          Ochratoxin trong 
 
- 
Luận văn thạc sĩ Nguyễn Thị Hà Bình


10
- .
- 
ochratoxin trong  
 .

Luận văn thạc sĩ Nguyễn Thị Hà Bình


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CHƢƠNG 1: TỔNG QUAN

1.1.Giới thiệu chung [14-15]
1.1.1.Định nghĩa
o 
            
              
             
 

 
 (
 


 Nam 
, n

 o

3  



Ochratoxin A


Ochratoxin B



H chratoxins
Luận văn thạc sĩ Nguyễn Thị Hà Bình


12

1.1.2.Tính chất hóa lý của Ochratoxins
Ochratoxins 

). Ochratoxins 
-25
0
ochratoxins 

. Ochratoxins 

Ochratoxin A . Ochratoxin
A 
0


-1
OTA 
a1
= 4,2-4,
a2
= 7,0-
7,3. Ochratoxin A TLC)
.


221
0
C.
ochratoxins: ochratoxins 
ribonucleic axit (tA   s   
 - tA
 
               
tARN   ochratoxin A       
 ochratoxin A  
A

 A

A
C
  o   
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13
gan v  
 

.
-               






- 

-  
o

-  


-  


- 


-     

- 
, protein, 
Luận văn thạc sĩ Nguyễn Thị Hà Bình


14
             



 



            




heo: -6  mg/

1.1.3. Giới hạn tồn dƣ tối đa cho phép (MRL)
-        


Ochratoxin A 




5

5

10

2

0,5

30

80

3
Luận văn thạc sĩ Nguyễn Thị Hà Bình


15
o
EN).
1.2 Các phƣơng pháp xác định
1.2.1 Phƣơng pháp ELISA (enzyme-linked immunosorbent assay).

           Ochratoxin-


   

  ochratoxin             
chratoxin- enzyme


    

    o          
ochratoxin           
Ochratoxin



, Lulu Feng, Ying Wang, Chenghua
He, Xichun Wang, Haibin Zhang [14

-
2,5-10 ppb. . 
 - 
, 
Luận văn thạc sĩ Nguyễn Thị Hà Bình


16
  Ochratoxin A   -23 ppb,       
Ochratoxin A -3 ppb.
     d Moulasa, Dua  
Prazeresb,  [32] 
n 
h  


     

1.2.2 Phƣơng pháp sắc ký khí (Gas chromatography-GC)
 
O

 
Ochra
[31] 
  chratoxin A 
ester 0-methyl ochratoxin A 

1.2.3. Phƣơng pháp sắc ký lỏng hiệu năng cao (HPLC) và cột ái lực miễn dịch
(IAC)
 
o

o
2

 

 . at
Luận văn thạc sĩ Nguyễn Thị Hà Bình


17
333 nm, 460 nm  
 a


  
-9 ppb, 
-90%.
27
2
c 
 . 
   , k     g    




1.2.4. Phƣơng pháp sắc ký lỏng khối phổ







-MS/MS




Luận văn thạc sĩ Nguyễn Thị Hà Bình


18

1 thng LC-MS/MS
1.2.4.1. Hệ thống sắc ký lỏng hiệu năng cao




ho
         
nhau.


2 n ca h thng sng.
Trong HPLC, 
 

-HPLC) v-HPLC)
Sắc ký lỏng
Ion hóa
Bộ phân
tích khối
Detector/ Lƣu
giữ số liệu
Luận văn thạc sĩ Nguyễn Thị Hà Bình

19
  

-NH
2
, -
n-

 
       C
18
H
37
      
methanol, axetonitr


          



  
            

                
cyclopentan, n-pentan, n-heptan, n-hexan, 2-chloropropan, cacbondisulfua
(CS
2
), chlorobutan, CCl

4
, toluene





 
-VIS),
         

Luận văn thạc sĩ Nguyễn Thị Hà Bình


20
1.2.4.2 Khối phổ (Mass Spectrometry)







3 ci ph MS.





 Nguồn ion

       

              



Detector







Luận văn thạc sĩ Nguyễn Thị Hà Bình


21
ionization             
chemical ionization  APCI).
a) Chế độ ion hóa đầu phun điện tử (ESI)





4: B ngu
 
-

 

2




2



 
o 
o  
+
.
Luận văn thạc sĩ Nguyễn Thị Hà Bình


22
o 
n+
, [M+Na

+
]
+

 
o 
o [M-H]
-
, [M-nH]
n-


          

b) Chế độ ion hoá hoá học ở áp suất khí quyển (APCI).

 


 

 Bộ phận phân tích khối



 
 - 







hu

+ 
Luận văn thạc sĩ Nguyễn Thị Hà Bình


23
+ 








5: B  cc


 



 Quadrupole Ion-Trap Mass Analyser)


 




ring electrode
end-cap electrode
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
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