Pesticides Identification at the Residue Level - ACS Publications

3 A Review of Enzymatic Techniques Used

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for Pesticide Residue Analysis D. C.

VILLENEUVE

F o o d and D r u g Directorate, Department of National Health and Welfare, Ottawa, Canada

A review

is presented

for pesticide

residue

on the use of enzymatic analysis.

-carboxylesterase procedures, of such theory their

techniques, behind

limits

vantages.

their

are use,

of detection, Other

enzymatic

techniques

Anticholinesterase which

described the

comprise with

different

and their

and the

reference methods

advantages

techniques

to

the

of

assay,

and

disad-

are also

*~r*he f a c t t h a t m a n y pesticides i n h i b i t e n z y m e s in vitro A

anti-

majority

discussed.

has l e d to t h e

i n t r o d u c t i o n of v a r i o u s a n a l y t i c a l m e t h o d s for the d e t e c t i o n a n d esti-

m a t i o n of p e s t i c i d e residues. T h i s p a p e r w i l l not d e s c r i b e i n d e t a i l a l l the e n z y m a t i c t e c h n i q u e s u s e d for p e s t i c i d e r e s i d u e analysis b u t r a t h e r w i l l a t t e m p t to c a t e g o r i z e a n d d e s c r i b e b r i e f l y the m a i n types w i t h r e f e r ence to t h e t h e o r y b e h i n d t h e i r use, t h e i r p r a c t i c a l a p p l i c a t i o n , a n d associated problems.

T h i s w i l l b e f o l l o w e d b y a b r i e f d i s c u s s i o n o n the

advantages a n d d i s a d v a n t a g e s of these t e c h n i q u e s .

Cholinesterase Techniques Theory. C h o l i n e s t e r a s e i n h i b i t i o n b y o r g a n o p h o s p h o r u s i n s e c t i c i d e s has b e e n the subject of s e v e r a l excellent r e v i e w s b y O ' B r i e n ( J , 2)

and

H e a t h ( 3 ) . T h e basis of toxic a c t i o n of o r g a n o p h o s p h a t e s a n d c a r b a m a t e s i n m a m m a l s is g e n e r a l l y associated w i t h t h e i r a b i l i t y to i n h i b i t c h o l i n esterase i n the c e n t r a l a n d p e r i p h e r a l n e r v o u s systems w h e r e i t p l a y s a n i m p o r t a n t r o l e i n the t r a n s m i s s i o n of n e r v e i m p u l s e s . O r g a n o p h o s p h a t e s a n d c a r b a m a t e s react w i t h cholinesterase i n a m a n n e r s i m i l a r to the r e a c t i o n of cholinesterase w i t h a c e t y l c h o l i n e a n d c a n b e d e p i c t e d as f o l l o w s : 27

In Pesticides Identification; Biros, F.; Advances in Chemistry; American Chemical Society: Washington, DC, 1971.

28

PESTICIDES

H 0 E O H + A X +± E O H . A X -> E O A - * E O H + \ X - + H+

IDENTIFICATION

2

w h e r e E O H represents cholinesterase

A" +

H+

(1)

( O H b e i n g a serine O H i n the

a c t i v e s i t e ) , A X is e i t h e r a c e t y l c h o l i n e o r a n o r g a n o p h o s p h a t e

or carba-


m a t e i n s e c t i c i d e , a n d X is the l e a v i n g g r o u p — i . e . , c h o l i n e i n a c e t y l c h o l i n e , p-nitrophenol i n paraoxon, or 1-naphthol i n c a r b a r y l ( 2 ) .

The symbol A

c a n designate e i t h e r the a c e t y l g r o u p , a d i a l k y l p h o s p h o r y l g r o u p , or a m e t h y l c a r b a m y l g r o u p . T h u s , the r e a c t i o n i n v o l v e s a c o m p l e x f o r m a t i o n f o l l o w e d b y p h o s p h o r y l a t i o n , a c e t y l a t i o n , o r c a r b a m y l a t i o n of the e n z y m e , a n d finally h y d r o l y s i s . I f the c o n c e n t r a t i o n of i n h i b i t o r is r e l a t i v e l y l a r g e (10~ o r 10" M ) as c o m p a r e d w i t h the c o n c e n t r a t i o n of substrate ( 1 0 " 5

6

10

M ) , the o v e r - a l l r e a c t i o n c a n b e c o n s i d e r e d a first-order b i m o l e c u l a r t y p e a n d c a n b e d e s c r i b e d i n terms of the f o l l o w i n g e q u a t i o n : log P = 2 -

g

(2)

t

w h e r e P is the p e r c e n t a c t i v i t y of the e n z y m e r e m a i n i n g after i n c u b a t i o n of I c o n c e n t r a t i o n o f i n h i b i t o r for t i m e t, a n d k is t h e b i m o l e c u l a r rate {

constant.

T h u s , f o r a n y v a l u e of t there is a c o n c e n t r a t i o n of i n h i b i t o r

w h i c h w o u l d r e d u c e P , the p e r c e n t a c t i v i t y , to 5 0 % . T h i s v a l u e is k n o w n as t h e ho, a n d because its v a l u e is often s m a l l , the n e g a t i v e l o g a r i t h m of the 7 , the p 7 , is u s e d . T a b l e I gives the J 50

5 0

5

0

a n d ph

of s e v e r a l o r g a n o -

0

phosphorus a n d carbamate insecticides, using h u m a n plasma cholinesterase as e n z y m e source. Table I. Cholinesterase-Inhibitive Effects of Several Organophosphate and Carbamate Insecticides 0

Quantity to Produce 50% Inhibition " 1

Mol.

Insecticide Coroxon DDVP DFP Guthoxon Parathion Paraoxon Ruelene Phosphamidon Carbaryl c

d

a 6 c d

Wt.

346.5 221.0 184.2 301.0 291.0 275.0 291.5 299.5 201.0

Molar 3.16 6.30 5.01 6.31 2.43 1.88 5.62 6.7 1.2

I

Concn.,

X 10X lO" X 10X 10X lO" X 10X lO" X 10X 10-'

7

6

8 6

6

8

4

7

Data taken from Giang and Hall (4), O'Brien (5), and Voss (6). Human plasma cholinesterase used as enzyme source. Oxygen analog of Co-Ral. Oxygen analog of Guthion.


pi 7.50 6.80 8.70 5.80 5.39 8.27 4.75 7.83 5.08

3.

F r o m the Z if

Enzymatic

V I L L E N E U V E

first-order

5 0

Techniques

for

Analysis

a n d t, one c a n c a l c u l a t e , u s i n g E q u a t i o n 2 , t h e ki v a l u e

k i n e t i c s are f o l l o w e d .

Main

( 7 ) has d e v e l o p e d

a kinetic

t r e a t m e n t t h a t d e t e r m i n e s b o t h the affinity of the i n h i b i t o r for the e n z y m e a n d the a b i l i t y to p h o s p h o r y l a t e the e n z y m e o n c e b i n d i n g has o c c u r r e d . W i t h this treatment, the ki f r o m E q u a t i o n 2 c a n n o t b e c o n s i d e r e d as a s i m p l e b i m o l e c u l a r rate constant b u t as a c o m b i n a t i o n of a u n i m o l e c u l a r Downloaded by KTH ROYAL INST OF TECHNOLOGY on November 18, 2015 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0104.ch003

rate constant a n d a n affinity constant. H o w e v e r , the m e a s u r e m e n t of the ki as o u t l i n e d i n E q u a t i o n 2 does p r o v i d e a satisfactory m e a s u r e of the i n h i b i t o r y p o t e n c y i n m o s t cases. Methods of Assessing Cholinesterase Inhibition. T h e i n c r e a s e d use of c h o l i n e s t e r a s e - i n h i b i t i n g i n s e c t i c i d e s has s t i m u l a t e d r e s e a r c h i n m a n y areas of scientific endeavor. in vivo

O n e s u c h a r e a has b e e n c o n c e r n e d w i t h the

t o x i c o l o g i c a l p r o p e r t i e s o f cholinesterase i n h i b i t o r s .

However,

t h e area of c o n c e r n h e r e is i n the field of a n a l y t i c a l c h e m i s t r y , w h e r e cholinesterases are u s e d as a t o o l for the q u a n t i t a t i v e d e t e r m i n a t i o n of u n k n o w n amounts of i n h i b i t o r s ( 8 ) .

S u c h p r o c e d u r e s are f r e q u e n t l y u s e d

for the analysis of c e r t a i n p e s t i c i d e residues a n d c a n be c a t e g o r i z e d i n t o the f o l l o w i n g types of m e t h o d s : P O T E N T I O M E T R I C

O R A P H

O n e s u c h m e t h o d , p r o b a b l y the

M E T H O D S .

first ever u s e d , w a s the one p u b l i s h e d b y G i a n g a n d H a l l (4)

for the

d e t e r m i n a t i o n of t e t r a e t h y l p y r o p h o s p h a t e ( T E P P ) a n d p a r a t h i o n i n p l a n t m a t e r i a l . O n e p r o b l e m was t h a t p a r a t h i o n w a s a w e a k i n h i b i t o r in vitro a n d r e q u i r e d c o n v e r s i o n to its o x y g e n a n a l o g to o b t a i n sufficient sensitivity.

T h i s w a s a c c o m p l i s h e d b y t r e a t i n g p a r a t h i o n w i t h a m i x t u r e of

c o n c e n t r a t e d a n d f u m i n g n i t r i c acids. It r e q u i r e d 4 2 . 5

o f p a r a t h i o n to

p r o d u c e 5 0 % i n h i b i t i o n , as c o m p a r e d w i t h 0 . 0 3 1 fig for p a r a o x o n .

The

m e t h o d consisted of e x t r a c t i n g the p e s t i c i d e f r o m the p l a n t , i n c u b a t i n g a n a l i q u o t of the e x t r a c t e d p e s t i c i d e w i t h a s t a n d a r d b o v i n e cholinesterase s o l u t i o n , d e t e r m i n i n g the r e m a i n i n g cholinesterase a c t i v i t y after i n c u b a t i o n , a n d c o m p a r i n g the p e r c e n t i n h i b i t i o n w i t h a s t a n d a r d c u r v e i n o r d e r to o b t a i n the q u a n t i t y of m a t e r i a l . C h o l i n e s t e r a s e a c t i v i t y w a s m e a s u r e d u s i n g a c e t y l c h o l i n e b r o m i d e as substrate a n d m e a s u r i n g t h e c h a n g e i n p H c a u s e d b y the release of a c e t i c a c i d .

T h e " p e r c e n t i n h i b i t i o n " ( 9 ) is

c a l c u l a t e d as f o l l o w s : A p H = p H (initial) % Inhibition = T I T R I M E T R I C

M E T H O D S .

1 —

p H (final)

A p H (sample) X A p H (control)

100

T h e s e m e t h o d s i n v o l v e the t i t r a t i o n of a c e t i c

a c i d l i b e r a t e d b y the h y d r o l y s i s of a c e t y l c h o l i n e , w h i c h is n o r m a l l y acc o m p l i s h e d w i t h a " p H - s t a t " so t h a t a constant p H is m a i n t a i n e d . T h e advantages of these m e t h o d s o v e r the A p H m e t h o d s are t h a t the m e a s u r e d


30

PESTICIDES

IDENTIFICATION

a c t i v i t y is not d i r e c t l y i n f l u e n c e d b y the buffer s t r e n g t h of the s o l u t i o n a n d that a constant p H is m a i n t a i n e d d u r i n g the e n z y m e r e a c t i o n .

(10)

T h i s t y p e of m e t h o d has b e e n u s e d d i r e c t l y for the d e t e r m i n a t i o n of p a r a t h i o n w h i l e other w o r k e r s u s e d a m i c r o t i t r i m e t r i c m e t h o d

to

obtain

greater s e n s i t i v i t y ( I I ) .


M A N O M E T R I C

M E T H O D S .

T h e s e m e t h o d s i n v o l v e the m e a s u r e m e n t of

c a r b o n d i o x i d e l i b e r a t e d f r o m the a c t i o n of the released acetic a c i d o n sodium bicarbonate.

D u B o i s a n d C o t t e r (in Ref. 10)

u s e d this t y p e of

m e t h o d to d e t e r m i n e D i p t e r e x i n m i l k . C O L O R I M E T R I C

M E T H O D S .

Measurement

of Unchanged

Acetylcholine.

T h i s c o l o r i m e t r i c t e c h n i q u e d e p e n d s o n m e a s u r i n g the u n c h a n g e d a c e t y l c h o l i n e w i t h h y d r o x y l a m i n e to p r o d u c e a c e t o h y d r o x a m i c a c i d w h i c h y i e l d s a p u r p l e color w i t h excess f e r r i c c h l o r i d e after a c i d i f i c a t i o n . C o o k o r i g i n a l l y a p p l i e d this m e t h o d to p e s t i c i d e r e s i d u e analysis o n

(12) paper

chromatograms. Indophenyl

A c o l o r i m e t r i c m e t h o d for the analysis of c h o -

Acetate.

l i n e s t e r a s e - i n h i b i t i n g insecticides u s i n g i n d o p h e n y l acetate as substrate was p u b l i s h e d b y A r c h e r a n d Z w e i g i n 1959 (13).

T h e procedure was

b a s e d o n the m e a s u r e m e n t of the h y d r o l y s i s p r o d u c t of i n d o p h e n y l acetate at 625 fx at a p H of 8.0.

T h r e e different e n z y m a t i c m e t h o d s , a p o -

t e n t i o m e t r i c , a p a p e r c h r o m a t o g r a p h i c , a n d the c o l o r i m e t r i c one tioned, were compared peaches.

G o o d agreement w a s s h o w n b y a l l three m e t h o d s .

H a n k e n s o n (17)

men-

b y d e t e r m i n i n g r e s i d u e levels of c a r b a r y l o n Beam and

u s e d the same m e t h o d to d e t e r m i n e k n o w n a m o u n t s of

Sevin, Trithion, parathion, malathion, G u t h i o n , Dipterex, and ronnel i n milk.

S e n s i t i v i t y r a n g e d f r o m 0.008 to 0.2 p p m .

Phenol

Red.

T h e first p u b l i c a t i o n s of a n a u t o m a t e d p r o c e d u r e

the m e a s u r e m e n t of cholinesterase i n h i b i t o r s are those of W i n t e r and Winter and Ferrari (15).

for (14)

T h e method employed an Autoanalyzer

i n s t r u m e n t a l system w h e r e i n the extracts c o n t a i n i n g the i n s e c t i c i d e w e r e i n c u b a t e d w i t h a s t a n d a r d cholinesterase s o l u t i o n at 37 ° C . A c o n t i n u o u s s a m p l e f r o m the i n c u b a t i o n b a t h is b u f f e r e d a n d m i x e d w i t h a c e t y l c h o l i n e i o d i d e . A f t e r a s e c o n d i n c u b a t i o n , the acetic a c i d released b y the a c t i o n of

the u n i n h i b i t e d cholinesterase

is m e a s u r e d

colorimetrically, using

p h e n o l r e d as the i n d i c a t o r . M o r e r e c e n t l y , F i s c h l et al. (16)

reported a

m e t h o d for r a p i d d e t e c t i o n of o r g a n i c p h o s p h a t e pesticides i n s e r u m . Strips of

filter

paper were impregnated w i t h a buffered

acetylcholine

substrate s o l u t i o n c o n t a i n i n g p h e n o l r e d as a n i n d i c a t o r . W h e n n o i n h i b i t i o n is present, the a c i d released f r o m the a c t i o n of turns the p a p e r y e l l o w .

cholinesterase

W h e n cholinesterase has b e e n i n h i b i t e d , the

p a p e r turns p i n k - t o - v i o l e t .


3.

Enzymatic

V I L L E N E U V E

Acetylthiocholine

Techniques

for

31

Analysis

A n o t h e r a u t o m a t e d p r o c e d u r e for c h o l i n -

Iodide.

esterase i n h i b i t i o n studies has b e e n u s e d b y L e v i n e et al. (18) V o s s (19).

and by

T h e m e t h o d uses a c e t y l t h i o c h o l i n e i o d i d e as substrate a n d

d i t h i o b i s n i t r o b e n z o i c a c i d ( D T N B ) . C h o l i n e s t e r a s e splits the substrate, a n d the t h i o c h o l i n e r e l e a s e d reduces the D T N B to the y e l l o w a n i o n of


t h i o n i t r o b e n z o i c a c i d , w h o s e a b s o r b a n c e is m e a s u r e d at 4 2 0 /*. Fluorogenic

Substrates.

method using a

fluorometric

G u i l b a u l t a n d K r a m e r (20)

published a

assay f o r a n t i c h o l i n e s t e r a s e c o m p o u n d s .

substrates u s e d w e r e nonfluorescent

compounds,

The

the a c e t y l a n d b u t y l

esters of 1 - a n d 2 - n a p h t h o l , w h i c h are h y d r o l y z e d b y cholinesterase to highly

fluorescent

m a t e r i a l s . T h e rate of c h a n g e

of

fluorescence

r e l a t e d to e n z y m e a c t i v i t y , a n d i n h i b i t i o n w a s m e a s u r e d b y rate of c h a n g e i n the p r o d u c t i o n of

was

decreased

fluorescence.

A s e m i q u a n t i t a t i v e test for the i d e n t i f i c a t i o n of c h o l i n e s t e r a s e - i n h i b i t i n g pesticides has b e e n d e s c r i b e d b y S c h u l t z m a n n a n d B a r t h e l

(21).

I n d o x y l acetate was u s e d as the substrate, i n c o n j u c t i o n w i t h a t w o - d i m e n s i o n a l t h i n - l a y e r c h r o m a t o g r a p h i c t e c h n i q u e . I t c o n s i s t e d of s p o t t i n g t h e c l e a n e d - u p samples o n a s i l i c a g e l t h i n - l a y e r p l a t e , d e v e l o p i n g t h e p l a t e i n the a p p r o p r i a t e solvent systems, a n d s p r a y i n g the p l a t e w i t h a horse s e r u m cholinesterase s o l u t i o n a n d i n d o x y l acetate. T h e c h o l i n e s t e r a s e - i n h i b i t i n g c o m p o u n d s a p p e a r e d as w h i t e spots o n a b l u e b a c k g r o u n d , a n d s e n s i t i v i t y w a s i n the s u b m i c r o g r a m r a n g e . A G A R - A G A R

D I F F U S I O N

T E C H N I Q U E S .

B e n y o n a n d S t o y d i n (22)

de-

s c r i b e d a p r o c e d u r e i n w h i c h a l a y e r of a g a r - a g a r 5 m m t h i c k c o n t a i n i n g cholinesterase a n d b r o m t h y m o l b l u e at a p H of 7 . 8 - 7 . 9 was u s e d .

A

2 - p r o p a n o l s o l u t i o n ( 0 . 1 m l ) of the i n s e c t i c i d e w a s a p p l i e d to a h o l e i n t h e agar a n d w a s a l l o w e d to diffuse for 1 8 h o u r s at r o o m t e m p e r a t u r e . T h e plate was then sprayed w i t h acetylcholine chloride solution, a n d the b l u e l a y e r t u r n e d y e l l o w w i t h i n 3 0 m i n u t e s except i n those w h e r e the e n z y m e w a s i n h i b i t e d , w h i c h r e m a i n e d as b l u e circles.

areas The

d i a m e t e r of these circles was p r o p o r t i o n a l to t h e a m o u n t of the i n h i b i t o r . A s little as 3 n g o f p a r a t h i o n c o u l d b e d e t e c t e d b y this t e c h n i q u e . F L U O R E S C E N T

25)

E Q U I L I B R I U M

PROBES.

have synthesized active-site-directed

H i m e l a n d co-workers fluorescent

(23,

24,

e q u i l i b r i u m probes

w h i c h are c o m p e t i t i v e i n h i b i t o r s of the a c t i v e site of cholinesterase e n zymes.

The

fluorescence

i n t e n s i t y of the p r o b e - e n z y m e

c o m p l e x is d e -

creased b y a n y f o r e i g n m o l e c u l e ( i n s e c t i c i d e ) w h i c h c o m p e t e s w i t h t h e equilibrium

fluorescent

p r o b e for the a c t i v e site of t h e e n z y m e or w h i c h

changes the e q u i l i b r i u m d y n a m i c s b y exo a r e a r e a c t i o n w i t h t h e e n z y m e . T h i s h i g h l y specific a n d sensitive s p e c t r o s c o p i c m e t h o d is b e i n g d e v e l o p e d as a n a n a l y t i c a l m e t h o d for i n s e c t i c i d e s

(26).


32

PESTICIDES

IDENTIFICATION

Carboxylesterase Inhibition I n the R e p o r t of the C o m m i s s i o n o n E n z y m e s ( 2 7 ) ,

Theory.

car-

boxylesterases are i n c l u d e d i n the g e n e r a l g r o u p " H y d r o l a s e s " ( E . C . 3 . ) and

more

specifically

in

the

category

carboxylic

ester

hydrolases

( E . C . 3 . 1 . 1 . 1 . ) . T h e s e e n z y m e s are r e s p o n s i b l e for c a t a l y z i n g the g e n e r a l Downloaded by KTH ROYAL INST OF TECHNOLOGY on November 18, 2015 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0104.ch003

r e a c t i o n , c a r b o x y l i c ester +

H 0 ^± a l c o h o l + 2

carboxylic acid.

These

e n z y m e s h a v e b e e n f o u n d i n tissues of m a n y species of insects, a n i m a l s , a n d plants ( 2 8 , 2 9 , 3 0 , 3 1 , 3 2 , 3 3 , 3 4 , 3 5 , 3 6 ) .

H o w e v e r , the distinction be-

t w e e n esterases a n d other h y d r o l y t i c e n z y m e s has b e c o m e less c l e a r i n the l i g h t of e v i d e n c e t h a t c e r t a i n p r o t e o l y t i c e n z y m e s

(trypsin, chymo-

t r y p s i n , t h r o m b i n ) c a n h y d r o l y z e c a r b o x y l esters a n d are i n h i b i t e d b y substances k n o w n to b e p o t e n t esterase i n h i b i t o r s (37,38).

A s w i t h cho-

linesterases, i n h i b i t i o n i n v o l v e s the r e a c t i o n of the p h o s p h a t e w i t h the e n z y m e to f o r m a n a c y l d e r i v a t i v e ( p h o s p h o r y l a t e d e n z y m e ) .

T h e phos-

p h o r y l a t e d e n z y m e is q u i t e stable a n d prevents the a c t i o n of the esterase o n other substrates. T h e p h o s p h o r y l a t i o n of t h e e n z y m e is the r a t e - l i m i t i n g step a n d d e p e n d s u p o n the " f i t " of the c o m p o u n d o n t h e e n z y m e a n d t h e a b i l i t y of the c o m p o u n d to p h o s p h o r y l a t e a serine or h i s t i d i n e at the enzyme's a c t i v e site. T h e d e t a i l e d m e c h a n i s m of t h e i r i n h i b i t i o n b y o r g a n o p h o s p h o r u s p e s t i c i d e s has b e e n d i s c u s s e d b y s e v e r a l w o r k e r s a n d p r o c e d u r e s h a v e b e e n w o r k e d o u t for d e t e r m i n i n g r a t e constants of this step (7, 39, 40,41). constant (ki)

It has b e e n suggested ( 7 ) t h a t the b i m o l e c u l a r rate

is the m o s t r e l i a b l e c r i t e r i o n to m e a s u r e the i n h i b i t o r y

p o w e r of a n o r g a n o p h o s p h a t e f o r a n esterase. H o w e v e r , c r i t e r i a s u c h as p e r c e n t i n h i b i t i o n a n d I values are c o n s i d e r e d to b e loosely d e f i n e d f u n c tions of the ki a n d m a y b e u s e d . Techniques. I n 1962, M c K i n l e y a n d R e a d (42)

d e v e l o p e d a n ester-

a s e - i n h i b i t i o n t e c h n i q u e for the d e t e c t i o n of o r g a n o p h o s p h a t e residues o n p a p e r c h r o m a t o g r a m s .

pesticide

T h e procedure involved conversion

of the t h i o p h o s p h a t e s w i t h b r o m i n e to y i e l d a c t i v e esterase i n h i b i t o r s , the i n h i b i t i o n b y the p e s t i c i d e of t h e esterases f r o m a b e e f l i v e r h o m o g e n a t e s p r a y e d o n t o the c h r o m a t o g r a m , the h y d r o l y s i s of t h e substrate ( a - n a p h t h y l acetate) w h i c h was s p r a y e d o n t o the p a p e r after the l i v e r

homo-

genate h a d d r i e d , a n d t h e d e v e l o p m e n t of a b a c k g r o u n d c o l o r b e t w e e n F a s t B l u e R R a n d the h y d r o l y s i s p r o d u c t , a - n a p h t h o l . T h i s t e c h n i q u e w a s a d a p t e d to t h i n l a y e r c h r o m a t o g r a p h y b y A c k e r m a n n (43).

T h e substrates u s e d w e r e 1-naphthylacetate, 2 - a z o b e n z e n e - l -

naphthylacetate, a n d indoxylacetate. r a n g e . A l a t e r s t u d y (44)

S e n s i t i v i t y w a s i n the n a n o g r a m

w a s c a r r i e d o u t u s i n g different a c t i v a t i o n t e c h -

n i q u e s i n a n a t t e m p t to m a k e the p r o c e d u r e m o r e sensitive. et al. (45)

Mendoza

u s e d i n d o x y l a n d s u b s t i t u t e d i n d o x y l a n d n a p h t h y l acetates

as substrates.

Indigo compounds

p r o d u c e d b y t h e h y d r o l y s i s of these


3.

Enzymatic

V I L L E N E U V E

Techniques

for

33

Analysis

substrates gave c o l o r e d b a c k g r o u n d s , l e a v i n g the sites of i n h i b i t i o n b y p e s t i c i d e s as w h i t e spots; the colors p r o d u c e d w e r e b l u e f o r i n d o x y l a n d 5 - b r o m o i n d o x y l acetates, t u r q u o i s e for 5 - b r o m o - 4 - c h l o r o i n d o x y l and

p i n k for 5 - b r o m o - 6 - c h l o r o i n d o x y l

acetate.

S e n s i t i v i t y of

acetate, detection

w a s i n the n a n o g r a m r a n g e , a n d t h e b a c k g r o u n d a n d spots w e r e stable for months.

Wales a n d co-workers

(46)

r e p o r t e d a p r o c e d u r e for t h e


s e m i q u a n t i t a t i v e d e t e r m i n a t i o n of some o r g a n o p h o s p h o r u s p e s t i c i d e r e s i dues i n p l a n t extracts u s i n g M e n d o z a s p r o c e d u r e (44).

Ackermann

(46)

e x p e r i m e n t e d w i t h different a c t i v a t i o n t e c h n i q u e s to m a k e his t h i n - l a y e r t e c h n i q u e m o r e sensitive. O t h e r w o r k e r s (47)

have developed a method

to estimate p a r a t h i o n , m a l a t h i o n , a n d d i a z i n o n i n lettuce u s i n g a c a r b o x y l e s t e r a s e - i n h i b i t i o n assay. T h e m e t h o d i n v o l v e d a n e x t r a c t i o n p r o c e d u r e , followed b y sweep codistillation and thin layer chromatography.

The

pesticides w e r e s c r a p e d off the t h i n l a y e r p l a t e a n d u s e d i n a c a r b o x y l esterase assay u s i n g o - n i t r o p h e n y l p r o p i o n a t e as substrate. S e n s i t i v i t y r a n g e d f r o m 1.0 p p m for p a r a t h i o n to 8.0 p p m for m a l a t h i o n a n d 0.75 p p m for d i a z i n o n .

Other Techniques DDT

i n h i b i t s c a r b o n i c a n h y d r a s e a n d has b e e n m a d e the basis of a

q u a n t i t a t i v e m e t h o d w h i c h c a n d e t e r m i n e as l i t t l e as 0.2 pg of D D T M o r e r e c e n t l y , G u i l b a u l t et al. (20)

(11).

p u b l i s h e d a m e t h o d f o r the d e t e r -

m i n a t i o n of m e t h y l p a r a t h i o n , a l d r i n , a n d h e p t a c h l o r b a s e d o n the i n h i b i t i o n of a c i d a n d a l k a l i n e phosphatases b y these substances. T h e substrate w a s u m b e l l i f e r o n e p h o s p h a t e w h i c h w a s c l e a v e d b y the phosphatases to the

fluorescent

compound

umbelliferone.

Decreased

fluorescence

was

u s e d as a d i r e c t m e a s u r e of the i n h i b i t o r , a n d the s e n s i t i v i t y w a s 5 p p m f o r m e t h y l p a r a t h i o n a n d a l d r i n a n d 50 p p m f o r h e p t a c h l o r . G e i k e

(48)

r e p o r t e d t h a t some o r g a n o c h l o r i n e insecticides w i l l i n h i b i t b o v i n e esterase after exposure to U V i r r a d i a t i o n . l i v e r esterase in

O r d i n a r i l y , these c o m p o u n d s

activate

vitro.

Advantages and Disadvantages of Enzyme Techniques for Residue Analysis T h e m e t h o d s r e f e r r e d to p r e v i o u s l y are s u m m a r i z e d i n T a b l e I I , a l o n g w i t h t h e i r r e p o r t e d l i m i t s of s e n s i t i v i t y a n d details o n t h e i r a p p l i c a t i o n to r e s i d u e analysis. T h e most o b v i o u s s h o r t c o m i n g of these m e t h o d s is t h a t v e r y f e w h a v e b e e n a p p l i e d to r e s i d u e analyses i n crops or foods. A m a j o r p r o b l e m i n the a p p l i c a t i o n of these m e t h o d s to r e s i d u e analysis is s a m p l e e x t r a c t i o n and

cleanup.

I t is w e l l k n o w n (8, 49, 51)

t h a t constituents of c e r t a i n


34

PESTICIDES

Table II. Method of

Assay


Potentiometric

Summary of the Different Methods

Ref. (4)

IDENTIFICATION

Pesticides

Used

Parathion, T E P P , Paraoxon

Titrimetric

(50)

Parathion

Manometric

(52)

Dipterex

Colorimetric

(12) (13) (17)

Systox Trithion, Sevin, Guthion, Thimet Sevin, Trithion, parathion, malathion, G u t h i o n , ronnel Thimet, parathion, diazinon, malathion Monocrotophos C - 8 3 5 3 (carbamate)

(15) (53) (6) Fluorogenic

(20) (54) (55) (36)

(8) Carboxylesterase

Systox, parathion D F P , methyl parathion, parathion, dimethyl chlorthiophosphate, diethyl chlorphosphate M e t h y l parathion, aldrin, heptachlor 29 O r g a n o p h o s p h o r u s a n d c a r b a m a t e i n s e c t i cides Birlane, dichlorvos mevinphos

(42)

22 O r g a n o p h o s p h a t e insecticides

(56)

32 O r g a n o p h o s p h o r u s a n d c a r b a m a t e i n s e c t i cides 8 Organophosphorus and carbamate insecticides 10 O r g a n o p h o s p h o r u s insecticides a n d S e v i n 7 O r g a n o p h o s p h o r u s insecticides Parathion, malathion and diazinon

(43) (44) (45) (47)

plants—i.e., tea, potatoes, t o b a c c o — r e q u i r e

f u r t h e r c l e a n u p b e c a u s e of

e n z y m e - i n h i b i t i n g i m p u r i t i e s . M a n y different p r o c e d u r e s h a v e b e e n u s e d f o r e x t r a c t i o n a n d c l e a n u p , a n d the selection of a s u i t a b l e m e t h o d w i l l d e p e n d o n w h a t c r o p or f o o d m a t e r i a l is b e i n g a n a l y z e d a n d the p e s t i c i d e or pesticides used. A n o t h e r d i s a d v a n t a g e of e n z y m e techniques is t h a t t h e y l a c k the specificity to d i s t i n g u i s h i n d i v i d u a l pesticides w h e n present as a m i x t u r e . M o s t of the m e t h o d s r e p o r t e d here ( T a b l e I I ) h a v e b e e n d e v e l o p e d for single c o m p o u n d s , a n d thus w h e r e m o r e t h a n one p e s t i c i d e is present, p r e l i m i n a r y separation t e c h n i q u e s are necessary.

Both paper and thin

l a y e r c h r o m a t o g r a p h i c t e c h n i q u e s d o not h a v e this d i s a d v a n t a g e a n d


3.

V I L L E N E U V E

Enzymatic

Techniques

for

35

Analysis

of Analysis Referred to in the T e x t Comments


Lower Limits of Detection 0.005-5.0 [xg/ml

T E P P residues detected o n lettuce a t levels of 3 p p m and up

1.0 m g / 1 0 cu. m . air

M e t h o d a p p l i e d t o t h e d e t e r m i n a t i o n of p a r a t h i o n in air

0.75 p p m

U s e d for t h e d e t e r m i n a t i o n of D i p t e r e x i n m i l k

0.60 p p m 0.001-0.1 t i g / m l 0.008-0.3 p p m

M e t h o d used for the e s t i m a t i o n of s y s t o x o n apples S e v i n d e t e r m i n e d as a residue o n peaches a t 35 p p m D e t e r m i n e d s i n g l y as residues i n m i l k

0.1-1.0 [ j L g / m l 0.02 p p m 0.2 p p m

Procedure applied to standards only Procedure applied to standards only A u t o a n a l y z e r procedure a p p l i e d t o apples, cabbage, rice, a n d w h e a t

1-10 [xg/ml 0.4-16 ( i g / m l

Procedure applied to standards only Procedure applied to standards only

0.05-1.0 t x g / m l 0 . 5 - 1 0 [xg/ml

Procedure applied to standards T h i n layer technique applied to standards only. V a r i o u s m e t h o d s of s a m p l e e x t r a c t i o n a n d c l e a n u p described M e v i n p h o s d e t e r m i n e d i n c r o p e x t r a c t s a t 0.05 p p m

0.01-0.3 [Lg/m\ 0 . 5 - 5 . 0 [Lg 0.01-3 [ A g 0.2-10 ng 0.001-0.1 [ig 0.2-4.0 p p m 0.75-8.0 p p m

Paper chromatographic technique applied to s t a n d ards only Paper chromatographic method applied to extracts of lettuce, s t r a w b e r r i e s , a n d apples T L C p r o c e d u r e ; a p p l i e d to s t a n d a r d s o n l y T L C procedure; applied to standards only T L C procedures; applied to potato extracts C o m b i n e d T L C - e n z y m e - i n h i b i t i o n procedure. I n secticides d e t e r m i n e d s i m u l t a n e o u s l y i n lettuce e x tracts

serve as excellent s c r e e n i n g t e c h n i q u e s .

H o w e v e r , b e f o r e the other e n -

z y m a t i c m e t h o d s c a n b e successfully a p p l i e d to the d e t e r m i n a t i o n of m u l t i - p e s t i c i d e residues, a p r e l i m i n a r y s e p a r a t i o n of the specific pesticides is essential. A t h i r d p r o b l e m is that m a n y o r g a n o p h o s p h o r u s pesticides are p o o r esterase i n h i b i t o r s in vitro

a n d n e e d to b e c o n v e r t e d to t h e i r o x y g e n

analogs i n o r d e r to o b t a i n sufficient i n h i b i t o r y p o t e n c y .

S o m e of the p r o -

cedures u s e d for this c o n v e r s i o n are s h o w n i n T a b l e I I I .

A g a i n , the

c h o i c e o f a s u i t a b l e p r o c e d u r e w i l l d e p e n d o n the t y p e o f assay u s e d a n d the pesticides i n q u e s t i o n . I n o u r laboratories, b r o m i n e v a p o r is u s e d


36

PESTICIDES

Table III.

Methods for Converting Organophosphorus Pesticides to Active Esterase Inhibitors Method


IDENTIFICATION

Reference

C o l d fuming nitric acid Dilute bromine water iV-Bromosuccinamide U V light H 0 - A c e t i c acid Bromine vapor Peracetic acid ra-Chloroperbenzoic acid H 0 2

2

2

2

(4) (9) (9, 21) (57) (17) (47) (37) (37) (37)

to a c c o m p l i s h a c t i v a t i o n of o r g a n o p h o s p h o r u s pesticides after s e p a r a t i o n on thin layer chromatograms.

H o w e v e r , the b r o m i n e also converts some

p l a n t constituents i n t o antiesterase c o m p o u n d s a n d thus a d d s a n e w source of interference. T h e r e are o t h e r d i s a d v a n t a g e s , s o m e of w h i c h are p e c u l i a r to the method employed.

F o r e x a m p l e , i n the case of the a u t o m a t e d analysis

d e s c r i b e d b y V o s s ( 8 ) , some of the p u m p t u b i n g u s e d w a s s u s c e p t i b l e to c o n t a m i n a t i o n b y c e r t a i n i n s e c t i c i d e s . O n e of t h e m o s t i m p o r t a n t advantages of these m e t h o d s is t h e i r sensitivity.

G e n e r a l l y , these m e t h o d s m e a s u r e s u b m i c r o g r a m q u a n t i t i e s

of the i n s e c t i c i d e i n q u e s t i o n a n d are m o r e sensitive t h a n most c h e m i c a l methods.

M o r e o v e r , e n z y m a t i c m e t h o d s c a n detect insecticides t h a t are

converted into metabolites w i t h a h i g h inhibitory potency. methods can be simple a n d rapid.

Enzymatic

A u t o m a t e d analyses, for

example,

p r o v i d e s i m p l e h i g h - p r e c i s i o n t e c h n i q u e s w i t h short i n c u b a t i o n p e r i o d s , h i g h s e n s i t i v i t y , a n d a d a p t a b i l i t y to r o u t i n e analyses. A s m e n t i o n e d p r e v i o u s l y , s u c h m e t h o d s are i n v a l u a b l e as s c r e e n i n g t e c h n i q u e s .

Summary A d e s c r i p t i o n of s e v e r a l anticholinesterase a n d anticarboxylesterase t e c h n i q u e s as t h e y are u s e d i n p e s t i c i d e r e s i d u e analysis has b e e n g i v e n . S o m e o t h e r e n z y m e t e c h n i q u e s h a v e also b e e n m e n t i o n e d , as w e l l as the advantages a n d disadvantages of these t e c h n i q u e s .

Literature Cited (1) (2) (3)

O'Brien, R. D . , "Toxic Phosphorus Esters," Ch. 3, Academic, N e w York, 1960. O'Brien, R. D., "Insecticides: A c t i o n and Metabolism," pp. 39-54, 8 6 - 9 5 , Academic, N e w York, 1967. Heath, D . F . , "Organophosphorus Poisons," Introductory Chapter, Pergamon, Oxford, 1961.



3.

V I L L E N E U V E

Enzymatic

Techniques

for

Analysis

37

(4) G i a n g , P . A., H a l l , S. A., A n a l . Chem. (1951) 2 3 , 1 8 3 0 - 4 . (5) O ' B r i e n , R . D., J. Agr. Food Chem. (1963) 11, 163. (6) Voss, G . , Bull. Environ. Contam. Toxicol. (1968) 3, 343. (7) M a i n , A . R., Science (1964) 144, 9 9 2 - 3 . (8) Voss, G . , Residue Rev. (1968) 2 3 , 7 1 - 9 5 . (9) Archer, T . E . , " E n z y m a t i c M e t h o d s " in " A n a l y t i c a l Methods for Pesticides, Plant G r o w t h Regulators and F o o d Additives," Vol. 1, C h . 14, G . Zweig, Ed., Academic, N e w York, 1963. (10) Gage, J. C., Advan. Pest Control Res. (1961) 4, 183-210. (11) Schechter, M. S., Hornstein, I., Advan. Pest Control Res. (1957) 1, 3 5 3 447. (12) Cook, J . W . , J. Assoc. Offic. Anal. Chemists (1954) 37, 561-4. (13) Archer, T . E., Zweig, G . , J. Agr. Food Chem. (1959) 7, 178-81. (14) W i n t e r , G . D., Ann. N. Y. Acad. Sci. (1960) 87, 875. (15) W i n t e r , G . D . , Ferrari, A . , Residue Rev. (1964) 5, 139. (16) F i s c h l , J . , Pinto, N., Gordon, C., Clin. Chem. (1968) 14, 3 7 1 - 3 . (17) B e a m , J. E., Hankenson, D. J., J. Dairy Sci. (1964) XLVII, 1297-1305. (18) L e v i n e , J . B . , Scheidt, R. A., Nelson, V. A., Technicon Symposium, " A u t o mation i n Analytical Chemistry," p. 582, N e w York, 1965. (19) Voss, G . , J. Econ. Entomol. (1966) 59, 1288. (20) Guilbault, G . G . , Kramer, D. N., Anal. Chem. (1965) 37, 1675-80. (21) Schultzmann, R. L., Barthel, W . F., J. Assoc. Offic. Anal. Chemists (1969) 52, 151-6. (22) Benyon, K . I., Stoydin, G . , Nature (1965) 208, 748-50. (23) H i m e l , C . M., M a y e r , R. T . , J. Georgia Entomol. Soc. (1970) 5, 31-8. (24) H i m e l , C . M., Mayer, R. T . , Cook, L. L., J. Polymer Sci. (1970) Pt. A-1, 8, 2219-30. (25) H i m e l , C . M., Aboul-Saad, W. G . , U k , S., J. Agr. Food Chem., i n press, 1971. (26) H i m e l , C . M., private communication, 1970. (27) D i x o n , M., W e b b , E . C., " E n z y m e s , " p. 732, Longmans, Green and C o . Ltd., 1965. (28) Barron, K . D., Bernsohn, J. I., Hess, A., J. Histochem. Cytochem. (1961) 9, 656-60. (29) Bernsohn, J., Barron, K . D., Hess, A., Proc. Soc. Exptl. Biol. Med. (1961) 108, 7 1 - 3 . (30) Cook, J. W . , Blake, J., Y i p , G., W i l l i a m s , M., J. Assoc. Offic. Agr. Chemists (1958) 4 1 , 399-407. (31) Ecobichon, D . J., K a l o w , W . , Can. J . Biochem. Physiol. (1961) 39, 1329-32. (32) Holmes, R. S., Masters, C . J., Biochem. Biophys. Acta (1968) 151, 147-58. (33) Lawrence, S. H., Melnick, P. J., W e i m e r , H. E., Proc. Soc. Exptl. Biol. Med. (1960) 105, 5 7 2 - 5 . (34) M a i n , A . R., B r a i d , P . E., Biochem. J. (1962) 84, 2 5 5 - 6 3 . (35) Norgaard, M. J., Montgomery, M. W . , Biochim. Biophys. Acta (1968) 151, 587-96. (36) Seume, F . W . , Casida, J. E., O'Brien, R. D., J. Agr. Food Chem. (1960) 8, 4 3 - 7 . (37) D i x o n , G . H., Neurath, H., Pechere, J. F., Ann. Rev. Biochem. (1958) 27, 489-532. (38) F r u t o n , J . S., Harvey Lecture Ser. (1955-56) 5 1 , 6 4 - 8 7 . (39) Aldridge, W . N., Biochem. J. (1950) 46, 451-60. (40) M a i n , A. R., Dauterman, W. C., Nature (1963) 196, 5 5 1 - 3 . (41) M a i n , A. R., Iverson, F., Biochem. J. (1966) 100, 5 2 5 - 3 1 . (42) M c K i n l e y , W . P . , Read, S. I., J. Assoc. Offic. Agr. Chemists (1962) 4 5 , 467-73.



38

PESTICIDES

IDENTIFICATION

(43) Ackermann, H., Nahrung (1966) 10, 273-4. (44) Ackermann, H., J. Chromatog. (1968) 36, 309-17. (45) M e n d o z a , C . E., Wales, P . J., M c L e o d , H. A., M c K i n l e y , W . P . , Analyst (1968) 9 3 , 3 4 - 8 . (46) Wales, P . J . , M e n d o z a , C . E., M c L e o d , H. A., M c K i n l e y , W . P . , Analyst (1968) 9 3 , 6 9 1 - 3 . (47) Villeneuve, D . C., Butterfield, A . G . , M c C u l l y , K . A . , Bull. Environ. Contam. Toxicol. (1969) 4, 232-9. (48) Geike, F., J. Chromatog. (1969) 44, 95-102. (49) Ackermann, H., Nahrung (1968) 12, 357-62. (50) B r o w n , H. V., Bush, A. F., Arch. Ind. Hyg. (1950) 1, 633-6. (51) M e n n , J . J . , M c B a i n , J. B . , Dennis, M. J., Nature (1964) 202, 697-8. (52) D u B o i s , K . P . , Gladys, J. C., Arch. Ind. Health (1955) 11, 53-60. (53) Voss, Gunther, J. Assoc. Offic. Anal. Chemists (1969) 52, 1027-34. (54) Guilbault, G . G . , Lubrano, G . J., Anal. Chim. Acta (1968) 4 3 , 2 5 3 - 6 1 . (55) Guilbault, G . C., Sadar, M. H., Zimmer, M., Anal. Chim. Acta (1969) 44, 361-7. (56) M c K i n l e y , W . P . , Johal, P . S., J. Assoc. Offic. Agr. Chemists (1963) 46, 840-2. (57) Ackermann, H., Arch. Toxikol. (1969) 24, 325-31. RECEIVED

June 12, 1970.


Pesticides Identification at the Residue Level - ACS Publications

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