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

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.