Pesticide Analytical Methodology - American Chemical Society

15 The Determination of 2,3,7,8-Tetrachlorodibenzo

Pesticide Analytical Methodology Downloaded from pubs.acs.org by UNIV OF CALIFORNIA SAN DIEGO on 03/06/16. For personal use only.

-p-dioxin in H u m a n M i l k L. A. SHADOFF Analytical Laboratories, Dow Chemical, Midland, MI 48640

Recent s t u d i e s have i n d i c a t e d that humans may be exposed to 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o - p - d i o x i n ( 2 , 3 , 7 , 8 - T C D D ) , a t o x i c and t e r a t o g e n i c substance i n l a b o r a t o r y animals ( 1 ) · A f r a c t i o n of beef f a t samples from c a t t l e known to have grazed on pasture t r e a t e d with h e r b i c i d e 2 , 4 , 5 - T (which contains t r a c e q u a n t i t i e s o f 2,3,7,8-TCDD) have been reported t o c o n t a i n low p a r t per t r i l l i o n (ppt) l e v e l s of 2,3,7,8-TCDD ( 2 , 3 ) ; Two s t u d i e s o f bovine milk reported no detectable c h l o r o d i o x i n s however ( 4 , 5 ) . Combustion processes have been reported to produce c h l o r i n a t e d d i o x i n s which enter the a i r as f l y ash and soot ( 6 , 7 . 8 ) . Three d i f f e r e n t s t u d i e s o f human milk have been c a r r i e d out by v a r i o u s workers t o determine i f humans c o n t a i n d e t e c t a b l e concentrations o f 2,3,7,8-TCDD ( 9 , 2 0 , 2 2 ) . In the f i r s t reported study (9j>, s i x samples of milk were obtained i n an area o f Texas where 2 , 4 , 5 - T i s used f o r rangeland management. The sample p r e p a r a t i o n scheme was c a u s t i c s a p o n i f i c a t i o n , e x t r a c t i o n , chromatography on s i l i c a followed by chromatography on alumina. The e x t r a c t was evaporated t o 1 0 - 2 0 μΐ and TCDD determined by gas chromatography/mass spec trometry (GC/MS). This technology i s s i m i l a r to t h a t reported f o r beef f a t sample p r e p a r a t i o n ( 2 , 3 ) . No 2,3,7,8-TCDD was detected with d e t e c t i o n l i m i t s o f 1-6 p p t . P r e l i m i n a r y r e s u l t s o f the determination of TCDD i n twenty-one human milk samples ( 1 0 ) i n d i c a t e d the p o s s i b l e presence o f TCDD i n three of the samples a t the 0 . 6 - 1 . 6 ppt l e v e l which was equal t o the d e t e c t i o n l i m i t . The sample p r e p a r a t i o n procedure ( 1 2 ) d i d not u t i l i z e a s a p o n f i c a t i o n step. S a p o n i f i c a t i o n had been used i n p r i o r procedures due t o the p o s s i b i l i t y t h a t TCDD was s t r o n g l y bound t o the matrix as has been the case f o r some p e s t i c i d e s ( 1 4 ) . In order to demon s t r a t e t h a t a l l the TCDD may be recovered from milk by e x t r a c t i o n , a comparison o f r e s u l t s between s a p o n i f i c a t i o n and extrac t i o n without s a p o n i f i c t i o n was performed. Such a t e s t must be

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performed f o r every matrix, however; recovery from milk does not i n s u r e t h a t i t may be recovered from l i v e r , f o r example. The advantage t o e l i m i n a t i n g s a p o n i f i c a t i o n from the sample p r e p a r a t i o n procedure i s t h a t fewer m a t e r i a l s w i l l be e x t r a c t e d from the matrix along with the TCDD, s i n c e many substances are indeed bound. The determination o f TCDD i n the f i n a l e x t r a c t was by h i g h r e s o l u t i o n mass spectrometry m u l t i p l e scan averaging. No gas chromatography was employed; the sample was v a p o r i z e d v i a the d i r e c t i n s e r t i o n probe (12,13). T h i s method i s i n h e r e n t l y more prone t o i n t e r f e r e n c e s than GC-MS (17,18); the response observed may not be due t o TCDD. The r e s u l t s do serve as an i n d i c a t i o n , however, t h a t i f TCDD i s present i n human m i l k i t i s a t 1 ppt o r l e s s . Thus any methods developed must be capable o f t h i s d e t e c t i o n l i m i t . A r e c e n t l y concluded study o f t h i r t e e n samples from the U.S. was a c o l l a b o r a t i v e e f f o r t sponsored by NIEHS. The samples were prepared and TCDD determined by three d i f f e r e n t l a b o r a t o r i e s . There was no confirmed d e t e c t i o n o f TCDD i n any of the samples (11). A summary o f the r e s u l t s o f completed s t u d i e s o f the determination o f TCDD i n human m i l k i s given i n Table I . EXPERIMENTAL CAUTION, 2,3,7,8-TCDD i s an extremely t o x i c substance which should be handled with care (13). The methodology used f o r the sample p r e p a r a t i o n i n our l a b o r a t o r y o f the NIEHS samples employed new technology i n many of the stages and i s an adaption o f t h a t reported f o r f i s h ( 8 ) . The i n i t i a l e x t r a c t i o n i n v o l v e s completely absorbing the sample onto c e l l u l o s e gauze (X-tube, Analytichem I n t e r n a t i o n a l ) . The m i l k i s not t r e a t e d i n any way except f o r the a d d i t i o n o f C-2,3,7,8-TCDD as an i n t e r n a l standard. The c a p a c i t y o f the gauze i s 15g o f milk and a t t h a t l o a d i n g , recovery experiments i n d i c a t e t h a t approximately 10% o f the TCDD i s e x t r a c t e d f o r each 10 ml o f hexane passed through the gauze a t ambient temp e r a t u r e . The volume o f e x t r a c t would be i n excess o f 100 ml. Therefore, the gauze i s p l a c e d i n an e x t r a c t i o n apparatus and hexane r e f l u x e d through the tube f o r a s u f f i c i e n t time t o pass a t l e a s t 1 £ through the gauze. The p l a s t i c container s u p p l i e d with the X-tube i s not s u i t a b l e f o r hot e x t r a c t i o n s i n c e i t deforms and contaminates the e x t r a c t with Ionol and low molecular weight polypropylene. A g l a s s thimble was constructed w i t h an o u t l e t a t the bottom o f 1 cm o f 0.5 m.m.i.d. tubing t o c o n t r o l the flow through the gauze. The approximately 75 ml o f hexane e x t r a c t i s t r a n s f e r r e d d i r e c t l y onto a column c o n t a i n i n g 6 g o f a c t i v a t e d s i l i c a ( B i o - S i l , B i o r a d , Inc.) coated with 25% w/w cone. H S 0 ( 8 j . T h i s column has a high c a p a c i t y f o r the f a t s from milk, l e a v i n g 13

2

4


15.

SHADOFF

2,3,7,8-Tetrachlorodibenzo-p-dioxin

in Human

Milk

279

TABLE I . Summary o f the r e s u l t s o f completed s t u d i e s on the Determination o f 2,3,7,8-TCDD i n human milk as o f March, 1979.

2,3,7,8-TCDD IN HUMAN MILK DONOR LOCATION

STUDY BY

RESULTS ( d e t e c t i o n l i m i t p p t )

Texas

Dow

6 spls

not detected (1-6)

Missouri

Harvard

6 spls

not detected

Texas

Harvard

2 spls

0.6-1.5 (0.6-1.6)

4 spls

not detected

1 spl

0.8-1.4 (0.7-1.5)

6 spls

not detected

(0.4-2.9) (0.3-1.6)

Oregon

Harvard

(0.7-3.3)

(1.2-1.9)

Massachusetts

Harvard

6 spls

not detected

Texas

NIEHS*

9 spls

not detected (0.4-4)

Kansas

NIEHS*

4 spls

not detected (1-10)

" C o l l a b o r a t i o n Study - Dow, Harvard, NIEHS(RTP).

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a r e s i d u e o f l e s s than 10 mg when l g o f f a t i s passed through the column. The progress through the column may be e a s i l y monitored s i n c e i t turns deep orange as the m a t e r i a l i s absorbed. T h i s column r e p l a c e s the s u l f u r i c a c i d e x t r a c t i o n employed i n p r e v i o u s procedures (12.15.16.17) (see F i g u r e 1). The e f f l u e n t from t h i s column p l u s a 30 ml r i n s e c o n t a i n i n g the TCDD i s passed d i r e c t l y onto a column c o n t a i n i n g 5g o f a c t i v a t e d b a s i c alumina (Biorad, Inc.) which traps the TCDD. The hexane p a s s i n g through the column i s d i s c a r d e d . The TCDD i s e l u t e d from the column with 10 ml o f methylene c h l o r i d e . T h i s alumina column t r a n s f e r s the TCDD from over 100 ml o f hexane t o 10 ml o f methylene c h l o r i d e a l l o w i n g a much s h o r t e r time f o r s o l v e n t evaporation and the use o f s m a l l e r glassware. Recovery o f TCDD a t t h i s stage i s i n excess o f 85%. The e l u a t e i s evaporated j u s t t o dryness, taken up i n hexane and p l a c e d on a column o f 10% AgN0 on a c t i v a t e d s i l i c a (8)(which removes DDE, many a l i p h a t i c halogenated substances and s u l f u r c o n t a i n i n g substances). The e l u a t e c o n t a i n i n g the TCDD drops d i r e c t l y onto a h i g h aspect r a t i o b a s i c alumina column ( 8 ) . PCB s are e l u t e d and TCDD r e t a i n e d on t h i s column by a d d i t i o n o f 50 ml o f 50% carbon t e t r a c h l o r i d e i n hexane. The TCDD i s then c o l l e c t e d by the a d d i t i o n o f 15 ml o f 50% methylene c h l o r i d e i n hexane. To t h i s stage the sample p r e p a r a t i o n i s s i m i l a r t o t h a t p r e v i o u s l y used t o determine TCDD i n environmental samples. The d e t e c t i o n l i m i t s r e p o r t e d were g e n e r a l l y i n excess o f 5 ppt (3,17) due t o i n t e r f e r e n c e s from substances not removed i n the sample p r e p a r a t i o n . An a d d i t i o n a l , h i g h l y e f f i c i e n t s e p a r a t i o n step was employed t o allow the lower d e t e c t i o n l i m i t s r e q u i r e d f o r meaningful determinations o f TCDD i n human m i l k . Reverse phase h i g h pressure l i q u i d chromatography (HPLC) was used t o f u r t h e r f r a c t i o n a t e the sample and add another dimension o f s p e c i f i c i t y (8,21). The e x t r a c t was evaported t o dryness and taken up i n CHC1 . The e n t i r e e x t r a c t was i n j e c t e d onto a DuPont Zorbax ODS column a t 40°C u s i n g 2 cc/min. CH 0H mobile phase. T y p i c a l chromatograms are shown i n F i g u r e 2. Note t h a t a c o n s i d e r a b l e amount o f U.V. absorbing m a t e r i a l present i n the e x t r a c t i s separated here. The TCDD f r a c t i o n i s e x t r a c t e d i n t o hexane a f t e r the a d d i t i o n o f water. After evaporation t o dryness the sample i s ready f o r the determination o f TCDD. Gas chromatography-low r e s o l u t i o n mass spectrometry was used t o determine i f TCDD i s i n the samples (12). The GC column used was a 6 f t . χ 2 mm i . d . g l a s s 2.5% BMBT l i q u i d c r y s t a l on 100/120 mesh Chromosorb WHP ( A l t e c h Assoc.) a t 225°C. In F i g u r e 3 i s shown the output from a standard and t y p i c a l human m i l k e x t r a c t run on an LKB-9000 w i t h a SI-150 (System I n d u s t r i e s ) data system equipped with a computer c o n t r o l l e d m u l t i p l e i o n d e t e c t o r (Ledland, I n c . ) . The e n t i r e e x t r a c t was 3

f

3

3

SHADOFF


in Human

Milk

281

HEXANE EXTRACTION FROM CELLULOSE GAUZE


2 5 % cone. SULFURIC ON ACTIVATED SILICA

I SOLVENT EXCHANGE 10% SILVER NITRATE ON ACTIVATED SILICA HIGH ASPECT RATIO BASIC ALUMINA

Figure 1. Sample preparation scheme for the determination of 2,3,7,8-TCDD in human milk as performed for NIEHS study

X REVERSE PHASE HPLC

500pg 2,37,8-TCOD

OOS-Zorbax 235nm

S pi. 22

—ι

4

Figure 2.

1

1

6

8 min.

High Pressure reverse-phase liquid chromatography trace of 2,3,7,8TCDD standard and a typical human milk extract

282

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METHODOLOGY

i n j e c t e d by adding 7 μΐ of iso-octane to the dry e x t r a c t i n a 0.3 ml cone shaped v i a l , washing down the w a l l s , and withdrawing as much as p o s s i b l e (the syringe needle was f i r s t f i l l e d with i s o - o c t a n e ) . Recovery measurements averaged 88 % e f f i c i e n c y o f removal from the v i a l (see Figure 3 ) .


RESULTS AND DISCUSSION As i n d i c a t e d i n Table I, s e v e r a l s t u d i e s have been performed to determine whether there i s d e t e c t a b l e concentrations o f 2,3,7,8-TCDD i n humans. Human milk was chosen because i t has a high f a t content where TCDD should r e s i d e s i n c e i t i s l y p o p h y l l i c , i t may be obtained i n l a r g e enough q u a n t i t i e s to allow s u f f i c i e n t sample f o r p a r t p e r t r i l l i o n determinations, and i t does not r e q u i r e v i o l a t i o n o f the body and the l e g a l problems a s s o c i a t e d with sample collection. The geographic areas were chosen i n a l l cases to determine i f the presence o f TCDD c o r r e l a t e s t o the use of 2,4,5-T h e r b i c i d e s which c o n t a i n t r a c e l e v e l s o f 2,3,7,8-TCDD. In no case was there a confirmed d e t e c t i o n of TCDD. The d e t e c t i o n l i m i t s achieved i n these s t u d i e s were i n the low p a r t per t r i l l i o n range with the Harvard study i n d i c a t i n g the p o s s i b l e presence o f TCDD a t or below 1 ppt s e t t i n g an upper l i m i t to the c o n c e n t r a t i o n o f TCDD which a method must be capable of detecting. The sample p r e p a r a t i o n methodology f o r the determination of TCDD a t these low l e v e l s i s an a c t i v e area o f development as i n d i c a t e d by the improved procedure reported here and used to analyze the milk f o r the NIEHS study. I t uses reagent modified adsorbants, a higher e f f i c i e n c y b a s i c alumina column than p r e v i o u s l y (19) and a new degree o f s e p a r a t i o n , reverse phase HPLC as an i n t e g r a l p a r t of the procedure. A l l o f these are d i r e c t e d towards improving the s p e c i f i c i t y o f the sample p r e p a r a t i o n f o r 2,3,7,8-TCDD. Increased s p e c i f i c i t y i s needed s i n c e the lowest d e t e c t i o n l i m i t achievable i n a l l previous s t u d i e s o f TCDD i n environmental samples has been d i c t a t e d by the presence o f other substances i n the samples which have not been removed by the sample p r e p a r a t i o n . Recently, a l l the isomers o f TCDD have been synthesized and separated (21). T h i s allows the e s t i m a t i o n o f TCDD isomer s p e c i f i c i t y o f the methods. The s e p a r a t i o n o f components by gas chromatography i s d i s c u s s e d by Jennings (22) who r e l a t e s r e l a t i v e r e t e n t i o n times, t h e o r e t a l p l a t e s and degree o f s e p a r a t i o n i n the formula (rearranged here to separate out the term a ) :


SHADOFF


in Human

Milk

283

Figure 3. Gas chromatography-low resolution mass spectrometry mass chromatograms for 2,3,7,8-TCDD standard and human milk extract (( ) 320; (- · -) 322; ( ; 334)

PESTICIDE

284

α =

Ν N-4R

α


Ν R

ANALYTICAL

METHODOLOGY

r e l a t i v e r e t e n t i o n times of the two components theoretical plates degree o f s e p a r a t i o n (1.0=completely separated)

With a good packed column o f 4000 t h e o r e t i c a l p l a t e s and a degree o f s e p a r a t i o n o f 0.5 (not separated) s u b s t i t u t e d i n t o the formula, a l l peaks with r e l a t i v e r e t e n t i o n i n d i c e s between 0.968 and 1.033 a r e i n d i c a t e d as not separated. Based on t h i s the GC peak measured as 2,3,7,8-TCDD may a l s o c o n t a i n 1,2,6,9-, 1,2,3,6-, 1,2,3,9-, 1,2,3,7-, 1,2,3,8-, and/or 1,2,3,4-TCDD (21). The reverse phase HPLC step reported here would separate the 1,2,6,9- and 1,2,3,4-TCDD isomers from the others. The a n a l y t i c a l accomplishments achieved i n the s t u d i e s o f 2,3,7,8-TCDD i n human milk are c o n s i d e r a b l e . The d e t e c t i o n l i m i t s are lower than f o r any other n o n - r a d i o a c t i v e compound with a high degree o f s e l e c t i v i t y gained through m u l t i - s t e p sample p r e p a r a t i o n and h i g h l y s p e c i f i c d e t e c t i o n . None the l e s s , i t i s an a c t i v e area o f r e s e a r c h i n t o the development o f more s p e c i f i c and s e n s i t i v e measurements.

Abstract Are humans contaminated with 2,3,7,8-TCDD? Several laboratories have been and are presently involved in determining this by examining human milk. Preliminary results have indicated that i t is necessary to achieve detection limits below one part per t r i l l i o n . The attempt to achieve this requires the development of very selective sample preparations capable of preserving picogram quantities of 2,3,7,8-TCDD. In general, they involve extraction, partition, and flow-through liquid chromatography steps. The prepared samples have been analyzed by the most sensitive and specific detectors available, mass spectrometers. The results to date and the methodology are discussed in detail.

15.

1.


2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

SHADOFF

2,3,7,8-Tetrachlorodibenzo-p-dioxin in Human Milk

LITERATURE CITED Schwetz, Β. Α., Norris, J. Μ., Sparschu, G. L . , Rowe, V. K. Gehring, P. J., Emerson, J. L . , Gerbig, C. G., Adv. Chem. Ser., 120, 55 (1973). Kocher, C. W., Mahle, Ν. Η., Hummel, R. Α., Shadoff, L. Α., Getzendaner, M. F . , Bull. Environ. Contam. Toxicol., 19, 229 (1978). Personal Communication, Ross, R. T., Dioxin Project Manager Office of Pesticide Programs, U. S. Environmental Protection Agency, December 16, 1975 and June 25, 1976, to Analytical Collaborators: Pest. Chem. News. 17 (June 23, 1976). Mahle, N. H., Higgins, H. S., Getzendaner, M. E . , Bull. Environ. Contam. Toxicol., 18, 123 (1977). Lamparski, L. L . , Mahle, Ν. Η., Shadoff, L. Α., J . Ag. Food Chem., 26, 1113 (1978). Olie, Κ., Vermeulen, P. L . , Hutzinger, O., Chemosphere, 1977, 455. Buser, H. R., Bosshardt, H. P., Rappe, C., ibid., 1978, 165. "The Trace Chemistries of Fire", Chlorinated Dioxin Task Force, Dow Chemical Co., 1978. Shadoff, L. Α., Hummel, R. Α., Lamparski, L. L . , Davidson, J. Η., Bull. Environ. Contam. Toxicol., 18, 478 (1977). Meselson, M., O'Keefe, P. W., Personal Communication to U. S. Congressman J. Weaver, January 26, 1977. Personal Communication, McKinney, J. D., 1979. O'Keefe, P. W. and Meselson, M., J.A.O.A.C., 61 621 (1978). "Summary of Safe Handling of 2,3,7,8-Tetrachlorodibenzo -p-dioxins in the Laboratory", Biochemical Research Laboratory, The Dow Chemical Company, Midland, MI 48640. Clark, D.E., Palmer, J.S., Radeleff, Rd., Crookshank, H.R., Farr, F. M., J . Ag. Food Chem., 23, 573 (1975). Baughmann, R. W., Meselson, Μ., Environ. Health Persp., 5, 27 (1973). Baughmann, R. W., Meselson, Μ., 166th National Meeting, American Chemical Society, Chicago, Illinois (1973), Abstract Pest. 55. Shadoff, L. Α., Hummel, R. Α., Biomed. Mass Spectrom., 5, 7 (1978). McKinney, J . D., "Chlorinated Phenoxy Acids and Their Dioxins", Ramel, C. (ed), Ecol. Bull. (Stockholm), 27, 53 (1978). Hummel, R. Α., J . Agric. Food Chem., 25, 1049 (1977). Pfeiffer, C. D., Nestrick, T. J., Kocher, C. W. Anal. Chem., 50, 800 (1978). Nestrick, T. J., Lamparski, L. L . , Stehl, R. Η., Anal. Chem., 51, 2273 (1979). Jennings, W., "Gas Chromatography with Glass Capillary Columns", Academic Press, New York, 1978, pp 13 ff.

RECEIVED March

30, 1980.

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Pesticide Analytical Methodology - American Chemical Society

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