Download Quantitation of Clonazepam and Its Major Metabolite 7

Journal of Analytical Toxicology, Vol. 24, October 2000
Quantitation of Clonazepam and Its Major Metabolite
7-Aminoclonazepam in Hair*
Adam Negrusz 1,*, Christine M. Moore 2, Jennifer L. Kern 1, Philip G. lanicak 3, Mary Jane Strong 3, and Naomi A. Levy 3
I Department of Pharmaceuticsand Pharmacodynamics, College of Pharmacy, University of Illinois at Chicago,
833 South Wood Street, Chicago, Illinois 60612; 2United StatesDrug TestingLaboratories, Inc., 1700 South Mount Prospect Road,
Des Plaines, Illinois 60018; and 3Department of Psychiatry, College of Medicine, University of Illinois at Chicago,
1601 West Taylor Street, Chicago, Illinois 60612
Abstract I
Introduction
Clonazepam (CLO) is an anticonvulsant benzodiazepine approved
by the Food and Drug Administration for use in the treatment
of seizures. It produces pharmacological effects (depression,
amnesia) similar to other compounds from the same therapeutic
class, and in combination with alcohol, its CNS-depressant action
can be significantly potentiated. As with some other
benzodiazepines, CLO is a drug possibly used in "date-rape"
situations. A method using solid-phaseextraction followed
by a highly sensitive negative chemical ionization gas
chromatography-mass spectrometry for the simultaneous
quantitation of CLO and its major metabolite 7-aminoclonazepam
(7-ACLO) in hair was developed and validated. The method
has potential application to alleged drug-facilitated rape
cases. To determine the feasibility of detecting 7-ACLO and
CLO in hair, specimens were collected from 10 psychiatric
patients treated with CLO, divided into 2-cm segments, and
analyzed. Standard curves for 7-ACI.O (1-1000 pg/mg)
and CLO (10-400 pg/mg) had correlation coefficients of 0.998.
All precision and accuracy values were within acceptable
limits. 7-ACLO was present in measurable quantities
(1.37-1267 pg/mg) in 9 out of 10 patient samples. CLO
concentrations in hair were much lower (10.7-180 pg/mg).
In 4 out of 10 cases, CLO was not detected in hair. Two patients
who had never been treated with CLO before received a single
2-rag dose of the drug. Approximately three weeks later,
hair samples were collected, and measurable quantities of
7-ACLO (4.8 pg/mg) were detected in the first segment (proximal)
of one of those samples, and traces of the drug were present
in the other sample. We concluded that the 7-ACLO is being
deposited in hair in much higher quantities than the parent
drug and remains there for extended periods of time. Our study
also indicates that it is possible to detect 7-ACLO after a single
dose of CLO as in the typical date-rape scenarios.
* This paper was presented at the 1999 Annual Meeting of the Society of Forensic Toxicologists
in San Juan, Puerto Rico.
Author to whom correspondence should be addressed.
61 4
Clonazepam (Klonopin TM, Clonex, Iktorivil, and Rivotril)
(CLO) is a benzodiazepine that exhibits many of the characteristic pharmacologic properties of the class. It has been shown
to decrease seizure activity, reduce anxiety, induce muscle
weakness, and even induce sleep or hypnosis. It has been approved by the Food and Drug Administration for use in the
treatment of seizures. However, physicians are also using CLO
in the treatment of anxiety, mania, panic disorders, and
schizophrenia because of its sedative and anxiolytic properties.
Chemically, CLO is 5-(2-chlorophenyl)-l,3-dihydro-7-nitro2H-1,4-benzodiazepin-2-one and has a molecular weight of
315 (1). It is available in tablets containing 0.5 rag, 1 rag, and
2 mg CLO. A single dose of CLO produces the desired effects in
as little as 1-2 h, with the half life of the parent drug being
18-50 h. CLO is absorbed and distributed rapidly after oral administration with peak plasma levels obtained within 1-4 h. It
is eliminated slowly, with an elimination half life of 30-40 h.
CLO is highly metabolized, with less than 2% of the parent drug
being excreted in the urine. The metabolism begins with the reduction of the 7-nitro group to the amine, and this amine is
then acetylated to the acetamide. The compounds can also be
hydroxylated at the C-3 position, which will result in their
elimination as either the glucuronide or sulfate conjugate.
In the past few years, considerable information about socalled "date-rape drugs" and drug-facilitated sexual assault has
accumulated, and in the same time an increase in the number
of scientific publications on the subject has been observed
(2-6). Because of its pharmacological effects, CLO has been
identified as a compound potentially used in drug-facilitated
rape (3,7,8) and has been classified as a Schedule IV substance.
Very recently, Raymon et al. (8) reported involvement of flunitrazepam (among other benzodiazepines) in driving-underthe-influence cases in South Florida between 1995 and 1998
and a significant rise in CLO cases after 1998. Drug-facilitated
sexual assault victims may not report the incident in a timely
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Journal of Analytical Toxicology, Vol. 24, October 2000
manner because of fear, shame, and other psychological reasons. It is not unusual for a urine or blood analysis to provide
a negative result because of the likelihood of a single, low-dose
ingestion, urine dilution, and the length of time since the alleged incident. Analysisof hair using highly sensitive detection
provides a solution to this problem. It is documented that 7nitro benzodiazepines metabolize to 7-amino compounds and
tend to incorporate into hair and remain there for much longer
periods of time than in either urine or in blood. Several forensically relevant benzodiazepines have been previously identified
in plasma (9), urine (10), and hair (11-15) using negative ion
chemical ionization gas chromatography-mass spectrometry
(NCI-GC-MS). Yegles et al. (16) reported concentrations of
several benzodiazepines in hair samples obtained from 21
corpses using electron ionization-GC-MS. Recently, LeBeau
et al. (3) suggested that hair may be a valuable specimen in the
cases of drug-facilitated sexual assault when reporting of the
crime was delayed. Negrusz et al. (17) described solid-phase extraction followed by highly sensitive NCI-GC-MS quantitation
of flunitrazepam and 7-aminoflunitrazepam in hair and applied it for the first time to the hair samples collected from alleged drug-facilitated rape victims.
The aim of study was to develop and validate a sensitive, precise, and accurate NCI-GC-MS method for the simultaneous
detection of 7-aminoclonazepam (7-ACLO)and CLO in human
hair with potential application to alleged drug-facilitated rape
victims. To test the developed methodology, we analyzed hair
samples collected from people treated with CLO.To our knowledge this is the first report on detection of CLO and its major
metabolite 7-ACLOin human hair.
Experimental
Subjectsand specimens
Ten subjects (4 women and 6 men, aged 26 to 66 years old)
who were already being treated with CLO for various medical
reasons in the Psychiatric Unit of the University of Illinois at
Chicago Hospital participated in this study. All hair samples
were collected between March and July 1999 using hair collection kits provided by the United States Drug Testing Laboratories, Inc. A single hair sample (equivalent to the thickness of a
pencil) was collected close to the scalp from the back of each
subject's head. The length of each hair sample was measured.
The samples were stored at room temperature until analysis.
Collection of a single hair sample from each subject was approved by the Institutional Review Board of the University of
Illinois at Chicago.
Instrumentation
The GC-MS system consisted of a Hewlett-Packard 6890
series injector, an HP 6890 series GC system, and an HP 5973
mass selective detector with positive and negative ion chemical
ionization capabilities (Hewlett Packard, Palo Alto, CA). An
HP-5MS capillary column (30 m • 250 IJm • 0.25 IJm) was used
for separation (Hewlett Packard, Palo Alto, CA). The hair pulverizer was acquired from Crescent (Lyons,IL), and the heating
block was from Fisher Scientific (Itasca, ILL The Vac-ElutTM
extraction manifold was from Analytical International (Varian,
Harbor City, CA),and vacuum oven model 5831 (Napco| was
purchased from Fisher Scientific (Itasca, IL). The centrifuge
model 5810 (Eppendorf-Netheler-Hinz GmbH, Germany) was
acquired from Brinkmann Instruments (Westbury, NY), and
the Meyer N-EVAP| analytical evaporator from Organomation
Assoc. (Northborough, MA). The Aerograph water bath sonicator was purchased from Varian.
Chemicals and reagents
CLO (1 mg/mL in methanol), 7-ACLO (100 IJg/mL in acetonitrile), and the deuterated internal standard diazepam-d5
(100 IJg/mL in methanol) were purchased from Radian International (Austin, TX). Methanol (high-performance liquid chromatography [HPLC] grade), hydrochloric acid (certified ACS
Plus), glacial acetic acid (HPLC grade), methylene chloride
(HPLC/GC-MS grade), isopropanol (HPLC grade), ethyl acetate (HPLC grade), and concentrated ammonium hydroxide
(certified ACS Plus) were bought from Fisher Scientific. Heptafluorobutyric anhydride (HFBA)was purchased from Campbell Supply Co. (Rockton, IL). The HCX Isolute| 200-mg,
10-mL columns (International Sorbent Technologies) were
purchased from Jones Chromatography (Lakewood, CO).
Standards and controls
The CLO (1 mg/mL in methanol) standard stock solution
was diluted to 100 IJg/mL, 101Jg/mL, 1 IJg/mL,and 0.2 lJg/mL.
The 7-ACLO(100 IJg/mLin acetonitrile) standard stock solution
was diluted to 10 IJg/mL, 1 IJg/mL, 0.1 IJg/mL, and 10 ng/mL.
The deuterated internal standard, diazepam-d5 (100 IJg/mL in
methanol), was diluted to 10 IJg/mL and 1 IJg/mL. All standards were diluted in their respective solvents.
An eight-point standard curve was made for 7-ACLO and a
five-point curve for CLO by using 50-mg aliquots of the negative
pulverized hair spiked with solutions of both drugs. The concentrations of the 7-ACLOin standard hair preparations were as
follows: 1, 5, 10, 50, 100, 200, 500, and 1000 pg/mg of hair. The
concentrations of CLO were 10, 50, 100, 200, and 400 pg/mg of
hair. In addition, two levels of controls were prepared. The low
controls for 7-ACLO (3 pg/mg) and CLO (30 pg/mg) were prepared by adding 15 IJL of 10 pg/IJLand 7.5 IJL of 200 pg/IJL,respectively,to 50-mg aliquots of negative hair. The high controls
for 7-ACLO(800 pg/mg) and CLO (300 pg/mg) were prepared by
adding 40 IJL of 1000 pg/IJLand 75 IJLof 200 pg/IJL,respectively,
to 50-mg aliquots of negative hair.
Analyticalprocedure
The hair samples were divided into approximately 2-cm segments and pulverized, and 50-mg aliquots were analyzed. In
one case, the hair sample was too short to be divided into segments. All other samples were divided into 2-8 segments. To
each hair sample, standard, and control preparations, 30 I~Lof
1-1Jg/mL of diazepam-d5 (600 pg/mg) was added. Methanol (3
mL) was added to the hair, and it was sonicated for 1 h. The
samples were centrifuged for 5 min at 400 x g, and the
methanol layer was decanted and transferred to clean test tubes
for storage in the refrigerator. To the remaining hair, 0.1N hydrochloric acid (3 mL) was added, and the specimens were incubated overnight at 55~ after being sealed and vortex mixed.
615
Journal of Analytical Toxicology, Vol. 24, October 2000
The test tubes were removed from the heating block and centrifuged for 5 rain at 400 x g. The methanol and acid were
combined, and 1.93M glacial acetic acid (1 mL) and deionized
water (9 mL) were added. Mixed-mode Isolute HCX solid-phase
extraction columns were conditioned with the following, never
allowing them to dry: methanol (3 mL), deionized water (3
mL), and 1.93M glacial acetic acid (1 mL). The sample was
then added to the column and drawn through slowly. The
column was allowed to dry for approximately 2 min. The bed of
the column was washed with 3 mL of deionized water (dried for
1-2 min), 1 mL of 0.1N hydrochloric acid (dried for 1-2 rain),
and 3 mL of methanol (dried for 5 min). The collection tubes
were placed in the rack, and the drugs were eluted using a
mixture of methylene chloride/isopropanol/ammonium hydroxide (78:20:2, v/v/v) (3 mL). The eluent was then evaporated to dryness using a stream of nitrogen.
The dry residue was reconstituted in 50 ~L of ethyl acetate
and transferred to autosampler vials. The extract was evaporated
to dryness in the vacuum oven at 60~ The samples were
derivatized using 50 I~L of HFBA at 60~ for 30 rain in the
sealed vials. After incubation, the derivatizing agent was evaporated in the vacuum oven at 60~ and the dry residue was reconstituted in 25 I~Lof ethyl acetate.
Chromatographicmethod
The injector was operated in the splitless mode at 240~
and the injection volume was 1 I~L. Ultra-high purity helium
(99.999%) was used as the carrier gas at a flow rate of 1.2
mL/min. The initial GC oven temperature of 60~ was held
for 1 rain, and then increased at a rate of 30~
until the
final temperature of 310~ was attained. The final temperature
~
was held for 3 min. The total run time for one injection was
12.33 rain. Methane (ultra high purity, 99.999%) was used as
reagent gas at an apparent pressure of 3.7 x 10-4 Torr in the ion
source (methane flow 3.25 mL/min.) The MS ion source temperature was 250~ and the quadrupole temperature was
106~ The electron multiplier voltage was set at +400V above
the NCI-tune voltage.
The mass selective chemical ionization detector was monitoring negative ions (NCI) in the selected ion monitoring (SIM)
mode. The solvent delay was 9 rain. The detector was turned off
after 11.1 rain. The following ions were monitored and used for
quantitation: m/z 315 and 279 for CLO, m/z 461 and 445 for 7ACLO, and rn/z 289 for diazepam-ds. The dwell time for all
ions was 20 ms. The single-taper deactivated liners with glass
wool were used. It was necessary to change the liner daily because CLO is particularly sensitive to active sites.
Precisionand accuracy
Quantitation of CLO and 7-ACLOwas performed by the internal standard method. An eight-point standard curve for 7ACLO and five-point standard curve for CLO were prepared by
linear least-square regression analysis of the ratio of the peak
area of 7-ACLOand CLO to the peak area of the internal standard, diazepam-ds. Peak-area ratios were determined for the
control hair preparations. Control concentrations were calculated from the standard curve values.
Intraday variability was ascertained by analyzing three replicates of low controls (30 pg/mg, 3 pg/mg) and four high controls (300 pg/mg, 800 pg/mg) for CLO and 7-ACLO, respectively.
Interday variability was ascertained over a period of eight weeks.
The mean measured concentrations and standard deviations
C
A
m
c
m
m
<
t0.12
0~o 0.~ 0.~o o.bo b.~ 0.~ O ~
Time
0.~ ~01oo10:~o t0~o ~o~o ~.~ 10~o 10'.w 10:~o 10~o 101~
Time (rain)
(min)
D
4000~
<
l~0Oq,
g0~
Time (rain)
Time
(mln)
Figure 1. Selected ion chromatograms of the blank hair (A, m/z461; B, m/z289), and blank hair spiked with 7-ACLO at a concentration of 3 pg/mg (C, m/z
461 ; D, m/z289) and CLO.
616
Journal of Analytical Toxicology, Vol. 24, October 2000
were calculated based on the inter- and intraday variability
populations.
The percent relative accuracy was calculated by the following
equation: [(Mean Measured Concentration - Theoretical Concentration)/Theoretical Concentration] x 100%. All data were
acquired and analyzed by HP software, Enhanced G1701BA
ChemStation (vet B.00.00 for Windows NT ver 4.0).
Results
The retention times of 7-ACLO, CLO, and diazepam-d~ were
approximately 10.10 min, 10.77 min, and 9.37 min, respect
A
tively. All chromatograms were recorded over the time range of
9.00 to 11.10 rain. Figure 1 shows selected ion chromatograms
(m/z 461, m/z 289) of the extract from blank hair (Figure 1A
and B), and blank hair spiked with 7-ACLO and diazepam-d5
(Figure 1C and D). The concentration of 7-ACLOwas 3 pg/mg.
Figure 2 shows the selected ion chromatograms (m/z 461) of
the extract from the proximal hair segment (Figure 2A) and
distal segment #8 (Figure 2B) collected from subject #9. The
concentration of 7-ACLO in segment #1 was 1267 pg/mg and in
segment #8, 70 pg/mg. CLO was detected in the first three segments of the hair sample collected from subject #9 (180, 32, and
27 pg/mg, respectively). The selected ion chromatogram (m/z
461) of the extract from hair sample (proximal segment) from
subject #2 is presented in Figure 3. This patient received a
single 2-rag dose of CLO approximately three weeks earlier.
The concentration of 7-ACLO in the proximal hair segment
was 4.8 pg/mg. The distal hair segment was negative for
i m
Clonazepam
A
o s-~
0.4-]
4
I
J
Time (mln)
0.3-I
~
B
lm
/jr
0.2~
t
w
i-
ra
I
4
o.i-I
u
/
m
.
m
m
-
\
Tim
/
oF
i
0
0.2
i
i
0.4
0.6
Amount ratio
(rain)
B
7-Amlnoclonazepam
Figure2. Selectedion chromatograms(m/z461) of the extractfromthe
proximal hair segment, 7-ACLOconcentration1267 pg/mg (A), and
distal segment, 7-ACLOconcentration70 pg/mg (B), collected from
'0
/
subject #9.
;0
;0
10.12
~~.~,~
Tlme (mln)
Figure 3. Selected ion chromatogram (m/z 461) of the extract from proximal hair segment collected from subject #2 (7-ACLO concentration
4.8 pg/mg).
~
s_
~
/
0
yJ
IE
/
:0
o
o
/
J
i
0.5
.
.
.
.
i
.
.
.
.
1
Amount ratio
i
1.5
Figure 4. Standard curves for CLO (A) and 7-ACLO (B).
617
Journal of Analytical Toxicology, Vol. 24, October 2000
7-ACLO. CLO was not detected in either of these segments.
Figure 4 presents the standard curves for CLO (Figure 4A) and
7-ACLO (Figure 4B). They were linear over the range of drugs
assayed (1 pg/mg to 1000 pg/mg for 7-ACLOand 10 pg/mg to
400 pg/mg for CLO) and had correlation coefficients of 0.998.
The limits of quantitation were 1 pg/mg for 7-ACLOand 10
pg/mg for CLO for 50-mg samples. Both limits of quantitation
were arbitrarily established to be the lowest concentrations on
the corresponding standard curves. The respective limits of
detection for CLO and 7-ACLOwere 5 pg/mg and 0.4 pglmg,
which were the lowest concentrations of drug at which the
signal-to-noise ratio was 3:1.
Tables I and II present the accuracy and precision of the CLO
and 7-ACLO control hair preparations, respectively. The intraday variability was determined by analyzing three low and
four high replicates of controls (30 and 300 pg/mg of hair for
CLO and 3 and 800 pg/mg of hair for 7oACLO)prepared in hair
on a single day. The interday variability was determined over a
period of 8 weeks on 10 separate days.
Figure 5 presents concentrations of 7-ACLOin hair samples
collected from subjects #1 and #2. Both subjects received a
single 2-mg dose of CLO approximately three weeks before
hair collection. For subject #1 only traces (belowquantitation
limit) of 7-ACLO were detected; for subject #2 4.8 pg/mg of
7-ACLO were detected. The concentrations of 7-ACLO in
remaining eight hair samples are presented in Figure 6. 7ACLOwas present in measurable quantities in 9 out of 10 hair
samples collected,and the concentration range was 1.37-1267
pg/mg. Figure 7 shows concentrations of parent drug, CLO, in
study samples. It was not detected in 4 out of 10 samples, and
the concentration range was 10.7-180.6 pg/mg.
Discussion
Clonazepam, together with flunitrazepam and nitrazepam,
belongs to the group of 7-nitro- 1,4-benzodiazepin-2-ones(1). In
the human body they all undergo complexbiotransformation,
which includesreduction of the nitro group to the amine (1,13).
All three benzodiazepines and their 7-amino metabolites are
1
2
/ ~
4
3
,+--
//
Y
r
Segments
Table I. Accuracy and Precision of Clonazepam Hair
Preparations (pg/mg)
proximal
distal
Figure 5. Concentrations of 7-ACLO in hair samples collected from subject #1 and #2.
Parameter
Theoretical concentration
Intraday variability
Mean measured
concentration (+ 5.D.)
% CV
% Relative accuracy
Interday variability
Mean measured
concentration (_+S,D.)
% CV
% Relative accuracy
Low control
High control
30
300
N=3
N=4
28,99 (_+2.14)
7./8
-3.37
N = 31
288.81 (_+46.26))
16.00
-3.73
"~1000
N= 29
) OllO
31.88 (_+9.06)
28.42
6.27
291.49 (-+ 73.40)
25.18
-2.84
1401)
L2 #3
3#4
c~ 1200
-,#s
g 81)I)
#6
6TI#7
[=#,
44111
o
o
9
I
2
4
3
Parameter
Theoretical concentration
Intraday variability
Mean measured
concentration (_+S.D.)
% CV
% Relative accuracy
Interday variability
Mean measured
concentration (__.S.D.)
% CV
% Relative accuracy
618
Low control
High control
3
N=3
800
N=4
5
6
7
di,tal
Figure 6. Concentrations of 7-ACLO in hair samples collected from subiects #3-10.
2oo ..... '
' [-,#l
iN#2
ill#3
~ 150"
3.23 (_+0.31)
9.60
7.67
805.96 (_+63.75)
7.91
0.74
N = 30
N = 23
3.24 (_+0.79)
24.45
7.67
836.73(_+ 138.84)
16.59
4.59
#111
8
Segments
|H'U~illlal
Table II. Accuracy and Precision of 7-Aminoclonazepam
Hair Preparations (pg/mg)
11#4
"[ 1 0 0 ~
]
),
"#S
/..'"
o
9 #7
im#8
=
i
proximal
!
; #g
21111
2
3
4
s 6 T
Segments
s
i~#9
m#1o
distil
Figure 7, Concentrations of CLO in all hair samples.
Journal of Analytical Toxicology, Vol. 24, October 2000
basic; therefore, they accumulate in hair and remain present for
extended periods of time. The presence of flunitrazepam and 7aminoflunitrazepam in hair in relatively high quantities has
been well documented (12-17). Cirimele et al. (13) found 26
hair specimens collected from corpses positive for 7-aminoflunitrazepam, flunitrazepam, or both. Flunitrazepam only was
found in 14 samples. All the specimens were positive for 7aminoflunitrazepam. Cirimele et al. (14) also described the determination of chronic flunitrazepam abuse by analysis of a
10-cm hair strand collected from a chronic drug user and divided into 3-cm segments. In another study (17), concentrations
of 7-aminoflunitrazepam in two postmortem hair samples were
found to be higher than corresponding flunitrazepam levels.
NCI-GC-MS offers much better sensitivity than the conventional EI-GC-MS and has been successfully applied to the detection of various benzodiazepines in human hair (12-15,17).
CLO and 7-ACLO, however, have not been included in those
studies. To our knowledge, this is the first report on simultaneous detection and sensitive quantitation of CLO and its major
metabolite 7-ACLOin segmented human hair.
The number of papers on detection of CLO and its metabolites,
including 7-ACLO, in biological specimens is very limited. De
Silva et al. (1) described serious difficultieswith GLC analysis of
blood and urine samples for CLO. Song et al. (9) reported several
analytical problems with the quantitative determination of CLO
in plasma using NCI-GC-MS, such as binding of the compound
to the analytical column, injection port, ion source and
quadrupole. In their study, CLO was found to be active even as
a TMS derivative.In our study, the limit of quantitation for CLO
was 10 pg/mg, and it was necessary to change the liner daily
(practically every 15 injections) in order to keep method validation parameters within acceptable limits because CLO is particularly sensitive to active sites. The standard curve for CLO was
linear overthe concentration range studied. The standard curve
for 7-ACLO had a wide linear concentration range (1
pg/mg-1000 pg/mg). Both standard curves had a correlation
coefficientof 0.998.The limit of quantitation for CLO established
in this study was considerably higher than previously described
for flunitrazepam in hair using similar analytical methodology
(17). In addition, our study revealed that the concentrations of
7-ACLOin hair are much higher than CLO. In subjects #9 and
#10, 7-ACLOwas found in the distal segments that were over 20
cm in length (Figure 5); the concentrations of 7-ACLOwere 70
and 15.3 pg/mg of hair, respectively.Assuming the average hair
growth of 0.8-1.3 cm per month, the presence of 7-ACLO in
these distal segments could represent over a 1.5-year history of
CLO use. This is not surprising because the majority of subjects
participating in the study were taking CLO for longer periods of
time. Out of 10 subjects, 2 who had never been treated with CLO
before received a single dose of the drug. In one case, only traces
of 7-ACLOwere found (subject #1). The analysis of the proximal
hair segment from subject #2 revealed the presence of measurable quantities of 7-ACLO (4.8 pg/mg). The distal segment was
negative for CLO metabolite. These results suggest that hair
may be a very important specimen in drug-facilitated sexual assault investigations when the use of CLO is suspected and considerable delay in reporting of the crime has occurred. Further
research is ongoing at this time.
Conclusions
The analytical method presented in this paper for the determination of CLO and its major metabolite 7-ACLOin hair employs solid-phase extraction and GC-NICI-MS, is extremely
sensitive, and allows accurate and precise quantitation of both
drugs in hair samples collected from CLO users. The 7-ACLO
concentrations in hair were higher than concentrations of the
parent drug, and 7-ACLOremains in hair for extended periods
of time. The developed methodology can possibly be applied to
hair samples collected after administration of a single dose of
the drug as in drug-facilitated sexual assault scenarios.
Acknowledgments
This project was partially supported under Award number
98-LB-VX-K020 from the Office of Justice Programs, National
Institute of Justice, U.S. Department of Justice. Points of view
in this paper are those of the authors and do not necessarily represent the official position of the U.S. Department of Justice.
The authors would like to thank medical personnel and staff of
the Psychiatric Unit of the Universityof Illinois at Chicago Hospital for help throughout the entire project.
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Manuscript received March 31, 2000;
revision received May 16, 2000.