An In Situ Fluorometric Method for the Analysis of Bayrusil in Foodstuffs by J.-G. ZAKREVSKYand V. MALLET"x" Universitd de Moncton, Moncton, New Brunswick, Canada

Bayrusil [O,O-diethylO-(2-quinoxalyl) phosphorothionate], for w h i c h the common name d i e t h q u i n a l p h i o n e has been proposed, was introduced in 1969 for the control of biting and sucking insects of field crops and fruits (SCHMIDT and HAMMAN 1969). A gas chromatographic procedure for the analysis of Bayrusil in foodstuffs has already been proposed (DRAGER 1969). The technique gives good quantitative results but additional steps are needed for the positive identification for the residues and therefore more time is required for the analysis. It was thought desirable to have a more simple method that could be used for the simultaneous identification and determination of residues of Bayrusil in plant materials at similar concentrations. Much attention has been devoted during the past few years to the analysis of o r g a n o p h o s p h o r o u s pesticides by in situ fluorometry. Earlier work was carried ou-~ w-i-~ chelate spray reagents following treatment of the c h r o m a t o g r a m w i t h bromine vapours (FREI and M A L L E T 1971). This work led to the analysis of azinphosmethyl in blueberries (FREI et al. 1971). The use of bromine vapour was e l i m i n a t e d when m e t a l - f l u o r o c h r o m i c indicators were discovered. BIDLEMAN et al. (1972) showed that o r g a n o p h o s p h o r o u s compounds appeared as yellow fluorescent spots on thin-layer chromatograms when sprayed with a palladiumcalcein complex. This m e t h o d of detection was used by MACNEIL et al. (1974) for the analysis of dimethoate and malathion in lettuce. Very recently, BRUN and MALLET (1973) showed t h a t some compounds could be made fluorescent on silica-gel layers simply by heating the c h r o m a t o g r a m in an oven. This detection technique was used successfully to determine Bayrusil in w a t e r (BRUN and MALLET 1974). In this study, the same detection technique is applied to the analysis of Bayrusil in various fruits and field crops. *To whom correspondence should be sent

633 Bulletin of Environmental Contamination & Toxicology, Vol. 13, No. 5 9 1975 by Springer.Verlag New York Inc.

EXPERIMENTAL Apparatus: A T u r n e r F l u o r o m e t e r M o d e l iii (G.K. T u r n e r A s s o c i a t e s ) e q u i p p e d w i t h a C a m a g TLC S c a n n e r was u s e d for q u a n t i t a t i v e work. The c h r o m a t o g r a m s w e r e s c a n n e d at a speed of 2 i n / m i n w i t h a c h a r t speed of 60 m m / m i n . The s u r f a c e u n d e r the p e a k s was m e a s u r e d w i t h a p l a n i m e t e r (A. Ott, K e m p t e n , Bayern, G.F.R.). E x c i t a t i o n f i l t e r # 7-60 (360 nm) and s e c o n d a r y filter # 2-A (415 nm), b o t h a v a i l a b l e from C o r n i n g Glass Work (New York, U.S.A.) w e r e u t i l i z e d . Reagents: B a y r u s i l was o b t a i n e d from C h e m a g r o C o r p o r a t i o n (Kansas City, M i s s o u r i ) as an a n a l y t i c a l s t a n d a r d (99.4%). A stock s o l u t i o n was p r e p a r e d i000 p p m (v/v) in n - h e x a n e from w h i c h d i l u t i o n s w e r e made. F l o r i s i l (60-100 mesh, F i s h e r S c i e n t i f i c Co., M o n t r e a l , Canada) was h e a t e d in an oven at 1 3 0 ~ for at least 48 hours, then d e a c t i v a t e d w i t h 3% w a t e r and s t o r e d in a d e s s i c a t o r u n t i l use. The thin l a y e r chrom a t o g r a p h i c p l a t e s (20 x 20 cm 2) w e r e p r e p a r e d 250 m i c r o n s thick f r o m 30 g of S I L I C A - G E L H (Brinkmann I n s t r u m e n t s L i m i t e d , Rexdale, Ontario) and 80 ml of 0.25 N KOH. The p l a t e s w e r e left to dry at r o o m t e m p e r a t u r e b e f o r e use. Extraction Procedure: i00 g of s a m p l e w e r e homog e n i z e d for five m i n u t e s w i t h 200 ml of a c e t o n i t r i l e in a W a r i n g Blendor. The m i x t u r e was f i l t e r e d w i t h v a c u u m on a B ~ c h n e r funnel t h r o u g h a W h a t m a n No. 42 f i l t e r paper. The r e s i d u e on the f i l t e r p a p e r was then r e - e x t r a c t e d w i t h i00 ml of a c e t o n i t r i l e and the m i x t u r e was f i l t e r e d as before. The B l e n d o r jar was r i n s e d w i t h a c e t o n i t r i l e (50 ml) w h i c h was then used to w a s h the f i l t e r cake. The c o m b i n e d e x t r a c t s w e r e t r a n s f e r r e d to a i 0 0 0 - m i r o u n d - b o t t o m e d flask and the a c e t o n i t r i l e was r e m o v e d on a r o t a r y v a c u u m e v a p o r a t o r at 35~ The r e m a i n i n g a q u e o u s s o l u t i o n was t r a n s f e r r e d to a g r a d u a t e d c y l i n d e r and d i l u t e d to i00 ml w i t h d i s t i l l e d water. It was then e x t r a c t e d t w i c e w i t h 1 2 5 - m i p o r t i o n s of n - h e x a n e in a 250-mi s e p a r a t o r y funnel and the c o m b i n e d e x t r a c t s w e r e d r i e d for five m i n u t e s w i t h 30 g of a n h y d r o u s s o d i u m sulfate. The h e x a n e e x t r a c t was t r a n s f e r r e d to a 5 0 0 - m l r o u n d - b o t t o m e d flask and the s o l v e n t was e v a p o r a t e d to a p p r o x i m a t e l y 5-10 ml on the r o t a r y e v a p o r a t o r at 35~ F l o r i s i l C o l u m n Cleanup: A p l u g of glass w o o l was p l a c e d into the b o t t o m of a c h r o m a t o g r a p h i c tube (20 mm, i.d.). A n h y d r o u s s o d i u m s u l f a t e (5 g) was added, f o l l o w e d by i0 g of F l o r i s i l . The F l o r i s i l l a y e r was t o p p e d w i t h 5 g of a n h y d r o u s s o d i u m sulfate. The c o l u m n

634

was then w a s h e d w i t h 30 ml of h e x a n e . The r e s i d u e from the h e x a n e e v a p o r a t i o n was t r a n s f e r r e d to the c o l u m n w i t h small p o r t i o n s of n - h e x a n e w h i c h w a s then d r a i n e d f r o m the column. 50 ml of 10% e t h e r in n - h e x a n e (v/v) w e r e p a s s e d t h r o u g h the c o l u m n and the e l u a t e was discarded. B a y r u s i l w a s e l u t e d f r o m the F l o r i s i l u s i n g i00 ml of 25% e t h y l e t h e r in n - h e x a n e . The e l u a t e was e v a p o r a t e d to less than 0.i ml, f i r s t in a 250-m! r o u n d - b o t t o m e d flask, and f i n a l l y into a 15-ml g r a d u a ted c e n t r i f u g e tube. The v o l u m e was then m a d e up to the 0.2 ml m a r k w i t h n - h e x a n e . Thin-LaYer Chromatography: A 20 ~i a l i q u o t of the s a m p l e was s p o t t e d on the r i g h t - h a n d side at the b o t t o m of a c h r o m a t o g r a p h i c plate. The p l a t e was d e v e l o p e d to I0 cm in a 5:1 m i x t u r e of n - h e x a n e in acetone. S t a n d a r d s w e r e then s p o t t e d on the free p o r t i o n of the c h r o m a t o g r a m atop the r i g h t - h a n d side and the p l a t e was p l a c e d in an oven at 100~ for 30 m i n u t e s . After cooling, it was d e v e l o p e d i0 cm in a i00:i0 m i x t u r e of c a r b o n t e t r a c h l o r i d e in m e t h a n o l . A f t e r r e m o v a l from the c h r o m a t o g r a p h i c c h a m b e r , the p l a t e was d r i e d w i t h a s t r e a m of w a r m air and the spots w e r e l o c a t e d u n d e r UV light, the f l u o r e s c e n c e of the spots was t h e n m e a sured.

RESULTS AND DISCUSSION V a r i o u s s o l v e n t s w e r e c o n s i d e r e d for e x t r a c t i n g B a y r u s i l f r o m the sample b u t a c e t o n i t r i l e was c h o s e n b e c a u s e of its lower s o l u b i l i t y t o w a r d s w a x e s and fats (GETZ, 1962). A l t h o u g h the w a t e r - h e x a n e p a r t i t i o n i n g of the e x t r a c t was v e r y e f f i c i e n t in r e m o v i n g a large a m o u n t of c o - e x t r a c t i v e s , it was not s u f f i c i e n t . Florisil c o l u m n c l e a n u p was d e e m e d n e c e s s a r y in o r d e r to m i n i m i z e s u b s t a n c e s w h i c h i n t e r f e r e d w i t h the i n s t r u m e n t a l detection. Even so, t w o - d i m e n s i o n a l c h r o m a t o g r a p h y of the e x t r a c t had to be done to s e p a r a t e B a y r u s i l f r o m interfering substances. W i t h the use of a p o l a r s o l v e n t system, m o s t of the i n t e r f e r i n g c o - e x t r a c t i v e s are e l u t e d to the top (Scheme i). The i m p r e g n a t i o n of the a d s o r b e n t w i t h the K O H was found to m i n i m i z e s p r e a d i n g of the spots and b a c k g r o u n d i r r e g u l a r i t i e s n o r m a l l y e n c o u n t e r e d w h e n u s i n g sprays. The f l u o r e s c e n c e o b t a i n e d f r o m B a y r u s i l is q u i t e s t a b l e (BRUN and M A L L E T 1974) and a d e c r e a s e in fluor e s c e n c e is not n o t i c e a b l e e v e n a f t e r a few days exp o s u r e to d a y l i g h t . As l i t t l e as 6 ng p e r spot of the p e s t i c i d e can be d e t e c t e d (BRUN and M A L L E T 1973)and

635

c a l i b r a t i o n c u r v e s are l i n e a r up to a c o n c e n t r a t i o n 10 ~g p e r spot (BRUN and M A L L E T , 1974).

of

R e s u l t s o b t a i n e d by a n a l y z i n g for B a y r u s i l in v a r i o u s c r o p s are g i v e n in Table I. R e c o v e r i e s are b e t t e r than those o b t a i n e d by. D R A G E R (1969) who w o r k e d at a c o n c e n t r a t i o n of 0.i ppm. R e s u l t s h i g h e r or l o w e r than 100% are w i t h i n the p r e c i s i o n of the t e c h n i q u e w h i c h is of the o r d e r of • (BRUN and M A L L E T , 1974). H o w e v e r , h i g h e r than n o r m a l r e c o v e r i e s m a y be

< 4J

(a')

(a) e

D~ 0 r~ > I

q3

Q O D

C~

I

+ a - Bayrusil

I st d e v e l o p m e n t from s a m p l e extract;

A - development Typical after heat

a' - B a y r u s i l standard after heat treatment Scheme 1 thin-layer chromatogram t r e a t m e n t and d e v e l o p m e n t Table

Recoveries

Substrate: % Recovery:

I

of B a y r u s i l in v a r i o u s at the 0.02 p p m level

apple

lettuce

tomatoes

105 102

109 i01

96 105

636

crops

cucumbers 98 107

beans i01 98

attributed in part to interferences of the b a c k g r o u n d w h i c h causes some difficulty in recording the chromatogram properly at very low concentrations. Reproducibility in the results is excellent compared to that reported by M A C N E I L et al. (1974) in their analysis of dimethoate and m a l a t h i o n in lettuce extract. It is also important to note that the method described in this study is done at a much lower concentration and the pesticide is spiked before extraction. When working at levels of 0.i0 ppm or more the column chromatography step can be omitted. The m e t h o d is then faster and more simple than that described by D R A G E R (1969). ACKNOWLEDGEMENTS This work was supported by a grant from Agriculture Canada. REFERENCES

BRUN, G.L. and V. Mallet, J. Chromatog., (1973).

8_~0, 117

BRUN, G.L. and V. Mallet, Bull. Environ. Contam. Toxicol., 12 (6) (1974). In print.

and

BIDLEMAN, T.F., B. Nowlan and R.W. Frei, Anal. Chim. Acta, 60, 13 (1972). DRAGER, G., Pflanzensch.

Nachr.

Bayer, 22, 318

(1969).

FREI, R.W. and V. Mallet, Intern. J. Environ. Anal. Chem., ~, 99 (1971). FREI, R.W., V. Mallet and M. Thi~baud, Intern. J. Environ. Anal. Chem., ~, 141 (1971). FREI, R.W. and P.E. Belliveau, Chromatographia,5, (1972). GETZ, M.E., J. Ass. Offic. Agric. Chem., 45, 393

296

(1962).

MACNEIL, J.D., B.L. MacLellan and R.W. Frei, J. Ass. Offic. Anal. Chem., 5~7, 165 (1974). SCHMIDT, K.-J. and I. Hamman, Pflanzensch, Nachr. Bayer, 22, 314 (1969).

637