an overview of biological markers of exposure.pdf

7/29/2019 An Overview of Biological Markers of Exposure.pdf

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J o u r n a l o f A n a l y t ic a l T o x i c o l o g y , V o l . 3 2 , J a n u a r y /F e b r u a r y 2 0 0 8

A n O v e r v i e w o f B io lo g ic a l M a r k e r s o f E x p o s u ret o C h e m ic a l W a r f a r e A g e n tsR o b in M . B l a c k *Defence Science and Technology Laboratory , Porton Do wn , Sal isbury, SP4 0JQ, Uni ted K ingdom

A b s t r a c t [An overview is given of b io logical markers of e x pos ur e to chemicalw a r f a r e agents. Metabol i tes, protein, and/or DN A adducts havebeen identif ied for m ost nerve agents a nd v e s i c a n ts and validated inexper imental animals or in a sm al l number of human exposures.For s e v e r a l a ge n ts , metabol i tes der ived from hydrolysisa reunsat is factory bioma rkers of exp osure because of background levelsin t h e h u m a n pop ulation. These are assumed o r e s u l t frome nv i r onme nta l e x pos ur e to comm ercial products that con tain thesehydrolysis produ cts or chem icals tha t a r e me ta bo l i ze d to them.I n the s e cases, m etabolites derived from glutathione pathways, orcovalent adducts with proteins or DN A, p r o v i d e m o r e definit ivebiomarkers. B iomarkers for cyanide and phosgene areunsat is factory as indicators of ch em ical warfare exposure becauseo f o t h e r s o u r c e s of these chem icals o r t h e i r metabolites.

I n t r o d u c t i o n

a r e s h o w n i n F i g u r e 1 . P h o s g e n e a n d h y d r o g e n c y a n i d e , b o t hu s e d a s C W a g e n t s i n W o r ld W a r I , r e m a i n o f c o n c e r n a s t o x i ci n d u s t r i a l c h e m i c a l s .

O r g a n o p h o s p h o r u s n e r v e a g e n t s , v e s i c a n t s , a n d p h o s g e n ea re e l ec tro p h i le s w i th v a ry in g d eg rees o f r eac ti v it y an d se l ect iv i t yt o w a r d s n u c l e o p h i l e s . In t h e b o d y t h e y r e a c t w i t h a r a n g e o f b i-o l o gi c al n u c l e o p h i l e s , f o r e x a m p le , w i t h w a t e r a n d g l u t a t h i o n eto fo rm f r ee m e tab o l it e s , w i th S H , O H , N H , an d C O 2 - am in o ac idr e s i d u e s o n p r o t e i n s t o f o r m p r o t e i n a d d u c t s , a n d , in t h e c a s e o fm u s t a r d s , w i t h N H a n d - P ( O ) O - r e s id u e s o n D N A . B e c a u s e o ft h e i r c h e m i c a l r ea c ti v it y , m o s t C W a g e n t s h a v e s h o r t l i f e t im e si n t h e b o d y a n d t h e r e f o r e h a v e v e r y l im i t e d u t i l it y a s m a r k e r s o fe x p o s u r e . T h e m a j o r f r a c t io n o f a n a b s o r be d d o s e i s m e t a b o l i z e do r c h e m i c a l ly h y d r o l y z e d a n d e l i m i n a t e d a s m o r e p o l a r m e t a b o -l it e s, p r e d o m i n a n t l y i n t h e u r i n e b u t w i t h a s m a l l f r a c ti o n i n t h ef e ce s . T h e r e m a i n d e r o f t h e a b s o rb e d d o s e i s a c c o u n t e d f o r b y c o -v a l e n t r e a c t io n s w i t h n u c l e o p h i l i c si te s o n m a c r o m o l e c u l e s ,m a i n l y p r o t e i n s ( e .g . , h e m o g l o b i n , a l b u m i n , c h o l i n e s t e r a s e s ) ,a n d D N A i n t h e c a s e o f m u s t a r d s .

T h e a n a l y si s o f b i o m e d i c a l s a m p l e s , s u c h a s u r i n e a n d b l o o d ,c a n p r o v i d e q u a l i ta t i v e a n d q u a n t i t a t iv e e v i d e n c e o f e x p o s u r e t oc h e m i c a l w a rf a re ( C W ) a g e n t s . T h e d e t e c t i o n o f fr e e m e t a b o l i te sa n d c o v a l e n t a d d u c t s w i t h m a c r o m o l e c u l e s p r o v id e s f o re n s i cev id en ce i n cases o f a l l eg a t i o n s o f m i l i t a ry o r t e r ro r i s t u se o f C Wa g e n t s . S u c h b i o m a r k e r s m a y a ls o b e u s e f u l f o r d i a g n o s t i c p u r -p o s e s , t o e n s u r e t h e a d m i n i s t r a t i o n o f a p p r o p r i a t e m e d i c a lc o u n t e r m e a s u r e s , a n d f o r m o n i t o r i n g e x p o s u r e in w o r k e r s e n -g a g e d i n d e m i l i t a r i z a t i o n a n d o t h e r d e f e n s i v e a c ti v i ti e s . T h i sp a p e r p r o v i d es a s h o r t o v e r v ie w o f t h e b i o m a r k e r s t h a t h a v eb e e n i d e n t i f ie d a n d d i s c u s s e s th e i r a d v a n t a g e s a n d l i m i t a t i o n s .O n l y s e l e c te d r e f e r e n c e s a r e g i v e n ; c o m p r e h e n s i v e l is t i n g s , to -g e t h e r w i t h m o r e d e t a i le d d i s c u s s i o n s o f a n a ly t ic a l m e t h o d s , a r ep r o v i d e d i n r e c e n t r e v i e w s ( 1 - 4 ) .

T y p e s o f B io m a r k e r ,T h e C W a g e n t s o f m o s t c o n c e r n a r e n e r v e a g e n t s a n d v e s i -

c a n t s ( b l i s t e r a g e n t s ) ; t h e i n c a p a c i t a n t 3 - q u i n u c l i d i n y l b e n z i -l a te ( B Z ) i s c o n s i d e r e d o f lo w e r i m p o r t a n c e . S o m e s t r u c t u r e s* E - m a i l : r m b l a c k @ d s t l . g o v .u k .

M e . . .O M e ~ . O M e z N . O9 p . . p ~R o ' P ~ F R O " ~ S C H ~ C H ~ N R ' E t O C Ns a r i n , R = i -P r V X , R ~ E t , R ' = i -P r t a b u ns o m a n , R = p i n a c o ly t R V X , R = i - B u , R ' = E tc y c l o s a r i n , R = c - h e xy t

s ~ CH 2 C H 2C l C H 2 C H 2 C C I P : h ' ~ ~R ~ N C H C I =C H - -A ~ OC H 2 C H 2 C I C H 2 C H 2 C l C I 0s u l fu r m u s t a r d H N - I , R = E t L e w ~ i t e 1 B ZH N - 2 , R = M e

H N - 3 , R = C I C H 2 C HF i g u r e I . S t r u c tu r e s o f t h e m a j o r c h e m i c a l w a r f a re a g e n t s .

S ~ C H 2C H 2O H ~ O S " c H 2 C H 2 O H \ O 2 $ ~ ' C H 2 C H 2 S C H 3 O 2 s ~ C H z C H 2 S O C H 3C H 2 C H 2 O H C H 2 C H 2 O H C H 2 C H 2 S O C H C H 2 C H z S O C H 3t h i o d i g l y c o l t h i o d i g l y c o l I % l y a s e m e t a b o l i t e ss u l r o x i d e

N H C O C H 3I. C H 2 C H 2 S C H 2 C H C O 2 H0 2 S \C H 2 C H 2 S C H 2 C H C O 2 HN H C O C H 3

1 l ' . s u l f o n y l b i s [ 2 - S - ( N - a c e t y l c y s t e i n y l ) e t ~ a n e ]F i g u r e 2 . S t r u c t u r e s o f s o m e m e t a b o l it e s o f s u l f u r m u s t a r d .

2 R e p r o d u c t io n p h o t o c o p y i n g )o f e d i t o r i a l c o n t e n t o f t h i s o u r n a l i s p r o h i b i te d w i t h o u t p u b l is h e r 'sp e r m i s s i o n .


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J o u r n a l o f A n a l y t i c a l T o x i c o l o g y , V o l . 3 2 , J a n u a r y / F e b r u a r y 2 0 0 8

Biomarkers of Vesicants and Ne rve A gentsM o s t a t t e n t i o n h a s b e e n f o c u s e d o n s u l f u r m u s t a r d a n d

n erv e ag en ts , f o r wh ic h d e ta iled m etab o l i sm s tu d ies h av e b eenu n d er tak en (5 ,6 ) .

Sulfur m usta rd has tw o relat ively non-select ive electroph il iccen te r s . I n i t ia l r eac t io n s w i th w ate r an d g lu ta th io n e , p lu s o x -id a t io n o n su l fu r , l ead to th e fo rm at io n o f man y m etab o l i testha t are excrete d in ur ine, 10 of wh ich were identif ied in the r at(5 ). Ch em ica l s t r u c tu r es o f so m e su l fu r mu s ta rd metab o l i tes

T a b l e I . M e t a b o l i te s a s B i o m a r k e rs o f C W A g e n t sA g e n t S a m p l e* B i o m a r k e r C o m m e n tsS u l fu r m u s t a r d u r i n e , b l o o d t h i o d i g ly c o l ,

t h i o d i g l y c o l s u l f o x i d e

H N - 1

u r i n e 1 3 - 1 y a s em e t a b o l it e s ( t w o )

u r i n e 1 , 1 - s u l f o n y l b i s[ 2 - S - ( N - a c e t y l c y s te i n y l )e t h a n e ]u r i n e N - e t h y l d i e t h a n o l a m i n e

H N - 2 u r i n e g / - m e t h y l d i e t h a n o l a m i n eH N - 3 u r in e t ri e th a n o l a m i n e

L e w i s it e I u r i n e c h l o r o v i n y l a r s o n o u sa c id ( C V A A )

S a r in u r i n e , b l o o d i s o p r o p y lm e t h y lp h o s p h o n i c a c id

T D G ( m i n o r ) a n d T D G O ( m a j o r ) a r e u ri n a ry m e t a b o l it e s in t h e r a t. T D G o c c u r su s u a lly a t < ~ 2 n g l m L a n d T D G O a t < 1 0 n g l m L in u r i n e o f n o n -e x p o s e d h u m a ns u b j e c t s , b u t w i t h o c c a s i o n a l o u t l ie r s . S i g n i fi c a n t l y h i g h e r le v e l s h a v e b e e nd e t e c t e d i n h u m a n c a s u a l ti e s o f m u s t a r d p o is o n i n g .M a j o r m e t a b o l it e s in t h e r a t. N o b a c k g r o u n d l e v e l s ( > -0 . 1 n g / m L ) i n > 1 3 0 h u m a ns a m p l e s . W h e n a n a l y z e d , h a v e b e e n d e t e c t e d in a l l s a m p l e s r o m h u m a n a c c i d e n t a la n d C W c a s u a l ti e s . T h e b i s - s u lf o x id e m a y b e p a r t ia l ly f o rm e d b y o x i d a t io n o ft h e m o n o s u l f o x id e d u r in g s a m p l e p re p a r a t io n .M a j o r u r i n a ry m e t a b o l it e i n t h e r a t b u t a p p e a r s o b e a m i n o r e x c r e ti o n p r o d u c t i nh u m a n s , b a s e d o n s a m p l e s r o m t h re e a c c i d e n t a l c a s u a l ti e s .M i n o r u r i n a r y m e t a b o l it e i n t h e r a t . N o b a c k g r o u n d l e v e ls d e t e c t e d ( > I n g l m L ) i n1 2 0 h u m a n u r i n e s a m p l e s ; n o s a m p l e s r o m h u m a n e x p o s u r e s . H a s c o m m e r c ia l u s e s .A s a b o v e . H N - 2 s t il l i n l im i te d u s e a s a n a n t i c a n c e r a g e n t .U r i n a r y m e t a b o l it e i n t h e r a t. D e t e c te d i n 4 7 % o f u r in e s a m p l e s f ro m 1 2 0n o n - e x p o s e d s u b j e c ts , s o m e t im e s a t h ig h l e v e ls . W i d e s p r e a d u s e i n d o m e s t ic a n di n d u s t r i a l p r o d u c t s .U r i n a r y m e t a b o l it e i n g u i n e a p ig s . N o b a c k g r o u n d l e v e ls d e t e c t e d in 1 2 0 h u m a ns a m p l e s; n o s a m p le s r o m h u m a n e x p o s u re s .M a j o r m e t a b o l it e i n a n i m a l s . D e t e c t e d in s a m p l e s f ro m h u m a n c a s u a l ti e s i nM a t s u m o t o a n d T o k y o .

u r in e , b l o o d m e t h y lp h o s p h o n i c a c i d ( M P A ) S e c o n d a r y h y d r o l y s is p r o d u c t t h a t m a y r e s u lt f ro m m e t a b o l is m o r s l o w h y d r o l y s iso f iP r M P A in t h e s a m p l e . D e t e c t e d in s a m p l e s r o m h u m a n c a s u a l t ie s n M a t s u m o t oa n d T o k y o . C o u l d a l s o a ri s e f ro m o t h e r n e r v e a g e n t s ( s o m a n , c y c l o s a r in , V X )a n d f r o m f ir e r e t a r d a n t s .

S o m a n u r in e , b l o o d p i n a c o l y l M P AC y c l o s a r in u r in e , b l o o d c y c l o h e x y l M P AV X u r in e , b l o o d e t h y l M P A

b lo o d M e S C H 2 C H 2 N ( iP r )

T a b u n u r in e M e 2 N - P ( O ) ( O E t ) O H ,H O - P ( O ) ( O E t ) C N

u r i n e E t O - P ( O ) ( O H )2

B Z u r i n e b e n z i li c a c i d , 3 - q u i n u c l id i n o l

P h o s g e n e u r i n eH C N u r in e 2 -a m i n o th i a z o li n e - 4 -

c a r b o x y l i c a c i d

M e t a b o l it e i n a n im a l s ; n o s a m p l e s f ro m h u m a n e x p o s u r e s .M e t a b o l it e i n a n im a l s ; n o s a m p l e s ro m h u m a n e x p o s u r e s .M e t a b o l it e i n a n i m a l s . D e t e c t e d in a h u m a n a s s a s s i n a t io n v i c t im . C o u l d a r is e f r o mo t h e r V a g e n t s o r t h e E t O a n a l o g u e o f s a r i n . C o n f ir m a t o r y f o r V X w h e n d e t e c t e dw i t h M e S C H 2 C H 2 N ( iP r ) .D e t e c t e d i n s e r u m o f a h u m a n a s s a s s in a t io n v i c ti m . C o n f ir m a t o r y f o r V X w h e nd e t e c t e d w i t h e t h y l M P A .A p p e a r to b e t o o u n s t a b l e t o b e u s e f u l b i o m a r k e r s .

U b i q u i to u s o c c u r r e n c e i n h u m a n u r i n e . A m e t a b o l it e c o m m o n t o s o m e p e s t ic i d e sa n d p l a s t i c iz e r s .A s s u m e d to b e m e t a b o l it e s b u t n o a n i m a l s t u d ie s r e p o r te d ; n o s a m p l e s f ro mh u m a n e x p o s u r e s.N o u r i n a r y m e t a b o l i t e s i d e n t i f i e d .M e t a b o l it e i n a n i m a l s . B a c k g r o u n d le v e l s o u n d i n u r in e f r o m 4 0 1 4 0 h u m a n s u b j e ct s ;s o u r c e s i n c lu d e t o b a c c o s m o k e a n d f o o d c o n s t it u e n t s .

* B io m e d i c a l s a m p le n w h i c h th e y h a v e b e e n s h o w n o r a r e a s s um e d o o c c u r .A l l r e p o r t e d b a c k g r o u n d t u d i e s h a v e b e e n n U . S . o r W e s t e rn E u r o p e a n o p u l a t i o n s .


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Journal of Analy t i ca l Tox ico logy , Vol . 32, Ja nuary /February2 0 0 8

are show n in Figure 2 . React ions w ith nucleo phil ic s i tes onhem oglob in (N-terminal val ines , h is t id ines , and aspart ic an dglutamic acid residues) (7,8), album in (cysteine-34) (9), keratin(aspartic and glutam ic acids) (10), and DNA (11) provide atleast s ix biom arkers tha t can be detec ted in appropriate sam-ples . Eleven biomarkers of sulfur mustard (metaboli tes andadducts) h ave been detected in cases of accidental or deliberatehum an exposure (1 ,12). Much debate has conce rned the va-l id i ty o f th iod ig lyco l (TDG ) and i t s su l fox ide (TDGO) asbiomarkers , derived from hydrolysis of sulfur mustard andsubs equ ent oxidation. Bo th occ ur at t race levels (Table I) inWes tern hum an popu la t ions (13 ,14) , source unkno wn, and inabnormally high concentrat ions in subjects who have beenexposed to sulfur mus tard (]5 ,16). In contrast, no backg roundlevels or in terference s have been detected for [3-1yase metabo-l i tes (13,17), which are derived from an ini t ial react ion ofsu l fu r mus tard w i th g lu ta th ione . These a re regarded as un -equivocal biom arkers of exposure to sulfur mu stard and havethe added advantage of sensi tive analyt ic me thods usin g bothgas ch rom atography- tandem mass spec t romet ry (GC-MS-MS)(]3,17) and l iquid chromatography (LC)-MS-MS (18). 1 ,1 ' -S u l f o n y l b i s [ 2 - S - ( N - a c e t y l c y s t e i n y l ) e t h a n e ] , also derived froman ini t ial rea ct ion with glu tathion e, is a majo r metabo li te inthe ra t (5) bu t appears to be a m inor metabo l i t e in hu ma nsbased on the analysis of sam ples from thre e accidental expo-sures (19,20).Much less is known on th e biological fate of ni troge n mu s-tards and Lewisi te; detailed m etabolism studies have no t beenreported. Hydrolysis produ cts of the thr ee n i trogen m ustards,N-m e t h y l d i e t h a n o l a m i n e , N-e t h y l d i e t h a n o l a m i n e , a n d t r i-ethanolamine (Figure 3), have been detected as excretion prod-uc ts in ra t s exposed to the agen ts , bu t appear to be minormetabol it es (21 ) . No ba ckground leve l s o f N-methy l and N -ethyldiethanolamines were detected [limit of dete ction (LOD)~1 ng/mL] in urine from 120 subjects but both have l imitedcom me rcial / industrial uses . Triethanolamine, the hydrolysisp roduct o f H N-3 , occurs a t var iab le and somet imes very h ighlevels in h um an urine (21). Nitrogen mustards w il l undoubtedlyalkylate a n um ber of residues on proteins , but to date, only theform ation of the HN -2 adduc t with th e cysteine-34 residue onalbum in has been identified (22). HN-2 reacts with the N-7 ofdeoxyguanosine residues in DNA as does sulfur m ustar d (23).The hydrolysis pro duc t of Lew isite 1 ,2-chlorovinylarsonousacid (CVAA) (Figure 3) has b een dete cted as its derivative with1 ,2 -e thaned i th io l in u r ine f rom exposed gu inea p igs (24) .Residues have been l iberated from h um an h em oglobin in vi troand guinea pig blood ex vivo by treatment with 1 ,3-dimer-capto-2-propanol (BAL) (25).N e r v e a g e n t s a r e m o r e s e l e c t i v e i n t h e i r r e a c t i o n s .

C H 2 C H 2 O H , O HR - - N C H C I = C H - - A sC H 2 C H 2 O H O H

R = E t , M e , H O C H 2 C H 2 C V A AF i g u r e 3. St ruc tures of m etabol i tes der ived f rom hydro lys is of n i t rogenmustards and Lew is ite 1.

Metabolism is dominated by chemical or enzyme mediatedhydrolysis (6); the a lkyl me thylp hos pho nic acids so producedare general ly regarded as unequivocal biom arkers of exposureas there are no kn ow n al ternat ive sources . Methylphosphonicacid (MPA), the secondary hydrolysis pro du ct and m inor ex-cret ion produc t , could possibly arise f rom fire retardants ortheir p recur sors . Isopropyl MPA, the hydrolysis prod uct ofsarin , w as detected in blood and urin e fr om casualties of theMatsumoto and Toky o terror is t at tacks (26). Tabun poses aproblem because i ts primary hydrolysis prod ucts are unstable,and there may be high backg round levels of i ts secondary hy-drolysis product ethyl phosphoric acid (27). Some metabo-l i tes of phosp honoflu oridate and V -type nerv e agents that areused as biomarkers are show n in Figure 4 .Major react ions with proteins appear to be l imited to theserin e residues in the active sites of acetylcholinesterase, thebiochemical target of nerve agents , and the related butyryl-cholinesterase prese nt in plasma (28) and, fo r the mor e reac-t iv e n e r v e a g e n t s ( p h o s p h o n o f l u o r i d a t e s a nd t a b u n ) ,tyrosine-411 o n albu min (29,30). The butyrylcholinesteraseadduct o f sar in w as de tec ted in Matsum oto and Tokyo casua l -t ies as a phosphonylated peptic n onap eptide (28) and as sarina f t e r r e a c t i v a t i o n w i t h f l u o r i d e (3 1 ) . A d i s a d v a n t a g e o fcholinesterases as biomarkers is that the organophosphorusresidues are pron e to aging, in which th e O-alkyl group iscleaved, thus losing key s tructural information; th is occurspar t i cu lar ly rap id ly wi th som an . The M e2N subs t i tuen t intabu n is prone to cleavage during en zym atic digest ion if de-tec ted as a no napep t ide . Also the en t i re o rga nophosphorusmo iety may be part ial ly displaced from no n-age d residues i ftherape utic oximes have been adminis tered. Albumin adducts ,wh ich are mu ch m ore s table with regard to ag ing or oxime dis-placem ent, have been identified in exp erime ntal animals butappear to be valuable biomarkers at h ighe r exposure levels incom parison to butyrylcholinesterase adducts (29). One point ofam biguity that arises from al l these nerve age nt biomarkers istha t the o rganophospho rus residue cou ld be der ived from asarin type nerve ag ent or a V -type nerve ag ent because thef luor ide o r 2 - (d ia lky lamino)e thy l th io g rou ps a re d i splaced(other leaving groups are m uc h less l ikely). A blood metaboli tederived from the 2-(di isopropylamino)ethylthio s ide chain ofVX was identified in a hu m an subject assassinated by percuta-neou s exposure to VX (32); i t is no t known if th is m etaboli te isfurth er elaborated before excretion in urin e.

D e s i ra b l e P r o p e r ti e s o f B i o m a r k e rsIf biomarke rs are to be used for forensic purposes, usually asunequivocal qualitative indicators of exposure, they need tom e e t a n u m b e r o f r e q u ir e m e n t s .

M e , , . O M e , , . O, P ~ , P , ~ M e S C H 2 C H 2 N ( i P r) ~R O O H H O O HR = i -Pr , p inacoly l , c -hexy l MP A VX metabol i teEt, i -BuF i g u r e 4. S t ruc tures of som e nerve agent me tabol i tes .


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Journal of A nalytical Toxicology, Vol. 32, January/February2008Lifetime

I n c a s e s o f a l le g e d m i l i t a r y u s e o f C W a g e n t s , h i s t o r i c a lp r e c e d e n c e ( 1 , 1 2 ) s u g g e s t s t h a t s a m p l e s a r e l i k e l y t o b e c o l -l e c t e d s e v e r a l d a y s , p o s s i b l y l o n g e r , a f t e r t h e i n c i d e n t , d e -p e n d i n g o n h o w q u i c k l y a n i n v e s t ig a t i n g te a m c a n g a i n a c c e s st o t h e c a s u a lt ie s . B i o m a r k e r s t h e r e f o r e n e e d t o b e d e t e c t a b l e a tl e a s t u p t o a w e e k a f t e r t h e e x p o s u r e , p r e f e r a b l y l o n g e r . I nm o s t c a s e s th i s e x c l u d e s t h e u s e o f th e o r i g i n a l a g e n t a s ab i o m a r k e r ( u n l e s s l a r g e q u a n t i t i e s a r e a b s o r b e d i n t o f a t t yt i s s u e ) . T h e p e r i o d o f d e t e c t a b i l i t y o f a b i o m a r k e r w i l l d e p e n do n a n u m b e r o f f a c t o r s , i n c l u d i n g i t s i n h e r e n t s ta b il it y , t h ee l i m i n a t i o n p r o f il e , a n d t h e L O D o f t h e a n a l y ti c a l m e t h o d .

A d isad v an tag e o f u r in a ry m e tab o l i t e s as b io m ark e r s i s t h a t t h em a j o r p o r t i o n ( ~ 9 0 % ) o f t h e m e t a b o l i t e s f o r m e d a r e e x c r e t e dw i t h i n t h e f i r s t 7 2 h a f t e r e x p o s u r e . F o r t h i s r e a s o n , L O D s < 1n g / m L a r e d e s i r a b l e f o r a p p l i c a t i o n i n i n v e s t i g a t i o n s i n t o a l -l e g e d C W u s e . A d d u c t s w i t h p r o t e i n s g e n e r a l l y p r o v i d e m u c hlo n g e r l iv ed b io lo g ica l m ark e r s , i n f av o rab le cases u p t o t h e l if e -t i m e o f th e m a c r o m o l e c u l e . F o r e x a m p l e , t h e a b u n d a n t b l o o dp r o t e i n s h e m o g l o b i n a n d a l b u m i n h a v e h a lf -l iv e s i n t h e b o d y o f~ 6 a n d 3 w e e k s , re s p ec t iv e ly . D N A a d d u c t s h a v e a m o r e l i m i t e dl i fe t i m e b e c a u s e o f th e i n t e r v e n t i o n o f r e p a i r p r o c e s s e s .

I n t h e e v e n t o f t e r r o r i s t u s e o f C W a g e n t s , s a m p l e s a r e l i k el yt o b e c o ll e c te d m u c h s o o n e r a f te r t h e e v e n t , a n d d e m a n d s o nb o t h t h e l if e t im e o f t h e b i o m a r k e r a n d t h e L O D s o f an a l y ti c a lm e t h o d s m a y b e l e ss . T h is w a s d e m o n s t r a t e d f o l lo w i n g th et e r r o r i s t r e l e a s e o f s a r i n i n t h e T o k y o s u b w a y , w h e n m o s t s a m -p i es w e r e c o l l e c t e d w i t h i n 2 4 h a n d b i o m a r k e r s w e r e d e t e c t a b l ei n s o m e c a s u a l ti e s a t r e l a ti v e ly h i g h c o n c e n t r a t io n s .

a g e n t . B a c k g r o u n d l ev e ls o f e t h y l p h o s p h o r i c a c i d ( F i g u r e 5 )( 27 ) r e s u l t f ro m e x p o s u r e t o o r g a n o p h o s p h o r u s p e s t i c id e s a n dp l a s t i c i z e r s t h a t a r e h y d r o l y z e d t o a c o m m o n e x c r e t i o np r o d u c t . T h i o d i g ly c o l a n d t r i e t h a n o l a m i n e h a v e c o m m e r c i a lu s e s a n d a r e p r e s e n t i n u r i n e f r o m m u c h o f t h e W e s te r n p o p -u l a t i o n ( i n w h i c h b a c k g r o u n d s t u d i e s h a v e b e e n u n d e r t a k e n ) .B a c k g r o u n d l e v el s o f t r i e t h a n o l a m i n e , w h i c h i s w id e l y u s e d i ni n d u s t r ia l a n d d o m e s t i c c le a n i n g p r o d u c t s a n d c o s m e t i c s , c a nb e v e ry h i g h ( 21 ). M e t a b o li te s d e r i v e d f ro m g l u t a t h i o n e p a t h -w a y s ( I3 - 1y a se m e t a b o l i t e s , N - a c e t y l c y s t e i n e c o n j u g a t e s ) , a n da d d u c t s w i t h p r o t e i n s a n d D N A , h a v e t h e a d v a n t a g e a sb i o m a r k e r s i n t h a t t h e i r f o r m a t i o n r e q u i r e s t h e r e a c t i o n o f a ne l e c t r o p h i l i c s p e c i e s w i t h a b i o l o g i c a l n u c l e o p h i l e , a n d a r em u c h l e ss l ik e l y t o o c c u r f r o m n o n - C W s o u r c e s .

I n t h e c a s e o f h y d r o g e n c y a n i d e, sm o k e ( i n c lu d i n g t h a t f r o mt o b a c c o ) a n d s o m e f o o d s a r e s o u r c e s o f e x p o s u r e . 2 - A m i n o t h -i a z o l i n e - 4 - c a rb o x y l i c a c i d , a m e t a b o l i t e o f c y a n i d e , i s f o u n d i nt h e u r i n e o f a l a r g e p e r c e n t a g e o f t h e p o p u l a t i o n ( 3 4 ). P h o s -g e n e m e t a b o l i s m i s c o m p l i c a t e d b e c a u s e i t h a s t w o l a bi le c h l o -r in e s , f o rm s m e t a b o l it e s i n c o m m o n w i t h c h l o r o f o r m , a n dm a y f o r m p r o t e i n a d d u c t s t h a t c o u l d a r i s e f r o m r e a c t i o n o fa m i n o a c i d r e s i d u e s w i t h c a r b o n d i o x i d e (3 5 ).

T h e a d v a n t a g e s , l i m i t a t io n s , a n d o c c u r r e n c e o f c u r r e n t l yk n o w n b i o m a r k e r s f o r n e r v e a g e n t s , v e si c a n ts , p h o s g e n e , h y -d r o g e n c y a n i d e , a n d B Z a re s u m m a r i z e d i n T a bl es I a n d I I ( f o rf u l l r e f e r e n c e s a n d d i s c u s s i o n s o f a n a l y t i c a l m e t h o d s , s e e r e f-e r en ces 1 - -4 ) .

Chemical stabil i tyB i o m a r k e r s s h o u l d h a v e g o o d s t a b il it y i n t h e b i o lo g i c a l m a -

t ri x , a n d n o t b e s u b j e c t t o h y d r o l y s i s o r o x i d a t io n . T h i s i s a p a r -t i c u l a r p r o b l e m w i t h t h e i n i ti a l h y d r o l y s i s p r o d u c t s o f t h en e r v e a g e n t t a b u n , w h i c h f u r t h e r h y d r o l y s e t o e t h y l p h o s -p h o r i c a c id , a u b i q u i t o u s e x c r e t i o n p r o d u c t ( F i g u r e 5 ) . S o m em e t a b o l i te s o f s u lf u r m u s t a r d a r e p r o n e t o o x i d a t io n o n s u lf u r.

A d d u c t s w i t h b u t y r y l c h o l i n e s t e r a s e m a y b e s u b j e c t t o s lo wr e a c t i v a t i o n ( e .g ., V X ) o r a g i n g . T h e l a t t e r o c c u r s v e r y r a p i d l yw i t h t h e s o m a n a d d u c t a n d m o r e s l ow l y w i t h o t h e r n e r v ea g e n t s . F o r t u n a t e l y , t h e f l u o r i d e r e a c t iv a t i o n m e t h o d i s so s e n -s i t i v e t h a t i t s t i l l s h o w s g o o d " r e t r o s p e c t i v i t y " f o r b u t y r y l -c h o l i n e s t e r a s e a d d u c t s ( 3 3 ) .No n- CW s o ur c e s o f b i o m a r ker s

T h e m o s t i m p o r t a n t f a c t o r f o r f o r e n s ic - ty p e a p p l i c a t io n s i st h a t t h e b i o m a r k e r s h o u l d p r e f e r a b ly b e a n u n e q u i v o c a l i n d i -c a t o r o f ex p o s u r e t o t h e C W a g e n t . A n u m b e r o f CW a g e n t h y -d r o ly s is p r o d u c t s a r e e x c r e t e d i n t h e u r i n e o f a l a rg e p e r c e n t a g eo f t h e h u m a n p o p u l a t i o n w h o h a v e n o t b e e n e x p o s e d t o t h e C W

M e 2 N " , O H 20 M e 2 N " .(D H O , O H O , O, , P ~" , , P + / P , ~ , ,P ,E t O C N E t O O H E t O C N E t O O Htabun

F i g u r e 5. Hy dro lyt ic pathway or tabun.

A n a l y t i c a l C o n s i d e r a t i o n s

e t h y l p h o s p h o r i c a c i d

T e c h n i q u e sI n m o s t s i t u a t i o n s f o r w h i c h b i o m e d i c a l s a m p l e a n a l y si s i sr e q u i r e d , a n a l y t e s a r e l i k e l y t o b e p r e s e n t a t lo w o r s u b - p a r t s -p e r - b il l io n c o n c e n t r a t i o n s . M S is t h e o n l y s p e c t r o m e t r i c t e c h -n i q u e t h a t u n i v e r s a l l y p r o v i d e s t h e r e q u i s i t e c o m b i n a t i o n o fs e n s i t i v i t y a n d s e l e c t i v i t y , u s u a l l y i n c o m b i n a t i o n w i t h G C o rL C , o r l e s s c o m m o n l y w i t h c a p i l la r y e l e c t r o p h o r e s i s . F o r a n a l -y s i s d o w n t o m i d t o l o w p a r t s - p e r - b i l l i o n l e v e l s , s i n g l e - s t a g eG C - M S o r L C - M S u s i n g s e l e c t e d i o n m o n i t o r i n g ( SI M ) (f ul ls c a n n i n g o n s o m e i n s t r u m e n t s ) m a y p r o v id e a d e q u a te l i m i t s o fd e t e c t i o n . F o r d e t e c t i o n a t l o w t o s u b - p a r t s - p e r - b i l l i o n l e v e ls ,a n d w i t h a h i g h e r d e g r e e o f c o n f i d e n c e , t a n d e m M S i s r e q u i r e d( o r h i g h - r e s o l u t i o n s i n g l e -s t a g e M S ) .

F o r m o s t u r i n a r y m e t a b o l i t e s , a n a l y s i s c a n b e p e r f o r m e du s i n g l e s s e x p e n s i v e b e n c h t o p i n s t r u m e n t a t i o n b y G C - M S o rG C - M S - M S ( io n t ra p s o r q u a d r u p o l e i n s t r u m e n t s ) . S o m e t h e r -m a l l y l ab i le m e t a b o l i t e s ( e .g ., N - a c e t y l c y s t e i n e c o n j u g a t e s ) r e -q u i r e L C - M S - M S ( 1 9 ) , a s d o m o s t m e t h o d s f o r i d e n t i f y i n g

p r o t e i n a n d D N A a d d u c t s ( 3) , u n l e s s th e a g e n tr e s i d u e c a n b e c h e m i c a l ly l i b e r at e d f r o m t h ep r o t e i n ( 2 5 , 3 1 , 3 6 - 3 8 ) .

I m m u n o a s s a y s a r e u s e f u l fo r r a p i ds c r e e n i n g o f l a rg e n u m b e r s o f s a m p l e s , b u tu n l e s s a n t i b o d i e s f o r t h e a n a l y t e a r e a v a il ab l e,t h e y r e q u i r e a c o n s i d e r a b l e e f f o r t t o d e v e l o p( 3 9 ) .


5/8

S e l e c t i v i t yA f e a t u r e o f m o s t a n a l y t i c a l m e t h o d s f o r C W a g e n t

b i o m a r k e r s i s t h a t t h e y a r e t a r g e t e d a t in d i v i d u a l o r c h e m i c a l l ys p e c i f ic c l a s s e s o f b i o m a r k e r . T h e h i s t o r y o f t h e i n c i d e n t , i n -c l u d i n g e y e w i t n e s s a c c o u n t s , s y m p t o m s i n c a s u a l t ie s , a n d r e -

J o u r n a l o f A n a l y t ic a l T o x i c o l o g y , V o l . 3 2 , J a n u a r y / F e b r u a r y 2 0 0 8

s u i t s o f a n y e n v i r o n m e n t a l a n a ly s is , i s t h e r e f o r e i m p o r t a n t i ng u i d i n g t h e a n a l y s is . A m o r e g e n e r i c m e t h o d h a s b e e n r e -por t ed f o r i den t i f y i ng bu t y r y l cho l i nes t e r a se i nh i b i t ed by expo-s u r e t o a n y o r g a n o p h o s p h o r u s n e r v e a g e n t o r p e s t i c i d e ( b u tw i t h o u t i d e n t i f y in g t h e a g e n t ) ( 4 0) .

T a b l e I I . P r o t e in a n d D N A A d d u c t s a s B i o m a r k e r s o f C W A g e n t sA g e n t S a m p l e * B i o m a r k e r C o m m e n t sS u l fu r m u s t a r d b l o o d : N - t e r m i n a l v a l in e D e m o n s t ra t e d i n a n i m a l s . N o b a c k g r o u n d l e v e ls i n a l im i te d n u m b e r o f h u m a n

h e m o g l o b i n N H - H E T E * d d u c t s a m p le s . D e t e c te d in s a m p l e s r o m h u m a n a c c id e n t a l a n d C W c a s u a lt ie s .b l o o d : h i s ti d in e N - H E T E a d d u c t A s a b o v e .h e m o g l o b i nb l o o d : a l b u m i n

H N - 2H N - 2L e w i s it e 1

S a r i n

b l o o d : a l b u m i na n d h e m o g l o b i n

c y s t e in e - 3 4 S - H E T E a d d u c t

a s p a r t ic a c i d l g l u t a m i ca c i d C O 2 - H E T E

b lo o d a n d N 7 - ( H E T E ) - 2 ' -s k in : D N A d e o x y g u a n o s i n eb l o o d : a lb u m i n c y s t e i n e - 3 4 S - H E T E a d d u c tb l o o d : D N A N 7 - 2 ' - d e o x y g u a n o s i n e a d d u c tb l o o d C V A A ( fr e e a n d b o u n d t o

h e m o g l o b i n )b l o o d : i P r M P - s e r i n e~ a d d u c tB u C h F _ / A C h E

S o m a n

C y c l o s a r i n

b l o o d : a l b u m i n

b l o o d :B u C h E / A C h E


b l o o d :B u C h E / A C h Eb l o o d : a l b u m i n

V X b l o o d :B u C h E / A C h E

T a b u n b l o o d :B u C h E / A C h E


i P r M P - t y r o s in e a d d u c t

M P ( O H ) - s e r in e a d d u c t *

p i n a c o l y lM P - t y r o s in e a d d u c t

c y c l o h e x y l M P - s e r in e a d d u c t

c - H e x M P - ty r o s in e a d d u c t

E t M P - s e r in e a d d u c t

M e 2 N ( E t O ) P ( O ) -s e r in e a d d u c t

M e 2 N ( E t O ) P ( O ) - ty r o s i n e a d d u c t

* 2 - H y d r o x y e t h y l t h i o e t h y l ,H O C H 2 C H 2 S C H 2 C H 2 - .t M P = M e P ( O ) - .

D e m o n s t ra t e d in a n i m a l s . N o b a c k g r o u n d l e v e l s d e t e c te d i n 8 0 h u m a n s a m p l e s .D e t e c t e d in s a m p l e s r o m h u m a n a c c id e n t a l a n d C W c a s u a lt ie s .D e t e c t e d a s T D G r e le a s e d b y h y d r o l y s is . D e m o n s t ra t e d n a n i m a l s . N o s i g n if ic a n tb a c k g r o u n d l e v e ls d e te c t e d i n a l i m i te d n u m b e r o f h u m a n s a m p le s . D e t e c t e d i na s i n g l e a c c i d e n t a l h u m a n e x p o s u r e .D e m o n s t ra t e d i n a n i m a l s . D e t e c t e d i n b l o o d f r o m h u m a n C W c a s u a lt ie s .N 7 - ( H E T E ) - g u a n i n e d e m o n s t r a t e d a s a s h o r t - l iv e d u r i n a r y m e t a b o l i te i n a n i m a l s .F o r m e d in h u m a n b l o o d i n v it ro ; n o s a m p l e s r o m h u m a n ex p o s u r e s .F o r m e d in h u m a n b l o o d i n v i tr o .D e m o n s t ra t e d i n th e r a t ; n o s a m p l e s r o m h u m a n e x p o s u re s .

D e m o n s t ra t e d in a n i m a l s . N o b a c k g r o u n d l e v e l s i n a l im i te d n u m b e r o f h u m a ns a m p l e s . D e t e c te d in s a m p l e s r o m h u m a n c a s u a l ti e s i n M a t s u m o t o a n d T o k y o .M a y b e d e t e c t e d a s a p h o s p h o n y l a te d n o n a p e p t id e , b y h y d r o l y ti c d i s p la c e m e n ta s i P r M P A , a n d m o s t s e n s i t iv e l y a s s a t i n a f t e r f lu o r i d e d i s p la c e m e n t .D e m o n s t ra t e d i n a n i m a l s a n d h u m a n p la s m a in v i t ro ; n o s a m p l e s r o mh u m a n e x p o s u re s .D e m o n s t ra t e d i n a n i m a l s a n d h u m a n p la s m a in v i tr o ; n o s a m p l e s r o m h u m a ne x p o s u r e s . I n d i c a t iv e o f a n a g e d n e r v e a g e n t r e s i d u e b u t d o e s n o t i d e n t i fy t h e a g e n t .N o t d i s p l a c e d b y f lu o r i d e .D e m o n s t ra t e d i n a n i m a l s a n d h u m a n p la s m a in v i tr o ; n o s a m p l e s r o m h u m a ne x p o s u r e s . D o e s n o t r a p i d l y a g e .D e m o n s t ra t e d i n a n im a l s a n d h u m a n p la s m a in v i t ro ; n o s a m p l e s r o m h u m a ne x p o s u r e s .D e m o n s t ra t e d i n a n im a l s a n d h u m a n p la s m a in v i t ro ; n o s a m p l e s r o m h u m a ne x p o s u r e s .D e m o n s t r a te d in a n i m a l s a n d h u m a n p la s m a in v i t r o . C o u l d a r i s e r o m o t h e r V a g e n t so r E t O a n a l o g u e o f s a r i n . C o n f ir m a t o r y f o r V X w h e n d e t e c te d w i thM e S C H 2 C H 2 N ( i P r ) 2 .D e m o n s t ra t e d i n a n im a l s a n d h u m a n p la s m a in v i t ro ; n o s a m p le s r o m h u m a ne x p o s u r e s . T e n d s o l o s e M e 2 N - P s u b s t it u e n t d u r in g d i g e s ti o n w h e n d e t e c te da s a n o n a p e p t i d e .D e m o n s t ra t e d i n a n i m a l s a n d h u m a n p l a s m a in v i t ro ; n o s a m p le s r o m h u m a ne x p o s u r e s .

N o n - a g e d a d d u c t w a s a p p a r e n t lyd e t e c t e d n g u i n e a p ig s b y f l u o r i d e d i s p la c e m e n tb u t w a s p r o b a b l y d e r i v e d r o m p h o s p h o n y l a te d l b u m i n ( 5 0 ) ; o w c o n c e n t ra t io n s f n o n -a g e da d d u c tw e r e d e t e c te d n r h e s u sm o n k e y sb y f lu o r i d e d i s p l a c e m e n t ,b u t B u C h E n t h i s s p e c ie sa g e sm o r e s l o w l y h a n i n h u m a n s ;s o m e n t e r a c t io n w i t h t h e le s s a c t i v e P ( + ) s o m e rm a y b e a c o n t r i b u t in g a c t o r (3 3 ) .


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J o u r n a l o f A n a l y t ic a l T o x i c o l o g y , V o l , 3 2 , J a n u a r y / F e b r u a r y 2 0 0 8

S a m p l e p r e p a r a tio nSample preparation is a key co mp onen t of biomedical sampleanalysis, particularly for protein adducts tha t require digestiont o s m a l l e r m o l e c u l e s . S o m e p ro t e i n s ( a l b u m i n , b u t y ry l -cho l ines te rase) can be se lec t ive ly i so la ted f rom b lood byaffinity-based solid-phase extraction (SPE) prior to digestion(9,40); alternatively blood ca n be crudely fractionated usin gprecipi tat ion techniques. Some analytes can be extracted di-rect ly from urine, b lood, plasma, or digests using l iquid-l iquide x t r a c t i o n , b u t S P E is m o re c o m m o n l y e m p l o y e d . E a r lyme thods ten de d to use C18 or C8 bonded s i l ica cartridges, oranion exchange for acidic analytes such as alkyl me thylpho s-phon ic acids . In rece nt years, there has been increasing use ofhydrophob ic-hydrophilic polymeric cartridges, whic h are oftenmore efficient for extracting partially polar analytes such asthiodiglycol (14,41). In a few exam ples, solid-phase m icroex-tract ion has provided very low LOD s (42). Molecularly im -printed polym ers (MIPs) are curre ntly unde r invest igat ion foraffinity-based SPE (43).Mo st metabo lites, particuiarly tho se derived from hydrolysis,are polar an d require derivat izat ion for GC-MS analysis (44).This can be a sou rce of error in t race analysis . Derivatizationusually require s isolat ion from a queou s media, which can re-sul t in s ignifica nt loss of analyte, and extraneous material ortraces of wa ter re ma ining in the residue may suppress deriva-tization. An adva ntage of derivatization is tha t it can be used toenhance detect ion. Conversion to perfluorinated derivat ivesand detect ion by negative ion chemical ionizat ion MS pro-vides very low LOD s for th iodig lycol (13,38,45) and alkylme thylp hosp hon ic acids (46). LC-M S-MS usually avoids theneed for derivatization, and in fav orable cases, it can also pro-vide low LOD s (18,47).A nu m be r of s trategies can be applied to the isolat ion andidentificat ion of protein adducts. Detect ion o f the ent ire pro-te in adduct i s poss ib le wi th modern MS techn iques , u s ingelectrospray or desorption ionization methods, for examp le, bu tLODs are mo dest . A co m m on approach is to selectively digestthe pro tein with enzym es, such as t rypsin, pepsin , or othe r pro-tease, to p rodu ce short-cha in peptides and de tect the alkylatedor phosphy la ted pep t ide us ing LC-MS-MS (9 ,28) . Al terna-t ively , the prote in may be digested to i ts con st i tue nt amin oacids usin g a protease (29) or 6M hydrochloric acid (37), al-though these me thods tend to p roduce a h igh chem ica l back-ground . In som e cases , part icularly wh ere ester l inkages arefo rmed , the bound moie ty may be d i splaced f rom the p ro te inb y h y d r o l y s i s ( e . g ., s u l f u r m u s t a r d a d d u c t s w i t h g l u -tamic/aspartic acid residues) (38), or fluoride ion in the c ase ofnerve agen t residues on th e serine of AChE /BuChE (31). The 2-hydroxyethylthioethyl residue fro m sulfur m usta rd on the N-term inal val ines of hem oglob in can be l iberated by select ivederivatizat ion with a modified Edma n reagen t (36,37). In eachof these exam ples the l iberated m oiety can be detected usings impler methodo logy (GC-MS or GC-MS-MS).Ana lyt ical standards

One of the obstacles to the broader application of biomedicalsample analysis is the acquisition of analytical standards. Theadvantage of urinary metaboli tes is that most are relat ively

simple molecules and are easily synthesized. Analytical stan-dards for prote in adducts, particularly if the ana lyte is an alky-l a t e d o r p h o s p h y l a t e d p e p t i d e , a r e m o re d e m a n d i n g . Anexpedient i s to use an incubate o f the CW agen t wi th w ho leblood, plasma or blood pro tein as a crude analytical s tandard,al tho ugh this requires access to the agent .C r it e r i a fo r i d e n t i fi c a t io n o f b i o m a r k e r sCriteria for the identification o f biomarke rs of CW age nts ina forensic context are curren tly being discussed in a num be r ofinternat iona l and nat iona l fora. The Laboratory of the Organ-isation for the Proh ibi t ion of Chemical Weapons (OPCW) hasto date only defined and exercised cri teria for analysis at p pmlevels in en v i ronme nta l samples, a t w h ich c oncen t ra t ions fu llscan data are obtainable. Identificat ion by two independenttechniqu es is required by the OPCW. It is l ikely that any agreedse t o f c r i t e r ia fo r iden t i f i ca t ion o f CW b iomarkers a t t racelevels wil l be broadly consis te nt with cri teria defined by regu-la to ry bod ies in o ther con tex ts , d rug tes t ing in spor t s andbanne d substances in animal products, for example. The W orldAnti-Doping Agency (WADA)accepts identification by GC-MSor LC -MS by selected ion m onito ring of three ions, providedthey are s t ruc tu ra l ly charac ter i s t i c and m eet def ined to le r -ances fo r re ten t ion t ime and ion ra t ios (48) . Al terna tively ,GC/LC -MS-M S is accepted if two transi t ions are m onito redus ing m ul t ip le reac t ion m oni to r ing , aga in sub jec t to def inedto lerances for re ten t ion t im e and ion ra tios. The EuropeanCo mm ission directive for bann ed substances in animal prod-ucts (49) uses a system of identification points whic h is broadlycons i s ten t wi th the WADAcriteria b ut w ith som e differences intolerances. If any individual metho d canno t mee t the req uire-m ents (e.g ., if only two selected ions o r one MRM transi t ion isavai lable), both systems al low identificat ion by mo re tha n oneanalytical m eth od (e.g., by using two derivatives, w o ionizationme thods, or GC and LC, subject to a n um be r of restrict ions).W here the quest ion is "has a subjec t been exposed to a part ic-ular CW agent?", the identification of more th an one biomarkershould offer an added degree of flexibility for identification, pro-vided the identification o f each bioma rker meets a min im umset of criteria.

C o n c l u s i o n sMetaboli tes and/or p rotein o r DNA adduc ts have been iden-t i fied for mos t nerve agen ts and vesicants and val idated in ex-per imen ta l an imals o r in a smal l num ber o f hum an exposures .Urinary metaboli tes and protein adducts derived from nitroge nmu stards, and urinary metabolites derived from the s ide chainof V agents are knowledge gaps.For several agents, metabolites derived from hydrolysis are

unsat isfactory biom arkers of CW exposure because of back-grou nd levels in the hu m an populat ion. In these cases , metabo-l i tes derived from gluta thione pathways, or covalent adductsw i t h p r o t e i n s o r D N A , g e n e r a l l y p r o v i d e u n e q u i v o c a lbiomarkers .No satisfactory biomarke rs ha ve been identified for phosgene


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and cyanide because of background levels of metabolites oradducts from oth er sources of exposure.Fu rther con sideration needs to b e given to criteria for un-equivocal identif ication of biomarkers of exposure to CWagents in forensic-type analysis.

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Journa l o f A na ly t i ca l Tox ico logy ,Vo l . 32, January/February 0083 3 . M . J . v a n d e r S c h a n s , M . P o l h u j s , C . v a n D i j k , C . E . A . M . D e g e n -hard t , K . P le i j s ie r , J .P . Lang enbe rg , an d H .P . 13enscho p. Ret ro -s p e c t i v e d e t e c t io n o f e x p o s u r e t o n e r v e a g e n ts : a n a l y s i s o fphosp hof luor ida tes o r ig in a t ing f rom f l u ro r ide- induced reac t i va t iono f p h o s p h y l a te d B u C h E . A r c h . T o x i c o l . 7 8 : 5 0 8 - 5 2 4 ( 2 0 0 4 ) .34 . B .A . Logu e, N .P . K i rsch ten , I . P e t r i kov ics , M .A . Mose r , G .A . R ock -w o o d , a n d S . I. B a s k i n . D e t e r m i n a t i o n o f t h e c y a n i d e m e t a b o l i te2 - a m i n o t h i a z o l i n e - 4 - c a r b o x y l i c a c id i n u r i n e a n d p la s m a b y g a sc 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 . J . C h r o m a t o g r . B 8 1 9 :

2 3 7 - 2 4 4 ( 2 0 0 5 ) .3 5 . D . N o o r t , A . G . H u l s t, A . F i d d e r , R . A . v a n G u r p , L . R A . d e J o n g ,a n d H . P . B e n s c h o p . In v i t r o a d d u c t f o r m a t io n o f p h o s g e n e w i t h a l -b u m i n a n d h e m o g l o b i n in h u m a n b l o o d . C h e m . R e s . T o x i c o l . 13 :7 1 9 - 7 2 6 ( 2 0 0 0 ) .36 . A . F idder , D . Noor t , A .L . de Jong, H .C . T rap , L .f fA . de Jong, andH . P . B e n s c h o p . M o n i t o r i n g o f i n v i t r o a n d i n v i v o e x p o s u r e t os u l f u r m u s t a r d b y G C / M S d e t e r m i n a t i o n o f t h e N - t e r m i n a l v a l i n ea d d u c t in h e m o g l o b i n a ft e r a m o d i f ie d E d m a n d e g r a d a t io n .C h e m . R e s . T o x i c o L 9 : 7 8 8 - 7 9 2 ( 1 9 9 6 ) .37 . R .M. B lack , R . J . C la rke , J .M. Har r i son , a nd R .W . Rea d. B io lo g ica lf a t e o f s u l p h u r m u s t a r d : i d e n t i f i c a t i o n o f v a l i n e a n d h i s t i d i n ea d d u c t s i n h a e m o g l o b i n f r o m c a s u a l t i e s o f s u l f u r m u s t a r d p o i -s o n i n g . X e n o b i o t i c a 2 7 : 4 9 9 - 5 1 2 ( 1 9 9 7 ) .3 8 . B . R . C a p a c i o , J .R . S m i th , M . T . D e L i o n , D . R . A n d e r s o n , J .S .G r a h a m , G . E . P la t o ff , a n d W . D . K o r t e . M o n i t o r i n g o f s u l f u r m u s -t a r d e x p o s u r e b y g a s c h r o m a t o g r a p h y - m a s s s p e c t ro m e t r y : a n a l -y s i s o f th i o d i g l y c o l c l e a v e d f r o m b l o o d p r o t e i n s . J . A n a l . T o x i c o l .2 8 : 3 0 6 - 3 1 1 ( 2 0 0 4 ) .39 . G .P . van der S chans , R .H . Mars -G roenen d i j k , L . f fA . d e Jong, H .P .B e n s c h o p , a n d D . N o o r t . S t a n d a r d o p e r a t i n g p r o c e d u r e f o ri m m u n o s l o t b l o t a s s a y o r a n a l y s i s o f D N A / s u l f u r m u s t a r d a d d u c t si n h u m a n b l o o d a n d s k in . J . A n a l . T o x i c o l . 2 8 : 3 1 6 - 3 1 9 ( 2 0 0 4 ) .4 0 . D . N o o r t , A . F i d d e r , M . J . v a n d e r S c h a n s , a n d A . G . H u l s t . V e r i f i-c a t i o n o f e x p o s u re t o o r g a n o p h o s p h a t e s : g e n e r i c m a s s s p e c t ro -m e t r i c m e t h o d f o r d e t e c t i o n o f h u m a n b u t y r y l c h o l i n e s t e r a s ea d d u c t s . A n a l . C h e m . 7 8 : 6 6 4 0 - 6 6 4 4 ( 2 0 0 6 ) .4 1 . J . R ic h e s , R . W . R e a d , a n d R . M . B la c k . A n a l ys i s o f t h e s u l p h u r m u s -t a r d m e t a b o l it e t h i o d i g l y c o [ i n u r i n e u s i n g is o t o p e - d i l u t i o n g a sc h r o m a t o g r a p h y - i o n t ra p t a n d e m m a s s s p e c tr o m e tr y . J . C h r o -

m a t o g r . B 8 4 5 : 1 1 4 - 1 2 0 ( 2 0 0 7 ) .4 2 . J . V . W o o t e n , D . L . A s h l e y , a n d A . M . C a l a fa t . Q u a n t i t a t i o n o f2 - c h l o r o v i n y l a r s o n o u s a c id i n h u m a n u r i n e b y a u t o m a te d s o l i d -p h a s e m i c r o e x t r a c t i o n - g a s c h r o m a t o g r a p h y - m a s s s p e c t ro m e t r y .J . C h r o m a t o g r . B 7 7 2 : 1 4 7 - 1 5 3 ( 2 0 0 2 ) .43 . S .M. M ou l lec , A . B ~ gos , V . P ichon, a nd 13 . B e l l i e r . S e lec t i ve ex -t r a c t i o n o f o r g a n o p h o s p h o r u s n e r v e a g e n t d e g ra d a t io n p r o d u c t sb y m o l e c u l a r l y i m p r i n t e d s o l i d - p h a s e e x t r a c t i o n . J . C h r o m a t o g r .A 1 1 0 8 : 7 - 1 3 ( 2 00 6 ).4 4 . R . M . B l a c k a n d R . M u i r . D e r i v a t i z a t i o n r e a c t i o n s i n t h e c h r o -m a t o g r a p h i c a n a l y s is o f c h e m i c a l w a r f a re a g e n t s a n d t h e i r d e g r a -d a t i o n p r o d u c t s. J . C h r o m a t o g r . A 1 0 0 0 : 2 5 3 - 2 8 1 ( 2 0 0 3 ) .4 5 . R . M . B l a c k a n d R . W . R e a d . D e t e c t i o n o f tr a c e l e v e l s o ft h i o d i g l y c o l i n b l o o d , p l as m a a n d u r i n e u s i n g ga s c h r o m a t o g -r a p h y - e l e c t r o n c a p t u r e n e g a t i v e i o n c h e m i c a l i o n i s a t i o n m a s sspec t romet ry . J . C h r o m a t o g r . 4 4 9 : 2 6 1 - 2 7 0 ( 1 9 8 8 ) .46 . S . - t L F redr i ksson , L . -G . Ham ma rs t r6m, L . Hen r i ksson , and H . - t~ .Lakso . T race de te rm ina t ion o f a l ky l methy lpho sphon ic a c ids in en -v i r o n m e n t a l a n d b i o l o g i c a l s a m p l e s u s i n g g a s c h r o m a t o g -r a p h y / n e g a ti v e -i o n c h e m i c a l i o n i z a t i o n m a s s s p e c t ro m e t r y a n dt a n d e m m a s s s p e c t ro m e t r y . J . M a s s S p e c t r o m . 3 0 : 1 1 3 3 - 1 1 4 3( 1 9 9 5 ) .4 7 . D . N o o r t , A . G . H u l s t , D . H . J ~ M . P l a t e n b u rg , M . P o l h u i js , a n dH . P . B e n s c h o p . Q u a n t i ta t i v e a n a l y s is o f O - i s o p r o p y l m e t h y l p h o s -p h o n i c a c i d i n s e r u m s a m p l e s o f J a p a n e se c i t iz e n s a l l e g e d l yexpose d t o sar in : es t imat ion o f i n t e rna l dosag e. A r c h . T o x i c o l . 7 2 :6 7 1 - 6 7 5 ( 1 9 9 8 ) .4 8 . W A D A ( W o r l d A n t i -D o p i n g A g e n c y ) . Id e n t i fi c a t io n c r i te r i a fo rq u a l i ta t i v e a s s a y s n c o r p o r a t i n g c h r o m a t o g r a p h y a n d m a s s s p e c -t r o m e t r y T D 2 0 0 3 1 D C R , 2 0 0 4 .4 9 . E u r o p e a n C o m m i s s io n . C o m m i s s io n d e c i s i o n o f 1 2 A u g u s t 2 0 0 2i m p l e m e n t i n g C o u n c i l D i r e c ti v e 9 6 /2 3 /E C c o n c e r n i n g t h e p e r -f o r m a n c e o f a n a l y t i c a l m e t h o d s a n d t h e i n t e r p r e ta t io n o f r e s u l ts .E u r o p e a n U n i o n D e c i s i o n 2 0 0 2 /6 5 7 /E C 1 7 . 8 .2 0 0 2 . O f f . J . E u r .C o m m u n . 2 2 1 : 8 - 3 6 ( 2 0 0 2 ) .5 0 . T . K . A d a m s , B . R . C a p a c i o , J . R . S m i t h , C . E . W h a l l e y , a n d W . D .K o r t e . T h e a p p l i c a t i o n o f th e f l u o r i d e r e a c ti v a ti o n p r o c e s s t o t h ed e t e c ti o n o f s a r in a n d s o m a n n e r v e a g e n t e x p o s u r e s i n b i o l o g i c a l

s a m p l e s . D r u g C h e m . T o x i c o l . 2 7 : 7 7 - 9 1 ( 2 0 0 4 ) .

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