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 .

2 5 , O c t o b e r 2 0 0 1
Technical Note I
A C o m p r e h e n s i v e S c r e e n f o r V o l a t i l e O r g a n i c
C o m p o u n d s i n B i o l o g i c a l F l u i d s
M a r y - E l l e n E . S h a r p
Toxicology Services, RCMPolice Forensic Laboratory Services - Regina, Box 6500, Regina, Saskatchewan, Canada S4P 3J7
[ A b s t r a c t
A h e a d s p a c e g a s c h r o m a t o g r a p h i c ( G C ) s c r e e n f o r c o m m o n vol at i l e
o r g a n i c c o m p o u n d s i n b i o l o g i c a l f l u i d s is r e p o r t e d . C o m m o n GC
p h a s e s , D B . 1 TM a n d D B - W A X T M , w i t h s p l i t i n j e c t i o n p r o v i d e
s e p a r a t i o n a n d i d e n t i f i c a t i o n o f m o r e t h a n 4 0 c o m p o u n d s i n a
s i n g l e 2 0 . r a i n r u n . I n a d d i t i o n , t h i s m e t h o d e a s i l y a c c o m m o d a t e s
q u a n t i t a t i o n . T h e s c r e e n d e t e c t s c o m m o n l y e n c o u n t e r e d v o l a t i l e
c o m p o u n d s a t l e v e l s b e l o w 4 rag%. A c o n t r o l m i x t u r e , p r o v i d i n g
q u a l i t a t i v e a n d s e m i q u a n t i t a t i v e i n f o r m a t i o n , is d e s c r i b e d . F o r
c o m p a r i s o n , e l u t i o n o f t h e v o l a t i l e s o n a s p e c i a l t y p h a s e , DB-
624 T M , is r e p o r t e d . T h i s m e t h o d is a n e x p a n s i o n a n d m o d i f i c a t i o n
of a s c r e e n t h a t h a d b e e n u s e d f o r m o r e t h a n 2 0 y e a r s . D u r i n g its
f i r s t y e a r o f u s e , t h e e x p a n d e d s c r e e n h a s p r o v e n t o b e
a d v a n t a g e o u s i n r o u t i n e f o r e n s i c c a s e w o r k .
I n t r o d u c t i o n
Headspace gas c h r o ma t o g r a p h y (HS-GC), as a f or ensi c t ool ,
h a s been evolving for t h e past hal f - cent ur y (1-7). Thi s me t h o d
c ont i nue s t o be t he s t at e- of - t he- ar t for analysis of a wi de vari et y
of bi ol ogi cal a n d n o n - b i o l o g i c a l s a mp l e s f or vol a t i l e o r g a n i c
c o mp o u n d s (VOC) ( 8- 14) . The p r e s e n t wor k was u n d e r t a k e n t o
e xpa nd the scope and i ncr eas e t h e det ect i on sensitivity provi ded
by a n est abl i shed, t h o u g h l i mi t e d , s c r e e n f or c o m m o n VOCs
(15). The or i gi nal s c r e e n , u s i n g d u a l - c o l u mn e l u t i o n o n DB-1
a n d DB-WAX, was k n o w n t o d e t e c t e t h a n o l , m e t h a n o l , i so-
propanol , n- pr opanol , a n d t ol ue ne . However, l i mi t s of de t e c t i on
h a d n o t been d e t e r mi n e d , a n d a l ack of i n t e r f e r e n c e was onl y
i nf er r ed t h r o u g h years of use. To e ns ur e a t h o r o u g h toxicological
analysis, a validated, c o mp r e h e n s i v e volatile s cr een was ne e de d
t o c o m p l e m e n t an e s t a bl i s he d, val i dat ed, a n d c o mp r e h e n s i v e
d r u g screen (16,17). I n a ddi t i on t o expandi ng t h e scope, i t was
necessary t o develop a me a n s of mo n i t o r i n g t h e qual i t at i ve a n d
quant i t at i ve i nt egr i t y of t h e vol at i l e screen. A c o n t r o l s o l u t i o n
c ont a i ni ng a mi xt ur e o f i mp o r t a n t VOCs was s ought . For t he s e
c o mp o u n d s , q u a n t i t a t i v e l i n e a r i t y was as s es s ed. Fi nal l y, t h e
speci f i ci t y o f a n e x p a n d e d s c r e e n d e ma n d e d s c r u t i n y . I t was
essent i al t o d e t e r mi n e wh e t h e r any of t he VOCs i nt er f er ed wi t h
t h e i d e n t i f i c a t i o n of a n o t h e r . To t h e s e e n d s , a n e s t a b l i s h e d
volatile s cr een was modi fi ed and r e t e n t i o n dat a for mo r e t h a n 50
VOCs wer e collected. Fol l owi ng t h e ori gi nal me t h o d , t o c onf i r m
volatile identity, e l u t i o n of t h e volatiles o n a specialty phase, DB-
624, is r epor t ed. Thus, a c ompr e he ns i ve HS-GC s cr een for r ou-
t i ne casework was validated.
M a t e r i a l s a n d M e t h o d s
Reagents, chemicals, and standard solutions
All volatile st andar ds wer e of h i g h puri t y. Aqueous s ol ut i ons of
e a c h vol a t i l e we r e p r e p a r e d a t 100 rag% i n wa t e r p u r i f i e d by
reverse os mos i s (less volatile, l at e- el ut i ng c o mp o u n d s r equi r ed a
c o n c e n t r a t i o n of 500 rag%). Most of t h e wat er - i ns ol ubl e c o m-
p o u n d s wer e r e nde r e d sol ubl e by addi ng t h e m t o a que ous sol u-
t i ons of e t h a n o l or n- pr opa not (200 rag%). Th e i nt e r na l s t andar d
(I.S.) was t ert i ary-but yl al cohol (t -but anol , BDH, Inc., Tor ont o,
ON, Canada), pr epar ed a t 75 ~g/ mL, i n a n aqueous s o l u t i o n con-
t a i n i n g s o d i u m f l uor i de (30 mg / mL ) a n d s o d i u m n i t r i t e (6.9
mg/ mL) .
Se l e c t e d vol at i l e c o mp o u n d s we r e mi xe d t o g e t h e r t o c r e a t e
t wo ' mi xed volatile' cont r ol s ol ut i ons , t o mo n i t o r HS-GC perfor-
ma n c e a n d t o provi de s emi quant i t at i ve i nf or mat i on. The c ompo-
n e n t s a n d c o n c e n t r a t i o n s i n t h e c o n t r o l s ol ut i ons ar e listed i n
Table I. The s t oc k s ol ut i on was pr epar ed by addi ng each compo-
n e n t t o a 100-mL vol ume t r i c flask t h a t was hal f filled wi t h water,
t h e n a ddi ng wa t e r t o v o l u me . Thi s s o l u t i o n was t r a ns f e r r e d t o
10-mL glass bot t l es wi t h foil-lined screw caps a n d s t or e d refrig-
er at ed (3~ u n t i l r equi r ed. Each of t h e 12 bot t l es was dat ed for
us e d u r i n g o n e m o n t h of t h e year. I mmedi at el y pr i or t o analysis,
t h e s t oc k bot t l e was allowed t o wa r m t o r o o m t e mpe r a t ur e , a n d
t h e ' HI ' a n d ' LO' c o n t r o l s o l u t i o n s we r e p r e p a r e d by d i l u t i n g
1- mL al i quot s t o 100 mL a n d 1 L, respectively, wi t h dei oni zed
water.
HS-GC
Sample preparation. Aqueous s ol ut i ons of each VOC (0.1 mL)
we r e t r ans f er r ed, wi t h I.S. (1.5 mL) , t o 20- mL heads pace vials
u s i n g a n a u t o ma t i c d i l u t e r / d i s p e n s e r ( Ha mi l t o n Microlab 900
ser i es, Ha mi l t o n Co., Reno, NV). Th e vi al s we r e i mme d i a t e l y
R e p r o d u c t i o n ( p h o t o c o p y i n 8 ) o f e d i t o r i a l c o n t e n t o f t h i s j o u r n a l i s p r o h i b i t e d w i t h o u t p u b l i s h e r ' s p e r m i s s i o n . 631
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 . 2 5 , O c t o b e r 2 0 0 1
crimp-sealed then placed in the autosampler carousel. For reten-
tion determinations, the VOC solutions were interspersed with
blanks (water). Retention times and overall detection (deemed to
be the appearance of a strong peak) were determined on two sep-
arate HS-GC systems as described.
Dual column screen--DB-1 and DB-WAX. A Varian TM 3400
GC (Varian Chromatography Systems, Walnut Creek, CA) with a
Tekmar TM 7050 Headspace Autosampler (Tekmar, Cincinnati,
OH) and a flame ionization detector (FID) was used. Injections
T a b l e I . T h e ' M i x e d V o l a t i l e ' C o n t r o l S o l u t i o n s : R e t e n t i o n T i m e s ,
P r e p a r a t i o n , a n d C o n c e n t r a t i o n s *
Stock S o lu tio n ~ C o n tro l Solutions ~
R etention Tim es (m in ) V o l. C onc. ' H I ' ' L O '
Com pound D B - 1 D B - W A X (mL) (g % ) (m g % ) (m g % )
Methanol 2.33 2 .3 6 5 4 4 0
Ethanol 3.39 2.75 5 4 4 0
Acetone 4 .1 4 1.86 5 4 4 0
Isopropanol 4 .4 6 2 .6 8 5 4 4 0
Ethyl ether 5.04 1.46 2 1.4 14
n-Propanol 6.98 4 .5 4 15 12 120
MIBKw 15.84 3.69 2 1.6 16
Toluene 17.14 4 .2 9 0.115 0.1 1
were split (approximately 20:1) and delivered through a ~/' con-
nector to two columns: a DB-1 (30 m x 0.32-mm i.d., 5.0-pro
film thickness, J&W Scientific, Inc., Folsom, CA) and a DB-WAX
(30 m x 0.32-mm i.d., 0.25-pro film thickness, J&W Scientific
Inc.). Samples were equilibrated at 70~ for 30 rain pri or to
injection. The temperature program was 40~ for 9 rain, then
ramped at 10~ to 150~ with no hold. The total run time
was 20 min. The injector and detector temperatures were 150
and 220~ respectively. The carrier gas was helium.
Single column comparison--DB-624. A
PerkinElmer TM AutoSystem XL GC-FID with an
HS-40 Automatic Headspace Sampler was used
(PerkinElmer Canada Inc., Markham, ON,
Canada). The injection was split (optimized at
approximately 20:1) onto a specialty column, DB-
624 (essentially a derivatized form of DB-1; 30 m x
0.53-mm i.d., 3.0-pm film thickness, J&W
4 Scientific). Samples were equilibrated at 60~ for
4
30 mi n prior to injection; the original equilibra-
4
4 tion temperature was found to provide adequate
1.4 sensitivity on this GC system. The temperature
12 program was 45~ for 5 rain, t he n ramped at
1.6 9~ to 90~ and held 5 rain. The total run
0.1 time was 15 min. The injector and detector tem-
peratures were 150 and 250~ respectively. The
carrier gas was helium.
* Retention times f o r t-butanol (I.S.) are 5.45 a n d 2.43 rain o n D B - 1 a n d DB-WAX, respectively.
~ N o m i n a l concentrations, based o n density a n d a d d e d v o l u m e o f each component, d i l u t e d t o 100 m L
* O n e m i l l i l i t e r o f stock solution is d i l u t e d t o 1 0 0 mL a n d 1 L t o p r e p a r e t h e ' H I ' a n d ' L O ' controls,
respectively.
w MIBK = methyl isobutyl ketone.
Mass spectrometric (MS) confirmation
HS-GC peak identities were confirmed by
A
- n
' ~ . ~ ~ ~ ~
" t i m e ( r a i n )
~ 2
-
1 5 . - 2
I O - ~
8
j ! c
2
' ' i / I , , , , , , , ....
T i m * ( r n l n )
! 8 ' ,
B ' D
3 5 -
' ~ ' T ' " r
. I O -
i i
I I ~ I I I!
T l r a 4 ( r a i n ) " [ l r a ~ ( n d n )
Figure 1. Headspace gas chromatograms o b t a in e d w i t h t w o c o n t r o l solutions, a t 1 0 - f o l d concentration differences ( t h e I.S., t-butanol, remains constant), designated
I I I I I I
' L O ' and ' H I ' : A , L O c o n t r o l s o l u t i o n - - D B - 1 c o l u m n ; B, L O c o n t r o l s o l u t i o n - - D B - W A X c o l u m n ; C, H I c o n t r o l s o l u t i o n - - D B - 1 c o l u m n ; and D , ' H I ' c o n t r o l s o l u -
t i o n - - D B - W A X c o l u m n . Retention t i m e s and concentrations are lis t e d i n T a b le I.
6 3 2
Journal of Analytical Toxicology, Vol. 25, October 2001
GC-MS. A He wl e t t - P a c k a r d 6890 GC wi t h a s p l i t / s p l i t l e s s
i nj ect or a n d equi pped wi t h an HP 5973 mass selective de t e c t or
was u s e d (Agilent Te c h n o l o g i e s , Pal o Alto, CA). Th e c o l u mn s
we r e DB-1 (30 m x 0. 25- r a m i.d., 0.25-1Jm f i l m t hi ckness) a n d
DB-WAX (15 m x 0 . 2 5 - mm i.d., 0.25-1Jm fi l m t hi ckness) .
Incorporation into forensic casework
To as s es s t h e e f f e c t i ve ne s s of t h e e xpa nde d s c r e e n a n d t h e
' mi xed volatile' c ont r ol s ol ut i ons , t he y were us e d i n r o u t i n e case-
wo r k f or a year. Al o n g wi t h a p p r o p r i a t e bl a nks , c o n t r o l s , a n d
externally prepared s t andar ds , repl i cat e s ampl es of t h e ' HI' and
'LO' c ont r ol s were s c r e e n e d wi t h ' bat ches' of mul t i pl e case s am-
pies. HS-GC results we r e cont i nual l y assessed, b o t h qualitatively
a n d qua nt i t a t i ve l y. On t h e basi s of r e t e n t i o n r e s u l t s f or t h e
n u me r o u s volatiles t e s t e d, i t was de t e r mi ne d t h a t GC-MS anal-
ysi s was n o t r equi r ed f or c o n f i r ma t i o n of vol at i l es d e t e c t e d i n
r o u t i n e casewor k. P r e c i s e q u a n t i t a t i o n of pos i t i ve cases p r o -
ceeded wi t h i ndependent l y pr epar ed calibrators.
Results a n d Discussion
C o n t r o l s o l u t i o n s
Th e c o m p o n e n t s o f t h e c o n t r o l s o l u t i o n s we r e s e l e c t e d
accor di ng t o forensic si gni f i cance a n d e l u t i o n t i me s ( Fi gur e 1,
Table I). Co n c e n t r a t i o n s i n t h e 'LO' c o n t r o l we r e d e s i g n e d t o
m o n i t o r m i n i m u m d e t e c t i o n s e n s i t i v i t y f or al l c o m p o n e n t s
e x c e p t n - p r o p a n o l . T h e r e l a t i ve l y h i g h c o n c e n t r a t i o n of n -
propanol was r equi r ed t o solubilize t ol uene. As expected, p u t r i d
bi ol ogi c a l s a mp l e s g e n e r a l l y c o n t a i n e d n - p r o p a n o l a t l evel s
below t h e 'LO' cont r ol . Et h e r a n d t o l u e n e levels i n t h e 'LO' con-
t r ol reflect t hos e t h a t ma y be s e e n i n t h e bl ood of abusers. Th e
r e ma i n i n g VOC c o n c e n t r a t i o n s wer e s et accor di ng t o l aborat ory
prot ocol .
The 10-fold c o n c e n t r a t i o n difference bet ween t h e ' HI' a n d 'LO'
c o n t r o l s ol ut i ons was ar bi t r ar y a n d f ound t o be advant ageous .
Ru n n i n g c o n t r o l s o l u t i o n s a t t wo widely spaced c o n c e n t r a t i o n
levels provi ded s e mi qua nt i t a t i ve es t i mat es for positive case s am-
pl es. I t is n o t e wo r t h y t h a t e a c h c o mp o n e n t di spl ayed a l i ne a r
r esponse ove r t he 10-fold c o n c e n t r a t i o n r a nge (See Sensi t i vi t y
a n d quant i t at i ve l i near i t y sect i on).
St or age of t h e s t o c k s o l u t i o n i n refri gerat ed, foil-lined, filled
gl ass b o t t l e s di d n o t p r o v i d e s at i s f act or y c o n t a i n m e n t of t h e
m o s t vol at i l e c o mp o n e n t s . E t h e r a n d t o l u e n e levels, s a mp l e d
f r o m s ucceedi ng bot t l es, decreased sl i ght l y over t i me. St or age of
t h e st ock sol ut i on, f r ozen, i n t i ght l y sealed specialized glass vials
wi l l be investigated. However, t h e s t or ed al i quot s, labeled f or a
s peci f i ed t i m e of u s e ( t o m i n i m i z e t h e n u m b e r of t i me s t h e
st orage vial is opened t o t h e air) alleviated t h e n e e d t o pr epar e a
compl ex s o l u t i o n for ever y bat ch of screens.
S p e c i f i c i t y , s e l e c t i v i t y , a n d stability
HS-GC d e t e c t i o n a n d r e t e n t i o n b e h a v i o r f or mo r e t h a n 50
VOCs are pr es ent ed i n Tables II a n d III. The d u a l - c o l u mn s cr een
provi ded i dent i fi cat i on of mo r e t h a n 40 c ompounds . GC r e t e n-
t i o n o n t h e DB-1 v e r s u s t h e DB-WAX c o l u mn s i ndi c a t e d t h a t
r e t e n t i o n behavi or is speci f i c f or t h e volatiles s t udi ed. Clearly,
T a b l e I I . H S - G C R e t e n t i o n T i m e o f V O C s *
DB-1 (rain) DB.WAX (min) DB-624 (min)
Compound Run program: 4 0 - 1 5 0 ~ 4 5 - 9 0 ~
Acetaldehyde 2.33 1.55 2.20
Methanol 2.33 2.36 2.31
Ethanol 3.39 2.75 3.02
Acetonitrile 3.76 3.61 3.86
Acetone 4.14 1.86 3.49
Propanol, 2- 4.46 2.68 3.66
Ethyl ether 5.04 1.46 3.15
Pentane 5.07 1.38 2.92
Butanol, tertiary 5.45 2.43 4.25
Methyl acetate 5.60 1.93 3.94
Methylene chloride 5.73 2.63 4.10
Propanol, n- 6.98 4.54 5.47
Methyl tertiary butyl ether 8.12 1.53 4.49
Methyl ethyl ketone 8.88 2.36 6.28
Hexane, isomer #2 9.46 1.36 4.52
Ethyl acetate 10.44 2.27 6.42
Hexane, n- 10.50 1.42 4.99
Chloroform 10.61 3.98 6.84
Butanol, iso-
[2-methyl-l-propanol] 11.27 6.49 7.70
Butanol, 4 - c h l o r o - l - t 11.31 2.10 6.76
Hexane, isomer # I 12.01 1.51 6.07
Trichloroethane, 111- 12.40 2.20 7.13
Chlorobutane, I - 12.51 1.96 7.32
Butanol, I - 12.94 8.99 8.84
Benzene 13.15 2.71 7.77
Cyclohexane 13.58 1.59 7.22
Pentanol, 3- 14.49 7.14 9.73
Trichloroethy[ene 14.69 3.47 8.90
Trimethylpentane, 224- 14.76 1.49 7.92
Heptane, n- 15.09 1.54 8.26
Formaldehyde* 15.42 2.38 10.48
Butanol, 3-methyl-l-* 15.72 11.55 11.09
Methyl-2-pentanone, 4- 15.84 3.69 10.87
Pentanol, I - I 16.77 12.77 12.29
Toluene 17.14 4.29 11.22
Hexanone, 2- 17.40 5.63 12.93
Butyl acetate, n- 18.16 5.36 13.29
Octane, n- 18.35 1.78 11.55
Pentanone, 4-OH-4-methyl
(HMP)w 18.94 17.24 > 15
Chlorobenzene 19.49 11.06 > 15
Xylene, m3 19.93 6.86 > 15
Ethylbenzene 19.93 6.92 > 15
Amyl acetate ~ 19.96 7.17 > 15
Xylene, o 3 > 20 7.19 > 15
Xylene, p - > 20 7.20 > 15
Amyl acetate, artifact > 20 10.01 > 15
Butanol, 4- chl or o- l - ,
artifact > 20 14.29 > 15
Octanolw > 20 18.25 > 15
Chloral hydra@ > 20 > 20 > 15
Ethchlorvynol > 20 > 20 > 15
Trichloroethanol, 222- > 20 > 20 > 15
* Retention time entries denote compounds readily detected at 100 mg%
given in order of retention on DB-I column.
Artifact peaks detected.
* Split peak.
s Primary peak is an artifact or breakdown product.
and are
633
n o n e of t hese c o mp o u n d s i nt er f er ed wi t h et hanol i dent i fi cat i on.
The I.S., t - but yl al cohol , was free f r om i nt er f er ence by all b u t t wo
c o mp o u n d s : me t h y l e n e chl or i de, wh i c h e l ut e d very closely o n
b o t h c o l u mn s , and me t ha nol , whi c h was poorl y resolved o n DB-
WAX ( t h o u g h i t was easily di s t i ngui s he d o n t h e DB-624 c ol umn) .
F u r t h e r mo r e , t h e pr es ence of a n i nt e r f e r e nt wo u l d be revealed
by a n a b e r r a n t I.S. peak area. Overall, t h e advant ages of u s i n g
t - but anol out we i gh t hes e few difficulties (14,15).
Th e ' HI' a n d 'LO' c o n t r o l s we r e assessed i n r o u t i n e f or ens i c
casework for a year. They provided a wi de-rangi ng, hi ghl y repro-
duci bl e qual i t at i ve c h e c k mo n i t o r i n g GC s epar at i on a n d selec-
t i vi t y. Re p e a t e d i n j e c t i o n s of t h e c o n t r o l mi x t u r e s , g e n e r a l l y
bi-weekly, reveal ed hi ghl y st abl e e l u t i o n t i me s . The ma x i mu m
deviations for any c o mp o u n d bet ween bat ched r u n s and wi t h i n a
r u n we r e 1% (0.04 r ai n) a n d 0.5% (0.02 mi n ) , r es pect i vel y.
F u r t h e r mo r e , r e t e n t i o n t i me s for t h e early e l u t i n g c ompounds ,
u n c h a n g e d f r om t h e ori gi nal me t h o d , have been absolutely stable
for a t least a decade (15). Ther e has been n o need t o use relative
r e t e nt i on t i mes; t h e ' clean' headspace i nj ect i ons have caused n o
al t er at i on i n c o l u mn charact eri st i cs over years of c o n t i n u i n g use.
A n u m b e r of t e mp e r a t u r e p r o g r a ms wer e i nvest i gat ed f or s ui t -
ability. I n a t t e mp t i n g t o d e t e c t a r a n g e of l ow-vol at i l i t y c o m-
p o u n d s s u c h as e t h y l e n e gl ycol , p r o p y l e n e gl ycol , h y d r o x y
but yr i c aci d anal ogues, and t h e l at e- el ut i ng c o mp o u n d s l i st ed i n
Table II, a h i g h - t e m p e r a t u r e p r o g r a m was i n v e s t i g a t e d
( 4 0 - 2 4 0 ~ a t 20~ Of t h e vol a t i l e s t e s t e d ( o t h e r t h a n
xyl ene a n d oc t a nol , wh i c h ar e d e t e c t e d o n DB-WAX anyway),
onl y chl or al hydrat e, et hchl or vynol , a n d t r i c h l o r o e t h a n o l wer e
r e a di l y d e t e c t e d a t 500 mg %. However, t h i s c o n c e n t r a t i o n
gr e a t l y exceeds t h e r a p e u t i c levels f or t h e s e c o m p o u n d s (18).
Be c a u s e t h e s e c o m p o u n d s a r e r eadi l y d e t e c t e d by m e t h o d s
empl oyed i n general d r u g s cr eeni ng (acidic-drug ext r act i on (17),
e l e c t r o n - c a p t u r e det ect i on) , a h i g h - t e mp e r a t u r e p r o g r a m was
deci dedl y w i t h o u t advant age. Th e be s t c o mp r o mi s e i n speed,
selectivity a n d compr ehens i ve s c r e e ni ng was achi eved wi t h t h e
40-150~ 20-rai n pr ogr am.
El ut i on of VOCs o n t h e specialized, DB-624 c o l u mn followed a
p a t t e r n s i mi l ar t o DB-1 e l ut i on (Table II). However, t h e specialty
c o l u mn pr ovi ded sel ect i vi t y t h a t was a dva nt a ge ous f or i dent i -
fying a n d q u a n t i t a t i n g t h e few c o mp o u n d s t h a t di d n o t display
u n i q u e e l u t i o n t i me s o n b o t h s c r e e ni ng c o l u mn s . For example,
me t h a n o l was clearly resolved f r om acet al dehyde and t h e I.S. o n
t h e DB-624 c o l u mn . To unequi vocal l y di s t i ngui s h t h e few com-
p o u n d s wi t h p o t e n t i a l i n t e r f e r a n t s , r e - a na l ys i s o n DB-624
c o l u mn or GC-MS wer e equally acceptable.
S e n s i t i v i t y a n d q u a n t i t a t i v e l i n e a r i t y
To achi eve adequat e s ens i t i v i t y , e q u i l i b r a t i o n t e m p e r a t u r e f o r
headspace s a mp l i n g was o p t i m i z e d at 70~ a 10~ i ncr ease f r o m
t h e o r i g i n a l m e t h o d ( 1 5 ) . T h e h i g h e r t e m p e r a t u r e p r o v i d e d
i m p r o v e d d e t e c t i o n s e n s i t i v i t y f o r m e t h a n o l i n p a r t i c u l a r , f r o m
8 mg % t o 3 mg %. A t h o r o u g h c o mp a r i s o n r eveal ed n o d e t e r i o -
r a t i o n i n t h e c h r o m a t o g r a m s ( n o ef f ec t o n bas el i ne o r noi se) , n o
loss o f q u a n t i t a t i v e l i n e a r i t y f o r c o m m o n VOCs ( i n c l u d i n g ace-
t o n e , e t h a n o l , e t h e r , i s o p r o p a n o l , m e t h a n o l , a n d t o l u e n e ) , a n d
n o a r t i f a c t peaks a r i s i n g f r o m b i o l o g i c a l f l u i d s . F i g u r e 2 depi c t s
t y p i c a l , c l ean c h r o m a t o g r a m s o b t a i n e d f r o m a b l o o d s a mp l e i n a
n e g a t i v e case.
6 3 4
Journal 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 . 2 5 , O c t o b e r 2 0 0 1
T a b l e I I I . H S - G C R e t e n t i o n T i m e o f V O C s *
DB-1 ( m in ) D B -W A X (m in) D B - 6 2 4 (min)
Compound Run program: 4 0 - 1 5 0 ~ 4 5 - 9 0 ~
Acetaldehyde 2.33 1.55 2.20
Acetone 4.14 1.86 3.49
Acetonitrile 3.76 3.61 3.86
Amyl acetate t 19.96 7.17 > 15
Amyl acetate, artifact > 20 10.01 > 15
Benzene 13.15 2.71 7.77
Butanol, 1- 12.94 8.99 8.84
Butanol, 3-methyl-l-* 15.72 11.55 11.09
Butanol, 4-chloro-l-, artifact > 20 14.29 > 15
Butanol, 4-chloro-l-~ 11.31 2.10 6.76
Butanol, iso-
[2-methyl-l-propanol] 11.27 6.49 7.70
Butanol, tertiary 5.45 2.43 4.25
Butyl acetate, n- 18.16 5.36 13.29
Chloral hydra@ > 20 > 20 > 15
Chlorobenzene 19.49 11.06 > 15
Chlorobutane, 1 - 12.51 1.96 7.32
Chloroform 10.61 3.98 6.84
Cyclohexane 13.58 1.59 7.22
Ethanol 3.39 2.75 3.02
Ethchlorvynol > 20 > 20 > 15
Ethyl acetate 10.44 2.27 6.42
Ethyl ether 5.04 1.46 3.15
Ethylbenzene 19.93 6.92 > 15
Formaldehyde* 15.42 2.38 10.48
Heptane, n- 15.09 1.54 8.26
Hexane, isomer//2 9.46 1.36 4.52
Hexane, isomer #1 12.01 1.51 6.07
Hexane, n- 10.50 1.42 4.99
Hexanone, 2- 17.40 5.63 12.93
Methanol 2.33 2.36 2.31
Methyl acetate 5.60 1.93 3.94
Methyl ethyl ketone 8.88 2.36 6.28
Methyl tertiary butyl ether 8.12 1.53 4.49
Methyl-2-pentanone, 4- 15.84 3.69 10.87
Methylene chloride 5.73 2.63 4.10
Octane, n- 18.35 1.78 11.55
Octanolw > 20 18.25 > 15
Pentane 5.07 1.38 2.92
Pentanol, 1 -~ 16.77 12.77 12.29
Pentanol, 3- 14.49 7.14 9.73
Pentanone, 4-OH-4-methyl
(HMP)w 18.94 17.24 > 15
Propanol, 2- 4.46 2.68 3.66
Propanol, n- 6.98 4.54 5.47
Toluene 17.14 4.29 11.22
Trichloroethane, 111- 12.40 2.20 7.13
Trichloroethano], 222- > 20 > 20 > 15
Trichloroethylene 14.69 3.47 8.90
Trimethylpentane, 224- 14.76 1.49 7.92
Xylene, m -t 19.93 6.86 > 15
Xylene, o -~ > 20 7.19 > 15
Xylene, p- > 20 7.20 > 15
* Retention time entries denote compounds readily detected at 100 mg%.
* Artifact peaks detected.
* Split peak.
w Primary peak is an artifact o r b r e a k d o w n product.
Journal of Analytical Toxicology, Vol. 25, October 2001
Quantitative linearity for all components of the control mix-
t ur e was excellent, as expected with flame detection. For each
component individually, multi-level calibration curves were con-
structed. Table IV lists the ranges over which each component
was tested. Furthermore, the 10-fold range between the 'HI' and
Table IV. The 'Mixed Volatile' Control Solutions:
Concentration Ranges Assessed* for Quantitative
Linearity (mg%)
D u a l C o l u m n Screen Single C o lu m n
V a r i a n G C P e r k i n E I m e r GC
Com pound DB-1 DE-WAX D E - 6 2 4
Methanol 4 - 5 0 NA t 10-50
Ethanol 3 - 5 0 0 3-500 4-500
Acetone 3 - 4 0 3- 40 NA
Isopropanol 3 - 5 0 4- 50 3- 50
Ethyl ether 3 . 5 - 7 0 3.5-70 3.5-70
n-Propanol 3 - 5 0 3- 50 3- 50
MIEK* 1. 5- 20 1.5-20 1.5-20
Toluene 0. 05- 4. 0 0.05-4.0 0.05-4.0
* Cited ranges are those tested using multiple calibrators for each component
individually; the wide linear range provided by FID would certainly allow a much
wider range for most components. All tested calibrators showed a linear response.
* NA = data not available.
* MIBK = methyl isobutyl ketone.
5- -
I o o - 5 .
I
'LO' controls was accompanied by a 10-fold difference in peak-
area ratios (component/I.S.).
Application in forensic casework
Detection results for the volatiles, in aqueous solution, apply
directly to analysis of biological fluids. Two factors that eliminate
significant matrix effects are analysis of headspace (as opposed to
direct injection) and the 15-fold dilution t hat occurs during
sample preparation (12,13,15,16). This screening method has
been applied to routine casework in this laboratory for a year. A
range of biological and non-biological exhibit material has been
analyzed during this time. Numerous putrefactive, ingested, and
inhaled volatile compounds have been detected. Most notable
among these are ethanol, toluene, and ethyl et her - - a novel
finding (19).
Conclusions
A
Time (mln)
B
I I I
, , , , i , , , , l
Time {ml~
F i g u r e 2 . Headspace gas chromatograms obtained with a blood sample in a negative case: DE-1
column (A) and DB-WAX column (E).
This HS-GC method provides a reliable, comprehensive screen
for the presence of volatile organic compounds in biological and
other fluids. The dual-column screen, DB-1 and D]]-WAX, pro-
vides detection and identification of more than 40 compounds.
Because numerous VOCs have been investigated with respect to
detection and selectivity, the implementation of this screen pro-
vides a simple and reliable identification of common volatiles.
For confirmation of the few compounds that do
not display unique retention times on both
columns, the use of a DB-624 col umn or HS-
GC-MS is recommended. The incorporation of
qualitative/semiquantitative control solutions,
containing of a range of forensically important
compounds provides the level of quality assur-
ance t hat is demanded in forensic work.
Furthermore, positive cases are easily quanti-
tated using the same system and conditions,
often with the benefit of a semiquantitative
result. This expanded, modified screen has
proven to be advantageous in routine forensic
casework. In the first year of use, numerous and
novel VOCs have been detected.
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