Mass Spectrometric Identification of Mirex Residues in Crude Extracts and in the Presence of Polychlorinated Biphenyls .,b by SOLANG UK, CHESTER M. HIMEL, and TOBIAS DIRKS

Department of Entomology University of Georgia Athens, Ga. 30601 Abstract

Mass s p e c t r o m e t r y using i0 eV e l e c t r o n i m p a c t was employed in the analysis of M i r e x r e s i d u e s in the p r e s ence of PCB's. Low v o l t a g e MS is a useful c o m p l e m e n t a r y tool for p o s i t i v e i d e n t i f i c a t i o n of both M i r e x and PCB r e s i d u e s in o r g a n i c extracts. The MS patterns of pure 2 , 4 , 5 , 2 ' , 5 ' - p e n t a c h l o r o and 2 , 5 , 2 ' , 5 ' - t e t r a c h l o r o b i p h e n y l and of m i x t u r e s of PCB isomers in A r o c l o r 1254 and 1260 are simple and d i s t i n g u i s h a b l e from the MS s p e c t r u m of Mirex. T h e r e is no o v e r l a p p i n g of ions (molecular or fragment) of PCB's w i t h those of Mirex. Each pure c h l o r o b i p h e n y l has its base peak at the m o l e c u l a r ion (M) in c o n t r a s t to M i r e x w h i c h exhibits the intense M/2 ion (m/e 270) as b a s e peak. In m i x t u r e s of PCB's, peak c l u s t e r s , 34 mass u n i t s apart, c o r r e s p o n d to C I 2 H I 0 _ x C I x w h e r e x = 4 to 9. A m i x t u r e of 500 ~g of M i r e x and 500 ~g of A r o e l o r 1254 added to 5 g of pork fat was d e t e c t e d by low v o l t a g e MS in the crude r e c o v e r y extract. A probe t e m p e r a t u r e of 90 or 100~ was s a t i s f a c t o r y for i d e n t i f i c a t i o n of each c o m p o n e n t in the mixture. However, w h e n the p r o b e t e m p e r a t u r e s r e a c h e d 140~ or above, i n t e r f e r e n c e by e x t r a n e o u s peaks b e c a m e m o r e s e r i o u s and m o s t of M i r e x and PCB's were depleted. The p r e s e n c e of t h e s e e x t r a n e o u s peaks indicated that the t r e a t m e n t of fat e x t r a c t s by either 24-hr stirring in c o n c e n t r a t e d H2SO 4 or by a s i n g l e f l o r i s i l column c h r o m a t o g r a p h y did not r e m o v e f a t t y r e s i dues s a t i s f a c t o r i l y . Thus, " s t a n d a r d " t e c h n i q u e s of Mirex r e s i d u e c l e a n u p may be i n a d e q u a t e .

Introduction

The w o r l d - w i d e i n d u s t r i a l use of p o l y c h l o r i n a t e d b i p h e n y l s (PCB's) (4) has made t h e m m a j o r p o t e n t i a l pollutants. They c o n c e r n e n v i r o n m e n t a l s c i e n t i s t s because their t o x i c o l o g i c a l p r o p e r t i e s are of a c h r o n i c nature. In addition, their c h e m i c a l c h a r a c t e r i s t i c s a S u p p o r t e d by a grant from the A g r i c u l t u r a l Service 660B (PP-ARS 539). b C o r r e s p o n d e n c e should be a d d r e s s e d to CMH, 97 Bulletin of Environmental Contamination & Toxicology, Vol 8, No. 2, 1972, ~) by Springer.Verlag New York Inc.

Research

create s u b s t a n t i a l d i f f i c u l t i e s in p e s t i c i d e r e s i d u e a n a l y s i s (2, 6, 9). For example, some of the GLC peaks of A r o c l o r 1254 and 1260 o v e r l a p or c o i n c i d e with M i r e x peak w h e n a n a l y z e d d o n QF-I + DC-200 c o l u m n (3) or on QF-I c or on 0V-210 (our l a b o r a t o r y ) . Mass s p e c t r o m e t r y is a m e a n s of p o s i t i v e l y d i s t i n g u i s h i n g M i r e x from PCB's. Mass spectral p a t t e r n s of Mirex and Kepone have been r e p o r t e d (10). The p r e s e n t paper r e p o r t s the progress of our study of a n a l y t i c a l m e t h o d o l o g y p e r t i n e n t to the c o n f i r m a t i o n of M i r e x r e s i d u e s in b i o l o g i c a l samples c o n t a i n i n g PCB's.

Ex per iment a I Sample

preparation

One, 5, 20, and 500 ~g of M i r e x e and of A r o c l o r 1254 f w e r e added to 5 g of pork fat. The sample was ground in a m o r t a r and pestle with the aid of p u r i f i e d sand in 4 p o r t i o n s of 5 ml of benzene. The b e n z e n e f r a c t i o n s w e r e c o m b i n e d and t r e a t e d w i t h an e q u i v o l u m e of c o n c e n t r a t e d H~SO~. A m o d i f i e d Craig c o u n t e r c u r r e n t d i s t r i b u t i o n cellZ(1) was used for the H?SO 4 t r e a t m e n t by phase partition. O v e r n i g h t stirring 6f the b e n z e n e e x t r a c t with H2S04 was also tested. The b e n z e n e layer was then separated, filtered over glass w o o l - N a g S 0 4 into a c o n i c a l - b o t t o m c e n t r i f u g e tube, and c a r e f u l l y c o n c e n t r a t e d w i t h a stream of dry air. W h e n the v o l u m e was at about 0.5 ml, the b e n z e n e was c a r e f u l l y w a s h e d into a Pasteur p i p e t t e that had the tip cut and sealed off to a l l o w the c o n c e n t r a t i o n to less t h a n 5 ~i. The r e s i d u e was t r a n s f e r r e d into a m e l t i n g point c a p i l l a r y tube w h i c h was then cut to 20 m m to fit the mass spectrom e t e r probe. The tube was p l u g g e d w i t h s p e c t r o s c o p i c a l l y inert, fine m e s h glass wool. Other samples of pure Mirex, pure c h l o r o b i p h e n y l isomers, or m i x t u r e s of M i r e x and A r o c l o r s were put d i r e c t l y into glass c a p i l l a r y tubes cut to 20 mm length, and p l u g g e d with s p e c t r o q u a l i t y glass wool.

Mass

was

Spectrometry A BelI-HowelI/CEC used. All samples

m o d e l 21-490 mass s p e c t r o m e t e r were i n t r o d u c e d into the i o n i z a t i o n

c 3% QF-I on C h r o m o s o r b G, AW-DMCS, d 3% 0V-210 on Supelooport, 100-120 e Poly Science Corp., Evanston, Iii. f M o n s a n t o Co., St. Louis, Mo. 63166

98

80-100 mesh. 60201

mesh.

chamber by means of a probe that can be h e a t e d independently of the source. Mass scans were taken at various probe temperatures for reasons discussed below. The source temperature was at 240~ and the sample pressure at about 1.5 x 10 -5 Torr. The bombarding energy was set at i0 eV since it gives simple mass spectra of Mirex suitable for i d e n t i f i c a t i o n (i0). The mass spectral pattern of Mirex was used as r e f e r e n c e in combination with isotopic ratios due to 37CI to facilitate peak identification.

Results

and Discussion

The f r a g m e n t a t i o n pattern of M i r e x has been discussed previously (I0). The mass spectral property of 2,4,5,2',5'-pentachlorobiphenyl is p r e s e n t e d in Figure i. Unless otherwise specifi~J, ion clusters due to chlorine isotopes (35CI and CI) are identified in the figures and discussion only by the m/e of parent peaks (P), i.e., peaks due to 35CI, and m o l e c u l a r ions are referred to as M. Under i0 eV e l e c t r o n impact, 2 , 5 , 2 ' , 5 ' - t e t r a c h l o r o b i p h e n y l showed b r e a k d o w n process similar to that of 2,4,5,2',5'-pentachlorobiphenyl. Both compounds produced 2 fragments by losing i CI (M-CI fragment) and 2 CI (M-2CI fragment). Whether the molecule lost I and 2 CI s i m u l t a n e o u s l y or the M-CI ion lost its second CI ([M-CI] - CI) is not clear. No other ion of significance was observed. Other chlorobiphenyl isomers (hexachloro, h e p t a e h l o r o b i p h e n y l , etc.) a p p a r e n t l y have similar f r a g m e n t a t i o n p r o p e r t y as mass scans of the A r o c l o r 1254 or 1260 p r o d u c e d no peaks other than those c o r r e s p o n d i n g to the m o l e c u l a r ion, the M-CI, and the M-2CI ions of the chlorobiphenyls. Biros, Walker, and M e d b e r y (2) r e p o r t e d comparable results of trichloro, hexachloro, and heptachlorobiphenyl if peaks other than the a f o r e d e s e r i b e d ones are disregarded. Though not mentioned, the authors o b v i o u s l y used much higher electron energy. The relative abundance of m o l e c u l a r ions of various isomers produced by successive mass scans w i t h a gradual increase of probe temperatures indicated that the major components of Aroclor 1260 are tetrachloro, pentachloro, hexachloro, heptachloro, octachloro, and a small amount of nonachlorobiphenyls. Under similar conditions, Aroclor 1254 produced similar mass spectra but negligible amounts of heptachloro and the absence of octachloro and nonachlorobiphenyls. When a mixture of Mirex and Aroclor was analyzed, their mass spectral patterns (produced by i0 eV electron impact) were completely separated from each other as

40

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io

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Figure i. Mass spectrum of 2 , 4 , 5 , 2 ' , 5 ' - p e n t a c h l o r o b i p h e n y l under i0 eV electron bombardment. M is the m o l e c u l a r ion. There are no other peaks of significance.

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shown in Figure 2. There were no peaks of importance pertaining to any of the components below m/e 200. Further differences in fragmentation behavior between Mirex and PCB's are that the former has its characteristic base peak at M/2 cluster (m/e 270) and quite small molecular ion peak (M), while PCB's have intense molecular ions as base peaks; the latter property is due to the 2 benzene rings (8). Recovery of a mixture of 500 ug of Mirex and 500 ~g of Aroclor 1254 added to 5 g pork fat was easily detected by low voltage MS in the crude extract after it was treated with concentrated H2S0 ~. The most favorable probe temperatures for obtalning good, clearly recognizable mass spectra were at 90-I00~ as illustrated in Figure 3. Despite numerous extraneous peaks from the fat particularly at m/e below 200, mass scans in this temperature range showed minimum interference with ions of Mirex and/or PCB's except at the following masses: 237.5, 241.5, 256.5, 270.5, 271.5, 360.5, and 361.5. Nevertheless, peak clusters due to CI isotopes at the aforementioned m/e regions remained clearly recognizable. The presence of extraneous peaks proved that the treatment of pork fat extracts with H2SO 4 either by simple binary phase partition or by continuous overnight stirring did not eliminate organic impurities. One sample from florisil column chromatography showed similar incomplete cleanup of organic contaminants. The results presented here show the feasibility of concentrating pesticide residue extracts, to ul-volume for positive confirmation by mass spectrometry. For relatively stable compounds with low vapor pressure such as Mirex, the sample in organic solvent such as benzene can be introduced into the glass capillary tube plugged with glass wool i to 2 weeks in advance before mass spectrometric testing. Thus, the sending of prepared samples to other laboratories where a mass spectrometer is available could be done without affecting the analysis. Thus, crude residues can be quantitated by EC-GLC then confirmed by mass spectrometry. Analytical sensitivity remains a problem since we were unable to detect the presence of Mirex and/or Aroclor 1254 in sample extracts containing i, 5, or 20 ug of each chemical. However, a standard preparation of 3.5 ug of pure Mirex gave highly satisfactory mass spectra during 5 repeated scans for about 5 min. Depending on the types of biological materials, better techniques of sample cleanup and of concentration to ~l-volume would certainly improve the sensitivity of mass spectrometric analysis. Hutzinger and Jamieson (5) reported the analysis of 1 ppm of 2,6-dichloro-4nitro-aniline (apparently a total amount of i00 ug) in

101

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400

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Figure 2. M a s s s p e c t r a of a m i x t u r e of M i r e x and A r o c l o r 1260 under 10 eV e l e c t r o n impact. T e t r a c h l o r o , p e n t a c h l o r o b i p h e n y l , etc., are r e f e r r e d to as 4 CBP and 5 CBP, etc. F r a g m e n t s are d e s i g n a t e d as M . W . - C I . The H e x a c h i o r o o y c l o p e n t a l e n e ion of the Mirex f r a g m e n t is c a l l e d M i r e x / 2 at m / e 270. T h e r e are no peaks of i m p o r t a n c e b e l o w ~/e 200.

260

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F i g u r e 3. P a r t i a l g a l v a n o m e t e r trace of mass spectra of p o r k fat e x t r a c t spiked w i t h 500 mg of M i r e x and of A r o c l o r 1254 at probe t e m p e r a t u r e of 90~ Peaks and p e a k c l u s t e r s not labelled are due to i n t e r f e r i n g m a t e r i a l s in the extract. There are n u m e r o u s peaks b e l o w m/e 200, but none p e r t a i n to M i r e x or Aroclor.

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crude peach extract with low r e s o l u t i o n mass spectrometry. Low voltage mass spectrometry (i0 eV or lower, i.e., slightly above ionization potential) should be a very useful complementary technique in the analysis of multiple pesticide residues as it produces, rather simple spectra. We also found that, under i0 eV, Dieldrin and DDE produced mass spectra easily distinguishable from each other as well as from those of Mirex and PCB's (unpublished data). Low voltage mass spectrometry in organic analysis has been briefly discussed by Roboz (7) who gave references to further details. Acknowledgments

We are indebted to Dr. R. E. Lowins, Dr. Fairwell Thomas, and John Craig, Department of Biochemistry, U n i v e r s i t y of Georgia, for their kind cooperation in making available the mass spectrometer. Our a p p r e c i a t i o n is also addressed to Dr. R. G. Webb, Southeast Water Laboratories, Athens, Ga., for providing pure samples of tetrachloro, and pentachlorobiphenyls, and to Miss Carol Lord of our laboratory for her technical assistance. References i.

2.

3. 4. 5. 6.

7. 8.

9 .

I0.

BEROZA, M., INSCOE, M. N., and BOWMAN, M. C. Residue Reviews, Vol. 30, p. 25 (1967), SpringerVerlag, New York BIROS, F. J., WALKER, A. C., and MEDBERY, A. Bull. Environ. Contam. Toxicol. 6, 385 (1970) GAUL, J. and CRUZ-LAGRANGE, P. Private Communication. Food and Drug Administration, New Orleans, La. (1971) GUSTAFSON, C. G. Environ. Sci. Teoh. I0, 814 (1970) HUTZINGER, O. and JAMIESON, W. D. Bull. Environ. Contam. Toxicol. 5, 587 (1971) REYNOLDS, L. M. Bull. Environ. Contam. Toxicol. 4, 128 (1969) ROBOZ, J. Introduction to Mass Spectrometry, p. 321 (1968), Interscience Publishers, New York SILVERSTEIN, R. M. and BASSLER, G. C. Spectrometric Identification of Organic Compounds, p. 13 (1967), John Wiley 6 Sons, New York SNYDER, D. and REINERT, R. Bull. Environ. Contam. Toxicol. 6, 385 (1971) UK, S., HIMEL, C. M., and DIRKS, T. F. Bull. Environ. Contam. Toxicol. 7(4), (1972)

104