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Multicomponent analysis method for PCDDs and PCDFs in biological samples
Chemosphere, Vol.15, Nos.9-12, pp 2109-2110, Printed in Great Britain
1986
0045-6535/86 $3.00 + .00 Pergamon Journals Ltd.
MULTICOMPONENT ANALYSIS METHOD FOR PCDDs and PCDFs IN BIOLOGICAL SAMPLES Juhani Tarhanen / a / ,
Jaakko P a a s i v i r t a
/a/
and J a a k k o S o i k k e l i
/ a / Department o f C h e m i s t r y , U n i v e r s i t y o f Kyllikinkatu 1 - 3 , SF-40100 J y v a s k y l a 10, /b/
NBC-Defence L a b o r a t o r y P.O.Box 8,
/b/
Jyv~skyl~ Finland
SF-02471Upinniemi, Finland
INTRODUCTION
P o l y c h l o r i n a t e d d i b e n z o - p - d l o x i n s (PCDD) and dibenzofurans ( P C D F ) have been d e t e c t e d i n t i s s u e s o f w i l d a n i m a l s / 1 - 3 / . More s o p h i s t i c a t e d a n a l y s i s p r o c e d u r e f o r t h e i r c l e a n up f r o m b i o l o g i c a l m a t e r i a l t h a n f r o m emissions was needed b e c a u s e o f i n t e r f e r e n c e s o f o t h e r m a t e r i a l s l i k e p o l y c h l o r o a r o m a t i c m e t h y l e t h e r s . These i n t e r f e r e n c e s will be d e s c r i b e d in another paper. In the present r e p o r t , o u r p r o c e d u r e f o r PCDD and PCDF c l e a n up w i l l be b r i e f l y reported.
EXPERIMENTAL
Our aim was t o d e v e l o p e an a d e q u a t e t r e a t m e n t of the sample f o r dual column CG/ECD d e t e r m i n a t i o n where GC/MS c o u l d s e r v e o n l y as a q u a l i t a t i v e verification t o o l . M o d i f i c a t i o n o f t h e c l e a n up p r o c e d u r e s o f Buser /4/ and S t a l l i n g e t a l . / 1 , 5 / was a d o p t e d . P r i o r any t r e a t m e n t o f t h e sample 1 , 2 , 3 , 4 - T C D D i s added t o make an internal standard for quantitation. Then t h e t i s s u e sample (5g) i s mixed w i t h 20 g o f d i s o d i u m s u l p h a t e and a l l o w e d t o s t a n d 48 h in erlenmeyer. The dried mixtur e is the ext ract ed in So×hlet apparatus 6 h with or ganic solvent mixture petroleum spirit-acetone-hexane-diethyl ether 9.0:5.5:2.5:1.0 (v/v/v/v). A f t e r e v a p o r a t i o n t h e r e s i d u e i s weighed and t h e n d i s s o l v e d in hexane. F a t i s removed f i r s t by s i l i c a g e l / c o n e , s u l p h u r i c a c i d p a s t e and t h e n by s h a k i n g w i t h cone. s u l p h u r i c a c i d . P o l y c h l o r i n a t e d h y d r o c a r b o n s (PCB, DDE, t o x a p h e n e , c h l o r d a n e s e t c . ) eluted off in a c t i v a t e d c a r b o n column / 1 , 5 / . P l a n a r a r o m a t i c e t h e r s t a k e n i n t o l u e n e by r e v e r s e d e l u t i o n f r o m the carbon column. Then sample is eluted t h r o u g h a a c t i v a t e d b a s i c a l u m i n a column f o l l o w i n g method o f Buser / 4 / where PCDDs and PCDFs a r e c o l l e c t e d as f r a c t i o n II.
are are the the
The f i n a l d e t e r m i n a t i o n i s done by a M i c r o m a t HRGC 412 gas chromatograph (Nordion, Espoo, Finland) equipped w i t h two q u a r t z c a p i l l a r y columns (0V-1701 and SE-54) and w i t h two ECDs.
2109
2110
I d e n t i t y and q u a n t i t a t i v e response oT each peak is conTirmed by 6C/ECD runs of a u t h e n t i c model compound m i x t u r e s and i n some cases by GC/MS. Pure PCDD and PCDF model compounds have come a v a i l a b l e by syntheses / 6 , 7 / . Number oT t h e s i n g l e pure compounds which have been used by us, thus Tar, is 14 Tor PCDDs and 15 Tot PCDFs. Recovery experiments have g i v e n r e s u l t s Trom 95 % (Tor TetraCDDs and CDFs) t o 55 % (Tot HeptaCPDs and CDFs). RESULTS AND DISCUSSION The samples where PCDD and PCDF components have been i d e n t i f i e d by us are Tish l i v e r o i l , salmon muscles and p e c t o r a l muscles oT w h i t e - t a i l e d e a g l e ( F i g . 1). cb
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1. Dual-column GC/ECD chromatogram oT PCDD/PCDF T r a c t i o n T r o m p e c t o r a l muscle o~ w h i t e - t a i l e d e a g l e ( H a l i a e t u s a l b i c i l l a ) Trom A r c h i p e l a g o o~ t h e B a l t i c Sea. The i d e n t i T i e d compounds are presented by Tormula and t h e i r peaks marked. ACKNOWLEDGEMENT
We are g r a t e f u l t o Maj and Tor Nessling Foundation Tot f i n a n c i a l s u p p o r t .
REFERENCES Io
2.
3.
4.
5. 6. 7.
C.Rappep H.R. Buser, D . L . S t a l l i n g , L.M.Smith and R.C.Dougherty. Nature 292 (1781) 524. D . L . S t a l l i n g , L.M.Smith, J . D . P e t t y , , J.W. Hogan, J . L . Johnson, C. Rappe and H.R.Buser. I n : Human and Environmental Risks o~ C h l o r i n a t e d D i o x i n s and Related Compounds; R.E.Tucker, A.L.Young and A . P . ~ r a y , Ed. Plenum Press9 New York (1983} p. 221. J . P a a s i v i r t a , K . H e i n o l a , T.Humppi, A . K a r j a l a i n e n , J . K n u u t i n e n , K.M&nt y k o s k i , R.Paukku, T.Piilola, K.Surma-Aho, J.Tarhanen, L.Welling, H.Vihonen and 3.S~rkk~. Chemosphere 14 (1985) 469. H.R.Buser. 3.Chromatogr. 107 (1975) 295. C.Rappe, D . L . S t a l l i n g , L.M.Smith and H.R.Buser. Manuscript (1981) A.Gar&. Doctor d i s s e r t a t i o n . Ume& U n i v e r s i t y , Sweden (1982). T.Humppi. Doctor d i s s e r t a t i o n : S y n t h e s e i s , i d e n t i f i c a t i o n and a n a l y sis of dime r i c i m p u r i t i e s of c h l o r o p h e n o l s . Department of Chemistry, U n i v e r s i t y of J y v ~ s k y l ~ . Research Report No. 23 (1985).
1986
0045-6535/86 $3.00 + .00 Pergamon Journals Ltd.
MULTICOMPONENT ANALYSIS METHOD FOR PCDDs and PCDFs IN BIOLOGICAL SAMPLES Juhani Tarhanen / a / ,
Jaakko P a a s i v i r t a
/a/
and J a a k k o S o i k k e l i
/ a / Department o f C h e m i s t r y , U n i v e r s i t y o f Kyllikinkatu 1 - 3 , SF-40100 J y v a s k y l a 10, /b/
NBC-Defence L a b o r a t o r y P.O.Box 8,
/b/
Jyv~skyl~ Finland
SF-02471Upinniemi, Finland
INTRODUCTION
P o l y c h l o r i n a t e d d i b e n z o - p - d l o x i n s (PCDD) and dibenzofurans ( P C D F ) have been d e t e c t e d i n t i s s u e s o f w i l d a n i m a l s / 1 - 3 / . More s o p h i s t i c a t e d a n a l y s i s p r o c e d u r e f o r t h e i r c l e a n up f r o m b i o l o g i c a l m a t e r i a l t h a n f r o m emissions was needed b e c a u s e o f i n t e r f e r e n c e s o f o t h e r m a t e r i a l s l i k e p o l y c h l o r o a r o m a t i c m e t h y l e t h e r s . These i n t e r f e r e n c e s will be d e s c r i b e d in another paper. In the present r e p o r t , o u r p r o c e d u r e f o r PCDD and PCDF c l e a n up w i l l be b r i e f l y reported.
EXPERIMENTAL
Our aim was t o d e v e l o p e an a d e q u a t e t r e a t m e n t of the sample f o r dual column CG/ECD d e t e r m i n a t i o n where GC/MS c o u l d s e r v e o n l y as a q u a l i t a t i v e verification t o o l . M o d i f i c a t i o n o f t h e c l e a n up p r o c e d u r e s o f Buser /4/ and S t a l l i n g e t a l . / 1 , 5 / was a d o p t e d . P r i o r any t r e a t m e n t o f t h e sample 1 , 2 , 3 , 4 - T C D D i s added t o make an internal standard for quantitation. Then t h e t i s s u e sample (5g) i s mixed w i t h 20 g o f d i s o d i u m s u l p h a t e and a l l o w e d t o s t a n d 48 h in erlenmeyer. The dried mixtur e is the ext ract ed in So×hlet apparatus 6 h with or ganic solvent mixture petroleum spirit-acetone-hexane-diethyl ether 9.0:5.5:2.5:1.0 (v/v/v/v). A f t e r e v a p o r a t i o n t h e r e s i d u e i s weighed and t h e n d i s s o l v e d in hexane. F a t i s removed f i r s t by s i l i c a g e l / c o n e , s u l p h u r i c a c i d p a s t e and t h e n by s h a k i n g w i t h cone. s u l p h u r i c a c i d . P o l y c h l o r i n a t e d h y d r o c a r b o n s (PCB, DDE, t o x a p h e n e , c h l o r d a n e s e t c . ) eluted off in a c t i v a t e d c a r b o n column / 1 , 5 / . P l a n a r a r o m a t i c e t h e r s t a k e n i n t o l u e n e by r e v e r s e d e l u t i o n f r o m the carbon column. Then sample is eluted t h r o u g h a a c t i v a t e d b a s i c a l u m i n a column f o l l o w i n g method o f Buser / 4 / where PCDDs and PCDFs a r e c o l l e c t e d as f r a c t i o n II.
are are the the
The f i n a l d e t e r m i n a t i o n i s done by a M i c r o m a t HRGC 412 gas chromatograph (Nordion, Espoo, Finland) equipped w i t h two q u a r t z c a p i l l a r y columns (0V-1701 and SE-54) and w i t h two ECDs.
2109
2110
I d e n t i t y and q u a n t i t a t i v e response oT each peak is conTirmed by 6C/ECD runs of a u t h e n t i c model compound m i x t u r e s and i n some cases by GC/MS. Pure PCDD and PCDF model compounds have come a v a i l a b l e by syntheses / 6 , 7 / . Number oT t h e s i n g l e pure compounds which have been used by us, thus Tar, is 14 Tor PCDDs and 15 Tot PCDFs. Recovery experiments have g i v e n r e s u l t s Trom 95 % (Tor TetraCDDs and CDFs) t o 55 % (Tot HeptaCPDs and CDFs). RESULTS AND DISCUSSION The samples where PCDD and PCDF components have been i d e n t i f i e d by us are Tish l i v e r o i l , salmon muscles and p e c t o r a l muscles oT w h i t e - t a i l e d e a g l e ( F i g . 1). cb
cl
Ill
|¥-17,|
t
f
C
o
l
¢1
cl
{i ¢1
I
II ~1 F9
Flg.
cl
1. Dual-column GC/ECD chromatogram oT PCDD/PCDF T r a c t i o n T r o m p e c t o r a l muscle o~ w h i t e - t a i l e d e a g l e ( H a l i a e t u s a l b i c i l l a ) Trom A r c h i p e l a g o o~ t h e B a l t i c Sea. The i d e n t i T i e d compounds are presented by Tormula and t h e i r peaks marked. ACKNOWLEDGEMENT
We are g r a t e f u l t o Maj and Tor Nessling Foundation Tot f i n a n c i a l s u p p o r t .
REFERENCES Io
2.
3.
4.
5. 6. 7.
C.Rappep H.R. Buser, D . L . S t a l l i n g , L.M.Smith and R.C.Dougherty. Nature 292 (1781) 524. D . L . S t a l l i n g , L.M.Smith, J . D . P e t t y , , J.W. Hogan, J . L . Johnson, C. Rappe and H.R.Buser. I n : Human and Environmental Risks o~ C h l o r i n a t e d D i o x i n s and Related Compounds; R.E.Tucker, A.L.Young and A . P . ~ r a y , Ed. Plenum Press9 New York (1983} p. 221. J . P a a s i v i r t a , K . H e i n o l a , T.Humppi, A . K a r j a l a i n e n , J . K n u u t i n e n , K.M&nt y k o s k i , R.Paukku, T.Piilola, K.Surma-Aho, J.Tarhanen, L.Welling, H.Vihonen and 3.S~rkk~. Chemosphere 14 (1985) 469. H.R.Buser. 3.Chromatogr. 107 (1975) 295. C.Rappe, D . L . S t a l l i n g , L.M.Smith and H.R.Buser. Manuscript (1981) A.Gar&. Doctor d i s s e r t a t i o n . Ume& U n i v e r s i t y , Sweden (1982). T.Humppi. Doctor d i s s e r t a t i o n : S y n t h e s e i s , i d e n t i f i c a t i o n and a n a l y sis of dime r i c i m p u r i t i e s of c h l o r o p h e n o l s . Department of Chemistry, U n i v e r s i t y of J y v ~ s k y l ~ . Research Report No. 23 (1985).