Technical Note ] Importance of Vacutainer Selection in Forensic ...

Journal of Analytical Toxicology, Vol. 25, July/August 2001
Importance of Vacutainer Selection in Forensic
Toxicological Analysis of Drugs of Abuse
Stefan W. Toennes* and Gerold F. Kauert
Institute of Forensic Toxicology, University of Frankfurt, Kennedyallee 104, D-60596 Frankfurt~Main, Germany
Abstract
The enzymatic degradation of cocaine in blood samples, even
during transport to a forensic laboratory, is a common problem in
toxicological analysis. This can be avoided by the use of blood-
sampling devices such as gray-top Vacutainers containing the
cholinesterase inhibitor sodium fluoride. In the present study,
which included 147 authentic cases, blood samples were
collected into two different tubes, one containing fluoride/oxalate
and one without stabilizing agents. In all cases, both samples
were analyzed for drugs of abuse using Abbott FPIA
immunoassays after precipitation and gas chromatography-mass
spectrometry (GC-MS) for quantitative analysis. The cannabinoid
immunoassay showed markedly lower values in the fluoride-
containing samples; this was investigated further and could be
explained by hemolysis of these samples. In addition, the
concentrations of 11 -nor-Ag-tetrahydrocannabinol-9-carboxylic
acid (THCCOOH) were lower in these samples. A stability study
with the THCCOOH acyl glucuronide showed that it is unstable
in unpreserved serum, which could explain our observation.
GC-MS quantitative data for amphetamine and derivatives,
opiates, Ag-tetrahydrocannabinol, and 11-hydroxy-Ag-
tetrahydrocannabinol were essentially identical; however, they
also differed substantially for cocaine, cocaethylene, ecgonine
methylester, and benzoylecgonine. Unexpectedly, the
concentrations of benzoylecgonine in unpreserved serum were
almost half as high as in the fluoride-containing samples.
Introduction
The rapid enzymatic degradation of cocaine in blood samples
even during transport to a laboratory is a well-known problem in
forensic toxicology. Through the use of blood samples collected
in gray-top Vacutainer tubes (Becton Dickinson), which contain
the anticoagulant potassium oxalate and the cholinesterase
inhibitor sodium fluoride, enzymatic cocaine catabolism can be
inhibited, at least for certain period of time (1-3). Con-
centrations of other drugs of abuse are reported to be unaffected
during storage (4-8). However, these studies were carried out
mostly with reference substances only or without controls in
unstabilized material.
* Author to whom correspondence should be addressed. E-maih toennes@em.uni-frankfurt,de.
Technical Note ]
In the present study, immunochemical and gas chromato-
graphic-mass spectrometric (GC-MS) analysis data of drugs of
abuse from blood samples collected in forensic cases of suspected
driving under the influence of drugs were compared. Blood sam-
ples were taken from the subjects using two Vacutainer tubes, one
free of additives and one containing fluoride/oxalate as stabilizing
agents (gray4op Vacutainer). This study design allowed the direct
comparison of the analytical results from the two types of blood
sampling tubes using authentic material. New information on the
stability of certain metabolites could be obtained.
Experimental
Chemicals, reference standards, and apparatus
The reference standards and their corresponding deuterated
internal standards were purchased from Radian (Promochem,
Wesel, Germany), except for (1)-9-carboxy-11-nor-A94etrahydro-
cannabinol glucuronide (THCCOOH-glucuronide) 10 lJg/mL
solution in methanol which was from Alltech (Deerfield, IL). The
derivatization reagents N-methyl-N-(tert-butyldimethyl-silyl)-
trifluoroacetamide (MTBSTFA), N-methyl-bis(trifluoroaceta-
mide) (MBTFA), and methyl iodide were from Sigma (Munich,
Germany). All other reagents and organic solvents were of analyt-
ical grade and from Merck (Darmstadt, Germany).
GC-MS analyses were performed on a Hewlett-Packard
(Waldbronn, Germany) GC-MS (HP 5890 series II GC, HP 6890
ALS, HP 5972 MSD) with an HP-5 MS capillary column (30 m x
0.25-ram i.d., 0.25-1Jm film thickness). The carrier gas was
helium with a flow rate of 1.0 mL/min. The MS conditions were
280~ transfer line temperature and 70 eV ionization energy.
Data analysis was performed on a Windows computer with HP
ChemStation software (Rev. C.03.00).
Samples and study design
In cooperation with the highway police (Hessen, Germany), a
study was performed using 147 cases of suspected driving under
drug influence. In each case, blood sampling was carried out
using two 10-mL Vacutainer tubes (Becton Dickinson,
Heidelberg, Germany): one contained 25 mg sodium fluoride and
20 mg potassium oxalate as stabilizing reagents (equipped with
gray rubber tops), and the other type was without additives (red
rubber tops). In all cases urine samples were also available. Upon
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission. 339

arrival in the laboratory, the Vacutainer tubes were centrifuged
for separation of serum (Vacutainer tubes with coagulated blood,
normal samples) or plasma (from blood in Vacutainer tubes con-
taining oxalate/fluoride additive, fluoride samples) and stored at
4~ until analysis which was carried out within two weeks.
Immunological screening was performed using the automated
AxSym analyzer (Abbott, Wiesbaden, Germany) with Abbott fluor-
escence-polarization immunoassays (FPIA) Amphetamine/
Methamphetamine II, Opiates U, Cannabinoids U, and Cocaine
Metabolite U; cutoff levels in urine were 300 ng/mL, 200 ng/mL,
25 ng/mL, and 200 ng/mL, respectively. Urine samples were ini-
tially analyzed immunologically, and if a positive result was
obtained, the serum and plasma samples were tested using the
same immunoassays. If at least one of the two samples tested pos-
itive, quantitative analysis with GC-MS was performed.
Immunological screening for drugs of abuse in serum/plasma
with Abbott FPIA
For protein separation, 400 tJL of serum/plasma was added drop-
wise to 400 IJL of acetone. The solution was vortex mixed and cen-
trifuged for 10 min at 1900 xg. The supernatant was analyzed by the
Abbott FPIA tests. According to the results of a previous study (9),
the following cutoffvalues were used: 40 ng/mL for Amphetamine/
Methamphetamine II, 40 ng/mL for Opiates U, 20 ng/mL for
Cannabinoids U, and 20 ng/mL for Cocaine Metabolite U.
Influence of the fluoride/oxalate additives on the FPIA results
Two blood samples were obtained from each of six healthy non-
drug users using the two different Vacutainer types (gray top and
red top). Each blood sample was split: one part was immediately
centrifuged for serum/plasma separation, and the other part was
stored for two days at 4~ to allow the development of hemolysis,
after which it was also centrifuged for serum/plasma separation.
From each of these 24 serum/plasma samples, 5001JL was added
dropwise to 500 IJL of acetone. After vortex mixing, the samples
were centrifuged for 10 min at 1900 xg. To 500 tJL of the super-
natant 25 IJL of a solution containing amphetamine (1000 ng),
benzoylecgonine (1000 ng), morphine (100 ng), and 11-nor-Ag-
tetrahydrocannabinol-9-carboxylic acid (THCCOOH) (40 rig) in
acetone was added. These preparations were analyzed in the
Abbott AxSym analyzer. The Student's t-test was applied to test for
significant differences.
Determination of basic analytes in serum/plasma by GC-MS
Serum samples (1 mL) were diluted with 4 mL of 0.1M phos-
phate buffer (pH 6.0), and 100 IJL of an internal standard solution
(1 ng/lJL of amphetamine-d5, 3,4-methylenedioxymetham-
phetamine-d5, morphine-d3, codeine-d3, cocaine-d3, benzoylec-
gonine-d3, and ecgonine methylester-d3 in acetonitrile) was
added. The samples were vortex mixed. The diluted samples were
extracted using 3-mL Bond Elut Certify HF 300-mg solid-phase
extraction (SPE) cartridges (Varian, Darmstadt, Germany) with
the extraction robot RapidTrace (Zymark, Idstein, Germany). The
extraction protocol was as follows: conditioning with 2 mL
methanol and 3 mL phosphate buffer, application of the sample
onto the column at 1 mL/min, rinsing with 2 mL 0.1M acetic acid
and 3 mL methanol at 1.5 mL/min, and elution of the analytes
with 3 mL of freshly prepared solution of methylene chloride/2-
340
Journal of Analytical Toxicology, Vol. 25, July/August 2001
propanol/concentrated aqueous ammonium hydroxide (80:20:2,
v/v/v) at I mL/min. The extracts were evaporated, the dry residue
was derivatized with 40 IJL MBTFA followed by derivatization with
30 IJL MTBSTFA, and 1 tJL of this solution was analyzed by
GC-MS. GC conditions were as follows: injection port tempera-
ture 260~ held at 60~ for 2 min, increased to 170~ at
20~ then to 310~ at 12~ and held for 5 min.
Quantitation of amphetamine, 3,4-methylenedioxymetham-
phetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA),
3,4-methylenedioxyethamphetamine (MDEA), morphine, 6-
monoacetylmorphine, codeine, dihydrocodeine, cocaine (COC),
cocaethylene, and ecgonine methylester (EME) as TFA derivatives
and benzoylecgonine (BZE) as tBDMS derivative was performed
in single ion monitoring (SIM) mode using internal standards.
Determination of cannabinoids in serum/plasma by GC-MS
For the determination of the cannabinoids A9-tetrahydro-
cannabinol (THC), 11-hydroxy-A94etrahydrocannabinol (THC-
OH), and THCCOOH, 1 mL of serum/plasma was mixed with 4
mL deionized water and 50 IJL of internal standard solution (0.5
ng/IJL of THC-d3 and THC-OH-d3 and 2 ng/IJL of THCCOOH-d3 in
methanol), vortex mixed, and centrifuged for 10 min at 1900 xg.
Manual SPE was performed using 3-mL Bakerbond C18 500-rag
cartridges (Baker, Griesheim, Germany) conditioned with 3 mL
methanol and 2 x 3 mL water on a vacuum manifold. The sam-
ples were slowly applied onto the columns (0.5 mL/min). The
columns were washed with 1 mL water, 1 mL 0.25M acetic acid,
and 1 mL water, and vacuum was applied for 5 min. The analytes
were eluted with 3 x 0.5 mL acetone, and the eluate was evapo-
rated to dryness. The residue was derivatized using methyl iodide,
re-extracted with isooctane, and evaporated again. The residue
was dissolved in 50 IJL isooctane, and 1 IJL was analyzed by
GC-MS. GC conditions were as follows: injection port tempera-
ture 250~ held at 100~ for 2 rain, increased to 310~ at
30~ and held for 6 rain. Quantitation ofTHC, THC-OH, and
THCCOOH as methyl derivatives was performed in the SIM mode
using internal standards.
Study on the stability of cocaine in unpreserved serum
TWenty milliliters of pooled blank serum (pH 7.5) was spiked
with cocaine to obtain 1 tJg/mL and incubated in a 25~ water
bath for 80 h. At selected times, 1-mL aliquots were analyzed for
COC, EME, and BZE by GC-MS.
Study on the stability of benzoylecgonine in
unpreserved serum
Ten milliliters of pooled blank serum (pH 7.5) was spiked with
BZE to obtain 1 IJg/mL and incubated in a 25~ water bath. At 0,
15, 23, and 38 h, two 1-mL aliquots were analyzed for BZE by
GC-MS.
Study on the stability of THCCOOH-glucuronide in
unpreserved serum and in fluoride/oxalate containing plasma
Blood from a healthy non-drug user was collected in vacu-
tainers with and without fluoride/oxalate and centrifuged for
serum/plasma separation. Aliquots of 5 mL of the unpreserved
serum and of the fluoride/oxalate plasma were each spiked with a
solution of THCCOOH-glucuronide to contain 37.8, 75.6, or

Journal of Analytical Toxicology, Vol. 25, July/August 2001
151.2 ng THCCOOH-glucuronide per milliliter of serum/plasma
(equivalent to 25, 50, or 100 ng/mL THCCOOH). The spiked sam-
ples were incubated in a 25~ water bath for 250 h, and at selected
times, 1-mL aliquots were analyzed for THCCOOH by GC-MS.
The residue of the unpreserved blood sample, which contained
the erythrocytes, was washed three times with aqueous 0.9%
sodium chloride solution to completely remove serum compo-
nents and was reconstituted with the sodium chloride solution to
the original volume of the blood sample. A 10-mL aliquot was
spiked with THCCOOH-glucuronide to contain 37.8 ng/mL
(equivalent to 25 ng/mL THCCOOH). The sample was incubated
18o Cocaine 350
~. 160 "st" ~" 300
140 E
~120 + ~ 250
8 1oo 8 200
2000
,~1500
1000
c
0
0 500
+ ,-
+ ++O ~_
0
29 pairs of samples
45 pairs of samples
Figure 1. Concentrations of cocaine, benzoylecgonine, ecgoninemethylester and THCCOOH measured
with GC-MS in serum from normal Vacutainers (O, ascending order) and in the corresponding
plasma from Vacutainers containing fluoride/oxalate (+).
150 1
Ecgonine methylestsr
:= 80 + ~ 150
O +
r
60
40 20 a,,~
0 ........
--[-
+ _t_t _[_ .st. _t_.t_
.'~..'~..'~.~.'~.C'~"i.'~..']..'].~.']..'~.~.'].
~
=.-
0
0 +
100
ooOO+ +
50
~_ .,~. ~i~+ +++ ++
0
19 pairs of samples
20 pairs of samples
7
3500
3000
,.~2500
Benzoylecgonine
+ ~
2501 THCCOOH
200
0
i'!
~2-
s-
o
,i
I_
c l-
G)
c
o
0
0 =,
xO
\
'x%\%
1~176 1
5o - .
. . . .
20 40 60 8
Incubation time in serum at 25~ (h)
Figure 2. Degradation of cocaine (I), 1000 ng/mL spiked) to benzoylecgonine (O), and ecgonine
methylester (A) in serum at 25~ The sum of the molar concentrations of cocaine, benzoylecgonine,
and ecgonine methylester at each time is given (9 together with the result of an exponential regres-
sion of these data (dashed line).
in a 25~ water bath for 250 h, and at selected times, 1-mL
aliquots were analyzed for THCCOOH by GC-MS.
Results and Discussion
The aim of the present study was to investigate possible differences
in results obtained by analyzing blood samples collected in
Vacutainer tubes with and without stabilizing additives. Such differences
have already been observed in the preanalytical phase. An
effect of the sodium fluoride was that all plasma
samples became markedly hemolytic, and the volo
umes of the plasma samples were higher because
o of the anticoagulant potassium oxalate.
#
Immunological screening for drugs of abuse in
serum/plasma with Abbott FPIA
To some extent, the results of the immunoas-
says in normal serum and fluoride plasma for
amphetamine and derivatives (79 cases), opiates
(40 cases), cannabinoids (65 cases), and cocaine
(46 cases) showed considerable differences. The
values for opiates and amphetamine did not differ
markedly in average, but in the case of the assay
Cocaine Metabolite U, the values in the fluoride
samples were, on average, twice as high as the
values in the normal samples. For the cannabi-
noid assay, the results of the fluoride samples
were, on average, only half as high as the values in
the normal samples, which caused a higher rate of
false negatives (28% vs. 5% in normal samples).
Influence of the additives on FPIA results
Drug-free serum/plasma samples were spiked
just before measurement to eliminate any differ-
ences due to sample preparation. The assays of all
six samples were reproducible with variation coef-
ficients of less than 8%. When serum/plasma was
separated immediately after blood sampling, none
of these samples was hemolytic, and no significant
differences in the FPIA values were observed, indi-
cating that the additives themselves did not affect
the measurement. This is in agreement with
results obtained by Robinson and Smith (10) for
an immunoassay of tricyclic antidepressants.
During storage for two days, hemolysis developed
in the fluoride/oxalate-containing blood samples.
FPIA values in the fluoride/oxalate-containing
samples before and after storage did not differ
except for the assay Cannabinoids U, where the
values obtained in the hemolytic samples were
significantly lower by 39% (p < 0.001). This effect
must therefore be attributed to hemolysis.
Quantitative analyses in serum/plasma by GC-MS
No differences could be observed during extrac-
tion and GC-MS analysis between the two sample
341

types; GC-MS signals of analytes and matrix compounds in SIM
mode showed no differences. No differences in the quantitative
results could be found for the analytes amphetamine (49 cases),
MDMA (23 cases), MDA (16 cases), morphine (14 cases), codeine
(13 cases), dihydrocodeine (3 cases), THC (35 cases), and THC-
OH (32 cases). However, clear differences were observed for
cocaine, its metabolites (29 cases), and THCCOOH (45 cases) as
shown in Figure 1.
Stability of cocaine and its metabolites in serum/plasma
In 29 cases, benzoylecgonine was determined as evidence of
previous cocaine use. Cocaine itself was not found in any of the
normal samples, but in 65% of the fluoride samples. Coc-
aethylene was present in two of the fluoride samples (8%), con-
firming that fluoride has a stabilizing effect on cocaine and
cocaethylene. It was surprising that the concentrations of ben-
zoylecgonine in the fluoride samples were almost twice as high
(compare immunological results) and those of ecgonine
methylester were almost half when compared to those of the
normal samples (Figure 1).
In serum without additives, cocaine is rapidly hydrolyzed enzy-
matically to ecgonine methylester as has been shown in various
studies (3) and in the present study (Figure 2). In contrast to
Isenschmid et al. (3), it was found that cocaine was also
hydrolyzed to benzoylecgonine, but only to a minor extent, which
is in accordance with Stewart et al. (11). Ecgonine methylester
and benzoylecgonine were also instable and were further
hydrolyzed to ecgonine. The overall degradation process could be
assessed by adding the molar concentrations of cocaine, ecgonine
methylester, and benzoylecgonine and performing an exponential
regression analysis which yielded a half-life of 15 h (equation:
y = 4.22 e 4)'0462 x x, regression coefficient 0.991, Figure 2).
The differences observed between the data obtained by ana-
lyzing the normal and the fluoride samples are mainly due to the
fluoride-mediated inhibition of the plasma cholinesterase
(PChE). Consequently, ecgonine methylester concentrations
were lower in the fluoride samples because enzymatic hydrolysis
of cocaine to ecgonine methylester was inhibited.
The literature reports on the stability of benzoylecgonine are
35%
0 25%
"o ~ 20%
P
15%
,o%
=
I-- 0%
0 50 I00 150 200 250
Incubation time (h)
Figure 3. Percentage of hydrolyzed THCCOOH-glucuronide in serum from
normal Vacutainers (O) and in plasma from Vacutainers containing
fluoride/oxalate (I-l) at 25~ For each type of material are shown the average
and standard deviation from three different concentrations of THCCOOHg[ucuronide
(37.8, 75.6, and 151.2 ng/mL spiked).
342
Duma[ of Analytical Toxicology, Vo[. 25, July/August 2001
controversial. An enzymatic hydrolysis of benzoylecgonine to
ecgonine like cocaine to ecgonine methylester could be expected;
however, some authors suggest that benzoylecgonine is stable in
unpreserved serum (3,8,12). Stewart et al. (11) found a constant,
but slow hydrolysis in unstabilized serum samples that was
explained by a low affinity of benzoylecgonine to the enzyme
PChE. Giorgi and Meeker (7) and Moody et al. (13) reported a sub-
stantial decrease of benzoylecgonine concentrations in blood sam-
ples stabilized with fluoride, but Baselt et al. (5) reported that
benzoylecgonine was stable in blood for one year in evacuated col-
lection tubes containing 100 mg sodium fluoride and 20 mg potas-
sium oxalate. Therefore, an experiment was performed in order to
test the stability of benzoylecgonine in unstabilized serum. A
sample containing benzoylecgonine in a concentration of 1000
ng/mL and incubated at 25~ exhibited a linear decrease of 13 ng
BZE/mL per hour (regression coefficient 0.997). The benzoylecgo-
nine concentration was reduced to almost half of the initial value
during the 38 h of incubation. Such a delay may be reached easily
when a blood sample is sent by mail. The observed differences of
the benzoylecgonine concentrations between the normal samples
and the fluoride samples can therefore be explained by the hydro-
lysis of benzoylecgonine in unpreserved samples.
Stability of THCCOOH-glucuronide in serum/plasma
The cannabinoid immunoassay is designed to measure THC-
COOH. The observation that the immunological values in the flu-
oride samples were about half as high as in the normal samples
could be explained by the finding that hemolysis in the fluoride
samples markedly affects FPIA measurement. Surprisingly, the
GC-MS confirmation analyses showed that, in 90% of the fluoride
samples, the THCCOOH concentrations were on average 32%
lower than in the normal samples. Previous studies suggested
that THC, THC-OH, and THCCOOI-I are stable in blood and
plasma during storage for several months (8,13-17). A possible
explanation for our finding would be a conjugate of THCCOOH
that is unstable, probably susceptible to degradation by serum
esterases. Such a metabolite could be the ester glucuronide of
THCCOOH (18) (THCCOOH-glucuronide). Therefore, we investi-
gated the stability of the THCCOOH-glucuronide in blood sam-
ples with and without stabilizing agents.
The THCCOOH-glucuronide was stable under the conditions of
the analytical procedure as immediately after spiking of the sam-
ples no THCCOOH was detectable. No THCCOOH developed
during the incubation of THCCOOH-glucuronide in suspended
erythrocytes, indicating that the erythrocytes had no influence on
the stability of the glucuronide. During incubation of THCCOOH-
glucuronide in serum or fluoride/oxalate containing plasma,
THCCOOH could be detected in increasing concentrations. The
absolute concentrations were dependent on the initial amount of
glucuronide but in each group (with or without fluoride/oxalate)
the THCCOOH concentration relative to the maximum theoret-
ical yield were reproducible (Figure 3). The concentrations at 139
or 236 h incubation time were significantly lower in the fluor-
ide/oxalate plasma samples than in the unstabilized serum sam-
ples (p < 0.01) proving that the addition of fluoride/oxalate had
indeed a stabilizing effect on the THC metabolite THCCOOH-glu-
curonide. This is probably the explanation for the lower concen-
trations of THCCOOH in the fluoride samples.

Journal of Analytical Toxicology, Vol. 25, July/August 2001
Conclusions
In authentic cases, two blood samples were collected in tubes
with and without the stabilizing agents fluoride and oxalate.
Analysis results of drugs of abuse using immunoassays and
GC-MS differed markedly for THCCOOH and cocaine and its
metabolites. The cannabinoid immunoassay was shown to be sus-
ceptible to hemolysis that developed in fluoride-containing sam-
ples. Furthermore, GC-MS quantitative analyses revealed lower
concentrations of THCCOOH in fluoride-containing samples,
which could be explained by the lower stability of the THCCOOH-
glucuronide in unpreserved serum. Degradation of cocaine and
cocaethylene to ecgonine esters was inhibited in fluoride-con-
taining samples, but the massive enzymatic hydrolysis of ben-
zoylecgonine that also occurred in the unstabilized samples was
unexpected.
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Manuscript received July 13, 2000;
revision received December 11, 2000.
343