Provisional Translation

from the Japanese Original

Specifications and Standards for Food, Food Additives, Etc.

M i n i s t r y o f H e a l t h a n d We l f a re N o t i f i c a t i o n N o . 3 7 0 , 1 9 5 9

T h e Mi n i s te r o f H e a lt h an d Wel f a r e h a s e s t a b l i sh e d t h e s p e cif i c a t i o n s an d
s t a n d a r d s f o r f o o d , f o o d ad d i t i v es g i ve n b e lo w, b a s ed o n A r t i c l e 1 0 an d A r t i c l e
11 P a r a g r ap h 1 o f t h e F o o d S a n ita t i o n L a w ( L a w N o . 2 3 3 , 1 9 4 7 ) .

Part I Food
Section A G e n e r a l C o m p o s i t i o n a l St a n d a r d s f o r Fo o d
1 . F o o d s s h a l l n o t c o n ta i n any a n t i b i ot ic s o r c h e mi c a l l y s y n t h e s iz e d
a n t ib a c te r i a l s u b s ta n c e s ( s u b s t an c e s o b t ain e d b y i n st i g a ti n g c h e mi c a l
r e a c t i o n s to e l e me n t s a n d /o r co mp o u n d s t h r o u g h c h e mi c a l me t h o d s ,
e x c e p t f o r d e co mp o s i t i o n ; t h i s a p p li e s h e r e i n a f t e r i n t h i s p a r a g r a p h ) ,
e x c e p t f o r th e f o l lo w i n g c a s e s :

( 1 ) W h e n t h e s u b s tan c e c o n c e rn e d i s id en t i c al t o th e f o o d ad d i ti v e
d e t e r mi n e d b y th e M i n i s t e r o f H e al t h , L a bo u r a nd We l f a re a s h a vi n g n o
p o t en t i al t o c a u s e d a ma g e t o h u ma n h e a lt h u n d e r A r t i c le 1 0 o f t h e F o o d
S a n i t a ti o n L a w ( L a w N o . 2 3 3 , 1 9 4 7 , h e r e in a f t e r “t h e L a w. ” )

( 2 ) W h e n co mp o s i t i o n a l s t an d a r d s a r e s e t f o r t h in 5 , 6 , 7 , 8 o r 9 b el o w f o r
the substance concerned.

( 3 ) W h e n th e f o o d p r o d u c t c o n c e rn e d h a s b e en ma n u f a c t u r e d o r p r o c e s s e d
u s i n g a f o o d in g r e d ie n t t h a t me e t s t h e c o mp o s i t i o n a l s tan d a r d s g i v en in
5 , 6 , 7 , 8 o r 9 b e l o w ( e x c e p t f o r f o o d s c o n ta i n in g a n t ib i o t i cs o r
c h e mi c a l l y s y n t h e s iz e d an t ib a c t e r i a l s u b s t a n ce s for which
c o mp o s i t i o n a l s t an d a rd s a r e n o t se t f o r t h in 5 , 6 , 7 , 8 o r 9 b e lo w. )

2.~4. ( O mi t t e d )

5 . F o o d s sh a l l n o t co n t a in s ub s ta n c e s ( in c lu d i ng su b s ta n c e s p r o du c e d b y
c h e mi c a l t r a n s f o r ma t i o n : t h i s a p p l i e s h e r e in a f t e r i n t h i s p a r a g r a p h ) u s ed
a s i n g r e d i en t s o f a g r ic u l tu r a l c h e mi c a l s a n d o t h e r c h e mi c a l s u b s t a n c e s
l i s t e d i n th e t ab l e i n ( 1 ) b e l o w. T h e ag r ic u l tu r a l c h e mi c a l s a n d o th e r
c h e mi c a l s u b s t a n c e s st a t e d a b o v e, h e r e a n d a l s o la t e r in th i s p a r ag r a p h ,
r e f e r t o s u b s t a n ce s u s e d f o r p u r p o s e s d e s ig n a te d b y t h e A g r i c u l t u r e ,
F o r e s t r y a n d F i s h e r i e s M i ni s t e r i al O r d i n an ce a c co r d i n g to th e p r o v i s io n

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o f P a r a g r a p h 3 o f A r t i c l e 2 o f t h e L a w C o n ce r n i n g S a f e t y A s s u r a n c e a n d
Q u a l i t y I mp r o v e me n t o f A g r i c u l t u r a l C h e mi c a l s an d F ee d s ( L a w N o . 3 5 ,
1 9 5 3 ) , w h i c h i s s t i p u l a t ed u n d e r P a r a g r a p h 1 o f A r t i c l e 1 - 2 o f t h e
A g r i c u l t u r al C h e mi c a l s R e g u l at i o n L a w ( L a w N o . 8 2 , 1 9 4 8 ) , w i t h s u ch
a i ms a s a d d i n g to , mi x i n g w i th , o r in f i l t ra t i n g in t o f e e d s ( t h e f e e d s
s t i p u la t ed u n d e r P a r ag r a p h 2 o f A r t i c l e 2 o f L a w N o . 3 5 ) , o r me d i c a l
p r o d u ct s to b e u s ed f o r a n i ma l s , w h i c h a r e s t i p u la t ed in P a r a g r a p h 1 o f
A r t i c l e 2 o f t h e P h a r ma c e u t i c a l A ff a i r s L a w ( L a w N o . 1 4 5 , 1 9 6 0 ) . I n
a s s o c i a t io n w i t h t h i s r e g u l at i o n , a s a mp l e o f t h e f o o d s l i s te d i n t he
“f o o d s ” c o lu mn i n t h e t a b l e in ( 2 ) b e l o w s h a l l be t e s te d u s i ng t he p a rt
l i s t e d i n th e “s a mp l e s ” c o l u mn i n t h e t ab l e b y th e t e s t i n g me t h o d s
d e s c r i b ed in ( 3 ) t o ( 1 6 ) b e l o w. N o i n g r e d ie n t s o f a g ri c u l tu r a l ch e mi c a l s
o r o t h e r che mi c a l s u b st a n c e s s ha ll b e d e t ec t ed i n t h e s e te s t s .

( 1 ) S u b s t a n c e s u s e d a s i n g r e d i en t s o f a g r ic u l tu r a l c h e mi c a l s a n d o t h er
c h e mi c a l s u b s t a n c e s th a t a r e s ti p u l a t ed to be “N o t d e t ec t e d ” i n f oo d s

1. 2, 4, 5-T
2 . A z o c yc lo t i n a nd c yh e x at i n
3 . A mi t r o l
4. Captafol
5 . C a r b ad ox
6 . C ou ma p h o s
7 . C hl o r a mp h e ni c ol
8. Chlorpromazine
9 . D i e t h y l st i l b e st r o l
1 0 . D i me t r i d a z o l e
11 . D a mi n o z i d e
1 2 . N i t r o f ur a n s
1 3 . P r o p h am
1 4 . Ma l a ch i t e g r e en
1 5 . Me t r o n i d a zo l e
1 6 . Ro n id az o l e

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Provisional Translation
from the Japanese Original

( 2 ) S a mp l e s

Food S a mp l e ma t e r i a l

Barley and buckwheat T h r e s h e d se e d s

W h e a t an d r y e H u s k e d s e ed s

R i c e ( b r o wn r i c e ) H u s k e d s e ed s

C o r n ( ma i z e ) S e e d s w i t h t h e h u s k s , th e s i lk a n d
t h e co r e s r e mo v e d
O t h e r c e r e al g r a i n s T h r e s h e d se e d s

Peas, beans (dry)(including butter S e e d s w i t h o u t th e p o d s

b e a n s , c o wb e a n s ( r e d b e a n s ) , l e n ti l ,
l i ma b e a n s , p e g ia , s u lta n i , s u l t ap y a
a n d w h it e b e a n s ) * , b r o a d b ea n s and
soybeans (dry))
Peanuts Wi t h t h e s h e l l s r e mo v e d

O t h e r l e g ume s / p u l s e s S e e d s w i t ho u t th e p o ds

A p r i c o t , mu me p l u m, c h e r r y,
J a p a n e s e p lu m ( i n c l u d in g p r u n e )
a n d n e c ta r i n e
Peach
O r a n g e ( i n c l u d i n g n av e l o r a n g e ) ,
g r a p e f r u i t , c i t r u s n a t su d a id a i
( w h o l e ) , l ime a n d l e mo n
C i t r u s n a t su d a id a i ( p u l p ) a n d u n sh u
o r a n g e ( p u lp )
C i t r u s n a t su d a id a i , p ee l s
Wi t h t h e p ed u n cl e an d t h e s e ed s
r e mo v e d
Wi t h t h e s k i n s a n d t h e s e e d s
r e mo v e d
Whole fruit
Wi t h t h e p ee l s r e mo v e d
Wi t h t h e c al y x e s r e mo v e d

Other citrus fruits Whole fruit

P e a r, J a p a n e s e p e a r, q u i n ce a n d Wi t h t h e b l o s s o m s c a r s , t h e c o r e s
a p p le a n d th e p e d u n c le s r e mo v e d
L o q u at Wi t h t h e p ed u n cl e s , th e s k i n s an d
t h e s e ed s r e mo v e d
Av o c a do a nd ma n g o Wi t h t h e s ee d s r e mo v e d

Kiwifruit Wi t h t h e s k i n s r e mo v e d

Guava Wi t h t h e c al y x e s r e mo v e d

Date Wi t h t h e c al y x e s a n d th e s e e d s
r e mo v e d

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Provisional Translation
from the Japanese Original
Pineapple
P a s s i o n f r u i t a n d p ap ay a
B a n a na
St r a w b e r r y, c r a n b e r r y, h u c k l e b e r r y,
b l a ck b e r r y a n d b l u eb e rr y
Raspberry
Other berries
J a p a n e s e pe r s i m mo n
Wa t e r me l o n , m a k u w a u r i me l o n an d
me l o n s
Grape
Other fruits
Tu r n i p ( r o o t s ) a n d J a p a n e s e r a d i s h
( r o o t s , i n clu d i ng r a di s h )
Tu r n i p ( l e a v e s ) , w a t e r c r e s s , k a l e ,
J a p a n e s e r ad i s h ( l e av es , i n c lu d in g
radish), and brussels sprouts
C a u l i f lo w e r a n d b r o c co l i
C a b b ag e an d C h in e s e ca b b ag e
Kyona and komatsuna (Japanese
mu s t a r d s p i n a c h )
Horseradish
Qing-geng-cai and other
c r u c i f e r o u s v e g et a bl e s
S w e e t p o t ato , k on j ac , ta r o , p o t a t o ,
y a m a n d o t h e r p o ta t o es
P u mp k i n ( i n c l u d i n g s q u a s h ) ,
c u c u mb e r ( i n c lu d in g g h e r k i n ) an d
o r i e n t al p i ck l i n g me l o n ( v e g et a b le )
O t h e r c u c u r b i t a c eo u s v e g et a b le s
A r t i c h o k e , e n d iv e an d c h i co r y
4/100
Wi t h t h e to p s r e mo v e d
Whole fruit
Wi t h t h e p ed i c e l s r e mo v e d
Wi t h t h e c al y x e s r e mo v e d
Whole fruit
Wi t h t h e c al y x e s r e mo v e d
Wi t h t h e c al y x e s a n d th e s e e d s
r e mo v e d
Wi t h t h e r in d s r e mo v e d
Wi t h t he p ed u n cl e s r emo v e d
E d i b le p o r t i o n s
Wi t h th e d ir t l i g h t l y r in s e d o ff w i t h
water
Wi t h t h e d ec a y e d l e av es r e mo v e d
Wi t h t h e l ea v e s r e mo v e d
A s a mp l e c o n s i s t in g o f o n e p o r t io n
f r o m e a c h o f f o u r h e ad s , w i t h eac h
h e a d e q ua l ly c u t i nt o fo u r p o r ti o ns ,
w i t h o u t t h e d e c a y e d o u t e r l e av e s
a n d th e co r e s
Wi t h t h e r o o t s a n d t h e d e c a y e d
l e a v e s r e mo v e d
R o o t s w i th t h e d i r t l ig h t l y r i n s e d
o ff w i t h w a t e r
E d i b le p o r t i o n s
Wi t h th e d ir t l i g h t l y r in s e d o ff w i t h
water
Wi t h t h e v in e s r e mo v e d
E d i b le p o r t i o n s
Wi t h t h e d ec a y e d l e av es r e mo v e d
Provisional Translation
from the Japanese Original

B u r d o ck a n d s a l s i f y
S h u n g i ku
L e t t u ce ( i n c l u d i n g c o s l e t tu c e an d
l e a f l e tt u c e)
O t h e r c o mp o s i t e v e g eta b l e s
A s a mp l e t h i n l y s l i ced t h en g r o u n d
w i t h a me a t g r i n d e r, t h e l e a v e s
h a v in g b e en r e mo v e d a n d th e d i r t
h a v in g b e en l i g h t l y r in s e d o ff w it h
water
Wi t h t h e r o o t s a n d t h e d e c a y e d
l e a v e s r e mo v e d
Wi t h t h e d ec a y e d o u t e r l e a v e s a n d
t h e co r e s r e mo v e d
E d i b le p o r t i o n s

S h i it a k e mu s h r o o m, b u t t o n E d i b le p o r t i o n s
mu s h r o o m a n d o t h e r mu s h r o o ms
C e l e r y, p a rs l e y a n d m it s u b a Wi t h t h e r o o t s a n d t h e d e c a y e d
l e a v e s r e mo v e d
Carrot and parsnip Wi t h th e d ir t l i g h t l y r in s e d o ff w i t h
water
O t h e r u mb e l l i f e r o u s v eg e t ab l e s E d i b le p o r t i o n s

To ma t o , e g g p l an t an d p i mi e n t o Wi t h t h e c al y x e s r e mo v e d
(sweet pepper)
O t h e r s o l a n c e o u s v e g eta b l e s E d i b le p o r t i o n s

Asparagus St e ms

O n i o n , g a r li c , w e l s h ( in c l ud i n g lee k ) Wi t h t h e o u t e r s k i n s an d t h e r o o t
a n d mu l t i p ly i n g o n i o n h a i r r e mo v e d
N i r a a n d o th e r l i l ia c eo u s v e g e t a b le s E d i b le p o r t i o n s

G r e e n s o y b e a n s , k i d n ey b e a n s Wi t h t h e p ed i c e l s r e mo v e d
( i m ma t u r e , w i t h p o d s ) a n d p e a s
( i m ma t u r e , w i t h p od s )
Okra Wi t h t h e c al y x e s r e mo v e d

Sugarcane Wi t h t he hus k s r e mo v e d

Ginger Wi t h t h e l ea v e s r e mo v e d , an d w i th
t h e d i r t l ig h t l y r i n s e d o ff w i t h
water
Sugar beet Wi t h th e d ir t l i g h t l y r in s e d o ff w i t h
water
Spinach Wi t h r e d r o o t s l e f t o n , a n d w i th t h e
r o o t h ai r an d th e d e c ay e d l e a v e s
r e mo v e d
B a mb o o s ho o t s an d o th e r v e g e t ab l e s E d i b le p o r t i o n s

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S e s a me s e e d s , r a p e s e e d s , s u n f l o w e r Seeds only
s e e d s , s a ff l o w e r s e e d s , c o t t o n s e e d s
a n d o t h e r oi l s e e d s
A l mo n d , g in k g o n u t , ch e s t n u t , Wi t h t h e h u s k s r e mo v e d
w a l n u t , p e ca n a n d o t h er n u t s
C a c a o b e an s a n d co ff e e b e an s B e a n s w i t h o u t th e p o d s

Te a Te a l ea v e s

Hop Dried flowers

O t h e r s p i c es a n d o t h e r h e r b s E d i b le p o r t i o n s

( 3 ) 2 , 4 , 5 - T a n a l yt ic al me t h o d
1. Apparatus
A g a s c h r oma t o g r a p h w i t h an e lec t r o n c a p tu r e d e t ec t o r ( G C - E C D ) a n d
a g a s ch r oma t o g r a p h /ma s s s p e c t r o me t e r ( G C / M S o r G C / M S / M S ) .
2 . R e ag e n t s a n d t e s t s o l u t io n s
I n a d d i t io n t o t h e r e ag e n t s a n d te s t s o lu t i o n s l i s t ed b e l o w, u s e th o s e
l i s t e d in S e c t i o n C R e a g e n ts /Te s t S o l u ti o n s , E t c . , P a r t I I F o o d
a d d it i v e s . R e a g en t s d e s i g n at e d as “s p e c i a l g r a d e ” i n th i s s e c ti o n mu s t
me e t t h e re q u i r e me n t s f o r “s p e c i a l g r a d e” s p e c i f ie d i n t h e J ap a n
I n d u s t r i a l St a n d a r d s fo r t h e r e ag e n t s .
A c e t o n it r i le : U s e a r o ta r y v a c u u m e v a p o r a t o r o n 3 0 0 mL o f a c e to n i t ri l e
t o c o n c e n t r a t e t o t h e p o i n t o f d r y n e s s . D i s s o l v e t h e r e s id u e i n 5 mL o f
n - h e x an e . F o r a n a l y s i s , i n j ec t 5 μ L o f th e so l u ti o n i n to a GC -ECD.
P e a k s o t h er t h a n t h a t o f n - h e x an e i n th e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
A c e t o n e: U s e a r o t a r y v a cu u m e v a p o r at o r o n 3 0 0 mL o f a c et o n e to
c o n c en t r a t e t o t h e p o in t o f d r y n e s s . D i s s o l v e t h e r e s i d u e i n 5 mL o f
n - h e x an e . F o r a n a l y s i s , i n j ec t 5 μ L o f th e so l u ti o n i n to a GC -ECD.
P e a k s o t h er t h a n t h a t o f n - h e x an e i n th e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
E t h e r : U s e a r o ta r y v a cu u m e v a p o r a t o r o n 3 0 0 mL o f e t h e r t o
c o n c en t r a t e t o t h e p o in t o f d r y n es s . D i s s o l v e t h e r e s i d u e i n 5 mL o f
n - h e x an e . F o r a n a l y s is , i n je c t 5 μ L o f t h e s o l u t i o n i n t o a GC -E C D .
P e a k s o t h er t h a n t h a t o f n - h e x an e i n t h e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
S o d i u m c h l o r i d e : S o d iu m c h l o r i de ( s p e c i a l g r a d e ) . I f t h e r e a g e n t i s
f o u n d to in c l u d e a n y s u b s t a nc e tha t ma y i n t e r f e r e w i t h t h e a n al ys i s o f

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Provisional Translation
from the Japanese Original

s u b s t a n ce s t o b e an a l yz e d f r o m a g r i c u l tu r a l c h e mi c a l s , w a s h w i t h a
s o l v e n t s u ch a s n - h e x a n e b e f o r e u s e .
S y n t h e t i c ma g n e s i u m s i l i c a t e (F l o r i s i l ) f o r c o l u mn c h r o ma t o g r a p h y :
H e a t f l o r i s i l ( 1 5 0 - 2 5 0 μ m i n p a r tic l e s iz e ) at 1 3 0 ° C f o r 1 2 h o u r s o r
l o n g e r. C o o l d o w n t o ro o m t e mp e r a t u r e in a d e s i c c a to r.
D i a t o ma c e o u s e a r t h : D i a t o ma c e o u s e a rt h f o r c h e mi c a l a n a l y s i s . .
E t h yl a c et at e : U s e a r o t a r y v a cu u m e v a p o ra t o r o n 3 0 0 mL o f e t h y l
a c e t a te t o co n c en t r a t e t o t h e p o in t o f d r y n e s s . D i s s o l v e th e r e s i d u e in
5 mL o f n - h e x an e . F o r a n a l y s i s , i n j e ct 5 μL o f t h e so l u ti o n i n to a
GC-ECD. P e a k s o t h e r t h a n th a t o f n -h e x an e in t h e r e s u lt i n g
c h r o ma t o g r a m s h o u l d b e a s h i g h a s o r l o w e r t h a n t he pe a k s o f γ - B H C
a t 2 x 10 - 1 1 g .
R e a g en t f o r b u t y l e s te r i f i c a t i o n : D i s s o l v e 1 0 g o f b o r o n t r i f l u o r i d e
e t h e r co mp l e x in 2 5 ml o f n - b u t an o l .
n - H e x a n e : U s e a r o t a r y v a c u u m e v a p o r at o r on 3 00 mL o f n - h e x a n e t o
e v a p o r a t e u n t i l 5 mL i s l e f t . F o r a n a l y s i s , i n j e c t 5 μ L o f t h e s o lu t io n
i n t o a G C - E C D . P e a k s o t h e r th an t h at o f n - h e x a n e i n t h e r e s u l t in g
c h r o ma t o g r a m s h o u l d b e a s h ig h a s o r lo w e r t h an t h e pe a k s o f γ - B H C
a t 2 x 10 - 1 1 g .
Wa t e r : D i s ti l l e d w a t e r. I f t h e d i s ti l l e d w a t e r i s f o u n d to i n c l u d e an y
s u b s t a n ce th a t ma y i n t e r f e r e w i t h a n a l y s i s o f t h e t a rg et co mp o s i t i o n a l
s u b s t a n ce s f r o m a g r i c u l t u r a l ch emi c a l s , w a s h w i t h a s o l v en t s u c h a s
n-hexane before use.
S o d i u m s u l f a t e ( a n h y d r o u s ) : S o d iu m s u l f a t e ( a n h y d r o u s) ( s p e c i a l g ra d e ) .
I f t h e r e a g e n t i s f o u n d t o in c lu d e a n y s u b st a n c e t h at ma y i n t e r f e r e
w i t h a n al y s i s o f t h e t a rg e t co mp o s i t i o n a l s u b st a n c e s f r o m a g r i c u l t u r al
c h e mi c a l s , w a s h w i t h a s o l v en t su c h a s n - h e x a n e b e f o r e u s e .
M e t h an o l: U s e a r o t a r y v a c u u m e v a p o r at o r on 3 00 mL o f me t h a n o l t o
c o n c en t r a t e t o t h e p o in t o f d r y n e s s . D i s s o l v e t h e r e s i d u e i n 5 mL o f
n - h e x an e . F o r a n a l y s i s , i n j ec t 5 μ L o f th e so l u ti o n i n to a GC -ECD.
P e a k s o t h er t h a n t h a t o f n - h e x an e i n th e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
3. Reference standard
2 , 4 , 5 - T: Thi s p r o du c t c o n ta i n s n o t l e s s t h an 9 8 % o f 2 , 4 , 5 - T, a n d i t s
me l t i n g p o in t i s 1 5 6 ° C.
4. Procedure
a . E x t r a ct io n

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i . C e r e a l g r a i n s , l eg u me s / p u l s e s an d s e ed s
We i g h 1 0. 0 g o f th e te s t s a mp l e , p r e v io u s l y g r o u n d s o a s to p a s s
t h r o u g h a s t a n d a r d me s h s i e v e ( 42 0 µ m) . A d d 2 0 ml o f w a t e r t o t he
o b t ai n ed s amp l e a n d l ea v e it t o s ta n d f o r t w o h o u r s .
T h e n , a d d 1 0 0 ml o f ac e t o n e an d 5 ml o f 4 mo l / l h y d r o c h lo r i c aci d ,
and h o mo g e n iz e the mi x t u r e for three mi n u t e s . Filter the
h o mo g e n i z e d s a mp l e b y s u c t io n i n to a r o ta r y v a c u u m e v a p o r a to r
through a filter paper c o v e r ed with a 1-cm thick layer of
d i a to ma c e o u s e a r t h . Co l l e ct t h e re s i d u e o n th e s u r f a c e o f t h e f i lt er
p a p e r. A d d 5 0 ml o f ac e t o n e an d h o mo g e n i z e t h e mi x t u r e f o r t h r e e
mi n u t e s . Re p e a t t he ab o v e p r o c ed u r e an d co mb i n e th e f i l t r a t e i n to
t h e r o ta r y v a c u u m e v a p o r a t o r, a n d co n ce n t r a t e t h e mi x t u r e t o
a p p r o x i ma t e l y 3 0 ml a t 4 0 ° C o r lo w e r.
Tr a n s f e r t h e c o n c en t ra t e d s o lu t io n t o a 3 0 0 - ml s e p a r a t i n g f u n n e l
a l r e a d y c on t a in i ng 10 0 ml o f 1 0 % s o d iu m c h l o r id e s o l u t io n . Wa sh
t h e eg g p l an t - s h a p ed fl a s k o f th e a b o v e r o ta r y v a c u u m e v a p o r a to r
w i t h 1 00 ml o f e th yl ac e t a t e a n d a d d t h e w as h i n g s t o th e s e p a r at in g
f u n n e l a b o v e . S h a k e th e mi x t u r e v i g o r o u s l y f o r f i v e mi n u t e s u si n g a
s h a k e r a nd l e a v e t h e f u n n e l t o st a n d , a n d th e n t r an s f e r t h e e t h y l
a c e t a te l a ye r t o a 3 0 0 -ml c o n i c a l f l a s k . A d d 5 0 ml o f e t h y l a c e t at e t o
t h e aq u eo u s l a y e r a n d r e p e a t t h e a b o v e p r o ce d u r e , a n d t h e n a d d th e
e t h y l a c e tat e l a y e r t o t h e c o n i ca l f l a s k a b o v e . A d d a n a d eq u ate
a mo u n t o f s o d i u m s u l f a t e ( a n h y d r o u s ) t o th e f l a s k an d l e a v e it to
s t a n d f o r 1 5 mi n u t e s w i t h o cc a s io n a l s h a k i n g . F i l te r t h e c o n t en t o f
t h e f l a s k i n t o a r o ta r y v a c u u m e v ap o r a t o r. Was h t h e f l a sk w i t h 2 0 ml
o f e t h y l a c e t a t e an d w a s h t w i c e t h e r e s i d u e o n t h e su r f a c e o f t h e
f i l t e r p ap e r w i t h t h e w a s h i n g s . A d d t h e wa s h i n g s i n to th e r o ta r y
v a c u u m e v a p o r a to r a n d c o n c e n t r at e t h e mi x t u re t o a p p r o x i ma t e l y 1
ml a t 4 0 ° C o r l o w e r, an d th e n e v ap o r a t e t h e s o l v e n t to n e a r d r y n e s s
i n a n i t r o g en s t r e a m a t r o o m t e mp e r a t u r e .
A d d 3 0 ml o f n - h ex a n e to th e d r ie d r e s id u e a n d t r a n s f er t h e mi x t u r e
t o a 10 0 - ml s e p a r a t in g f u n n e l , an d th e n ad d 3 0 ml o f n - h e x an e -
saturated a c e t o n i t r i le. Shake th e mi x t u r e v i g o r o u sl y for five
mi n u t e s u s i n g a s h a k e r a n d le av e t h e f u n n e l to s t an d , a n d th en
t r a n s f e r t h e a c e t o n i t r i le l a y e r t o a 2 0 0 - ml s e p a r a t i n g f u n n e l . A d d 3 0
ml o f n - h e x a n e - s a tu r a t e d a c et o n it r i l e t o t h e n - h e x an e l a y e r a n d
r e p e a t t h e a b o ve p r oce d u r e t w i c e, a n d th e n c o mb i n e th e a c et o n i t r il e

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l a ye r t o t h e s e p a r a t in g f u n n e l a b o v e . A d d 5 0 ml o f n - h e x a n e-
s a t u r a t e d ac e t o n i t r i l e i n t o t h e s ep a r a t i n g f u n n e l a n d s h a k e l i g h t ly,
a n d th e n l ea v e th e f u n n e l to s t a n d . Tr a n s f e r t h e a ce t o n i t r i l e l a y e r
i n t o a r o tar y v a c u u m e v ap o r a t o r a n d co n ce n t r a t e t h e mi x t u r e to
a p p r o x i ma t e l y 1 ml a t 4 0 ° C o r l o w e r, a n d t h e n ev a p o r a te t h e s o lv en t
t o n ea r d r yn e s s i n a n it r o g e n st r e a m a t r o o m t e mp e r a t u r e .
i i . F r u i t , v eg e t ab l e s , ma t c h a a n d h o p s
I n c a s e o f f r u i t a n d ve g e t ab l e s , w e i g h ac c ur a t e l y a b out 1 k g o f th e
t e s t s a mp l e a n d a d d an a p p r op r i a te a mo u n t of w a t e r t o th e s a mp l e , i f
r e q u i r e d . H o mo g e n i ze the mi x t u r e an d me s u r e out a s a mp l e
e q u iv a l en t t o 2 0 . 0 g .
I n c a s e o f ma t c h a , w e i g h 5 . 0 0 g o f t h e t e s t s a mp l e a n d a d d 2 0 ml o f
w a t e r, a n d t h e n l e av e i t t o s t a n d f o r t w o h o u r s .
I n c a s e o f h o p s , w e ig h 5 .0 0 g o f t h e te s t s amp l e , p r e v io u s l y g r o un d ,
a n d ad d 2 0 ml o f w a t e r, a n d t h en le a v e it to st a n d f o r t wo h o u r s .
T h e n , a d d 1 0 0 ml o f ac e t o n e an d 5 ml o f 4 mo l / l h y d r o c h lo r i c aci d ,
and h o mo g e n iz e the mi x t u r e for three mi n u t e s . Filter the
h o mo g e n i z e d s a mp l e b y s u c t io n i n to a r o ta r y v a c u u m e v a p o r a to r
through a filter paper c o v e r ed with a 1-cm thick layer of
d i a to ma c e o u s e a r t h . Co l l e ct t h e re s i d u e o n th e s u r f a c e o f t h e f i lt er
p a p e r an d a d d 5 0 ml o f a c e to n e , a n d t h en h o mo g e n i z e t h e mi x t u r e
f o r t h r e e mi n u t e s . R e p e a t th e a b o v e p r o ce d u r e a n d c o mb i n e t h e
f i l t r a t e in t o t h e r o t a r y v a c u u m e va p o r a to r, an d th e n c o n c e n t r a t e th e
mi x t u r e t o a p p r o x i ma t e l y 3 0 ml a t 4 0 ° C o r lo w e r.
Tr a n s f e r t h e c o n c e n t r a t e d so l ut io n to a 3 0 0 - ml s e p a r a t i n g f u n n e l
a l r e a d y c on t a in i ng 1 0 0 ml o f 1 0 % s o d i u m c h l o r id e s o l u t io n . Wa sh
t h e e g g p lan t - s h a p ed f l a s k o f t h e a b o v e r o ta r y v a c u u m e v a p o r a t o r
w i t h 1 00 ml o f e t h yl ac e t a t e a n d a d d th e w as h i n g s to th e s e p a r a tin g
f u n n e l ab ov e . S h a k e th e mi x t u r e v i g o r o u s l y f o r f i v e mi n u t e s u s in g a
s h a k e r a n d l e a v e th e f u n n e l to s t a n d , an d th e n t r a n s f er t h e e t h y l
a c e t a te l a ye r t o a 3 0 0 - ml c o n i c a l f l a s k . A d d 5 0 ml o f e t h y l a c e t a te t o
t h e a qu e ous l a ye r a n d r e p e a t t h e a b o v e p r o c e d u r e t w ic e , a n d th en
c o mb i n e th e e t h y l ac et a t e la y e r t o t h e c o n i ca l f l a s k ab o v e . A d d an
a d e q u a t e amo u n t o f so d i u m s u l f a t e ( a nh ydr o u s ) t o t he f l a s k a nd
l e a v e i t t o s t a n d f o r 1 5 mi n u t e s w i t h o c c a s io n a l s h ak i n g . F i l te r t h e
c o n te n t o f th e f l a s k in to a r o t a r y v a c u u m e v a p o r a t o r. Wa s h th e f l a sk
w i t h 2 0 ml o f e th y l a c e t a te an d w a s h t wi c e th e r e si d u e o n t h e

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s u r f a c e o f th e f i l t e r p ap e r w i t h th e w a s h i n g s . A d d t h e wa s h i n g s i n to
t h e r o t a r y v a c u u m e v a p o r a t o r a n d co n ce n t r a t e th e mi x t u r e t o
a p p r o x i ma t e l y 1 ml a t 4 0 ° C o r l o w e r, a n d th e n e v a p o r ate t h e s o l v en t
t o n ea r d r yn e s s i n a n it r o g e n st r e a m a t r o o m t e mp e r a t u r e .
i i i . Te a s o th e r t h an ma t c h a
We i g h 9 .0 0 g o f t h e te s t s a mp l e a n d s o ak i t i n 5 4 0 ml o f w a t e r a t
1 0 0 ° C , an d t h e n l e av e i t t o s t an d a t r o o m t e mp e r a t u r e f o r f i v e
mi n u t e s . Tr a n s f e r 3 6 0 ml o f t h e c o o l ed f i l t r at e i n to a 5 0 0 - ml c o n i c a l
f l a s k a n d ad d 1 8 g o f s o d i u m c h l o r i d e an d 4 mo l / l h y d r o c h lo r i c a ci d
t o a d ju s t the p H t o 1 o r l o w e r. Tr a n s f e r t h e s o l u t io n to a 1 , 0 0 0 -ml
s e p a r a t i ng f u n n e l a l r ea d y c o n t a i n i n g 1 0 0 ml o f e t h y l ace t a t e . S h a k e
t h e mi x t u r e v i g o r o u s l y f o r f i v e mi n u t e s u s i n g a s h ak e r an d le a v e th e
f u n n e l to s ta n d , an d t h e n t r a n s f e r t h e e th y l a c e t a t e l a y e r t o a 3 0 0 - ml
c o n ic a l f l as k . A d d 1 0 0 ml o f e t h y l a c e t a te to t h e aq u eo u s l a y e r a nd
r e p e a t th e a b o v e p r o ce d u r e t w i ce , a n d co mb i n e t h e e th y l a c e t at e
l a ye r t o t he c o n ic a l f l as k a b ov e . A d d a n ad eq u a te a mo u n t o f s odium
s u l f a t e ( an h y d r o u s ) t o t h e f l a s k an d l e av e i t t o s ta n d f o r 1 5 mi n u t e s
w i t h o c ca s io n a l s h a k in g . F i l t e r t h e c o n t e n t o f t h e f l a s k i n t o a r o t a ry
v a c u u m e v a p o r a to r. Wa s h t h e f l as k w i t h 2 0 ml o f e t h y l a c e t a t e an d
w a s h t w i c e t h e r e s i d u e o n t h e s u r fa c e o f t h e f i l t e r p a p e r w i t h t h e
w a s h i n g s . A d d t h e w as h i n g s i n to t h e r o t a r y v a c u u m e v a p o r a to r a n d
c o n c en t r a t e t h e mi x t u r e t o a p p r o x i ma t e l y 1 ml a t 4 0 ° C o r l o w e r, an d
t h e n ev a p o r a t e t h e s o l v e n t t o n ea r d r y n e s s i n a n i t r o g e n s t r e a m a t
r o o m t e mp e r a t u r e .
i v. F o o d s o t h e r t h a n th o s e l i s t ed in i to i ii a b o v e
O b t a i n t h e e x t r a c t s acc o r d i n g to t h e me t h o d s d e s c r i b e d i n i o r i i .
b. Hydrolysis
D i s s o l v e t he r e s i d u e ob t a in e d b y t h e e x t r a c t io n d e s c r ib ed i n 4 - a in 2 0
ml o f me t h a n o l a n d t ra n s f e r t h e mi x t u r e t o a 1 00 - ml e g g p la n t - s h ap e d
f l a s k , a n d t h e n a d d 1 0 ml o f 1 .5 mo l / l s o d i u m h y d r o x i d e s o l u t io n .
A t t a c h a r ef l u x co n d en s e r t o t h e fl a s k a n d h e a t t h e m i x t u r e fo r 3 0
mi n u t e s i n a w a t e r b a th a t 8 0 ° C, a n d t h e n a ll o w i t t o co o l . Tr a n s fe r
t h e so l u t i o n in t o a r o ta r y v a c u u m e v ap o r a to r a n d r e mo v e mo s t o f th e
me t h a n o l at 4 0 ° C o r l o w e r. F i l t e r t h e r e s i d u e b y s u c t i o n in t o a 3 0 0 -ml
s e p a r a t i n g f u n n e l ( I ) t h r o u g h a g l a s s f i l t e r ( p o r e s i z e G 3 ) . Wa s h t h e
r e s i d u e o n t h e g l a s s f il t e r w i t h a s ma l l a mo u n t o f a c et o n e an d w a te r
a n d a d d th e w a s h i n g s to t he s ep a r a t i ng f u nn el a bo v e , and t he n a dd 5 0

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ml o f e t h e r a n d 1 0 0 ml o f 1 0 % s o d i u m c h l o r i d e s o l u tio n . S h a k e th e
mi x t u r e v i g o r o u s l y fo r f i v e mi n u t e s u s in g a s h a k e r a n d l e av e it to
s t a n d . Tr a ns f e r t h e aqu e o u s l a y e r t o a 3 0 0 -ml s e p a r a t in g f u n n el (I I )
a n d a d d 4 mo l / l h y d r o c h l o r i c a c id t o a d ju s t t h e p H t o 1 o r l o w e r. A d d
5 0 ml o f e t h y l a c e t at e t o t h e a d j u s t e d s o lu t io n a n d s h a k e t h e mi x t u r e
v i g o r o u s l y f o r f i v e mi n u t e s u s i n g a s h ak e r, an d th e n l eav e it to s t an d .
Tr a n s f e r t h e e t h yl a c e ta t e l a y e r t o a 3 0 0 - ml c o n i ca l f l a s k . A d d 5 0 ml
o f e t h y l a ce t a t e t o t h e a q u e o u s l ay e r a n d r e p e a t th e a b o v e p r o c e d u r e ,
a n d th e n co mb i n e t h e e t h y l a c e ta t e l a y e r t o t h e c o n i c al f l a s k a b o v e.
A d d a n ad eq u a te a mo u n t o f s od i um s u l f a t e ( a n h yd r o u s ) t o th e f l a sk
a n d l e av e it t o s t an d fo r 1 5 mi n ut e s w i t h o cc a s i o n a l s h a k i n g . F i lte r
t h e co n te n t o f t h e f las k i n to a ro t a r y v a c u u m e v a p o ra t o r. Wa s h t h e
f l a s k w i t h 2 0 ml o f e t h y l a c e t a te a n d w a s h t w i c e t h e re s i d u e o n th e
s u r f a c e o f t h e f il t e r p a p e r w i t h th e w a s h in g s . A d d t h e w a s h i n g s in t o
the rotary v a cu u m e v a p o r a t o r and co n c e n t r a t e th e mi x t u r e to
a p p r o xi ma t e l y 1 ml a t 4 0 ° C o r lo w e r.
c . B u t y l e s te r i f i c a t i o n
Tr a n s f e r t h e s o l u t io n o b t ai n ed b y t h e h y d r o ly s i s d e s c r i b e d i n 4 - b to a
2 0 - ml e g g p l a n t - s h ap e d f l a s k an d t h e n e v a p o r a t e t h e s o l v e n t to n ea r
dryness in a nitrogen s t r e a m a t r o o m t e mp e r a t u r e . A f t e r the
d e s i c c at i o n , a d d 1 ml o f a b u t y l e st e r i f i c a te d a g e n t t o t h e r e s i d u e .
A t t a c h a r e f l u x co n d e n s e r t o th e e g g p l a n t - s h a p e d f l a s k d e s c r ib e d
a b o ve an d h e a t t h e mi x t u r e f o r 3 0 mi n u t e s in a w a te r b a t h at 9 0 ° C,
a n d a ll o w i t t o co o l . Tr a n s f e r t h e mi x t u r e t o a 2 0 0 - ml s e p a r a t i n g
f u n n e l a l r ea d y c o nt a ini n g 5 0 ml o f 1 0 % s o d iu m c h l o r i d e s o l u t i o n an d
5 0 ml o f n - h e x an e an d s h a k e t he mi x t u r e v i go r o u s l y f o r f i v e mi n u t e s
u s i n g a s h ak e r, a n d th en l ea v e i t to s t a n d . Tr a n s f e r t h e n - h e x an e l aye r
t o a 2 0 0 - ml c o n i c al f la s k a nd add 50 ml o f n - h e x an e to t h e aq u eo u s
l a ye r a n d r e p e a t th e ab o v e p r o c ed u r e , a n d th e n c o mb i n e t h e n - h ex a n e
l a y e r t o t h e c o n ic a l f la s k a b ov e . A d d a n a d eq u a te a mo u n t o f s o d iu m
s u l f a t e ( a n h y d r o u s ) t o t h e f l a s k an d l e av e i t t o s t a n d fo r 1 5 mi n u t e s
w i t h o cc a s io n a l s h a k i n g . F i l t e r t h e c o n t e n t o f t h e f l a s k i n t o a r o ta r y
v a c u u m e v a p o r a to r. Wa s h t h e f l a sk w i t h 1 0 ml o f n - h e x a n e an d wa s h
t h e r e s i d u e o n t h e s u rf a c e o f t h e f i l t e r p a p e r w i t h t h e wa s h i n g s . Ad d
t h e w a s h i n g s t o t h e r o t a r y v a c u u m e v a p o r a t o r a n d co n c en t r a t e t h e
mi x t u r e t o a p p r o x i ma t e l y 2 ml a t 4 0 ° C o r lo w e r.
d . C l ea n - u p

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A d d 5 g o f f l o r i s i l f o r c o l u mn c h r o ma t o g r a p h y s u s p e n d ed i n n - h ex an e
i n t o a c h ro ma t o g r a p h t u b e ( 1 5 m m i n i n n e r d i a me t e r, 3 0 0 mm i n
l e n gt h ) a nd a d d ov e r ap p r o x i ma t e l y 5 g o f s o d i u m s u l f a t e ( a n h y d r o u s )
i n t o th e c o l u mn . S p i l l o u t t h e n -h e x an e u n ti l o n l y a s ma l l a mo u n t
r e ma i n s o n t h e p a ck i ng o f t h e co l u mn a n d p o u r t h e s o l u tio n o b t ai n ed
b y t h e b u t y l e s t e r i f i c at i o n d e s c r ib e d in 4 - c i n t o t h e c o lu mn . P o u r 5 0
ml o f e t h e r / n - h e x a n e (1 : 1 9 ) i n to th e co l u mn a n d d i s c a r d t h e e ff l u e n t .
P o u r 1 5 0 ml o f e t h e r / n - h e x a n e (3 :1 7 ) i n to t h e c o lu mn a n d c o l l e ct th e
e l u a te i n to a r o t a r y v a c u u m e v ap o r a t o r. C o n c e n t r a t e th e mi x t u r e t o
a p p r o xi ma t e l y 1 ml a t 4 0 ° C o r lo w e r a n d e v a p o r a t e t h e s o l v e n t to n e a r
d r y n e s s i n a n i t r o g en s t r e a m a t r o o m t e mp e r a t u r e . D i s s o l v e t h e
r e s i d u e i n n - h e x an e t o ma k e e x a ct l y 1 0 ml o f s o l u t io n , w h i c h i s u se d
a s t h e s a mp l e s o lu t i o n .
5 . D e t e r mi n a t io n
a . Q u a l i ta t iv e t e s t s
P e r f o r m q u a l i t at i v e t es t s u n d e r th e f o l l o w in g c o n d i t io n s . Te s t r e s u l t s
o b t ai n ed mu s t b e t h e s a me a s t h o s e o b t a i n ed f o r t h e r e f e r e n c e
s t a n d a r d u n d e r t h e p r o c e d u r e d e s cr i b e d in 4 -c .
Te s t i n g c o n d i t io n s
C o l u mn : A s i l i c a te g l as s c a p i ll a r y c o lu mn ( 0 . 2 5 mm i n i n n e r d i a me t e r,
3 0 m i n len g t h ) c o a te d w i th 5 % p h e n y l me t h y l s i l ic o n e f o r g a s
c h r o ma t o g r a p h y t o a th i c kn e s s o f 0 . 2 5 µ m.
C o l u mn t e mp e r a t u r e : H o l d t h e c o l u mn t e mp e r a t u r e a t 5 0 ° C f o r o n e
mi n u t e , f o ll o w e d b y an i n c r e a s e o f 2 5 ° C ev er y mi n u t e u n t i l r e ac h in g
1 2 5 ° C , a f t er w h i c h in cr e a s e t h e te mp e r a t u r e b y 1 0 ° C ev e r y mi n u t e
u n t il r e a c h in g 3 0 0 ° C , a n d h o ld f o r f i v e mi n u t e s .
I n l e t t e mp e r a t u r e : 2 6 0 ° C
D e t e c t o r : Op e r a t e at 3 0 0 ° C .
Gas flow rate: Use nitrogen or helium as the carrier gas. Adjust the
f l o w r a t e s o t h a t n - b u t y l ( 2 , 4 ,5 - tr i c h l o r o p h en o x y ) a c et at e f l o w s o u t
i n a p p r o x ima t e l y 1 5 mi n u t e s .
b . Q u a n ti t at i v e t e s t s
D e t e r mi n e t h e q u a n ti t y f r o m t h e t e s t r e su l t s o b t a in e d u n d e r th e
c o n d i t io n s d e s c r i b ed in 5 - a u si n g e i t he r t h e p e a k h e ig h t o r p e ak a re a
me t h o d .
c . C o n f i r ma t i o n t e s t s
P e r f o r m g a s c h r o ma t o g r a p h y / ma s s s p e c t ro me t r y u n d e r t h e s am e

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c o n d i t io n s a s t h o s e d e s c r i b e d i n 5 - a . Te s t r e s u l t s o b ta i n ed f o r th e
r e f e r e n c e s t a n d a r d mu s t b e th e s a me a s t h o s e o b t a in e d u n d e r th e
p r o c e d u r e d e s c r i b ed in 4 - c . D e t er mi n e t h e q u a n t i t y u si n g e it h e r th e
p e a k h e ig h t o r p e ak a re a me t h o d , i f r e q u i r e d .

( 4 ) A n a l yt i c a l me t h o d f o r a z o c y c lo t i n a n d c y h e x at i n
1. Apparatus
A g a s c h r o ma t o g r a p h w i t h a f l a me p h o t o me t r i c d e t e c t o r ( G C - F P D ,
i n t e r f e r e n ce f i l t e r f o r t i n d e t e r mi n a t io n , w a v e l e n g t h : 6 1 0 n m) a n d a g a s
c h r o ma t o g r a p h / ma s s s p e c t r o me t e r ( G C / M S o r G C / M S / M S ) .
2 . R e ag e n t s a n d t e s t s o l u t io n s
I n a d d i t io n t o t h e r e ag e n t s a n d te s t s o lu t i o n s l i s t ed b e l o w, u s e th o s e
l i s t e d in S e c t i o n C R e a g e n ts /Te s t S o l u ti o n s , E t c . , P a r t I I F o o d
a d d it i v e s . R e a g en t s d e s i g n at e d as “s p e c i a l g r a d e ” i n th i s s e c ti o n mu s t
me e t t h e re q u i r e me n t s f o r “s p e c i a l g r a d e” s p e c i f ie d i n t h e J ap a n
I n d u s t r i a l St a n d a r d s fo r t h e r e ag e n t s .
A c e t o n it r i le : U s e a r o ta r y v a c u u m e v a p o r a t o r o n 3 0 0 mL o f a c e to n i t ri l e
t o c o n c e n t r a t e t o t h e p o i n t o f d r y n e s s . D i s s o l v e t h e r e s id u e i n 5 mL o f
n - h e x an e . F o r a n a l y s i s , i n j ec t 5 μ L o f th e so l u ti o n i n to a GC -ECD.
P e a k s o t h er t h a n t h a t o f n - h e x an e i n th e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
A c e t o n e: U s e a r o t a r y v a c u u m e v ap o r at o r o n 3 0 0 mL o f a c e t o n e t o
c o n c en t r a t e t o t h e p o in t o f d r y n es s . D i s s o l v e t h e r e s i d u e in 5 mL o f
n - h e x an e . F o r a n a l y s i s , i n j e c t 5 μ L o f t h e so l u ti o n i n to a G C - E C D .
P e a k s o th er t h a n t h a t o f n - h e x an e in th e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
3 mo l / l e th y l ma g n e s i u m b r o mi d e - e t h e r e a l s o l u t io n : 3 mo l / l e t h y l
ma g n e s i u m b r o mi d e - e t h e r e a l s o l u t i o n .
E t h e r : U s e a r o t a r y v a c u u m e v a p o r a t o r o n 3 0 0 mL o f e t h e r t o
c o n c en t r a t e t o t h e p o in t o f d r y n e s s . D i s s o l v e t h e r e s i d u e i n 5 mL o f
n - h e x an e . F o r a n a l y s i s , i n j ec t 5 μ L o f th e so l u ti o n i n to a GC -ECD.
P e a k s o t h er t h a n t h a t o f n - h e x an e i n th e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
S o d i u m c h l o r i d e : S o diu m c h l o r i d e ( s p e c i a l g r a d e ) . I f t h e r e a g en t i s
f o u n d to i nc l u de a n y s u b s t a n c e t h a t ma y i n t er f e r e w i t h t h e a n al y s i s o f
s u b s t a n ce s t o b e a n al y z e d f r o m a g r i c u l tu r a l c h e mi c a l s , w a s h w i t h a
s o l v e n t s u ch a s n - h e x a n e b e f o r e u s e .
S y n t h e t i c ma g n e s i u m s i l i c at e (F l o r i s i l ) f o r c o l u mn c h r o ma t o g r a p h y:

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H e a t f l o r i s il ( 1 5 0 -2 5 0 μ m i n p a r t ic l e s i ze ) a t 1 3 0 ° C f o r 1 2 h o u r s o r
l o n g e r. C o o l d o w n t o ro o m t e mp e r a t u r e in a d e s i c c a to r.
D i a t o ma c e o u s e a r t h : D i a t o ma c e o u s e a r t h f o r c h e mi c a l a n a l y s i s .
C y h e x a t in s t a n d a r d s o l u t io n : A d d a c e ti c a ci d / e th y l a ce t a t e ( 1 :9 9 ) t o
1 0 . 0 mg o f c y h e x a ti n t o ma k e a 1 0 0 - ml s o l u t i o n . Tak e o u t 1 0 ml o f
t h e s o l u t io n a n d a d d n - h e x a n e t o ma k e 1 0 0 ml o f mi x .
S o d i u m d o d e c y l s u l f a t e : S od i u m d o d ec yl s u lf a t e w i t h a p u r it y o f 8 5%
o r h i g h e r.
n - H e x a n e : U s e a r o t a r y v a c u u m e v a p o r at o r on 3 00 mL o f n - h e x a n e t o
e v a p o r a t e u n t i l 5 mL i s l e f t . F o r a n a l y s i s , i n j e c t 5 μ L o f t h e s o lu t io n
i n t o a G C - E C D . P e a k s o t h e r th an t h at o f n - h e x a n e i n t h e r e s u l t in g
c h r o ma t o g r a m s h o u l d b e a s h ig h a s o r lo w e r t h an t h e pe a k s o f γ - B H C
a t 2 x 10 - 1 1 g .
Wa t e r : D i s ti l l e d w a t e r. I f t h e d i s ti l l e d w a t e r i s f o u n d to i n c l u d e an y
s u b s t a n ce th a t ma y i n t e r f e r e w i t h a n a l y s i s o f t h e t a rg et co mp o s i t i o n a l
s u b s t a n ce s f r o m a g r i c u l t u r a l ch emi c a l s , w a s h w i t h a s o l v en t s u c h a s
n-hexane before use.
S o d i u m s u l f a t e ( a n h y d r o u s ) : S o d iu m s u l f a t e ( a n h y d r o u s) ( s p e c i a l g ra d e ) .
I f t h e r e a g e n t i s f o u n d t o in c lu d e a n y s u b st a n c e t h at ma y i n t e r f e r e
w i t h a n al y s i s o f t h e t a rg e t co mp o s i t i o n a l s u b st a n c e s f r o m a g r i c u l t u r al
c h e mi c a l s , w a s h w i t h a s o l v en t su c h a s n - h e x a n e b e f o r e u s e .
3. Reference standard
C yh e x a t in : T h i s p r o duc t c o n t a in s n o t l e s s th a n 9 9 % o f c y h e x a t in , a n d
i t s me l t i n g p o i n t i s 1 9 5 - 1 9 8 ° C .
4. Procedure
a . E x t r a ct io n
i . L e g u me s / p u l s e s a n d s e e d s
We i g h 1 0 .0 g o f t he te s t s a mp l e , p r e v io u s l y g r o u n d so a s to p a s s
t h r o u g h a st a n d a r d me s h s i e v e ( 4 2 0 µ m) . A d d 2 0 ml o f w a t e r t o t h e
o b t ai n ed s amp l e a n d l ea v e it t o s ta n d f o r t w o h o u r s .
T h e n , a d d 1 0 0 ml o f a c e t on e / a ce ti c a c id ( 99 :1 ) a n d h o mo g e ni z e t h e
mi x t u r e f o r t h r e e mi n u t e s . F i l t e r th e h o mo g e n i z ed s a mp l e b y s u c t i o n
i n t o a r o ta r y v a c u u m e v a p o r at o r th r o u g h a f il t e r p ap e r co v e r e d w i th
a 1 - c m t h i c k l a y e r o f di a t o ma c e o u s e a r t h . C o ll e c t th e r e s i d u e o n th e
s u r f a c e o f t h e f i lt e r p a p e r an d a d d 5 0 ml o f a c e to n e / a c e ti c aci d
( 9 9 : 1 ) , an d t h e n h o mo g e n iz e th e mi x t u r e f o r t h r e e mi n u t e s . R e pe a t
t h e ab o v e p r o c e d u r e and c o mb i n e th e f i l t r a te in t o t h e r o t ar y v a c u u m

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e v a p o r a t o r, a n d th e n co n c en t r a t e t h e mi x t u r e t o a p p r o x ima t e l y 3 0 ml
a t 4 0 °C o r l o w e r.
Tr a s f e r t h e c on c e nt r a te d s o l u ti o n t o a 5 0 0 - ml s e p a r a t in g f u n n el
a l r e a d y c on t a in i ng 20 0 ml o f 1 0 % s o d iu m c h l o r id e s o l u t io n . Wa sh
t h e eg g p l an t - s h a p ed fl a s k o f th e a b o v e r o ta r y v a c u u m e v a p o r a to r
w i t h 1 00 ml o f n - h e x a n e a n d ad d th e w a s h i n g s t o th e s e p a r a t in g
f u n n e l a b o v e . S h a k e th e mi x t u r e v i g o r o u s l y f o r f i v e mi n u t e s u si n g a
s h a k e r a n d l e a v e i t t o s t a n d , a n d th e n t r an s f e r t h e n - h ex a n e la y e r t o
a 3 0 0 - ml c o n i ca l f l a sk . A d d 5 0 ml o f n - h e x a n e t o t h e a q u eo u s l aye r
a n d r ep e a t t h e a b o v e p r o c e d u r e , an d t h en add th e n - h e x a n e l a y e r to
t h e c o ni c a l f l a s k a b ov e . A d d a n ad e q u a t e a mo u n t o f s o d i u m s u l f a t e
( a n h y d r o u s) t o t h e f l as k a n d l e av e i t t o s tan d f o r 1 5 mi n u t e s w i t h
o c c a s io n a l s h a k i n g . F i l t e r t h e c o n te n t o f t h e f l a s k i n t o a r o t ar y
v a c u u m e v a p o r a t o r. Was h t h e f l a s k w i t h 2 0 ml o f n - h e x a n e a n d w a s h
t w i c e t h e r es i d u e o n t h e s u r f a c e o f t h e f i lt e r p a p e r w i t h th e w a s h i n g s .
A d d t h e w a s h i n g s i nt o t h e r o t a r y v a c u u m e v a p o r a t o r a n d r e mo v e t h e
n - h e x an e at 4 0 ° C o r l ow e r.
A d d 2 0 ml o f n - h e x a n e t o t h e r e si d u e a n d t ra n s f e r t h e mi x t u r e t o a
1 0 0 - ml s e p a r a t i n g f u n n e l , a n d t h e n ad d 4 0 ml o f n - h e x a n e - s a t u r a ted
a c e t o n i t r i le. S h a k e t h e mi x t u r e v i g o r o u s l y f o r f i v e mi n u t e s u s i n g a
s h a k e r an d l e a v e i t to s t a n d , a n d t h e n t r a n s fe r t h e a c eto n i t r il e l a y e r
i n t o th e r o ta r y v a c u u m e v a p o r at o r. A d d 4 0 ml o f n - h e x an e - s a t u r a t ed
a c e t o n i t r i le t o th e n - h e x a n e l a y er a n d r e p ea t t h e a b o v e p r o c ed u re
t w i c e , an d t h en c o mb i n e t h e ac e t o n i t r i l e l a y e r i n to t h e r o t ar y
v a c u u m e v a p o r a t o r a b o v e . R e mo v e t h e a c e to n it r i l e a t 4 0 °C o r l o w e r.
D i s s o l v e t h e r e s i d u e i n n - h ex a n e t o ma k e e x a c t l y 5 ml o f s o l u ti o n .
i i . C e r e al g r a i n s , f r u i t a n d v e g et ab l e s
I n c a s e o f ce r e a l g r a i n s , w e i g h 1 0 . 0 g o f th e t e s t s a mp l e , p r e v i o u sly
g r o u n d s o a s t o p a s s th r o u g h a s ta n d a r d me s h s i e v e ( 4 2 0 µ m) . A d d
2 0 ml o f w a t e r t o th e o b t ai n ed s amp l e a n d le a v e i t t o st a n d f o r t wo
hours.
I n c a s e o f f r u i t a n d v e g e t ab l e s , w e i g h a c cu r a t e l y 1 k g o f t h e t e st
s a mp l e a n d a d d a n app r o p r i a t e amo u n t o f w a t e r t o t he s a mp l e , i f
r e q u i r e d . H o mo g e n i ze t h e mi x t u r e a n d me a s u r e o u t a s a mp l e
e q u iv a l en t t o 2 0 . 0 g .
T h e n , a d d 1 0 0 ml o f ac e t o n e / a ce ti c a ci d ( 9 9 :1 ) a n d h o mo g e n i z e t h e
mi x t u r e f o r t h r e e mi n u t e s . F i l t e r th e h o mo g e n i z ed s a mp l e b y s u c t io n

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i n t o a r o t a ry v a c u u m e v a p o r at o r th r o u g h a f il t e r p a p e r co v e r e d w i th
a 1 - c m t h i ck l a y e r o f di a t o ma c e o u s e a r t h . Co ll e c t th e r e si d u e o n t h e
s u r f a c e o f t h e f il t e r p a p e r a n d a d d 5 0 ml o f a ce t o n e / a c e ti c a ci d
( 9 9 : 1 ) , a n d t h en h o mo g e n iz e th e mi x t u r e f o r t h r e e mi n u t e s . R ep e at
t h e a b o v e p r o c e d u r e a n d c o mb i n e th e f i l t r at e in t o t h e r o t ar y v a c u u m
e v a p o r a t o r, a n d t h e n co n c en t r a t e t h e mi x t u r e t o a p p r o x ima t e l y 3 0 ml
a t 4 0 °C o r l o w e r.
Tr a n s f e r t h e c o n c en t ra t e d s o lu t io n t o a 5 0 0 - ml s e p a r a t i n g f u n n e l
a l r e a d y c on t a in i ng 20 0 ml o f 1 0 % s o d iu m c h l o r id e s o l u t io n . Wa sh
t h e eg g p l an t - s h a p ed fl a s k o f th e a b o v e r o ta r y v a c u u m e v a p o r a to r
w i t h 1 00 ml o f n - h e x a n e a n d ad d th e w a s h i n g s t o th e s e p a r a t in g
f u n n e l a b o v e . S h a k e th e mi x t u r e v i g o r o u s l y f o r f i v e mi n u t e s u si n g a
s h a k e r a n d l e a v e i t t o s t a n d , a n d th e n t r an s f e r t h e n - h ex a n e la y e r t o
a 3 0 0 - ml c o n i ca l f l a sk . A d d 5 0 ml o f n - h e x a n e t o t h e a q u eo u s l aye r
a n d r e p ea t t h e a b o v e p r o c e d u r e t w i c e , an d t h e n co mb i n e t h e
n - h e x an e l ay e r t o th e co n i ca l f l a sk a b o v e . A d d an a d eq u a t e a mo u n t
o f s o d iu m s u l f a t e ( an h y d r o u s ) t o t h e f l a s k an d l e a v e it t o s t an d f o r
1 5 mi n u t e s w i t h o c c a si o n al s h ak in g . F i lt e r th e co n te n t o f t h e f l a sk
i n t o a r o t ar y v a c u u m e v a p o r a t o r. Wa s h t h e f l a s k w i th 2 0 ml o f
n - h e x an e , an d w a s h tw i c e t h e re s i d u e o n t h e su r f a c e o f th e f i lte r
p a p e r w i t h t h e w a s h i ng s . A d d t h e w a s h i n g s i n t o th e r o t a r y v a c u u m
e v a p o r a t o r a n d r e mo v e th e n - h e x a n e a t 4 0 ° C o r l o w e r, a n d th e n
d i s s o l v e t h e r e s i d u e i n n - h ex a n e t o ma k e e x a c t l y 1 0 ml o f s o l u ti o n .
i i i . Ma t ch a a n d h o p s
I n c a s e o f ma t c h a , w e i g h 5 . 0 0 g o f t h e t e s t s a mp l e a n d a d d 2 0 ml o f
w a t e r, a n d t h e n l e av e i t t o s t a n d f o r t w o h o u r s .
I n c a s e o f h o p s , w e ig h 5 .0 0 g o f t h e te s t s amp l e , p r e v io u s l y g r o un d ,
a n d ad d 2 0 ml o f w a t e r, a n d t h en le a v e it to st a n d f o r t wo h o u r s .
T h e n , a d d 1 0 0 ml o f ac e t o n e / a ce ti c a ci d ( 9 9 :1 ) a n d h o mo g e n i z e t h e
mi x t u r e f o r t h r e e mi n u t e s . F i l t e r th e h o mo g e n i z ed s a mp l e b y s u c t io n
i n t o a r o t a ry v a c u u m e v a p o r at o r th r o u g h a f il t e r p a p e r co v e r e d w i th
a 1 - c m t h i ck l a y e r o f di a t o ma c e o u s e a r t h . Co ll e c t th e r e si d u e o n t h e
s u r f a c e o f t h e f il t e r p a p e r a n d a d d 5 0 ml o f a ce t o n e / a c e ti c a ci d
( 9 9 : 1 ) , a n d t h en h o mo g e n iz e th e mi x t u r e f o r t h r e e mi n u t e s . R ep e at
t h e a b o v e p r o c e d u r e a n d c o mb i n e th e f i l t r at e in t o t h e r o t ar y v a c u u m
e v a p o r a t o r, a n d t h e n co n c en t r a t e t h e mi x t u r e t o a p p r o x ima t e l y 3 0 ml
a t 4 0 °C o r l o w e r.

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Tr a n s f e r t h e c o n c en t ra t e d s o lu t io n t o a 5 0 0 - ml s e p a r a t i n g f u n n e l
a l r e a d y c on t a in i ng 20 0 ml o f 1 0 % s o d iu m c h l o r id e s o l u t io n . Wa sh
t h e eg g p l an t - s h a p ed fl a s k o f th e a b o v e r o ta r y v a c u u m e v a p o r a to r
w i t h 1 00 ml o f n - h e x a n e a n d ad d th e w a s h i n g s t o th e s e p a r a t in g
f u n n e l a b o v e . S h a k e th e mi x t u r e v i g o r o u s l y f o r f i v e mi n u t e s u si n g a
s h a k e r a n d l e a v e i t t o s t a n d , a n d th e n t r an s f e r t h e n - h ex a n e la y e r t o
a 3 0 0 - ml c o n i ca l f l a sk . A d d 5 0 ml o f n - h e x a n e t o t h e a q u eo u s l aye r
a n d r e p ea t t h e a b o v e p r o c e d u r e t w i c e , an d t h e n co mb i n e t h e
n - h e x an e l ay e r t o th e co n i ca l f l a sk a b o v e . A d d an a d eq u a t e a mo u n t
o f s o d iu m s u l f a t e ( an h y d r o u s ) t o t h e f l a s k an d l e a v e it t o s t an d f o r
1 5 mi n u t e s w i t h o c c a si o n al s h ak in g . F i lt e r th e co n te n t o f t h e f l a sk
i n t o a r o t ar y v a c u u m e v a p o r a t o r. Wa s h t h e f l a s k w i th 2 0 ml o f
n - h e x an e an d w a s h tw i c e t h e r e s i d u e on t h e s u r f a c e o f t h e f i l te r
p a p e r w i t h t h e w a s h i ng s . A d d t h e w a s h i n g s i n t o th e r o t a r y v a c u u m
e v a p o r a t o r a n d co n cen t r a t e th e mi x t u r e t o a p p r o xi ma t e l y 5 ml a t
4 0 ° C o r l ow e r, a n d th e n a d d n -h e x an e to ma k e e x a c tl y 1 0 ml o f
s o l u t io n .
i v. Te a s o t h e r t h a n ma t c h a
We i g h 9 .0 0 g o f t h e te s t s a mp l e a n d s o ak i t i n 5 4 0 ml o f w a t e r a t
1 0 0 ° C , an d t h e n l e av e i t t o s t an d a t r o o m t e mp e r a t u r e f o r f i v e
mi n u t e s . Tr a n s f r e r 3 6 0 ml o f t h e c o o l e d f i l t r a t e i n to a 5 0 0 - ml
s e p a r a t i ng f u n n e l an d a d d 3 0 g o f s o d i u m c h l o r i d e , 2 ml o f 2 %
s o d i u m d o d e c y l s u l f a te s o lu t io n a n d 1 00 ml o f n -h e x an e . S h a k e t h e
mi x t u r e v ig o r o u s l y f o r f i v e mi n u t e s u s in g a s h a k e r a n d l ea v e i t to
s t a n d , a n d th e n t r a n s f er t h e n - h exa n e l a ye r to a 30 0 - ml c o n ic a l f l a sk .
A d d 1 0 0 ml o f n - h e x an e t o th e aq u e ou s l a ye r a n d r e p ea t t h e ab o ve
p r o c e d u r e tw i c e , a n d th e n c o mb i n e t h e n - h exa n e l a ye r to t h e co n ica l
f l a s k a b ov e. A d d a n ad e q u at e a mo u n t o f s o d iu m s u l f a t e ( a n h y d ro u s)
t o th e f l a sk a n d l ea v e i t t o s t an d f o r 1 5 mi n u te s with occasional
s h a k i n g . F i l t e r t h e co n t en t o f t h e f l a s k i n t o a r o ta r y v a c u u m
e v a p o r a t o r. Wa s h t h e f l a s k w i t h 2 0 ml o f n - h e x an e an d w a s h t w i ce
t h e r e s id u e o n th e su r f a c e o f t h e f i l t e r p a p e r w i t h t h e w as h i n g s . A d d
t h e w a s h in g s i n to th e r o t a r y v a c u u m e v a p o r a t o r a n d r e mo v e
n - h e x an e a t 4 0 ° C o r l ow e r. D i s s o l v e t h e r e s idu e i n n - h e x a n e t o ma k e
e x a c tl y 6 ml o f s o l u t i o n .
v. F o o d s o th e r t h an t h o s e l i s t ed in i to i v a b o v e
O b t a i n t h e e x t r a c t s a cc o r d i n g t o t he me t h o ds d e s c r i be d i n i , ii , o r

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iii.
b . E t h y l at io n
Tr a n s f e r 1 ml ( 2 ml f o r c e r e a l g r a i n s , t e a s an d h o p s ) o f t h e s o lu t io n
o b t ai n ed b y t h e ex t r a c ti o n d e s c r i be d in 4 - a to a 5 0 - ml t e s t t ub e w i t h a
g l a s s s t o p p e r a n d ad d 1 ml ( 2 ml f o r c e r e a l g r a i n s , t e a s a n d h o p s ) o f a
3 mo l / l et h y l ma g n e s i u m b r o mi d e - e t h e r e a l s o l u t io n , a n d t h en l ea v e i t
t o s t an d f o r 2 0 mi n u t e s a t r o o m t e mp e r a t u r e .
T h e n , a d d 1 0 ml o f 0 . 5 ml / l s u l f u r i c a ci d g r a d u a l l y an d a d d 1 0 ml o f
w a t e r t o b e mi x e d . A d d 1 0 ml o f n - h e x an e to t h e mi x t u r e a n d s h a k e
t h e mi x t u r e v i go r o u s ly f o r o n e mi n u t e , a n d t h en l e av e i t to s ta n d .
Tr a n s f e r t h e n - h e x a n e l a ye r t o a 5 0 - ml c o ni c a l f l a s k. A d d 5 ml o f
n - h e x an e to t h e a qu eo u s l a y e r a n d r e p e a t th e a b o v e p r o c e d u r e , an d
t h e n c o mb i n e th e n - h ex a n e l a y e r t o th e c o n ic a l f l a s k ab o v e. A d d a n
a d e qu a t e amo u n t o f s o d i u m s u l f a t e ( a n h y d ro u s ) t o th e f l a s k a n d l ea v e
i t to s t an d f o r 1 5 mi n u t e s w i th o c c a s i o n a l s h a k in g . F i lt e r t h e co n te n t
o f t h e f l a sk i n t o a r o tar y v a c u u m e v a p o r a t o r. Wa s h t h e fl a s k w i t h 5 m l
of n-hexane and wash the residue on the surface of the filter paper
w i t h t h e w a s h i n g s . A d d t h e w a s h in g s t o t h e ro t a r y v a c u u m e v a p o r a to r
a n d co n ce nt r a t e t he mi x t u r e to 2 ml a t 4 0 ° C o r l o w e r.
c . C l e an - u p
A d d 5 g o f f l o r i s i l f o r c o l u mn c h r o ma t o g r a p h y s u s p e n d ed i n n - h ex an e
i n t o a c h ro ma t o g r a p h t u b e ( 1 5 m m i n i n n e r d i a me t e r, 3 0 0 m m i n
l e n gt h ) a nd a d d ov e r ap p r o x i ma t e l y 5 g o f s o d i u m s u l f a t e ( a n h y d r o u s )
i n t o th e c o l u mn . S p i l l o u t t h e n -h e x an e u n ti l o n l y a s ma l l a mo u n t
r e ma i n s o n t h e p a ck i ng o f t h e co l u mn a n d p o u r t h e s o l u tio n o b t ai n ed
b y t h e e t h yl a t i o n d e s cr i b e d i n 4 - b i n to t h e c o l u mn . T h e n , w a s h th e
e g g p l a n t - s h a p e d f l a s k o f t h e r o t a ry v a c u u m e v a p o r at o r w i t h 15 ml o f
n - h e x an e an d t r a n s f e r r h e w a s h in g s to t h e c o l u mn , a n d th e n c o l le c t
t h e e lu a t e i n t o th e r o t a r y v a c u u m e v a p o r a t o r. T h e n , p o u r 5 0 ml o f
e t h e r / n - h e x a n e ( 1 :9 9 ) i n t o t h e col u mn a n d co l l e ct t h e e l u a te i n th e
r o t a r y v a c uu m e v a p o r at o r, a nd t h en r e mo v e t h e e th e r a nd n - h e x a n e a t
4 0 ° C o r l o w e r. D i s s o l v e th e r e s id u e in n - h e x a n e t o ma k e e x ac t l y 2 m l
o f s o l u ti o n , w h i c h i s u s e d a s t h e sa mp l e s o l u ti o n .
5 . D e t e r mi n a t io n
a . Q u a l i ta t iv e t e s t s
P e r f o r m q u a l i t at i v e t es t s u n d e r th e f o l l o w in g c o n d i t io n s . Te s t r e s u l t s
o b t ai n ed mu s t b e t h e s a me a s t h o s e o b t a i n ed f o r t h e c y h ex a t in

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r e f e r e n c e s t a n da r d u nd e r t h e p r o c e d u r e d e s cr i b e d in 4 -b . A z o c y c lo tin
i s mo d i f i e d b y e t h yl a t io n in t o t h e s a me s u b s t a n c e a s c y h e x a t in .
Te s t i n g c o n d i t io n s
C o l u mn : A s i l i c a te g l as s c a p i l l a r y c o l u mn ( 0 . 3 2 mm- 0 . 5 3 mm i n i n n e r
d i a me t e r, 3 0 m i n l en g t h ) c o at e d w i t h 5 % p h e n y l me t h y l s i l i co n e f o r
g a s c h r o ma t o g r a p h y t o a t h ic k n e s s o f 1 . 5 µ m.
C o l u mn t emp e r a t u r e : H o l d th e co l u mn t e mp e r a t u r e a t 1 2 0 ° C f o r t w o
mi n u t e s , f o l l o w e d b y a n in c r ea s e o f 1 0 ° C e v e r y mi n u t e u n ti l
r e a c h i n g 2 0 0 ° C , a f t e r w h i c h in c r ea s e t h e t e mp e r a t u r e b y 2 0 ° C e v e ry
mi n u t e u nt il r e a c h in g 3 0 0 ° C , a n d h o ld f o r f i v e mi n u t e s .
I n l e t t e mp e r a t u r e : 2 8 0 ° C
D e t e c t o r : Op e r a t e at 3 0 0 ° C .
G a s f l o w r a t e : U s e h el i u m a s t h e c a r r i e r g a s . A d j u s t th e f l o w r a t e s o
t h a t c yh e x at i n f lo w s o u t in ap p r o x i ma t e l y 1 3 - 1 5 mi n u t e s . A d j u s t th e
f l o w r a t e s o f a i r a n d h y d r o g e n to o p t i ma l c o n d i ti o n s .
b . Q u a n ti t at i v e t e s t s
D e t e r mi n e t h e q u an t i ty f r o m t h e t e s t r e s u lt s o b ta i n e d u n d e r th e s ame
c o n d i t io n s d e s c r i b ed in 5 - a u si n g e i t he r t h e p e a k h e ig h t o r p e ak a re a
me t h o d .
c . C o n f i r ma t i o n t e s t s
P e r f o r m g a s c h r o ma t o g r a p h y / ma s s s p e c t ro me t r y u n d e r t h e s am e
c o n d i t io n s d e s c r i b ed in 5 - a . Te s t r e s u l t s o b t a i n ed f o r t h e r e f e r e n c e
s t a n d a r d mu s t b e s a me a s t h o s e o b ta i n e d u n d e r t h e p r o c e d u r e
d e s c r i b ed in 4 - b . D e t er mi n e t h e q u a n ti t y u s in g e i th e r th e p ea k h ei g h t
o r p e a k a r ea me t h o d , if r e q u i r e d .

( 5 ) A mi t r o l e a n a l y t ic a l me t h o d
1. Apparatus
A h i g h - p e r fo r ma n c e l iq u i d ch r o ma t o g r ap h wi t h a f l u o r es c e n c e d e tec t o r
( H P L C - F L ) a n d a l iq u i d ch r o ma t o g ra p h / ma s s s p e c t r o m e t e r ( L C / M S o r
LC/MS/MS).
2 . R e ag e n t s a n d t e s t s o l u t io n s
I n a d d i t io n t o t h e r e ag e n t s a n d te s t s o lu t i o n s l i s t ed b e l o w, u s e th o s e
l i s t e d in S e c t i o n C R e a g e n ts /Te s t S o l u ti o n s , E t c . , P a r t I I F o o d
a d d it i v e s .
A c e t o n e: Us e a r o ta r y v a cu u m e v a p o r at o r o n 3 0 0 mL o f a ce t o n e t o
c o n c en t r a t e t o t h e p o in t o f d r y n e s s . D i s s o l v e t h e r e s i d u e i n 5 mL o f
n - h e x an e . F o r a n a l y s i s , i n j ec t 5 μ L o f th e so l u ti o n i n to a GC -ECD.

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P e a k s o t h er t h a n t h a t o f n - h e x an e i n th e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
E t h a n o l : Us e a r o t a r y v a c uu m e v a p o r at o r o n 30 0 mL o f e t ha n ol t o
c o n c en t r a t e t o t h e p o in t o f d r y n es s . D i s s o l v e t h e r e s i d u e i n 5 mL o f
n - h e x an e . F o r a n a l y s i s , i n j e ct 5 μ L o f th e s o l u t i o n i n to a g a s
c h r o ma t o g r a p h e qu i ppe d w i t h a n e l e c t r o n c a p tu r e d e te c t o r. P e a k s
o t h e r t h a n t h a t o f n - h e x a n e i n the r e s u l t i ng c h r o ma t o g r a m s h o u l d b e
a s h i g h a s o r l o w e r t h a n t h e p ea k s o f γ - B H C a t 2 x 10 - 1 1 g .
D i a t o ma c e o u s e a r t h : D i a t o ma c e o u s e a r t h f o r c h e mi c a l a n a l y s i s .
A c e t i c a ci d b u ff e r s o lu t i o n : A d d 0 . 0 5 mo l / l s o d i u m a c e t a t e s o l u ti o n t o
8 0 0 ml o f 0 . 0 5 mo l / l a c e t i c a ci d so l u ti o n to ma k e a 1 , 0 0 0 - ml s o l u t i o n .
We a k l y a c id i c c at i o n e x c h an g e r e s i n : Wa s h t h e w e a k ly a c i d i c cat i o n
e x c h an g e re s i n p r o d u c e d f o r c o lu mn c h o r o ma t o g r a p h y w i t h 1 mo l / l
h y d r o c h l o r ic a c i d , s ec o n dl y w i t h a 2 . 8 % a q u eo u s am mo n i a , a n d
t h i r d l y w i th 1 mo l / l h y d r o c h l o r ic a c i d ag a in . T h e r e a f te r, w a s h t h e
w a s h i n g s w i t h w a t e r u n t i l t h e y b ec o me n e u t ra l .
F l u o r e s c a mi n : A r e a g en t w i t h a p u r i t y o f 9 8 % o r h i g h e r.
Wa t e r : D i s ti l l e d w a t e r. I f t h e d i s ti l l e d w a t e r i s f o u n d to i n c l u d e an y
s u b s t a n ce th a t ma y i n t e r f e r e w i t h a n a l y s i s o f t h e t a rg et co mp o s i t i o n a l
s u b s t a n ce s f r o m a g r i c u l t u r a l ch emi c a l s , w a s h w i t h a s o l v en t s u c h a s
n-hexane before use.
P h o s p h a te b u ff e r s o l uti o n : A d d 1 0 % p h o s p h o r i c a ci d so l u t i o n t o a 0 .0 5
ml / l mo n o s o d i u m p h o s p h a te s o lu ti o n to ad j us t t h e p H to 3 . 0.
3. Reference standard
A mi t r o l e : T h i s p r o duc t c o n t a in s n o t le s s t h an 9 8 % o f ami t r o l e , a n d i t s
me l t i n g po in t i s 15 7 - 15 9 ° C .
4. Procedure
a . E x t r a ct io n
i . C e r e a l g r a i n s , l eg u me s / p u l s e s , se e d s , f r u i t , v e g et a b le s , ma t c h a a n d
hops
I n c a s e o f c e r e a l g r ain s , l eg u me s /p u l s e s an d s e e d s , w e i g h 3 0 .0 g o f
t h e t e s t s amp l e , p r e v io u s l y g r o u n d s o a s t o p a s s th r o u g h a s t an d ar d
me s h s i e v e ( 4 2 0 µ m) .
I n c a s e o f f r u i t a n d v e g e t ab l e s , w e i g h a c c u r a t e l y a b o u t 1 k g . o f th e
t e s t s a mp l e a n d ad d a n a p p r o p r i a te a mo u n t o f w a t e r t o th e s a mp l e , i f
r e q u i r e d . H o mo g e n i ze t h e mi x t u r e a n d me a s u r e o u t a s a mp l e
e q u iv a l en t t o 3 0 . 0 g .

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I n c a s e o f ma t c h a , w e i g h 3 0 .0 g o f t h e te s t s a mp l e .
I n c a s e o f h o p s , w e ig h 3 0. 0 g o f t h e te s t s amp l e , p r e v io u s l y g r o un d .
T h e n , ad d 8 0 ml o f e t h a n o l a n d h o mo g e n i z e t h e mi x t u r e f o r t h r e e
mi n u t e s . F i l t e r th e h o mo g e n i z ed s a mp l e b y s u c t i o n in t o a r o t a ry
v a c u u m e v a p o r a t o r t h ro u g h a f i l t er p a p e r c o v e r e d w i t h a 1 - c m t h i c k
l a y e r o f d i a t o ma c e o u s e a r t h , a n d t h en t r an s f e r t h e f i l t r a t e to a
2 0 0 - ml g r a d u a te d c yl in d e r. C o l lec t t h e r e s i d u e o n th e su r f a c e o f t h e
f i l t e r p a p e r a n d ad d 4 0 ml o f 6 0 % e t h an o l , a n d t h en h o mo g e n i z e th e
mi x t u r e f o r t h r e e mi n u t e s . R e p e at t h e a b o v e p r o c e d u r e a n d c o mb i n e
t h e f i l t r a t e t o t h e g r ad u a te d c y l in d e r a b o v e, a n d t h e n me a s u r e t h e
a mo u n t o f f i l t r a t e .
Tr a n s f e r 1 0 ml o f t h e a b o v e f i l t ra t e t o a 2 0 0 - ml r o u n d b o t to m f l a s k
a n d ad d 1 ml o f h y d r o g e n p e ro x i de s o lut i o n . A t t a ch a r e f l u x
c o n d e n s e r to t h e f la s k a n d h e a t t h e mi x t u r e f o r 3 0 mi n u t e s i n a w a t er
b a t h a t 7 5 °C , a n d t h e n a l l o w it t o c o o l.
i i . Tea s e x ce p t ma t c h a
We i g h 10 . 0 g o f t e s t s amp l e a n d so a k it i n 6 0 0 ml o f w a t e r a t 1 0 0 ° C,
a n d t h en le a v e i t t o s t a n d a t r o o m t e mp e r a t u r e f o r fi v e mi n u t es .
Tr a n s f e r 1 2 ml o f t h e c o o l ed f il t r a t e i n to a 2 0 0 - ml r o u n d b o tt o m
f l a s k a n d ad d 1 ml o f h yd r o g e n p e r o x id e so l u ti o n. A t t a c h a r e f lux
c o n d e n s e r to t h e f la s k a n d h e a t t h e mi x t u r e f o r 3 0 mi n u t e s i n a w a t er
b a t h a t 7 5 °C , a n d t h e n a l l o w it t o c o o l.
i i i . F o o d s ex c e p t t h o s e l i s t e d i n i a n d ii a b o v e
O b t a i n t h e e x t r a c t s acc o r d i n g to t h e me t h o d s d e s c r i b e d i n i .
b . C l ea n - u p
A d d 1 ml o f s t r o n g l y ac i d i c c at i o n e x ch a n g e re s i n ( 0 .0 6 3 - 0 . 1 5 6 µ m i n
p a r t i c le s i ze ) s u s p e n d e d i n w a t e r i n t o a ch r o ma t o g r a p h t u b e ( 1 0 m m
i n i n ne r d i ame t e r, 3 0 0 m m i n l e n g t h ) a n d d i sc a r d t h e w at e r u n t i l o n l y
a s ma l l a mo u n t r e ma i n s o n th e p a c k in g o f th e c o l u mn . P o u r 5 ml o f
w a t e r i n to th e co l u mn a n d d i s c a r d t h e e ff l u en t . P o u r t h e s o l u t io n i n to
t h e c ol u mn o b t ai n ed by t h e e x t r ac t i o n d e s c ri b e d i n 4 - a. T h e n , w as h
t h e ab ov e r o u n d b ot to m f l a s k w i t h 1 0 ml o f w a t e r an d t r an s f e r t h e
w a s h i n g s in t o th e co lu mn , a n d th e n d i s c a r d t h e e ff l u en t . P o u r 1 2 ml
o f 2 . 8 % a q u e o u s a mm o n i a i n t o th e c o lu mn a n d co l l ec t th e e lu a te in a
r o t a r y v a c u u m e v a p o r a t o r. A d d 3 0 ml o f n - p r o p a n o l a n d r e mo v e t h e
w a t e r a n d n - p r o p a n o l a t 4 5 ° C o r l o w e r. D i s s o l v e t h e r es i d u e i n 5 m l
o f w a t e r.

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A d d 5 ml o f w e a k l y a c i d ic c a t io n e x ch a n g e r e s i n ( 0 . 3 3 - 0 . 5 0 µm i n
p a r t i c le s i ze ) s u s p e n d e d i n w a t e r i n t o a ch r o ma t o g r a p h t u b e ( 1 0 m m
i n i n ne r d i ame t e r, 3 0 0 m m i n l e n g t h ) a n d d i sc a r d t h e w at e r u n t i l o n l y
a s ma l l a mo u n t r e ma i n s o n t h e p a c k in g o f th e c o l u mn . P o u r 1 0 ml o f
w a t e r i n t o t h e c ol umn a n d d is c a r d t h e e ff l u e n t . Po u r t h e ab o v e
s o l u t io n in to th e c o lu mn an d d i s car d t h e e ff l u e n t . P o u r 5 0 ml o f w a t e r
i n t o t he co lu mn a n d d is c a r d t h e e ff l u e n t . P o u r 3 5 ml o f 2 . 8 % a q u e o u s
a mmo n i a i n t o t h e c o lu mn a n d c o l l e ct t h e e l u a te i n a r o t a r y v a c u u m
e v a po r a t o r. A d d 1 00 ml o f n -p r o p a n o l an d r e mo v e t h e aq u eo u s
a mmo n i a a n d n - p r o p an o l at 4 5 ° C o r l o w e r.
c . D e r i v a t iz a t i o n
A d d 2 ml o f a c e t i c ac id b u ff e r s ol u t io n to t he r e s i d u e to d i s s o lv e i t
w e l l . A d d 1 0 0 µ l o f 0 . 2 5 % f l uo r e s c a mi n - a c e t o ne s o l u ti o n to 1 ml o f
t h e ab o v e s o l u t io n a n d s h a k e the mi x tu r e w e l l , a n d th e n l e a v e it t o
s t a n d f o r o n e h o u r. A d d 0 .5 ml o f 0 . 0 5 mo l / l s o d i u m b o r a t e s o l u t i o n
t o u s e a s t h e s a mp l e s o l u t io n .
5 . D e t e r mi n a t io n
a . Q u a l i ta t iv e t e s t s
P e r f o r m q u a l i t at i v e t es t s u n d e r th e f o l l o w in g c o n d i t io n s . Te s t r e s u l t s
o b t ai n ed mu s t b e t h e s a me a s t h o s e o b t a i n ed f o r t h e r e f e r e n c e
s t a n d a r d u n d e r t h e p r o c e d u r e d e s cr i b e d in 4 -c .
Te s t i n g c o n d i t io n s
C o l u mn p a c k i ng : O c t ad e c y l s i l a n e- b o n d ed s i li c a g e l ( 5 µm i n p a r t i cl e
size).
C o l u mn : A s t a i n l e s s t u b e ( 4 .6 m m i n i n n e r d i a me t e r, 1 5 0 mm i n
length).
C o l u mn t e mp e r a t u r e : 4 0 ° C
D e t e c t o r : O p e r a t e w i t h a n ex c i ta t i o n w a v el e n g t h o f 3 8 0 n m a n d a
f l u o r e s c e n t w a v e l e n g th o f 4 8 4 n m.
M o b il e p ha s e : U s e a c e t o n i t r i le/ p h o sp h at e b u ff e r so l u ti o n ( 3 :7 ) .
A d j u s t th e f l o w r a t e so th a t a mi t r o l e f l o w s o u t i n a pp r o x i ma t e l y 1 5
mi n u t e s .
b . Q u a n ti t at i v e t e s t s
D e t e r mi n e t h e q u an t i ty f r o m t h e t e s t r e s u lt s o b ta i n e d u n d e r th e s ame
c o n d i t io n s d e s c r i b ed in 5 - a u si n g e i t he r t h e p e a k h e ig h t o r p e ak a re a
me t h o d .
c . C o n f i r ma t i o n t e s t s

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P e r f o r m l i q u i d c h r o ma t o g r a p h y/ ma s s s p e c t r o me t r y u n d e r t h e sa me
c o n d i t io n s d e s c r i b ed in 5 - a . Te s t r e s u l t s o b t a i n ed f o r t h e r e f e r e n c e
s t a n d a r d mu s t b e th e s a me a s t h o s e o b t a in e d u n d e r t h e p r o c e d u r e
d e s c r i b ed in 4 - c . D e t er mi n e t h e qu a n ti t y u s in g e i th e r the p ea k h ei gh t
o r p e a k a r ea me t h o d , if r e q u i r e d .

( 6 ) C a p t a f ol a n al yt i c a l me t h o d
1. Apparatus
A g a s c h r oma t o g r a p h w i t h an e lec t r o n c a p tu r e d e t ec t o r ( G C - E C D ) a n d
a g a s ch r oma t o g r a p h /ma s s s p e c t r o me t e r ( G C / M S o r G C / M S / M S ) .
2 . R e ag e n t s a n d t e s t s o l u t io n s
I n a d d i t io n t o t h e r e ag e n t s a n d te s t s o lu t i o n s l i s t ed b e l o w, u s e th o s e
l i s t e d in S e c t i o n C R e a g e n ts /Te s t S o l u ti o n s , E t c . , P a r t I I F o o d
a d d it i v e s . R e a g en t s d e s i g n at e d as “s p e c i a l g r a d e ” i n th i s s e c ti o n mu s t
me e t t h e re q u i r e me n t s f o r “s p e c i a l g r a d e” s p e c i f ie d i n t h e J ap a n
I n d u s t r i a l St a n d a r d s fo r t h e r e ag e n t s .
A c e t o n it r i le : U s e a r o t a r y v a c u u m e v a p o r a t o r o n 3 0 0 mL o f a c e t o n i t ri l e
t o c o n c e n t r a t e t o t h e p o i n t o f d r y n e s s . D i s s o l v e t h e r e s id u e i n 5 mL o f
n - h e x an e . F o r a n a l y s i s , i n j ec t 5 μ L o f th e so l u ti o n i n to a GC -ECD.
P e a k s o t h er t h a n t h a t o f n - h e x an e i n th e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
A c e t o n e: U s e a r o t a r y v a c u u m e v ap o r at o r o n 3 0 0 mL o f a c e to n e t o
c o n c en t r a t e t o t h e p o in t o f d r y n es s . D i s s o l v e t h e r e s i d u e i n 5 mL o f
n - h e x an e . F o r a n a l y s is , i n j ec t 5 μ L o f t h e s o l u ti o n i n to a G C - E CD .
P e a k s o t h er t h a n th a t o f n - h e x an e i n t h e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
S o d i u m c h l o r i d e : S o diu m c h l o r i d e ( s p e c i a l g r a d e ) . I f t h e r e a g en t i s
f o u n d to i nc l u de a n y s u b s t a n c e t h a t ma y i n t er f e r e w i t h t h e a n al y s i s o f
s u b s t a n ce s t o b e a n al y z e d f r o m a g r i c u l tu r a l c h e mi c a l s , w a s h w i t h a
s o l v e n t s u ch a s n - h e x a n e b e f o r e u s e .
S y n t h e t i c ma g n e s i u m s i l i c at e (F l o r i s i l ) f o r c o l u mn c h r o ma t o g r a p h y:
H e a t f l o r i s il ( 1 5 0 -2 5 0 μ m i n p a r t ic l e s i ze ) a t 1 3 0 ° C f o r 1 2 h o u r s o r
l o n g e r. C o o l d o w n t o ro o m t e mp e r a t u r e in a d e s i c c a to r.
D i a t o ma c e o u s e a r t h : D i a t o ma c e o u s e a r t h f o r c h e mi c a l a n a l y s i s .
E t h y l a c et at e : U s e a ro t a r y v a c u u m e v a p o ra t o r o n 3 0 0 mL o f e th y l
a c e t a te t o co n c en t r a t e t o t h e p o i n t o f d r y n e s s . D i s s o l v e th e r e s i d u e in
5 mL o f n - h e x an e . F o r a n a l y s i s , i n j e c t 5 μL o f t h e s o l u t i o n i n t o a
GC-ECD. P e a k s o t h e r t h a n th a t o f n - h e x an e i n t h e r e s u lt i n g

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c h r o ma t o g r a m s h o u l d b e a s h ig h a s o r lo w e r t h an t h e pe a k s o f γ - B H C
a t 2 x 10 - 1 1 g .
n - H e x a n e : U s e a r o t a r y v a c u u m e v a p o r at o r on 3 00 mL o f n - h e x a n e t o
e v a p o r a t e u n t i l 5 mL i s l e f t . F o r a n a l y s i s , i n j e c t 5 μ L o f t h e s o lu t io n
i n t o a G C - E C D . P e a k s o t h e r th an t h at o f n- h e x a n e i n t h e r e s u l t in g
c h r o ma t o g r a m s h o u l d b e a s h ig h a s o r lo w e r t h an t h e pe a k s o f γ - B H C
a t 2 x 10 - 1 1 g .
Wa t e r : D i s ti l l e d w a t e r. I f t h e d i s ti l l e d w a t e r i s f o u n d to i n c l u d e an y
s u b s t a n ce th a t ma y i n t e r f e r e w i t h a n a l y s i s o f t h e t a rg et co mp o s i t i o n a l
s u b s t a n ce s f r o m a g r i c u l t u r a l ch emi c a l s , w a s h w i t h a s o l v en t s u c h a s
n-hexane before use.
S o d i u m s u l f a t e ( a n h y d r o u s ) : S o d iu m s u l f a t e ( a n h y d r o u s) ( s p e c i a l g ra d e ) .
I f t h e r e a g e n t i s f o u n d t o in c lu d e a n y s u b st a n c e t h at ma y i n t e r f e r e
w i t h a n al y s i s o f t h e t a rg e t co mp o s i t i o n a l s u b st a n c e s f r o m a g r i c u l t u r al
c h e mi c a l s , w a s h w i t h a s o l v en t su c h a s n - h e x a n e b e f o r e u s e .
3. Reference standard
C a p t a f o l : Th i s p r o d u c t c o n ta i n s no t l e s s th a n 9 8 % o f c ap t a f o l , a n d i t s
me l t i n g po in t i s 15 9 - 16 1 ° C .
4. Procedure
a . E x t r a ct io n
i . C e r e a l g r a i n s , l eg u me s / p u l s e s an d s e ed s
We i g h 1 0. 0 g o f th e te s t s a mp l e , p r e v io u s l y g r o u n d s o a s to p a s s
t h r o u g h a s t a n d a r d me s h s i e v e ( 4 2 0 µm) . A d d 2 0 ml o f 3 %
p h o s ph o r i c a c i d s o lu t io n t o t h e o b ta i n ed s a mp l e a n d l e a v e i t t o s t an d
for two hours.
T h e n , a d d 1 0 0 ml a c e t o n e an d h o mo g e n i z e t h e mi x t u r e f o r t h r ee
mi n u t e s F i l t e r t h e h o mo g e n i z e d s a mp l e b y s u c t i o n in t o a r o t a ry
v a c u u m e v ap o r a t o r t h ro u g h a f i l t er p a p e r c o v e r e d w i t h a 1 - c m t h i c k
l a y e r o f d ia t o ma c e o u s e a r t h . Co ll e c t t h e r e si d u e o n th e s u r f a c e o f
t h e f il t e r p a p e r an d a d d 5 0 ml o f a c e t o n e , a n d th e n h o mo g e n i z e t h e
mi x t u r e f o r t h r e e mi n u t e s . R ep e a t t h e a b o v e p r o c e d u r e an d co mb i n e
t h e f i l t r at e i n t o t h e r o ta r y v a c u u m e v a p o r a t o r, a n d t h en c o n c en t r a t e
t h e mi x t u r e t o ap p r o xima t e l y 3 0 ml a t 4 0 ° C o r l o w e r.
Tr a n s f e r t h e c o n c en t ra t e d s o lu t io n t o a 3 0 0 - ml s e p a r a t i n g f u n n e l
a l r e a d y c on t a in i ng 10 0 ml o f 1 0 % s o d iu m c h l o r id e s o l u t io n . Wa sh
t h e eg g p l an t - s h a p ed fl a s k o f t h e a b o v e r o ta r y v a c u u m e v a p o r a to r
w i t h 1 00 ml o f n - h e x a n e ,a n d ad d t h e w a s h i n g s to t h e s e p a r a t ing

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f u n n e l a b o v e . S h a k e th e mi x t u r e v i g o r o u s l y f o r f i v e mi n u t e s u s i n g a
s h a k e r an d l e a v e i t t o s t a n d , a n d th e n t r a n s f e r t h e n - h e x a n e l a y e r t o
a 3 0 0 - ml c o n i ca l f l a sk . A d d 50 ml o f n - h e x a n e t o t h e a q u eo u s l aye r
a n d r e p ea t t h e a b o v e p r o c e d u r e , a n d ad d th e n - h e x an e l a y e r t o t h e
c o n ic a l f l as k a b o v e . A d d a n ad e q u at e a mo u n t o f s o d i u m s u l f a t e
( a n h y d r o u s) t o t h e f l as k a n d l e av e i t t o s t an d f o r 1 5 mi n u t e s w i t h
o c c a s io n a l s h a k i n g . F i l t e r t h e c o n te n t o f t h e f l a s k i n t o a r o t ar y
v a c u u m e v a p o r a t o r. Was h t h e f l a s k w i t h 2 0 ml o f n - h e x a n e a n d w a s h
t w i c e t h e r es i d u e o n t h e s u r f a c e o f t h e f il t e r p a p e r w i t h th e w a s h i n g s .
A d d t h e w as h i n g s in t o t h e r o t a r y v a c u u m e v a p o r a t o r a n d r e mo v e t h e
n - h e x an e at 4 0 ° C o r l ow e r.
A d d 3 0 ml o f n - h e x a n e t o t h e r e si d u e a n d t ra n s f e r t h e mi x t u r e t o a
1 0 0 - ml s e p a r a t i n g f u n n e l , a n d t h e n a d d 3 0 ml o f n - h e x a n e - s a t u r a te d
a c e t o n i t r i l. S h a k e t h e mi x t u r e v ig o ro u s l y f o r f i v e mi n u t e s u s i n g a
s h a k e r an d l e a v e it to s t a n d , a n d t h e n t r a n s fe r t h e a ce to n i t r il e l a y e r
i n t o th e r o ta r y v a c u u m e v a p o r a t o r. A d d 3 0 ml o f n - h e x an e - s a t u r a t ed
a c e t o n i t r i le t o t h e n - h e x a n e l a y er a n d r e p ea t t h e ab o v e p r o c ed u re
t w i c e , an d t h en co mb i n e t h e ac e t o n i t r i l e l a y e r i n to t h e r o t ar y
v a c u u m e v a p o r a t o r ab o v e . R e mo v e t h e a ce t o n i t r i l e a t 4 0 ° C o r l o w e r
a n d d i s s o l ve t h e r e s idu e i n 5 ml o f n - h e x an e .
i i . F r u i t , v eg e t ab l e s , ma t c h a a n d h o p s
I n c a s e o f f r u i t a n d v e g e t ab l e s , w e i g h a c cu r a t e l y 1 k g o f t h e t e st
s a mp l e and add 500 ml of 10% phosphoric ac i d s o l u tio n .
H o mo g e n i ze t h e mi x t u r e a n d me s u r e o u t a s a m p l e e q u iv a l e n t t o 2 0 . 0 g .
I n c a s e o f ma t c h a , w e i g h 5 .0 0 g o f t h e t e t sa mp l e a n d a d d 2 0 ml o f
3 % ph o s pho r i c ac i d so l u ti o n , and th e n l e av e it to sta n d fo r t w o
hours.
I n c a s e o f h o p s , w e ig h 5 .0 0 g o f t h e te s t s amp l e , p r e v io u s l y g r o un d ,
a n d a d d 2 0 ml o f 3 % p h o s p h o r i c a c id s o l u tio n , a n d th en l ea v e i t to
s t a n d f o r tw o h o u r s .
T h e n , a d d 1 0 0 ml o f a c e t on e and h o mo g e n iz e t he mi x t u r e f o r t h re e
mi n u t e s . F i l t e r th e h o mo g e n i z ed s a mp l e b y s u c t i o n in t o a r o t a ry
v a c u u m e v a p o r a to r t h r o u g h a f i l te r p a p e r co v e r e d w i t h a 1 - c m thick
l a y e r o f d ia t o ma c e o u s e a r t h . Co ll e c t th e r e si d u e o n t h e s u r f a c e o f
t h e f i l t e r p a p e r a n d a d d 5 0 ml o f a c e t o n e , a n d t h e n h o mo g e n i z e t h e
mi x t u r e f o r t h r e e mi n u t e s . R p e at t h e ab o v e p r o c e d u r e a n d co mb i n e
t h e f i lt r a t e i n t o t h e r o ta r y v a c u u m e v a p o r a t o r, a n d t h e n c o n c en t r a t e

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t h e mi x t u r e t o ap p r o xima t e l y 3 0 ml a t 4 0 ° C o r l o w e r.
Tr a n s f e r t h e c o n c en t ra t e d s o lu t io n t o a 3 0 0 - ml s e p a r a t i n g f u n n e l
a l r e a d y c on t a in i ng 10 0 ml o f 1 0 % s o d iu m c h l o r id e s o l u t io n . Wa sh
t h e eg g p l an t - s h a p ed fl a s k o f th e a b o v e r o ta r y v a c u u m e v a p o r a to r
w i t h 1 00 ml o f n - h e x a n e a n d ad d th e w a s h i n g s t o th e s e p a r a t in g
f u n n e l a b o v e . S h a k e th e mi x t u r e v i g o r o u s l y f o r f i v e mi n u t e s u si n g a
s h a k e r a n d l e a v e i t t o s t a n d , a n d th e n t r an s f e r t h e n - h ex a n e la y e r t o
a 3 0 0 - ml c o n i ca l f l a sk . A d d 5 0 ml o f n - h e x a n e t o t h e a q u eo u s l aye r
a n d r e p ea t t h e a b o v e p r o c e d u r e t w i c e , an d t h e n co mb i n e t h e
n - h e x an e l ay e r t o th e co n i ca l f l a sk a b o v e . A d d an a d eq u a t e a mo u n t
o f s o d iu m s u l f a t e ( an h y d r o u s ) t o t h e f l a s k an d l e a v e it t o s t an d f o r
1 5 mi n u t e s w i t h o c c a si o n al s h ak in g . F i lt e r th e co n te n t o f t h e f l a sk
i n t o a r o t ar y v a c u u m e v a p o r a t o r. Wa s h t h e f l a s k w i th 2 0 ml o f
n - h e x an e an d w a s h tw i c e t h e r e s i d u e on t h e s u r f a c e o f t h e f i l te r
p a p e r. A d d t h e w a s h in g s i n t o th e r o t a r y v a c u u m e v a p o r a to r a n d
r e mo v e t h e n - h e x a n e at 4 0 ° C o r lo w e r. D i s s o l v e t h e r es i d u e i n 5 ml
o f n - h e x an e .
i i i . Te a s exc e p t ma t c h a
We i g h 9 .0 0 g o f t h e te s t s a mp l e a n d s o a k i t i n 5 4 0 ml o f w a t e r a t
1 0 0 ° C , a nd t h en le a v e i t t o s t an d at r o o m t e mp e r a t u r e f o r f i v e
mi n u t e s . Tra n s f e r 3 60 ml o f t h e co o l ed f i lt r at e in t o a 5 0 0 - ml c o n i ca l
flask.
A d d 3 0 ml o f p ho s p h o r i c a c id , 1 0 0 ml o f a c e to n e an d 2 ml o f
s a t u r a t e d le a d a ce t a te s o l u ti o n to t h e a b o v e c o n i c a l f l a s k , a n d th e n
l e a v e th e mi x t u r e t o s ta n d f o r o n e h o u r at r o o m t e mp e r a t u r e . F i lt e r
t h e mi x t u r e b y s u ct i o n i n to a 1 , 00 0 - ml s e p ar a t i n g f u nne l th r o u gh a
f i l t e r p a p e r c o v e r ed w i t h a 1 - c m t h i ck l a y e r o f d i a to ma c e o u s ea r t h .
Wa s h t h e ab o v e co n i ca l f l a s k w it h 5 0 ml o f a c e t o n e an d w a s h t h e
residue on the surface of the filter paper with the washings. Add the
w a s h i n g s in t o th e a b o v e s e p a r a t i n g f u n n el a n d a l so a d d 3 0 g o f
s o d i u m c h l o r i d e an d 1 0 0 ml o f n - h e x an e . S h a k e t h e mi x t u r e
v i g o r o u s l y f o r f i v e mi n u t e s u s i n g a s h a k e r an d l ea v e i t t o s t a nd , and
t h e n t r a n s f e r t h e n - h ex a n e l a y e r t o a 3 0 0 - ml c o n ic a l f l a s k . A d d 1 0 0
ml o f n - h e x a n e t o t h e aq u e o u s l a y e r a n d r e p ea t t h e a b o v e p r o c e d u r e,
a n d th e n a d d t h e n - h e xa n e l a ye r to t h e c o ni ca l f l a s k a bo v e . A d d an
a d e q u a t e amo u n t o f s o d i u m s u l f a t e ( a n h ydr o u s ) t o t h e f l a s k a nd
l e a v e it t o s t a n d f o r 1 5 mi n u t e s w i t h o c c a s io n a l s h ak i n g . F i l t e r t h e

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c o n te n t o f th e f l a s k i n to a r o t a r y v a c u u m e v a p o r a t o r. Wa s h t h e f l a s k
w i t h 2 0 ml o f n - h e x an e a n d w a s h t w i c e t h e re s i d u e o n th e s u r f a c e o f
t h e f il t e r p a p e r w i t h th e w a s h in g s . A d d t h e w a s h i n g s in t o t h e r o tar y
v a c uu m e v a p o r a to r a n d r e mo v e t h e n - h e xa n e a t 40 °C o r l o w e r.
D i s s o l v e t he r e s i d u e in 5 ml o f n - h e x a n e.
i v. F o o d s ex c e p t t h o s e l i st e d i n i t o i ii a b o v e
O b t a i n t h e e x t r a c t s acc o r d i n g to t h e me t h o d s d e s c r i b e d i n i o r i i .
b . C l ea n - u p
A d d 5 g o f f l o r i s i l f o r c o l u mn c h r o ma t o g r a p h y s u s p e n d ed i n n - h ex an e
i n t o a c h ro ma t o g r a p h t u b e ( 1 5 m m i n i n n e r d i a me t e r, 3 0 0 m m i n
l e n gt h ) a nd a d d ov e r ap p r o x i ma t e l y 5 g o f s o d i u m s u l f a t e ( a n h y d r o u s )
i n t o th e c o l u mn . S p i l l o u t t h e n -h e x an e u n ti l o n l y a s ma l l a mo u n t
r e ma i n s o n t h e p a ck i ng o f t h e co l u mn a n d p o u r t h e s o l u tio n o b t ai n ed
b y t h e ex t ra c t i o n d e s cr i b e d in 4 - a i n to t he c ol u mn . T h e n, p o u r 1 0 0 m l
o f n - h e x an e i n to t h e co l u mn a n d d i s c a r d th e e ff l u e n t . P o u r 1 50 ml o f
e t h y l a c e tat e / n - h ex a n e ( 1 : 9 ) i n t o t h e c o lu mn a n d co l l ec t t h e el u at e
i n t o a r o tar y v a c u u m e v a p o r a t o r, a n d t h en r o mo v e t h e e t h y l a c e tat e
a n d n - he x an e a t 40 ° C o r l o w e r. D i s s o l v e t h e r e s i d u e in n - h e x a n e t o
ma k e e x a c tl y 5 ml o f s o l u ti o n, w hi c h i s u s e d a s t h e s a mp l e s o lu t i o n .
5 . D e t e r mi n a t io n
a . Q u a l i ta t iv e t e s t s
P e r f o r m q u a l i t at i v e t es t s u n d e r th e f o l l o w in g c o n d i t io n s . Te s t r e s u l t s
o b t ai n ed mu s t b e t h e s a me a s t h o s e o b t a i n ed f o r t h e r e f e r e n c e
standard.
Te s t i n g c o n d i t io n s 1
C o l u mn : A s i l i c at e g la s s c a p i ll a ry c o l u mn ( 0 . 2 5 mm i n n e r d i a me t e r,
10-30 m in le n g th ) c o a te d with me t h y l s i l i co n e for gas
c h r o ma t o g r a p h y t o a th i c kn e s s o f 1 . 5 µ m.
C o l u mn t e mp e r a t u r e : H o l d t h e c o l u mn t e mp e r a t u r e a t 5 0 ° C f o r o n e
mi n u t e , f o ll o w e d b y an i n c r e a s e o f 2 5 ° C ev er y mi n u t e u n t i l r e ac h in g
1 7 5 ° C , a f t er w h i c h in cr e a s e t h e te mp e r a t u r e b y 1 0 ° C ev e r y mi n u t e
u n t il r e a c h in g 3 0 0 ° C , a n d h o ld f o r f i v e mi n u t e s .
I n l e t t e mp e r a t u r e : 2 3 0 ° C
D e t e c t o r : Op e r a t e at 3 0 0 ° C
G a s f l o w r a t e : U s e h el i u m a s t h e c a r r i e r g a s . A d j u s t t h e f l o w r a t e t o
t h e o p t i ma l c o n d i t io n .
Te s t i n g c o n d i t io n s 2

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C o l u mn : A s i l i c a te g l as s c a p i ll a r y c o lu mn ( 0 . 2 5 mm i n i n n e r d i a me t e r,
1 0 - 3 0 m i n l e n g th ) c o a t e d w i t h 5 % p h e n y l me t h y l s i l i c o n e f o r g as
c h r o ma t o g r a p h y t o a th i c kn e s s o f 0 . 2 5 µ m.
C o l u mn t e mp e r a t u r e : H o l d t h e c o l u mn t e mp e r a t u r e a t 5 0 ° C f o r o n e
mi n u t e , f o ll o w e d b y an i n c r e a s e o f 2 5 ° C ev er y mi n u t e u n t i l r e ac h in g
1 2 5 ° C , a f t er w h i c h in cr e a s e t h e te mp e r a t u r e b y 1 0 ° C ev e r y mi n u t e
u n t il r e a c h in g 3 0 0 ° C , a n d h o ld f o r t h r e e mi n u t e s .
I n l e t t e mp e r a t u r e : 2 3 0 ° C
D e t e c t o r : Op e r a t e at 3 0 0 ° C
G a s f l o w r a t e : U s e h el i u m a s t h e c a r r i e r g a s . A d j u s t t h e f l o w r a t e t o
t h e o p t i ma l c o n d i t io n .
b . Q u a n ti t at i v e t e s t s
D e t e r mi n e t h e q u an t i ty f r o m t h e t e s t r e s u lt s o b ta i n e d u n d e r th e s ame
c o n d i t io n s d e s c r i b ed in 5 - a u si n g e i t he r t h e p e a k h e ig h t o r p e ak a re a
me t h o d .
c . C o n f i r ma t i o n t e s t s
P e r f o r m g a s c h r o ma t o g r a p h y / ma s s s p e c t ro me t r y u n d e r t h e s am e
c o n d i t io n s d e s c r i b ed in 5 - a . Te s t r e s u l t s o bt ai n e d mu s t b e t h e s a me a s
t h o s e o b t a in e d f o r t h e r e f e r e n c e s t a nd a r d . D e t e r mi n e t h e q u an t ity
u s i n g e i th er t h e p ea k h e i gh t o r pe a k a r e a me t h od , i f r eq u i r e d .

( 7 ) C a r b a d o x an a l y t i ca l me t h o d
A n a l yz e q ui n o xa l in e - 2- c a r b o x y l i c a c i d .
1. Apparatus
A h i g h - p er f o r ma n c e l i q u id ch r o ma t o g r a p h with an u l t r av io l e t
s p e c t r o p h o to me t r i c d e te c t o r ( H P L C -U V ) a n d a l iq u i d ch r o ma t o g r a p h /
m a s s s p e c t r o me t e r ( L C / M S o r L C / M S / M S )
2 . R e ag e n t s a n d t e s t s o l u t io n s
I n a d d i t io n t o t h e r e ag e n t s a n d te s t s o lu t i o n s l i s t ed b e l o w, u s e th o s e
l i s t e d in S e c t i o n C R e a g e n ts /Te s t S o l u ti o n s , E t c . , P a r t I I F o o d
a d d it i v e s . R e a g en t s d e s i g n at e d as “s p e c i a l g r a d e ” i n th i s s e c ti o n mu s t
me e t t h e re q u i r e me n t s f o r “s p e c i a l g r a d e” s p e c i f ie d i n t h e J ap a n
I n d u s t r i a l St a n d a r d s fo r t h e r e ag e n t s .
O c t a d e c y l si l a n e - b o n d e d s i l ic a g e l cartridge c o l u mn ( 5 0 0 mg ) : A
p o l y e t h y l en e c o l u mn o f 8 - 9 mm i n i nn e r d i ame t e r p a c k e d w i t h 5 0 0 mg
o f o c t a d ecy l s i l a n e - b o n d e d s i li c a g e l , o r a c o l u mn e q u i v al e n t to t h e
s p e c i f i e d o n e i n s e p a ra t i o n c ap a b i l i t y.
St r o n g l y b a s i c an i o n e x ch a n g er cartridg e c o l u mn ( 3 6 0 mg ) : A

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p o l ye t h yl en e co l u mn o f 8 - 9 mm i n i n n e r d i ame t e r p a c k e d w i t h 3 6 0 mg
o f t r i me t h y l a mi n o a c r yl a mi d e c o p o l y me r s i l a n e - b o n d ed s i l i c a g e l , o r
a c o lu mn e q u i v a l en t to t h e s p e c if i e d o n e i n s e p a r a t i o n c a p a b i l it y.
Wa t e r : Wa te r p r o d u ced f o r l i q u i d c h r o ma t o g r a p h y.
S o d i u m s u l f a t e ( a n h y d r o u s ) : S o d iu m s u l f a t e ( a n h y d r o u s) ( s p e c i a l g r a d e ) .
I f t h e r e a g e n t i s f o u n d t o i n c l u d e a n y s u b st a n c e t h a t ma y i n t e r f e r e
w i t h an a l y s i s o f th e t a rg e t c o mp o s i t i o n a l s u b st a n c e s f r o m a g r i cu l tu r a l
c h e mi c a l s , w a s h w i t h a s o l v en t su c h a s n - h e x a n e b e f o r e u s e .
P h o s p h a te b u ff e r s o l u t io n (p H 2.5): Dissolve mo n o p o ta s s i u m
d i h y d r o g en mo n o p h o sp h a te ( 1 . 36 g ) i n w a t e r t o ma k e a 8 0 0 -ml
s o l u t io n , an d ad d p hos p h o r i c a c id t o a d ju s t t h e p H t o 2 . 5 . A d d w a t e r
t o t h e a d j u s t e d s o lu t io n t o ma k e a 1 , 0 0 0 - ml s o l u ti o n .
3. Reference standard
Q u i n o x a l in e - 2 - c a r b o x y l i c a c i d : T h i s p r o d u c t c o n t a in s n o t le s s t h a n
9 9 % o f q uin o x al i ne - 2 - c a r b o x y l i c a c i d , a n d its me l t i n g p o i n t i s 2 0 8 °C .
4. Procedure
a . E x t r a ct io n
We i g h 5. 0 0 g o f t h e te s t s a mp l e , p r e v i o u s l y g r o u n d . . F o r mu s c l e ,
r e mo v e t h e f a t l a y e r as mu c h a s p o s s i b l e b e fo r e g r i n di ng .
T h e n , a d d 1 0 0 o f me t h a n ol / 0. 3 % me t a p h o s ph o r i c a c i d s o l u t io n ( 3: 7 )
a n d h o mo g e n i z e t h e mi x t u r e . F i l t e r th e h o mo g e n i z ed s a mp l e b y
s u c t i o n in to a r o t a r y v a cu u m e va p o r at o r t h r o u g h a f i l t e r p a p e r
c o v e r e d w it h a 2 - mm t h i c k l a y e r o f d i a t o ma c e o u s e a r t h . Wa s h t h e
r e s i d u e o n th e s u r f a c e o f t h e f i l t e r p ap e r w i t h 1 0 ml o f m e t h an o l /0 . 3 %
me t a p h o s p h o r i c ac i d s o l u t io n ( 3: 7 ) a nd f i l te r t h e w a sh i n g s ,i n to t h e
r o t a r y v a c u u m e v a p o r at o r. C o n c e n t ra t e th e mi x t u r e t o 3 0 ml a t 4 5 °C
o r l o w e r a n d a d d 0 .1 ml o f p h o s p h o r i c a c id.
b . C l ea n - u p
i . O c t a d ec y l s i l a n e - b o n d e d s il i c a g e l c o l u mn c h r o ma t o g r a p h y
A d d 5 ml o f me t h an o l t o a n o c t ad e c yls i l a n e - bo n de d s i li c a g el
cartridge c o l u mn ( 5 0 0 mg ) f o l l o w e d b y 1 0 ml o f w a t e r a n d d i s c a rd
t h e e ff l u e nt. P o u r t h e s o l u t io n o b t a i n ed b y th e ex t r a c tio n d e s c r i b ed
i n 4 - a in to th e c o lu mn f o l l o w e d b y 2 0 ml o f p hos p h a t e bu ff e r
s o l u t io n ( pH 2 . 5 ) a nd d i s c a r d t h e e ff l u e n t . Po u r 1 0 ml o f me t h a n o l
i n t o th e c o lu mn a n d c o l l e c t t h e elu a t e i n a r o t a r y v a c u u m e v a p o r a to r,
a n d t h e n r emo v e t h e me t h an o l a t 40 ° C o r l o w e r. D i s s o l v e t h e r e s i d ue
i n 5 ml o f w a t e r.

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i i . St r o n g l y b a s i c an i o n e x ch a ng er c o l u mn c h r o ma t o g r a p h y
P o u r 5 ml o f w a t e r i nt o a s t r o n g ly b a s i c an io n ex c h a n g e r cartridg e
c o l u mn ( 3 6 0 mg ) a n d d i s c a r d t h e e ff l ue n t . P o u r t h e so l ut i on
o b t ai n ed in 4 - b - i i n to t h e c o lu mn f o l lo w e d b y 2 ml o f w a t e r, 1 0 ml
o f e t h a no l a n d 5 ml o f w a t e r, a n d d i s c a r d t h e e ff l u e n t . P o u r 5 ml o f
0 . 1 mo l / l h y d r o c h l o r i c a c i d i n to th e c o lu mn a n d c o l l e ct t h e e l u a t e in
a 5 0 - ml t e s t t u b e . A d d 3 ml o f 3 mo l / l h y d r o c h lo r i c a ci d a n d 1 5 ml
o f e t h yl a ce t a t e a n d sh a k e t h e mi x t u r e v i g o r o u s l y f o r f i v e mi n u t es
u s i n g a s h ak e r, a n d the n l e av e i t t o s t a n d . Tr a n s f e r t h e e t h y l a c e ta te
l a ye r i n t o a 1 0 0 - ml c o n i c a l f la s k . A d d 1 5 ml o f e t h yl a ce t a t e t o th e
a q u eo u s l ay e r i n t h e te s t t ub e a nd r e p ea t the a bo v e p ro c e du r e , and
t h e n ad d t h e e t h y l a c et a t e l a y e r t o t h e c o n i ca l f l a s k a b o v e . A d d an
a d e q u a t e amo u n t o f s o d i u m s u l f a t e ( a n h ydr o u s ) t o t h e f l a s k a nd
l e a v e it t o s t a n d f o r 1 5 mi n u t e s w i t h o c c a s io n a l s h ak i n g . F i l t e r t h e
c o n te n t o f th e f l a s k i n to a r o t a r y v a c u u m e v a p o r a t o r a n d r e mo v e t h e
e t h y l ac e t at e at 4 0 ° C o r lo w e r. D i s s o l v e th e r e s i d u e in 1 .0 ml o f
a c e t on i t r i le/ p h o sp h at e b u ff e r s o l u t i o n ( p H 2 .5 ) ( 1 : 4 ) , w h i c h i s u s ed
a s t h e s a mp l e s o lu t i o n .
5 . D e t e r mi n a t io n
a . Q u a l i ta t iv e t e s t s
P e r f o r m q u a l i t at i v e t es t s u n d e r th e f o l l o w in g c o n d i t io n s . Te s t r e s u l t s
o b t ai n ed mu s t b e t h e s a me a s t h o s e o b t a i n ed f o r t h e r e f e r e n c e
standard.
Te s t i n g c o n d i t io n s
C o l u mn p a c k i ng : O c t ad e c y l s i l a n e- b o n d ed s i li c a g e l ( 5 µm i n p a r t i cl e
size).
C o l u mn : A s t a i n l e s s tu b e ( 4 . 0 - 6 .0 mm i n i n n e r d i a me t e r, 1 5 0 mm i n
length).
C o l u mn t e mp e r a t u r e : 4 0 ° C
D e t e c t o r : Op e r a t e w i th a n ab s o r p ti o n w a v e l en g t h o f 2 4 5 n m.
M o b il e ph as e : U s e a ce t o n i t r i l e/ p h o s p h a t e b u ff e r s o l u ti o n ( p H 2 .5 )
( 1 : 4 ) . A d j u s t t h e f l o w r a t e s o t h a t q u in o xa li n e - 2 - c a r b ox yl i c a c i d
f l o w s o u t in a p p r o x i ma t e l y 1 0 mi n u t e s .
b . Q u a n ti t at i v e t e s t s
D e t e r mi n e t h e q u an t ity f r o m t h e t e s t r e s u lt s o b t a i n e d u n d e r th e s ame
c o n d i t io n s d e s c r i b ed in 5 - a u si n g e i t he r t h e p e a k h e ig h t o r p e ak a re a
me t h o d .

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c . C o n f i r ma t i o n t e s t s
P e r f o r m l i q u i d ch r o ma t o g r a p h y / ma s s s p e c t r o me t r y u n d e r t h e sa me
c o n di t io n s d e s c r i be d i n 5 - a , b u t t h e mo b i le p h a se should be
a c e t o n i t r i l/w a t e r ( 1 : 4 ) . Te s t r e s u l t s o b t a i n ed mu s t b e t h e s a me a s
t h o s e o b t a in e d f o r t h e r e f e r e n c e s t a nd a r d . D e t e r mi n e t h e q u an t ity
u s i n g e i th er t h e p ea k h e i gh t o r pe a k a r e a me t h od , i f r eq u i r e d .

( 8 ) C o u ma p h o s a na l yt ic a l me t h o d
1. Apparatus
A gas c h r o ma t o g r a p h with an a l k al i f la me i o n i za t i on d et ec t o r
( G C - F I D ) , a f l a me p h o t o me t r i c d e t e c to r ( G C - F P D , i n te r f e r e n c e f i l t e r
for p h o s ph o r u s d e te r mi n a t i o n , wavelength: 526 n m) , or a
h i g h l y - s e n s i t i v e n it r o g e n p h o sp ho r u s d e te c to r ( G C - N P D ) , a n d a g a s
c h r o ma t o g r a p h / ma s s s p e c t r o me t e r ( G C / M S o r G C / M S / M S ) .
2 . R e ag e n t s a n d t e s t s o l u t io n s
I n a d d i t io n t o t h e r e ag e n t s a n d te s t s o lu t i o n s l i s t ed b e l o w, u s e th o s e
l i s t e d in S e c t i o n C R e a g e n ts /Te s t S o l u ti o n s , E t c . , P a r t I I F o o d
a d d it i v e s . R e a g en t s d e s i g n at e d as “s p e c i a l g r a d e ” i n th i s s e c ti o n mu s t
me e t t h e re q u i r e me n t s f o r “s p e c i a l g r a d e” s p e c i f ie d i n t h e J ap a n
I n d u s t r i a l St a n d a r d s fo r t h e r e ag e n t s .
A c e t o n it r i le : U s e a r o t a r y v a c u u m e v a p o r a t o r o n 3 0 0 mL o f a c e t o n i t ri l e
t o c o n c e n t r a t e t o t h e p o i n t o f d r y n e s s . D i s s o l v e t h e r e s id u e i n 5 mL o f
n - h e x an e . F o r a n a l y s i s , i n j ec t 5 μ L o f th e so l u ti o n i n to a GC -ECD.
P e a k s o t h er t h a n t h a t o f n - h e x an e i n th e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
A c e t o n e: U s e a r o t a r y v a c u u m e v ap o r at o r o n 3 0 0 mL o f a c e to n e t o
c o n c en t r a t e t o t h e p o in t o f d r y n e s s . D i s s o l v e t h e r e s i d u e i n 5 mL o f
n - h e x an e . F o r a n a l y s i s , i n j ec t 5 μ L o f th e so l u ti o n i n to a GC -ECD.
P e a k s o t h er t h a n t h a t o f n - h e x an e i n th e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
S o d i u m c h l o r i d e : S o diu m c h l o r i d e ( s p e c i a l g r a d e ) . I f t h e r e a g en t i s
f o u n d to i nc l u de a n y s u b s t a n c e t h a t ma y i n t er f e r e w i t h t h e a n al y s i s o f
s u b s t a n ce s t o b e a n al y z e d f r o m a g r i c u l tu r a l c h e mi c a l s , w a s h w i t h a
s o l v e n t s u ch a s n - h e x a n e b e f o r e u s e .
S i l i c a g el f o r c o l u mn c h r o ma t o g r a p h y ( 6 3 - 2 0 0 µ m i n p a r t i c l e siz e ) :
H e a t s i l i ca g e l ma d e f o r c o l u mn c h r o ma t o g r a p h y ( 6 3 - 2 0 0 µ m i n
p a r t i c le s i ze ) a t 1 30 ° C f o r 1 2 h o u r s o r lo n g e r. C o o l d o w n t o r o o m
t e mp e r a t u r e i n a d e s i cc a t o r.

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D i a t o ma c e o u s e a r t h : D i a t o ma c e o u s e a r t h f o r c h e mi c a l a n a l y s i s .
E t h y l a c et at e : U s e a ro t a r y v a c u u m e v a p o ra t o r o n 3 0 0 mL o f e th y l
a c e t a te t o co n c en t r a t e t o t h e p o i n t o f d r y n e s s . D i s s o l v e th e r e s i d u e in
5 mL o f n - h e x an e . F o r a n a l y s i s , i n j e c t 5 μL o f t h e s o l u t i o n i n t o a
GC-ECD. P e a k s o t h e r t h a n th a t o f n - h e x an e i n t h e r e s u lt i n g
c h r o ma t o g r a m s h o u l d b e a s h ig h a s o r lo w e r t h an t h e pe a k s o f γ - B H C
a t 2 x 10 - 1 1 g .
n - H e x a n e : U s e a r o t a r y v a c u u m e v a p o r at o r on 3 00 mL o f n - h e x a n e t o
e v a p o r a t e u n t i l 5 mL i s l e f t . F o r a n a l y s i s , i n j e c t 5 μ L o f t h e s o lu t io n
i n t o a G C - E C D . P e a k s o t h e r th an t h at o f n- h e x a n e i n t h e r e s u l t in g
c h r o ma t o g r a m s h o u l d b e a s h ig h a s o r lo w e r t h an t h e pe a k s o f γ - B H C
a t 2 x 10 - 1 1 g .
Wa t e r : D i s ti l l e d w a t e r. I f t h e d i s ti l l e d w a t e r i s f o u n d to i n c l u d e an y
s u b s t a n ce th a t ma y i n t e r f e r e w i t h a n a l y s i s o f t h e t a rg et co mp o s i t i o n a l
s u b s t a n ce s f r o m a g r i c u l t u r a l ch emi c a l s , w a s h w i t h a s o l v en t s u c h a s
n-hexane before use.
S o d i u m s u l f a t e ( a n h y d r o u s ) : S o d iu m s u l f a t e ( a n h y d r o u s) ( s p e c i a l g r a d e ) .
I f t h e r e a g e n t i s f o u n d t o in c lu d e a n y s u b st a n c e t h at ma y i n t e r f e r e
w i t h a n al y s i s o f t h e t a rg e t co mp o s i t i o n a l s u b st a n c e s f r o m a g r i c u l t u r al
c h e mi c a l s , w a s h w i t h a s o l v en t su c h a s n - h e x a n e b e f o r e u s e .
3. Reference standard
C o u ma p h o s : T h i s p r o d u c t c on t ai ns n o t l e s s t h a n 9 8 % o f c o u ma p h o s ,
a n d it s me l t i n g p o i n t is 9 5 ° C .
4. Procedure
a . E x t r a ct io n
i . C e r e a l g r a i n s , l eg u me s / p u l s e s an d s e ed s
We i g h 1 0. 0 g o f th e te s t s a mp l e , p r e v io u s l y g r o u n d s o a s to p a s s
t h r o u g h a s t a n d a r d me s h s i e v e ( 42 0 µ m) . A d d 2 0 ml o f w a t e r t o t he
o b t ai n ed s amp l e a n d l ea v e it t o s ta n d f o r t w o h o u r s .
T h e n , a d d 1 0 0 ml o f a c e t on e and h o mo g e n iz e t he mi x t u r e f o r t h re e
mi n u t e s . F i l t e r th e h o mo g e n i z ed s a mp l e b y s u c t i o n in t o a r o ta r y
v a c u u m e v a p o r a t o r t h ro u g h a f i l t er p a p e r c o v e r e d w i t h a 1 - c m t h i c k
l a y e r o f d i a t o ma c e o u s e a r t h . C o ll e c t t h e r e s i d u e o n t h e s u r f a c e o f
t h e f i l t e r p a p e r a n d ad d 5 0 ml o f a c e t o n e , a n d t h e n h o mo g e n i z e th e
mi x t u r e f o r t h r e e mi n u t e s . R e p e at t h e a b o v e p r o c e d u r e a n d c o mb i n e
t h e f i lt r a t e i n t o t h e r o ta r y v a c u u m e v ap o r a t o r, a n d t h e n c o n c en t r a te
t h e mi x t u r e t o ap p r o xima t e l y 3 0 ml a t 4 0 ° C o r l o w e r.

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Tr a n s f e r t h e c o n c en t ra t e d s o lu t io n t o a 3 0 0 - ml s e p a r a t i n g f u n n e l
a l r e a d y co n t a in i n g 1 0 0 ml o f sa t u r a t ed s od i u m c h l o r i d e s o l ut i on .
Wa s h t h e e g gp l an t - s h a p e d f l as k o f t h e a b o v e r o t a r y v a c u u m
e v a po r a t o r w i t h 1 00 ml o f e t h y l ac e t a t e/ n - h ex a n e ( 1 :4 ) a n d ad d th e
w a s h i n g s to t h e s epa r a t i n g fun n e l a b ove . S h a k e t h e mi x t u r e
v i g o r o u s l y f o r f i v e mi n u t e s u s i n g a s h a k e r an d l ea v e i t t o s t an d , an d
t h e n t r an s f e r e t h y l a c e t a t e an d n - h e x a n e l a y e r s t o a 3 0 0 - ml c o n i c al
f l a s k . A d d 5 0 ml o f e t h y l a c e tat e / n - h ex a ne ( 1 : 4 ) t o th e a q ue o u s
l a ye r a n d r e p e a t th e ab o v e p r o c ed u r e , a n d th e n a d d e t h y l a c e t at e an d
n - h e x an e lay e r s t o t h e c o n ic a l f l a sk a b o v e . Ad d an a d e q u a t e a mo u n t
o f s o d iu m s u l f a t e ( an h y d r o u s ) t o t h e f l a s k an d l e a v e it t o s t an d f o r
1 5 mi n u t e s with occasional s h a k i n g . F i lt e r t h e c o n t en t o f t h e f l a sk
i n t o a r o t ar y v a c u u m e v a p o r a t o r. Wa s h t h e f l a s k w i th 2 0 ml o f
n - h e x an e an d w a s h tw i c e t h e r e s i d u e o n t h e s u r f a c e o f t h e f i l te r
p a p e r w i t h t h e w a s h i ng s . A d d t h e w a s h i n g s i n t o th e r o t a r y v a c u u m
e v a p o r a t o r a n d r e mo v e t h e et h y l a c e t at e a n d n - h ex a n e a t 4 0 °C o r
l o w e r.
A d d 30 ml o f n - h e x a n e t o t h e r e si d u e a n d t ra n s f e r t h e mi x t u r e t o a
1 0 0 - ml s e p a r a t i n g f u n n e l , a n d t h e n ad d 3 0 ml o f n - h e x a n e - s a t u r a ted
a c e t o n i t r i le. S h a k e t h e mi x t u r e v i g o r o u s l y f o r f i v e mi n u t e s u s i n g a
s h a k e r an d l e a v e i t to s t a n d , a n d t h e n t r a n s fe r t h e a c eto n i t r il e l a y e r
t o t h e r o t a ry v a c uu m e v a p o r at o r. A d d 3 0 ml o f n - h e x an e - s a t u r a t ed
a c e t o n i t r i le t o th e n - h e x a n e l a y er a n d r e p ea t t h e a b o v e p r o c ed u re
t w i c e , an d t h en c o mb i n e t h e ac e t o n i t r i l e l a y e r i n to t h e r o t ar y
v a c u u m e v a p o r a to r. Re mo v e t h e a c e t o n i t r i le a t 4 0 ° C o r l o w e r a n d
d i s s o l v e t h e r e s i d u e in 5 - ml o f a c e t o n e / n - hex a n e ( 1: 1 ) .
i i . F r u i t , v eg e t ab l e s , tea s a n d h o p s
I n c a s e o f f r u i t a n d v e g e t ab l e s , w e i g h a c cu r a t e l y 1 k g o f t h e t e st
s a mp l e a nd ad d a n a p p r o p r i at e a mo u n t o f w a te r, i f r e q u i re d .
H o mo g e n i ze t h e mi x t u r e a n d me s u r e o u t a s a m p l e e q u iv a l e n t t o 2 0 . 0 g .
I n c a s e o f t e a s , w e ig h 5 . 0 0 g o f t h e t e s t s amp l e a n d ad d 2 0 ml o f
w a t e r, a n d t h e n l e av e i t t o s t a n d f o r t w o h o u r s .
I n c a s e o f h o p s , w e ig h 5 .0 0 g o f t h e te s t s amp l e , p r e v io u s l y g r o un d ,
a n d ad d 2 0 ml o f w a t e r, a n d t h en le a v e it to st a n d f o r t wo h o u r s .
T h e n , a d d 1 0 0 ml o f a c e t on e and h o mo g e n iz e t he mi x t u r e f o r t h re e
mi n u t e s . F i l t e r th e h o mo g e n i z ed s a mp l e b y s u c t i o n in t o a r o t a ry
v a c u u m e v a p o r a t o r t h ro u g h a f i l t er p a p e r c o v e r e d w i t h a 1 - c m t h i c k

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l a y e r o f d i a t o ma c e o u s e a r t h . C o ll e c t t h e r e s i d u e o n t h e s u r f a c e o f
t h e f i l t e r p a p e r a n d a d d 5 0 ml o f a c e t o n e a n d h o mo g e n i z e th e
mi x t u r e f o r t h r e e mi n u t e s . R e p e at t h e a b o v e p r o c e d u r e a n d c o mb i n e
t h e f i lt r a t e i n t o t h e r o ta r y v a c u u m e v a p o r a t o r, a n d t h e n c o n c en t r a t e
t h e mi x t u r e t o ap p r o x ima t e l y 3 0 ml a t 40℃ o r l o w e r.
Tr a n s f e r t h e c o n c en t ra t e d s o lu t io n t o a 3 0 0 - ml s e p a r a t i n g f u n n e l
a l r e a d y con t a in i ng 10 0 ml o f s a t u r a t ed s o d i u m c h l o r i d e so l u t i o n .
Wa s h t h e e g g p l a n t - s h a p e d f l a s k o f t h e a b o v e r o t a r y v a c u u m
e v a po r a t o r w i t h 1 00 ml o f e t h y l ac e t a t e/ n - h ex a n e ( 1 :4 ) a n d a d d t h e
w a s h i n g s to t h e s ep a r a t i n g f u n n e l ab o v e . S h a k e t h e mi x t u r e
v i g o r o u s l y f o r f i v e mi n u t e s u s in g a s h a k e r an d l e av e the i t t o s t and ,
a n d th e n t r a n s f e r t h e e t h y l a c et at e a n d n - h ex a n e l a y e r s t o a 3 0 0 -ml
c o n ic a l f l as k . A d d 5 0 ml o f e t h y l a c e t a te/n - h e x a n e ( 1 : 4 ) t o t h e
a q u eo u s l aye r a n d r e p e a t th e ab o v e p r o c ed u r e t w i c e, a n d t h e n
c o mb i n e the e th y l a c eta t e / n - h e x an e l y e r s t o th e co n ic a l f l a s k a b o v e.
A d d a n ad eq u a te a mo u n t o f s o d i u m s u l f a t e ( a n h y d r o u s ) t o t h e f l a s k
a n d l e a v e it t o s ta n d f o r 1 5 mi n u t e s w i t h o cc a s i o n a l s h a k i n g . F i l ter
t h e c o n t en t o f t h e f l a sk i n to a r o ta r y v a c u u m e v a p o r a t o r. Wa s h t h e
f l a s k w i t h 2 0 ml o f n - h e x a n e a nd w a s h t w ic e t he r e s id u e o n th e
s u r f a c e o f th e f i l te r p ap e r. A d d t h e w a s h i n g s i n t o t h e r o ta r y v a c u u m
e v a p o r a t o r a n d r e mo v e t h e et h y l a c e t a te a n d n - h e x a n e a t 4 0 ° C o r
l o w e r. D i s s o l v e t h e r es i d u e i n 5 ml o f a c e t o n e / n - h e x an e ( 1 : 1 ) .
i i i . F o o d s ex c e p t t h o s e l i s t e d i n i a n d ii a b o v e
O b t a i n s th e e x t r a c t s a cc o r d i n g to t h e me t h o d s d e s c r i b e d i n i o r i i .
b . C l ea n - u p
A d d 5 g of s i l i c a g el f o r c o l u mn c h r o ma t o g r a p h y ( 6 3 - 2 0 0 µ m i n
p a r t i c le si z e ) s u s p e n d ed in a c e to n e/n - h e x an e (1:1) into a
c h r o ma t o g r a p h t u b e ( 1 5 m m i n i n n e r d ia me t e r, 3 0 0 mm i n l en g th ) an d
a d d o v e r ap p r o x i ma t e l y 5 g o f so d i u m s u l f a t e ( a n h y d r o u s ) i n t o th e
c o l u mn . P o u r a c e t o n e /n - h e x an e (1 : 1 ) i n t o th e c o l u mn u n t il o n l y a
s ma l l a mo u n t r e ma i n s o n th e p a c k in g o f th e co l u mn a n d p o u r t h e
s o l u t io n o b t a i n ed b y t h e e x t r a ct io n d e s c r i b ed i n 4 - a i n to t h e c o l u mn .
P o u r 1 00 ml o f ac e to n e/ n - h e x a n e ( 1 : 1 ) in to th e c o l u mn a n d co l le c t th e
e l u a te in t o a r o t a r y v ac u u m e v a p o r a t o r, an d t h en r e mo v e th e a ce to n e
a n d n - h ex an e at 4 0 ° C o r lo w e r. D i s s o l v e t h e r e s i d u e in a ce t o n e to
ma k e e x a c tl y 5 ml o f s o l u ti o n, w hi c h i s u s e d a s t h e s a mp l e s o lu t i o n .
5 . D e t e r mi n a t io n

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a . Q u a l i ta t iv e t e s t s
P e r f o r m q u a l i t at i v e t es t s u n d e r th e f o l l o w in g c o n d i t io n s . Te s t r e s u l t s
o b t ai n ed mu s t b e t h e s a me a s t h o s e o b t a i n ed f o r t h e r e f e r e n c e
standard.
Te s t i n g c o n d i t io n s 1
C o l u mn : A s i l i c a te g l as s c a p i ll a r y c o lu mn ( 0 . 5 3 mm i n i n n e r d i a me t e r,
10-30 m in le n g th ) c o a te d with me t h y l s i l i co n e for gas
c h r o ma t o g r a p h y t o a th i c kn e s s o f 1 . 5 µ m
C o l u mn t e mp e r a t u r e : H o l d t h e c o l u mn t e mp e r a t u r e a t 8 0 ° C f o r o n e
mi n u t e , f o ll o w e d b y an in c r e a s e o f 8 ° C e v e ry mi n u t e u n t i l r e a c h i n g
2 5 0 ° C , a n d h o ld f o r f i v e mi n u t e s .
I n l e t t e mp e r a t u r e : 2 3 0 ° C
D e t e c t o r : Op e r a t e at 2 8 0 ° C
G a s f l o w r a t e : U s e h el i u m a s t h e c a r r i e r g a s . A d j u s t t h e f l o w r a t e t o
t h e o pt i ma l c on d it i on . A d j u s t th e f l o w s o f ai r a n d h y d r o g e n to t h e
o p t i ma l c o n d i ti o n s .
Te s t i n g c o n d i t io n s 2
C o l u mn : A s i l i c a te g l as s c a p i ll a r y c o lu mn ( 0 . 3 2 mm i n i n n e r d i a me t e r,
1 0 - 3 0 m i n l e n g t h ) co at e d w i th 5 0% t r i f l u o r o p r o p y l me t h y l s i l i co n e
f o r g a s c h ro ma t o g r a p hy t o a t h i c k n e s s o f 0 . 2 5 µ m
C o l u mn t e mp e r a t u r e : H o l d t h e c o l u mn t e mp e r a t u r e a t 7 0 ° C f o r o n e
mi n u t e , f o ll o w e d b y an i n c r e a s e o f 2 5 ° C ev er y mi n u t e u n t i l r e ac h in g
1 2 5 ° C , a f t er w h i c h in cr e a s e t h e te mp e r a t u r e b y 1 0 ° C ev e r y mi n u t e
u n t il r e a c h in g 2 3 5 ° C , a n d h o ld f o r 1 2 mi n u t es .
I n l e t t e mp e r a t u r e : 2 3 0 ° C
D e t e c t o r : Op e r a t e at 2 8 0 ° C
Gas flow rate: Use helium as the carrier gas. Adjust the flow rate
o p t i ma l c o n d i ti o n. A d j u s t t h e fl o w s o f a ir a n d h y d ro g e n t o t h e
o p t i ma l c o n d i ti o n s .
b . Q u a n ti t at i v e t e s t s
D e t e r mi n e t h e q u an t i ty f r o m t h e t e s t r e s u lt s o b ta i n e d u n d e r th e s ame
c o n d i t io n s d e s c r i b ed in 5 - a u si n g e i t he r t h e p e a k h e ig h t o r p e ak a re a
me t h o d .
c . C o n f i r ma t i o n t e s t s
P e r f o r m g a s c h r o ma t o g r a p h y / ma s s s p e c t ro me t r y u n d e r t h e s am e
c o n d i t io n s d e s c r i b ed in 5 - a . Te s t r e s u l t s o bt ai n e d mu s t b e t h e s a me a s
t h o s e o b t a in e d f o r t h e r e f e r e n c e s t a nd a r d . D e t e r mi n e t h e q u an t ity

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u s i n g e i th er t h e p ea k h e i g h t o r p e a k a r e a me t h o d , i f r eq u i r e d .

( 9 ) C h l o r amp h e n i c o l a n a l yt i ca l me t h o d
1. Apparatus
A l i q u i d c h r o ma t o g r a p h / ma s s s p e c t r o me t e r ( L C / M S o r L C / M S / M S )
2 . R e ag e n t s a n d t e s t s o l u t io n s
I n a d d i t io n t o t h e r e ag e n t s a n d te s t s o l u t i o n s l i s t ed b e l o w, u s e th o se
l i s t e d i n S e c t i o n C R e a g e n ts /Te s t S o l u ti o n s , E t c . , P a r t I I F o o d
a d d it i v e s .
A c e t o n it r i le : A c e t o n i tr i l e p r o d u ce d f o r l i q u id c h r o ma t o g r a p h y.
D i v i n y l b en z e n e - N - v inyl p y r r o l i d i n e c o p o l yme r c a r t r i d g e co l u mn ( 6 0
mg ) : A p o l ye t h yl e n e co l u mn o f 1 2 - 1 3 mm i n in n e r d i ame t e r p a c k e d
w i t h 60 mg o f d i vi ny l b e n z en -N - v i n y l p y r r o l i d o n e c o p o l y me r, o r a
c o l u mn e q u i v a l en t to th e s p e ci f i ed o n e i n s ep a r a t i o n c ap a b il i t y.
D i v i n y l b en z e n e - N - v in y l p y r r o l i d i n e c o p o l y me r c a r t r i d g e c o lu mn ( 2 0 0
mg ) : A p o l ye t h yl e n e co l u mn o f 1 2 - 1 3 mm i n in n e r d i ame t e r p a c k e d
w i t h 2 0 0 mg o f d i v in y l b e n ze n -N - v i n y l p y r r o l i d o n e co p o l y me r, o r a
c o l u mn e q u i v a l en t to th e s p e ci f i ed o n e i n s ep a r a t i o n c ap a b il i t y.
Wa t e r : Wa te r p r o d u ced f o r l i q u i d c h r o ma t o g r a p h y.
3. Reference standard
C h l o r a mp h e n i co l : Th i s product c o n ta i n s not less than 99% of
c h l o r a mp h e n i c o l , a n d it s d e c o mp o s i t i o n p o i n t i s 2 0 8 ° C .
4 . P r o c e du r e
a . E x t r a ct io n
i. Honey
We i g h 5 .00 g o f t h e t e s t s a mp l e , p r e v io u s l y h o mo g e n i z ed , a n d
d i s s o l v e i t i n 2 0 ml o f w a t e r.
i i . R o y a l j el l y
We i g h 1. 0 0 g o f t h e t es t s a mp l e , p r e v i o u s l y h o mo g e n i z e d , a d d 6 0 ml
o f me t h a n o l / 1 % me t a p h o sp h o r ic a c i d ( 3 :2 ) , a n d h o mo g e n i z e th e
mi x t u r e a g a i n . F i l te r t h e h o mo g e n iz e d s amp l e b y s u c t io n in t o a
r o t a r y v a cu u m e v a p o r a t o r t h r o u g h a f il t e r p a p e r co v e r e d w i t h a
2 - mm t h i c k l a ye r o f d i a t o ma c e o u s e a r t h . Wa s h t h e r e si d u e o n t h e
f i l t e r p a p e r w i t h 1 5 ml o f me t h a n o l /1 % me t a p h o sp h o r ic a c i d ( 3 : 2 )
a n d f i l te r th e w a s h i n g s b y s u c t i on . C o mb i n e t h e f i l t r at e s i n to t h e
r o t a r y v a c u u m e v a p o r a t o r a n d co n c en t r a t e t h e mi x t u r e t o 2 ml a t
4 5 º C o r lo w e r.
i i i . F o o d s o t h e r t h a n th o s e g iv e n in s e c t io n s i a n d i i

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We i g h 5 . 00 g o f t he te s t s a mp l e , p r e v i o u s l y g r o u n d . Fo r mu s c l e ,
r e mo v e t h e f a t l a y e r as mu c h a s p o s s i b le b ef o r e g r i n d in g . A d d 1 0 0
ml o f me t h a n o l/ 1 % me t a p h o s p h o r i c a c i d ( 3 :2 ) a n d h o mo g e n i z e th e
mi x t u r e a g a i n . F i l t e r t h e h o mo g e n iz e d s amp l e b y s u c t io n i n t o a
r o t a r y v a c u u m e v a p o r a t o r t h r o u g h a f i lt e r p a p e r co v e r e d w i t h a
2 - mm t h i c k l a ye r o f d i a t o ma c e o u s e a r t h . Wa s h t h e r e si d u e o n t h e
f i l t e r p a p e r w i t h 1 0 ml o f me t h a n o l /1 % me t a p h o sp h o r ic a c i d ( 3 : 2 )
a n d f i l te r t h e w a s h i n g s b y s u c t i on . C o mb i n e t h e f i l t r at e s i n to t h e
r o t a r y v a c u u m e v a p o r a t o r a n d co c e n t r a t e th e mi x t u r e t o 3 0 ml a t
4 5 º C o r lo w e r.
b. Clean-up
i. Honey
P o u r 5 ml o f me t h a n o l a n d 5 ml o f w a t e r i n t o a d iv i n yl b e n z e n e -N-
v i n y l p y r r o l i d i n e co p o ly me r c a r t r i d g e c o l u mn ( 6 0 mg ) a n d d i s c a r d
t h e e ff l u e nt. P o u r t h e s o l u t io n o b t a i n ed b y th e ex t r a c tio n d e s c r i b ed
i n 4 - a in t o t h e c o lu mn f o l lo w e d b y 5 ml o f 2 0 % ( v o l ) me t h an o l , an d
d i s c a r d th e e ff l u e n t . P o u r 6 ml o f 6 0 % ( v ol ) me t h a n o l a n d co l l ect
t h e e l u a t e in t o a r o t a r y v a cu u m e va p o r a to r a n d r e mo v e t h e me t h a n o l
a n d w a te r a t 4 5 ° C o r l o w e r. D i s s o l v e th e r e s i d u e in 1 .0 ml o f
a c e t o n i t r i le/ w a t e r ( 3 : 7 ) , w h i c h i s u s e d a s th e s a mp l e s o l u t i o n .
i i . R o y a l j e ll y
P o u r 1 0 ml o f me t h a n ol an d 1 0 ml o f w a te r i n t o a d i v i n y lb e n z en e -N -
v i n y l p y r r o l i d i n e c o p o ly me r c a r t r i d g e c o l u mn ( 2 0 0 mg ) a n d d i sc a r d
t h e e ff l u e n t. P o u r t h e s o l u t io n obt a i n ed b y t h e e x t r a ct io n d e s c r i bed
i n 4 - a i n to t h e c o lu mn f o l l o w e d b y 4 ml o f w a t e r a n d 4 ml o f 5 %
( v o l ) me t h a n o l an d d i s c a r d t h e e ff l u e n t . Po u r 1 0 ml o f 6 0 % ( v o l )
me t h a n o l an d c o l l e ct th e e l u a t e in t o a r o t a r y v a c u u m e v a p o r a t o r, an d
r e mo v e t h e me t h a n o l a n d w a t e r a t 4 5 ° C o r l o w e r. D i s s o l v e t he
r e s i d u e in 1 . 0 ml o f a c e t o n i t r i l e/w a t e r ( 3 : 7 ) , w h i c h i s u s e d a s t h e
s a mp l e s o l u t i o n .
i i i . F o o d s o t h e r t h a n th o s e g iv e n in s e c t io n s i a n d i i
P o u r 5 ml o f me t h a n o l a n d 5 ml o f w a t e r i n t o a d iv i n yl b e n z e n e -N-
v i n y l p y r r o l i d i n e co p o ly me r c a r t r i d g e c o l u mn ( 6 0 mg ) a n d d i s c a r d
t h e e ff l u e nt. P o u r t h e s o l u t io n o b t a i n ed b y th e ex t r a c tio n d e s c r i b ed
i n 4 - a in t o t h e co l u mn f o l l o w e d b y 1 0 ml o f w a t e r, a n d d i s c a r d th e
e ff l u e n t . P o u r 1 0 ml o f me t h a n o l a n d co l le c t t h e el u a te in t o a r o t a r y
v a c u u m e v a p o r a to r a n d r e mo v e t h e me t h an o l a t 4 0 °C o r l o w e r.

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D i s s o l v e t h e r e s i d u e in 1 . 0 ml o f a c e t o n i t r i le / w a t e r ( 3 : 7 ) , w h i c h is
u s e d a s th e s a mp l e s o l u t i o n .
5 . D e t e r mi n a t io n
a . Q u a l i ta t iv e t e s t s
P e r f o r m q u a l i t at i v e t es t s u n d e r th e f o l lo w i n g c o n d i ti o n s . Te s t r e s u l t s
o b t ai n ed mu s t b e t h e s a me a s t h o s e o b t a i n ed f o r t h e r e f e r e n c e
standard.
Te s t i n g c o n d i t io n s
C o l u mn p ac k i n g : O c ta d e c y l s i l an i z e d s i l ic a g e l ( 2 – 5 µ m i n p a r ti c l e
size).
C o l u mn : A s t a i n l e s s s t e e l tu b e ( 2 . 0 – 6 . 0 m m i n i n n e r d i a me t e r,
1 0 0– 2 50 mm i n l e n g th ) .
C o l u mn t e mp e r a t u r e : 4 0 ° C
M o b il e p h as e : U s e a ce t o n i t r i l e/ 1 0 mmo l / l a m mo n i u m f o r ma t e ( 3 : 7 ) .
Adjust the flow rate so that c h l o r a mp h e n ic o l f lo w s out in
a p p r o xi ma t e l y 5 mi n u te s .
b . Q u a n ti t at i v e t e s t s
D e t e r mi n e t h e q u an t i ty f r o m t h e t e s t r e s u lt s o b ta i n e d u n d e r th e s ame
c o n d i t io n s d e s c r i b ed in 5 - a u si n g e i t he r t h e p e a k h e ig h t o r p e ak a re a
me t h o d .

( 1 0 ) C h l o r p r o ma z i n e an a l y t i c al me t h o d
1. Apparatus
A l i q u i d c h r o ma t o g r a p h / t an d e m m a s s s p e c t r o me t e r ( L C / M S / M S ) .
2 . R e ag e n t s a n d t e s t s o l u t io n s
I n a d d i t io n t o t h e r e ag e n t s a n d te s t s o lu t i o n s l i s t ed b e l o w, u s e th o s e
l i s t e d in S e c t i o n C R e a g e n ts /Te s t S o l u ti o n s , E t c . , P a r t I I F o o d
a d d it i v e s .
A c e t o n it r i le : A c e t o n i tr i l e p r o d u ce d f o r l i q u id c h r o ma t o g r a p h y.
St r o n g l y ac i d i c c a ti on e x c h an g e r c a r t r i d g e c o lu mn ( 5 0 0 mg ) : A
p o l y e t h y l en e c o l u mn o f 8 - 9 mm i n i nn e r d i ame t e r p a c k e d w i t h 5 0 0 mg
o f b e n ze n es u l f o n y l p ro p y l s i l a n e- b o n d ed s il i c a g e l , o r a c o l u mn
e q u iv a l en t t o t he s p e cif i e d o n e i n s e p a r a t i o n c a p a b i l it y.
Wa t e r : Wa te r p r o d u ced f o r l i q u i d c h r o ma t o g r a p h y.
M e t h an o l: M e t h an o l p r o d u c ed f o r l i q u i d c h ro ma t o g r a p h y.
3. Reference standard
C h l o r p r o ma z i n e h yd r o c h lo r i d e : T h i s p r o d u c t c o n t ai n s n o t l e s s th a n
9 8 % o f ch l o r p r o ma z i n e h y d r o ch lo r i d e , a n d it s me l t i n g p o i n t i s

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1 9 4 - 19 6 ° C .
4. Procedure
a . E x t r a ct io n
We i g h 5 .0 0 g o f t h e t es t s a mp l e , p r e v i o u s l y g r o u n d , a n d h o mo g e n i z e
i t w i th 2 5 ml o f e t h y l a c e t a te an d 1 ml o f 4 mo l / l p o t a ss i u m c a r b o n a t e
s o l u t io n . Ce n t r i f u g e th e mi x t u r e a t 3 ,0 0 0 r p m f o r f i v e mi n u t e s A n d
t r a n s f e r t h e e th y l a ce t at e la y e r i n to a r o t a r y v a c u u m e v a p o r a to r. A d d
2 5 ml o f e t h y l a c e t at e t o th e r e s id u e an d r e p e a t t h e a b o v e p r o c ed u r e
a n d t r a n s f e r t h e et h y l a c e t a te l a ye r i n t o t h e r o t a r y v a c u u m e v a p o r a to r,
a n d th e n r emo v e t h e e th y l a c e t at e a t 4 0 ° C o r lo w e r. A d d 3 0 ml e a c h o f
a c e t o n i t r i le a n d a c et o n i t r i l e - s a tu r a t e d n - h ex a n e t o t h e r e s i d u e a n d
s h a k e th e mi x t u r e v ig o r o u s l y f o r f i v e mi n u t e s u s ing a s h a k e r a n d
l e a v e i t t o s t a n d , an d t h e n t r a n s f e r t h e a c eto n i t r il e l a ye r t o a 1 0 0 - ml
s e p a r a t i ng f u n n e l . A dd 3 0 ml o f a c e to n i t r ile - s a t u r a t e d n - h e x an e a n d
r e p e a t t h e a b o v e p r o ce d u r e . C o l le c t t h e a cet o n it r i l e l ay e r i n t o t h e
r o t a r y v a cu u m e v a p o r a t o r a n d r e mo v e t h e a c e to n it r i le a t 4 0 ° C o r
l o w e r. Dissolve the residue in 10 ml of me t h a n o l/ 1 . 2 %
me t a p h o s p h o r i c a c i d s o l u t io n ( 2 : 3 ) a n d f i lte r t h e mi x t u r e t h r o u g h a
c o t to n p l ug.
b . C l ea n - u p
P o u r 3 ml o f me t h a n o l f o l l o w e d b y 3 ml o f w a t e r i n t o a s t r o n g l y
a c i d ic c at io n ex c ha n ge r c a r t r i d g e c o lu mn ( 5 0 0 mg ) a n d d i s c a r d t h e
e ff l u e n t . Po u r t h e s o lu t i on o bt a in e d b y t h e e x t r a c ti o n d e s c r i b ed in 4 - a
i n t o th e c ol u mn f o l l ow e d b y 5 ml o f w a t e r a n d d i s c ar d t h e e ff l u e n t .
Pour 15 ml of me t h a n o l / 0 . 1 mo l / l d i p o t a ss i u m hydrogen
o r t h o p h o s p h a t e s o lu t io n ( 9 : 1 ) t o t h e c o lu mn a n d co l le c t t h e el ua te
i n t o a r o t ar y v a c u u m e v a p o r a t o r, a n d th en r e mo v e th e w a t e r an d
me t h a n o l a t 4 0 ° C o r l o w e r. D i s s o l v e th e r e s i d u e in 1 . 0 ml o f me t h a n o l ,
w h i c h i s u s e d a s th e s amp l e s o l u t io n .
5 . D e t e r mi n a t io n
a . Q u a l i ta t iv e t e s t s
P e r f o r m q u a l i t at i v e t es t s u n d e r th e f o l l o w in g c o n d i t io n s . Te s t r e s u l t s
o b t ai n ed mu s t b e t h e s a me a s t h o s e o b t a i n ed f o r t h e r e f e r e n c e
standard.
Te s t i n g c o n d i t io n s
C o l u mn p a c k i n g : O c ta d e c y l s i l an e - b o n d e d s i l i c a g e l ( 2 - 5 µ m in
p a r t i c le s i ze ) .

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C o l u mn : A s t a i n l e s s t ub e ( 2 .0 - 6 . 0 m m i n i n n er d i a me t e r, 1 0 0 - 2 5 0 m m
i n l en g th ) .
C o l u mn t e mp e r a t u r e : 4 0 ° C
M o b il e p h as e : U s e a c et o n it r i l e / f o r mi c a c i d /w a t e r ( 5 0 0 :1 : 5 0 0 ) . A d ju s t
t h e f l o w r a t e s o t h at ch l o r p r o ma z i n e f l o w s o u t i n ap p r o x i ma t e l y 1 5
mi n u t e s .

b . Q u a n ti t at i v e t e s t s
D e t e r mi n e t h e q u an t i ty f r o m t h e t e s t r e s u lt s o b ta i n e d u n d e r th e s ame
c o n d i t io n s d e s c r i b ed in 5 - a u si n g e i t he r t h e p e a k h e ig h t o r p e ak a re a
me t h o d .

( 11 ) D i e t h yl s t i l b e s t r o l a n a l y t i ca l me t h o d
1. Apparatus
A l i q u i d c hr o ma t o g r a p h / t an d e m m a s s s p e c t r ome t e r ( L C / M S / M S )
2 . R e ag e n t s a n d t e s t s o l u t io n s
I n a d d i t io n t o t h e r e ag e n t s a n d te s t s o lu t i o n s l i s t ed b e l o w, u s e th o s e
l i s t e d in S e c t i o n C R e a g e n ts /Te s t S o l u ti o n s , E t c . , P a r t I I F o o d
a d d it i v e s . R e a g en t s d e s i g n at e d as “s p e c i a l g r a d e ” i n th i s s e c ti o n mu s t
me e t t h e re q u i r e me n t s f o r “s p e c i a l g r a d e” s p e c i f ie d i n t h e J ap a n
I n d u s t r i a l St a n d a r d s fo r t h e r e ag e n t s .
A c e t o n it r i le : A c e t o n i tr i l e p r o d u ce d f o r l i q u id c h r o ma t o g r a p h y.
G l u c u r o n id a s e s o l u t io n : S h o u ld c o n ta i n 1 0 0 , 0 0 0 u n it s/ ml o f b e t a - D -
g l u cu r o n ida s e e x t r a cte d f r o m H e l i x p o m a t ia a n d r e f i n ed .
D i c h l o r o me t h a n e : D i c h l o r o me t h a n e ( s p e c i a l g r a d e ) .
We a k l y b as i c anion e x ch a n g er cartridge c o l u mn (500 mg ) : A
p o l y e t h y l en e c o l u mn o f 8 - 9 mm i n i nn e r d i ame t e r p a c k e d w i t h 5 0 0 mg
o f d i e t h yl ami n o p r o p yl w e a k l y b a s i c a n i o n e x c h an g e r p r o d u ce d f o r
c o l u mn c h r o ma t o g r a p h y, o r a c o l u mn e q u i v a le n t t o t h e sp e c i f i ed o n e
i n s e p a r at io n c ap a b i l it y.
Wa t e r : Wa te r p r o d u ced f o r l i q u i d c h r o ma t o g r a p h y.
S o d i u m s u l f a t e ( a n h y d r o u s ) : S o d iu m s u l f a t e ( a n h y d r o u s) ( s p e c i a l g ra d e ) .
I f t h e r e a g e n t i s f o u n d t o in c lu d e a n y s u b st a n c e t h at ma y i n t e r f e r e
w i t h a n al y s i s o f t h e t a rg e t co mp o s i t i o n a l s u b st a n c e s f r o m a g r i c u l t u r al
c h e mi c a l s , w a s h w i t h a s o l v en t su c h a s d i ch l o r o me t h a n e b e f o r e u se . .
3. Reference standard
D i e t h yl s t i lb e s t r o l : Th i s p r o d u ct contains not le s s than 99% of
d i e th y l s t i l b e s t r o l , a nd i t s d e co mp o s i t i o n p o in t i s 2 0 8 ° C.
4. Procedure

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a . E x t r a ct io n
We i g h 5 .0 0 g o f t h e t es t s a mp l e , p r e v i o u s l y g r o u n d , a n d h o mo g e n i z e
i t w i t h 11 ml o f 0 . 0 4 mo l / l s o d iu m a c e t a t e s o l u ti o n , a n d t h en ad d
a c e t i c a c i d t o a d ju s t t he p H t o 4 . 2 5 - 4 . 7 5 . A d d 1 0 0 µl o f g l u cu r o n id a s e
s o l u t io n to t h e mi x t u r e a n d l ea v e it t o s t an d fo r 1 4 h ou r s a t 3 7 ° C . Ad d
1 6 ml o f a c e t o n i t r i le a n d s h a k e t he mi x t u r e v ig o r o u s l y f o r f i v e
mi n u t e s u s in g a s h a k e r. C en t r i f u g e th e mi x t u r e a t 3 , 0 0 0 r p m f o r f i v e
mi n u t e s a n d t r a n s f e r th e s u p e r n a ta n t i n to a r o t a r y v a cu u m e v a p o r a to r.
A d d 1 6 ml o f a ce t o n i t r i l e t o t h e r e s id u e a n d r e p e a t th e ab o v e
p r o c e d u r e tw i c e a n d t ra n s f e r t h e su p e r n a ta n t i n t o th e r o t a r y v a c u u m
e v a p o r a t o r, a n d t h en co n c en t r a t e t h e s o l u t i o n to a p p r o x i ma t e l y 3 ml
a t 50 ° C o r l o w e r. A d d 5 0 ml o f d i c h l o r o me t h a n e a n d 2 0 0 ml o f 5 %
s o d i u m c h l o r i d e s ol u tio n to th e co n c en t r a t ed s o l u ti o n , an d th e n s h ak e
it v i go r o us l y for five mi n u te s using a s h a k e r. C o l l e ct the
d i c h l o r o me t h a n e l a y e r a n d de h yd ra t e w i t h sod i u m s u l f a t e ( a n h yd r ou s ) .
A d d 5 0 ml o f d i ch l o ro me t h a n e to th e a qu eo u s la ye r an d r ep e a t th e
a b o v e p r o ce d u r e t w i ce, a n d t h en c o mb i n e th e d i ch l o r o me t h a n e l ay e r.
A d d a n a d eq u a te a mo u n t o f s o d i um s u l f a t e ( a n h yd r o u s ) a n d l e av e i t t o
s t a n d f o r 1 5 mi n u t e s w i t h o c c a s io n a l s h ak i n g . F i l t e r t h e c o n t en t in t o a
r o t a r y v a c u u m e v a p o r at o r a n d r e mo v e t h e d i ch l o r o me t h a n e at 4 0 ° C o r
l o w e r. D i s s o l v e t h e r es i d u e i s i n 4 ml o f n - h e x a n e/ b en ze n e ( 3 :1 ) .
b . C l ea n - u p
P o u r 1 0 ml o f n - h e x a n e / b e n z e n e ( 3 : 1 ) i n t o a w e a k l y b a s i c a n i o n
e x c h an g e r c a r t r i d g e co l u mn ( 5 0 0 mg ) a n d d i s c a r d t he e ff l u e n t . Po u r
t h e s o lu t io n o b ta i n e d b y t h e e xt r a c t i on d es c r i b e d in 4 - a i n to t he
c o l u mn f o l l o w e d b y 2 ml o f n - h e x an e /b e n z e n e ( 3 : 1 ) a n d 4 ml o f
d i c h l o r o me t h a n e , an d d i s c a r d the e ff l u en t . P o u r 8 ml o f d i ch l or o
me t h a n e / me t h an o l ( 9 :1 ) i n to th e c o l u mn a n d c o ll e c t t h e e l u at e i n to a
r o t a r y v a c u u m e v a p o r at o r, a n d t h en r e mo v e t h e d i ch l o r ome t h a n e a nd
me t h a n o l a t 4 0 ° C o r l o w e r. D i s s o l v e th e r e s i du e i n 0 . 5 ml o f
a c e t o n i t r i le/ w a t e r ( 1 : 1 ) , w h i c h i s u s e d a s th e s a mp l e s o l u t i o n .
5 . D e t e r mi n a t io n
a . Q u a l i ta t iv e t e s t s
P e r f o r m q u a l i t at i v e t es t s u n d e r th e f o l l o w in g c o n d i t io n s . Te s t r e s u l t s
o b t ai n ed mu s t b e t h e s a me a s t h o s e o b t a i n ed f o r t h e r e f e r e n c e
standard.
Te s t i n g c o n d i t io n s

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C o l u mn p a c k i n g : O c ta d e c y l s i l an e - b o n d e d s i l i c a g e l ( 2 - 5 µ m in
p a r t i c le s i ze ) .
C o l u mn : A s t a i n l e s s t ub e ( 2 .0 - 6 . 0 m m i n i n n er d i a me t e r, 1 0 0 - 2 5 0 m m
i n l en g th ) .
C o l u mn t e mp e r a t u r e : 4 0 ° C
M o b il e p has e : U s e a c e t o n it r i l e /0 .0 0 2 mo l / l a m mo n i u m a c e t a t e ( 1 : 1 ).
Adjust the flow rate so that d i e th y l s t il b e s t r o l f lo w s out in
a p p r o x i ma t e l y s e v e n mi n u t e s .
b . Q u a n ti t at i v e t e s t s
D e t e r mi n e t h e q u an t i ty f r o m t h e t e s t r e s u lt s o b ta i n e d u n d e r th e s ame
c o n d i t io n s d e s c r i b ed in 5 - a u si n g e i t he r t h e p e a k h e ig h t o r p e ak a re a
me t h o d .

( 1 2 ) A n a l yt i c a l me t h o d f o r d i me t r i d a z o l e , me t r o n i d az o l e a n d r o n i d azo l e
1. Apparatus
A l i q u i d c hr o ma t o g r a p h / t an d e m m a s s s p e c t r ome t e r ( L C / M S / M S )
2 . R e ag e n t s a n d t e s t s o l u t io n s
I n a d d i t io n t o t h e r e ag e n t s a n d te s t s o lu t i o n s l i s t ed b e l o w, u s e th o s e
l i s t e d in S e c t i o n C R e a g e n ts /Te s t S o l u ti o n s , E t c . , P a r t I I F o o d
a d d it i v e s .
A c e t o n it r i le : A c e t o n i tr i l e p r o d u ce d f o r l i q u id c h r o ma t o g r a p h y.
M e t h an o l: M e t h an o l p r o d u c ed f o r l i q u i d c h ro ma t o g r a p h y.
Wa t e r : Wa te r p r o d u ced f o r l i q u i d c h r o ma t o g r a p h y.
3. Reference standard
D i me t r i d a z o l e : This p r o d u ct c o n t ai n s not less than 99% of
d i me t r i d a zo l e , an d i t s me l t i n g p o i n t i s 1 3 8 -1 3 9 ° C .
M e t r o n id a zo l e : This p r o du c t c o nt a in s not less t h an 99% of
me t r o n i d a zo l e , an d i t s me l t i n g p o i n t i s 1 5 8 -1 6 0 ° C .
R o n id a z ol e: T h i s p r odu c t co n ta i n s n o t le s s th a n 9 9 % o f r o n i d az o le, a n d
i t s me l t i n g p o i n t i s 1 6 7 - 1 6 9 ° C .
4. Procedure
We i g h 5 . 0 0 g o f t h e t es t s a mp l e , p r e v i o u s l y g r o u n d , an d h o mo g e n iz e i t
w i t h 2 0 ml o f a c e to n i t ri l e . C en t r i fu g e th e mi x t u r e a t 3 , 0 0 0 r p m f o r f i v e
mi n u t e s a n d t r a n s f e r t h e a ce t o n it r i l e l a y e r t o a 1 0 0 -ml s e p a r a t i n g
f u n n e l . A dd 2 0 ml o f n - h e x an e an d s h ak e t h e mi x t u r e v i g o r o u sl y, a n d
t h e n l ea v e i t t o s t a n d . Tr a n s f e r t h e a ce t o n i t r i l e l a y er i n t o a r o t a r y
v a c u u m e v a p o r a to r a n d a d d 5 ml o f n - p r o p an o l , an d th e n r e mo v e t h e
a c e t o n i t r i le a n d n - p r o p a n o l a t 4 0 ° C o r l o w er. D i s s o l v e t h e r e s id u e i n

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1 . 0 ml o f me t h a n o l an d f i l te r t he mi x t u r e th r o u g h a me mb r a n e f i l t e r
w i t h a p o r e s i z e o f 0 . 2 µ m, w h i c h i s u s e d a s t h e s a mp l e s o l u t io n .
5 . D e t e r mi n a t io n
a . Q u a l i ta t iv e t e s t s
P e r f o r m q u a l i t at i v e t es t s u n d e r th e f o l l o w in g c o n d i t io n s . Te s t r e s u l t s
o b t ai n ed mu s t b e t h e s a me a s t h o s e o b t a i n ed f o r t h e r e f e r e n c e
standard.
Te s t i n g c o n d i t io n s
C o l u mn p a c k i n g : O c ta d e c y l s i l an e - b o n d e d s i l i c a g e l ( 2 - 5 µ m in
p a r t i c le s i ze ) .
C o l u mn : A s t a i n l e s s t ub e ( 2 .0 - 6 . 0 m m i n i n n er d i a me t e r, 1 0 0 - 2 5 0 m m
i n l en g th ) .
C o l u mn t e mp e r a t u r e : 4 0 ° C
M o b il e p h as e : U s e a c e t o n it r i l / w a te r ( 1 : 9) . A d j u s t t h e f l o w r a t e s o t h a t
d i me t r i d a zo l e , me t r o n i d az o l e an d r o n i d a zo l e f l o w o u t in 4 - 1 0
mi n u t e s .
b . Q u a n ti t at i v e t e s t s
D e t e r mi n e t h e q u an t i ty f r o m t h e t e s t r e s u lt s o b ta i n e d u n d e r th e s ame
c o n d i t io n s d e s c r i b ed in 5 - a u si n g e i t he r t h e p e a k h e ig h t o r p e ak a re a
me t h o d .

( 1 3 ) D a mi n o z i d e a n a l y t i c a l me t h o d
1. Apparatus
A gas c h r o ma t o g r a p h with an a l k al i f la me i o n i za t i on d et ec t o r
( G C - F I D ) o r h i g h l y - s e n s i t i v e n i t ro g e n p h o sp h o r u s d e te ct o r ( G C - N P D ) ,
a g a s c h r o ma t o g r a p h /ma s s s p e c t r o me t e r ( G C / M S o r GC / M S / M S ) , a n d
s t e a m d i s t i ll a t i o n a p p ar a t u s . T h e st e a m d i s t i lla t i o n ap p a ra t u s s h o u l d b e
ma d e o f g la s s a n d r oug h l y a s s h o w n i n th e fo l l o w in g f ig u r e :

A : 1 , 00 0 - ml r o un d b o tt o m f l a s k
(for steam generation)
B : 1 ,0 00 - ml r o un d b o tt o m f l a s k
( f o r d i s t i l l at i o n )
C : D i s t i l la ti o n t r ap
D : C o o l in g t u b e
E : 1 0 0 - ml c o n ic a l f l a sk

2 . R e ag e n t s a n d t e s t s o l u t io n s
I n a d d i t io n t o t h e r e ag e n t s a n d te s t s o lu t i o n s l i s t ed b e l o w, u s e th o s e

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l i s t e d in S e c t i o n C R e a g e n ts /Te s t S o l u ti o n s , E t c . , P a r t I I F o o d
a d d it i v e s . R e a g en t s d e s i g n at e d as “s p e c i a l g r a d e ” i n th i s s e c ti o n mu s t
me e t t h e re q u i r e me n t s f o r “s p e c i a l g r a d e” s p e c i f ie d i n t h e J ap a n
I n d u s t r i a l St a n d a r d s fo r t h e r e ag e n t s .
A c e t o n e: U s e a r o t a r y v a c u u m e v ap o r at o r o n 3 0 0 mL o f a c e to n e t o
c o n c en t r a t e t o t h e p o in t o f d r y n e s s . D i s s o l v e t h e r e s i d u e i n 5 mL o f
n - h e x an e . F o r a n a l y s i s , i n j ec t 5 μ L o f th e so l u ti o n i n to a GC -ECD.
P e a k s o t h er t h a n t h a t o f n - h e x an e i n th e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
B a s i c a lu mi n a ca r t r i d g e c o lu mn ( 1 , 7 1 0 mg ) : A p o l y e t h y l e n e co l u mn o f
8 - 9 mm i n i n n e r d ia me t e r p a c k ed w i t h 1 ,7 1 0 mg o f b a s i c a l u mi n a , o r a
c o l u mn e q u i v a l en t to th e s p e ci f i ed o n e i n s ep a r a t i o n c ap a b il i t y.
S i l i c o n e f o r d e f o a mi n g : S i l i co n e p r o d u ce d fo r d e f o a mi n g .
ο - N i t r o b e nz a l d eh yd e : ο - N i t r o b e n z a l d eh y d e (s p e c i a l g r ad e ) .
1 % ο - N i t r o b e n z a l d e h y d e - me t h a n o l s o l u t i o n : D is s o l v e ο - n i tr o b e n z a l d e h y d e
( 1 0 0 mg ) i n 1 0 ml o f me t h a n o l . P r e p a r e i mme d i a t e l y b e f o r e u s e .
P h e n o lp h th a l e i n r e ag en t : D i s s o l ve p h e no lp ht h a l ei n ( 1 g ) i n 1 00 ml o f
e t h an o l .
n - H e x a n e : U s e a r o t a r y v a c u u m e v a p o r at o r on 3 00 mL o f n - h e x a n e t o
e v a p o r a t e u n t i l 5 mL i s l e f t . F o r a n a l y s i s , i n j e c t 5 μ L o f t h e s o lu t io n
i n t o a G C - E C D . P e a k s o t h e r th an t h at o f n - h e x a n e i n t h e r e s u l t in g
c h r o ma t o g r a m s h o u l d b e a s h ig h a s o r lo w e r t h an t h e pe a k s o f γ - B H C
a t 2 x 10 - 1 1 g .
Wa t e r : D i s ti l l e d w a t e r. I f t h e d i s ti l l e d w a t e r i s f o u n d to i n c l u d e an y
s u b s t a n ce th a t ma y i n t e r f e r e w i t h a n a l y s i s o f t h e t a rg et co mp o s i t i o n a l
s u b s t a n ce s f r o m a g r i c u l t u r a l ch emi c a l s , w a s h w i t h a s o l v en t s u c h a s
n-hexane before use.
M e t h an o l: U s e a r o t a r y v a c u u m e v a p o r at o r on 3 00 mL o f me t h a n o l t o
c o n c en t r a t e t o t h e p o in t o f d r y n e s s . D i s s o l v e t h e r e s i d u e i n 5 mL o f
n - h e x an e . F o r a n a l y s i s , i n j ec t 5 μ L o f th e so l u ti o n i n to a GC -ECD.
P e a k s o t h er t h a n t h a t o f n - h e x an e i n th e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
P h o s p h a te b u ff e r s o l uti o n ( p H 5 ) : D i s s o l v e mo n o p o t a s si u m d i h y d r o g e n
mo n o p h o s p h a t e ( 1 3 .1 5 g ) a n d d i p o t a s s i u m h y d r o g e n o rt h o p h o s p h at e
( 0 . 5 9 g ) i n w a t e r t o ma k e a 1 0 0 - ml s o l u t i o n .
3. Reference standard
D i me t h yl h y d r a z i n e: Th i s p r o d u ct c o n ta i n s n o t l e s s t h a n 9 7 % o f

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1 , 1 - d i me t h yl h yd r a z i ne , a n d i t s b o i l in g p o in t i s 6 2 - 6 4 °C .
4. Procedure
a . E x t r a ct io n
i . C e r e a l g r a i n s , l eg u me s / p u l s e s , se e d s , f r u i t , v e g et a b le s , ma t c h a a n d
hops
I n c a s e c e r e a l g r a i n s , l e g u me s / p u l s e s a n d s ee d s , w e i g h 5 . 0 g o f t e st
s a mp l e s s o a s t o p a s s t h r o u g h a s t a n d a r d me s h s i e v e ( 4 2 0 µ m) .
I n c a s e f ru i t a n d v eg e t ab l e s , w e i g h ac c u r a t e l y 1 k g o f t h e te s t
s a mp l e a n d a d d a n app r o p r i a t e amo u n t o f w a t e r t o t he s a mp l e , i f
r e q u i r e d . H o mo g e n i ze t h e mi x t u r e a n d me a s u r e o u t a s a mp l e
e q u iv a l en t t o 1 0 . 0 g .
I n c a s e o f ma t c h a , w e i g h 5 . 0 g o f t h e t e s t s a mp l e .
I n c a s e o f h o p s , w e ig h 5 . 0 g o f th e t e s t s a mp l e , p r e v i o u s l y g r o u n d .
T h e n , a d d 8 0 ml o f w a t e r to t h e o b t a i n ed s a mp l e a n d s h a k e i t
v i g o r o u s l y f o r 3 0 mi n u t e s , an d th e n f i lt e r th e mi x t u r e b y s u c t i o n
t h r o u g h a g l a s s f i b e r fi l t e r. Co l l ec t th e r e s i d u e o n t h e f il t e r an d a d d
4 0 ml o f w a t e r, a n d t h e n s h a k e t h e mi x t u r e f o r f i v e mi n u t e s . R ep e at
t h e ab o v e p r o c e d u r e a n d co mb i n e t he f i l t ra t e t o a 1 ,0 0 0 - ml r o u n d
b o t to m f l a s k ( f o r d i s t i ll a t i o n ) .
i i . Tea s e x ce p t ma t c h a
We i g h 6 .0 g o f t h e t es t s a mp l e a n d s o ak i t i n 3 6 0 ml o f w a t e r a t
1 0 0 ° C , an d t h e n l e av e i t t o s t an d a t r o o m t e mp e r a t u r e f o r f i v e
mi n u t e s . Tr a n s f e r 1 2 0 ml o f t h e c o o l ed f i l t r at e i n to a 1 ,0 0 0 - ml r o u n d
b o t to m f l a s k ( f o r d i s t i ll a t i o n ) .
i i i . F o o d s ex c e p t t h o s e l i s t e d i n i a n d ii a b o v e
O b t a i n t h e e x t r a c t s acc o r d i n g to t h e me t h o d s d e s c r i b e d i n i .
b . D i s t i l la t io n
A d d 6 0 g of s o d i u m h y d r o x i d e ( 6 5 g f o r v e g e t a b le s a n d f r u i t ) b i t b y
b i t in t o th e r o u n d b o t to m f l a s k d e s c r i b e d ab o v e w i t h t h e f l a s k w a t e r
c o o li n g . A d d 1 - 2 d r o p s o f d e fo a mi n g s i li c o n e t o th e d i s s o l v e d
s o l u t io n a n d a t ta c h t h e f l a s k t o a d is t i ll a t io n a p p a r a t u s i mme d i a t el y.
S e p a r a t e l y, a t t ac h a 1 0 0 - ml c o n i ca l f l a sk c o n t ai n in g 5 ml o f
p h o s ph a t e b u ff e r s o l ut i o n ( p H 5 ) a n d o n e d r o p o f p h e n o lp h th a l ei n
r e a g e n t to a s t e a m d i s t i l l e r a n d h e a t a 1 , 0 0 0 - ml r o u n d b o t to m f l a s k
( f o r s t e a m g e n e r a ti o n ). D i s t i l l th e s o l u t i o n u n t il t h e d is t i l l a te c o me s
t o 4 5 ml a n d c o n f i r m t h a t t h e d i s ti l l a te r e ma i n s c o l o r l e ss . A d j u s t th e
h e a t e r s o as t o c o mp l e t e t h e d i s ti ll a t i o n in a b o u t 1 5 mi n u t e s .

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c . D e r i v a t iz a t i o n
A d d 1 ml o f 1 % ο - n i t ro b e n z a ld e h y d e - me t h a n o l s o l u ti o n t o t h e a b o v e
d i s t i ll a t e an d s h ak e th e mi x t u r e , a n d th e n le a v e it t o s t a n d f o r tw o
h o u r s at 3 0 ° C . A d d 5 0 ml o f n - h ex a n e a nd sh a k e t he mi x t u r e f o r f iv e
mi n u t e s , a n d t h en l e av e i t t o s t an d . C o l l e ct t h e n - h e xan e l a ye r a nd
f i l t e r i n a 2 0 0 - ml e g g p la n t - s h ap e d f l a s k t h r o u g h a l i q u i d p h as e
s e p a r a t i o n f i l t e r p ap e r. A d d 5 0 ml o f n -h e x an e to th e aq u e o u s la y e r
a n d r e p e at t h e a b o v e p r o c e d u r e , an d t h en c o mb i n e t h e n - h e x an e l ay e r
t o th e e g g p l a n t - s h a p ed f l a s k a bo v e . Wa s h t h e r e s id u e o n t he f i lt e r
with 10 ml of n - h e x an e and c o mb i n e the washings to the
e g g p l a n t - s h a p e d f l a s k a b o v e , a n d t h e n r e mo v e t h e n - h ex a n e a t 4 0 °C
o r l o w e r. D i s s o l v e t h e r e s i d u e in 5 ml o f a c et o n e/ n - h ex a n e ( 1 :1 9 ) .
d . C l ea n - u p
P o u r 1 0 ml o f a c e to n e/ n - h e x a n e (1 : 1 9 ) i n to a b a s i c a lu mi n a c a r t r i d g e
c o l u mn ( 1 , 7 1 0 mg ) a n d d i s c a r d t h e e ff l u e n t . P o u r t h e s o lu t io n
o b t ai n ed in 4 - c in t o t h e c o lu mn f o l l o w e d b y 1 0 ml o f a c e t o n e /
n - h e x an e (1:19) and c o l l e ct th e eluate into a rotary vacuum
e v a p o r a t o r, a n d t h e n r e mo v e t h e a c e t o n e an d n - h ex a n e a t 4 0 ° C o r
l o w e r. D i s s o l v e t h e r e s i d u e in a c e t o n e to ma k e e x a c t l y 5 ml o f
s o l u t io n , wh i c h i s u s ed a s t h e s amp l e s o l u t io n .
5 . D e t e r mi n a t io n
a . Q u a l i ta t iv e t e s t s
P e r f o r m q u a l i t at i v e t es t s u n d e r th e f o l l o w in g c o n d i t io n s . Te s t r e s u l t s
o b t ai n ed mu s t b e t h e s a me a s t h o s e o b t a i n ed f o r t h e r e f e r e n c e
s t a n d a r d u n d e r t h e p r o c e d u r e d e s cr i b e d in 4 -c .
Te s t i n g c o n d i t io n s
C o l u mn : A s i l i c a te g l as s c a p i ll a r y c o lu mn ( 0 . 2 5 mm i n i n n e r d i a me t e r,
1 0 - 3 0 m i n l e n g th ) c o a t e d w i t h 5 % p h e n y l me t h y l s i l i c o n e f o r g as
c h r o ma t o g r a p h y t o a th i c kn e s s o f 0 . 2 5 µ m
C o l u mn t e mp e r a t u r e : H o l d t h e c o l u mn t e mp e r a t u r e a t 6 0 ° C f o r t w o
mi n u t e s , f o l l o w e d b y a n in c r ea s e o f 1 0 ° C e v e r y mi n u t e u n ti l
r e a c h i n g 2 8 0 ° C , a n d h o l d f o r f i v e mi n u t e s .
I n l e t t e mp e r a t u r e : 2 8 0 ° C
D e t e c t o r : Op e r a t e at 2 8 0 ° C .
G a s f l o w r a t e : U s e h el i u m a s t h e c a r r i e r g a s . A d j u s t th e f l o w r a t e s o
t h a t di me t h yl h yd r a z in e d e r i v at iv e s f l o w o u t i n a p p r o x i ma t e l y 1 3
mi n u t e s . A d j u s t th e f l o w s o f a i r a n d h y d r o g e n to th e o p t i ma l

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c o n d i t io n s .
b . Q u a n ti t at i v e t e s t s
D e t e r mi n e t h e co n te n t o f d i me t h y l h y d r a z i n e f r o m t h e t e s t r e s u lt s
o b t ai n ed un d e r th e s ame c o n d i tio n s d e s c r i b e d in 5 - a u s i n g e i t h e r t h e
p e a k he i gh t o r p e a k a r e a me t h o d . F u r t h e r mo r e , d e t e r mi n e t h e c o n t en t
o f d a mi n o z i d e u si n g th e f o l lo w i n g f o r mu l a :
D a mi n o z i d e c o n t e n t (p p m) = 2 . 6 7 x d i me t h y l h y d r a z i n e c o n t e n t (p p m )

c . C o n f i r ma t i o n t e s t s
P e r f o r m g a s c h r o ma t o g r a p h y / ma s s s p e c t ro me t r y u n d e r t h e s am e
c o n d i t io n s d e s c r i b ed in 5 - a . Te s t r e s u l t s o b t a i n ed f o r t h e r e f e r e n c e
s t a n d a r d mu s t b e th e s a me a s t h o s e o b t a in e d u n d e r t h e p r o c e d u r e
d e s c r i b ed in 4 - c . D e t er mi n e t h e qu a n ti t y u s in g e i th e r the p ea k h ei gh t
o r p e a k a r ea me t h o d , if r e q u i r e d .
d . C a li b r a t io n cu r v e
A s s a y a s o l u t i on o f d ime t h y l h y d r a z i n e ( 1 ml ) c o mb i n e d w i t h 5 ml o f
p h o s p h a t e b u ff e r s o l u t i o n ( p H 5 ) a n d 4 0 ml o f w a t e r w i t h t h e s am e
p r o c e d u r e d e s c r i b ed in 4 - c .

( 1 4 ) N i t r o f u r a n s a n al y t i c a l me t h o d
Analyse 3 - a mi n o - 2 - o x a z o l i d in o n e , 1 - a mi n o h y d an t o in , 3 - a mi n o - 5 -
mo r p h o l i n o me t h y l - 2 - o x a z o l i d in o n e a n d s e mi c a r b a z id e .
1. Apparatus
A l i q u i d c hr o ma t o g r a p h / t an d e m m a s s s p e c t r ome t e r ( L C / M S / M S )
2 . R e ag e n t s a n d t e s t s o l u t io n s
I n a d d i t io n t o t h e r e ag e n t s a n d te s t s o lu t i o n s l i s t ed b e l o w, u s e th o s e
l i s t e d in S e c t i o n C R e a g e n ts /Te s t S o l u ti o n s , E t c . , P a r t I I F o o d
a d d it i v e s . R e a g en t s d e s i g n at e d as “s p e c i a l g r a d e ” i n th i s s e c ti o n mu s t
me e t t h e re q u i r e me n t s f o r “s p e c i a l g r a d e” s p e c i f ie d i n t h e J ap a n
I n d u s t r i a l St a n d a r d s fo r t h e r e ag e n t s .
A c e t o n it r i le : A c e t o n i tr i l e p r o d u ce d f o r l i q u id c h r o ma t o g r a p h y.
P o r o u s d i at o ma c e o u s e a r t h co l u mn ( t o h o ld 2 0 ml o f s o l u t io n ): A
p o l y e t h y l e n e c o l u mn o f 2 0 - 3 0 m m i n i n n e r d i a me t e r p a c k e d w i t h
g r a n u la r porous d ia t o ma c e o u s earth p r o d u ce d for c o lu mn
c h r o ma t o g r a p h y t h a t c a n ho ld 2 0 ml o f s o l u t io n , or a c o lu mn
e q u iv a l en t t o t he s p e cif i e d o n e i n s e p a r a t i o n c a p a b i l it y.
ο - N i t r o b e nz a l d eh yd e : o - N i t r o b e n z a l d eh y d e (s p e c i a l g r ad e ) .
Wa t e r : Wa te r p r o d u ced f o r l i q u i d c h r o ma t o g r a p h y.

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3. Reference standard
3 - a mi n o - 2 - o x a zo l id i n o n e : T h i s p r o d u ct co n ta i n s n o t l es s t h a n 9 9 % o f
3 - a mi n o - 2 - o x a zo l id i n o n e , a n d i t s d e c o mp o s i t i o n p o i n t is 6 5 - 6 7 ° C .
1 - a mi n o h yd a n to i n h yd r o c h lo r i d e: T h i s p r o d u c t co n ta i n s n o t l e s s th an
9 0 % o f 1 - ami n o h y d a n to i n h y d r o ch l o ri d e , an d i t s d e c o mp o s i t io n p o i n t
i s 2 0 1 - 20 5 °C .
3 - a mi n o - 5 - mo r p h o l i n o me t h yl - 2 - o x a z o l i d in o n e : Th i s p r o d u c t c o n t a i n s
n o t le s s t h an 9 9 % o f 3 - a mi n o - 5 - mo r p h o l i n o m e t h y l - 2 - o x a z o l i d i n o n e a n d
i t s d e c o mp o s i t i o n p o in t i s 11 5 - 1 2 0 ° C .
S e mi c a r b a z i d e h y d r o ch l o r i d e : T h i s p r o d u ct c o n ta i n s n o t l e s s t h an 9 9 %
o f s e mi c a r b a z id e h yd r o c h l o r id e , a n d i t s d e c o mp o s i t i o n p o i n t i s
1 7 5 - 17 7 ° C .
4. Procedure
a . E x t r a ct io n
We i g h 5 .0 0 g o f t h e t es t s a mp l e , p r e v i o u s l y g r o u n d , a n d h o mo g e n i z e
i t w i t h 7 0 ml o f 0 . 1 m o l / l h y d r o ch l o r i c a ci d . C e n t r i f u g e t h e mi x t u r e at
2 , 5 00 r p m f o r f i v e mi n u t e s a n d co l l e ct th e su p e r n at a n t, a n d th e n ad d
0 . 1 mo l / l h y d r o c h l o r i c a c i d t o ma k e 1 0 0 ml o f s o l u ti o n .
b . D e r i v a t iz a t i on
Tr a n s f e r 10 ml o f t h e s o l u t io n o b t a i n ed b y th e e x t r a c t io n d e s c r i b ed in
4 - a a n d ad d 0 .4 ml o f 0 . 0 5 mo l / l ο - n i tr o b e n z al d eh y d e - d i me t h y l
s u l f o x id e s o l u ti o n, a nd t h en l e a v e i t t o s t an d f o r 1 6 h o u r s a t 3 7 °C .
A d d 5 ml o f 0 . 1 mo l / l d i p o t a s s i u m h y d r o g e n o r t h o p h o s p h a t e s o l u t i o n
t o th e s o lut i o n a n d ad d ap p r o x ima t e l y 0 . 8 ml o f a 1 mo l / l s o d iu m
h yd r o x i d e s o l u t io n t o a d ju s t t h e p H t o 7 - 8 . W h e n s o me r e s i d u e
r e ma i n s i n t h e s o lu t io n , c e n t r i f u g e a t 2 , 5 0 0 r p m f o r f i v e mi n u t e s a n d
c o l l ec t th e s u p e r n a ta n t.
b . C l ea n - u p
P o u r t h e s ol u t io n ob t ain e d b y t h e d e r i v a ti z a tio n d e s c r ib ed i n 4 - b i n to
a p o r o u s d i a t o ma c e o u s e a r t h c o lu mn ( t o h o l d 20 ml o f s o l u t io n) .
L e a v e th e c o l u mn t o s t a n d f o r f iv e mi n u t e s a n d ad d 1 0 0 ml o f e t h y l
a c e t a te . C o ll e c t th e el u a t e i n to a r o t a r y v a c u u m e v a p o r a t o r a n d
r e mo v e t h e e t h y l a c et a te a t 4 0 ° C o r l o w e r. D i s s o l v e th e re s i d u e i n 1 .0
ml o f a c e t o n i t r i l e/ w a t er ( 1 : 1 ) , w h ic h i s u s ed a s t h e s a mp l e s o lu t i o n .
5 . D e t e r mi n a t io n
a . Q u a l i ta t iv e t e s t s
P e r f o r m q u a l i t at i v e t es t s u n d e r th e f o l l o w in g c o n d i t io n s . Te s t r e s u l t s

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o b t ai n ed mu s t b e t h e s a me a s t h o s e o b t a i n ed f o r t h e r e f e r e n c e
s t a n d a r d s u n d e r t h e p ro c e d u r e d es c r i b e d in 4 - b a n d 4 - c. .
Te s t i n g c o n d i t io n s
C o l u mn p a c k i n g : O c ta d e c y l s i l an e - b o n d e d s i l i c a g e l ( 2 - 5 µ m in
p a r t i c le s i ze ) .
C o l u mn : A s t a i n l e s s t ub e ( 2 .0 - 6 . 0 m m i n i n n er d i a me t e r, 1 0 0 - 2 5 0 m m
i n l en g th ) .
C o l u mn t e mp e r a t u r e : 4 0 ° C
M o b il e p h as e : U s e a mi x t u r e o f a c e to n it r i le a n d 0 .1 % a c e ti c ac i d
s o l u t io n . Cr e a t e a c on c e n t r a ti o n g r a d i en t o f 1 : 4 to 4 : 1 acetonitril e/
0.1% aceti c acid sol u tion i n 1 5 mi n u t e s . A d j u s t t h e f l o w r a t e s o
that 3 - a mi n o - 2 - o x az ol i d in o n e fl o w s out in a p p r o x i ma t e l y 12
mi n u t e s .
b . Q u a n ti t at i v e t e s t s
D e t e r mi n e t h e q u an t i ty f r o m t h e t e s t r e s u lt s o b ta i n e d u n d e r th e s ame
c o n d i t io n s d e s c r i b ed in 5 - a u si n g e i t he r t h e p e a k h e ig h t o r p e ak a re a
me t h o d .

( 1 5 ) P r o p h a m a n a l y t i c a l me t h o d
1. Apparatus
A g a s c h r o ma t o g r a p h /ma s s s p e c t r o me t e r ( G C / M S o r G C / M S / M S )
2 . R e ag e n t s a n d t e s t s o l u t io n s
I n a d d i t io n t o t h e r e ag e n t s a n d te s t s o lu t i o n s l i s t ed b e l o w, u s e th o s e
l i s t e d in S e c t i o n C R e a g e n ts /Te s t S o l u ti o n s , E t c . , P a r t I I F o o d
a d d it i v e s . R e a g en t s d e s i g n at e d as “s p e c i a l g r a d e ” i n th i s s e c ti o n mu s t
me e t t h e re q u i r e me n t s f o r “s p e c i a l g r a d e” s p e c i f ie d i n t h e J ap a n
I n d u s t r i a l St a n d a r d s fo r t h e r e ag e n t s .
A c e t o n it r i le : U s e a r o t a r y v a c u u m e v a p o r a t o r o n 3 0 0 mL o f a c e t o n i t ri l e
t o c o n c e n t r a t e t o t h e p o i n t o f d r y n e s s . D i s s o l v e t h e r e s id u e i n 5 mL o f
n - h e x an e . F o r a n a l y s i s , i n j ec t 5 μ L o f th e so l u ti o n i n to a GC -ECD.
P e a k s o t h er t h a n t h a t o f n - h e x an e i n th e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
A c e t o n e: U s e a r o t a r y v a c u u m e v ap o r at o r o n 3 0 0 mL o f a c e to n e t o
c o n c en t r a t e t o t h e p o in t o f d r y n e s s . D i s s o l v e t h e r e s i d u e i n 5 mL o f
n - h e x an e . F o r a n a l y s i s , i n j ec t 5 μ L o f th e so l u ti o n i n to a GC -ECD.
P e a k s o t h er t h a n t h a t o f n - h e x an e i n th e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
S o d i u m c h l o r i d e : S o diu m c h l o r i d e ( s p e c i a l g r a d e ) . I f t h e r e a g en t i s

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f o u n d to i nc l u de a n y s u b s t a n c e t h a t ma y i n t er f e r e w i t h t h e a n al y s i s o f
s u b s t a n ce s t o b e a n al y z e d f r o m a g r i c u l tu r a l c h e mi c a l s , w a s h w i t h a
s o l v e n t s u ch a s n - h e x a n e b e f o r e u s e .
O c t a d e c y l si l a n e - b o n d e d s il i c a g e l c a r t r i d g e co l u mn ( 1 , 0 0 0 mg ) : A
p o l y e t h y l e n e c o l u mn o f 1 2 - 1 3 m m i n i n n e r d i a me t e r p a c k e d w i t h
1,000 mg of o c ta d e c y l s i l a n e -b o n d e d si li c a gel, or a c o l u mn
e q u iv a l en t t o t he s p e cif i e d o n e i n s e p a r a t i o n c a p a b i l it y.
G r a p h i t e ca r b o n an d a mi n o p r o pyl s i l a n e - b on d e d si l i c a g e l l a mi n a t ed
c a r t r i d g e co l u mn ( 5 0 0 mg / 5 0 0 mg ) : A p o l ye t h yl e n e co lu mn o f 1 2 - 1 3
m m i n i n n e r d i a me t e r p a ck e d w it h 5 0 0 mg e a c h o f g r a p h it e c a r b o n
a n d a mi n o p r o p yl s i l a n e- b o n d ed si li c a g e l , o r a c o l u mn e q u i v a l en t to
t h e s p ec i f i ed o n e i n s ep a r a t i o n c ap a b il i t y.
D i a t o ma c e o u s e a r t h : D i a t o ma c e o u s e a r t h f o r c h e mi c a l a n a l y s i s .
To l ue n e : U s e a r o t a r y v a cu u m e v a p o r at o r o n 3 0 0 mL o f t o l u en e to
c o n c en t r a t e t o t h e p o in t o f d r y n e s s . D i s s o l v e t h e r e s i d u e i n 5 mL o f
n - h e x an e . F o r a n a l y s i s , i n j ec t 5 μ L o f th e so l u ti o n i n to a GC -ECD.
P e a k s o t h er t h a n t h a t o f n - h e x an e i n th e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
n - H e x a n e : U s e a r o t a r y v a c u u m e v a p o r at o r on 3 00 mL o f n - h e x a n e t o
e v a p o r a t e u n t i l 5 mL i s l e f t . F o r a n a l y s i s , i n j e c t 5 μ L o f t h e s o lu t io n
i n t o a G C - E C D . P e a k s o t h e r th an t h at o f n - h e x a n e i n t h e r e s u l t in g
c h r o ma t o g r a m s h o u l d b e a s h ig h a s o r lo w e r t h an t h e pe a k s o f γ - B H C
a t 2 x 10 - 1 1 g .
Wa t e r : D i s ti l l e d w a t e r. I f t h e d i s ti l l e d w a t e r i s f o u n d to i n c l u d e an y
s u b s t a n ce th a t ma y i n t e r f e r e w i t h a n a l y s i s o f t h e t a rg et co mp o s i t i o n a l
s u b s t a n ce s f r o m a g r i c u l t u r a l ch emi c a l s , w a s h w i t h a s o l v en t s u c h a s
n-hexane before use.
S o d i u m s u l f a t e ( a n h y d r o u s ) : S o d iu m s u l f a t e ( a n h y d r o u s) ( s p e c i a l g ra d e ) .
I f t h e r e a g e n t i s f o u n d t o in c lu d e a n y s u b st a n c e t h at ma y i n t e r f e r e
w i t h a n al y s i s o f t h e t a rg e t co mp o s i t i o n a l s u b st a n c e s f r o m a g r i c u l t u r al
c h e mi c a l s , w a s h w i t h a s o l v en t su c h a s n - h e x a n e b e f o r e u s e .
0 . 5 mo l / l p h o s p h a t e b u ff e r s o l ut i o n ( p H 7. 0 ) : D i s s o l v e 1 3 6. 8 5 g o f
secondary sodium phosphate (dodecahydrate) and 17.92 g of sodium
p h o s p h a t e mo n o b a s i c ( d i h yd r a te ) i n w a te r t o ma k e a 1 ,0 0 0- ml
s o l u t io n .
3. Reference standard
P r o p h a m: T h i s p r o d u ct c on t a in s no t le s s th a n 9 9 % o f p ro p h a m, a n d i t s

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me l t i n g p o in t i s 8 7 ° C .
4. Procedure
a . E x t r a ct io n
i . C e r e a l g r a i n s , l eg u me s / p u l s e s an d s e ed s
We i g h 1 0 .0 g o f te s t s a mp l e s , p r e v i o u s ly g r o u n d so a s t o p as s
t h r o u g h a s t a n d a r d me s h s i e v e ( 42 0 µ m) . A d d 2 0 ml o f w a t e r t o t he
o b t ai n ed s amp l e a n d l ea v e it t o s ta n d f o r 15 mi n u t e s .
T h e n , ad d 5 0 ml o f a c e t on i t r i l e a n d ho mo g e n i z e th e mi x t u r e f o r
t h r e e mi n u t e s . F i l t e r th e h o mo g e n i z ed s a mp l e b y s u c t io n t h r o u g h a
f i l t e r p a p e r c o v e r ed w i t h a 1 - c m t h i ck l a y e r o f d i a to ma c e o u s ea r t h .
C o l l ec t t h e r e s i d u e o n t h e s u r f a ce o f t h e f i lt e r p a p e r an d a d d 2 0 ml
o f a c et o ni tr i l e , an d t h e n h o mo g e n i z e t h e mi x t u r e f o r t h r e e mi n u t e s.
R e p e at th e a b o v e p r o ce d u r e a n d c o mb i n e t h e f i l t r a te , an d t h en a d d
a c e t on i t r i le t o ma k e a 1 0 0 - ml s o l u t i o n .
Tr a n s f e r 2 0 ml o f t h e a b o v e s o l u ti o n to a 1 0 0 - ml s e p a r a t i n g f u n n el
a n d a d d 1 0 g o f so d iu m c h l o r i d e a n d 2 0 ml o f 0 . 5 mo l / l p h o s p h a t e
b u ff e r s o l u t i o n ( p H 7 . 0 ) . S h a k e t h e mi x t u r e v i g o r o u s l y f o r 1 0
mi n u t e s u s in g a s h a k er a n d l e a v e i t to s t an d , a n d t h e n c o l l ec t th e
a c e t o n i t r i le l a y e r.
P o u r 1 0 ml o f ac e to n itr i l e in t o an o ct a d ec yl s i l a n e - bo nde d s il i c a gel
c a r t r i d g e co l u mn ( 1 , 0 0 0 mg ) a n d d i s c a r d t h e e ff l u e n t . P o u r t h e
a b o v e a c eto n i t r il e l a ye r i n t o t h e c o l u mn a n d c o l le c t th e e l u at e in a
5 0 - ml c o n ic a l f l a s k Po u r 2 ml o f a c e t o n i t r il e i n t o th e c o l u mn a n d
c o mb i n e t h e e l u at e t o t h e f l a s k a b o v e . A d d a n a d eq u at e a mo u n t o f
s o d i u m s u l f a t e ( a n h y d r o u s ) t o t h e f l a s k a n d l e a v e it t o s t a n d f o r 1 5
mi n u t e s w i t h o c c a s io n al s h a k in g . F i l t e r t h e co n t en t o f t h e f l a s k i n t o
a r o t a r y v a c u u m e v a p o r a to r. Wa s h t h e f l a s k w i t h 1 0 ml o f
a c e t o n i t r i le a n d w a s h t w i c e th e r e s i d u e o n th e s u r f a c e o f t h e f i lt er
p a p e r w i t h . t h e w a s h in g s . A d d t h e w a s h i n g s in t o th e r o ta r y v a c u u m
e v a p o r a t o r a n d r e mo v e t h e a ce t o n i t r i l e a t 4 0 ° C o r lo w er. D i s s o l v e
t h e r e s i d u e i n 2 ml o f t o l u e n e /a c et o n it r i l ( 1 :3 ) .
i i . F r u i t , v eg e t ab l e s , tea s a n d h o p s
I n c a s e o f f r u i t a n d v e g e t ab l e s , w e i g h a c cu r a t e l y 1 k g o f t h e t e st
s a mp l e a n d a d d a n app r o p r i a t e amo u n t o f w a t e r t o t he s a mp l e , i f
r e q u i r e d . H o mo g e n i ze t h e mi x t u r e a n d me a s u r e o u t a s a mp l e
e q u iv a l en t t o 2 0 . 0 g .
I n c a s e o f t e a s a n d ho p s , w e i g h 5 .0 0 g o f t h e t e s t s a mp l e , p r e v i o u s ly

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g r o u n d , a n d a d d 2 0 ml o f w a t e r, a n d t he n l e a v e i t t o s t a n d f o r 15
mi n u t e s .
T h e n , ad d 5 0 ml o f a c e t on i t r i l e a n d ho mo g e n i z e th e mi x t u r e f o r
t h r e e mi n u t e s . F i l t e r th e h o mo g e n i z ed s a mp l e b y s u c t io n t h r o u g h a
f i l t e r p a p e r c o v e r ed w i t h a 1 - c m t h i ck l a y e r o f d i a to ma c e o u s ea r t h .
C o l l ec t t h e r e s i d u e o n t h e s u r f a ce o f t h e f i lt e r p a p e r an d a d d 2 0 ml
o f a c et o ni tr i l e , an d t h e n h o mo g e n i z e t h e mi x t u r e f o r t h r e e mi n u t e s.
R e p e at th e a b o v e p r o ce d u r e a n d c o mb i n e t h e f i l t r a te , an d t h en a d d
a c e t on i t r i le t o ma k e a 1 0 0 - ml s o l u t i o n .
Tr a n s f e r 2 0 ml o f t h e a b o v e s o l u ti o n to a 1 0 0 - ml s e p a r a t i n g f u n n el
a n d a d d 1 0 g o f so d iu m c h l o r i d e a n d 2 0 ml o f 0 . 5 mo l / l p h o s p h a t e
b u ff e r s o l u t i o n ( p H 7 . 0 ) . S h a k e t h e mi x t u r e v i g o r o u s l y f o r 1 0
mi n u t e s u s in g a s h a k er a n d l e a v e i t to s t an d , a n d t h e n c o l l ec t th e
a c e t o n i t r i le l a y e r i n a 5 0 - ml c o n ic a l f l a s k . A d d a n ad eq u a te a mo u n t
o f s o d iu m s u l f a t e ( an h y d r o u s ) t o t h e f l a s k an d l e a v e it t o s t an d f o r
1 5 mi n u t e s w i t h o c c a si o n al s h ak in g . F i lt e r th e co n te n t o f t h e f l a sk
i n t o a r o t ar y v a c u u m e v a p o r a t o r. Wa s h t h e f l a s k w i th 1 0 ml o f
a c e t o n i t r i le a n d w a s h t w i c e th e r e s i d u e o n th e s u r f a c e o f t h e f i lt er
p a p e r w i t h t h e w a s h i n g s . A d d t h e w a s h i n g s i n t o th e r o t a r y v a c u u m
e v a p o r a t o r a n d r e mo v e t h e a ce t o n i t r i l e a t 4 0 ° C o r lo w er. D i s s o l v e
t h e r e s i d u e i n 2 ml o f t o l u e n e /a c et o n it r i l ( 1 :3 ) .
i i i . F o o d s ex c e p t t h o s e l i s t e d i n i a n d ii a b o v e
O b t a i n t h e e x t r a c t s acc o r d i n g to t h e me t h o d s d e s c r i b e d i n i o r i i .
b . C l ea n - u p
P o u r 1 0 ml o f t o l u e n e/ a c e to n it r i le ( 1 : 3 ) i n to a g r a p h i te c a r b o n an d
a mi n o p r o p y l s i l a n e - b o n d e d s i li c a g e l l a mi n a t e d c a r t r i d g e c o l u mn ( 5 0 0
mg / 5 0 0 mg ) a n d d i s c ar d t h e e ff l u e n t . P o u r t h e s o l u ti o n o b t a in e d b y
t h e ex t r a c tio n d e s c r i b ed i n 4 - a i n to t he co l umn f o l l o w e d b y 20 ml o f
t o l u e n e /a c et o n it r i l e ( 1 : 3 ) . C o l le ct t h e e lu a te i n t o a r o t a r y v a c u u m
e v a p o r a t o r a n d r e mo v e t h e to l u en e a n d ac e to n i t r i l e a t 4 0 ° C o r l o we r.
D i s s o l v e t h e r e s i d u e in a ce t o n e /n - h e x a n e ( 1 :1 ) t o ma k e e x a c tl y 2 m l
( 1 ml f o r c e r e a l g r a in s , l eg u me s / p u l s e s , se e d s , te a s a n d h o p s ) o f
s o l u t io n , wh i c h i s u s ed a s t h e s amp l e s o l u t io n .
5 . D e t e r mi n a t io n
a . Q u a l i ta t iv e t e s t s
P e r f o r m q u a l i t at i v e t es t s u n d e r th e f o l l o w in g c o n d i t io n s . Te s t r e s u l t s
o b t ai n ed mu s t b e t h e s a me a s t h o s e o b t a i n ed f o r t h e r e f e r e n c e

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standard.
Te s t i n g c o n d i t io n s
C o l u mn : A s i l i c a te g l as s c a p i ll a r y c o lu mn ( 0 . 2 5 mm i n i n n e r d i a me t e r,
3 0 m i n len g t h ) c o a te d w i th 5 % p h e n y l me t h y l s i l ic o n e f o r g a s
c h r o ma t o g r a p h y t o a th i c kn e s s o f 0 . 2 5 µ m
C o l u mn t e mp e r a t u r e : H o l d t h e c o l u mn t e mp e r a t u r e a t 5 0 ° C f o r o n e
mi n u t e , f o ll o w e d b y an i n c r e a s e o f 2 5 ° C ev er y mi n u t e u n t i l r e ac h in g
1 2 5 ° C , a f t er w h i c h in cr e a s e t h e te mp e r a t u r e b y 1 0 ° C ev e r y mi n u t e
u n t il r e a c h in g 3 0 0 ° C , a n d h o ld f o r 6 . 5 mi n u t e s .
I n l e t t e mp e r a t u r e : 2 5 0 ° C
D e t e c t o r : Op e r a t e at 2 8 0 ° C
G a s f l o w r a t e : U s e h el i u m a s t h e c a r r i e r g a s . A d j u s t t h e f l o w r a t e t o
t h e o p t i ma l c o n d i t io n s.
I n j e c t io n me t h o d : S p lit l e s s i n je c ti o n me t h o d .
M e a s u r e d ma s s s i z e ( m/ z ) : 9 3 , 13 7 an d 17 9
b . Q u a n ti t at i v e t e s t s
D e t e r mi n e t h e q u an t i ty f r o m t h e t e s t r e s u lt s o b ta i n e d u n d e r th e s ame
c o n d i t io n s d e s c r i b ed in 5 - a u si n g e i t he r t h e p e a k h e ig h t o r p e ak a re a
me t h o d .

( 1 6 ) M a l a ch i t e g r e e n a n a l y t i ca l me t h o d
A n a l y z e ma l a c h it e g r ee n a n d l eu co ma l a c h i t e g r e e n .
1. Apparatus
A l i q u i d c h r o ma t o g r a p h / ma s s s p e c t r o me t e r ( L C / M S o r L C / M S / M S )
2 . R e ag e n t s a n d t e s t s o l u t io n s
I n a d d i t io n t o t h e r e ag e n t s a n d te s t s o lu t i o n s l i s t ed b e l o w, u s e th o s e
l i s t e d in S e c t i o n C R e a g e nts /Te s t To l u t i o n s , E t c. , P a r t I I F o o d
A d d it i v e s . R e a g en t s d e s i g n at e d as “s p e c i a l g r a d e ” i n th i s s e ct i o n mu s t
me e t t h e re q u i r e me n t s f o r “s p e c i a l g r a d e” s p e c i f ie d i n t h e J ap a n
I n d u s t r i a l S t a n d a r d s fo r t h e r e ag e n t s .
A c e t o n it r i le : A c e t o n i tr i l e p r o d u ce d f o r l i q u id c h r o ma t o g r a p h y.
A m mo n i u m f o r ma t e : A m mo n i u m f o r ma t e ( s p e c i a l g r a d e ) .
St r o n g l y ac i d i c c a ti on e x c h an g e r c a r t r i d g e c o lu mn ( 5 0 0 mg ) : A
p o l y e t h y l en e c o l u mn o f 8 - 9 mm i n i nn e r d i ame t e r p a c k e d w i t h 5 0 0 mg
o f b e n z en es u l f o n y l p r o p yl s i l a ni ze d s i l i c a g el , o r a c o l umn e q u i v a le n t
t o t h e s p e cif i e d o n e i n s e p a r a t i o n c a p a b i l it y.
C i t r i c a ci d – p h o s p h at e b u ff e r ( p H 3 . 0 )
S o l u t io n 1: D i s s o l v e 6 3 . 0 g o f c i t r ic a c i d in w a t e r t o ma k e 1 , 0 0 0 mL o f

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s o l u t io n .
S o l u t io n 2 : D i s s o l v e 2 1 5 g o f d i so d i u m p h o sp h a te in w a t e r t o ma k e
1 , 0 00 mL o f s o l u t io n .
M i x S o lu t io n 1 a n d S o l u ti o n 2 an d ad j u s t th e p H t o 3 .0 .
D i c h l o r o me t h a n e : D i c h l o r o me t h a n e ( s p e c i a l g r a d e ) .
Wa t e r : Wa te r p r o d u ced f o r l i q u i d c h r o ma t o g r a p h y.
S o d i u m s u l f a t e ( a n h y d r o u s ) : S o d iu m s u l f a t e ( a n h y d r o u s) ( s p e c i a l g ra d e ) .
W h e n i t co n t ai n s s u b s t a n ce s t ha t ma y i n t e r f e r e w i t h t h e a n a l y s i s ,
w a s h i t w i th e t h y l ac et a t e b e f o r e u s i n g .
3. Reference standard
M a l a ch i t e G r e e n O x a l a t e : T h i s pr o d u c t co n ta i n s n o t les s t h a n 9 9% o f
ma l a c h i t e g r e e n o x a l a te , a n d i t s d e c o mp o s i t i o n p o i n t i s 1 6 4 º C .
L e u c o ma l a c h i t e G r e e n : T h i s p ro d u ct co n ta i n s n o t les s t h a n 9 9 % o f
l e u co ma l a c h i t e g r e e n , a n d it s d e co mp o s i t i o n p o in t i s 1 0 3 º C .
4. Procedure
a . E x t r a ct io n
We i g h 5 .0 0 g o f t h e t es t s a mp l e , p r e v i o u s l y g r o u n d , a n d h o mo g e n i z e
i t w i t h 1 0 mL o f c i t r ic a c id - p h o s p h ate bu ff e r ( p H 3 . 0 ) . A d d 1 5 mL o f
a c e t o n i t r i le a n d s h a k e t h e mi x t u r e v i g o r o u s l y f o r f i v e mi n u t e s u s in g a
s h a k e r. C en t r i f u g e th e mi x t u r e a t 3 , 00 0 rp m f o r f i v e mi n u t e s a n d
c o l l ec t th e a c e t o n i t r i le- w a t e r l a y e r. A d d 1 5 mL o f a ce t o n i t r i l e to th e
r e s i d u e a n d r e p e a t th e a b o v e p ro c e d u r e , a n d t h e n c o mb i n e t h e
a c e t o n i t r i le l a y e r w i t h t h e a ce t o n it r i l e - w a t e r l a y e r.
A d d 5 mL o f n - h e x a n e t o t h e comb i n e d l a y e r s a n d s h a k e t h e mi x t u r e
v i g o r o u s l y f o r f i v e mi n u t e s u s i n g a s h a k e r an d le a v e i t t o s t an d , an d
t h e n c o ll e ct t h e a c e to n i t r i l e - w a t e r l a y e r. A d d 5 mL o f n - h e x an e to th e
c o l l ec t ed la y e r a n d r e p e a t t h e a b o v e p r o c e d u r e t o c o l le c t th e
a c e t o n i t r i le- w a t e r l a y e r.
A d d 5 0 mL o f 2 0 % s o d i u m c h l o r i d e an d 1 0 mL o f d i c hlo r o me t h a n e t o
t h e c o ll e c ted l a ye r an d s h a k e it v ig o r o u s l y f o r f i v e mi n u t e s u s in g t h e
s h a k e r a n d l e a v e i t t o s t an d , a n d th e n c o l l ec t th e a c e t o n i t r il e -
d i c h l o r o me t h a n e l a y e r.
A d d a n a d eq u a te a mo u n t o f s o d i um s u l f a t e ( a n h yd r o u s ) a n d l e av e i t t o
s t a n d f o r 1 5 mi n u t e s w i t h o c c as i o n a l s h ak i n g , a n d th e n f il t e r t h e
mi x t u r e .
b . C l ea n - u p
P o u r 5 mL o f a c e t o n it r i l e i nt o a s t r o n g l y ac i d i c c a t ion e x ch a ng er

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c a r t r i d g e co l u mn ( 5 0 0 mg ) a n d d is c a r d t h e e ff l u e n t . P o u r t h e s o lu t io n
o b t ai n ed b y t h e e x t r a cti o n d e s c r i b e d in 4 – a in t o t h e c o lu mn f o l l o w e d
b y 5 mL o f a c e t o n i t ri l e a n d d i s c a rd t h e e ff l u e n t . P o u r 1 0 mL o f
a c e t o n i t r i le/ a mmo n i a w a t e r ( 9 : 1 ) a n d c o ll e ct t h e e lu a t e i n t o a r o t ar y
v a c u u m e v a p o r a to r, a n d t h e n r emo v e t h e ac e t o n i t r i l e a n d a mmo n i a
w a t e r a t 4 0 º C o r l o w e r. D i s s o l v e t h e r e s i d u e in 1 . 0 mL o f a c e t o n i t r i le ,
w h i c h i s u s e d a s th e s amp l e s o l u t io n .
5 . D e t e r mi n a t io n
a . Q u a l i ta t iv e t e s t s
P e r f o r m q u a l i t at i v e t es t s u n d e r th e f o l l o w in g c o n d i t io n s . Te s t r e s u l t s
o b t ai n ed mu s t b e t h e s a me a s t h o s e o b t a i n ed f o r t h e r e f e r e n c e
standards.
Te s t i n g c o n d i t io n s
C o l u mn p a c k in g : O c t a d ec y l s i la n e - b o n d ed s i l i ca g el (2-5µm in
p a r t i c le s i ze ) .
C o l u mn : A s t a i n l e s s t ub e ( 2 .0 - 6 . 0 m m i n i n n er d i a me t e r, 1 0 0 - 2 5 0 m m
i n l en g th ) .
C o l u mn t e mp e r a t u r e : 4 0 ° C
M o b il e phase: Use a mi x t u r e of acetonitrile an d 0.01 mo l / L
a mmo n i u m f o r ma t e . C r e a t e a co n c en t r a t io n g r a d i e n t o f 1 : 9 t o 1 : 0
a c e t o n i t r i le/ 0 . 0 1 mo l / L a m mo n i u m f o r ma t e i n 2 0 mi n u t e s , an d
ma i n t a i n a r a t i o o f 1 :0 f o r 1 0 mi n u t e s . A d j u st t h e f l o w r a t e s o t h a t
ma l a c h i t e g r e e n f l o w s o u t in ap p ro x i ma t e l y 1 0 mi n u t e s .
b . Q u a n ti t at i v e t e s t s
D e t e r mi n e t h e q u an t i ty f r o m t h e t e s t r e s u lt s o b ta i n e d u n d e r th e s ame
c o n d i t io n s d e s c r i b ed in 5 - a u si n g e i t he r t h e p e a k h e ig h t o r p e ak a re a
me t h o d .

6 . R e g a r d l es s o f t h e p r o v i s io n s i n 5 , i n g r ed i en t s o f a g r i cu l t u r a l c h emi c a l s
a n d o th e r ch e mi c a l s u b s t a n ce s l i s t e d i n th e f i r s t c o l u mn o f t h e t a b le i n ( 1 )
mu s t n o t b e c o n t a in e d in f o o d s a t le v e l s e x c eed i n g th e limi t s s t i p u la t e d i n
t h e t h i r d c o l u mn o f t h e s a me t a b l e a c c o r d i n g t o th e f oo d c a t e g o r i e s s h o w n
i n t h e s e co n d c o lu mn o f t he s a me t a b l e. I n a s s o ci a t i on w i th t h i s
r e g u l a ti o n, f o o d s l i s t ed i n th e “f o o d s ” c o l u mn i n t h e ta b l e i n ( 2 ) b e lo w
s h a l l b e t e st e d u s i n g th e p a r t li s te d in t h e “s a mp l e s ” c o l u mn i n th e t ab l e
a s a s a mp l e . I n ad d i t i o n , in th e s u b s ta n c e s u s e d a s i n g r e d ie n t s o f
a g r i c u lt u r al ch e mi c a l s a n d o t h e r c h e mi c a l s u b s t a n c e s li s t e d i n th e f i r s t

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c o l u mn o f t h e t a b le i n ( 1 ) b e l o w, w h i c h a r e s t i p u la t e d t o b e “ N o t
d e t e ct e d ” in t h e th i r d c o l u mn o f t h e s a me t a b l e in th e fo o d s l i s t ed i n t h e
s e c o n d co lu mn o f t he s a me t a b l e , n o in g r e d ie n t o f ag r i cu l t u r a l ch emi c a l s
o r o t h e r ch e mi c a l s u b s t a n ce s s h a l l b e d ete c t e d w h en t h e s e f o o d s a r e
t e s t e d u s i n g t h e t e s t me t h o d s s t ip u l at e d i n (3 ) t o ( 1 0 ) be l o w.

( 1 ) T h e ma x i mu m r e s i d u e l i mi t s o f s u b s t a n c e s u s e d a s i n g r e d ien t s o f
a g r i c u lt u r al c h e mi c a l s i n f o o d s ( M R L s L i s t )

( 2 ) S a mp l e s

Food S a mp l e ma t e r i a l

Barley and buckwheat T h r e s h e d se e d s

W h e a t an d r y e H u s k e d s e ed s

R i c e ( b r o wn r i c e ) H u s k e d s e ed s

C o r n ( ma i z e ) S e e d s w i t h t h e h u s k , th e s i lk a n d
t h e co r e s r e mo v e d
O t h e r c e r e al g r a i n s T h r e s h e d se e d s

P e a s , b e a n s ( d r y) * , b r o a d b ea n s an d S e e d s w i t h o u t th e p o d s
soybeans (dry)
Peanuts, dry Wi t h t he s he l l s r e mo v e d

O t h e r l e g u me s / p u l s e s
A p r i c o t , mu me p l u m, c h e r r y,
J a p a n e s e p lu m ( i n c l u d in g p r u n e )
a n d n e c ta r i n e
Peach
O r a n g e ( i n c l u d i n g n av e l o r a n g e ) ,
g r a p e f r u i t , c i t r u s na t su d a id a i
( w h o l e ) , l ime a n d l e mo n
C i t r u s n a t su d a id a i ( p u l p ) a n d u n sh u
o r a n g e ( p u lp )
C i t r u s n a t su d a id a i , p ee l s
S e e d s w i t h o u t th e p o d s
Wi t h t h e p ed u n cl e s a n d t h e s e e d s
r e mo v e d
Wi t h t h e s k i n s a n d t h e s e e d s
r e mo v e d
Whole fruit
Wi t h t h e p ee l s r e mo v e d
Wi t h t h e c al y x e s r e mo v e d

Other citrus fruits Whole fruit

P e a r, J a p a n e s e p e a r, q u i n ce a n d Wi t h t h e b l o s s o m s c a r s , t h e c o r e s
a p p le a n d th e p e d u n c le s r e mo v e d
L o q u at Wi t h t he p ed u n cl e s , the s k i n s an d
t h e s e ed s r e mo v e d
Av o c a do a nd ma n g o Wi t h t h e s ee d s r e mo v e d

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Kiwifruit Wi t h t h e s k i n s r e mo v e d

Guava Wi t h t h e c al y x e s r e mo v e d

Date Wi t h t h e c al y x e s a n d th e s e e d s
r e mo v e d
Pineapple Wi t h t h e to p s r e mo v e d

P a s s i o n f r u i t a n d p ap ay a Whole fruit

B a n a na Wi t h t h e p ed i c e l s r e mo v e d

St r a w b e r r y, c r a n b e r r y, h u c k l e b e r r y, Wi t h t h e c al y x e s r e mo v e d
b l a ck b e r r y a n d b l u eb e rr y
Raspberry Whole fruit

Other berries Wi t h t h e c al y x e s r e mo v e d

J a p a n e s e p e r s i m mo n Wi t h t he c al yx e s a n d th e s e e d s
r e mo v e d
Wa t e r me l o n , m a k u w a u r i me l o n an d Wi t h t h e r in d s r e mo v e d
me l o n s
Grape Wi t h t he p ed u n cl e s r emo v e d

Other fruits E d i b le p o r t i o n s

Tu r n i p ( r o o t s ) a n d J a p a n e s e r a d i s h
( r o o t s , i n clu d i ng r a di s h )
Tu r n i p ( l e a v e s ) , w a t e r c r e s s , k a l e ,
J a p a n e s e r ad i s h ( l e av es , i n c lu d in g
radish) and brussels sprouts
C a u l i f lo w e r a n d b r o c co l i
Wi t h th e d ir t l i g h t l y r in s e d o ff w i t h
water
Wi t h t h e d ec a y e d l e av es r e mo v e d
Wi t h t h e l ea v e s r e mo v e d

C a b b ag e an d C h in e s e ca b b ag e
Kyona and komatsuna (Japanese
mu s t a r d s p i n a c h )
Horseradish
Qing-geng-cai and other
c r u c i f e r o u s v e g et a bl e s
S w e e t p o t ato , k on j ac , ta r o , p o t a t o ,
y a m a n d o t h e r p o ta t o es
A s a mp l e c o n s i s t in g o f o n e p o r t io n
f r o m e a c h o f f o u r h e ad s , w i t h eac h
h e a d e q ua l ly c u t i nt o fo u r p o r ti o ns ,
w i t h o u t t h e d e c a y e d o u t e r l e av e s
a n d th e co r e s .
Wi t h t h e r o o t s a n d t h e d e c a y e d
l e a v e s r e mo v e d
R o o t s w i th t h e d i r t l ig h t l y r i n s e d
o ff w i t h w a t e r
E d i b le p o r t i o n s
Wi t h th e d ir t l i g h t l y r in s e d o ff w i t h
water

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P u mp k i n ( i n c l u d i n g s q u a s h ) ,
c u c u mb e r ( i n c lu d in g g h e r k i n ) an d
o r i e n t al p i ck l i n g me l o n ( v e g et a b le )
O t h e r c u c u r b i t a c eo u s v e g et a b le s
A r t i c h o k e , e n d iv e an d c h i co r y
B u r d o ck a nd s a l s i f y
S h u n g i ku
L e t t u ce ( i n c l u d i n g c o s l e t tu c e an d
l e a f l e tt u c e)
O t h e r c o mp o s i t e v e g eta b l e s
S h i it a k e mu s h r o o m, b u t t o n
mu s h r o o m a n d o t h e r mu s h r o o ms
C e l e r y, p a rs l e y a n d m it s u b a
Carrot and parsnip
O t h e r u mb e l l i f e r o u s v eg e t ab l e s
To ma t o , e g g p l an t an d p i mi e n t o
(sweet pepper)
O t h e r s o l a n c e o u s v e g eta b l e s
Asparagus
O n i o n , g a r li c , w e l s h ( in c l ud i n g
l e e k ) an d mu l t i p l y i n g o n io n
N i r a a n d o th e r l i l ia c eo u s
v e g et a b le s
G r e e n s o y b e a n s , k i d n ey b e a n s ( w i t h
p o d s , i mma t u r e ) a n d p e a s ( w i t h
p o d s , i mma t u r e )
Okra
Sugarcane
Ginger
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Wi t h t h e v in e s r e mo v e d
E d i b le p o r t i o n s
Wi t h t h e d ec a y e d l e av es r e mo v e d
A s a mp l e t h i n l y s l i ced t h en g r o u n d
w i t h a me a t g r i n d e r, t h e l e a v e s
h a v in g b e en r e mo v e d a n d th e d i r t
h a v in g b e en l i g h t l y r in s e d o ff w it h
water
Wi t h t h e r o o t s a n d t h e d e c a y e d
l e a v e s r e mo v e d
Wi t h t h e d ec a y e d o u t e r l e a v e s a n d
t h e co r e s r e mo v e d
E d i b le p o r t i o n s
E d i b le p o r t i o n s
Wi t h t h e r o o t s a n d t h e d e c a y e d
l e a v e s r e mo v e d
Wi t h t h e d ir t l i g h t l y r i n s e d o ff
E d i b le p o r t i o n s
Wi t h t h e c al y x e s r e mo v e d
E d i b le p o r t i o n s
St e ms
Wi t h t h e o u t e r s k i n s an d t h e r o o t
h a i r r e mo v e d
E d i b le p o r t i o n s
Wi t h t h e p ed i c e l s r e mo v e d
Wi t h t h e c al y x e s r e mo v e d
Wi t h t he hus k s r e mo v e d
Wi t h t h e l ea v e s r e mo v e d , an d w i th
t h e d i r t l ig h t l y r i n s e d o ff w i t h
water
Provisional Translation
from the Japanese Original

Sugar beet
Spinach
B a mb o o s h o o t s an d o th e r
v e g et a b le s
S e s a me s e e d s , r a p e s e e d s , s u n f l o w e r
s e e d s , s a ff l o w e r s e e d s , c o t t o n s e e d
a n d o t h e r oi l s e e d s
A l mo n d , g in k g o n u t , ch e s t n u t ,
w a l n u t , p e ca n a n d o t h er n u t s
C a c a o b e an s a n d co ff e e b e an s
Wi t h th e d ir t l i g h t l y r in s e d o ff w i t h
water
Wi t h r e d r o o t s l e f t o n , a n d w i th t h e
r o o t h ai r an d th e d e c ay e d l e a v e s
r e mo v e d
E d i b le p o r t i o n s
Seeds only
Wi t h t h e h u s k s r e mo v e d
B e a n s w i t h o u t th e p o d s

Te a Te a l ea v e s

Hop Dried flowers

O t h e r s p i c es a n d o t h e r h e r b s E d i b le p o r t i o n s

( 3 ) 2 , 4 , 5 - T a n a l yt i ca l me t h o d
Perform according to 5 (3).

( 4 ) A mi t r o l a n a l yt i ca l me t h o d
Perform according to 5 (5).

( 5 ) A n a l yt i c a l me t h o d f o r a l d r i n , e n d r i n a n d d i e ld r i n
1. Apparatus
A g a s c h r oma t o g r a p h w i t h an e lec t r o n c a p tu r e d e t ec t o r ( G C - E C D ) a n d
a g a s ch r o ma t o g r a p h /ma s s s p e c t r o me t e r ( G C / M S o r G C / M S / M S )
2 . R e ag e n t s a n d t e s t s o l u t io n s
I n a d d i t io n t o t h e r e ag e n t s a n d te s t s o lu t i o n s l i s t ed b e l o w, u s e th o s e
l i s t e d in S e c t i o n C R e a g e n ts /Te s t S o l u ti o n s , E t c . , P a r t I I F o o d
a d d it i v e s . R e a g en t s d e s i g n at e d as “s p e c i a l g r a d e ” i n th i s s e c ti o n mu s t
me e t t h e re q u i r e me n t s f o r “s p e c i a l g r a d e” s p e c i f ie d i n t h e J ap a n
I n d u s t r i a l St a n d a r d s fo r t h e r e ag e n t s .
A c e t o n it r i le : U s e a r o ta r y v a c u u m e v a p o r a t o r o n 3 0 0 mL o f a c e to n i t ri l e
t o c o n c e n t r a t e t o t h e p o i n t o f d r y n e s s . D i s s o l v e t h e r e s id u e i n 5 mL o f
n - h e x an e . F o r a n a l y s i s , i n j ec t 5 μ L o f th e so l u ti o n i n to a GC -ECD.
P e a k s o t h er t h a n t h a t o f n - h e x an e i n th e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
A c e t o n e: U s e a r o t a r y v a cu u m e v a p o r at o r o n 3 0 0 mL o f a c et o n e to

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c o n c en t r a t e t o t h e p o in t o f d r y n e s s . D i s s o l v e t h e r e s i d u e i n 5 mL o f
n - h e x an e . F o r a n a l y s i s , i n j ec t 5 μ L o f th e so l u ti o n i n to a GC -ECD.
P e a k s o t h er t h a n t h a t o f n - h e x an e i n th e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
E t h e r : U s e a r o t a r y v a c u u m e v a p o r a t o r o n 30 0 mL o f d i e t h yl e th e r t o
c o n c en t r a t e t o t h e p o in t o f d r y n e s s . D i s s o l v e t h e r e s i d u e i n 5 mL o f
n - h e x an e . F o r a n a l y s i s , i n j ec t 5 μ L o f th e so l u ti o n i n to a GC -ECD.
P e a k s o t h er t h a n t h a t o f n - h e x an e i n th e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
S o d i u m c h l o r i d e : S o d iu m c h l o r i de ( s p e c i a l g r a d e ) . I f t h e r e a g e n t i s
f o u n d to i nc l u de a n y s u b s t a n c e t h a t ma y i n t er f e r e w i t h t h e a n al y s i s o f
s u b s t a n ce s t o b e a n al y z e d f r o m a g r i c u l tu r a l c h e mi c a l s , w a s h w i t h a
s o l v e n t s u ch a s n - h e x a n e b e f o r e u s e .
S y n t h e t i c ma g n e s i u m s i l i c a t e (F l o r i s i l ) f o r c o l u mn c h r o ma t o g r a p h y :
H e a t f l o r i s il ( 1 5 0 -2 5 0 μ m i n p a r t ic l e s i ze ) a t 1 3 0 ° C f o r 1 2 h o u r s o r
l o n g e r. C o o l d o w n t o ro o m t e mp e r a t u r e in a d e s i c c a to r.
D i a t o ma c e o u s e a r t h : D i a t o ma c e o u s e a r t h f o r c h e mi c a l a n a l y s i s .
n - H e x a n e : U s e a r o t a r y v a c u u m e v a p o r at o r on 3 00 mL o f n - h e x a n e to
e v a p o r a t e u n t i l 5 mL i s l e f t . F o r a n a l y s i s , i n j e c t 5 μ L o f t h e s o lu t io n
i n t o a G C - E C D . P e a k s o t h e r th an t h at o f n- h e x a n e i n t h e r e s u l t in g
c h r o ma t o g r a m s h o u l d b e a s h ig h a s o r lo w e r t h an t h e pe a k s o f γ - B H C
a t 2 x 10 - 1 1 g .
Wa t e r : D i s ti l l e d w a t e r. I f t h e d i s ti l l e d w a t e r i s f o u n d to in c l u d e an y
s u b s t a n ce th a t ma y i n t e r f e r e w i t h a n a l y s i s o f t h e t a rg et co mp o s i t i o n a l
s u b s t a n ce s f r o m a g r i c u l t u r a l ch emi c a l s , w a s h w i t h a s o l v en t s u c h a s
n-hexane before use.
S o d i u m s u l f a t e ( a n h y d r o u s ) : S o d iu m s u l f a t e ( a n h y d r o u s) ( s p e c i a l g r a d e ) .
I f t h e r e a g e n t i s f o u n d t o in c lu d e a n y s u b st a n c e t h at ma y i n t e r f e r e
w i t h a n al y s i s o f t h e t a rg e t co mp o s i t i o n a l s u b st a n c e s f r o m a g r i c u l t u r al
c h e mi c a l s , w a s h w i t h a s o l v en t su c h a s n - h e x a n e b e f o r e u s e .
3. Reference standard
A l d r i n : T h i s p r o d u ct c o n ta i n s no t l e s s t h an 9 7 % o f a l d r i n , an d i t s
me l t i n g po in t i s 10 2 - 10 4 ° C .
E n d r i n : T h i s p r o d u c t c o n ta i n s n o t l e s s th an 9 8 % o f e n d r i n , a n d i t s
d e c o mp o s i t i o n p o in t i s 2 0 0 ° C.
D i e l d r i n : Th i s p r od u ct c o n t a in s n o t le s s t h an 9 8 % o f d i e l d r i n , an d i t s
me l t i n g po in t i s 17 7 - 17 9 ° C .

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4. Procedure
a . E x t r a ct io n
i . C e r e a l g r a i n s , l eg u me s / p u l s e s an d s e ed s
We i g h 1 0 .0 g o f te s t s a mp l e s , p r e v i o u s ly g r o u n d so a s t o p as s
t h r o u g h a s t a n d a r d me s h s i e v e ( 42 0 µ m) . A d d 2 0 ml o f w a t e r t o t he
o b t ai n ed s amp l e a n d l ea v e it t o s ta n d f o r t w o h o u r s .
T h e n , a d d 1 0 0 ml o f a c e t on e and h o mo g e n iz e t he mi x t u r e f o r t h re e
mi n u t e s . F i l t e r th e h o mo g e n i z ed s a mp l e b y s u c t i o n in t o a r o t a ry
v a c u u m e v a p o r a t o r t h ro u g h a f i l t er p a p e r c o v e r e d w i t h a 1 - c m t h i c k
l a y e r o f d i a t o ma c e o u s e a r t h . C o ll e c t t h e r e s i d u e o n t h e s u r f a c e o f
t h e f i l t e r p a p e r a n d a d d 5 0 ml o f a c e t o n e , a n d t h e n h o mo g e n i z e t h e
mi x t u r e f o r t h r e e mi n u t e s . R e p e at t h e a b o v e p r o c e d u r e a n d c o mb i n e
t h e f i lt r a t e i n t o t h e r o ta r y v a c u u m e v a p o r a t o r, a n d t h e n c o n c en t r a t e
t h e mi x t u r e t o ap p r o xima t e l y 3 0 ml a t 4 0 ° C o r l o w e r.
Tr a n s f e r t h e c o n c en t ra t e d s o lu t io n t o a 3 0 0 - ml s e p a r a t i n g f u n n e l
a l r e a d y c on t a in i ng 10 0 ml o f 1 0 % s o d iu m c h l o r id e s o l u t io n . Wa sh
t h e eg g p l an t - s h a p ed fl a s k o f th e a b o v e r o ta r y v a c u u m e v a p o r a to r
w i t h 1 00 ml o f n - h e x a n e a n d ad d th e w a s h i n g s t o th e s e p a r a t in g
f u n n e l a b o v e . S h a k e th e mi x t u r e v i g o r o u s l y f o r f i v e mi n u t e s u si n g a
s h a k e r a n d l e a v e i t t o s t a n d , a n d th e n t r an s f e r t h e n - h ex a n e la y e r t o
a 3 0 0 - ml c o n i ca l f l a sk . A d d 5 0 ml o f n - h e x a n e t o t h e a q u eo u s l aye r
a n d r ep e a t t h e a b o v e p r o c e d u r e , an d t h en add th e n - h e x a n e l a y e r to
t h e c o ni c a l f l a s k a b ov e . A d d a n ad e q u a t e a mo u n t o f s o d i u m s u l f a t e
( a n h y d r o u s) t o th e f l as k a nd l ea ve i t t o s t a nd f o r 15 mi n u te s with
occasional s h a k in g . F i l t e r t h e c o n te n t o f t h e f l a s k i n t o a r o t ar y
v a c u u m e v a p o r a t o r. Was h t h e f l a s k w i t h 2 0 ml o f n - h e x a n e a n d w a s h
t w i c e t h e r es i d u e o n t h e s u r f a c e o f t h e f i lt e r p a p e r w i t h th e w a s h i n g s .
A d d t h e w as h i n g i n to th e r o t a r y va c u u m e v a p o r a t o r an d r e mo v e t he
n - h e x an e at 4 0 ° C o r l ow e r.
A d d 2 0 ml o f n - h e x a n e t o t h e r e si d u e a n d t ra n s f e r t h e mi x t u r e t o a
1 0 0 - ml s e p a r a t i n g f u n n e l , a n d t h e n ad d 4 0 ml o f n - h e x a n e - s a t u r a ted
a c e t o n i t r i le. S h a k e t h e mi x t u r e v i g o r o u s l y f o r f i v e mi n u t e s u s i n g a
s h a k e r an d l e a v e i t to s t a n d , a n d t h e n t r a n s fe r t h e a c eto n i t r il e l a y e r
i n t o th e r o ta r y v a c u u m e v a p o r at o r. A d d 4 0 ml o f n - h e x an e - s a t u r a t ed
a c e t o n i t r i le t o th e n - h e x a n e l a y er a n d r e p ea t t h e a b o v e p r o c ed u re
t w i c e , an d t h en c o mb i n e t h e ac e t o n i t r i l e l a y e r i n to t h e r o t ar y
v a c u u m e v a p o r a to r. Re mo v e t h e a c e t o n i t r i le a t 4 0 ° C o r l o w e r a n d

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d i s s o l v e t h e r e s i d u e i n n - h ex a n e t o ma k e e x a c t l y 5 ml o f s o l u ti o n .
i i . F r u i t , v eg e t ab l e s , ma t c h a
I n c a s e o f f r u i t a n d v e g e t ab l e s , w e i g h a c cu r a t e l y 1 k g o f t h e t e st
s a mp l e a n d a d d a n app r o p r i a t e amo u n t o f w a t e r t o t he s a mp l e , i f
r e q u i r e d . H o mo g e n i ze t h e mi x t u r e a n d me a s u r e o u t a s a mp l e
e q u iv a l en t t o 2 0 . 0 g .
I n c a s e o f ma t c h a , w e i g h 5 . 0 0 g o f t h e t e s t s a mp l e a n d a d d 2 0 ml o f
w a t e r, a n d t h e n l e av e i t t o s t a n d f o r t w o h o u r s .
T h e n , a d d 1 0 0 ml o f a c e t on e and h o mo g e n iz e t he mi x t u r e f o r t h re e
mi n u t e s . F i l t e r th e h o mo g e n i z ed s a mp l e b y s u c t i o n in t o a r o t a ry
v a c u u m e v a p o r a t o r t h ro u g h a f i l t er p a p e r c o v e r e d w i t h a 1 - c m t h i c k
l a y e r o f d i a t o ma c e o u s e a r t h . C o ll e c t t h e r e s i d u e o n t h e s u r f a c e o f
t h e f i l t e r p a p e r a n d a d d 5 0 ml o f a c e t o n e , a n d t h e n h o mo g e n i z e t h e
mi x t u r e f o r t h r e e mi n u t e s . R e p e at t h e a b o v e p r o c e d u r e a n d c o mb i n e
t h e f i lt r a t e i n t o t h e r o ta r y v a c u u m e v a p o r a t o r, a n d t h e n c o n c en t r a t e
t h e mi x t u r e t o ap p r o xima t e l y 3 0 ml a t 4 0 ° C o r l o w e r.
Tr a n s f e r t h e c o n c en t ra t e d s o lu t io n t o a 3 0 0 - ml s e p a r a t i n g f u n n e l
a l r e a d y c on t a in i ng 10 0 ml o f 1 0 % s o d iu m c h l o r id e s o l u t io n . Wa sh
t h e eg g p l an t - s h a p ed fl a s k o f th e a b o v e r o ta r y v a c u u m e v a p o r a to r
w i t h 1 00 ml o f n - h e x a n e a n d ad d th e w a s h i n g s t o th e s e p a r a t in g
f u n n e l a b o v e . S h a k e th e mi x t u r e v i g o r o u s l y f o r f i v e mi n u t e s u si n g a
s h a k e r a n d l e a v e i t t o s t a n d , a n d th e n t r an s f e r t h e n - h ex a n e la y e r t o
a 3 0 0 - ml c o n i ca l f l a sk . A d d 5 0 ml o f n - h e x a n e t o t h e a q u eo u s l aye r
a n d r e p ea t t h e a b o v e p r o c e d u r e t w i c e , an d t h e n co mb i n e t h e
n - h e x an e l ay e r t o th e co n i ca l f l a sk a b o v e . A d d an a d eq u a t e a mo u n t
o f s o d iu m s u l f a t e ( an h y d r o u s ) t o t h e f l a s k an d l e a v e it t o s t an d f o r
1 5 mi n u t e s with occasional s h a k i n g . F il t e r t h e co n te n t o f t h e f l a sk
i n t o a r o t ar y v a c u u m e v a p o r a t o r. Wa s h t h e f l a s k w i th 2 0 ml o f
n - h e x an e an d w a s h tw i c e t h e r e s i d u e on t h e s u r f a c e o f t h e f i l te r
p a p e r. A d d t h e w a s h in g s i n t o th e r o t a r y v a c u u m e v a p o r a to r a n d
r e mo v e t h e n - h ex a n e a t 4 0 ° C o r l o w e r. D is s o l v e t h e r e s i d u e in
n - h e x an e to ma k e e x a ct l y 1 0 ml o f s o l u t io n .
i i i . Te a s ( l imi t e d t o u n f e r me n t e d te a ) o t h e r t h a n ma t c h a
We i g h 9 .0 0 g o f t h e te s t s a mp l e a n d s o ak i t i n 5 4 0 ml o f w a t e r a t
1 0 0 ° C , a nd t h e l e ave i t to s t an d a t r o o m t e mp e r a t u r e f o r f i v e
mi n u t e s . Tr a n s f e r 3 6 0 ml o f t h e c o o l ed f i l t r at e i n to a 5 0 0 - ml c o n i c a l
f l a s k . A d d 1 0 0 ml o f a c e to n e an d 2 ml o f s a t u r a t ed l e a d a c e ta te

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s o l u t io n t o t h e ab o ve f l a s k a n d l ea v e i t t o s tan d f o r o n e h o u r a t r o o m
t e mp e r a t u r e . F i l t e r t he mi x t u e b y s u c t i o n in to a 1 ,0 0 0 -ml s e p a r a t i n g
f u n n e l th r o u g h a f i l ter p a p e r c o v e r e d w i t h a 1 - c m t h i c k l a y e r o f
d i a to ma c e o u s e a r t h . Wa s h t h e ab o v e c o n i ca l f l a s k w it h 5 0 ml o f
a c e t o n e an d w a s h t h e r e s i d u e o n t h e s u r f a c e o f t h e f i lt er p a p e r w i th
t h e w a s h i ng s . A d d t h e w a s h i n g s i n t o th e ab o v e s ep a ra t i n g f u n n el
a n d a l s o a dd 3 0 g o f so d i u m c h l o r i d e a n d 1 0 0 ml o f n - h e x a n e. S h ak e
t h e mi x t u r e v i go r o u s l y f o r f i v e mi n u te s a n d l e a v e i t t o s t a n d , a n d
t h e n t r a n s f e r t h e n - h ex a n e l a y e r t o a 3 0 0 - ml c o n ic a l f l a s k . A d d 1 0 0
ml o f n - h e x a n e t o t h e aq u e o u s l a y e r a n d r e p ea t t h e a b o v e p r o c e d u r e,
a n d th e n a d d t h e n - h e xa n e l a ye r to t h e c o ni ca l f l a s k a bo v e . A d d an
a d e q u a t e amo u n t o f as o d i u m s u l f a t e ( a n h ydr o u s ) t o the f l a s k an d
l e a v e it t o s t a n d f o r 1 5 mi n u t e s w i t h o c c a s io n a l s h ak i n g . F i l t e r t h e
c o n te n t o f th e f l a s k i n to a r o t a r y v a c u u m e v a p o r a t o r. Wa s h t h e f l a s k
2 0 ml o f n - h e x an e an d w a s h t w i ce t h e r e s id u e o n t h e su r f a c e o f th e
f i l t e r p ap e r w i t h t h e w a s h i n g s . A d d t h e wa s h i n g s i n to th e r o ta r y
v a c uu m e v a p o r a to r a n d r e mo v e t h e n - h e xa n e a t 40 °C o r l o w e r.
D i s s o l v e t h e r e s i d u e i n n - h ex a n e t o ma k e e x a c t l y 5 ml o f s o l u ti o n .
b . C l ea n - u p
A d d 1 0 g o f f l o r i s il f o r c o lu mn c h r o ma t o g r ap h y s u s p e n d e d i n
n - h e x an e in t o a c h r o ma t o g r a p h t u b e ( 15 mm i n i n n e r d i a me t e r, 3 0 0
m m i n l e n g t h ) a n d ad d o v e r a p p r o x i ma t e l y 5 g o f s o d i u m s u l f a t e
( a n h y d r o u s) i n t o th e co l u mn . S p i l l o u t t h e n -h e x an e u n t il o n l y a s ma l l
a mo u n t r ema i n s o n th e p a c k in g o f t h e c o lu mn a n d p o u r 2 ml o f t h e
s o l u t io n o b t a i n ed b y t h e e x t r a ct io n d e s c r i b ed i n 4 - a i n to t h e c o l u mn .
P o u r 2 0 0 ml o f e t h e r / n - h e x a n e (3 :1 7 ) i n to t h e c o lu mn a n d c o l l e ct th e
e l u a te in t o a r o t a r y v ac u u m e v a p o r a t o r, an d t h e n r e mo v e t h e e th e r a n d
n - h e x an e a t 4 0 ° C o r lo w e r. D i s s o l v e t he r e s id u e in n - h ex a n e t o ma k e
e x a c tl y 2 ml o f s o l u t i o n , w h i c h i s u s e d a s t h e s a mp l e s o l u t io n .
5 . D e t e r mi n a t io n
a . Q u a l i ta t iv e t e s t s
P e r f o r m q u a l i t at i v e t es t s u n d e r th e f o l l o w in g c o n d i t io n s . Te s t r e s u l t s
o b t ai n ed mu s t b e t h e s a me a s t h o s e o b t a i n ed f o r t h e r e f e r e n c e
standard.
Te s t i n g c o n d i t io n s 1
C o l u mn : A s i l i c a te g l as s c a p i ll a r y c o lu mn ( 0 . 2 5 mm i n i n n e r d i a me t e r,
10-30 m in le n g th ) c o a te d with me t h y l s i l i co n e for gas

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c h r o ma t o g r a p h y t o a th i c kn e s s o f 0 . 2 5 µ m.
C o l u mn t e mp e r a t u r e : H o l d t h e c o l u mn t e mp e r a t u r e a t 5 0 ° C f o r o n e
mi n u t e , f o ll o w e d b y an i n c r e a s e o f 2 5 ° C ev er y mi n u t e u n t i l r e ac h in g
1 7 5 ° C , a f t er w h i c h in cr e a s e t h e te mp e r a t u r e b y 1 0 ° C ev e r y mi n u t e
u n t il r e a c h in g 3 0 0 ° C , a n d h o ld f o r f i v e mi n u t e s .
I n l e t t e mp e r a t u r e : 2 3 0 ° C
D e t e c t o r : Op e r a t e at 3 0 0 ° C
G a s f l o w r a t e : U s e h el i u m a s t h e c a r r i e r g a s . A d j u s t th e f l o w r a t e s o
t h a t a l d r in f l o w s o u t in a pp r o x i ma t e l y 1 0 mi n u t e s .
Te s t i n g c o n d i t io n s 2
C o l u mn : A s i l i c a te g l as s c a p i ll a r y c o lu mn ( 0 . 2 5 mm i n i n n e r d i a me t e r,
1 0 - 3 0 m i n l en g th ) c o a t ed w i th 1 4 % c y an o p r o p y l p h e n y l - me t h y l
s i l i c o n e f o r g a s c h r o ma t o g r ap h y to a th i c k n es s o f 0 . 2 5 µ m. .
C o l u mn t e mp e r a t u r e : H o l d t h e c o l u mn t e mp e r a t u r e a t 8 0 ° C f o r t w o
mi n u t e s , f o l l o w e d by a n i n c r ea s e o f 3 0 ° C e v e r y mi n u t e u n ti l
r e a c h i n g 1 9 0 ° C , a f ter w h i c h in c re a s e t h e t e mp e r a t u r e b y 3 . 6 °C
e v e r y mi n u t e u n t i l r e ac h i n g 2 5 0 °C , a n d h o l d f o r e i g h t mi n u t e s .
I n l e t t e mp e r a t u r e : 2 3 0 ° C
D e t e c t o r : Op e r a t e at 3 0 0 ° C
G a s f l o w r a t e : U s e h el i u m a s t h e c a r r i e r g a s . A d j u s t th e f l o w r a t e s o
t h a t a l d r in f l o w s o u t in a pp r o x i ma t e l y 1 0 mi n u t e s .
b . Q u a n ti t at i v e t e s t s
D e t e r mi n e t h e q u a n ti t y f r o m t h e t e s t r e su l t s o b t a in e d u n d e r th e
c o n d i t io n s d e s c r i b ed in 5 - a u si n g e i t he r t h e p e a k h e ig h t o r p e ak a re a
me t h o d .
c . C o n f i r ma t i o n t e s t s
P e r f o r m g a s c h r o ma t o g r a p h y / ma s s s p e c t r o me t r y u n d e r t h e co n d i t io n s
d e s c r i b ed in 5 - a . Te st r e s u l t s o b t a i n ed mu s t b e th e s ame a s t h o s e
o b t ai n ed f o r t h e r e f e r e n c e s t a n d a r d . D e t er mi n e t h e q u a n t i t y u si n g
e i t h e r th e pe a k he i gh t o r p e a k a r ea me t h o d , if r e q u i r e d .

( 6 ) C a p t a f ol a n al yt i c a l me t h o d
Perform according to 5 (6).

( 7 ) Q u i n o x a l i n e - 2 - c a rb o x y l i c a c id a n al yt i c al me t h o d
P e r f o r me d a c c o r d in g to 5 ( 7 ) .

( 8 ) C y h e x at i n an a l y t i ca l me t h o d
P e r f o r me d a c c o r d in g to 5 ( 4 ) .

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( 9 ) D a mi n o z i d e an a l y ti c a l me t h o d
P e r f o r me d a c c o r d in g to 5 ( 1 3 ) .

( 1 0 ) A n a l y t i c a l me t h o d f o r t r i a z o p h o s a n d p a r a t h io n
1. Apparatus
A gas c h r o ma t o g r a p h with an a l k al i f la me i o n i za t i on d et ec t o r
( G C - F I D ) , a f l a me p h o t o me t r i c d e t e c to r ( G C - F P D , i n te r f e r e n c e f i l t e r
for p h o s ph o r u s d e te r mi n a t i o n , wavelength: 526 n m) , or a
h i g h l y - s e n s i t i v e n it r o g e n p h o sp ho r u s d e te c to r ( G C - N P D ) , a n d a g a s
c h r o ma t o g r a p h / ma s s s p e c t r o me t e r ( G C / M S o r G C / M S / M S )
2 . R e ag e n t s a n d t e s t s o l u t io n s
I n a d d i t io n t o t h e r e ag e n t s a n d te s t s o lu t i o n s l i s t ed b e l o w, u s e th o s e
l i s t e d in S e c t i o n C R e a g e n ts /Te s t S o l u ti o n s , E t c . , P a r t I I F o o d
a d d it i v e s . R e a g en t s d e s i g n at e d as “s p e c i a l g r a d e ” i n th i s s e c ti o n mu s t
me e t t h e re q u i r e me n t s f o r “s p e c i a l g r a d e” s p e c i f ie d i n t h e J ap a n
I n d u s t r i a l St a n d a r d s fo r t h e r e ag e n t s .
A c e t o n it r i le : U s e a r o t a r y v a c u u m e v a p o r a t o r o n 3 0 0 mL o f a c e t o n i t ri l e
t o c o n c e n t r a t e t o t h e p o i n t o f d r y n e s s . D i s s o l v e t h e r e s id u e i n 5 mL o f
n - h e x an e . F o r a n a l y s i s , i n j ec t 5 μ L o f th e so l u ti o n i n to a GC -ECD.
P e a k s o t h er t h a n t h a t o f n - h e x an e i n th e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
A c e t o n e: U s e a r o t a r y v a c u u m e v ap o r at o r o n 3 0 0 mL o f a c e to n e t o
c o n c en t r a t e t o t h e p o in t o f d r y n e s s . D i s s o l v e t h e r e s i d u e i n 5 mL o f
n - h e x an e . F o r a n a l y s i s , i n j ec t 5 μ L o f th e so l u ti o n i n to a GC -ECD.
P e a k s o t h er t h a n t h a t o f n - h e x an e i n th e r e s u l t i n g ch r o ma t o g r a m
s h o u ld b e as h i g h a s o r l o w e r t h an t h e p e a k s o f γ - B H C a t 2 x 1 0 - 1 1 g .
S o d i u m c h l o r i d e : S o diu m c h l o r i d e ( s p e c i a l g r a d e ) . I f t h e r e a g en t i s
f o u n d to i nc l u de a n y s u b s t a n c e t h a t ma y i n t er f e r e w i t h t h e a n al y s i s o f
s u b s t a n ce s t o b e a n al y z e d f r o m a g r i c u l tu r a l c h e mi c a l s , w a s h w i t h a
s o l v e n t s u ch a s n - h e x a n e b e f o r e u s e .
S i l i c a g el f o r c o l u mn c h r o ma t o g r a p h y ( 6 3 - 2 0 0 µ m i n p a r t i c l e s iz e ) :
H e a t s i l i c a g e l ma d e f o r c o l u mn c h r o ma t o g r a p h y ( 6 3 - 2 0 0 µ m i n
p a r t i c le s i ze ) a t 1 3 0 °C f o r 1 2 h o u r s o r lo n g e r. C o o l d o w n to r o o m
t e mp e r a t u r e i n a d e s i cc a t o r.
D i a t o ma c e o u s e a r t h : D i a t o ma c e o u s e a r t h f o r c h e mi c a l a n a l y s i s .
E t h y l a c e tat e : U s e a r o t a r y v a c u u m e v a p o r a t o r o n 3 0 0 mL o f e t h y l
a c e t a te to co n c en t r a t e t o th e p o i n t o f d r y n e s s . D i s s o l v e th e r e s i d u e in
5 mL o f n - h e x an e . F o r a n a l y s i s , i n je c t 5 μL o f t h e s o l u ti o n i n to a

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GC-ECD. Peaks o th e r than th a t o f n - h e x an e in t h e r e s u lt i n g

c h r o ma t o g r a m s h o u l d b e a s hi g h a s o r l o w e r t h a n t h e pe a k s o f γ - B H C
a t 2 x 10 - 1 1 g .
n - H e x a n e : U s e a r o t a r y v a c u u m e v a p o r at o r o n 3 0 0 mL o f n - h e x a n e t o
e v a p o r a t e u n t i l 5 mL i s l e f t . F o r a n a l y s i s , i n j e c t 5 μL o f t h e s o l u t io n
i n t o a G C - E C D . P e a k s o t h e r t h an t h at o f n- h e x a n e i n t h e r e s u lt in g
c h r o ma t o g r a m s h o u l d b e a s h i gh a s o r l o w e r t h an th e pe a k s o f γ - B H C
a t 2 x 10 - 1 1 g .
Wa t e r : D i s t i l l e d w a t e r. I f t h e d i s ti l l e d w a t e r i s f o u n d to i n cl u d e an y
s u b s t a n ce th a t ma y i n t e r f e r e w i t h a n a l y s i s o f t h e t a rg e t co mp o s i t i o n al
s u b s t a n ce s f r o m a g r i c u l t u r a l c h emi c a l s , w a s h w i t h a so l v en t su c h a s
n-hexane before use.
S o d i u m s u l f a t e ( a n h y d r o u s ) : S o d iu m s u l f a t e ( a n h yd r o u s) ( s p e c i a l g ra d e ) .
I f t h e r e a g e n t i s f o u n d t o in c l u d e a n y s u b s t a n c e t h a t ma y i n t e r f e r e
w i t h a n al y s i s o f t h e t a rg e t c o mp o si t i o n a l s u b s t a n c e s f r o m a g r i c u lt u r al
c h e mi c a l s , w a s h w i t h a s o l v en t su c h a s n - h e x a n e b e f o r e u s e .
3. Reference standard
Tr i a z o ph o s: T hi s p r odu c t c o n t a in s n o t le s s th a n 9 8 % o f t r i a zo p h o s, a n d
i t s me l t i n g p o i n t i s 0 -5 ° C .
P a r a t h i o n : T h i s p r o d u c t c o n t a in s n o t l e s s t h a n 9 7 % o f p a r a t h io n , a n d
i t s b oi l in g p o i nt i s 375 ° C .
4. Procedure
a . E x t r a ct io n
i . C e r e a l g r a i n s a n d l eg u me s / p u l s e s
We i g h 1 0 .0 g o f t e s t s amp l e , p r e v io u s l y g r o u n d s o a s to p a s s t h r o u g h
a s t a n d a r d me s h s i e v e ( 4 2 0 µ m) . A d d 2 0 ml o f w a t e r t o t h e o b t a in ed
s a mp l e a n d l e a v e i t t o s t a n d f o r tw o h o u r s .
T h e n , a d d 1 0 0 ml o f a c e t on e and h o mo g e n iz e t he mi x t u r e f o r t h re e
mi n u t e s . F i l t e r th e h o mo g e n i z ed s a mp l e b y s u c t i o n in t o a r o t a ry
v a c u u m e v a p o r a t o r t h ro u g h a f i l t er p a p e r c o v e r e d w i t h a 1 - c m t h i c k
l a y e r o f d i a t o ma c e o u s e a r t h . C o ll e c t t h e r e s i d u e o n t h e s u r f a c e o f
t h e f i l t e r p a p e r a n d a d d 5 0 ml o f a c e t o n e , a n d t h e n h o mo g e n i z e t h e
mi x t u r e f o r t h r e e mi n u t e s . R e p e at t h e a b o v e p r o c e d u r e a n d c o mb i n e
t h e f i lt r a t e i n t o t h e r o ta r y v a c u u m e v a p o r a t o r, a n d t h e n c o n c en t r a t e
t h e mi x t u e t o a p p r o x ima t e l y 3 0 ml a t 4 0℃ o r l o w e r.
Tr a n s f e r t h e c o n c en t ra t e d s o lu t io n t o a 3 0 0 - ml s e p a r a t i n g f u n n e l
a l r e a d y con t a in i ng 10 0 ml o f s a t u r a t ed s o d i u m c h l o r i d e so l u t i o n .

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Wa s h t h e e g g p l a n t - s h a p e d f l a s k o f t h e a b o v e r o t a r y v a c u u m
e v a po r a t o r w i t h 1 00 ml o f e t h y l ac e t a t e/ n - h ex a n e ( 1 :4 ) a n d a d d t h e
w a s h i n g s to t h e s ep a r a t i n g f u n n e l ab o v e . S h a k e t h e mi x t u r e
v i g o r o u s l y f o r f i v e mi n u t e s u s i n g a s h a k e r an d l ea v e i t t o s t a nd , and
t h e n t r a n s fe r t h e l a y e r s o f e t h y l a c e t a t e an d n - h e x an e t o a 3 0 0 -ml
c o n ic a l f l as k . A d d 5 0 ml o f e t h y l a c e t a te/n - h e x a n e ( 1 : 4 ) t o t h e
a q u eo u s l ay e r a n d r e p e a t th e ab o v e p r o c ed u r e , a nd th e n a dd t he
l a y e r s o f e th y l a c e ta t e a n d n - h e xan e to th e co n i ca l f l a sk a bo v e. A d d
a n a d e q u a t e a mo u n t o f s o d i u m s u l f a t e ( a n h y d r o u s ) t o t h e f l a s k a n d
l e a v e i t t o s t a n d f o r 1 5 mi n u t e s w i t h o cc a s io n a l s h a k i n g . F i l t e r t h e
c o n te n t o f th e f l a s k i n to a r o t a r y v a c u u m e v a p o r a t o r. Wa s h t h e f l a s k
w i t h 2 0 ml o f n - h e x an e a n d w a s h t w i c e t h e re s i d u e o n th e s u r f a c e o f
t h e f i l t e r p a p e r w i t h th e w a s h i n g s . A d d t h e w a s h i n g i n to t h e r o t a ry
v a c u u m e v a p o r a to r a n d r e mo v e th e e th y l a ce t a t e a n d n - h e x a n e at
4 0 ° C o r lo w e r.
A d d 3 0 ml o f n - h e x an e t he r e s id u e an d t r a n s f e r t h e mi x t u r e t o a
1 0 0 - ml s e p a r a t i n g f u n n e l , a n d t h e n ad d 3 0 ml o f n - h e x a n e - s a t u r a ted
a c e t o n i t r i le. S h a k e t h e mi x t u r e v i g o r o u s l y f o r f i v e mi n u t e s u s i n g a
s h a k e r an d l e a v e i t to s t a n d , a n d t h e n t r a n s fe r t h e a c eto n i t r il e l a y e r
i n t o th e r o ta r y v a c u u m e v a p o r at o r. A d d 3 0 ml o f n - h e x an e - s a t u r a t ed
a c e t o n i t r i le t o th e n - h e x a n e l a y er a n d r e p ea t t h e a b o v e p r o c ed u re
t w i c e , an d t h en c o mb i n e t h e ac e t o n i t r i l e l a y e r i n to t h e r o t ar y
v a c u u m e v a p o r a t o r ab o v e . R e mo v e t h e a c et o n i t r i l e a t 4 0 ° C o r l o w e r
a n d d i s s o l v e t h e r e s id u e i n 5 ml o f a c e to n e /n - h e x an e (1 : 1 ) .
i i . F r u i t an d v eg e t ab l es
We i g h ac cu r a t e l y 1 k g o f t h e t es t s a mp l e a n d ad d an a p p r o p r ia te
a mo u n t o f w a t e r t o t he s a mp l e , if r e q u i r e d . H o mo g e n i ze th e mi x t u r e
a n d me a s u r e o u t a s a mp l e eq u iv a le n t to 2 0 . 0 g .
T h e n , a d d 1 0 0 ml o f a c e t on e and h o mo g e n iz e t he mi x t u r e f o r t h re e
mi n u t e s . F i l t e r th e h o mo g e n i z ed s a mp l e b y s u c t i o n in t o a r o t a ry
v a c u u m e v a p o r a to r u si n g f i l t e r p ap e r c o v er e d w i t h a 1 - c m t h i c k
l a y e r o f d i a t o ma c e o u s e a r t h . C o ll e c t t h e r e s i d u e o n t h e s u r f a c e o f
t h e f i l t e r p a p e r a n d a d d 5 0 ml o f a c e t o n e , a n d t h e n h o mo g e n i z e t h e
mi x t u r e f o r t h r e e mi n u t e s . R e p e at t h e a b o v e p r o c e d u r e a n d c o mb i n e
t h e f i lt r a t e i n t o t h e r o ta r y v a c u u m e v a p o r a t o r, a n d t h e n c o n c en t r a t e
t h e mi x t u r e t o ap p r o xima t e l y 3 0 ml a t 4 0 ° C o r l o w e r.
Tr a n s f e r t h e c o n c en t ra t e d s o lu t io n t o a 3 0 0 - ml s e p a r a t i n g f u n n e l

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a l r e a d y con t a in i ng 10 0 ml o f s a t u r a t ed s o d i u m c h l o r i d e so l u t i o n .
Wa s h t h e e g g p l a n t - s h a p e d f l a s k o f t h e a b o v e r o t a r y v a c u u m
e v a po r a t o r w i t h 1 00 ml o f e t h y l ac e t a t e/ n - h ex a n e ( 1 :4 ) a n d a d d t h e
washing to the s e pa r a t i n g f un n e l a bo ve . Shake the mi x t u r e
v i g o r o u s l y f o r f i v e mi n u t e s u s i n g a s h a k e r an d l ea v e i t t o s t a nd , and
t h e n t r a n s fe r t h e l a y e r s o f e t h y l a c e t a t e an d n - h e x an e t o a 3 0 0 -ml
c o n ic a l f l as k . A d d 5 0 ml o f e t h y l a c e t a te/n - h e x a n e ( 1 : 4 ) t o t h e
a q u eo u s l aye r a n d r e p e a t th e ab o v e p r o c ed u r e t w i c e, a n d t h e n
c o mb i n e the e th y l a c et a t e a n d n -h e x an e l a y e r t o t h e co n i ca l f l a sk
a b o v e . A d d a n a d e q u a te a mo u n t o f s o d i u m s u l f a t e ( a n h ydr o u s ) t o t h e
f l a s k a n d le a v e it t o st a n d f o r 1 5 mi n u t e s w i t h o c c a s io n a l s h ak i n g .
F i l t e r t h e co n t en t o f t h e f l a s k i n to a r o t a r y v a c u u m e v a p o r a t o r. Wa s h
t h e f l a s k w i t h 2 0 ml o f n - h e x a n e a n d w a s h tw i c e t h e r es i d u e o n t h e
s u r f a c e o f th e f i l te r p ap e r. A d d t h e w a s h i n g s i n t o t h e r o ta r y v a c u u m
e v a p o r a t o r a n d r e mo v e t h e et h y l a c e t a te a n d n - h e x a n e a t 4 0 ° C o r
l o w e r. D i s s o l v e t h e r es i d u e i n 5 ml o f a c e t o n e / n - h e x an e ( 1 : 1 ) .
b . C l ea n - u p
A d d 5 g of s i l i c a g el f o r c o l u mn c h r o ma t o g r a p h y ( 6 3 - 2 0 0 µ m i n
p a r t i c le si z e ) s u s p e n d ed in a c e to n e/n - h e x an e (1:1) into a
c h r o ma t o g r a p h t u b e ( 1 5 m m i n i n n e r d ia me t e r, 3 0 0 mm i n l en g th ) an d
a d d o v e r ap p r o x i ma t e l y 5 g o f s o d i u m s u l f a t e ( a n h yd r o u s ) t o th e
c o l u mn . P o u r a c e t o n e a n d n - h e x an e ( 1 : 1 ) i n to t h e co l u mn u n ti l o n l y a
s ma l l a mo u n t r e ma i n s o n th e p a ck i n g o f t h e co l u mn . P o u r t h e s o l u tio n
o b t ai n ed b y t h e ex t r ac t i on de s c r i b e d i n 4 - a i n to th e c o l u mn f o l l o w e d
b y 1 0 0 ml o f a c e to n e / n - h ex a n e ( 1 : 1 ) a n d c o l l e ct th e e lu a t e in t o a
r o t a r y v a c u u m e v a p o r a t o r, a n d t h en r e mo v e t h e a c e to n e a n d n - h ex an e
a t 4 0 ° C o r l o w e r. D i s s o l v e t h e r es i d u e in a ce t o n e t o ma k e ex a c tl y 5
ml o f s o l u t i o n , w h i c h i s u s e d a s th e s a mp l e s o l u ti o n .
5 . D e t e r mi n a t io n
a . Q u a l i ta t iv e t e s t s
P e r f o r m q u a l i t at i v e t es t s u n d e r th e f o l l o w in g c o n d i t io n s . Te s t r e s u l t s
o b t ai n ed mu s t b e t h e s a me a s t h o s e o b t a i n ed f o r t h e r e f e r e n c e
standard.
Te s t i n g c o n d i t io n s 1
C o l u mn : A s i l i c a te g l as s c a p i ll a r y c o lu mn ( 0 . 5 3 mm i n i n n e r d i a me t e r,
10-30 m in le n g th ) c o a te d with me t h y l s i l i co n e for gas
c h r o ma t o g r a p h y t o a th i c kn e s s o f 1 . 5 µ m.

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C o l u mn t e mp e r a t u r e : H o l d t h e c o l u mn t e mp e r a t u r e a t 8 0 ° C f o r o n e
mi n u t e , f o ll o w e d b y an in c r e a s e o f 8 ° C e v e ry mi n u t e u n t i l r e a c h i n g
2 5 0 ° C , a n d h o ld f o r f i v e mi n u t e s .
I n l e t t e mp e r a t u r e : 2 3 0 ° C
D e t e c t o r : Op e r a t e at 2 8 0 ° C
G a s f l o w r a t e : U s e h el i u m a s t h e c a r r i e r g a s. A d j u s t t h e f l o w r a t e to
t h e o p t i ma l c o n d i t io n . A d j u s t t h e f l o w s o f a i r a n d h yd r o g e n t o the
o p t i ma l c o n d i ti o n s .
Te s t i n g c o n d i t io n s 2
C o l u mn : A s i l i c a te g l as s c a p i ll a r y c o lu mn ( 0 . 3 2 mm i n i n n e r d i a me t e r,
1 0 - 3 0 m i n l e n g t h ) c o at e d w i th 5 0 % t r i f l u o r o p r o p y l me t h y l s i l i co n e
f o r g a s c h ro ma t o g r a p h y t o a t h i c k n e s s o f 0 . 2 5 µ m.
C o l u mn t e mp e r a t u r e : H o l d t h e c o l u mn t e mp e r a t u r e a t 7 0 ° C f o r o n e
mi n u t e , f o ll o w e d b y an i n c r e a s e o f 2 5 ° C ev er y mi n u t e u n t i l r e ac h in g
1 2 5 ° C , a f t er w h i c h in cr e a s e t h e te mp e r a t u r e b y 1 0 ° C ev e r y mi n u t e
u n t il r e a c h in g 2 3 5 ° C , a n d h o ld f o r 1 2 mi n u t es .
I n l e t t e mp e r a t u r e : 2 3 0 ° C
D e t e c t o r : Op e r a t e at 2 8 0 ° C
G a s f l o w r a t e : U s e h el i u m a s t h e c a r r i e r g a s . A d j u s t t h e f l o w r a t e t o
t h e o pt i ma l c on d it i on . A d j u s t th e f l o w s o f ai r a n d h y d r o g e n to t h e
o p t i ma l c o n d i ti o n s .
b . Q u a n ti t at i v e t e s t s
D e t e r mi n e t h e q u a n ti t y f r o m t h e t e s t r e su l t s o b t a in e d u n d e r th e
c o n d i t io n s d e s c r i b ed in 5 - a u si n g e i t he r t h e p e a k h e ig h t o r p e ak a re a
me t h o d .
c . C o n f i r ma t i o n t e s t s
P e r f o r m g a s c h r o ma t o g r a p h y / ma s s s p e c t r o me t r y u n d e r t h e co n d i t io n s
d e s c r i b ed in 5 - a . Te st r e s u l t s o b t a i n ed mu s t b e th e s ame a s t h o s e
o b t ai n ed f o r t h e r e f e r e n c e s t a n d a r d . D e t er mi n e t h e q u a n t i t y u si n g
e i t h e r th e pe a k he i gh t o r p e a k a r ea me t h o d , if r e q u i r e d .

7 . I n a d dit i o n t o the s u b s t a n ce s s t i p u la te d u n d e r 6 , i n g r e d ie n t s o f
a g r i c u lt u r al c h e mi c a l s a n d o th e r c h e mi c a l s u b s t a n c e s li s t e d i n th e f i r s t
c o l u mn o f t h e ta b le i n ( 1 ) mu s t n o t b e c o n ta i n ed in f o o d s a t l e v el s
e x c e ed i n g t h e li mi t s s t i p u la t ed i n t h e t h i r d c o lu mn o f t h e s a me t a b l e
a c c o r d in g to t h e f o o d c a t e g o r i e s s h o w n in the s e c o n d c o l u mn o f t h e s a me
t a b l e. C o n ce r n i n g th i s , f o o d s l i s t ed i n t he “f o o d s ” c ol u mn i n t he t a bl e i n

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( 2 ) s h a l l b e t e s t ed u si n g t h e p a rt l i s t ed in t h e “s a mp l e s ” c o lu mn i n t h e
t a b l e a s a s a mp l e . I n a d d it i o n , in t h e s u b s ta n c e s u s ed a s i n g r e d ie n t s o f
a g r i c u lt u r al c h e mi c a l s a n d o th e r c h e mi c a l s u b s t a n c e s li s t e d i n th e f i r s t
c o l u mn o f t h e t ab l e in ( 1 ) , w h i c h a r e s t i p u l at e d t o b e “N o t d e t e ct ed ” in
t h e th i r d co l u mn o f t h e s a me t a b l e in th e f o o d s l i s ted i n t h e se c o n d
c o l u mn o f t h e s a me t a b l e , n o i n g r ed i e nt o f a g ri c u l tu r a l che mi c a l s o r o t h e r
c h e mi c a l s u b s t a n c e s sh a l l b e d ete c t e d w h e n t h e s e f o o d s a r e t e s te d u s i n g
t h e te s t me t h o d s s ti p u l a t e d i n ( 3 ) t o ( 8 ) . ”

( 1 ) T h e ma x i mu m r e s i d u e l i mi t s o f s u b s t a n c e s u s e d a s i n g r e d ien t s o f
a g r i c u lt u r al c h e mi c a l s i n f o o d s ( P ro v i s i o n a l M R L s L i s t )

( 2 ) S a mp l e s

Food S a mp l e ma t e r i a l

Barley and buckwheat T h r e s h e d se e d s

W h e a t an d r y e H u s k e d s e ed s

R i c e ( b r o wn r i c e ) H u s k e d s e ed s

C o r n ( ma i z e ) S e e d s w i t h t h e h u s k , th e s i lk a n d
t h e co r e s r e mo v e d
O t h e r c e r e al g r a i n s T h r e s h e d se e d s

P e a s , b e a n s ( d r y) * , b r o a d b ea n s an d S e e d s w i t h o u t th e p o d s
soybeans (dry)
Peanuts, dry Wi t h t he s he l l s r e mo v e d

O t h e r l e g u me s / p u l s e s S e e d s w i t h o u t th e p o d s

A p r i c o t , mu me p l u m, c h e r r y, Wi t h t h e p ed u n cl e s a n d t h e s e e d s
J a p a n e s e p lu m ( i n c l u d in g p r u n e ) r e mo v e d
a n d n e c ta r i n e

Peach Wi t h t h e s k i n s a n d t h e s e e d s
r e mo v e d
O r a n g e ( i n c l u d i n g n av e l o r a n g e ) , Whole fruit
g r a p e f r u i t , c i t r u s na t su d a id a i
( w h o l e ) , l ime a n d l e mo n
C i t r u s n a t su d a id a i ( p u l p ) a n d u n sh u Wi t h t h e p ee l s r e mo v e d
o r a n g e ( p u lp )

C i t r u s n a t su d a id a i , p ee l s Wi t h t h e c al y x e s r e mo v e d

Other citrus fruits Whole fruit

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P e a r, J a p a n e s e p e a r, q u i n ce a n d Wi t h t h e b l o s s o m s c a r s , t h e c o r e s
a p p le a n d th e p e d u n c le s r e mo v e d

L o q u at Wi t h t he p ed u n cl e s , the s k i n s an d
t h e s e ed s r e mo v e d
Av o c a do a nd ma n g o Wi t h t h e s ee d s r e mo v e d

Kiwifruit Wi t h t h e s k i n s r e mo v e d

Guava Wi t h t h e c al y x e s r e mo v e d

Date Wi t h t h e c al y x e s a n d th e s e e d s
r e mo v e d
Pineapple Wi t h t h e to p s r e mo v e d

P a s s i o n f r u i t a n d p ap ay a Whole fruit

B a n a na Wi t h t h e p ed i c e l s r e mo v e d

St r a w b e r r y, c r a n b e r r y, h u c k l e b e r r y, Wi t h t h e c al y x e s r e mo v e d
b l a ck b e r r y a n d b l u eb e rr y

Raspberry Whole fruit

Other berries Wi t h t h e c al y x e s r e mo v e d

J a p a n e s e p e r s i m mo n Wi t h t he c al yx e s a n d th e s e e d s
r e mo v e d

Wa t e r me l o n , m a k u w a u r i me l o n an d Wi t h t h e r in d s r e mo v e d
me l o n s
Grape Wi t h t he p ed u n cl e s r emo v e d

Other fruits E d i b le p o r t i o n s

Tu r n i p ( r o o t s ) a n d J a p a n e s e r a d i s h Wi t h th e d ir t l i g h t l y r in s e d o ff w i t h
( r o o t s , i n clu d i ng r a di s h ) water

Tu r n i p ( l e a v e s ) , w a t e r c r e s s , k a l e , Wi t h t h e d ec a y e d l e av es r e mo v e d
J a p a n e s e r ad i s h ( l e av es , i n c lu d in g
radish) and brussels sprouts

C a u l i f lo w e r a n d b r o c co l i
C a b b ag e an d C h in e s e ca b b ag e
Kyona and komatsuna (Japanese
mu s t a r d s p i n a c h )
Wi t h t h e l ea v e s r e mo v e d
A s a mp l e c o n s i s t in g o f o n e p o r t io n
f r o m e a c h o f f o u r h e ad s , w i t h eac h
h e a d e q ua l ly c u t i nt o fo u r p o r ti o ns ,
w i t h o u t t h e d e c a y e d o u t e r l e av e s
a n d th e co r e s
Wi t h t h e r o o t s a n d t h e d e c a y e d
l e a v e s r e mo v e d

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Horseradish R o o t s w i th t h e d i r t l ig h t l y r i n s e d
o ff w i t h w a t e r

Qing-geng-cai and other E d i b le p o r t i o n s

c r u c i f e r o u s v e g et a bl e s

S w e e t p o t ato , k on j ac , ta r o , p o t a t o , Wi t h th e d ir t l i g h t l y r in s e d o ff w i t h
y a m a n d o t h e r p o ta t o es water

P u mp k i n ( i n c l u d i n g s q u a s h ) , Wi t h t h e v in e s r e mo v e d
c u c u mb e r ( i n c lu d in g g h e r k i n ) an d
o r i e n t al p i ck l i n g me l o n ( v e g et a b le )

O t h e r c u c u r b i t a c eo u s v e g et a b le s E d i b le p o r t i o n s

A r t i c h o k e , e n d iv e an d c h i co r y Wi t h t h e d ec a y e d l e av es r e mo v e d

B u r d o ck a nd s a l s i f y A s a mp l e t h i n l y s l i ced t h en g r o u n d
w i t h a me a t g r i n d e r, t h e l e a v e s
h a v in g b e en r e mo v e d a n d th e d i r t
h a v in g b e en l i g h t l y r in s e d o ff w it h
water
S h u n g i ku Wi t h t h e r o o t s a n d t h e d e c a y e d
l e a v e s r e mo v e d
L e t t u ce ( i n c l u d i n g c o s l e t tu c e an d Wi t h t h e d ec a y e d o u t e r l e a v e s a n d
l e a f l e tt u c e) t h e co r e s r e mo v e d
O t h e r c o mp o s i t e v e g eta b l e s E d i b le p o r t i o n s

S h i it a k e mu s h r o o m, b u t t o n E d i b le p o r t i o n s
mu s h r o o m a n d o t h e r mu s h r o o ms

C e l e r y, p a rs l e y a n d m it s u b a Wi t h t h e r o o t s a n d t h e d e c a y e d
l e a v e s r e mo v e d
Carrot and parsnip Wi t h t h e d ir t l i g h t l y r i n s e d o ff

O t h e r u mb e l l i f e r o u s v eg e t ab l e s E d i b le p o r t i o n s

To ma t o , e g g p l an t an d p i mi e n t o Wi t h t h e c al y x e s r e mo v e d
(sweet pepper)

O t h e r s o l a n c e o u s v e g eta b l e s E d i b le p o r t i o n s

Asparagus St e ms

O n i o n , g a r li c , w e l s h ( in c l ud i n g Wi t h t h e o u t e r s k i n s an d t h e r o o t
l e e k ) an d mu l t i p l y i n g o n io n h a i r r e mo v e d

N i r a a n d o th e r l i l ia c eo u s E d i b le p o r t i o n s
v e g et a b le s

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G r e e n s o y b e a n s , k i d n ey b e a n s ( w i t h Wi t h t h e p ed i c e l s r e mo v e d
p o d s , i mma t u r e ) a n d p e a s ( w i t h
p o d s , i mma t u r e )

Okra Wi t h t h e c al y x e s r e mo v e d

Sugarcane Wi t h t he hus k s r e mo v e d

Ginger Wi t h t h e l ea v e s r e mo v e d , an d w i th
t h e d i r t l ig h t l y r i n s e d o ff w i t h
water

Sugar beet Wi t h th e d ir t l i g h t l y r in s e d o ff w i t h
water

Spinach Wi t h r e d r o o t s l e f t o n , a n d w i th t h e
r o o t h ai r an d th e d e c ay e d l e a v e s
r e mo v e d

B a mb o o s h o o t s an d o th e r E d i b le p o r t i o n s
v e g et a b le s

S e s a me s e e d s , r a p e s e e d s , s u n f l o w e r Seeds only
s e e d s , s a ff l o w e r s e e d s , c o t t o n s e e d
a n d o t h e r oi l s e e d s

A l mo n d , g in k g o n u t , ch e s t n u t , Wi t h t h e h u s k s r e mo v e d
w a l n u t , p e ca n a n d o t h er n u t s

C a c a o b e an s a n d co ff e e b e an s B e a n s w i t h o u t th e p o d s

Te a Te a l ea v e s

Hop Dried flowers

O t h e r s p i c es a n d o t h e r h e r b s E d i b le p o r t i o n s

( 3 ) A n a l yt i c a l me t h o d f o r a l d r i n , e n d r i n a n d d i e ld r i n
Perform according to 6 (5).

( 4 ) C l e n b u te r o l a n a l y t ic a l me t h o d
1. Apparatus
A l i q u i d c hr o ma t o g r a p h / t an d e m m a s s s p e c t r ome t e r ( L C / M S / M S )
2 . R e ag e n t s a n d t e s t s o l u t io n s
I n a d d i t io n t o t h e r e ag e n t s a n d te s t s o lu t i o n s l i s t ed b e l o w, u s e th o s e
l i s t e d in S e c t i o n C R e a g e n ts /Te s t S o l u ti o n s , E t c . , P a r t I I F o o d
a d d it i v e s .
A c e t o n it r i le : A c e t o n i tr i l e p r o d u ce d f o r l i q u id c h r o ma t o g r a p h y.

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St r o n g l y ac i d i c c at i on ex c h an g e r c a r t r i d g e c o l u mn ( 5 0 0 mg ) : A
p o l ye t h yl en e co l u mn o f 8 - 9 mm i n i n n e r d i ame t e r p a c k e d w i t h 5 0 0 mg
o f b en z e n es u l f o n i l p ro p yl s i la ne - b o n de d s i l i c a g el , o r a co lumn
e q u iv a l en t t o t h e s p e cif i e d o n e s ep a r a t i o n c ap a b il i t y.
Wa t e r : Wa te r p r o d u ced f o r l i q u i d c h r o ma t o g r a p h y.
M e t h an o l: M e t h an o l p r o d u c ed f o r l i q u i d c h ro ma t o g r a p h y.
3. Reference standard
C l e n bu t e r ol h yd r o ch l or i d e : Th i s p r o d u ct co n t a in s n o t le s s t h an 9 8 % o f
c l e nb u t e r ol h yd r o c h lo r i d e , an d i t s me l t i n g p o i n t i s 1 7 4 -1 7 6 ° C .
4. Procedure
a . E x t r a ct io n
We i g h 5 .0 0 g o f t h e t es t s a mp l e , p r e v i o u s l y g r o u n d , a n d h o mo g e n i z e
i t w i th 2 5 ml o f e t h y l a c e t a te an d 1 ml o f 4 mo l / l p o t a ss i u m c a r b o n a t e
s o l u t io n . Ce n t r i f u g e th e mi x t u r e a t 3 ,0 0 0 rp m f o r f i v e mi n u t e s a n d
c o l l ec t th e e t h y l a c et at e l a y e r i n to a r o t a r y v a c u u m e v a p o r a to r. A d d
2 5 ml o f e t h y l a c e t at e t o th e r e s id u e an d r e p e a t t h e a b o v e p r o c ed u r e
a n d co mb i n e t h e et h y l a c e t a te l a ye r, a n d th e n r e mo v e t h e e t h y l a c e tat e
a t 4 0 ° C o r l o w e r. A d d 3 0 ml e a c h o f a c e to n i t r il e an d a ce t o n i t ri l e -
s a t u r a t e d n - h e x a n e to t h e r e s id u e a n d s h ak e t h e mi x t u r e v ig o r o u sly
f o r f i v e mi n u t e s u s in g a s h a k e r, a n d th e n l ea v e i t to s ta n d . Tr a n s fe r
t h e a ce t o n it r i l e l a y e r to a 1 00 - ml s e p a r a t i ng f u n n e l a n d a d d 30 ml o f
a c e t o n i t r i le- s a t u r a t e d n - h e x an e . R e p e at th e a b o v e p ro c e d u r e an d
c o l l ec t t h e a c e t o n i t r i le l a y e r i n to t h e r o t a r y v a c u u m e v a p o r a to r, an d
t h e n r e mo v e t h e a c e to n i t r i l e at 4 0 ° C o r l o w e r. D i s s o l v e t h e r e s i d u e i n
1 0 ml o f me t h an o l /1 . 2% me t a p h o s p h o r i c a c id s o lu t io n ( 2 : 3 ) an d f i lt e r
t h e mi x t u r e t h r o ug h a c o t to n p l u g .
b . C l ea n - u p
P o u r 3 ml o f me t h a n o l f o l l o w e d b y 3 ml o f w a t e r i n t o a s t r o n g l y
a c i d ic c at io n ex c ha n ge r c a r t r i d g e c o lu mn ( 5 0 0 mg ) a n d d i s c a r d t h e
e ff l u e n t . Po u r t h e s o lu t i on o bt a in e d b y t h e e x t r a c ti o n d e s c r i b ed in 4 - a
i n t o th e c ol u mn f o l l ow e d b y 5 ml o f w a t e r a n d d i s c ar d t h e e ff l u e n t .
P o u r 1 5 ml o f me t h a n o l / 0 . 1 mo l / l d ip o t a s s i u m h y d r o g e n o r th o
p h o s ph a t e so l u ti o n ( 9 :1 ) i n to th e c o l u mn a n d c o ll e c t t h e e l u a t e i n to a
r o t a r y v a c uu m e v a p o r at o r, a n d t h en r e mo v e t h e w a t e r a n d me t h a n o l a t
4 0 ° C o r l o w e r. D i s s o l v e t h e r e s id u e i n 1 . 0 ml o f me t h a n o l , w h ich i s
u s e d a s th e s a mp l e s o l u t i o n .
5 . D e t e r mi n a t io n

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a . Q u a l i ta t iv e t e s t s
P e r f o r m q u a l i t at i v e t es t s u n d e r th e f o l l o w in g c o n d i t io n s . Te s t r e s u l t s
o b t ai n ed mu s t b e t h e s a me a s t h o s e o b t a i n ed f o r t h e r e f e r e n c e
standard.
Te s t i n g c o n d i t io n s
C o l u mn p a c k i n g : O c ta d e c y l s i l an e - b o n d e d s i l i c a g e l ( 2 - 5 µ m in
p a r t i c le s i ze ) .
C o l u mn : A s t a i n l e s s t ub e ( 2 .0 - 6 . 0 m m i n i n n er d i a me t e r, 1 0 0 - 2 5 0 m m
i n l en g th ) .
C o l u mn t e mp e r a t u r e : 4 0 ° C
M o b il e p h as e : U s e a c et o n it r i l e / f o r mi c a c i d /w a t e r ( 5 0 0 :1 : 5 0 0 ) . A d ju s t
t h e f lo w r a t e s o t h a t c l e n b u t e r o l f l o w s o u t in ap p ro x i ma t e l y 1 0
mi n u t e s .
b . Q u a n ti t at i v e t e s t s
D e t e r mi n e t h e q u a n t i ty f r o m t h e t e s t r e s u l t s o b t a i n ed u n d e r t h e
c o n d i t io n s d e s c r i b ed in 5 - a u si n g e i t he r t h e p e a k h e ig h t o r p e ak a re a
me t h o d .

( 5 ) D e x a me t h a s o n e a n al y t i c a l me t h o d
1. Apparatus
A l i q u i d c hr o ma t o g r a p h / t an d e m m a s s s p e c t r ome t e r ( L C / M S / M S )
2 . R e ag e n t s a n d t e s t s o l u t io n s
I n a d d i t io n t o t h e r e ag e n t s a n d te s t s o lu t i o n s l i s t ed b e l o w, u s e th o s e
l i s t e d in S e c t i o n C R e a g e n ts /Te s t S o l u ti o n s , E t c . , P a r t I I F o o d
a d d it i v e s .
A c e t o n it r i le : A c e t o n i tr i l e p r o d u ce d f o r l i q u id c h r o ma t o g r a p h y.
O c t a d e c y l si l a n e - b o n d e d s il i c a g e l c a r t r i dg e co lu mn ( 3 6 0 mg ) : A
p o l y e t h y l en e c o l u mn o f 8 - 9 mm i n i nn e r d i ame t e r p a c k e d w i t h 3 6 0 mg
o f o c t a d ecy l s i l a n e - b o n d e d s i li c a g e l , o r a c o l u mn e q u i v al e n t to t h e
s p e c i f i e d o n e i n s e p a ra t i o n c ap a b i l i t y.
S y n t h e t i c ma g n e s i u m s i l i c at e (F l o r i s i l ) f o r c o l u mn c h r o ma t o g r a p h y:
H e a t f l o r i s il ( 1 5 0 -2 5 0 μ m i n p a r t ic l e s i ze ) a t 1 3 0 ° C f o r 1 2 h o u r s o r
l o n g e r. C o o l d o w n t o ro o m t e mp e r a t u r e in a d e s i c c a to r.
Wa t e r : Wa te r p r o d u ced f o r l i q u i d c h r o ma t o g r a p h y.
M e t h an o l: M e t h an o l p r o d u c ed f o r l i q u i d c h ro ma t o g r a p h y.
P h o s p h a te b u ff e r s o l u t i o n (p H 5 .0 ) :
S o l u t io n 1: D i s s o l v e 2 7 . 2 g o f mo n o p o t as s i u m d i h y d r o g e n mo n o
p h o s p h a t e in w a t e r t o ma k e a 1 , 00 0 ml o f s o l u t io n .

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S o l u t io n 2 : D i s s o l v e 3 . 4 8 g o f d ip o t a s s iu m h yd r o g e n or t h o p ho s p ha te
i n w a t e r t o ma k e a 1 0 0 ml o f s o l u ti o n .
M i x S o lu t io n 1 a n d S o l u ti o n 2 an d ad j u s t th e p H t o 5 .0 .
3. Reference standard
D e x a me t h a s o n e : T h is product c o n t a in s not le s s t h an 99% of
d e x a me t h a s o n e , a n d i t s me l t i n g p o i n t i s 2 6 2 -2 6 4 ° C .
4. Procedure
a . E x t r a ct io n
We i g h 5 .0 0 g o f t h e t es t s a mp l e , p r e v i o u s l y g r o u n d , a n d h o mo g e n i z e
i t w i t h 3 0 ml o f 9 5 % a c e t o n i t r i le s o l u t io n . C e n t r i f u g e t h e mi x t u r e a t
2 , 5 00 r p m f o r f i v e mi n u t e s a n d co l l e ct th e ac e t on i t r i l e l a ye r. A d d 3 0
ml o f 9 5 % a c e t o n i t r i le s o l u ti o n to t h e r e s i d u e an d r ep e a t th e ab o v e
p r o c e d u r e , a n d th e n c o l l e c t t h e a ce t o n i t r i l e la y e r.
b . C l ea n - u p
i . F l o r i s i l fo r c o l u mn c h r o ma t o g r a p h y
A d d 8 g o f f l o r i s i l f o r c o l u mn c h r o m a t o g r ap h y s u s p e n d e d in
a c e t o n i t r i le i n t o a ch r o ma t o g r a p h t u b e ( 1 5 m m i n i n n e r d i a me t e r,
3 0 0 mm i n l e n g t h ) . S p i l l o u t t h e a c et o n i t ri l e u n ti l o n l y a s ma l l
a mo u n t r e ma i n s o n t h e p a ck i n g o f t h e c o lu mn a n d p o u r 1 0 0 ml o f
a c e t o n i t r i le, a n d t h en d i s c a r d t h e e ff l u e n t . P o u r t h e s o l u ti o n
o b t ai n ed b y t h e ex t r a cti o n d e s c r i be d i n 4 - a int o th e co l umn f o l l o w e d
b y 3 0 ml o f a c e t o n it r i l e a n d th e n c o l l e ct t h e e l u a te i n t o a 3 0 0 - ml
s e p a r a t i ng f u n n e l . A d d 5 0 ml o f n - h e x an e th e f u n n e l an d s h a k e i t
v i g o r o u s l y u s i n g a s h a k e r f o r t h r e e mi n u t e s , a n d t h en l e a v e i t to
s t a n d . C o l le c t th e a c e to n i t r il e la y e r i n to a r o t a r y v a c u u m e v a p o r a to r
a n d r e mo v e t h e a c et o n it r i l e at 4 0 °C o r lo w e r. D i s s o l v e th e r e s i d u e in
4 ml o f p h os p h a t e b u ffe r s o l u t io n ( p H 5 . 0 ) a n d a d d 6 ml o f w a t e r.
i i . O c t a d e cy l s i l a n e - b o n d e d s i l i ca g e l co l u mn c h r o ma t o g r a p h y
P o u r 1 0 ml o f me t h a n o l , 1 0 ml o f w a t e r a n d 2 ml o f p h o sp h a te b u ff e r
s o l u t io n (pH 5.0) in t o the o c t a d ec y l s i la n e - b o n d ed s i l i ca g el
c a r t r i d g e co l u mn ( 3 6 0 mg ) i n t ha t o r d e r, an d di s c a r d t h e e ff l ue nt .
P o u r t h e s o l u ti o n o b ta i n ed b y c h r o ma t o g r a p h y d e s c r ib e d in 4 - b- i
i n t o t h e c olu mn f o l l o w e d b y 5 ml o f p h o sp h at e bu ff e r s o l u ti o n ( p H
5 . 0 ) a nd 10 ml o f 2 5 % me t h a n o l s o lu t io n , . a n d t h en d i s c a r d th e
e ff l u e n t . Po u r 1 0 ml o f 6 0 % a c et o n it r i l e s ol u t io n i nt o t h e c o lu mn
a n d c o l l e ct t h e e l u at e i n t o a r o ta r y v a c u u m e v a p o r a t o r, a n d t h en
r e mo v e t h e a c e to n i t r i le a n d w a t er a t 4 0 ° C o r l o w e r. D i s s o l v e th e

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r e s i d u e i n 0 . 5 ml o f 1 0 % a c e to n it r i l e s o l u t io n , w h i c h is u s e d a s t h e
s a mp l e s o l u t i o n .
5 . D e t e r mi n a t io n
a . Q u a l i ta t iv e t e s t s
P e r f o r m q u a l i t at i v e t es t s u n d e r th e f o l l o w in g c o n d i t io n s . Te s t r e s u l t s
o b t ai n ed mu s t b e t h e s a me a s t h o s e o b t a i n ed f o r t h e r e f e r e n c e
standard.
Te s t i n g c o n d i t io n s
C o l u mn p a c k i n g : O c ta d e c y l s i l an e - b o n d e d s i l i c a g e l ( 2 - 5 µ m in
p a r t i c le s i ze ) .
C o l u mn : A s t a i n l e s s t ub e ( 2 .0 - 6 . 0 m m i n i n n er d i a me t e r, 1 0 0 - 2 5 0 m m
i n l en g th ) .
C o l u mn t e mp e r a t u r e : 4 0 ° C
M o b il e p h a s e : U s e a c e t o n i t r i le/ f o r mi c a c i d / w a t e r (1 , 2 0 0 : 1 : 8 0 0 ) .
Adjust t he flow rate so t h at d e x a me t h a s o n e flows out in
a p p r o x i ma t e l y 7 - 1 0 mi n u t e s .
b . Q u a n ti t at i v e t e s t s
D e t e r mi n e t h e q u a n ti t y f r o m t h e t e s t r e su l t s o b t a in e d u n d e r th e
c o n d i t io n s d e s c r i b ed in 5 - a u si n g e i t he r t h e p e a k h e ig h t o r p e ak a re a
me t h o d .

( 6 ) A n a l yt i c a l me t h o d f o r t r i a z o p h o s a n d p a r a t h io n
P e r f o r m a c c o r d i ng to 6 ( 1 0 ) .

( 7 ) A n a l y t i c a l me t h o d f o r a l p h a - t re n b o l o n e an d b e t a - t r en b o lo n e
1. Apparatus
A h i g h - p er f o r ma n c e l i q u id ch r o ma t o g r a p h with an u l t r av io l e t
s p e c t r o p h o to me t r i c d e te c t o r ( H P L C -U V ) a n d a l iq u i d ch r o ma t o g r a p h /
m a s s s p e c t r o me t e r ( L C / M S o r L C / M S / M S )
2 . R e ag e n t s a n d t e s t s o l u t io n s
I n a d d i t io n t o t h e r e ag e n t s a n d te s t s o lu t i o n s l i s t ed b e l o w, u s e th o s e
l i s t e d in S e c t i o n C R e a g e n ts /Te s t S o l u ti o n s , E t c . , P a r t I I F o o d
a d d it i v e s . R e a g en t s d e s i g n at e d as “s p e c i a l g r a d e ” i n th i s s e c ti o n mu s t
me e t t h e re q u i r e me n t s f o r “s p e c i a l g r a d e” s p e c i f ie d i n t h e J ap a n
I n d u s t r i a l St a n d a r d s fo r t h e r e ag e n t s .
We a k l y b as i c anion e x ch a n g er cartridge c o l u mn (500 mg ) : A
p o l y e t h y l en e c o l u mn o f 8 - 9 mm i n i nn e r d i ame t e r p a c k e d w i t h 5 0 0 mg
o f d i e t h yl ami n o p r o p yl w e a k l y b a s i c a n i o n e x c h an g e r p r o d u ce d f o r

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c o l u mn c h r o ma t o g r a p h y, o r a c o l u mn e q u i v a le n t t o t h e sp e c i f i ed o n e
i n s e p a r at io n c ap a b i l it y.
H y d r o x y p r o p y l d e x t r an f o r c o l u mn c h r o ma t o g r a p h y : D e x t r a n ( 2 5 - 1 0 0
µ m i n p a r t ic l e s i ze ) ch e mi c a l l y b o n d e d w i th a h y d r o x y p r o p y l g r o u p
p r o d u ce d fo r c o l u mn c h r o ma t o g r a p h y.
D i c h l o r o me t h a n e : D i c h l o r o me t h a n e ( s p e c i a l g r a d e ) .
3. Reference standard
A l p h a - t r e nb o l on e : Th i s p r o d u ct c o n ta i n s not less than 94% of
a l p h a - t r e n b o l o n e , an d i t s me l t i n g p o i n t i s 11 0 ° C .
B e t a - t r e n bo l o ne : Th is p r o d u ct contains not less t h an 99% of
b e t a - t r e n b o l o n e , a n d i ts me l t i n g p o i n t i s 1 8 6 ° C .
4 . P r o c e du r e
a . E x t r a ct io n
We i g h 5 .0 0 g o u t o f t e s t s a mp l e , p r e v io us l y g r o u n d. F o r mu s c l e ,
r e mo v e t h e f a t l a y e r as mu c h a s p o s s i b l e b e fo r e g r i n di ng .
A d d 2 0 ml o f a c e to n i t ri l e / me t h a n o l ( 4 :1 ) a n d h o mo g e n iz e t h e mi x t u r e
a g a in . Ce n t r i f u g e a t 2 , 6 00 r p m f o r f i v e mi n u t e s a nd co l l ec t th e
s u p e r n a ta n t in a 1 0 0 - ml separating f un n e l . Add 20 ml of
a c e t o n i t r i le/ me t h a n o l ( 4 : 1 ) t o th e p r e ci p ita t e an d cen t r i f u g e t h e
mi x t u r e u n d e r t h e a b o v e c o nd i ti on s , a n d t hen c o mb i n e th e a c e to n itr i l e
a n d me t h a n o l l a ye r to t h e s e p ar a t i n g f u n n e l a b o v e . A d d 2 0 ml o f
w a t e r- s a t u r a t e d n - h exa n e t o t h e f u n n e l a n d s h a k e i t v i g o r o u sl y f o r
f i v e mi n u t es , a n d t h e n l e a v e i t t o s t a n d . Tr an s f e r t h e a c et o n it r i l e an d
me t h a n o l la y e r t o a 2 0 0 - ml s e p a r a t i n g f u n n e l . A d d 4 0 ml e a c h o f 5 %
s o d i u m s u l f a t e s o lu t ion an d d i ch lo r o me t h a n e t o t h e f u n n e l a n d s h ak e
i t v i go r o u s ly f o r f i v e mi n u t e s a n d l e a v e i t to s t a n d , a n d t h en t r a n s f e r
t h e di c hl o ro me t h a n e la y e r i n t o a r o t a r y v a cu u m e v a p o r a t o r. A d d 1 5
ml o f d i c h l o r o me t h a n e t o th e aq u e o u s l a ye r a n d r e p e a t t h e a b o v e
p r o c e d u r e a n d c o mb i n e th e d i ch l o r o me t h a n e la y e r i n t o t h e r o ta r y
v a c u u m e v a p o r a to r, an d t h en r emo v e t h e d i c h lo r o me t h a n e a t 4 0 °C o r
l o w e r. D i s s o l v e t h e r es i d u e i n 1 ml o f n - h e x a n e /b e n ze n e ( 3 : 1 ) .
b . C l ea n - u p
i . We a k l y b a s i c a n i o n e x c h an g e r c o l u mn c h r o ma t o g r a p h y
P o u r 6 ml o f n - h e x an e / b e n z en e ( 3 : 1 ) i n t o a w e a k l y b a s i c a n io n
e x c h an g e r c a r t r i d g e co l u mn ( 5 0 0 mg ) a n d d is c a r d t h e e ff l u e n t . P o ur
t h e th e s o lu t i o n o b t a in e d b y th e e x t r a c ti o n d e s c r i b ed in 4 - a i nt o the
c o l u mn f o l l o w e d b y 2 ml o f n - h e x a n e /b e n ze ne ( 3 : 1 ) a n d d i s c a r d t he

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e ff l u e n t . P o u r 3 ml o f d i ch lo r o me t h a n e /me t h a n o l ( 9 : 1 ) i n to th e
c o l u mn a n d c o l l e ct th e e lu a t e in t o a ro t a r y v a c u u m e v a p o r a t o r, a nd
t h e n e va p o ra t e t h e s o lv e n t t o n e a r d r y n e s s u n d e r n it r o g en a t 4 0 ° C o r
l o w e r. D i s s o l v e t h e r es i d u e i n 0 .5 ml o f b e n z e n e / me t h a n o l ( 1 7 : 3 ) .
i i . H yd r o x yp r o p yl d ex tr a n f o r c o lu mn c h r o ma t o g r ap h y
A d d h y d r o x y p r o p y l d e x t r a n f o r c o l u mn c h r o ma t o g r a p hy s u s p e n d ed
i n b e nz e n e/me t h a n o l ( 1 7 : 3 ) , p r e v i o u s l y le f t t o s ta n d f o r 1 2 h o u r s ,
i n t o a p o l y e t h y l e n e c o l u mn ( 6 mm i n i n n e r d i a me t e r ) s o a s t o o b tai n
a 1 20 - mm l o n g l a ye r o f d ex t r an . S p i ll o u t t h e b en ze n e / me t h a n o l
( 1 7 : 3 ) u n t il o n l y a sma l l a mo u n t r e ma i n s o n th e p ac k i n g o f th e
c o l u mn . P o u r t h e s o lu ti o n ob t ai n ed b y c h r o ma t o g r ap h y d e s c r i b ed in
4 - b - i i n to t h i s c o l u mn f o l l o w e d b y 1 0 ml o f b e n z en e / me t h a n o l
( 1 7 : 3 ) . D i sc a r d 2 .0 ml o f th e f i r s t e ff l u e n t a n d co l l ec t th e n e x t
e l u a te i n to a r o t a r y v a c u u m e va p o r a to r, an d t h en e va p o r a t e th e
s o l v e n t to n e a r d r y n es s u n d e r n it r o g e n a t 4 0 ° C o r l o we r. D i s s o l v e
t h e r e s i d u e i n 0 .5 ml o f a c e t o n i t r ile / w a t e r ( 1 :1 ) , w h i c h i s u s e d a s th e
s a mp l e s o l u t i o n .
5 . D e t e r mi n a t io n
a . Q u a l i ta t iv e t e s t s
P e r f o m q u al i t a ti v e t e s ts u n d e r t h e f o l l o w i n g c o n d i t io n s . Te s t r e s u lts
o b t ai n ed mu s t b e t h e s a me a s t h o s e o b t a i n ed f o r t h e r e f e r e n c e
standard.
Te s t i n g c o n d i t io n s
C o l u mn p a c k i ng : O c t ad e c y l s i l a n e- b o n d ed s i li c a g e l ( 5 µm i n p a r t i cl e
size).
C o l u mn : A s t a i n l e s s tu b e ( 4 . 0 - 6 .0 mm i n i n n e r d i a me t e r, 1 5 0 mm i n
length).
C o l u mn t e mp e r a t u r e : 4 0 ° C
D e t e c t o r : Op e r a t e w i th a n ab s o r p ti o n w a v e l en g t h o f 3 4 0 n m.
M o b il e p h as e : U s e ace t o n i t r i l e/w a t e r ( 5 : 6 ). A d j u s t th e f l o w r a te s o
t h a t a l p h a - tr e n b o lo n e fl o w s o u t i n a p p r ox i ma t e l y 1 0 mi n u t e s .
b . Q u a n ti t at i v e t e s t s
D e t e r mi n e t h e q u a n ti t y f r o m t h e t e s t r e su l t s o b t a in e d u n d e r th e
c o n d i t io n s d e s c r i b ed in 5 - a u si n g e i t he r t h e p e a k h e ig h t o r p e ak a re a
me t h o d .
c . C o n f i r ma t i o n t e s t s
Perform l i q u id c h ro ma t o g r a p h y / ma s s s p e c t r o me t r y under th e

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c o n d i t io n s d e s c r i b ed in 5 - a . Te s t r e s u l t s o bt ai n e d mu s t b e t h e s a me a s
t h o s e o b t a in e d f o r t h e r e f e r e n c e s t a nd a r d . D e t e r mi n e t h e q u an t ity
u s i n g e i th er t h e p ea k h e i gh t o r pe a k a r e a me t h od , i f r eq u i r e d .

( 8 ) E t h y l e n e d i b r o mi d e a n a l yt ic a l me t h o d
1. Apparatus
A g a s c h r o ma t o g r a p h w i t h a n e lec t r o n c ap t u r e d e t e c to r ( G C - E C D ) , a
g a s c h r o ma t o g r ap h / ma s s s p e c t r o me t e r ( G C / M S o r G C / M S / M S ) a n d
D e a n St a r k d i s t i ll a t io n a p p a r at u s
T h e D e a n St a r k d i s t il l a t io n ap p a r a t u s i s r o u g h l y a s s h o w n i n th e
following figure:

A : D i s t i l l a ti o n f l a sk
B : C o o l in g t u b e
C : D i s t i l la ti o n t r ap

2 . R e ag e n t s a n d t e s t s o l u t io n s
I n a d d i t io n t o t h e r e ag e n t s a n d te s t s o lu t i o n s l i s t ed b e l o w, u s e th o s e
l i s t e d in S e c t i o n C R e a g e n ts /Te s t S o l u ti o n s , E t c . , P a r t I I F o o d
a d d it i v e s .
S y n t h e t i c ma g n e s i u m s i l i c at e (F l o r i s i l ) f o r c o l u mn c h r o ma t o g r a p h y:
H e a t f l o r i s il ( 1 5 0 -2 5 0 μ m i n p a r t ic l e s i ze ) a t 1 3 0 ° C f o r 1 2 h o u r s o r
l o n g e r. C o o l d o w n t o ro o m t e mp e r a t u r e in a d e s i c c a to r.
S i l i c o n e f o r d e f o a mi n g : S i l i co n e p r o d u ce d fo r d e f o a mi n g .
n - H e x a n e : U s e a r o t a r y v a c u u m e v a p o r at o r on 3 00 mL o f n - h e x a n e t o
e v a p o r a t e u n t i l 5 mL i s l e f t . F o r a n a l y s i s , i n j e c t 5 μ L o f t h e s o lu t io n
i n t o a G C - E C D . P e a k s o t h e r th an t h at o f n - h e x a n e i n t h e r e s u l t in g
c h r o ma t o g r a m s h o u l d b e a s h ig h a s o r lo w e r t h an t h e pe a k s o f γ - B H C
a t 2 x 10 - 1 1 g .
Wa t e r : D i s ti l l e d w a t e r. I f t h e d i s ti l l e d w a t e r i s f o u n d to i n c l u d e an y
s u b s t a n ce th a t ma y i n t e r f e r e w i t h a n a l y s i s o f t h e t a rg et co mp o s i t i o n a l
s u b s t a n ce s f r o m a g r i c u l t u r a l ch emi c a l s , w a s h w i t h a s o l v en t s u c h a s
n-hexane before use.
3. Reference standard
E t h y l e n e d i b r o mi d e : T h i s p ro d u c t co n ta i n s n o t l e s s t h a n 9 9 % o f

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e t h yl e n e d ib r o mi d e , an d i t s b o il in g p o i n t i s 1 3 1 .5 ° C .
4. Procedure
a . E x t r a ct io n
H o mo g e n i ze a b ou t 1 k g o f a t e st s a mp l e a n d t r a n s f e r 1 0 0 g o f th e
h o mo g e n i z e d s a mp l e in t o a 1 , 0 0 0 - ml d i s t i l l at i o n f la s k , a n d th e n ad d
2 0 0 ml o f w a t e r a n d 10 ml o f n - h e x a n e to th e f l a s k . A d d a f e w d r o p s
o f d e f o a mi n g s il i co n e a n d b o i l in g s t o n e s to th e mi x t u r e a n d a t ta c h th e
f l a s k t o th e D e a n St a r k d i s t i l lat i o n ap p ar a t u s t o b e h e at e d an d
r e f l u x e d f o r o n e h o u r. A f t e r c o o l in g , r e mo v e mo s t o f t h e w a t e r i n th e
d i s t i ll a t io n t r a p a n d f il t e r t h e n - h e x a n e l a y e r i n t o a 1 0 - ml me a s u r i n g
f l a s k t h r o u g h a l i q u id p h a s e s e pa r a t i o n f i l te r p a p e r. Wa s h t h e tr a p
w i t h a s ma l l a mo u n t o f n - h e x a n e an d f i l t e r t h e w a s h i n g s t h r o u g h t h e
f i l t e r p ap e r a b o v e . C o mb i n e t h e f i l t r a t e w i th th e n - h ex a n e l a y e r t o
ma k e e x a c tl y a 1 0 - ml s o l u t io n .
b . C l ea n - u p
Tr a n s f e r t h e s o l u ti o n o b t ai n ed b y t h e ex t r a c ti o n d e s c r i b e d i n 4 - a t o a
1 0 - ml t e s t tu b e w it h a g l a s s s t o p p e r. A d d a p p r o x i ma t e l y 1 g o f f l o r is i l
f o r c o l u mn c h r o ma t o g r a p h y t o th e t e s t tu b e a n d s h ak e it v i g o r o u s ly,
a n d t h en l ea v e i t t o s ta n d a t r o o m t e mp e r a t u r e f o r ab o u t 1 5 mi n u te s .
C o l l ec t a b o u t 5 ml o f s u p e r n a ta n t i n a n o th e r t e s t t u b e w i t h a g l a s s
s t o p p e r, w h i c h i s u s e d a s t h e s a mp l e s o lu t i o n .
5 . D e t e r mi n a t io n
a . Q u a l i ta t iv e t e s t s
P e r f o r m q u a l i t at i v e t es t s u n d e r th e f o l l o w in g c o n d i t io n s . Te s t r e s u l t s
o b t ai n ed mu s t b e t h e s a me a s t h o s e o b t a i n ed f o r t h e r e f e r e n c e
standard.
Te s t i n g c o n d i t io n s
C o l u mn : A s i l i c a te g l as s c a p i ll a r y c o lu mn ( 0 . 3 2 mm i n i n n e r d i a me t e r,
l e n gt h i n 30 m) c o a t ed w i th 6 % c y a n o p r o p yl p h en y l - me t h y l s i li c o n e
f o r g a s c h ro ma t o g r a p h y t o a t h i c k n e s s o f 1 . 8 µ m.
C o l u mn t e mp e r a t u r e : H o l d t h e c o l u mn t e mp e r a t u r e a t 5 0 ° C f o r t w o
mi n u t e , f o ll o w e d b y an in c r e a s e o f 5 ° C e v e ry mi n u t e u n t i l r e a c h i n g
110 ° C , a f t er w h i c h i n cr e a s e t h e te mp e r a t u r e b y 3 0 ° C ev e r y mi n u t e
u n t il r e a c h in g 2 6 0 ° C , a n d h o ld f o r f i v e mi n u t e s .
I n l e t t e mp e r a t u r e : 2 5 0 ° C
D e t e c t o r : Op e r a t e at 3 0 0 ° C
G a s f l o w r a t e : U s e h el i u m a s t h e c a r r i e r g a s . A d j u s t th e f l o w r a t e s o

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t h a t e t h y l en e d ib r o mi d e f l o w s o ut i n ap p r oxi ma t e l y e i g h t mi n u t e s .
b . Q u a n ti t at i v e t e s t s
D e t e r mi n e t h e q u a n ti t y f r o m t h e t e s t r e su l t s o b t a in e d u n d e r th e
c o n d i t io n s d e s c r i b ed in 5 - a u si n g e i t he r t h e p e a k h e ig h t o r p e ak a re a
me t h o d .
c . C o n f i r ma t i o n t e s t s
P e r f o r m g a s c h r o ma t o g r a p h y / ma s s s p e c t r o me t r y u n d e r t h e co n d i t io n s
d e s c r i b ed in 5 - a . Te st r e s u l t s o b t a i n ed mu s t b e th e s ame a s t h o s e
o b t ai n ed f o r t h e r e f e r e n c e s t a n d a r d . D e t er mi n e t h e q u a n t i t y u si n g
e i t h e r th e pe a k he i gh t o r p e a k a r ea me t h o d , if r e q u i r e d .

8 . I n c a s e s w h e r e a s u b s t a n c e u s e d a s a n i n g r e d i e n t o f a n a g r i c u l tu r a l
c h e mi c a l ( e x c lu d in g s u b s t a n ce s d et e r mi n e d b y t h e M i n i s te r o f H e a l th ,
L a b o u r an d We l f a r e a s h a v in g n o p o t e n ti a l t o c au s e d a ma g e t o h u ma n
h e a l th u n d e r P a r a g r a p h 3 o f A r t i c l e 11 o f t h e L aw ) , f o r w h i c h no
c o mp o s i t i o n a l st a n d a r d s a r e s ti p u l a t ed i n 5 to 7 , i s t h e s a me a s th e
s u b s t a n ce n a t u r a ll y co n t ai n ed in t h e f o o d , t h e a mo u n t o f th e s u b s t a n ce
c o n ta i n ed in t h e f o o d s h a l l n o t ex c e e d th e amo u n t n o r ma l l y c o n t a i n ed i n
t h e f o o d . Th i s , h o w e v e r, d o e s n ot a p p l y t o f o o d s c o nta i n in g s u b st a n c es
t h a t ma y c a u s e d a ma g e t o h u ma n h e a l t h a t th e a mo u n t n o rma l l y c o n t a i n e d .

9 . I n c a s e s w h e r e s u b st a n c e s u s e d a s i n g r e d ie n t s o f ag r ic u l tu r a l ch e mi c a l s
a n d o th e r ch e mi c a l s u b s t a n ce s th a t a r e l i s ted in t h e f i r s t c o l u mn o f t h e
f o l l o w i n g ta b l e a r e c o n t a in e d in a f o o d , t h e a mo u n t o f t h e s u b s t an c e s
s h a l l n o t ex c e e d t h e a mo u n t s t ip u l at e d i n t h e th i r d co l u mn o f t h e s a me
t a b l e a c co r d i n g t o t he f o o d ca t e g o r i e s s h o w n i n th e s e co n d co l u mn o f t h e
s a me t a b l e .

T h e ma x i mu m r e s i d u e l i mi t s o f s u b s t a n c e s u s e d a s i n g r e d ie n t s o f
a g r i c u lt u r al c h e mi c a l s i n f o o d s ( e x c l u d i n g f o o d s l i st e d i n th e s e c o n d
c o l u mn o f t h e t ab l e i n 6 ( 1 ) a n d th e s e c o n d co l u mn o f t h e t a b l e in 7 ( 1 ) ) :

C o l u mn 1 C o l u mn 2 C o l u mn 3

AZINPHOS-METHYL O t h e r s p i c es , d r i e d 0.5 ppm

A C E P H AT E O t h e r s p i c es , d r i e d ( e x c e p t t h e 0 . 2 p p m
f r u i t o f S a n s h o ( J a p a n es e
pepper))

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from the Japanese Original

AMITRAZ C o t to n s e ed o i l ( e xc e pt r e f i n e d 0 . 0 5 p p m
c o t to n s e ed o i l an d co t to n s e e d
s a l a d o i l t h a t me e t t h e J a p a n e s e
A g r i c u l t u r a l St a n d a r d s ( J A S )
f o r E d i b l e Ve g e t ab l e F a t s a n d
O i l s , a n d o t h e r e d i b l e o i l s th a t
me e t s t a n d a r d s e q u i v al e n t t o o r
s t r i c t e r t h an J A S )

ALDICARB P e a n u t o i l ( l i mi t e d t o r e f i n e d 0.01 ppm

peanut oil and peanut salad oil
t h a t me e t t h e J A S f o r E d i b le
Ve g e t ab l e F a t s a n d O il s , a n d
o t h e r ed i b le o i l s t h a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )
C o t to n s e ed o i l ( l i mi t e d t o 0.01 ppm
r e f i n e d co t to n s e e d o il a n d
c o t to n s e ed s a l a d o i l th a t me e t
t h e J A S f o r E d i b le Veg e t a b le
F a t s a n d O il s , a n d o th er e d i b le
o i l s th a t me e t s t an d a rd s
e q u iv a l en t t o o r s t r i c te r t h a n
JAS)
IPRODIONE O t h e r s p i c es , d r i e d ( l imi t e d t o 0.05 ppm
seeds)
O t h e r s p i c es , d r i e d ( l imi t e d t o 0.1 ppm
r o o t s o r r h iz o me s )

IMIDACLOPRID W h e a t f l o u r ( e x c e p t wh o l e 0.03 ppm

grain)

W h e a t b r an 0.3 ppm

ETHION O t h e r s p i c es , d r i e d ( l imi t e d t o 5 ppm

fruits)

O t h e r s p i c es , d r i e d ( l imi t e d t o 3 ppm
seeds)
O t h e r s p i c es , d r i e d ( l imi t e d t o 0.3 ppm
r o o t s o r r h iz o me s )

ETHEPHON Raisin 5 ppm

Fig, dried 10 ppm

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Provisional Translation
from the Japanese Original

E N D O S U L FA N C o t to n s e ed o i l ( e xc e pt r e f i n e d 0 . 5 p p m
c o t to n s e ed o i l an d co t to n s e e d
s a l a d o i l th a t me e t t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )
O t h e r s p i c es , d r i e d ( l imi t e d t o 5 ppm
fruits)
O t h e r s p i c es , d r i e d ( l imi t e d t o 1 ppm
seeds)
O t h e r s p i c es , d r i e d ( l imi t e d t o 0.5 ppm
r o o t s o r r h iz o me s )
C A R B A RY L R i c e b r an 170 ppm

M i l l ed r i c e 1 ppm

W h e a t f l o u r ( e x c e p t wh o l e 0.2 ppm
grain)

Wheat germ 1 ppm

W h e a t b r an 2 ppm

C o r n o i l ( ex c e p t ed i b le c o r n o il 0 . 1 p p m
t h a t me e t s t h e J A S f o r E d i b le
Ve g e t ab l e F a t s a n d O il s , a n d
o t h e r ed i b le o i l s t h a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )

S o y b e a n o il ( e x c ep t ed i b le 0.2 ppm
s o y b e a n o i l t h a t me e t s t h e J A S
f o r E d i b l e Ve g e t ab l e F a t s a n d
O i l s , a n d o t h e r e d i b l e o i l s th a t
me e t s t a n d a r d s e q u i v al e n t t o o r
s t r i c t e r t h an J A S )
To ma t o j u ic e 3 ppm

To ma t o p a st e 10 ppm

E d i b le o li v e o i l ( li mi t e d t o 25 ppm
v i rg in o il )
Sunflower oil (except edible 0.05 ppm
s u n f l o w e r o i l th a t me e t s t h e
J A S f o r E d i b l e Ve g et ab l e F a t s
a n d O i l s , an d o t h e r ed i b l e o i l s
t h a t me e t s t a n d a r d s e q u i v al e n t
t o o r s t r i c te r t h a n J A S )

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Provisional Translation
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Q U I N TO Z E N E O t h e r s p i c es , d r i e d ( l imi t e d t o 0.02 ppm

fruits)
O t h e r s p i c es , d r i e d ( l imi t e d t o 0.1 ppm
seeds)
O t h e r s p i c es , d r i e d ( l imi t e d t o 2 ppm
r o o t s o r r h iz o me s )

G LY P H O S AT E C o t to n s e ed o i l ( l i mi t e d t o 0.05 ppm
r e f i n e d co t to n s e e d o il a n d
c o t to n s e ed s a l a d o i l th a t me e t
t h e J A S f o r E d i b le Veg e t a b le
F a t s a n d O il s , a n d o th er e d i b le
o i l s th a t me e t s t an d a rd s
e q u iv a l en t t o o r s t r i c te r t h a n
JAS)
C o t to n s e ed o i l ( e xc e pt r e f i n e d 0 . 0 5 p p m
c o t to n s e ed o i l an d co t to n s e e d
s a l a d o i l th a t me e t t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )

G L U F O S I N AT E Sunflower oil (except edible 0.05 ppm

s u n f l o w e r o i l th a t me e t s t h e
J A S f o r E d i b l e Ve g et ab l e F a t s
a n d O i l s , an d o t h e r ed i b l e o i l s
t h a t me e t s t a n d a r d s e q u i v al e n t
t o o r s t r i c te r t h a n J A S )
R a p e s e ed o i l ( e x c ep t re f i n e d 0.05 ppm
r a p e s e e d o il a n d r ap e se e d s al a d
o i l th a t me e t t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )

KRESOXIM-METHYL Raisin 2 ppm

E d i b le o li v e o i l ( li mi t e d t o 0.7 ppm
v i rg in o li ve o i l )

CLETHODIM S o y b e a n o il ( l i mi t e d to e d ib l e 0 . 5 p p m
s o y b e a n o i l t h a t me e t s t h e J A S
f o r E d i b l e Ve g e t ab l e F a t s a n d
O i l s , a n d o t h e r e d i b l e o i l s th a t
me e t s t a n d a r d s e q u i v al e n t t o o r
s t r i c t e r t h an J A S )

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Provisional Translation
from the Japanese Original

S o y b e a n o il ( e x c ep t ed i b le 1 ppm
s o y b e a n o i l t h a t me e t s t h e J A S
f o r E d i b l e Ve g e t ab l e F a t s a n d
O i l s , a n d o t h e r e d i b l e o i l s th a t
me e t s t a n d a r d s e q u i v al e n t t o o r
s t r i c t e r t h an J A S )

Sunflower oil (except edible 0.1 ppm

s u n f l o w e r o i l th a t me e t s t h e
J A S f o r E d i b l e Ve g et ab l e F a t s
a n d O i l s , an d o t h e r ed i b l e o i l s
t h a t me e t s t a n d a r d s e q u i v al e n t
t o o r s t r i c te r t h a n J A S )

C o t to n s e ed o i l ( l i mi t e d t o 0.5 ppm
r e f i n e d co t to n s e e d o il a n d
c o t to n s e ed s a l a d o i l th a t me e t
t h e J A S f o r E d i b le Veg e t a b le
F a t s a n d O il s , a n d o th er e d i b le
o i l s th a t me e t s t an d a rd s
e q u iv a l en t t o o r s t r i c te r t h a n
JAS)

C o t to n s e ed o i l ( e xc e pt r e f i n e d 0 . 5 p p m
c o t to n s e ed o i l an d co t to n s e ed
s a l a d o i l th a t me e t t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )

R a p e s e ed o i l ( l i mi t e d t o 0.5 ppm
r e f i n e d r a pe s e e d oi l an d
r a p e s e e d s al a d o i l t h at me e t t h e
J A S f o r E d i b l e Ve g et ab l e F a t s
a n d O i l s , an d o t h e r ed i b l e o i l s
t h a t me e t s t a n d a r d s e q u i v al e n t
t o o r s t r i c te r t h a n J A S )

R a p e s e ed o i l ( e x c ep t re f i n e d 0.5 ppm
r a p e s e e d o il a n d r ap e se e d s al a d
o i l th a t me e t t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )
CHLORDANE M i l l ed r i c e 0.02 ppm

86/100
Provisional Translation
from the Japanese Original

S o y b e a n o il ( l i mi t e d to e d ib l e 0 . 0 2 p p m
s o y b e a n o i l t h a t me e t s t h e J A S
f o r E d i b l e Ve g e t ab l e F a t s a n d
O i l s , a n d o t h e r e d i b l e o i l s th a t
me e t s t a n d a r d s e q u i v al e n t t o o r
s t r i c t e r t h an J A S )
S o y b e a n o il ( e x c ep t ed i b le 0.05 ppm
s o y b e a n o i l t h a t me e t s t h e J A S
f o r E d i b l e Ve g e t ab l e F a t s a n d
O i l s , a n d o t h e r e d i b l e o i l s th a t
me e t s t a n d a r d s e q u i v al e n t t o o r
s t r i c t e r t h an J A S )
C o t to n s e ed o i l ( e xc e pt r e f i n e d 0 . 0 5 p p m
c o t to n s e ed o i l an d co t to n s e e d
s a l a d o i l th a t me e t t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )
L i n s e e d o i l ( e x c e pt r e f i n e d 0.05 ppm
l i n s e ed o il )

CHLORPYRIFOS W h e a t f l o u r ( e x c e p t wh o l e 0.1 ppm

grain)

C o r n o i l ( limi t e d t o e d i b l e c o r n 0 . 2 p p m
o i l th a t me e t s t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )
S o y b e a n o il ( l i mi t e d to e d ib l e 0 . 0 5 p p m
s o y b e a n o i l t h a t me e t s t h e J A S
f o r E d i b l e Ve g e t ab l e F a t s a n d
O i l s , a n d o t h e r e d i b l e o i l s th a t
me e t s t a n d a r d s e q u i v al e n t t o o r
s t r i c t e r t h an J A S )
C o t to n s e ed o i l ( l i mi t e d t o 0.05 ppm
r e f i n e d co t to n s e e d o il a n d
c o t to n s e ed s a l a d o i l th a t me e t
t h e J A S f o r E d i b le Veg e t a b le
F a t s a n d O il s , a n d o th er e d i b le
o i l s th a t me e t s t an d a rd s
e q u iv a l en t t o o r s t r i c te r t h a n
JAS)

87/100
Provisional Translation
from the Japanese Original

C o t to n s e ed o i l ( e xc e pt r e f i n e d 0 . 0 5 p p m
c o t to n s e ed o i l an d co t to n s e e d
s a l a d o i l th a t me e t t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )
O t h e r s p i c es , d r i e d ( l imi t e d t o 1 ppm
fruits)
O t h e r s p i c es , d r i e d ( l imi t e d t o 5 ppm
seeds)
O t h e r s p i c es , d r i e d ( l imi t e d t o 1 ppm
r o o t s o r r h iz o me s )
C H L O R P Y R I F O S - M E T H Y L W h e a t f l o u r ( e x c e p t wh o l e 2 ppm
grain)

W h e a t b r an 20 ppm

O t h e r s p i c es , d r i e d ( l imi t e d t o 0.3 ppm

fruits)
O t h e r s p i c es , d r i e d ( l imi t e d t o 1 ppm
seeds)
O t h e r s p i c es , d r i e d ( l imi t e d t o 5 ppm
r o o t s o r r h iz o me s )

C H L O R M E Q U AT W h e a t f l o u r ( l i mi t e d t o w h o l e 5 ppm
grain)

W h e a t f l o u r ( e x c e p t wh o l e 2 ppm
grain)

W h e a t b r an 10 ppm

Ry e f l o u r ( l i mi t e d t o w h o l e 4 ppm
grain)
Ry e f l o u r ( e x c e p t w h o l e g r a in ) 3 p p m

Ry e b r a n 10 ppm

F E N B U TAT I N O X I D E
R a p e s e ed o i l ( e x c ep t re f i n e d 0.1 ppm
r a p e s e e d o il a n d r ap e se e d s al a d
o i l th a t me e t t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )
P l u m, d r i e d 1 0 pp m

Raisin 20 ppm

88/100
Provisional Translation
from the Japanese Original

D I C H L O RV O S a n d N A L E D W h e a t f l o u r ( l i mi t e d t o w h o l e 2 ppm
(as total) grain)
W h e a t f l o u r ( e x c e p t wh o l e 1 ppm
grain)

W h e a t b r an 10 ppm

Wheat germ 10 ppm

O t h e r s p i c es , d r i e d 0.1 ppm

D I Q U AT M i l l ed r i c e 0.2 ppm

W h e a t f l o u r ( l i mi t e d t o w h o l e 2 ppm
grain)

W h e a t f l o u r ( e x c e p t wh o l e 0.5 ppm
grain)

W h e a t b r an 5 ppm

Ve g e t ab l e oi l ( e x c ep t re f i n e d 0.05 ppm
v e g et a b le o i l )

DICOFOL P l u m, d r i e d 3 ppm

C o t to n s e ed o i l ( l i mi t e d t o 0.5 ppm
r e f i n e d co t to n s e e d o il a n d
c o t to n s e ed s a l a d o i l th a t me e t
t h e J A S f o r E d i b le Veg e t a b le
F a t s a n d O il s , a n d o th er e d i b le
o i l s th a t me e t s t an d a rd s
e q u iv a l en t t o o r s t r i c te r t h a n
JAS)

C o t to n s e ed o i l ( e xc e pt r e f i n e d 0 . 5 p p m
c o t to n s e ed o i l an d co t to n s e e d
s a l a d o i l th a t me e t t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )

O t h e r s p i c es , d r i e d ( l imi t e d t o 0.1 ppm

fruits)

O t h e r s p i c es , d r i e d ( l imi t e d t o 0.05 ppm

seeds)

O t h e r s p i c es , d r i e d ( l imi t e d t o 0.1 ppm

r o o t s o r r h iz o me s )

D I S U L F O TO N O t h e r s p i c es , d r i e d 0.05 ppm

89/100
Provisional Translation
from the Japanese Original

CYHALOTHRIN C o t to n s e ed o i l ( l i mi t e d t o 0.02 ppm

r e f i n e d co t to n s e e d o il a n d
c o t to n s e ed s a l a d o i l th a t me e t
t h e J A S f o r E d i b le Veg e t a b le
F a t s a n d O il s , a n d o th er e d i b le
o i l s th a t me e t s t an d a rd s
e q u iv a l en t t o o r s t r i c te r t h a n
JAS)
C o t to n s e ed o i l ( e xc e pt r e f i n e d 0 . 0 2 p p m
c o t to n s e ed o i l an d co t to n s e e d
s a l a d o i l th a t me e t t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )

DIPHENYLAMINE Apple juice 0.5 ppm

CYPRODINIL W h e a t b r an 2 ppm

P l u m, d r i e d 5 ppm

Raisin 5 ppm

CYPERMETHRIN O t h e r s p i c es , d r i e d ( l imi t e d t o 0.1 ppm

fruits)
O t h e r s p i c es , d r i e d ( l imi t e d t o 0.2 ppm
r o o t s o r r h iz o me s )
Ve g e t ab l e o i l ( l i mi t e d t o 0.5 ppm
r e f i n e d v e g e t a b le o il )
DIMETHIPIN C o t to n s e ed o i l ( l i mi t e d t o 0.1 ppm
r e f i n e d co t to n s e e d o il a n d
c o t to n s e ed s a l a d o i l th a t me e t
t h e J A S f o r E d i b le Veg e t a b le
F a t s a n d O il s , a n d o th er e d i b le
o i l s th a t me e t s t an d a rd s
e q u iv a l en t t o o r s t r i c te r t h a n
JAS)
C o t to n s e ed o i l ( e xc e pt r e f i n e d 0 . 1 p p m
c o t to n s e ed o i l an d co t to n s e e d
s a l a d o i l th a t me e t t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )

D I M E T H O AT E E d i b le o li v e o i l ( ex c ep t v i rg i n 0.05 ppm
oil)
O t h e r s p i c es , d r i e d ( l imi t e d t o 0.5 ppm
fruits)

90/100
Provisional Translation
from the Japanese Original

O t h e r s p i c es , d r i e d ( l imi t e d t o 5 ppm
seeds)
O t h e r s p i c es , d r i e d ( l imi t e d t o 0.1 ppm
r o o t s o r r h iz o me s )

BROMIDE W h e a t f l o u r ( l i mi t e d t o w h o l e 50 ppm
grain)

P e a c h , d r i ed 50 ppm

P l u m, d r i e d 20 ppm

Raisin 100 ppm

Date, dried 100 ppm

Fig, dried 250 ppm

Fruits (dried fruits except 30 ppm

p e a c h , p lu m, g r a p e , d a t e a n d
fig)
O t h e r h e r b s, d r i e d 400 ppm

SPINOSAD W h e a t b r an 2 ppm

Raisin 1 ppm

C o t to n s e ed o i l ( l i mi t e d t o 0.01 ppm
r e f i n e d co t to n s e e d o il a n d
c o t to n s e ed s a l a d o i l th a t me e t
t h e J A S f o r E d i b le Veg e t a b le
F a t s a n d O il s , a n d o th er e d i b le
o i l s th a t me e t s t an d a rd s
e q u iv a l en t t o o r s t r i c te r t h a n
JAS)
C o t to n s e ed o i l ( e xc e pt r e f i n e d 0 . 0 1 p p m
c o t to n s e ed o i l an d co t to n s e e d
s a l a d o i l th a t me e t t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )

DIAZINON P l u m, d r i e d 2 ppm

O t h e r s p i c es , d r i e d ( l imi t e d t o 0.1 ppm

fruits)

O t h e r s p i c es , d r i e d ( l imi t e d t o 5 ppm
seeds)

O t h e r s p i c es , d r i e d ( l imi t e d t o 0.5 ppm

r o o t s o r r h iz o me s )

91/100
Provisional Translation
from the Japanese Original

THIODICARB and W h e a t f l o u r ( e x c e p t wh o l e 0.03 ppm

METHOMYL grain)

Wheat germ 2 ppm

W h e a t b r an 3 ppm

C o r n o i l ( limi t e d t o e d i b l e c o r n 0 . 0 2 p p m
o i l th a t me e t s t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )
S o y b e a n o il ( l i mi t e d to e d ib l e 0 . 2 p p m
s o y b e a n o i l t h a t me e t s t h e J A S
f o r E d i b l e Ve g e t ab l e F a t s a n d
O i l s , a n d o t h e r e d i b l e o i l s th a t
me e t s t a n d a r d s e q u i v al e n t t o o r
s t r i c t e r t h an J A S )
S o y b e a n o il ( e x c ep t ed i b le 0.2 ppm
s o y b e a n o i l t h a t me e t s t h e J A S
f o r E d i b l e Ve g e t ab l e F a t s a n d
O i l s , a n d o t h e r e d i b l e o i l s th a t
me e t s t a n d a r d s e q u i v al e n t t o o r
s t r i c t e r t h an J A S )
C o t to n s e ed o i l ( l i mi t e d t o 0.04 ppm
r e f i n e d co t to n s e e d o il a n d
c o t to n s e ed s a l a d o i l th a t me e t
t h e J A S f o r E d i b le Veg e t a b le
F a t s a n d O il s , a n d o th er e d i b le
o i l s th a t me e t s t an d a rd s
e q u iv a l en t t o o r s t r i c te r t h a n
JAS)

TEBUCONAZOLE Raisin 3 ppm

TEBUFENOZIDE Raisin 2 ppm

D E LTA M E T H R I N a n d W h e a t f l o u r ( l i mi t e d t o w h o l e 2 ppm
TRALOMETHRIN (as total) grain)
W h e a t f l o u r ( e x c e p t wh o l e 0.3 ppm
grain)

W h e a t b r an 5 ppm
TERBUFOS R a p e s e ed o i l ( e x c ep t re f i n e d 0.05 ppm
r a p e s e e d o il a n d r ap e se e d s al a d
o i l th a t me e t t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )

92/100
Provisional Translation
from the Japanese Original

PA R A Q U AT S u n f l o w e r o i l ( l i mi t e d t o 0.05 ppm
e d i b l e s u n f l o w e r o i l th a t me e t s
t h e J A S f o r E d i b le Veg e t a b le
F a t s a n d O il s , a n d o th er e d i b le
o i l s th a t me e t s t an d a rd s
e q u iv a l en t t o o r s t r i c te r t h a n
JAS)
Sunflower oil (except edible 0.05 ppm
s u n f l o w e r o i l th a t me e t s t h e
J A S f o r E d i b l e Ve g et ab l e F a t s
a n d O i l s , an d o t h e r ed i b l e o i l s
t h a t me e t s t a n d a r d s e q u i v al e n t
t o o r s t r i c te r t h a n J A S )
C o t to n s e ed o i l ( l i mi t e d t o 0.05 ppm
r e f i n e d co t to n s e e d o il a n d
c o t to n s e ed s a l a d o i l th a t me e t
t h e J A S f o r E d i b le Veg e t a b le
F a t s a n d O il s , a n d o th er e d i b le
o i l s th a t me e t s t an d a rd s
e q u iv a l en t t o o r s t r i c te r t h a n
JAS)
PA R AT H I O N O t h e r s p i c es , d r i e d ( l imi t e d t o 0.2 ppm
fruits)
O t h e r s p i c es , d r i e d ( l imi t e d t o 0.1 ppm
seeds)
O t h e r s p i c es , d r i e d ( l imi t e d t o 0.2 ppm
r o o t s o r r h iz o me s )
PA R AT H I O N - M E T H Y L Raisin 1 ppm
O t h e r s p i c es , d r i e d ( l imi t e d t o 5 ppm
fruits)
O t h e r s p i c es , d r i e d ( l imi t e d t o 5 ppm
seeds)
O t h e r s p i c es , d r i e d ( l imi t e d t o 3 ppm
r o o t s o r r h iz o me s )
BIORESMETHRIN W h e a t f l o u r ( l i mi t e d t o w h o l e 1 ppm
grain)
W h e a t f l o u r ( e x c e p t wh o l e 1 ppm
grain)
Wheat germ 3 ppm
W h e a t b r an 5 ppm
BIFENTHRIN W h e a t f l o u r ( l i mi t e d t o w h o l e 0.5 ppm
grain)
W h e a t f l o u r ( e x c e p t wh o l e 0.2 ppm
grain)

93/100
Provisional Translation
from the Japanese Original

W h e a t b r an 2 ppm

P I P E R O N Y L B U TO X I D E W h e a t f l o u r ( l i mi t e d t o w h o l e 30 ppm
grain)

W h e a t f l o u r ( e x c e p t wh o l e 10 ppm
grain)
Wheat germ 90 ppm

W h e a t b r an 80 ppm

C o r n o i l ( ex c e p t ed i b le c o r n o il 8 0 p p m
t h a t me e t s t h e J A S f o r E d i b le
Ve g e t ab l e F a t s a n d O il s , a n d
o t h e r ed i b le o i l s t h a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )
To ma t o j u ic e 0.3 ppm

J u i c e o f ci tr u s f r u i t s 0.05 ppm

F r u i t s , d r i ed 0.2 ppm

PYRIPROXYFEN C o t to n s e ed o i l ( l i mi t e d t o 0.01 ppm

r e f i n e d co t to n s e e d o il a n d
c o t to n s e ed s a l a d o i l th a t me e t
t h e J A S f o r E d i b le Veg e t a b le
F a t s a n d O il s , a n d o th er e d i b le
o i l s th a t me e t s t an d a rd s
e q u iv a l en t t o o r s t r i c te r t h a n
JAS)
C o t to n s e ed o i l ( e xc e pt r e f i n e d 0 . 0 1 p p m
c o t to n s e ed o i l an d co t to n s e e d
s a l a d o i l th a t me e t t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )

PIRIMICARB O t h e r s p i c es , d r i e d ( l imi t e d t o 5 ppm

seeds)

PIRIMIPHOS-METHYL W h e a t b r an 15 ppm

O t h e r s p i c es , d r i e d ( l imi t e d t o 0.5 ppm

fruits)
O t h e r s p i c es , d r i e d ( l imi t e d t o 3 ppm
seeds)

PYRETHRINS F r u i t s , d r i ed 0.2 ppm

VINCLOZOLIN O t h e r s p i c es , d r i e d 0.05 ppm

94/100
Provisional Translation
from the Japanese Original

FA M O X A D O N E W h e a t b r an 0.2 ppm

Raisin 5 ppm

FENAMIPHOS Peanut oil (except refined 0.05 ppm

peanut oil and peanut salad oil
t h a t me e t t h e J A S f o r E d i b le
Ve g e t ab l e F a t s a n d O il s , a n d
o t h e r ed i b le o i l s t h a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )
C o t to n s e ed o i l ( e xc e pt r e f i n e d 0 . 0 5 p p m
c o t to n s e ed o i l an d co t to n s e e d
s a l a d o i l th a t me e t t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )

FENITROTHION W h e a t f l o u r ( l i mi t e d t o w h o l e 5 ppm
grain)
W h e a t b r an 20 ppm

O t h e r s p i c es , d r i e d ( l imi t e d t o 1 ppm
fruits)
O t h e r s p i c es , d r i e d ( l imi t e d t o 7 ppm
seeds)
O t h e r s p i c es , d r i e d ( l imi t e d t o 0.1 ppm
r o o t s o r r h iz o me s )

FENTHION E d i b le o li v e o i l ( li mi t e d t o 1 ppm
v i rg in o il )

P H E N T H O AT E O t h e r s p i c es , d r i e d ( l imi t e d t o 7 ppm
seeds)

F E N VA L E R AT E W h e a t f l o u r ( l i mi t e d t o w h o l e 2 ppm
grain)

W h e a t f l o u r ( e x c e p t wh o l e 0.2 ppm
grain)

W h e a t b r an 5 ppm

C o t to n s e ed o i l ( l i mi t e d t o 0.1 ppm
r e f i n e d co t to n s e e d o il a n d
c o t to n s e ed s a l a d o i l th a t me e t
t h e J A S f o r E d i b le Veg e t a b le
F a t s a n d O il s , a n d o th er e d i b le
o i l s th a t me e t s t an d a rd s
e q u iv a l en t t o o r s t r i c te r t h a n
JAS)

95/100
Provisional Translation
from the Japanese Original

C o t to n s e ed o i l ( e xc e pt r e f i n e d 0 . 1 p p m
c o t to n s e ed o i l an d co t to n s e e d
s a l a d o i l th a t me e t t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )

F E N P R O PAT H R I N C o t to n s e ed o i l ( e xc e pt r e f i n e d 3 p p m
c o t to n s e ed o i l an d co t to n s e e d
s a l a d o i l th a t me e t t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )

FLUDIOXONIL N i r a , d r i ed 50 ppm

B a s i l , d r ied 50 ppm

FLUSILAZOLE Raisin 1 ppm

F L U TO L A N I L R i c e b r an 10 ppm

M i l l ed r i c e 1 ppm

PROCHLORAZ W h e a t b r an 7 ppm

S u n f l o w e r o i l ( l i mi t e d t o 1 ppm
e d i b l e s u n f l o w e r o i l th a t me e t s
t h e J A S f o r E d i b le Veg e t a b le
F a t s a n d O il s , a n d o th er e d i b le
o i l s th a t me e t s t an d a rd s
e q u iv a l en t t o o r s t r i c te r t h a n
JAS)
PROCYMIDONE S u n f l o w e r o i l ( l i mi t e d t o 0.5 ppm
e d i b l e s u n f l o w e r o i l th a t me e t s
t h e J A S f o r E d i b le Veg e t a b le
F a t s a n d O il s , a n d o th er e d i b le
o i l s th a t me e t s t an d a rd s
e q u iv a l en t t o o r s t r i c te r t h a n
JAS)

P R O PA R G I T E Corn flour 0.2 ppm

C o r n o i l ( limi t e d t o e d i b l e c o r n 0 . 5 p p m
o i l th a t me e t s t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )

96/100
Provisional Translation
from the Japanese Original

C o r n o i l ( ex c e p t ed i b le c o r n o il 0 . 7 p p m
t h a t me e t s t h e J A S f o r E d i b le
Ve g e t ab l e F a t s a n d O il s , a n d
o t h e r ed i b le o i l s t h a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )
P e a n u t o i l ( l i mi t e d t o r e f i n e d 0.3 ppm
peanut oil and peanut salad oil
t h a t me e t t h e J A S f o r E d i b le
Ve g e t ab l e F a t s a n d O il s , a n d
o t h e r ed i b le o i l s t h a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )
Peanut oil (except refined 0.3 ppm
peanut oil and peanut salad oil
t h a t me e t t h e J A S f o r E d i b le
Ve g e t ab l e F a t s a n d O il s , a n d
o t h e r ed i b le o i l s t h a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )
O r a n g e j u ice 0.3 ppm

A p p l e ju i c e 0.2 ppm

G r a p e j u i ce 1 ppm

Raisin 12 ppm

C o t to n s e ed o i l ( l i mi t e d t o 0.2 ppm
r e f i n e d co t to n s e e d o il a n d
c o t to n s e ed s a l a d o i l th a t me e t
t h e J A S f o r E d i b le Veg e t a b le
F a t s a n d O il s , a n d o th er e d i b le
o i l s th a t me e t s t an d a rd s
e q u iv a l en t t o o r s t r i c te r t h a n
JAS)
PROFENOFOS C o t to n s e ed o i l ( l i mi t e d t o 0.05 ppm
r e f i n e d co t to n s e e d o il a n d
c o t to n s e ed s a l a d o i l th a t me e t
t h e J A S f o r E d i b le Veg e t a b le
F a t s a n d O il s , a n d o th er e d i b le
o i l s th a t me e t s t an d a rd s
e q u iv a l en t t o o r s t r i c te r t h a n
JAS)
H E P TA C H L O R S o y b e a n o il ( l i mi t e d to e d ib l e 0 . 0 2 p p m
s o y b e a n o i l t h a t me e t s t h e J A S
f o r E d i b l e Ve g e t ab l e F a t s a n d
O i l s , a n d o t h e r e d i b l e o i l s th a t
me e t s t a n d a r d s e q u i v al e n t t o o r
s t r i c t e r t h an J A S )

97/100
Provisional Translation
from the Japanese Original

S o y b e a n o il ( e x c ep t ed i b le 0.5 ppm
s o y b e a n o i l t h a t me e t s t h e J A S
f o r E d i b l e Ve g e t ab l e F a t s a n d
O i l s , a n d o t h e r e d i b l e o i l s th a t
me e t s t a n d a r d s e q u i v al e n t t o o r
s t r i c t e r t h an J A S )
PERMETHRIN W h e a t f l o u r ( l i mi t e d t o w h o l e 2 ppm
grain)
W h e a t f l o u r ( e x c e p t wh o l e 0.5 ppm
grain)
Wheat germ 2 ppm

W h e a t b r an 5 ppm

S o y b e a n o il ( e x c ep t ed i b le 0.1 ppm
s o y b e a n o i l t h a t me e t s t h e J A S
f o r E d i b l e Ve g e t ab l e F a t s a n d
O i l s , a n d o t h e r e d i b l e o i l s th a t
me e t s t a n d a r d s e q u i v al e n t t o o r
s t r i c t e r t h an J A S )
S u n f l o w e r o i l ( l i mi t e d t o 1 ppm
e d i b l e s u n f l o w e r o i l th a t me e t s
t h e J A S f o r E d i b le Veg e t a b le
F a t s a n d O il s , a n d o th er e d i b le
o i l s th a t me e t s t an d a rd s
e q u iv a l en t t o o r s t r i c te r t h a n
JAS)
Sunflower oil (except edible 1 ppm
s u n f l o w e r o i l th a t me e t s t h e
J A S f o r E d i b l e Ve g et ab l e F a t s
a n d O i l s , an d o t h e r ed i b l e o i l s
t h a t me e t s t a n d a r d s e q u i v al e n t
t o o r s t r i c te r t h a n J A S )
C o t to n s e ed o i l ( l i mi t e d t o 0.1 ppm
r e f i n e d co t to n s e e d o il a n d
c o t to n s e ed s a l a d o i l th a t me e t
t h e J A S f o r E d i b le Veg e t a b le
F a t s a n d O il s , a n d o th er e d i b le
o i l s th a t me e t s t an d a rd s
e q u iv a l en t t o o r s t r i c te r t h a n
JAS)

O t h e r s p i c es , d r i e d 0.05 ppm
PENCONAZOLE Raisin 0 . 5 pp m
PHOSALONE O t h e r s p i c es , d r i e d ( l imi t e d t o 2 ppm
fruits)
O t h e r s p i c es , d r i e d ( l imi t e d t o 2 ppm
seeds)

98/100
Provisional Translation
from the Japanese Original

O t h e r s p i c es , d r i e d ( l imi t e d t o 3 ppm
r o o t s o r r h iz o me s )

P H O R AT E P e a n u t o i l ( l i mi t e d t o r e f i n e d 0.05 ppm
peanut oil and peanut salad oil
t h a t me e t t h e J A S f o r E d i b le
Ve g e t ab l e F a t s a n d O il s , a n d
o t h e r ed i b le o i l s t h a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )
Peanut oil (except refined 0.05 ppm
peanut oil and peanut salad oil
t h a t me e t t h e J A S f o r E d i b le
Ve g e t ab l e F a t s a n d O il s , a n d
o t h e r ed i b le o i l s t h a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )
O t h e r s p i c es , d r i e d ( l imi t e d t o 0.1 ppm
fruits)
O t h e r s p i c es , d r i e d ( l imi t e d t o 0.5 ppm
seeds)
O t h e r s p i c es , d r i e d ( l imi t e d t o 0.1 ppm
r o o t s o r r h iz o me s )

M A L AT H I O N To ma t o j u ic e 0.01 ppm
O t h e r s p i c es , d r i e d ( l imi t e d t o 1 ppm
fruits)
O t h e r s p i c es , d r i e d ( l imi t e d t o 2 ppm
seeds)

O t h e r s p i c es , d r i e d ( l imi t e d t o 0.5 ppm

r o o t s o r r h iz o me s )

METHAMIDOPHOS O t h e r s p i c es , d r i e d ( e x c e p t t h e 0 . 1 p p m
f r u i t o f S a n s h o ( J a p a n es e
pepper))

M E TA L A X Y L a n d O t h e r s p i c es , d r i e d ( l imi t e d t o 5 ppm
MEFENOXAM seeds)

M E T H I D AT H I O N E d i b le o li v e o i l ( li mi t e d t o 2 ppm
v i rg in o il )
C o t to n s e ed o i l ( e xc e pt r e f i n e d 2 p p m
c o t to n s e ed o i l an d co t to n s e e d
s a l a d o i l th a t me e t t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )

99/100
Provisional Translation
from the Japanese Original

METHOXYFENOZIDE P l u m, d r i e d 2 ppm
Raisin 3 ppm
METHOPRENE W h e a t f l o u r ( l i mi t e d t o w h o l e 5 ppm
grain)
W h e a t f l o u r ( e x c e p t wh o l e 2 ppm
grain)
W h e a t b r an 10 ppm
C o r n o i l ( limi t e d t o e d i b l e c o r n 0 . 2 p p m
o i l th a t me e t s t h e J A S f o r
E d i b le Ve g e t a b le F a t s a n d O i l s ,
a n d o t h e r ed i b le o il s th a t me e t
s t a n d a r d s eq u i v a l en t to o r
s t r i c t e r t h an J A S )
HYDROGEN PHOSPHIDE Ve g e t a b l e s , d r i e d 0.01 ppm
F r u i t s , d r i ed 0.01 ppm

1 0 . I n a ddi t i on t o the p r o v i s io n s o f 6 o r 9 , w h e n f o o d p r o d u c t s a r e
ma n u f a c t u r e d o r p r o ces s e d u s in g f o o d s f o r wh i c h c o mp o s i t i o n a l st an d a r d s
a r e s p e ci f i e d in 6 t h r o u g h 9 , th e f oo d s us e d mu s t c o mp l y w i t h t he
standards given in 6 through 9.

100/100