Pesticide Analytical Methodology - ACS Publications - American

14 Distribution of Pesticides in H u m a n Organs as Determined by Quantitative T h i n - L a y e r Downloaded via UNIV OF CALIFORNIA SANTA BARBARA on July 20, 2018 at 19:40:27 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Chromatography S. N. T E W A R I

1

State Forensic Science Laboratory, Government of Uttar Pradesh Lucknow (India)

To c o n t r o l t h e heavy l o s s e s o f c r o p s from i n s e c t s and o t h e r p e s t s , t h e use o f d i f f e r e n t p e s t i c i d e s has become e x t e n s i v e , and w i t h t h e p a s s i n g o f t i m e , more o f t h e s e compounds a r e b e i n g d e v e l o p e d and p r o d u c e d on a c o m m e r c i a l s c a l e . At p r e s e n t , a p a r t from t h e i n o r g a n i c compounds s u c h as z i n c p h o s p h i d e and a r s e n i o u s o x i d e , s e v e r a l h u n d r e d o r g a n i c compounds, h a v i n g w i d e l y d i f f e r e n t c h e m i c a l c o n s t i t u e n t s and p r o p e r t i e s , a r e b e i n g u s e d t h r o u g h o u t the w o r l d . The f r e q u e n t use o f t h e s e o r g a n i c p e s t i c i d e s has r e s u l t e d i n t h e i r widespread d i s t r i b u t i o n i n the environment. Often they are found i n unwanted p l a c e s , t h e r e b y c a u s i n g p o l l u t i o n p r o b l e m s . B e c a u s e t h e s e p e s t i c i d e s a r e t o x i c n o t o n l y t o i n s e c t s and p e s t s b u t a l s o , i n v a r y i n g d e g r e e s , t o h i g h e r a n i m a l s and m a n , t h e s e c h e m i c a l s have c r e a t e d a p u b l i c h e a l t h m e n a c e . Today many c o m m e r c i a l p r o d u c t s a r e a v a i l a b l e as home remedi e s a g a i n s t f l i e s , t i c k s , b u g s , r a t s and o t h e r i n s e c t s and p e s t s under t h e t r a d e names o f ' F l i t ' , T i c k - 2 0 , B u g m a r , D a l f , B u g t o n and many m o r e . These p e s t i c i d e s a r e a l s o a v a i l a b l e i n c o n c e n t r a ted s o l u t i o n s , powders, a e r o s o l s , e m u l s i o n s , e t c . f o r a g r i c u l tural purposes. Due t o t h e i r e a s y a v a i l a b i l i t y and r a p i d t o x i c a c t i o n , t h e s e p e s t i c i d e s have been m i s u s e d as h o m i c i d a l a g e n t s and a c t e d as a c c i d e n t a l p o i s o n s . The m i s u s e has become a p r o b l e m f o r t h e t o x i c o l o g i s t , e s p e c i a l l y i n I n d i a , where p o i s o n i n g s by t h e u s e o f p e s t i c i d e s a r e i n c r e a s i n g a n n u a l l y . The i n v e s t i g a t i o n o f t h e s e a c c i d e n t a l d e a t h s has become a g r e a t c h a l l e n g e t o t h e forensic toxicologist. In o r d e r t o e s t a b l i s h t h a t t h e d e a t h o f a v i c t i m i s due t o t h e i n g e s t i o n o f a s p e c i f i c p o i s o n , t h e a b s o l u t e p r o o f needed i s t h e d e t e c t i o n and i d e n t i f i c a t i o n o f t h e p o i s o n i n v a r i o u s o r g a n s of the body. Poisonous substances are r a r e l y d i s t r i b u t e d evenly i n body t i s s u e s b u t t e n d t o a c c u m u l a t e i n some p a r t i c u l a r o r g a n due t o t h e i r c h e m i c a l n a t u r e and m e t a b o l i c c o n v e r s i o n s . There''Director, State Forensic Science Laboratory, P r a d e s h Lucknow ( I n d i a ) .

Govt, of

0-8412-0581-7/80/47-136-259$05.00/0 © 1980 American Chemical Society Harvey et al.; Pesticide Analytical Methodology ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

Uttar

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f o r e t o s e a r c h f o r a p o i s o n a b o u t w h i c h no d e t a i l e d i n f o r m a t i o n i s a v a i l a b l e , i n c l u d i n g a s t o how a n d when i t was a d m i n i s t e r e d , a f u l l a n a l y s i s o f a v a r i e t y o f autopsy m a t e r i a l i s r e q u i r e d . This a n a l y s i s p r o v i d e s t h e d a t a on t h e d i s t r i b u t i o n o f t h e p o i s o n i n d i f f e r e n t o r g a n s and may be o f g r e a t v a l u e i n d e d u c i n g t h e p r o b a b l e o r i g i n a l d o s e i n g e s t e d a n d a l s o t h e t i m e , a n d t o some e x t e n t , the nature o f a d m i n i s t r a t i o n o f the poison. These d a t a may a l s o h e l p t o g i v e a c l u e r e g a r d i n g t h e cause o f d e a t h . However, f r o m e x p e r i e n c e i t has been f o u n d t h a t t h e knowledge o f ( 1 ) t h e a c t i o n o f t h e p e s t i c i d a l p o i s o n under c o n s i d e r a t i o n , ( 2 ) c l i n i c a l c o u r s e o f t h e p o i s o n i n g a n d ( 3 ) t h e postmortem f i n d i n g s i s o f great importance i n the i n t e r p r e t a t i o n o f the r e s u l t s obtained from chemical a n a l y s i s o f t h e m a t e r i a l r e c e i v e d . T h e r e a r e o c c a s i o n s i n w h i c h d e a t h i s u n d o u b t e d l y due t o a p e s t i c i d e , and y e t t h e c h e m i c a l a n a l y s i s f a i l s t o r e v e a l i t s p r e s e n c e o r shows o n l y i n s i g n i f i c a n t t r a c e s . T h i s may be due t o the r a p i d m e t a b o l i c conversion o f the p a r t i c u l a r p e s t i c i d e . This i s the case w i t h p a r a t h i o n which i s r a p i d l y converted t o p a r a n i t r o p h e n o l due t o i n v i v o h y d r o l y s i s . Pesticides, especially t h o s e t h a t a r e c o m p a r a t i v e l y l e s s t o x i c t o mammals, s u c h a s d i c h l o r o v o s , e t c , may be e x c r e t e d r a p i d l y a n d , t h e r e f o r e , may p e r h a p s be f o u n d i n t h e u r i n e , t h o u g h n o t i n t h e t i s s u e s . The s t u d y o f t h e d i s t r i b u t i o n o f a p o i s o n , p o s t m o r t e m , p r e s e n t s a complex problem. The e s t i m a t i o n o f t h e p o i s o n i n t h e b l o o d p r e s e n t s a c o m p l e x p r o b l e m due t o t h e p u t r e f a c t i v e changes i n b l o o d and t h e minute q u a n t i t y a v a i l a b l e f o r a n a l y s i s . Distrib u t i o n o f i n s e c t i c i d a l p o i s o n s from t h e b l o o d t o t h e t i s s u e s and body f l u i d s t a k e s p l a c e w i t h some r e c i r c u l a t i o n t o t h e b l o o d ( l _ ) . The body t i s s u e s a r e g e n e r a l l y s u b m i t t e d t o a t o x i c o l o g i c a l l a b o r a t o r y i n s u f f i c i e n t q u a n t i t i e s ; hence t h e s e s p e c i m e n s a r e better suited for quantitative analysis. The d e t e c t i o n a n d d e t e r m i n a t i o n o f t h e unchanged i n s e c t i c i d e i n t h e s t o m a c h , i n t e s t i n e , l i v e r , k i d n e y , s p l e e n , l u n g s , b r a i n , and i n o t h e r t i s s u e s and body f l u i d s a r e u s e f u l i n d i c a t o r s o f d i r e c t i n s e c t i c i d e action. I n o r d e r t o r e a c h a c o n c l u s i o n when and how a n d , a n d t o some e x t e n t , how much o f a p a r t i c u l a r i n s e c t i c i d e was a d m i n i s t e r e d , t h e f o r m and q u a n t i t y i n w h i c h t h e i n s e c t i c i d e e x i s t s i n d i f f e r e n t t i s s u e s a n d i n b l o o d a n d u r i n e may g i v e v a l u a b l e c l u e s . I n d e e d , a n i n t e l l i g e n t d e d u c t i o n i s a l s o b a s e d on t h e knowledge o f m e t a b o l i c pathways a n d f o r m a t i o n o f o t h e r d e r i v a t i v e s . Thus, as p a r a t h i o n i s e x c r e t e d i n t h e u r i n e u l t i m a t e l y as p - n i t r o p h e n o l , u r i n a r y p - n i t r o p h e n o l l e v e l s may i n d i c a t e t h e e x t e n t o f exposure t o p a r a t h i o n ( 2 j . In c a s e s o f m i l d a n d m o d e r a t e e x p o s u r e , t h e e x c r e t e d p - n i t r o p h e n o l i n u r i n e was f o u n d t o be o f t h e o r d e r o f 0 . 0 5 7 t o 0 . 3 2 2 mg. p e r c e n t . In s e v e r e a n d f a t a l c a s e s o f p o i s o n i n g by p a r a t h i o n , t h e l e v e l o f p - n i t r o p h e n o l i n u r i n e was f r o m 0 . 1 6 t o 1.16 mg. p e r c e n t . para-Nitrophenol thus i s r a p i d l y e x c r e t e d i n u r i n e a n d no l o n g e r d e t e c t e d 4 8 h o u r s a f t e r the exposure t o the p e s t i c i d e .

Harvey et al.; Pesticide Analytical Methodology ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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H e y n d r i c k x e t . aj_. (_3) have p o i n t e d o u t t h a t t h e p e s t i c i d e a b s o r b e d by mammals i s t r a n s p o r t e d i n t o m i l k . A c a s e i n p o i n t i s a one y e a r o l d c h i l d who s u f f e r e d f r o m c h r o n i c p a r a t h i o n p o i s o n i n g a f t e r d r i n k i n g t h e m i l k o f a cow f e d on p a r a t h i o n - c o n t a m inated water. A r e v i e w o f 41 c a s e s o f 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 p o i s o n i n g and t h e q u a n t i t a t i v e d a t a have been r e p o r t e d by Heyndrickx(4,j)). Lewi η e t . a h ( 6 ) have r e p o r t e d t h e d i s t r i b u t i o n d a t a f r o m some c a s e s o f m a l a t h i o n p o i s o n i n g u s i n g GLC f o r t h e d e t e r m i n a t i o n o f i n s e c t i c i d e s e x t r a c t e d from d i f f e r e n t autopsy tissues. The d a t a o b t a i n e d by t h e s e w o r k e r s a r e g i v e n i n T a b l e I. The d i s t r i b u t i o n o f s u m i t h i o n , d i m e t h o a t e , e n d r i n and o t h e r o r g a n o c h l o r o - and 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 have a l s o been r e p o r t e d by t h e a u t h o r and c o - w o r k e r s (7-12^). Once t h e p e s t i c i d e i s d e t e c t e d i n t h e t i s s u e s and body f l u i d s , c a r e f u l c o n s i d e r a t i o n i s g i v e n t o the f o l l o w i n g d a t a : (1) t h e amounts o f t h e p e s t i c i d e f o u n d i n v a r i o u s body f l u i d s and t i s s u e s e x a m i n e d ; ( 2 ) how r a p i d l y t h e p o i s o n i s a b s o r b e d ; (3) how r a p i d l y and a t what c o n c e n t r a t i o n i t e x e r t s i t s e f f e c t s ; a n d , ( 4 ) how t h e d i s t r i b u t i o n t h r o u g h t h e t i s s u e s and body f l u i d s v a r i e s w i t h t i m e and r o u t e o f a c c e s s . This information i s e s s e n t i a l , and a d e t a i l e d d e s c r i p t i o n o f s t u d i e s on t h e d i s t r i b u t i o n o f p e s t i c i d e s i n human t i s s u e s p e r t a i n i n g t o h o m i c i d e , s u i c i d e and a c c i d e n t a l p o i s o n i n g cases i s d e s c r i b e d below: Table I : Amount o f m a l a t h i o n e s t i m a t e d i n d i f f e r e n t b i o l o g i c a l m a t e r i a l s from p o i s o n c a s e s , (from Lewin, e t . a l . ( 6 ) ) . Malathion Case No.

Stomach

1. 2. 3. 4. 5. 6. 7.

992 172 1296 31 357 600 5400

(mg./lOOg.) p r e s e n t Liver 166 130 170 26

i n the: Blood 188 29 99 10

- 0.24

- 0.50 0.067

To s t u d y t h e d i s t r i b u t i o n o f p e s t i c i d e s , t h e amount o f t h e p e s t i c i d e s p r e s e n t i n d i f f e r e n t a u t o p s y t i s s u e s and o t h e r b i o l o g i c a l m a t e r i a l s have been e s t i m a t e d q u a n t i t a t i v e l y i n f a t a l c a s e s o f p o i s o n i n g o f b o t h male and f e m a l e s u b j e c t s . The c a s e s were o f (1) s u i c i d e s i n which l a r g e excess o f the p o i s o n i s g e n e r a l l y taken t o ensure quick a c t i o n ; (2) homicides i n which t h e p o i s o n i s a d m i n i s t e r e d by m i x i n g i t i n f o o d o r d r i n k , and (3) a c c i d e n t a l p o i s o n i n g s where l a r g e d o s e s o f p e s t i c i d e s a r e t a k e n up w h i l e s p r a y i n g .


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The methods o f e x t r a c t i o n o f t h e p e s t i c i d e s f r o m t i s s u e s a s r e p o r t e d by t h e a u t h o r and coworkers (13-15) a r e d e s c r i b e d b e l o w : I s o l a t i o n o f 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 From Body T i s s u e s . Twenty grams o f t h e t i s s u e i s m a c e r a t e d i n t o a f i n e s l u r r y b y m i x i n g i t w i t h e q u a l amounts o f a n h y d r o u s s o d i u m s u l f a t e a n d p l a c i n g i t i n t o a c o n i c a l f l a s k f i t t e d w i t h an a i r c o n d e n s e r . F i f t y m i l l i l i t e r s o f n-hexane i s a d d e d , and t h e f l a s k i s heated on a b o i l i n g w a t e r b a t h f o r o n e h o u r . A f t e r c o o l i n g , t h e n hexane l a y e r i s f i l t e r e d . The e x t r a c t i o n o f t h e r e s i d u a l s l u r r y i s repeated twice with twenty-five m i l l i l i t e r portions o f n hexane. The f i l t e r e d n - h e x a n e f r a c t i o n s a r e c o m b i n e d a n d t r a n s fered to a separatory funnel. The hexane l a y e r i s v i g o r o u s l y s h a k e n f o r f i v e m i n u t e s s u c c e s s i v e l y w i t h 15 m l - , 10 m l - , a n d 10 m l - p o r t i o n s o f a c e t o n i t r i l e p r e v i o u s l y s a t u r a t e d w i t h n-hexane. The a c e t o n i t r i l e l a y e r s a r e m i x e d a n d p o u r e d i n t o a n o t h e r s e p a r a tory funnel. I n c a s e s o f d i m e t h o a t e , D a s a n i t , phosphamidon a n d DDVP p o i s o n i n g s , t h e t h r e e a c e t o n i t r i l e f r a c t i o n s a r e combined and e v a p o r a t e d t o d r y n e s s , a n d t h e r e s i d u e i s t a k e n up i n one m i l l i l i t e r o f acetone. Further extraction of acetonitrile i s a v o i d e d a s t h i s r e s u l t s i n a p p r e c i a b l e l o s s o f p e s t i c i d e s . The a c e t o n i t r i l e l a y e r i s d i l u t e d w i t h w a t e r t o t e n times o f i t s o r i g i n a l volume. T w e n t y - f i v e m l . o f s a t u r a t e d sodium s u l f a t e s o l u t i o n i s added t o t h i s s o l u t i o n a n d e x t r a c t e d t h r e e t i m e s w i t h t w e n t y - f i v e m i l l i l i t e r - p o r t i o n s o f n-hexane p r e v i o u s l y s a t u r a t e d with d i s t i l l e d water. The s e p a r a t e d n - h e x a n e l a y e r s a r e c o m b i n e d and t h e n c o n c e n t r a t e d t o two m i l l i l i t e r s on a warm w a t e r b a t h . The r e m a i n i n g s o l v e n t i s removed by p a s s i n g a c u r r e n t o f d r y a i r o v e r i t . The r e s i d u e i s t h e n d i s s o l v e d i n one m i l l i l i t e r o f acetone. The e x t r a c t i o n f r o m n o n - f a t t y t i s s u e s i s p e r f o r m e d a s f o l lows: The t i s s u e sample (20 g . ) i s f i n e l y m i x e d a n d a c i d i f i e d w i t h one m i l l i l i t e r o f 5N s u l f u r i c a c i d , t h e n t r a n s f e r r e d t o a conical flask. To t h i s , t e n grams o f a n h y d r o u s s o d i u m s u l f a t e and f i f t y m i l l i l i t e r s o f a c e t o n e a r e a d d e d , a n d t h e m i x t u r e i s r e f l u x e d on a warm w a t e r b a t h f o r t h i r t y m i n u t e s a n d t h e n f i l t e r ed. The r e s i d u a l m a t e r i a l i s f u r t h e r e x t r a c t e d t w i c e w i t h f i f t y m i l l i l i t e r - p o r t i o n s o f acetone. The a c e t o n e l a y e r s a r e c o m b i n e d , c o n c e n t r a t e d b y e v a p o r a t i o n on a warm w a t e r b a t h t o a b o u t t w e n t y f i v e m i l l i l i t e r s and then t r a n s f e r r e d t o a s e p a r a t o r y f u n n e l . The a c e t o n e s o l u t i o n i s d i l u t e d w i t h f o u r t i m e s t h e volume o f water. Twenty m i l l i l i t e r s o f s a t u r a t e d aqueous s o d i u m s u l f a t e i s added a n d t h e n e x t r a c t e d t h r e e t i m e s b y s h a k i n g f o r 5 m i n u t e s with twenty-five m i l l i l i t e r - p o r t i o n s of chloroform. The c h l o r o f o r m e x t r a c t s a r e combined a n d s h a k e n w i t h a s m a l l amount o f a c t i v a t e d c h a r c o a l f o r d e c o l o r i z a t i o n a n d t h e n f i l t e r e d t h r o u g h Whatman f i l t e r p a p e r . The c h a r c o a l on t h e f i l t e r p a p e r i s washed w i t h t w e n t y m i l l i l i t e r s o f c h l o r o f o r m , a n d t h e w a s h i n g s are c o l l e c t e d together with the chloroform e x t r a c t . The c o m b i n e d c h l o r o f o r m e x t r a c t i s d r i e d by p a s s i n g i t t h r o u g h a column o f


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a n h y d r o u s s o d i u m s u l f a t e and t h e n e v a p o r a t e d j u s t t o d r y n e s s on a warm w a t e r b a t h . The r e m a i n i n g s o l v e n t i s removed by p a s s i n g a c u r r e n t of dry a i r over i t . T h i s r e s i d u e i s d i s s o l v e d i n one mi 11 i l i t e r o f a c e t o n e . Carbamate p e s t i c i d e s a r e a l s o e x t r a c t e d by t h i s m e t h o d . I s o l a t i o n of Organochloro I n s e c t i c i d e s . Twenty grams o f t i s s u e i s m a c e r a t e d i n t o a f i n e s l u r r y and t r a n s f e r r e d t o a c o n i c a l f l a s k f i t t e d w i t h a condenser. T h i r t y grams o f a n h y d r o u s sodium s u l f a t e and f i f t y m i l l i l i t e r s o f acetone a r e added. The c o n i c a l f l a s k i s p l a c e d on a b o i l i n g w a t e r b a t h a t 80° f o r h a l f an h o u r . The f l a s k i s removed f r o m t h e w a t e r b a t h , c o o l e d t o room t e m p e r a t u r e , and t h e c o n t e n t s a r e f i l t e r e d . The p r o c e s s i s repeated twice with twenty-five m i H i l i t e r - p o r t i o n s of acetone. The f i l t e r e d a c e t o n e l a y e r s a r e combined and t r a n s f e r e d t o a séparatory f u n n e l . One h u n d r e d m i l l i l i t e r s o f d i s t i l l e d w a t e r and t w e n t y m i l l i l i t e r s o f a s a t u r a t e d aqueous s o l u t i o n o f s o d i u m s u l f a t e i s added t o t h e a c e t o n e l a y e r . The s o l u t i o n i s t h e n extracted with chloroform previously saturated with d i s t i l l e d w a t e r u s i n g t w e n t y and two t e n m i l l i l i t e r - p o r t i o n s . The e x t r a c t i o n i s c o m p l e t e d by s l o w l y s h a k i n g f o r f i v e m i n u t e s (20 t o 25 shakings per m i n u t e ) . The s e p a r a t e d c h l o r o f o r m l a y e r s a r e c o m b i n e d and p l a c e d i n a n o t h e r 250-ml s e p a r a t o r y f u n n e l . Twenty m i l l i l i t e r s o f 5% aqueous p o t a s s i u m h y d r o x i d e s o l u t i o n i s added and s h a k e n f o r one m i n u t e . The c h l o r o f o r m l a y e r i s s e p a r a t e d by d i s c a r d i n g t h e aqueous l a y e r a n d a g a i n s h a k e n f o r one m i n u t e w i t h t w e n t y mi H i l i t e r s o f 5% KÛH. The s e p a r a t e d c h l o r o f o r m l a y e r i s now washed w i t h f i v e 20 m l . p o r t i o n s o f d i s t i l l e d w a t e r by s l o w l y s h a k i n g i t f o r one m i n u t e a t each t u r n . The washed c h l o r o f o r m l a y e r i s d r i e d by p a s s i n g t h r o u g h a l a y e r o f a n h y d r o u s s o d i u m s u l f a t e p r e v i o u s l y wetted with dry chloroform. The o r g a n i c s o l v e n t i s t h e n c o n c e n t r a t e d up t o two m i l l i l i t e r s on a warm w a t e r b a t h a t 6 0 ° , and t h e r e m a i n i n g s o l v e n t i s e v a p o r a t e d t o d r y n e s s i n a c u r r e n t of dry a i r . The r e s i d u e w h i c h c o n t a i n s t h e p e s t i c i d e i s t h e n d i s s o l v e d i n one m i l l i l i t e r o f a c e t o n e . Methods o f A n a l y s i s o f P e s t i c i d e s . The e x t r a c t e d r e s i d u e o b t a i n e d a f t e r i s o l a t i o n f r o m t i s s u e s and o t h e r b i o l o g i c a l m a t e r i a l s i s s u b j e c t e d t o q u a l i t a t i v e and 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 o f the p e s t i c i d e s . S o m e t i m e s , t h e amount o f m a t e r i a l a v a i l a b l e i s so s m a l l t h a t t h e c o l o r i m e t r i c and o t h e r a l l i e d methods c a n n o t be s u c c e s s f u l l y a p p l i e d as some o f t h e r e s i d u e i s l i k e l y t o be l o s t d u r i n g t h e p u r i f i c a t i o n t e c h n i q u e . Furthermore, these p u r i f i c a t i o n techniques required for spectrophotometry, c o l o r i m e t r y , and o t h e r s o p h i s t i c a t e d i n s t r u m e n t a l methods a r e a p p r e c i a b l y t i m e c o n s u m i n g . T h e r e f o r e , o t h e r t e c h n i q u e s were s o u g h t f o r 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 p e s t i c i d e s . T h i n l a y e r chromat o g r a p h i c (TLC) t e c h n i q u e s were f o u n d t o be most s u i t a b l e f o r toxicological analysis of pesticides. Randerath(lJ5) s t a t e d t h a t


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a c c u r a t e r e s u l t s i n q u a n t i t a t i v e TLC were o b t a i n e d by r e m o v a l o f t h e s u b s t a n c e t o be a n a l y z e d f r o m t h e a d s o r b e n t u s i n g s u i t a b l e solvents f o r elution. C h o u 1 i s ( 1 7 , 1 8 ) has a l s o advocated t h e e l u t i o n technique f o r q u a n t i t a t i v e analyses. The e l u t i o n t e c h n i q u e i s n o r m a l l y used i n a s s o c i a t i o n w i t h g r a v i m e t r i c , s p e c t r o p h o t o m e t r y , c o l o r i m e t r i c , f l u o r o m e t r i c and other e s t i m a t i o n methods(19). The g r a v i m e t r i c method b e i n g u n r e l i a b l e and y i e l d i n g g e n e r a l l y h i g h r e s u l t s i s n o t s u i t a b l e for toxicological analysis. The a c c u r a c y o f t h e f l u o r o m e t r i c method i s g e n e r a l l y c o n s i d e r e d t o be l o w . R e g a r d l e s s , t h e f l u o r o m e t r i c method c a n o n l y be a p p l i e d t o t h o s e s u b s t a n c e s w h i c h f l u o r e s c e under t h e assay c o n d i t i o n s o r which f l u o r e s c e a f t e r r e a c t i n g w i t h some s u i t a b l e r e a g e n t . C o l o r i m e t r i c methods a r e a p p l i c a b l e o n l y t o those s u b s t a n c e s which form c o l o r e d s o l u t i o n s w i t h a c h r o m o g e n i c r e a g e n t , t h e a b s o r b a n c e o f w h i c h c a n be measured using a spectrophotometer o r c o l o r i m e t e r . These e l u t i o n methods p r e s e n t s e v e r a l d i s a d v a n t a g e s f o r t h e f o l l o w i n g r e a s o n s : ( i ) I n a c c u r a t e m a r k i n g o f t h e s p o t t o be e l u t e d f r o m TLC p l a t e , ( i i ) Incomplete d e s o r p t i o n o f t h e s u b s t a n c e i n t h e s o l v e n t used for elution. ( i i i ) I n t e r f e r e n c e due t o c e r t a i n s u b s t a n c e s e x t r a c t e d f r o m t h e adsorbents. For these r e a s o n s , Purdy and Truter(2fJ) and s e v e r a l o t h e r w o r k e r s have s t r e s s e d methods by w h i c h measurement o f t h e a r e a o f t h e d e v e l o p e d c o l o r e d s p o t on TLC p l a t e s c a n be u s e d d i r e c t l y f o r quantitative analysis. Methods f o r m e a s u r i n g t h e s p o t a r e a may be c l a s s i f i e d as f o l l o w s : 1. V i s u a l comparison o f spot i n t e n s i t y and/or s i z e t o a s t a n d a r d amount o f t h e same p e s t i c i d e r u n on t h e same TLC p l a t e . This p r o v i d e s a n e s t i m a t i o n o f t h e s u b s t a n c e w i t h a p p r o x i m a t e l y 20% a c c u r a c y . A d e t a i l e d d e s c r i p t i o n o f t h i s method o f v i s u a l e s t i m a t i o n o f t h i n l a y e r c h r o m a t o g r a p h s has been g i v e n by J o h n s o n ( 2 1 ) , who h a s v e r y w i s e l y commented t h a t i t i s b e t t e r t o be " a p p r o x i mately r i g h t than p r e c i s e l y wrong". 2. The a r e a o f t h e s p o t may be t r a c e d o n g r a p h p a p e r , a n d t h e s q u a r e s on t h e p a p e r c o u n t e d a n d compared w i t h t h o s e o f known amounts o f a s t a n d a r d p e s t i c i d e . T h i s method g i v e s s l i g h t l y b e t t e r r e s u l t s than 1., but l a c k s accuracy. 3. S c a n n i n g t h e d e v e l o p e d c o l o r e d s p o t on t h e t h i n l a y e r chromatographic plate with the help o f a p h o t o e l e c t r i c d e n s i tometer. G a e n s h i r t and Polderman(22) have mentioned t h a t t h e e r r o r s o f t h e 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 o f a p e s t i c i d e by p l a n i m e t r i c measurements o f t h e a r e a o f t h e s p o t s a r e g e n e r a l l y h i g h a s a r e s u l t o f v a r i a t i o n s i n t h e shape o f t h e s p o t a n d d i f f i c u l t i e s i n defining the outline o f the spot. M o r r i s o n and C h a t t e n ( 2 3 ) and O s w a l d a n d F l u e c k ( 2 4 ) have p o i n t e d o u t t h e same d i f f i c u l t i e s i n t h e v i s u a l measurement o f t h e a r e a o f t h e s p o t . Quantitative a n a l y s i s on TLC w i t h a f l y i n g s p o t d e n s i t o m e t e r has been s u g g e s t ed by S c h u t e a n d c o - w o r k e r s ( 2 5 ) . D a l l a s ( 2 6 ) i n 1965 h a s d e s -


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c r i b e d t h e use o f a " C h r o m a s c a n " f o r s c a n n i n g c o l o r e d s u b s t a n c e s on a d s o r b e n t s s p r e a d on g l a s s p l a t e s . Genest(27) used a P h o t o v o l t d e n s i t o m e t e r f o r d e t e r m i n a t i o n o f d r u g s on T L C . Later, B l u n d e n and c o - w o r k e r s ( 2 8 ) , S h e l l a r d ( 2 9 ) and Thomas e t a h (30) a l s o r e p o r t e d on t h e use o f a d e n s i t o m e t e r f o r q u a n t i t a t i o n . S h e l l a r d and A l a m ( 3 1 J i n 1968 have made a d e t a i l e d s t u d y o f t h e d e n s i t o m e t r i c method f o r TLC. S e v e r a l w o r k e r s have d e s c r i b e d t h e t h i n l a y e r c h r o m a t o g r a p h i c technique f o r the d e t e r m i n a t i o n o f organophosphorus p e s t i c i d e s ( 3 2 - 4 4 ) a s w e l l as c h l o r i n a t e d p e s t i c i d e s ( 4 5 - 5 6 ) . V y l e g z h a n i n a and Kalrrrykova(57) have d e s c r i b e d t h e use o f TLC f o r d e t e r m i n a t i o n o f DDVP i n p l a n t s , s o i l and w a t e r . Determin a t i o n o f 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 i n r i v e r w a t e r and e f f l u e n t s have a l s o been used by A s k e w , e t a h ( 5 8 ) . Z y c i n s k i ( 5 9 j has d e t e r m i n e d t h e c o n c e n t r a t i o n o f m a l a t h i o n , DDVP a n d o t h e r 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 i n w a t e r by d i r e c t measurement on TLC plates. TLC d e t e r m i n a t i o n o f s e v e r a l 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 s u c h as f e n i t r o t h i o n , f e n t h i o n , and m a l a t h i o n i n s o i l has been s u g g e s t e d by Cywi η s ka-Smo t e r ( 6 0 ) u s i n g Congo r e d s p r a y r e a g e n t f o r t h e d e v e l o p m e n t o f t h e s p o t s on T L C . Several workers ( 6 1 , 6 2 , 6 3 , 6 4 ) have u t i l i z e d t h e e s t e r a s e i n h i b i t i o n t e c h n i q u e f o r t h e l o c a t i o n o f t h e 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 on TLC and t h e i r d i r e c t measurement on t h e p l a t e s . Routine determination of 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 by TLC has a l s o been s u g g e s t e d by A n t o i n e and M e e s ( 3 9 ) . They have s u g g e s t e d p a l l a d i u m c h l o r i d e s p r a y o r 4 - ( 4 - n i t r o b e n z y l ) p y r i d i n e f o r l o c a t i o n o f t h e s p o t s on t h e TLC p l a t e and c o m p a r i s o n o f i n t e n s i t y and a r e a o f t h e d e v e l oped c o l o r e d s p o t s w i t h t h o s e o f known amounts o f t h e i n s e c t i c i d e s on t h e same TLC p l a t e . He11omann(65) has u s e d b o v i n e l i v e r e x t r a c t as s p r a y f o l l o w e d by c o m p a r i s o n o f t h e d e v e l o p e d y e l l o w c o l o r e d soot f o r the 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 . Kosmatyi e t aj_. (66) have u s e d b r o m o p h e n o l - b l u e / s i l v e r n i t r a t e as c h r o m o g e n i c r e a a e n t f o r t h e d e t e r m i n a t i o n o f d i a z i n o n . The a u t h o r ( 6 7 , 6 8 , 6 9 ) has r e p o r t e d t h e d e n s i t o m e t r i c d e t e r m i n a t i o n o f m i c r o gram amounts o f 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 i n t i s s u e s . D e t e r m i n a t i o n o f d i m e t h o a t e and m a l a t h i o n u s i n g p a l l a d i u m c h l o r i d e s p r a y on TLC p l a t e was d e s c r i b e d by M a c N e i l a n d c o workers (70). 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 organophosphorus p e s t i c i d e s by TLC d e n s i t o m e t r y 71J a n d TLC f l u o r o m e t r y ( 7 2 ) have a l s o been d e s c r i b e d . I v e y and 0 e h l e r ( 7 3 ) have d e s c r i b e d t h e d e t e r m i n a t i o n o f 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 s i n t i s s u e s a n d urine of c a t t l e . G e t z ( 7 4 ) has d e s c r i b e d t h e q u a n t i t a t i v e e s t i m a t i o n o f c h l o r i n a t e d p e s t i c i d e s by d e n s i t o m e t r y . Dobbins(75^) has used t h e d e n s i t o m e t r i c method f o r q u a n t i t a t i o n o f c h l o r i n a t e d p e s t i c i d e s using s i l v e r n i t r a t e spray reagent. Q u a n t i t a t i v e TLC t e c h n i q u e f o r o r g a n o c h l o r o p e s t i c i d e s was a l s o s u g g e s t e d by M a c N e i l e t a h ( 5 1 J and L a k s h i n a r a y a n a and c o - w o r k e r s ( 7 6 ) . Quan t i t a t i o n o f c a r b a m a t e p e s t i c i d e s by f l u o r e s c e n c e d e n s i t o m e t r y on TLC was d e s c r i b e d by some o f t h e w o r k e r s ( 7 7 , 7 8 , 7 9 , 8 0 ) . Z w e i g and Sherma(81) made an e x c e l l e n t r e v i e w on t h e q u a l i -


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t a t i v e and q u a n t i t a t i v e aspects o f t h i n l a y e r chromatography. In 1 9 7 7 , a s e l e c t i v e r e v i e w on p e s t i c i d e r e s i d u e s was w r i t t e n by Thornburg(82). From a r e v i e w o f t h e a v a i l a b l e l i t e r a t u r e i t i s c l e a r t h a t methods b a s e d on t h i n l a y e r c h r o m a t o g r a p h y a r e f r e q u e n t l y b e i n g used b y v a r i o u s w o r k e r s i n t h e f i e l d f o r t h e q u a n t i t a t i o n o f pesticides. I n t h e p r e s e n t p a p e r t h e q u a n t i t a t i v e TLC t e c h n i q u e using densitometry i s described i n d e t a i l f o r the determination of p e s t i c i d e s i n v a r i o u s t i s s u e s p e r t a i n i n g to a c t u a l cases o f poisoning. P r e p a r a t i o n o f Thin Layer

Plates

G l a s s p l a t e s ( 2 0 x 2 0 c m . ) a r e w e l l c l e a n e d w i t h non g r e a s y s o l v e n t a n d a r r a n g e d on t h e a l i g n i n g t r a y . T w e n t y - f i v e grams o f the adsorbent s i l i c a gel-G (or alumina with calcium s u l f a t e b i n d e r ) a r e mixed w i t h f i f t y m i l l i l i t e r s o f d i s t i l l e d water i n a 250 m l - f l a s k a n d g e n t l y s h a k e n t o f o r m a homogeneous s l u r r y . T h i s s l u r r y i s t r a n s f e r r e d i m m e d i a t e l y ( w i t h i n s i x t y seconds o f mixing adsorbent w i t h water) t o the a p p l i c a t o r . The s p r e a d e r i s a d j u s t e d f o r a 250-um t h i c k l a y e r ( o r o t h e r s u i t a b l e t h i c k n e s s ) b e f o r e h a n d , and a t h i n l a y e r o f t h e a d s o r b e n t i s c o a t e d on t h e plates. I n t h i s way a homogeneous 250-ym t h i c k l a y e r o f t h e absorbent i s applied to the glass p l a t e s . The p l a t e s a r e d r i e d a t room t e m p e r a t u r e a n d t h e n a c t i v a t e d i n a n oven a t 110° f o r 30 minutes. Storage. The p l a t e s a r e s t o r e d i n a d e s i c c a t o r o v e r a n h y drous c a l c i u m s u l f a t e and p r o t e c t e d from t h e l a b o r a t o r y fumes. A p p l i c a t i o n o f samples. An a l i q u o t ( a b o u t one t o t e n y l ) o f s o l u t i o n o f t h e sample i s s p o t t e d on t h e p l a t e w i t h a c a p i l l a r y d r o p p e r o r m i c r o p i p e t t e 1.5 cm. f r o m one e n d o f t h e p l a t e . Care must be t a k e n t o a v o i d d i f f u s i o n o f t h e s p o t ; m o r e o v e r , t h e a r e a o f t h e s p o t s h o u l d be r e s t r i c t e d t o a t w o - o r t h r e e m i l l i m e t e r diameter. To e n s u r e t h i s , t h e s o l u t i o n s h o u l d be a p p l i e d i n s t a g e s a n d d r i e d i n a s t r e a m o f warm a i r . The l a y e r on t h e p l a t e s h o u l d n o t be p e n e t r a t e d d u r i n g s p o t t i n g . M u l t i p l e spots should be a p p l i e d on t h e p l a t e a t l e a s t one c e n t i m e t e r a p a r t . Development o f t h e p l a t e . A f t e r ensuring that the applied spots are d r y , the spotted plate i s placed i n a developing chamber c o n t a i n i n g t h e s o l v e n t s y s t e m and t h e chamber i s c l o s e d w i t h a n a i r - t i g h t l i d . The s o l v e n t s h o u l d be a b o u t 0 . 5 t o 1 c m . deep i n t h e d e v e l o p i n g c h a m b e r . The chamber i s s a t u r a t e d w i t h the s o l v e n t vapors f o r a p a r t i c u l a r s e p a r a t i o n before i n s e r t i o n o f t h e p l a t e i n t o t h e chamber. The c h r o m a t o g r a m i s a l l o w e d t o run by a s c e n d i n g technique a t ambient temperature. The s o l v e n t i s g e n e r a l l y a l l o w e d t o r u n t e n o r f i f t e e n c e n t i m e t e r s up t h e p l a t e from t h e s t a r t i n g l i n e t o a pre-marked l i n e . The p l a t e


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the

Systems

P u r e s o l v e n t s o f A n a l a b g r a d e s h o u l d be u s e d i n T L C . The v a r i o u s s o l v e n t s possess d i f f e r e n t e l u t i n g p r o p e r t i e s . Further, because o f the d i f f e r e n c e s i n p o l a r i t y of the o r g a n i c i n s e c t i c i d e s and v a r i a t i o n s i n t h e i r w a t e r s o l u b i l i t y , s e v e r a l solvent s y s t e m s a r e t e s t e d f o r s e p a r a t i o n on t h e TLC p l a t e . The l a r g e number o f s o l v e n t s y s t e m s l i s t e d i n T a b l e II f o r o r g a n o p h o s p h o r u s o r g a n o c h l o r i n e and c a r b a m a t e i n s e c t i c i d e s p r o v i d e s a t i s f a c t o r y a l t e r n a t i v e systems f o r the s e p a r a t i o n o f the group of i n s e c t i c i d e s under c o n s i d e r a t i o n . Location of

the

Spots

A f t e r s e p a r a t i o n , t h e p l a t e i s examined f o r t h e p o s i t i o n o f t h e s p o t s on t h e p l a t e . Some o f t h e compounds a r e d e t e c t e d on t h e p l a t e by v i e w i n g t h e p l a t e u n d e r u l t r a v i o l e t l i g h t (254 nm). F o r l o c a t i o n o f t h e s p o t s o f i n s e c t i c i d e s on t h e TLC p l a t e d i f f e r e n t chromogenic reagents are used. S u i t a b l e l o c a t i o n reagents for detecting organophosphorus-, o r g a n o c h l o r o - and c a r b a m a t e i n s e c t i c i d e s on TLC p l a t e s a r e g i v e n below. D e t a i l s o f t h e i r c o m p o s i t i o n and t h e t r e a t m e n t o f t h e p l a t e n e c e s s a r y w i t h each r e a g e n t a r e a l s o g i v e n . Organophosphate. For the e s t i m a t i o n of phosphoric a c i d e s t e r s , t h e d e v e l o p e d p l a t e i s s p r a y e d w i t h 0.25% s o l u t i o n o f p a l l a d i u m c h l o r i d e i n 0 . 1 Ν HC1 and d r i e d i n a i r . After exactly ten m i n u t e s , the area o f the yellow-brown c o l o r e d spot developed i s m e a s u r e d and compared w i t h t h a t o f t h e s p o t s o f s t a n d a r d i n s e c t i c i d e s o l u t i o n s run a l o n g s i d e . Organochloride. For the e s t i m a t i o n o f c h l o r i n e c o n t a i n i n g i n s e c t i c i d e s , t h e d e v e l o p e d p l a t e i s s p r a y e d w i t h a 1.0% e t h a n o l i c s i l v e r n i t r a t e s o l u t i o n containing f i v e m i l l i l i t e r s of concentra t e d ammonia s o l u t i o n . A f t e r d r y i n g t h e s p r a y e d p l a t e i n a i r , i t i s i r r a d i a t e d w i t h U . V . l i g h t (366 nm) f o r t e n m i n u t e s . After e x a c t l y t e n m i n u t e s , the a r e a o f the d e v e l o p e d s p o t s i s measured and compared w i t h t h a t o f t h e c o n t r o l i n s e c t i c i d e r u n on t h e same TLC p l a t e . Carbamate. For carbamate p e s t i c i d e s the p l a t e i s sprayed w i t h 1% e t h a n o l i c s o l u t i o n o f F a s t B l u e - B , d r i e d f o r t h i r t y m i n u t e s , and t h e n s p r a y e d w i t h a 20% aqueous s o l u t i o n o f s o d i u m h y d r o x i d e , and a g a i n d r i e d a t room t e m p e r a t u r e . Different c o l o r e d s p o t s a r e o b t a i n e d f o r v a r i o u s carbamate p e s t i c i d e s . A f t e r e x a c t l y t e n m i n u t e s , t h e s p o t i s s c a n n e d by d e n s i t o m e t r y . Rf

Values The m o s t i m p o r t a n t f a c t o r

i n chromatography

for

the


identi-

PESTICIDE A N A L Y T I C A L

268

Table

I I : S o l v e n t S y s t e m s f o r TLC S e p a r a t i o n o f

Organo-Phosphorus

METHODOLOGY

Pesticides

Pesticides: Ratio

(i) (ii) (iii) (iv) (v) (vi) (vii) (viii) (ix) (x)

Cyclohexane : L i q u i d P a r a f f i n Cyclohexane : Chloroform Cyclohexane : Acetone : A c e t o n i t r i l e . . Cyclohexane : Acetone : Ethanol n-Hexane : A c e t o n e Benzene Petroleum Ether : Acetone D i e t h y l Ether : Methanol Chloroform : L i q u i d P a r a f f i n Carbon T e t r a c h l o r i d e : E t h y l a c e t a t e . . .

Chlorinated Organic

(9:1) (7:3) (17:2:1) (95:1:4) (17:3) (7:3) (3:1) (9:1) (3:2)

Pesticides: Ratio


n-Hexane : A c e t o n e (9:1) n-Hexane : L i q u i d P a r a f f i n (39:1) n-Hexane : B u t a n o n e (39:1) n-Hexane : Dioxan (49:1) Cyclohexane : Acetone (99:1) C y c l o h e x a n e : D i m e t h y l Formamide . . . . (19:1) Cyclohexane : L i q u i d P a r a f f i n (4:1) Cyclohexane : Methylene D i c h l o r i d e . . . (9:1) L i g h t Petroleum : Chloroform (97:3) L i g h t Petroleum : A c e t i c Acid (19:1)

Carbamate

Pesticides: Ratio


Benzene : E t h y l - M e t h y l K e t o n e (9:1) Benzene : A c e t o n e (19:1) Benzene : n - H e x a n e (17:1) Benzene n-Hexane : D i e t h y l e t h e r (3:1) Cyclohexane : E t h y l - M e t h y l Ketone . . . . ( 9 : 1 ) Petroleum Ether (40-60°) : Chloroform . . (19:1) Chloroform Chloroform : Dichloromethane (99:1) C h l o r o f o r m : Methanol (9:1)


14.

TEWARi

Pesticides

in Human

269

Organs

f i c a t i o n o f s e p a r a t e d substances i s the Rf v a l u e . However, the e x a c t R f v a l u e depends on s e v e r a l f a c t o r s s u c h as t h e a d s o r b e n t , t h i c k n e s s o f t h e l a y e r on t h e p l a t e , a c t i v a t i o n o f t h e p l a t e , c o m p o s i t i o n of the s o l v e n t system, s a t u r a t i o n o f the d e v e l o p i n g chamber w i t h t h e s o l v e n t , and t e m p e r a t u r e a t w h i c h t h e s e p a r a t i o n i s carried out. A c t u a l l y , t h i n l a y e r chromatography i s a compara t i v e a r t r a t h e r t h a n an a b s o l u t e t e c h n i q u e as commented b y Clarke(83). A c c o r d i n g t o Zweig and S h e r m a ( 8 4 ) an a g r e e m e n t o f a b o u t two m i l l i m e t e r s i n t h e m i g r a t i o n d i s t a n c e s o f t h e unknown and s t a n d a r d s p o t i s c o n s i d e r e d a d e q u a t e f o r t e n t a t i v e i d e n t i f i c a t i o n , s i n c e t h e sample s p o t may be a f f e c t e d s l i g h t l y by t h e c o e x t r a c t i v e s d e s p i t e thorough c l e a n - u p s t e p s . To overcome t h e d i f f i c u l t y due t o n o n - r e p r o d u c i b i l i t y o f R f v a l u e s , a r e f e r e n c e s t a n d a r d s h o u l d be r u n t o g e t h e r on t h e same p l a t e a l o n g w i t h t h e unknown m a t e r i a l . I t makes c o m p a r i s o n o f R f v a l u e s much e a s i e r and a l s o e l i m i n a t e s t h e c h a n c e s o f e r r o r due t o any i r r e g u l a r i t y d u r i n g t h e s e p a r a t i o n . Estimation of Pesticides

on TLC P l a t e s

Using

Densitometry

Densitometry i s a technique o f d i r e c t d e t e r m i n a t i o n o f the c o n c e n t r a t i o n o f t h e c o l o r e d d e r i v a t i v e on t h e c h r o m a t o g r a m by absorptometry. The c h r o m a t o g r a m i s s c a n n e d w i t h a beam o f f i l t e r e d l i g h t , a p o r t i o n o f w h i c h i s a b s o r b e d by t h e c o l o r e d s p o t and t h e r e m a i n d e r i s t r a n s m i t t e d by t h e s p o t on t h e c h r o m a t ogram and measured p h o t o e l e c t r i c a l ^ . In d e n s i t o m e t r i c m e a s u r e m e n t s , t h e c o l o r e d s p o t i s s c a n n e d along the l i n e o f s o l v e n t development. The g a l v a n o m e t e r i s a t f i r s t s e t a t z e r o on a p o r t i o n o f t h e s p r a y e d and d r i e d TLC p l a t e n e a r t h e s p o t t o be s c a n n e d . The TLC p l a t e i s s l i g h t l y s h i f t e d ( s a y 0 . 5 mm), and t h e t r a n s m i t t e d l i g h t t h r o u g h t h e s p o t i s measured i n terms o f e x t i n c t i o n . The s p o t i s s u c c e s s i v e l y s c a n n e d , and t h e g a l v a n o m e t e r r e c o r d s t h e maximum a b s o r b a n c e a t t h e peak point. When t h e beam o f l i g h t r e t u r n s t o t h e b l a n k b a c k g r o u n d o f the t h i n l a y e r p l a t e , the galvanometer again reads z e r o . A curve i s p l o t t e d of e x t i n c t i o n versus scanning l e n g t h o f the s p o t . The a r e a o f t h e e x t i n c t i o n c u r v e i s measured f o r e a c h s p o t o f d i f f e r ent concentrations. This curve area i s p r o p o r t i o n a l to the c o n c e n t r a t i o n o f the s u b s t a n c e p r e s e n t i n each s p o t . D e n s i t o m e t r i c s c a n n i n g o f t h e TLC s p o t i s more a c c u r a t e and s e n s i t i v e f o r 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 than o t h e r methods. Data o f

D i s t r i b u t i o n of

Pesticides

in

Tissues

The amount o f t h e p e s t i c i d e s p r e s e n t i n d i f f e r e n t a u t o p s l e d t i s s u e s r e c e i v e d f r o m f a t a l c a s e s o f p o i s o n i n g have been e s t i m ated q u a n t i t a t i v e l y i n our l a b o r a t o r y . In a c c i d e n t a l p o i s o n i n g c a s e s w h e r e v a p o r s o f i n s e c t i c i d e s a r e i n h a l e d , no i n s e c t i c i d e i s l i k e l y t o be d e t e c t e d i n t h e s t o m a c h o r i n t e s t i n e b u t c o n s i d e r a b l e amounts may be d e t e c t e d i n t h e l u n g s , b r a i n , and b l o o d .


270

PESTICIDE

ANALYTICAL

METHODOLOGY

The d a t a r e g a r d i n g t h e amount o f a p a r t i c u l a r p e s t i c i d e f o u n d i n v a r i o u s d i f f e r e n t t i s s u e s a n d body f l u i d s u s i n g d e n s i t o m e t r y o n TLC p l a t e s i s r e c o r d e d i n T a b l e s I I I a n d I V · The age o f t h e v i c t i m s a n d o t h e r p a r t i c u l a r s o b t a i n e d from t h e r e c o r d s o f i n vestigating officers are also given. Discussion From t h e p e r u s a l o f d a t a r e c o r d e d i n T a b l e s I I I a n d I V , i t i s evident that i n s e c t i c i d e s are d i s t r i b u t e d i n p r a c t i c a l l y a l l the v i s c e r a l t i s s u e s . H o w e v e r , t h e amount o f i n s e c t i c i d e i n d i f f e r e n t body t i s s u e s depends o n t h e t i m e p e r i o d between i n g e s t i o n o f t h e i n s e c t i c i d e and d e a t h . A t t h e i n i t i a l o n s e t o f symptoms, t h e m a j o r i t y o f t h e i n s e c t i c i d e s i s s t i l l i n t h e stomach. But i n c a s e s where d e a t h i s p r o l o n g e d , t h e i n s e c t i c i d e a c c u m u l a t e s i n o t h e r t i s s e s , e . g . l i v e r and k i d n e y . By c o n d u c t i n g c o n t r o l e x p e r i m e n t s on w h i t e r a t s w h i c h were f e d l e t h a l d o s e s o f some o f t h e 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 ( p a r a t h i o n and m a l a t h i o n ) i t was o b s e r v e d t h a t t h e s e i n s e c t i c i d e s t e n d e d t o accumul a t e i n t h e body f a t , a n d a s m a l l amount was e x c r e t e d i n t h e urine. A s m a l l amount was a l s o e l i m i n a t e d t h r o u g h e x c r e t a . A h i g h e r c o n c e n t r a t i o n o f t h e i n s e c t i c i d e i n t h e stomach i s i n d i c a t i v e t h a t t h e death o f t h e v i c t i m took place w i t h i n a s h o r t p e r i o d , b e c a u s e t h e i n s e c t i c i d e c o u l d n o t even be c o m p l e t e l y e l i m i n a t e d from t h e stomach. A c o m p a r a t i v e l y l a r g e c o n c e n t r a t i o n o f p e s t i c i d e i n l i v e r t i s s u e i s s u g g e s t i v e o f a d e l a y i n death from t h e t i m e o f administration of the insecticide. H o w e v e r , i t s h o u l d be k e p t i n mind t h a t t h e t r a n s p o r t o f i n s e c t i c i d e t o stomach f r o m o t h e r body t i s s u e s a l s o depends on t h e m e d i a by w h i c h t h e i n s e c t i c i d e i s administered. I f t h e i n s e c t i c i d e i s mixed w i t h m i l k , a l c o h o l , o r l i p i d o i l , t h e t o x i c i t y o f i n s e c t i c i d e i s i n c r e a s e d due t o i t s ready a b s o r p t i o n through t h e g a s t r o - i n t e s t i n a l t r a c t . In such c a s e s , t h e t i m e r e q u i r e d i s much l e s s , a n d d e a t h o c c u r s w i t h i n one o r two h o u r s o f i n g e s t i o n o f t h e i n s e c t i c i d e a s i n t h e c a s e shown i n s e r i a l numbers 3 , 1 1 , 1 2 , 1 6 , 1 9 , and 20 i n T a b l e IV w h e r e t h e i n s e c t i c i d e was a d m i n i s t e r e d by m i x i n g i t w i t h l i q u o r o r mil k. Stomach wash s a m p l e s i n n o n f a t a l c a s e s were f o u n d t o c o n t a i n l a r g e q u a n t i t i e s o f i n s e c t i c i d e , t h e r e b y i n d i c a t i n g t h a t t h e major p a r t o f t h e i n s e c t i c i d e was e x c r e t e d by t h e b o d y . O n l y t r a c e amounts o f p e s t i c i d e s w o u l d have r e m a i n e d i n t h e body w h i c h d i d n o t p r o v e fatal. The d i s t r i b u t i o n p a t t e r n i n d i f f e r e n t body o r g a n s i s p r a c t i c a l l y t h e same i n a l l c a s e s examined f o r p a r a t h i o n p o i s o n i n g , a s d e a t h g e n e r a l l y o c c u r r e d w i t h i n an hour o r two from a d m i n i s t r a t i o n o f t h e poison. The d i s t r i b u t i o n d a t a o f a c a s e o f p o i s o n i n g by phosphamidon r e c o r d e d i n s e r i a l number 24 i n T a b l e I I I i n d i c a t e s t h a t c o m p a r a t i v e l y l a r g e amounts o f phosphamidon was d e t e c t e d i n t h e l u n g



Time period before death

lia hrs. 2 hrs. 1 hrs. 2 hrs. Us hrs. λΗ hrs. 4 hrs. 8 hrs. Zh h r s . 5 hrs. 3 hrs. 2 hrs. 1 hrs. Recovered Recovered 24 hrs. 16 h r s . 2h hrs. 3 hrs. λΗ hrs. \h h r s . Recovered Recovered 6 hrs. unknown

Sex & Age

M-25 F-50 F-3 F-22 M-40 M-28 F-25 M-28 F-18 M-32 M-61 F-22 M-43 F-25 F-18 M-39 F-27 M-35 M-54 F-18 M-28 M-22 F-19 M-50 M-24

8.75 in bread 6.00 in bread in bread 5.13 in bread 6.88 6.38 in liquor in liquor 10.26 unknown 9.31 46.88 oral unknown in liquor 75.00 126.40 oral 12.5 oral in liquor 84.6 oral oral 0.84 unknown 3.25 oral 6.35 unknown 18.30 unknown with Zn3P2 16.40 with ΖΠ3Ρ2 9.54 oral oral 0.094 spray in 1iquor 14.60

-

0.25 4.32

-

0.11

-

2.84 3.60 4.34 8.19

-

4.00 4.50 4.13 5.13 4.80 7.24 6.51 2.31 2.13 6.00 7.40 12.50 8.32 2.20 4.75 3.84 4.54 3.22 5.44

-

16.38

-

28.40

-

11.63

-

6.25 4.88 4.88 5.63 5.36 8.40 4.39

-

-

0.25 3.84

-

1.64 2.50 2.68 2.50 2.84 3.69

-

2.86 6.82 6.25 6.28

0.13 2.96

-

0.94 4.38 3.20 2.50 1.80 3.44

-

1.34 6.20 4.17 4.20

-

3.88 2.30 3.42 1.42 1.67

2.08 2.38

3.75 3.88 2.63 4.78 4.63 6.80 1.69 2.86

-

0.35

-

0.13

-

-

-

0.31

-

0.32

-

-

1.06

2.75

_

_

3.13 3.18 4.08 3.50 5.80 1.47

_

2.50 2.88 4.10 3.13 5.24 0.90

-

61.40 32.8

-

-

184.8 252.0 8160.0 28.4

25.00 8.38 65.00 84.20

-

2.60 24.50

-

_

Concentration (milligram per cent of tissue/material) Mode of ingestion StomachlIntestine!LiverlKidney|Spleen Lung 1 Heart!BrainStomach Wash

In addition, subject number 22 contained 9320 mg per cent pesticide in the vomi tus.

Ethyl Parathion Ethyl Parathion Ethyl Parathion Ethyl Parathion Ethyl Parathion Ethyl Parathion Methyl Parathion Malathion Malathion Malathion Sumithion Sumithion Sumithion Sumithion Sumithion Dimethoate Dimethoate Diazinon Diazinon Meta-systox Meta-systox Baytex Phosphamidon Phosphamidon DDVP

Insecticide

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

III

DISTRIBUTION OF ORGANOPHOSPHORUS INSECTICIDES IN DIFFERENT BODY TISSUES AND FLUIDS FROM CASES OF HUMAN POISONING

Table


Endrin Aldrin Endrin Endrin Endrin Aldrin Endrin Endrin Endrin BHC Endrin Endrin Endrin Endrin Aldrin Endrin DDT Endrin BHC Endrin Endrin

F-25 F-17 M-3 F-22 M-30 M-35 F-38 M-37 F-40 F-31 M-33 M-29 F-28 F-42 M-35 M-32 M-28 F-28 F-31 F-25 F-24

Sex & Age

hrs. hrs. hrs. hrs. hrs. hrs. hrs. 2h hrs. 3H hrs. 2h hrs. Us hrs. 1 hrs. 5 hrs. 6 hrs. -\h hrs. 1* hrs. 5* hrs. Us hrs. 4^ hrs. 2 hrs. 3k hrs.

4 3% 1% 1% 5 4 5

Time period before death unknown oral in milk unknown oral oral unknown unknown unknown unknown in 1iquor in milk unknown unknown oral in liquor unknown unknown in 1iquor in liquor unknown 1.04 4.46 2.90 3.75 3.47 1.94 5.81 4.88 6.38 5.04 6.29 3.84 81.00 6.08 60.00 7.09 61.00 13.88

2.63

14.50

25.00 3.01 22.50 8.06

_

22.00

1.59

3.19 1.31 2.56 2.88 1.69 3.68 4.38 1.94 1.56

1.33

66.00

-

0.99

1.38 0.61 1.19 1.31 1.28 30.00 2.98 12.5 3.01 20.30 4.91

-

0.84 2.13 1.34 2.81 1.19 4.80 1.18 3.50 2.38 1.56 1.31 1.75 1.06 1 .94 1.44 2.88 1.72 48.00 45.00 4.91 3.17 22.5 12.5 4.06 3.55 49.00 25.00 10.91 6.59

1.20

2.17

0.54

1.25 2.31

5.17

0.75

4.00 5.69

20.00

32.15

25.00

1.79 0.79 1.05

0.56

1.08

22.50 1.72 -

---

0.38 0.57

1.06

0.55 0.43 0.68 -

0.85

0.55 0.25 0.37 -

0.35

3.17 4.69 -

7.88

Concentration ( m i l l i ç ram per cent of tissue/material) Mode of ingestion StomachlIntestinelLiver|Kidney] Spleen|Lung|Heart|Blood|Urine|Stomach Wash

In addition, 8.12, 3.25, 20.00 mg per cent was found in the vomitus of subject numbers 2,9, and 20 respectively. Also, subject number 2 showed 6.38 mg per cent pesticide in the stomach contents.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

Insecticide

DISTRIBUTION OF ORGANOCHLORIDE INSECTICIES IN DIFFERENT BODY TISSUES AND FLuiDS FROM CASES OF,HUMAN POISONING

Table IV

ο

§

X

H

m

Ο >

H

> r

>

m

Ο

Ο

H

C/3

m

Ν)

to ο

14.

TEWARi

Pesticides

in Human

Organs

273

and b r a i n t i s s u e s a n d o n l y v e r y s m a l l amounts o f phosphamidon c o u l d be f o u n d i n t h e s t o m a c h . These f i n d i n g s s u g g e s t t h a t phosphamidon was most p r o b a b l y a d m i n i s t e r e d t h r o u g h i n h a l a t i o n r a t h e r t h a n by m o u t h . The d i s t r i b u t i o n d a t a was s u p p o r t e d b y t h e r e p o r t o f t h e i n v e s t i g a t i o n o f f i c e r t h a t t h e v i c t i m was a c c i d e n t a l l y exposed t o t h e i n s e c t i c i d e vapor d u r i n g s p r a y i n g o f h i s agricultural field. S i m i l a r l y , l a r g e e x c e s s o f s u m i t h i o n p r e s e n t i n s t o m a c h wash s a m p l e s a n d i n t h e stomach t i s s u e s i s r e c o r d e d i n s e r i a l numbers 1 1 , 1 3 , 1 4 , a n d 15 i n T a b l e I I I . T h i s i s s u g g e s t i v e o f s u i c i d a l p o i s o n i n g by o r a l c o n s u m p t i o n o f T i k - 2 0 , a h o u s e h o l d i n s e c t i c i d a l preparation containing sumithion. Data r e c o r d e d i n T a b l e s I I I a n d IV shows t h e amount o f t h e i n s e c t i c i d e i n t h e unchanged f o r m . H o w e v e r , most o f t h e t i s s u e s also contain the metabolite o f the i n s e c t i c i d e . From t h e a b o v e discussion i t i s evident that the study o f the d i s t r i b u t i o n o f p e s t i c i d e s i n d i f f e r e n t t i s s u e s and body f l u i d s c a n h e l p i n r e s o l v i n g t h e q u e s t i o n s o f how and when t h e p e s t i c i d e was a d m i n istered. Furthermore, the clue regarding the nature o f poisoning w h e t h e r s u i c i d a l , h o m i c i d a l o r a c c i d e n t a l c a n a l s o be o b t a i n e d by careful study o f the d i s t r i b u t i o n pattern o f the i n s e c t i c i d e s i n body t i s s u e s and f l u i d s d e r i v e d by q u a n t i t a t i v e T L C .

Abstract The absolute proof of fatal cases of poisoning is the detection of the poison in different organs of the body. Due to the chemical nature of the poison and its metabolism, the suspected chemical is rarely evenly distributed in body tissues but tends to accumulate in some particular organ. Hence, to conduct a thorough forensic investigation, it is necessary to analyze different visceral tissues qualitatively and quantitatively for poison residues. In this paper, the quantitative TLC technique using densitometry for the determination of traces of pesticides has been described in detail. The concentration of pesticides in various human organs from poisoning cases has been recorded as well as data on tissue distribution. The distribution pattern of several important pesticides, like parathion, malathion, sumithion, and endrin has been studied. The results of these studies have aided in solving the questions in the probable causes of death or poisoning-- how and when the pesticide was probably administered, and i f death occurred by suicide, homicide or accident. Several actual cases of poisoning and subsequent tissue analyses by quantitative TLC have been described. Acknowledgement E d i t i n g and f i n a l t y p i n g o f t h i s c h a p t e r was done by Douglas S m i t h , L i n d a A . K e o l a P e r e i r a a n d G u n t e r Z w e i g , t o whom t h e editors are greatly indebted.


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