9 Applications of Combined Gas Chromatography-Mass Spectrometry to Pesticide Residue Identifications
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F R A N C I S J . BIROS Perrine Primate Research Branch, D i v i s i o n of Pesticide Chemistry a n d Toxicology, F o o d and D r u g Administration, U. S. D e p t . of Health, Education, and Welfare, Perrine, F l a . 33157
Several
individual
and multiresidue
analytical
available
for gas chromatographic-mass
firmation
and identification
examples
considered
intact and derivatized
phenolic
phenol and 1-naphthyl secticide
metabolic
-diethyl O-methyl
of pesticide
in this report
residues
chloroacetate,
pesticides
type, phenoxyalkanoic
acid herbicide
studies
rinated
involving
biphenyl
methodology
2,4-D
residues. directly
and environmental
nique.
Evaluation
identity
definitive
method-
and
polychlo-
residue column
analytical technology human,
by the combined
of mass spectral
fragmentation
and conclusive
confirmation
of unknown
cyclodiene
and
to the analysis of
substrates
as well as characterization
metabolites
2,4,5-T,
Conventional
applicable
animal, provides
and
phosphate,
and
exposure
inO,O-
S-methyl
O-methyl
of the DDT
and gas chromatographic
are, in general,
including
O,O-diethyl
O,O-diethyl
of
pentachloro-
organophosphorus
several organochlorine ology
Specific
the analysis
such as
products
phosphorothionate, and
are con-
residues.
include
and hydrolytic
phosphorothiolate,
methods
spectrometric
of residues
tech-
pathways of and
residue their
structure.
T T n e q u i v o c a l i d e n t i f i c a t i o n of p e s t i c i d e s , p e s t i c i d e m e t a b o l i t e s , a n d ^ other c h e m i c a l e n v i r o n m e n t a l p o l l u t a n t s i n m o s t cases r e q u i r e s m o r e evidence than can be p r o v i d e d b y a single chromatographic method. Seve r a l a p p r o a c h e s m a y b e u t i l i z e d to secure firm p r o o f of i d e n t i t y . F o r e x a m p l e , s u p p o r t i v e c h r o m a t o g r a p h i c d a t a s u c h as r e l a t i v e r e t e n t i o n 132
Biros; Pesticides Identification Advances in Chemistry; American Chemical Society: Washington, DC, 1971.
9.
BIROS
Gas Chromatography—Mass
133
Spectrometry
values o n t w o gas c h r o m a t o g r a p h i c c o l u m n s of d i f f e r i n g characteristics m a y b e o b t a i n e d . O f f u r t h e r v a l u e is i n f o r m a t i o n d e r i v e d b y means of t h i n - l a y e r c h r o m a t o g r a p h y (R
f
v a l u e s ) , the solvent p a r t i t i o n i n g c h a r a c -
teristics of the r e s i d u e of interest ( p - v a l u e s ) , a n d the response b e h a v i o r o n several m o r e o r less specific gas c h r o m a t o g r a p h i c detectors.
In addi-
t i o n , s p e c t r o m e t r i c t e c h n i q u e s s u c h as i n f r a r e d a n d u l t r a v i o l e t spectrop h o t o m e t r y m a y b e a p p l i e d to p r o v i d e e v e n less a m b i g u o u s p r o o f
of
r e s i d u e s t r u c t u r a l i d e n t i t y . T h e v a l u e of mass s p e c t r o m e t r y as a t o o l for p r o v i d i n g t h e s t r u c t u r a l i d e n t i t y of c o m p l e x o r g a n i c m o l e c u l e s has b e c o m e w e l l r e c o g n i z e d i n recent years. M a s s spectra f u r n i s h i n f o r m a t i o n Downloaded by FUDAN UNIV on January 4, 2017 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0104.ch009
c o n c e r n i n g the s t r u c t u r a l a r r a n g e m e n t of atoms w i t h i n a m o l e c u l e o n the basis of the m o d e of f r a g m e n t a t i o n of the c o m p o u n d as a r a d i c a l i o n , usually produced b y electron bombardment.
F r a g m e n t a t i o n patterns r e -
s u l t i n g f r o m b o n d fission a n d r e a r r a n g e m e n t of atoms are h i g h l y d i a g n o s t i c a n d c h a r a c t e r i s t i c of the o r i g i n a l m o l e c u l a r structure. I n t e r p r e t a t i o n s are u s u a l l y m a d e o n the basis of e m p i r i c a l correlations, c o m p a r i s o n w i t h the mass spectra of s t a n d a r d m a t e r i a l s , or d e t a i l e d studies of i o n d e c o m p o sition mechanisms.
T h e theories a n d p r i n c i p l e s g o v e r n i n g the i n t e r p r e -
t a t i o n of mass spectra h a v e b e e n discussed i n m a n y reference texts a n d comprehensive reviews ( J , 2 ) . T h e p o t e n t i a l u t i l i t y of mass s p e c t r o m e t r y i n e v a l u a t i n g the m e t a b o l i c p a t h w a y s of p e s t i c i d a l c h e m i c a l s b y p r o v i d i n g the s t r u c t u r a l i d e n t i t y of metabolites w a s stated as e a r l y as 1962 i n a r e v i e w b y G u n t h e r M o r e r e c e n t l y , several r e v i e w s (4, 5, 6)
(3).
h a v e c o n s i d e r e d the r o l e of mass
s p e c t r o m e t r y i n c h e m i c a l s t r u c t u r e evaluations w i t h s p e c i a l reference to p e s t i c i d e r e s i d u e analysis. C o r r e l a t i o n s of mass s p e c t r a l f r a g m e n t a t i o n p a t h w a y s w i t h the s t r u c ture of pesticides are r e q u i r e d to o b t a i n b a c k g r o u n d i n f o r m a t i o n u s e f u l i n the i n t e r p r e t a t i o n of the mass spectra of u n k n o w n p e s t i c i d e m e t a b o l i t e s a n d other c o n v e r s i o n p r o d u c t s . F o r t u n a t e l y , the c h e m i c a l m o d e s of f r a g m e n t a t i o n of a l a r g e n u m b e r of p e s t i c i d a l c o m p o u n d s h a v e b e e n determined
(7).
T h i s i n f o r m a t i o n is p a r t i c u l a r l y u s e f u l i n those instances
w h e n o n l y m i c r o g r a m q u a n t i t i e s of m a t e r i a l s are a v a i l a b l e for c h a r a c t e r i z a t i o n ; mass s p e c t r o m e t r y offers one of the best i n s t r u m e n t a l a p proaches i n v i e w of the l o w s e n s i t i v i t y of s u c h c o m p l e m e n t a r y t e c h n i q u e s as n u c l e a r m a g n e t i c resonance spectrometry.
T h e investigator may then
r e l y o n this single source of i n f o r m a t i o n for a l l the s t r u c t u r a l d a t a . A n a d d i t i o n a l a d v a n t a g e of the mass s p e c t r o m e t r i c a p p r o a c h is the a v a i l a b i l i t y of c o m b i n e d gas c h r o m a t o g r a p h - m a s s spectrometer instruments w h i c h p e r m i t h i g h sensitivity analyses of m u l t i c o m p o n e n t m i x t u r e s . M a s s s p e c t r o m e t r y , of course, has b e e n w i d e l y e m p l o y e d as a n i n d i v i d u a l t e c h n i q u e for analysis of residues a n d metabolites i s o l a t e d b y c o n v e n t i o n a l separation t e c h n i q u e s s u c h as t h i n - l a y e r , l i q u i d , p a p e r , g a s - l i q u i d , a n d
Biros; Pesticides Identification Advances in Chemistry; American Chemical Society: Washington, DC, 1971.
134
PESTICIDES
column chromatography.
IDENTIFICATION
I n these instances, s p e c i a l i z e d t e c h n i q u e s are
e m p l o y e d f o r the efficient t r a p p i n g a n d c o l l e c t i o n of i n d i v i d u a l gas c h r o m a t o g r a p h i c peaks or t h i n - l a y e r c h r o m a t o g r a p h i c spots for
subsequent
mass s p e c t r a l analysis b y s t a n d a r d s a m p l e v a c u u m i n t r o d u c t i o n m e t h o d s . R e c e n t reports h a v e f o c u s e d a t t e n t i o n o n the n e e d for c o n f i r m i n g p e s t i c i d e residues, p a r t i c u l a r l y those i s o l a t e d f r o m e n v i r o n m e n t a l samples (8, 9 ) , a n d f o r c h e m i c a l i d e n t i t y i n u l t r a m i c r o a n a l y s i s i n g e n e r a l . O f t h e s p e c t r o m e t r i c t e c h n i q u e s a v a i l a b l e for c o n f i r m a t i o n of residues, the c o m b i n e d gas c h r o m a t o g r a p h i c - m a s s s p e c t r o m e t r i c a p p r o a c h affords
many
advantages i n c l u d i n g r e l a t i v e l y h i g h s e n s i t i v i t y , e l i m i n a t i o n of the necesDownloaded by FUDAN UNIV on January 4, 2017 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0104.ch009
sity for i s o l a t i n g m i n u t e q u a n t i t i e s of p u r e samples w i t h s t a n d a r d c h r o m a t o g r a p h i c m e t h o d s , a n d a greater c e r t a i n t y i n i d e n t i f i c a t i o n of a n e l u t e d c o m p o n e n t t h a n that a c h i e v e d b y c o n v e n t i o n a l detector response c h a r a c teristics a n d gas c h r o m a t o g r a p h i c r e t e n t i o n times alone.
B e c a u s e of the
definitive s t r u c t u r a l i n f o r m a t i o n o b t a i n e d b y mass s p e c t r o m e t r y , p r o p e r use of the t e c h n i q u e w o u l d o b v i a t e t h e necessity for the a p p l i c a t i o n of t w o or m o r e different c a r e f u l l y selected t e c h n i q u e s for c h a r a c t e r i z i n g p e s t i c i d e residues
(8).
A n a d d i t i o n a l o b v i o u s a d v a n t a g e w o u l d b e the c h a r a c t e r i z a t i o n a n d i d e n t i f i c a t i o n of f r e q u e n t l y e n c o u n t e r e d , u n k n o w n c o m p o n e n t s of extracts, w h i c h m a y represent p e s t i c i d a l m e t a b o l i t e s , p h o t o c h e m i c a l a n d
other
" w e a t h e r e d " residues, n o n p e s t i c i d a l c h e m i c a l p o l l u t a n t s , or co-extractive interferences w h o s e i d e n t i t y m a y be r e q u i r e d . T h i s r e p o r t presents a d i s c u s s i o n of recent a p p l i c a t i o n s of c o m b i n e d gas c h r o m a t o g r a p h y - m a s s s p e c t r o m e t r y to analysis of p e s t i c i d e residues isolated from h u m a n , animal, a n d environmental media. Emphasis w i l l be p l a c e d o n a n a l y t i c a l t e c h n i q u e s , r e l a t e d gas c h r o m a t o g r a p h i c c o l u m n technology,
a n d analysis of f r a g m e n t a t i o n p a t h w a y s p e r t i n e n t to
the
i d e n t i f i c a t i o n of p e s t i c i d e residues.
Experimental T h e mass s p e c t r o m e t r i c analyses i n i t i a l l y r e p o r t e d i n this c o m m u n i c a t i o n w e r e p e r f o r m e d w i t h a d o u b l e f o c u s i n g l o w r e s o l u t i o n mass spect r o m e t e r ; M o d e l 270, P e r k i n - E l m e r C o r p . , N o r w a l k , C o n n . , c o u p l e d t h r o u g h a W a t s o n - B i e m a n n t y p e of m o l e c u l a r separator w i t h a gas c h r o m a t o g r a p h i c system. ( C o m m e r c i a l sources a n d t r a d e names are p r o v i d e d for i d e n t i f i c a t i o n o n l y . T h e i r m e n t i o n does not constitute endorsem e n t b y the P u b l i c H e a l t h S e r v i c e or b y the U . S. D e p t . of H e a l t h , Education, and Welfare.) C o i l e d glass gas c h r o m a t o g r a p h i c c o l u m n s w e r e e m p l o y e d except w h e r e o t h e r w i s e i n d i c a t e d . P r o g r a m m e d t e m p e r a t u r e analyses w e r e m a d e w i t h the i n i t i a l a n d final o v e n c o n d i t i o n s as w e l l as t h e p r o g r a m rate g i v e n i n the discussion. T h e m o l e c u l a r separator a n d gas i n l e t temperatures w e r e m a i n t a i n e d at 200° a n d 2 1 0 ° C , r e s p e c t i v e l y . A l l mass spectra w e r e
Biros; Pesticides Identification Advances in Chemistry; American Chemical Society: Washington, DC, 1971.
9.
BIROS
Gas Chromatography—Mass
135
Spectrometry
r e c o r d e d at 80 e V e l e c t r o n e n e r g y w i t h 2400 V a c c e l e r a t i n g v o l t a g e ; t h e filament e m i s s i o n c u r r e n t w a s 100 /*a. C h r o m a t o g r a m s w e r e r e c o r d e d f r o m the t o t a l i o n c u r r e n t m o n i t o r l o c a t e d b e t w e e n t h e electrostatic a n d m a g n e t i c a n a l y z e r sectors. H e l i u m c a r r i e r gas w a s a p p r o x i m a t e l y 10 m l p e r m i n for the p a c k e d c h r o m a t o g r a p h i c c o l u m n s a n d a p p r o x i m a t e l y 2 m l p e r m i n for the c a p i l l a r y c o l u m n s . Injector t e m p e r a t u r e w a s m a i n t a i n e d at 165 ° C . M a s s s p e c t r a w e r e s c a n n e d m a g n e t i c a l l y over t h e r a n g e of interest at a rate of either 3 or 10 sec p e r decade. T h e c o m b i n e d gas c h r o m a t o g r a p h - m a s s spectrometer system has b e e n d e s c r i b e d i n m o r e d e t a i l elsewhere ( 1 0 ) .
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Phenolic Residues C o n f i r m a t i o n of the i d e n t i t y of p e n t a c h l o r o p h e n o l ( P C P ) i n samples of h u m a n b l o o d , u r i n e , tissue, c l o t h i n g , a n d b e d d i n g m a t e r i a l s has b e e n reported
(II),
utilizing combined
gas c h r o m a t o g r a p h i c - m a s s
spectro-
m e t r i c analysis of hexane extracts of these substrates. T h i s r e p o r t c o n s t i t u t e d a d e t a i l e d a c c o u n t of t h e a n a l y t i c a l aspects o f a n e p i d e m i c of i n f a n t deaths associated w i t h the i n g e s t i o n of this c o m p o u n d , w h i c h w a s the a c t i v e i n g r e d i e n t of a m i l d e w p r e v e n t a t i v e , the suspected source of exposure.
A n a l y s i s of the hexane extracts w a s
p e r f o r m e d o n a 1.0 m X 2.5 m m c o i l e d glass gas c h r o m a t o g r a p h i c c o l u m n packed with 3 %
D E G S and 2 %
concentrated phosphoric acid.
Under
these c o n d i t i o n s , analysis c o u l d b e p e r f o r m e d w i t h o u t p r i o r d e r i v a t i z a t i o n of the P C P . A l l s a m p l e spectra gave f o u r i d e n t i c a l m a j o r ions w h e n c o m p a r e d w i t h P C P s t a n d a r d mass s p e c t r u m o b t a i n e d at 20 e V . T h u s , i d e n t i t y of the residues of P C P was c o n f i r m e d o n the basis of the o b s e r v a t i o n of t h e m o l e c u l a r i o n p e a k at m / e 264, a f r a g m e n t i o n of m / e 229, o w i n g t o e l i m i n a t i o n of a C I a t o m , a m a j o r f r a g m e n t r e s u l t i n g f r o m the c o n s e c u t i v e loss of H C 1 a n d C O at m / e 200, a n d finally a c h a r a c t e r i s t i c f r a g m e n t of
0
2
4
6
8
1
0
1
2
1
4
0
2
4
6
8
H>
U
ft
Figure I . Total ion current chromatograms of (A) standard 1-naphthyl chloroacetate (1 fig) and (B) 1-naphthyl chloroacetate isolated from human urine Programmed temperature conditions: two minutes at 165°C, isothermal at 185°C
to 185°C
at
Biros; Pesticides Identification Advances in Chemistry; American Chemical Society: Washington, DC, 1971.
5°C/min,
136
PESTICIDES
IDENTIFICATION
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-CHCICO
B
Figure
m/e
2.
Electron
/.89
impact-induced fragmentation chloroacetate
scheme for
1-naphthyl
165 u n d o u b t e d l y r e s u l t i n g f r o m loss of a c h l o r i n e a t o m f r o m the
m / e 200 i o n . D u r i n g the course o f a n a l y t i c a l m e t h o d studies i n v o l v i n g t h e d e v e l o p m e n t of p r o c e d u r e s for the d e t e c t i o n a n d q u a n t i t a t i o n of 1 - n a p h t h o l , a m a j o r m e t a b o l i t e of c a r b a r y l , i n h u m a n a n d a n i m a l u r i n e ( 1 2 ) ,
the
d e r i v a t i z e d r e s i d u e , 1 - n a p h t h y l chloroacetate, w a s c o n f i r m e d b y gas c h r o matography-mass spectrometry. l a t i o n of
T h e a n a l y t i c a l p r o c e d u r e f o r t h e iso-
1 - n a p h t h o l residues i n v o l v e d a c i d h y d r o l y s i s of
1-naphthol
conjugates i n t h e u r i n e , b e n z e n e e x t r a c t i o n , d e r i v a t i z a t i o n w i t h c h l o r o acetic a n h y d r i d e a n d p y r i d i n e , a n d r e m o v a l of i n t e r f e r i n g m a t e r i a l i n urine b y c o l u m n chromatography using silica gel. 1 - N a p h t h y l chloroacetate w a s e l u t e d w i t h 6 0 % b e n z e n e - h e x a n e .
P r o g r a m m e d t e m p e r a t u r e gas
c h r o m a t o g r a p h i c analysis w i t h a c o i l e d glass c o l u m n , 4 ft X 1 / 8 i n c h o.d., p a c k e d w i t h 2 % S E - 3 0 o n 6 0 / 8 0 m e s h G a s C h r o m Q , w a s u s e d to c o n f i r m residues of 1 - n a p h t h o l as the chloroacetate ester i n h u m a n u r i n e of i n d i v i d u a l s o c c u p a t i o n a l l y e x p o s e d to c a r b a r y l ( F i g u r e 1 ) .
D i a g n o s t i c mass
s p e c t r a l peaks o b s e r v e d for 1 - n a p h t h y l chloroacetate i n c l u d e d the m o l e c u l a r i o n of m / e 220, a base p e a k f r a g m e n t w h i c h m a y b e f o r m u l a t e d as a 1 - n a p h t h o l r a d i c a l i o n ( o r a n i s o m e r ) f o u n d at m / e
144 a r i s i n g f r o m
h y d r o g e n r e a r r a n g e m e n t a n d loss of — C H C I C O , a r e l a t i v e l y w e a k p e a k at m / e 127 f o r m e d either b y e l i m i n a t i o n of — O C O C H C l f r o m t h e m o 2
l e c u l a r i o n a n d / o r b y loss of w a t e r f r o m t h e m / e 145 f r a g m e n t , a n d
finally,
other peaks c h a r a c t e r i s t i c of the f r a g m e n t a t i o n of 1 - n a p h t h o l at m / e
Biros; Pesticides Identification Advances in Chemistry; American Chemical Society: Washington, DC, 1971.
115
9.
BIROS
Gas Chromatography—Mass
137
Spectrometry
a n d m / e 116 a r i s i n g f r o m e x p u l s i o n of C H O a n d C O , r e s p e c t i v e l y , a n d a p e a k at m / e 89 p r e s u m a b l y r e s u l t i n g f r o m a C H 7
5
+
ion formed b y the
loss of acetylene ( C H ) f r o m t h e m / e 115 f r a g m e n t ( F i g u r e 2 ) . 2
2
Organophosphorus Pesticides I n c o n n e c t i o n w i t h the d e v e l o p m e n t o f a n a n a l y t i c a l m e t h o d f o r t h e d e t e r m i n a t i o n of o r g a n o p h o s p h o r u s
(13)
pesticides i n h u m a n b l o o d
a n d u r i n e , mass s p e c t r a l c o n f i r m a t i o n of a series of m e t h y l a t e d a n d e t h y l a t e d d e r i v a t i v e s of t h e h y d r o l y t i c a n d m e t a b o l i c p r o d u c t s of these i n s e c t i Downloaded by FUDAN UNIV on January 4, 2017 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0104.ch009
cides w a s r e q u i r e d . T h e u r i n e o f a n i n d i v i d u a l o c c u p a t i o n a l l y
exposed
to p a r a t h i o n w a s e x t r a c t e d w i t h a 1:1 ( v / v ) solvent m i x t u r e of a c e t o n i t r i l e a n d d i e t h y l ether. S i m u l t a n e o u s l y , t h e i n t a c t o r g a n o p h o s p h o r u s i n s e c t i cides w e r e h y d r o l y z e d b y a d d i n g a p o r t i o n o f 5 N h y d r o c h l o r i c a c i d t o
O
2
4
6
8
K>
12
O
2
4
6
8
10
12
14
14
16
Figure 3. Total ion current chromatograms of (A) standard mixture of methylated dialkylphosphateSy phosphorothioates, and phosphorodithioates (2 fig) and (B) human urine extract containing methylated hydrolytic and metabolic products of parathion Programmed temperature conditions: five minutes at 75°C, to 120°C at 5°C/min, isothermal at 120°C
Biros; Pesticides Identification Advances in Chemistry; American Chemical Society: Washington, DC, 1971.
138
PESTICIDES
IDENTIFICATION
the u r i n e . F o l l o w i n g e x t r a c t i o n , the o r g a n i c l a y e r w a s c o n c e n t r a t e d a n d t h e residues i n s o l u t i o n d e r i v a t i z e d b y means of d i a z o m e t h a n e . T h e m i n e extract was t h e n s u b j e c t e d to a c l e a n - u p p r o c e d u r e e m p l o y i n g s i l i c a g e l c o l u m n c h r o m a t o g r a p h y (14),
and benzene ( I ) , 4 0 %
e t h y l acetate i n
b e n z e n e ( I I ) , a n d 8 0 % e t h y l acetate i n b e n z e n e ( I I I ) as e l u t i n g solvents. C o n c e n t r a t i o n of t h e c h r o m a t o g r a p h i c c o l u m n eluents p r o v i d e d the u r i n e extract w h i c h w a s subjected to p r o g r a m m e d t e m p e r a t u r e analyses o n a c o i l e d glass c a p i l l a r y c o l u m n , 110 ft X 0.025 i n c h i . d . , c o a t e d w i t h V e r s a m i d 900 c o n t a i n i n g 5 % I g e p a l C O - 8 8 0 . T h e r e s u l t i n g t o t a l i o n c u r r e n t c h r o m a t o g r a m is s h o w n i n F i g u r e 3. A l s o i l l u s t r a t e d is the t o t a l i o n c u r r e n t Downloaded by FUDAN UNIV on January 4, 2017 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0104.ch009
trace o b t a i n e d b y analysis of a series of m e t h y l a t e d d i a l k y l
phosphate
standard materials. T h e m e t a b o l i c a n d / o r h y d r o l y t i c p r o d u c t s of p a r a t h i o n e n c o u n t e r e d as residues i n t h e u r i n e i n c l u d e b o t h d i e t h y l p h o s p h o r i c a c i d a n d d i e t h y l p h o s p h o r o t h i o i c a c i d , most p r o b a b l y as t h e i r salts ( p o t a s s i u m or s o d i u m ) . D e r i v a t i z a t i o n of these residues w i t h d i a z o m e t h a n e w o u l d result i n the f o r m a t i o n of three t r i a l k y l phosphate c o m p o u n d s , n a m e l y , 0 , 0 - d i e t h y l O-methyl thionate
phosphate (DEMMTP),
(DEMMPTh).
(DEMMP), and
0,0-diethyl
0,0-diethyl
O-methyl
S-methyl
phosphoro-
phosphorothiolate
E a r l i e r ( 1 5 ) , it h a d b e e n s h o w n b y c o m b i n e d gas c h r o -
m a t o g r a p h y - m a s s s p e c t r o m e t r y a n d other a n a l y t i c a l d a t a that a l a t e r e l u t i n g major p r o d u c t
(ca.
85%)
of the m e t h y l a t i o n of d i e t h y l p h o s -
p h o r o t h i o i c a c i d f o r m e d u n d e r the c o n d i t i o n s of the a n a l y t i c a l m e t h o d was
DEMMPTh,
DEMMTP.
and
the
minor
product
formed
(ca.
15%)
was
A c c o r d i n g l y , a l l three t r i a l k y l phosphates w e r e o b s e r v e d a n d
c o n f i r m e d b y mass s p e c t r o m e t r y i n the analysis of the h u m a n u r i n e extract. Sufficient i n t e r n a l b o n d energy differences are associated w i t h the i s o m e r i c structures D E M M P T h
and D E M M T P
that q u a l i t a t i v e l y a n d
q u a n t i t a t i v e l y d i s s i m i l a r f r a g m e n t a t i o n patterns are o b s e r v e d for
both
isomers as c a n b e seen f r o m t h e mass spectra of these c o m p o u n d s s h o w n i n F i g u r e 4. T h e mass s p e c t r u m of D E M M T P is c h a r a c t e r i z e d b y b o t h p h o s p h o r u s - o x y g e n a n d c a r b o n - o x y g e n b o n d fission as e v i d e n c e d b y the f o r m a t i o n of f r a g m e n t ions at m / e 140 ( M - G > H 0 ) a n d m / e 156 4
(M-C H ). 2
4
S i n g l e a n d d o u b l e h y d r o g e n rearrangements a c c o m p a n y the loss of the e t h y l substituent. S u b s e q u e n t f r a g m e n t a t i o n reactions n o t e d w e r e s i m i l a r b o n d fission i n v o l v i n g e l i m i n a t i o n of the r e m a i n i n g a l k y l substituents as a n e t h y l e n e m o l e c u l e or a n e t h o x y l r a d i c a l . A r e l a t i v e l y intense peak, for e x a m p l e , of m / e 111 [ C H * O P ( : S ) - O H ] , appears to b e f o r m e d b y c o n +
secutive loss of ethylene ( t o f o r m the m / e 156 i o n ) a n d a n e t h o x y l s u b stituent f r o m t h e m o l e c u l a r i o n as e v i d e n c e d b y metastable peaks at m / e 132.26 a n d m / e 78.98 o b s e r v e d for these t w o processes. Significant f r a g m e n t ions w e r e also f o r m e d t h r o u g h loss of b o t h s u l f u r atoms or s u l f h y d r y l
Biros; Pesticides Identification Advances in Chemistry; American Chemical Society: Washington, DC, 1971.
9.
BIROS
Gas Chromatography—Mass
139
Spectrometry CH C M
100
t
79 . CH^OPOH
/
N
CH CH 0 {
90
2
/
,SHI HO'
Figure 5. Electron impact-induced fragmentation scheme for O O-diethyl O-methyl phosphorothionate (DEMMTP) y
C¥H 0 j>
T
N /
2
CH CH 0 3
2
-C H 0 2
.»
/
m/t 184 2
3
2
3
3
HO
~]t
HO' m/t 112 -C2H4
CH CH 0-P 3
2
2
m/t 138 CH CH 0^ / / " | P-SCH HO' m/t 156
4
1 3
2
CH CH 0. "It >-0H« CH CH 0'
4
-C H 2
CH CH0-P-SCHj 0-P-SCH ' n / i 140 3
-C H
-it
4
•/•
3
^0H
110
2
x
3
CH0 2
5
0 H0-P-SCH
3
m/t 111
m/t 126
CH CH=0-P=0 M/I 91 3
Figure
6. Electron impact-induced fragmentation scheme for ethyl S-methyl phosphorothiolate (DEMMPTh)
0,0-di-
Biros; Pesticides Identification Advances in Chemistry; American Chemical Society: Washington, DC, 1971.
u
9.
BIROS
Gas Chromatography—Mass
compounds process A
141
Spectrometry
h a v e also b e e n o b s e r v e d
to u n d e r g o
this r e a r r a n g e m e n t
(18). somewhat more complex dissociative scheme was observed
DEMMPTh
(Figure 6).
T h e molecular i o n of this c o m p o u n d
poses b y fission of a c a r b o n - o x y g e n b o n d ( m / e
156, M - C H ) , 2
for
decom-
4
phos-
p h o r u s - o x y g e n b o n d ( m / e 140, M - C H 0 ) , a n d the p h o s p h o r u s - s u l f u r 2
b o n d ( m / e 138, M - C H S ) . 2
4
T h e s t r u c t u r e of t h e m / e 156 f r a g m e n t ( F i g -
ure 6) permits a subsequent identical triple decomposition scheme to y i e l d fragments o f m / e 128 ( 1 5 6 - C H ) , m / e 109 ( 1 5 6 - C H S ) , a n d m / e 2
111 ( 1 5 6 - C H 0 ) . 2
5
4
3
A n o t h e r significant r e a c t i o n o b s e r v e d w a s the e l i m i -
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n a t i o n o f w a t e r w h i c h , for e x a m p l e , l e d to t h e f o r m a t i o n of a n intense i o n f r a g m e n t at m / e 91 ( 1 0 9 - H O ) , c h a r a c t e r i s t i c o f t h e e l e c t r o n i m p a c t 2
f r a g m e n t a t i o n of esters. T h e d e c o m p o s i t i o n of D E M M P u n d e r e l e c t r o n i m p a c t results i n t h e f o r m a t i o n of significant f r a g m e n t ions of m / e 141, m / e 113, a n d m / e 9 5 , a c c o r d i n g to the d i s s o c i a t i v e s c h e m e i l l u s t r a t e d i n F i g u r e 7.
Organochlorine Pesticides C h l o r i n a t e d h y d r o c a r b o n p e s t i c i d e residues i n h u m a n a d i p o s e tissue a n d l i v e r tissue samples h a v e b e e n i d e n t i f i e d b y mass s p e c t r o m e t r y c o u pled with
gas c h r o m a t o g r a p h y .
A
g e n e r a l , extensive
extraction a n d
c l e a n u p p r o c e d u r e a d a p t e d f r o m e x i s t i n g m e t h o d s w a s u s e d to isolate
Figure
7.
Electron
impact-induced fragmentation scheme for O-methyl phosphate (DEMMP)
O O-diethyl
Biros; Pesticides Identification Advances in Chemistry; American Chemical Society: Washington, DC, 1971.
y
142
PESTICIDES
IDENTIFICATION
a n d p u r i f y the o r g a n o c h l o r i n e residues. T h e tissue samples w e r e e x t r a c t e d w i t h a c e t o n i t r i l e o r p e t r o l e u m ether, f o l l o w e d b y l i q u i d - l i q u i d p a r t i t i o n i n g o f t h e extract b e t w e e n hexane or p e t r o l e u m ether a n d a c e t o n i t r i l e . C o l u m n chromatography and cleanup on Florisil provided t w o individual fractions ( 1 9 ) , w h i c h w e r e t h e n c o n c e n t r a t e d a n d subjected t o f u r t h e r c l e a n u p a n d p e s t i c i d e r e s i d u e f r a c t i o n a t i o n b y means o f s i l i c a g e l c o l u m n c h r o m a t o g r a p h y e m p l o y i n g a p u b l i s h e d p r o c e d u r e (20).
T h e pesticide
residues w e r e separated b y t h e s i l i c a g e l c h r o m a t o g r a p h i c step i n t o t w o fractions w i t h sufficient r e s o l u t i o n o n t h e gas c h r o m a t o g r a p h i c c o l u m n for t h e i d e n t i f i c a t i o n o f seven p e s t i c i d e residues f r o m t h e a d i p o s e tissue Downloaded by FUDAN UNIV on January 4, 2017 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0104.ch009
s a m p l e a n d six p e s t i c i d e residues f r o m t h e l i v e r tissue sample. i n c l u d e d : /?- a n d y - h e x a c h l o r o c y c l o h e x a n e
(HCH),
These
1,4,5,6,7,8,8-hepta-
chloro-2,3-epoxy-2,3,3a,4,7,7a-hexahydro-4,7-methanoindene
(heptachlor
e p o x i d e ) , 1,2,3,4,10,10 - h e x a c h l o r o - 6,7 - e p o x y - l,4,4a,5,6,7,8,8a - o c t a h y d r o 1,4 - endo,exo - 5,7 - d i m e t h a n o n a p h t h a l e n e p h e n y l ) -1,1-dichloroethylene
(dieldrin),
(p,p'-DDE),
2,2 - b i s (p - c h l o r o -
2,2-bis ( p - c h l o r o p h e n y l ) -1,1-
dichloroethane ( p , p ' - D D D ) , a n d l,l-bis(p-chlorophenyl)-2,2,2-trichloroethane
a n d 1- ( o - c h l o r o p h e n y l ) - 1 - ( p - c h l o r o p h e n y l ) -2,2,2-trichloroethane
( p , p ' - D D T a n d o , p ' - D D T , respectively). T h e concentration of the pesticides i n tissue r a n g e d f r o m 0.073 to 28.7 p p m . G a s c h r o m a t o g r a p h i c separations w e r e p e r f o r m e d o n a 4 ft X 1 / 8 i n c h o.d. c o i l e d glass c o l u m n packed w i t h 3 % O V - 2 1 0 on 80/100 mesh Chromosorb W ( H P ) . F i g u r e 8 illustrates t h e t o t a l i o n c u r r e n t c h r o m a t o g r a m o b t a i n e d b y analysis of t h e a d i p o s e tissue extract r e p r e s e n t i n g 5 grams of tissue. I n s t r u m e n t a l s e n s i t i v i t y w a s sufficient t o i d e n t i f y residues at c o n c e n trations of 0.30 p p m . C o m p u t e r e n h a n c e m e n t t e c h n i q u e s (21) p e r m i t t e d i d e n t i f i c a t i o n of h e p t a c h l o r e p o x i d e residues at a l e v e l of 0.073 p p m .
__,
0
,
2
-
l
4
6
8
i
10 12
14
MINUTES
~6
2
4
6
8
10
12 14 MINUTES
Figure 8. Total ion current chromatograms of extracts containing organochh rine pesticide residues isolated from adipose tissue (A) fraction 1 and (B) fraction 2 Programmed temperature conditions: one minute at 150°C, min, isothermal at 200°C
to 200°C
at 5°CI
Biros; Pesticides Identification Advances in Chemistry; American Chemical Society: Washington, DC, 1971.
9.
BIROS
Gas Chromatography—Mass
Spectrometry
143
D e f i n i t i v e c o n f i r m a t i o n of p e s t i c i d e residues w a s o b t a i n e d b y c o m p a r i s o n of p a r e n t a n d f r a g m e n t i o n intensities a n d mass n u m b e r s of e l u t e d p e s t i cides a n d reference pesticides. T a b l e I lists the residues e n c o u n t e r e d a n d t h e mass n u m b e r s a n d intensities of t h e c h a r a c t e r i s t i c fragments e m p l o y e d for i d e n t i f i c a t i o n i n the a d i p o s e tissue s a m p l e . T h e mass s p e c t r a l f r a g m e n t a t i o n patterns for a l l t h e c o m p o u n d s i n c l u d e d i n T a b l e I w i t h t h e e x c e p t i o n of / 2 - H C H h a v e b e e n a d e q u a t e l y d i s c u s s e d b y other i n v e s t i -
Downloaded by FUDAN UNIV on January 4, 2017 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0104.ch009
gators
(7).
Table I. Gas Chromatographic Peak Identities and Characteristic Mass Spectral Fragments and Intensities of Some Organochlorine Pesticide Residues Isolated From H u m a n Adipose Tissue Peak
No.
Pesticide p,p'-DDE
1
2
3
o,p'-DDT
p,p'-DDT
£-HCH
5
6
H e p t a c h l o r epoxide
Dieldrin
p,p'-DDD
Characteristic Mass Spectral and Intensities
Peaks
m / e 246 ( 1 0 0 % ) , 318 ( 8 3 % ) , 316 ( M o l e c u l a r i o n , 6 6 % ) , 248 ( 5 8 % ) , 320 ( 4 1 % ) , 176 ( 4 1 % ) , 210 ( 1 6 % ) . m / e 2 3 5 ( 1 0 0 % ) , 2 3 7 ( 7 0 % ) , 165 ( 4 0 % ) , 75 ( 2 0 % ) , 199 ( 1 8 % ) , 246 ( 1 8 % ) , 352 (Molecular ion, 3%). m / e 235 ( 1 0 0 % ) , 237 ( 7 2 % ) , 165 ( 4 8 % ) , 75 ( 2 2 % ) , 50 ( 1 8 % ) , 51 ( 9 % ) , 352 (Molecular ion, 2 % ) . m / e 109 ( 1 0 0 % ) , 2 1 9 ( 8 5 % ) , 181 ( 7 8 % ) , 183 ( 8 0 % ) , 111 ( 6 2 % ) , 193 ( 6 2 % ) , 288 ( M o l e c u l a r i o n , 4 % ) . m / e 81 ( 1 0 0 % ) , 353 ( 8 4 % ) , 355 ( 7 6 % ) , 351 ( 4 8 % ) , 357 ( 3 5 % ) , 237 ( 3 3 % ) , 386 ( M o l e c u l a r i o n , 8 % ) . m / e 79 ( 1 0 0 % ) , 108 ( 1 9 % ) , 263 ( 1 8 % ) , 277 ( 1 9 % ) , 279 ( 1 6 % ) , 345 ( 7 % ) , 378 (Molecular ion, 3%). m / e 2 3 5 ( 1 0 0 % ) , 2 3 7 ( 6 6 % ) , 165 ( 5 8 % ) , 75 ( 2 1 % ) , 82 ( 1 6 % ) , 88 ( 1 6 % ) , 318 (Molecular ion, 5%).
S o m e difficulties w e r e e n c o u n t e r e d i n the absolute c o n f i r m a t i o n of c o n f i g u r a t i o n a l a n d s t r u c t u r a l isomers w h e r e mass s p e c t r a l p e a k intensities a n d mass values of c h a r a c t e r i s t i c f r a g m e n t ions w e r e too s i m i l a r u n d e r the c o n d i t i o n s of the analysis. I n those instances, h o w e v e r , i d e n t i f i c a t i o n w a s r e a d i l y m a d e o n the basis of gas c h r o m a t o g r a p h i c r e t e n t i o n t i m e d a t a . T h e e s t i m a t e d l o w e r l i m i t of d e t e c t a b i l i t y of o r g a n o c h l o r i n e p e s t i c i d e residues b y this a n a l y t i c a l scheme a n d i n s t r u m e n t c o n f i g u r a t i o n w a s d e t e r m i n e d to b e a p p r o x i m a t e l y 0.05 to 0.1 p p m .
Biros; Pesticides Identification Advances in Chemistry; American Chemical Society: Washington, DC, 1971.
144
PESTICIDES
IDENTIFICATION
Herbicide Residues C o m b i n e d m e t h o d o l o g y a n d m e t a b o l i s m studies i n v o l v i n g t h e h e r b i cides 2,4-dichlorophenoxyacetic a c i d ( 2 , 4 - D ) a n d 2,4,5-trichlorophenoxya c e t i c a c i d ( 2 , 4 , 5 - T ) f u r n i s h e d a n o p p o r t u n i t y f o r the a p p l i c a t i o n o f the gas c h r o m a t o g r a p h i o - m a s s s p e c t r o m e t r i c t e c h n i q u e t o the c o n f i r m a t i o n of d e r i v a t i z e d i n t a c t residues a n d m e t a b o l i t e s a n d t h e s t r u c t u r a l c h a r a c terization o f u n k n o w n metabolites isolated from rat urine. T h e analytical technique, briefly summarized, involves a c i d hydrolysis of phenolic conjugates, e x t r a c t i o n o f free p h e n o l s a n d acids w i t h b e n z e n e , extract c o n -
Downloaded by FUDAN UNIV on January 4, 2017 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0104.ch009
centration
and
derivatization with
diazoethane,
and
chromatographic cleanup a n d separation b y silica gel employing benzene-hexane as eluents ( 2 2 ) .
finally,
column
chromatography
a n d b e n z e n e - e t h y l acetate solvent
systems
P r o g r a m m e d t e m p e r a t u r e gas c h r o m a t o g r a p h i c analyses
of t h e u r i n e extracts w i t h a 7 ft X 1 / 8 i n c h o.d. a l u m i n u m c o l u m n p a c k e d w i t h 2 0 % O V - 1 0 1 on 6 0 / 8 0 mesh Gas C h r o m Q p r o v i d e d the total ion c u r r e n t c h r o m a t o g r a m s s h o w n i n F i g u r e 9. T h e mass s p e c t r a l f r a g m e n t a t i o n d a t a w e r e u s e d t o c o n f i r m the presence o f 2 , 4 - D a n d 2,4,5-T as t h e e t h y l esters a n d t r i c h l o r o p h e n o l ( T C P ) as the e t h y l ether i n the s e v e r a l
"12.4.5-TCP
•
2
4
•
t
M
1
2
W
t
t
t
«
2
*
2
2
2
4
Figure 9. Total ion current chromatograms of animal urine extracts containing (A) 2,4-D, (B) 2,4,5-T, and (C) 2,4,5-trichlorophenol and an isomer of trichlorodihydroxybenzene, peak 2 (as ethyl esters and mono- and diethyl ethers, respectively) Programmed temperature conditions: five minutes at 180° C, to 220° C at 5°C/min, isothermal at 220°C
Biros; Pesticides Identification Advances in Chemistry; American Chemical Society: Washington, DC, 1971.
9.
BIROS
Gas Chromatography—Mass
Spectrometry
^
^
145
OCRjCOOCijClj
• / • 213
— I -C«2=CH 2
CI
• / , 24S
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-COOCt • / • "5
»
C H 0Cl7 6
•/
4
t
162
Figure 10. Electron impact-induced fragmentation scheme for 2,4-dichlorophenoxyacetic acid, ethyl ester analyses p e r f o r m e d .
T h e diagnostic fragmentation pattern for 2,4-D
e t h y l ester i n c l u d e d peaks at m / e 248, m o l e c u l a r i o n ; m / e 213, e l i m i n a t i o n of a C I a t o m ; m / e 185, c o n s e c u t i v e e l i m i n a t i o n o f a C I a t o m a n d e t h y l e n e ( C H ) f r o m t h e p a r e n t i o n species; m / e 175, loss o f t h e c a r b o e t h o x y l 2
4
f u n c t i o n f r o m t h e m o l e c u l a r i o n ; a n d finally, a p e a k a t m / e 162 p r e s u m ably arising from a dichlorophenol or dichlorocyclohexadienone-type
rad-
i c a l i o n ( F i g u r e 1 0 ) . A n analogous f r a g m e n t a t i o n s c h e m e w a s o b s e r v e d for t h e e t h y l ester o f 2,4,5-T: m / e 282, m o l e c u l a r i o n ; m / e 2 4 7 , loss o f c h l o r i n e a t o m ; m / e 219, consecutive e l i m i n a t i o n o f a c h l o r i n e a t o m a n d a m o l e c u l e o f e t h y l e n e f r o m t h e p a r e n t i o n ; m / e 209, d e c o m p o s i t i o n o f the p a r e n t i o n w i t h e l i m i n a t i o n o f t h e c a r b o e t h o x y l g r o u p ; a n d m/e
196, c o r r e s p o n d i n g
finally,
to a trichlorophenol or trichlorocyclohexa-
d i e n o n e - t y p e r a d i c a l i o n species. I n a d d i t i o n to t h e i n t a c t residues o f 2,4,5-T excreted i n t h e r a t u r i n e , mass s p e c t r a l e v i d e n c e w a s o b t a i n e d f o r t h e presence o f t h e m e t a b o l i t e T C P a n d a t r i c h l o r o d i h y d r o x y b e n z e n e isomer, w h i c h w e r e o b s e r v e d as t h e i r m o n o - a n d d i e t h y l ether d e r i v a t i v e s , r e s p e c t i v e l y . T h u s , T C P e t h y l ether d i s p l a y e d a m o l e c u l a r i o n at m / e 224 a n d c h a r a c t e r i s t i c fragments at m / e 196 a n d m / e 160 o w i n g to consecutive e l i m i n a t i o n o f e t h y l e n e a n d H C 1 f r o m t h e p a r e n t i o n . T h e f r a g m e n t a t i o n p a t t e r n o f 2,4,5-tric h l o r o p e n o l w a s o b s e r v e d t o e x h i b i t s i m i l a r b e h a v i o r b e l o w m / e 196.
Biros; Pesticides Identification Advances in Chemistry; American Chemical Society: Washington, DC, 1971.
146
•
PESTICIDES
J
4
6
8
10 U
M
16 O
minutes
20 22 24 2^
~0
2
4
6
8
IDENTIFICATION
10121416182022242628
minutes
Downloaded by FUDAN UNIV on January 4, 2017 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0104.ch009
Figure 11. Total ion current chromatograms of (A) standard Aroclor 1260 mixture of polychlorinated biphenyls. Programmed temperature analysis: two minutes at 200°C, to 230°C/min, isothermal at 230°C. (B) Human adipose tissue extract. Programmed temperature analysis: two minutes at 190°C, to 230°C at 5°C/min, isothermal at 230°C.
T h e mass s p e c t r a l e v i d e n c e f o r t h e t r i c h l o r o d i h y d r o x y b e n z e r r e d i e t h y l ether i n c l u d e d a m o l e c u l a r i o n o f m / e 286 a n d significant f r a g m e n t ions at m / e 240, m / e 212, a n d m / e 176 a r i s i n g f r o m the c o n s e c u t i v e e l i m i n a t i o n o f t w o m o l e c u l e s o f e t h y l e n e a n d one m o l e c u l e o f H C 1 .
Polychlorinated Biphenyls C o n f i r m a t o r y e v i d e n c e f o r the presence o f p o l y c h l o r i n a t e d b i p h e n y l s ( P C B ) i n fish, seabirds, c o n i f e r needles, a n d h u m a n d e p o t f a t has b e e n o b t a i n e d b y gas c h r o m a t o g r a p h y - m a s s s p e c t r o m e t r y (23, 24). T h e mass s p e c t r a l b e h a v i o r o f standards o f P C B m a t e r i a l s ( A r o c l o r 1254 a n d 1260) a n d P C B c o m p o u n d s i s o l a t e d f r o m h u m a n a d i p o s e tissue u t i l i z i n g s t a n d ard analytical methodology
(19) h a s b e e n r e p o r t e d r e c e n t l y ( 2 5 ) .
illustration of the total i o n current chromatogram
An
obtained b y pro-
g r a m m e d t e m p e r a t u r e analysis o f a m i x t u r e of s t a n d a r d P C B c o m p o u n d s ( A r o c l o r 1260) o n a 100 ft X 0.02 i n c h i . d . stainless steel c a p i l l a r y c o l u m n c o a t e d w i t h O V - 1 s i l i c o n e o i l is d e p i c t e d i n F i g u r e 11. M o s t c o m p o n e n t s of the A r o c l o r s t a n d a r d p r o d u c e d mass spectra w i t h m o l e c u l a r i o n groups of h i g h i n t e n s i t y . I n a d d i t i o n , t h e c h a r a c t e r i s t i c isotopic d i s t r i b u t i o n patterns c o r r e s p o n d i n g t o t h e n u m b e r o f c h l o r i n e atoms i n the p a r e n t i o n a n d c h l o r i n e - c o n t a i n i n g f r a g m e n t ions w e r e o b s e r v e d .
Other noteworthy
features o f t h e mass spectra w e r e t h e r e l a t i v e l y intense i o n fragments r e s u l t i n g f r o m consecutive loss o f c h l o r i n e atoms f r o m the p a r e n t i o n a n d the presence o f intense d o u b l y c h a r g e d fragments w i t h i n the mass spectra of most o f the P C B c o m p o u n d s .
T h u s , i t was possible to establish t h e
m o l e c u l a r w e i g h t a n d n u m b e r o f c h l o r i n e atoms associated w i t h major numbered component
i n t h e c h r o m a t o g r a m o f F i g u r e 11.
each The
s t a n d a r d A r o c l o r 1260, o n this basis, w a s s h o w n t o consist o f at least t w o p e n t a c h l o r o b i p h e n y l s , f o u r h e x a c h l o r o b i p h e n y l s , six h e p t a c h l o r o b i phenyls, t w o octachlorobiphenyls, t w o nonachlorobiphenyls, a n d decachlorobiphenyl.
Biros; Pesticides Identification Advances in Chemistry; American Chemical Society: Washington, DC, 1971.
9.
BIROS
Gas Chromatography—Mass
147
Spectrometry
A l t h o u g h i t w a s a p p a r e n t that several of the m a j o r peaks i n the c h r o m a t o g r a m of the s t a n d a r d P C B m a t e r i a l w e r e i n fact c o m p o s e d of m o r e t h a n a single c o m p o n e n t , n o f u r t h e r a t t e m p t w a s m a d e to elaborate o n the i d e n t i t y or n o n i d e n t i t y of the i n d i v i d u a l c o m p o n e n t s of e a c h of these peaks. S t r u c t u r a l studies of t h e P C B c o m p o n e n t s m u s t necessarily i n v o l v e the a p p l i c a t i o n of c o m p l e x s e p a r a t i o n p r o c e d u r e s c o u p l e d w i t h d e t a i l e d s p e c t r o m e t r i c studies of e a c h i s o l a t e d c o m p o n e n t .
F i g u r e 11 also i n c l u d e s
the t o t a l i o n c u r r e n t c h r o m a t o g r a m of a h u m a n a d i p o s e tissue extract w h i c h w a s s h o w n to c o n t a i n traces of P C B c o m p o u n d s ( p e a k s a t h r o u g h Downloaded by FUDAN UNIV on January 4, 2017 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0104.ch009
/) w h o s e mass spectra w e r e consistent w i t h those f o u n d for A r o c l o r 1260, n a m e l y , peaks 6 a n d 9 t h r o u g h 17. PCB identified
materials i n utilizing
field-collected
combined
gas
b a l d eagle specimens chromatography-mass
have
spectrometry
techniques i n conjunction w i t h thin-layer chromatographic of s t a n d a r d m a t e r i a l s as w e l l as tissue extracts ( 2 6 ) .
been
separations
A t o t a l of 19 P C B
c o m p o u n d s w e r e d e t e c t e d i n the field samples. A n a l y s e s w e r e p e r f o r m e d e m p l o y i n g a s p i r a l glass gas c h r o m a t o g r a p h i c c o l u m n , 9 ft X
0.25 i n c h
o.d., p a c k e d w i t h 1 % S E - 3 0 o n 1 0 0 / 1 2 0 m e s h G a s C h r o m Q . M a s s spectra of the m a j o r c o m p o n e n t s of A r o c l o r 1254 P C B s t a n d a r d w e r e p r e s e n t e d i n this r e p o r t as w e l l as r e l a t i v e r e t e n t i o n d a t a for the i n d i v i d u a l c o m ponents.
Discussion O f a l l the systems w h i c h h a v e b e e n u t i l i z e d for the analysis of p e s t i c i d e residues, c o m b i n e d gas c h r o m a t o g r a p h y - m a s s s p e c t r o m e t r y
affords
a p a r t i c u l a r l y u s e f u l a p p r o a c h because p o s i t i v e i d e n t i f i c a t i o n of the c o m ponents o f a m i x t u r e c a n b e m a d e w i t h o u t p r i o r s e p a r a t i o n at sensitivities c o m p a t i b l e w i t h the l i m i t e d q u a n t i t i e s of residues g e n e r a l l y a v a i l a b l e . F r o m the results of recent studies i n v o l v i n g the a p p l i c a t i o n of this t e c h n i q u e , it has b e e n d e m o n s t r a t e d that a v a i l a b l e r e s i d u e a n a l y t i c a l m e t h o d s p r o v i d e efficient i s o l a t i o n a n d a d e q u a t e c l e a n u p of extracts of h u m a n , a n i m a l , a n d e n v i r o n m e n t a l m e d i a i n most cases to p e r m i t gas c h r o m a t o graphic—mass s p e c t r o m e t r i c analyses w i t h m a x i m u m confidence.
Addi-
t i o n a l l y , i t has b e e n s h o w n t h a t this c o m b i n e d t e c h n i q u e w i l l c o n v e n i e n t l y p r o v i d e d e f i n i t i v e a n d c o n c l u s i v e c o n f i r m a t i o n of r e s i d u e i d e n t i t y as w e l l as c h a r a c t e r i z a t i o n of residues a n d t h e i r metabolites of u n k n o w n s t r u c t u r e . I n a d d i t i o n to the a p p l i c a t i o n s r e p o r t e d here, gas c h r o m a t o g r a p h y mass s p e c t r o m e t r y has b e e n e m p l o y e d i n the c h a r a c t e r i z a t i o n of p h o t o c h e m i c a l d e g r a d a t i o n p r o d u c t s of p , p ' - D D T a n d p , p ' - D D E ( 2 7 ) , s y n t h e t i c i n t e r m e d i a t e s i n p , p ' - D D T m e t a b o l i s m studies ( 2 8 ) , a n d t r a n s f o r m a t i o n p r o d u c t s of h e r b i c i d a l c h l o r o a n i l i n e s i n soil
(29).
Biros; Pesticides Identification Advances in Chemistry; American Chemical Society: Washington, DC, 1971.
148
PESTICIDES
IDENTIFICATION
F r o m a t e c h n i q u e s t a n d p o i n t , a d d i t i o n a l studies s h o u l d b e m a d e o n i m p r o v e m e n t s of i n s t r u m e n t a l s e n s i t i v i t y t o a l l o w r o u t i n e c o n f i r m a t i o n of p e s t i c i d e residues at the n a n o g r a m a n d p o s s i b l y t h e p i c o g r a m l e v e l . S o m e gains h a v e a l r e a d y b e e n r e p o r t e d i n this a r e a , u t i l i z i n g e n h a n c e m e n t t e c h n i q u e s (21), m e t h o d s (4).
p u l s e h e i g h t analysis (30, 31),
computer a n d other
Studies documenting c o m p o u n d y i e l d a n d enrichment fac-
tors f o r the s e v e r a l classes of o r g a n i c pesticides a n d v a r i o u s gas c h r o m a tographic—mass
spectrometric
i n t e r f a c i a l systems
under
a variety
of
o p e r a t i n g c o n d i t i o n s are r e q u i r e d . T e c h n o l o g i c a l studies s y s t e m a t i c a l l y d e s i g n e d to e x p l o i t the advantages of c a p i l l a r y a n d other gas c h r o m a Downloaded by FUDAN UNIV on January 4, 2017 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0104.ch009
t o g r a p h i c c o l u m n s for p e s t i c i d e r e s i d u e analysis are also n e e d e d .
Finally,
d e v e l o p m e n t of a c o m p l e t e r e s i d u e analysis system e m p l o y i n g e i t h e r a n e l e c t r o n c a p t u r e or flame i o n i z a t i o n detector a n d s p l i t t e r a r r a n g e m e n t i n t a n d e m w i t h the m o l e c u l a r separator p e r m i t t i n g s i m u l t a n e o u s mass spect r o m e t r i c i d e n t i f i c a t i o n a n d d e t e c t i o n a n d q u a n t i t a t i o n of p e s t i c i d e r e s i dues f r o m a s i n g l e gas c h r o m a t o g r a p h i c i n j e c t i o n w o u l d offer
obvious
advantages. T h e r e p o r t e d a p p l i c a t i o n s of c o m b i n e d
gas
chromatography-mass
s p e c t r o m e t r y to the analysis of p e s t i c i d e residues h a v e b e e n i n c r e a s i n g i n f r e q u e n c y i n recent years. W i t h d e v e l o p m e n t s i n i n s t r u m e n t t e c h n o l o g y a n d a v a i l a b i l i t y of i n s t r u m e n t a t i o n , i t c a n b e e x p e c t e d that the t e c h n i q u e w i l l b e r e l i e d u p o n to a n e v e n greater extent i n t h e f u t u r e .
Literature Cited (1) (2) (3) (4) (5) (6) (7) (8) (9)
Biemann, K . , "Mass Spectrometry: Organic C h e m i c a l Applications," 1st ed., p. 46 ff., M c G r a w - H i l l , N e w York, 1962. Budzikiewicz, H., Djerassi, C . , W i l l i a m s , D . H., "Mass Spectrometry of Organic Compounds," p. 6 ff., H o l d e n - D a y , San Francisco, 1967. Gunther, F. A . , 'Instrumentation i n Pesticide Residue Determinations," Advan. Pest Control Res. (1962) 5, 191-319. Biros, F . J., "Recent Applications of Mass Spectrometry and C o m b i n e d Gas Chromatography-Mass Spectrometry to Pesticide Residue A n a l y sis," Residue Rev. (1971) i n press. Kantner, T . R., M u m m a , R. O., "Application of Mass Spectroscopy to Pesticide Residue Analysis," Residue Rev. (1966) 16, 138-51. Westlake, W. E., Gunther, F. A., "Advances i n Gas Chromatographic D e tectors Illustrated from Applications to Pesticide Residue Evaluations," Residue Rev. (1967) 18, 175-217. Sphon, J . A., D a m i c o , J . N., " T h e Mass Spectra of Some Chlorinated Aromatic Pesticidal Compounds," Org. Mass Spectr. (1970) 3, 5 1 - 6 2 and references cited therein. Robinson, J., Richardson, A., Elgar, K . E., " C h e m i c a l Identity i n U l t r a microanalysis," 152nd National Meeting, ACS, New York, September 1966, paper # A - 0 7 5 . Schechter, M. S., " T h e N e e d for Confirmation," Pestic. Monit. J. (1968) 2, 1.
Biros; Pesticides Identification Advances in Chemistry; American Chemical Society: Washington, DC, 1971.
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9.
BIROS
Gas Chromatography—Mass
Spectrometry
149
(10) Biros, F . J., Walker, A . C., "Pesticide Residue Analysis in Human Tissue by Combined Gas Chromatography-Mass Spectrometry," J. Agr. Food Chem. (1970) 18, 425-9. (11) Barthel, W . F . , Curley, A., Thrasher, C. L., Sedlak, V . A., "Determination of Pentachlorophenol in Blood, Urine, Tissue, and Clothing," J. Assoc. Offic. Anal. Chemists (1969) 52, 294-8. (12) Shafik, M . T., Sullivan, H . , Enos, H . F . , " A Method for the Determination of 1-Naphthol in Urine," Bull. Environ. Contam. Toxicol. (1971) 6, 34-9. (13) Shafik, M . T . , Enos, H . F . , "Determination of Metabolic and Hydrolytic Products of Organophosphorus Pesticide Chemicals in Human Blood and Urine," J. Agr. Food Chem. (1969) 17, 1186-9. (14) Shafik, M . T . , Bradway, D., " A Cleanup Procedure for the Determination of Low Levels of Alkyl Phosphates, Thiophosphates, and Dithiophosphates in Rat Urine," Joint ACS-Chem. Inst. Canada Intern. Meeting, Toronto, May 1970, paper #PEST 054. (15) Shafik, M . T . , Bradway, D . , Biros, F . J., Enos, H . F . , "Characterization of Alkylation Products of Diethyl Phosphorothioate," J. Agr. Food Chem. (1970) 18, 1174-5. (16) Cooks, R. G., Gerrard, A . F . , "Electron Impact Induced Rearrangements in Compounds Having the P=S Bond," J. Chem. Soc. B (1968) 132733. (17) Jorg, J. R., Houriet, R., Spiteller, G., "Massenspektren von Pflanzenschutzmitteln," Monatsh. Chem. (1966) 97, 1064-87. (18) Biros, F . J., Ross, R. T., "Fragmentation Processes in the Mass Spectra of Trialkylphosphates, Phosphorothionates, Phosphorothiolates, and Phosphorodithioates," 18th Conf. Mass Spectr. Allied Topics, San Francisco, June 1970, paper #G3. (19) Food and Drug Administration, "Pesticide Analytical Manual," Vol. I, Sec. 211, General Methods for Fatty Foods, U.S. Dept. of Health, Education, and Welfare, Washington, D . C., revised July 1969. (20) Kadoum, A. M . , "Application of the Rapid Micromethod of Sample Cleanup for Gas Chromatographic Analysis of Common Organic Pesticides in Ground Water, Soil, Plant, and Animal Extracts," Bull. Environ. Contam. Toxicol. (1968) 3, 65-70. (21) Biros, F . J., "Enhancement of Mass Spectral Data by Means of a Time Averaging Computer," Anal. Chem. (1970) 42, 537-40. (22) Shafik, M . T., Sullivan, H . , " A Method for the Determination of Low Level Exposure to 2,4-D and 2,4,5-T," 7th Inter-Am. Conf. Toxicol. Occupational Med., Miami, August 1970; J. Environ. Anal. Chem. (1971) in press. (23) Koeman, J. H . , ten Noever de Brauw, M . C., de Vos, R. H . , "Chlorinated Biphenyls in Fish, Mussels, and Birds from the River Rhine and the Netherlands Coastal Area," Nature (1969) 221, 1126-8. (24) Widmark, G., "Possible Interference by Chlorinated Biphenyls," J. Assoc. Offic. Anal. Chemists (1967) 50, 1069. (25) Biros, F . J., Walker, A . C., Medbery, A., "Polychlorinated Biphenyls in Human Adipose Tissue," Bull. Environ. Contam. Toxicol. (1970) 5, 317-23. (26) Bagley, G. E., Reichel, W . L . , Cromartie, E., "Identification of Polychlorinated Biphenyls in Two Bald Eagles by Combined Gas Liquid Chromatography-Mass Spectrometry," J. Assoc. Offic. Anal. Chemists (1970) 53, 251-61. (27) Plimmer, J. R., Klingebiel, U . I., Hummer, B. E., "Photo-oxidation of D D T and DDE," Science (1970) 167, 67-9.
Biros; Pesticides Identification Advances in Chemistry; American Chemical Society: Washington, DC, 1971.
150 (28) (29) (30) (31)
PESTICIDES
IDENTIFICATION
M c K i n n e y , J. D., Boozer, E . L., Hopkins, H. P . , Suggs, J . E., "Synthesis and Reactions of a Proposed DDT Metabolite, 2,2-bis(p-Chlorophenyl) Acetaldehyde," Experientia (1969) 25, 897-8. Kearney, P. C., Plimmer, J. R., Guardia, F . B . , " M i x e d Chloroazobenzene Formation i n S o i l , " J. Agr. Food Chem. (1969) 17, 1418-9. Bergstedt, L., W i d m a r k , G . , "Repetitive Scanning i n Gas Chromatograp h y - M a s s Spectrometry," Chromatographia (1970) 3, 5 9 - 6 3 . W i d m a r k , G., Bergstedt, L., Laveskog, A., "Mass Spectrometry of Organochlorine Compounds and Attempts to Reach Positive Identification," I.U.P.A.C. Conference, Appl. Chem. Div., Sittingbourne, Kent, U.K., October 1968. September 3,
1970.
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RECEIVED
Biros; Pesticides Identification Advances in Chemistry; American Chemical Society: Washington, DC, 1971.