Journal of Analytical Toxicology, Vol. 33, October 2009
Urinary Detection Times and Excretion Patterns
of Flunitrazepam and its Metabolites After
A Single Oral Dose
Malin Forsman, Ingrid Nyström, Markus Roman, Liselotte Berglund, Johan Ahlner, and Robert Kronstrand*
National Board of Forensic Medicine, Department of Forensic Genetics and Forensic Toxicology, Linköping, Sweden
Abstract
We investigated the excretion profiles of flunitrazepam
metabolites in urine after a single dose. Sixteen volunteers
received either 0.5 or 2.0 mg flunitrazepam. Urine samples were
collected after 2, 4, 6, 8, 12, 24, 48, 72, 96, 120, 240, and 336 h.
Samples were screened using CEDIA (300 µg/L cutoff) and
quantitated using liquid chromatography–tandem mass
spectrometry. The cutoff was 0.5 µg/L for flunitrazepam,
N-desmethylflunitrazepam, 7-aminoflunitrazepam,
7-aminodesmethylflunitrazepam, 7-acetamidoflunitrazepam,
and 7-acetamidodesmethylflunitrazepam. None of the subjects
receiving 0.5 mg were screened positive, and only 23 of 102
samples from the subjects given 2.0 mg were positive with CEDIA.
The predominant metabolites were 7-aminoflunitrazepam and
7-aminodesmethylflunitrazepam. For all subjects given the low
dose, 7-aminoflunitrazepam was detected up to 120 h, and for
two subjects for more than 240 h. Seven subjects given the high
dose were positive up to 240 h for 7-aminoflunitrazepam. We
conclude that the ratio 7-aminodesmethylflunitrazepam to
7-aminoflunitrazepam increased with time, independent of dose,
and may be used to estimate the time of intake. For some low-dose
subjects, the metabolite concentrations in the early samples were
low and a chromatographic method may fail to detect the intake.
We think laboratories should consider this when advising police
and hospitals about sampling as well as when they set up strategies
for analysis.
to the enhanced transmission of the signal substance γaminobutyric acid (GABA) in the CNS caused by flunitrazepam. GABA diminishes the transmission of several important signal substances such as noradrenaline, serotonin,
dopamine, and acetylcholine (1). Flunitrazepam is a low dose
sedative with therapeutic doses ranging from 0.5 to 2 mg and
is used in short term treatment of moderate insomnia and as
a premedication for minor surgical procedures (1,2).
Flunitrazepam is quickly absorbed by the small intestine
after oral administration and reaches a maximum plasma concentration within 20–30 min with an elimination half-life of
13–19 h. The clinical effect varies between 4 and 8 h depending on the dosage given. It is metabolized almost completely
in the liver and yields several metabolites. The metabolites
Introduction
Flunitrazepam, along with nitrazepam and clonazepam, is a
member of the 7-nitrobenzodiazepines. In addition to the nitro
group on position 7, it has a fluorine atom on the 2' position
of the phenol ring. Besides its strong hypnotic effect, it also
exerts sedative, anxiolytic, muscle relaxant, and anticonvulsant effects, as seen with other benzodiazepines. This is due
* Author to whom correspondence should be addressed: Robert Kronstrand, National Board of
Forensic Medicine, Department of Forensic Genetics and Forensic Toxicology, Artillerigatan
12, SE 587 58, Linköping, Sweden. E-mail: [email protected].
Figure 1. Structures of flunitrazepam and metabolites. Hydroxymetabolites may also be further conjugated. Abbreviations: Flu, flunitrazepam;
dmFlu, N-desmethylflunitrazepam; 7AFlu, 7-aminoflunitrazepam; 7AdmFlu, 7-aminodesmethylflunitrazepam; 7AcFlu, 7-acetamidoflunitrazepam;
7AcdmFlu, 7-acetamidodesmethylflunitrazepam; 3OHFlu, 3-hydroxyflunitrazepam; 3OH7AFlu, 3-hydroxy-7-aminoflunitrazepam; and 3OH7AcFlu, 3-hydroxy-7-acetamidoflunitrazepam. Hydroxymetabolites were
not included in the analytical method.
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
491
Journal of Analytical Toxicology, Vol. 33, October 2009
7-aminoflunitrazepam, 3-hydroxyflunitrazepam, 3-hydroxy-7aminoflunitrazepam, 7-aminodesmethylflunitrazepam, 7-acetamidoflunitrazepam, 3-hydroxy-7-acetamidoflunitrazepam,
7-acetamidodesmethylflunitrazepam, and N-desmethylflunitrazepam have all been identified in plasma or urine. The
metabolites may also be conjugated prior to renal excretion
(1). Structures for flunitrazepam and metabolites are shown in
Figure 1.
In recent years, reports of drug-facilitated sexual assault
(DFSA) have increased, and benzodiazepines such as flunitrazepam may be used to incapacitate the victim. An increased
CNS depressant effect is seen when flunitrazepam is combined
with alcohol. Therefore, even a small dose added to the victim’s drink may induce drowsiness. The result is a semiconscious or unconscious victim that often suffers from anterograde amnesia (2,3). The fear of being mistrusted by the police
due to impaired memory may lead to several hours or days of
delay before reporting the incident. This time delay makes it
difficult to obtain positive blood and urine samples when investigating the alleged incident.
Several dosing studies have been reported based on single oral
doses of flunitrazepam using gas chromatography coupled with
mass spectrometry (GC–MS) (4–9) or high-performance liquid
chromatography (HPLC) (10) as the confirmation method. In a
study performed by Salamone et al. (5), six volunteers were
given 1 or 4 mg flunitrazepam. For all subjects, 7-aminoflunitrazepam was detected with GC–MS throughout the 72 h sample period with peak concentrations for the low dose after 12–60
h (2–58 µg/L) and after 8–60 h (2–118 µg/L) for the high dose.
In a similar study by Negrusz et al. (6), they investigated the
elimination of 7-aminoflunitrazepam in urine after administration of 2 mg flunitrazepam (n = 10). Using a very sensitive
method, GC–MS with negative chemical ionization, 7-aminoflunitrazepam was detected up to 28 days with peak concentrations
after 6 h (70–518 µg/L). Studies reported by the French Society
of Analytical Toxicology (SFTA) (11) showed an increase in urinary detection time for 7-aminoflunitrazepam, the major
metabolite of flunitrazepam, when using LC–MS–MS compared
to immunoassays such as fluorescence-polarization immunoassay and CEDIA, or other chromatographic techniques
such as LC–diode-array detection (DAD), GC–MS, and LC–MS.
After a single oral dose of 1 mg flunitrazepam, 7-aminoflunitrazepam was detectable up to 48 h with GC–MS and 96 h with
LC–MS, whereas LC–MS–MS gave positive results up to 6 days
after ingestion. This shows the great sensitivity achieved with
LC–MS–MS that is necessary for the detection of low dosage
drugs such as flunitrazepam.
Jourdil et al. (12) reported a positive urine sample from a
DFSA victim using LC–electrospray (ESI)-MS. The sample was
obtained 4–6 h after the incident and contained 11.9 µg/L 7aminoflunitrazepam. Cheze et al. (13) also reported a positive
urine sample obtained from a DFSA victim. Eighty-four hours
after the alleged incident, 2.3 µg/L 7-aminoflunitrazepam was
detected using LC–ESI-MS–MS.
In summary, previous methods have focused the detection
on the metabolite 7-aminoflunitrazepam; however, urinary
concentrations vary tremendously and can seldom be used to
estimate the time of intake. The concentration depends on fac-
492
tors such as dose, urine production, and fluid intake among
others. Providing an estimate of the time of intake may be important, especially in DFSA cases where the involuntary drug
administration is thought to have happened at a certain time.
We have previously reported the use of the metabolite ratio
(for buprenorphine) to estimate time of intake (14).
The aim of this study was to investigate excretion patterns
for several metabolites after a single dose of flunitrazepam.
Two doses were used to examine if metabolite ratios were dosedependent and if these ratios could be used to estimate the
time of intake. In the light of the existing literature, a chromatographic method using LC–MS–MS was developed to be
able to follow the excretion over several days.
Materials and Methods
Chemicals and reagents
The standards flunitrazepam, 7-aminoflunitrazepam,
desmethylflunitrazepam, flunitrazepam-d7, and 7-aminoflunitrazepam-d7 were obtained from Cerilliant (Round Rock,
TX). 7-Aminodesmethylflunitrazepam, 7-acetamidoflunitrazepam, and 7-acetamidodesmethylflunitrazepam were gifts
from Roche (Stockholm, Sweden). Organic and inorganic
chemicals were of gradient-grade or better and purchased
from Merck (Darmstadt, Germany). Deionized water was purified by a Millipore gradient A system from Millipore AB
(Sundbyberg, Sweden). Enzymatic hydrolysis of urine samples
was performed using β-glucuronidase (E. coli K12, 200 U/mL)
purchased from Roche (Mannheim, Germany). Drug-free
urine, used as matrix for controls and calibrators, were obtained from laboratory personnel. Each batch was tested with
immunoassays and analyzed with the confirmation method
before use. A hydrolysis control was obtained from a healthy
nonmedicating laboratory personnel member who voluntarily ingested a 0.5-mg dose of flunitrazepam under controlled
conditions (not part of the study). Urine samples taken 12 and
22 h post-dose were pooled and frozen in 1-mL aliquots. Stock
solutions for calibrators and controls were made separately in
acetonitrile. Working solutions for both calibrators and controls were prepared separately at 0.05 and 1.0 mg/L.
Study design
The study was approved by the Regional Research Ethics
Committee in Linköping, Sweden (#M2-08). Through advertising, 16 healthy volunteers were recruited to the study after having signed an informed consent form and a health declaration.
Eleven females aged 20 to 31 years (mean 25) and 5 males aged
21 to 32 years (mean 26) participated. Their mean body mass
indexes were 21.8 and 24.6, respectively. The subject demographics are shown in Table I. There were no specific inclusion
criteria. Exclusion criterions for participating in the study were
ongoing medication with benzodiazepines, opiates or opioids,
or a documented abuse of any of these substances or alcohol.
Ongoing medication with erythromycin, antidepressants, antipsychotics, and antihistamines were also considered exclusion
criteria, as were pregnancy, nursing, and participation in
Journal of Analytical Toxicology, Vol. 33, October 2009
another study during the time of this study. A single oral dose
of either 0.5 or 2.0 mg flunitrazepam (Flunitrazepam, NM
Merck) was given to the subjects in a randomized fashion. The
dose given to each subject is shown in Table I. Prior to dosing,
urine samples were collected from each subject; thereafter,
urine samples were provided at approximately 2, 4, 6, 8, 12, 24,
48, 72, 96, 120, 240, and 336 h post-dose. The subjects remained
at the clinic and were monitored in case of adverse effects for approximately 8–10 h following drug administration. Urine samples from day 1 and onwards were collected at home, stored refrigerated according to our instruction, and delivered to the
laboratory on a daily basis.
Samples
The urine samples were collected in plastic bottles and
stored refrigerated (4°C) prior to aliquoting. A 1-mL aliquot
was subjected to immunoassay, and one 1-mL aliquot and
one 2-mL aliquot were frozen in 10-mL plastic tubes for
LC–MS–MS analysis. Twenty-five milliliters of the remaining
urine were transferred into plastic tubes for further storage at
–20°C. The urine samples were first screened for benzodiazepines using CEDIA reagent with a 300 µg/L cutoff, and the
urine creatinine concentration was determined with the Jaffé
method. Both analyses were performed on an ADVIA 1650
from Bayer AB (Gothenburg, Sweden).
1.0 and 100 µg/L. The negative control sample consisted of
1 mL drug-free urine and was prepared as an authentic sample,
as was the hydrolysis control. To 1 mL of urine was added 50 µL
of internal standard] flunitrazepam-d7 (100 µg/L) and 7aminoflunitrazepam-d7 (400 µg/L)], 40 µL of β-glucuronidase,
and 1 mL of 50 mM acetate buffer (pH 6.0). Enzymatic hydrolysis was performed by incubation at room temperature for 30
min. Before extraction another 2 mL of 50 mM acetate buffer
(pH 6.0) was added to the urine sample. For the solid-phase extraction, BondElut 130 mg Certify Columns (Varian, Palo Alto,
CA) were activated and conditioned with 2 mL of methanol followed by 2 mL of 50 mM acetate buffer (pH 6.0). The sample
was drawn through the column by gravity, which then was
rinsed with 2 mL of deionized water and 1 mL of 1 M acetic acid.
The column was dried for 20 min (10 in. Hg), and then washed
with 1 mL of methanol and dried for 1 min. The analytes were
eluted with a 2 mL mixture of dichloromethane/2-propanol
(80:20, v/v) containing 2% ammonia (25%). The eluate was
evaporated in a GeneVac DD-4 (Genevac, Ipswich, U.K.) at 40°C
and 5 psi nitrogen for 25–30 min. The residue was reconstituted
in a 200-µL mixture of 20 mM ammoniumformiate
buffer/acetonitrile (80:20, v/v). A 1-µL aliquot was injected into
the chromatographic system.
Samples were analyzed in batches of 12 authentic samples
along with two positive controls, one negative control, and one
hydrolysis control.
Extraction and quantification
The calibration curve and the two positive control samples
were made by addition of standard or control solutions to 1 mL
of drug-free urine that then were treated as authentic samples.
Calibrator concentrations were 0.25, 0.5, 5.0, 20, 50, 75, 100,
150, and 200 µg/L. If a sample had concentrations above the
highest calibrator, it was re-analyzed with less sample volume.
The two positive control samples had a final concentration of
Instrumentation
The LC–MS–MS system consisted of a Waters ACQUITY
Table II. Retention Times, MRM Transitions, MS–MS
Parameters, Internal Standard, and Qualifier Target Ratio
for Each Analyte
Table I. Demographics of the 16 Subjects and the
Individual Doses Given
Subject
FP 1
FP 2
FP 3
FP 4
FP 5
FP 6
FP 7
FP 8
FP 9
FP 10
FP 11
FP 12
FP 13
FP 14
FP 15
FP 16
Sex
(Male/Female)
F
F
M
M
M
M
F
F
F
F
F
F
F
F
F
M
* BMI = body mass index.
Age Height
(years) (cm)
31
26
32
31
25
21
22
22
26
24
22
28
28
22
20
21
165
158
172
172
169
182
159
168
162
163
168
169
168
174
167
176
Weight
(kg)
BMI*
Dose
(mg)
57
50
75
72
70
86
50
56
59
65
75
63
58
68
58
70
20.9
20.0
25.4
24.3
24.5
26.0
19.8
19.8
22.5
24.5
26.6
22.1
20.5
22.5
20.8
22.6
0.5
2.0
2.0
0.5
2.0
0.5
0.5
2.0
0.5
2.0
0.5
2.0
0.5
2.0
2.0
0.5
Analyte
Rt
(min)
Flu
Flu Qualifier
dmFlu
dmFlu Qualifier
7AFlu
7AFlu Qualifier
7AdmFlu
7AdmFlu Qualifier
7AcFlu
7AcFlu Qualifier
7AcdmFlu
7AcdmFlu Qualifier
Flu-d7
7AFlu-d7
1.68
1.51
1.17
0.97
1.21
1.04
1.67
1.16
MRM
Transition*
(m/z)
Qualifier
Target
DP† EP CE CXP Ratio
Q1 Q3 (V) (V) (V) (V) (%)
314
314
300
300
284
284
270
270
326
326
312
312
321
291
268
239
254
225
135
227
121
222
219
227
205
121
275
138
90
90
90
90
99
99
85
85
93
93
45
45
93
99
15
15
9
9
15
15
8
8
7
7
6
6
9
9
37
48
36
49
38
36
41
35
41
42
47
52
38
41
15
15
15
15
10
15
8
17
12
18
11
5
15
11
30
25
55
40
95
90
* Dwell time was 10 ms for all transitions.
† Abbreviations: DP, declustering potential; EP, entrance potential; CE, collision
energy; CXP, collision cell exit potential; Flu, flunitrazepam; dmFlu,
N-desmethylflunitrazepam; 7AFlu, 7-aminoflunitrazepam; 7AdmFlu,
7-aminodesmethylflunitrazepam; 7AcFlu, 7-acetamidoflunitrazepam; and
7AcdmFlu, 7-acetamidodesmethylflunitrazepam.
493
Journal of Analytical Toxicology, Vol. 33, October 2009
UPLC® (ultra-performance liquid chromatography) with a Binary Solvent Manager, Sample Manager, and Column Manager
(Waters, Milford, MA) connected to an API 4000™ triplequadrupole instrument (Applied Biosystems/MDS Sciex,
Stockholm, Sweden) equipped with an electrospray interface
(TURBO V™ source, TurboIonSpray® probe) operating in the
multiple reaction monitoring (MRM) mode. Ion spray voltage
was set to 5500 V. Nitrogen was used as nebulizer gas (345
kPa), heater gas (517 kPa at 500°C), curtain gas (207 kPa), and
as collision-activated dissociation gas (set on 5). UPLC was performed using an ACQUITY UPLC® ethylene-bridged hybrid
(BEH) C18 column (1.7 µm, 50 × 2.1 mm, Waters) preceded by
a 0.2-µm column filter (Waters) operated at 0.6 mL/min with
a total run time of 3 min. Mobile phase A consisted of 0.05%
formic acid in 10 mM ammoniumformiate and phase B of
0.05% formic acid in acetonitrile. The chromatographic system was run in a linear gradient from 5 to 65% phase B in 2
min, then up to 95% phase B for 0.5 min followed by a 0.5 min
equilibration with 95% phase A. The injection volume was 1
µL, and the Column Manager temperature was set to 60°C. Instrument control, integration, and calculation were performed
using Analyst™ 1.4.2 software. Quadratic regression with 1/x
weighting was used for the calibration curves. The final MRM
method included 14 transitions, the two most intense transitions for each analyte, with a dwell time of 10 ms for each transition resulting in a total scan time of 0.21 s. The mass spectrometric details of the method are listed in Table II. Criteria
for identification were based on a qualifier ratio within 20% of
the target ratio.
area of set 2 with set 1. Extraction recovery was calculated by
dividing the mean area of set 3 with set 2. The within-day and between-day imprecision were estimated by analysis of control
samples at 1.0 and 100 µg/L, as well as an authentic sample (the
hydrolysis control) in the same batch (n = 5) and on different
days during a period of four weeks (n = 15).
Results
An extracted ion chromatogram of a 20 µg/L calibrator is
shown in Figure 2. The sub-2 micron particles and the high
flow rate resulted in a short elution time with the first analyte
eluting at 0.97 min and the last at 1.68 min. Calibration curves
were best fitted to quadratic models. The solid-phase extraction procedure had high recoveries for the amino- and acetamidometabolites, whereas flunitrazepam and desmethylflunitrazepam had lower recoveries (Table III). Despite the
solid-phase extraction method including a 100% methanol
wash, matrix effects were observed (Table III) and appeared dependent on urine creatinine. The close retention time span
and the use of deuterated internal standards partly compensated for matrix effects in the quantitative results. The accu-
Validation experiments
Ion suppression was studied by post-column infusion of every
analyte individually at 250 µg/L for flunitrazepam and 7aminoflunitrazepam and 500 µg/L for the other four metabolites (15). Infusion was done at a flow rate of 10 µL/min with a
Harvard Apparatus 11 Plus syringe pump (Applied Biosystems/MDS Sciex, Stockholm, Sweden). The most intense MRM
transition for each analyte was recorded. Drug-free urine samFigure 2. Extracted and overlayed ion chromatograms for a 20 µg/L calibrator. Peak identification: 1, 7AdmFlu; 2, 7AcdmFlu; 3, 7AFlu-d7; 4,
ples from five subjects with varying creatinine concentrations
7AFlu; 5, 7AcFlu; 6, dmFlu; 7, Flu-d7; and 8, Flu. The concentrations
(0.4–5.3 g/L) were extracted according to the assay and injected
for the internal standards are 20 µg/L (7AFlu-d7) and 5 µg/L (Flu-d7). Both
with the same chromatographic conditions used in the final
are shown with dashed traces.
method. Quantitative matrix effects together with extraction recovery was also investigated by determination
of peak areas of the analytes in three different
Table III. Matrix Effects and Extraction Recovery at 50 µg/L
sets of samples (16). One set (set 1) consisting
of neat standards [50 µg/L in 20 mM ammoFlu
dmFlu
7AFlu
7AdmFlu 7AcFlu 7AcdmFlu
nium formate buffer/acetonitrile (80:20, v/v)],
†
Creatinine ME* ER ME ER ME ER ME ER ME ER ME ER
one prepared in blank matrix extracts from five
Sample
(g/L)
(%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%)
different sources and spiked (50 µg/L) after extraction (set 2), and one prepared in blank ma1
0.4
28
80
6 70 18 91 26 85 26 108 23 97
trix from the same sources but spiked before
2
1.0
14
51 15 60 21 83 35 89 22 82 24 87
extraction (set 3). The same urine samples that
3
2.2
25
56 18 63 25 77 43 98 50 86 33 81
were used for post-column infusion experi4
3.0
33
47 25 58 51 88 57 90 76 79 45 77
ments were also used for quantitative matrix ef5
5.3
34
55 34 67 59 95 68 93 75 97 57 108
fects and extraction recovery experiments. The
Mean
27
58 20 64 35 86 46 90 50 92 36 89
analyte peak areas were used to estimate the
* ME = Matrix effects.
quantitative matrix effects by dividing the mean
† ER = Extraction recovery.
494
Journal of Analytical Toxicology, Vol. 33, October 2009
racy and the within-day and between-day imprecision are presented in Tables IV–V.
A total of 204 urine samples were collected over a period of
14 days after dosing. Actual sampling time was in a 10% range
of the scheduled sampling time. Only one subject could not
provide the urine samples at 2 and 4 h after dose due to adverse effects (dizziness and nausea), and two subjects forgot to
provide a urine sample at 12 h post-dose. Even though pharmacological effects were not included in the scope of this
study, we observed that several subjects encountered adverse
(or normal) effects of the drug, especially drowsiness and an-
terograde amnesia; however, 8 h after administration, most
subjects showed few signs of impairment. The adverse effects
were more pronounced after the high dose.
In addition to the LC–MS–MS method, all samples were subject to immunochemical screening using the CEDIA reagent at
the proposed cutoff at 300 µg/L. None of the samples from subjects given 0.5 mg flunitrazepam presented with a positive
screening result. Twenty-three of those from subjects receiving
Table IV. Within-Day Imprecision (n = 5)
Low (1.0 µg/L) High (100 µg/L)
Analyte
Flu
dmFlu
7AFlu
7AdmFlu
7AcFlu
7AcdmFlu
Accuracy CV
(%)
(%)
106
106
106
104
103
109
11.2
7.1
2.1
7.7
8.4
7.0
Authentic Sample*
Accuracy CV Mean Accuracy CV
(%)
(%) (µg/L)
(%)
(%)
94
101
96
98
103
103
9.9 < 0.5
7.8
0.73
3.1 140
6.7 23.0
3.9
2.43
2.3 13.6
—
—
—
—
—
—
—
13.3
6.5
11.2
6.2
13.4
Figure 4. Mean metabolite excretion profiles after 0.5 mg single dose
corrected for creatinine concentration (n = 8). Error bars represent standard error of the mean (s.e.m.).
* Hydrolysis control as described in the Chemicals and reagents section.
Table V. Between-Day Imprecision (n = 15)
Low (1.0 µg/L) High (100 µg/L)
Analyte
Flu
dmFlu
7AFlu
7AdmFlu
7AcFlu
7AcdmFlu
Accuracy CV
(%)
(%)
104
114
97
103
91
92
13.8
16.0
7.7
12.9
14.9
10.6
Authentic Sample*
Accuracy CV Mean Accuracy CV
(%)
(%) (µg/L)
(%)
(%)
104
110
95
102
89
88
12.5
14.3
5.5
7.3
10.8
8.6
< 0.5
0.80
138
19.6
2.53
13.1
—
—
—
—
—
—
—
23.2
6.4
12.8
15.2
13.2
* Hydrolysis control as described in the Chemicals and reagents section.
Figure 3. Mean excretion profiles of 7-aminoflunitrazepam corrected for
creatinine concentration. N = 8 for 0.5-mg dose and n = 8 for 2.0 mg
dose. Error bars represent standard error of the mean (s.e.m.).
Figure 5. Mean metabolite excretion profiles after 2.0 mg single dose
corrected for creatinine concentration (n = 8). Error bars represent standard error of the mean (s.e.m.).
Figure 6. Mean 7-aminodesmethylflunitrazepam to 7-aminoflunitrazepam ratios for the 16 subjects during 240 h. The insert graph shows
the mean ratios during the first 8 h (n = 8 for 0.5 mg dose and n = 8 for
2.0 mg dose). Error bars represent standard error of the mean (s.e.m).
X-axis is scheduled time for sampling.
495
Journal of Analytical Toxicology, Vol. 33, October 2009
Table VI. Results From Each Subject Given 0.5 mg Flunitrazepam
Time
(h)
Flu
(µg/L)
7AFlu
(µg/L)
dmFlu
(µg/L)
FP 1
0.0
2.0
3.9
6.0
8.0
12.0
23.0
46.5
70.5
94.0
118.5
240.3
335.0
–†
–
0.8
–
–
–
–
–
–
–
–
–
–
0.0
0.6
7.8
6.5
3.7
87
90
43
33
26
10
–
–
–
–
–
–
–
0.5
–
–
–
–
–
–
–
–
–
–
0.7
0.7
7.7
17
16
22
19
14
1.0
–
–
–
–
–
0.6
1.1
1.1
–
–
–
–
–
–
FP 4
0.0
2.0
4.0
6.0
8.0
12.0
22.8
46.8
70.8
94.8
116.8
239.0
335.0
–
–
1.9
1.0
–
–
–
–
–
–
–
–
–
–
0.5
5.0
5.9
4.8
41
67
29
15
15
4.7
0.6
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1.0
1.4
4.6
13
10
8.7
9.0
3.7
–
–
FP 6
0.0
2.3
4.3
6.3
8.3
12.0
24.3
48.8
72.8
96.3
120.3
243.0
336.3
–
–
1.1
0.8
0.5
n.a.‡
–
–
–
–
–
–
–
–
0.7
13
33
43
n.a.
54
13
22
13
6.3
0.5
–
–
–
–
0.5
–
n.a.
–
–
–
–
–
–
–
FP 7
0.0
2.3
4.3
6.3
8.3
12.3
22.3
48.3
72.3
96.3
120.3
240.3
334.3
–
–
–
0.6
–
–
–
–
–
–
–
–
–
–
0.5
3.0
6.6
9.7
31
3.4
11
8.9
8.6
0.7
–
–
–
–
–
–
–
0.5
–
–
–
–
–
–
–
Subject
* Absorbance rate in comparison to calibrator’s (positive number = above cutoff).
† – = negative result.
‡ n.a. = not available because no sample was obtained.
§ Sample was confirmed positive for oxazepam from ingestion outside the study.
496
7AdmFlu
(µg/L)
7AcFlu
(µg/L)
7AcdmFlu
(µg/L)
CEDIA*
Creatinine
(g/L)
–
–
–
–
0.7
3.9
6.1
4.5
7.2
7.6
4.4
–
–
–131
–149
–129
–133
–131
–12
0
–69
–71
–90
–115
–150
–152
5.27
0.24
0.47
0.38
0.51
1.86
2.17
1.43
2.19
3.01
2.50
2.36
1.53
–
–
1.0
1.3
1.2
2.7
3.3
1.3
0.8
0.7
–
–
–
–
–
0.7
1.7
1.4
5.8
17
13
7.2
6.4
3.1
–
–
–144
–149
–136
–128
–131
–78
–35
–99
–125
–127
–150
–155
–169
2.72
0.44
0.40
0.63
0.59
1.29
2.66
1.83
1.95
2.60
1.29
2.18
1.18
–
–
0.8
1.7
3.2
n.a.
11
6.0
8.3
5.6
4.6
–
–
–
–
–
1.3
1.6
n.a.
1.4
–
–
–
–
–
–
–
–
–
1.4
2.3
n.a.
8.4
3.7
3.9
2.2
1.7
–
–
–155
–150
–128
–91
–76
n.a.
–59
–132
–121
–126
–143
–154
–164
0.01
0.20
0.67
1.63
2.03
n.a.
2.55
1.37
2.17
2.41
2.34
1.44
2.37
–
–
0.7
2.3
3.8
10
2.8
8.7
7.6
7.9
1.8
–
–
–
–
0.6
1.7
2.0
2.4
0.6
–
–
–
–
–
–
–
–
0.6
2.3
4.0
11
2.2
3.9
4.6
3.9
0.7
–
–
–149
–149
–128
–101
–83
–53
–141
–132
–131
–89
174§
–59
76
0.14
0.10
0.28
0.51
0.73
1.27
0.17
0.59
0.59
0.58
0.26
0.14
0.65
Journal of Analytical Toxicology, Vol. 33, October 2009
Table VI. (Continued) Results From Each Subject Given 0.5 mg Flunitrazepam
Time
(h)
Flu
(µg/L)
7AFlu
(µg/L)
dmFlu
(µg/L)
7AdmFlu
(µg/L)
7AcFlu
(µg/L)
FP 9
0.0
2.0
4.0
6.0
8.0
11.6
24.6
47.6
72.6
96.0
120.0
239.6
335.6
–†
0.5
1.1
0.5
–
–
–
–
–
–
–
–
–
–
5.3
7.7
6.7
13
50
54
23
15
5.5
1.8
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0.7
1.2
1.6
3.7
9.8
18
12
12
6.3
2.8
0.5
–
–
–
–
–
0.8
0.8
0.6
–
–
–
–
–
–
FP 11
0.0
2.0
4.0
6.0
8.0
12.0
23.6
47.0
70.6
93.0
119.3
239.3
335.4
–
–
0.5
–
–
–
–
–
–
–
–
–
–
–
2.0
11
27
13
43
52
19
14
5.4
2.4
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1.4
3.8
2.6
7.3
15
9.7
8.1
4.5
2.5
–
–
FP 13
0.0
2.0
4.0
6.0
8.0
12.0
23.0
47.6
71.6
95.3
119.6
239.4
335.0
–
–
0.6
0.8
–
n.a.‡
–
–
–
–
–
–
–
–
0.9
2.8
19
5.8
n.a.
100
48
16
5.1
3.5
–
–
–
–
–
–
–
n.a.
0.8
0.5
–
–
–
–
–
0.0
2.2
4.2
6.2
8.2
11.9
22.9
46.9
70.9
95.4
118.9
239.3
335.0
–
0.7
–
–
–
–
–
–
–
–
–
–
–
–
4.4
4.7
8.2
12
5.1
38
23
8.2
1.7
1.3
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Subject
FP 16
7AcdmFlu
(µg/L)
CEDIA*
Creatinine
(g/L)
–
–
0.5
0.7
2.3
3.3
4.2
3.2
3.9
1.6
1.2
–
–
–153
–142
–133
–133
–123
–86
–82
–117
–126
–149
–156
–167
–166
1.00
0.61
0.37
0.43
0.90
1.02
0.97
1.07
1.59
0.96
0.67
1.42
0.83
–
0.5
3.1
3.9
2.7
3.4
3.2
1.9
0.7
–
–
–
–
–
–
1.8
4.6
3.8
9.0
20
12
9.1
6.7
2.1
–
–
–157
–157
–135
–115
–137
–98
–80
–123
–139
–146
–159
–165
–165
1.19
0.19
0.58
0.84
0.49
1.16
1.86
1.59
1.39
1.54
0.90
2.34
1.87
–
–
–
1.3
1.2
n.a.
14
9.5
5.3
1.9
1.9
–
–
–
–
0.8
3.0
2.4
n.a.
6.6
2.7
0.9
–
–
–
–
–
–
0.9
3.5
4.3
n.a.
41
25
10
3.5
2.5
–
–
–148
–160
–148
–122
–136
n.a.
–27
–74
–135
–154
–153
–165
–143
1.80
0.14
0.30
0.62
0.45
n.a.
1.90
2.89
1.64
0.91
1.00
1.39
2.08
–
–
0.8
1.9
2.8
1.3
7.5
8.8
4.2
1.2
1.0
–
–
–
1.1
1.9
4.2
6.9
2.2
6.3
3.8
1.5
–
–
–
–
–
0.9
1.7
4.7
9.6
3.8
28
30
13
2.8
2.3
–
–
–165
–158
–158
–148
–134
–158
–81
–111
–148
–161
–158
–171
–174
2.18
0.79
0.46
0.78
1.32
0.29
1.82
2.24
1.82
0.77
1.24
1.01
2.55
* Absorbance rate in comparison to calibrator’s (positive number = above cutoff).
† – = negative result.
‡ n.a. = not available because no sample was obtained.
497
Journal of Analytical Toxicology, Vol. 33, October 2009
Table VII. Results From Each Subject Given 2.0 mg Flunitrazepam
Time
(h)
Flu
(µg/L)
7AFlu
(µg/L)
dmFlu
(µg/L)
7AdmFlu
(µg/L)
7AcFlu
(µg/L)
7AcdmFlu
(µg/L)
CEDIA*
Creatinine
(g/L)
FP 2
0.0
1.7
3.9
6.0
8.0
13.0
25.0
49.0
72.5
96.4
130.0
240.3
336.5
–†
3.4
2.5
1.8
1.0
1.2
1.0
–
–
–
–
–
–
–
6.5
12
16
18
195
334
148
84
24
12
0.6
–
–
–
–
–
–
1.4
2.5
1.0
1.3
–
–
–
–
–
–
1.9
5.1
8.4
44
95
61
33
13
10
1.1
–
–
1.8
9.2
20
21
40
59
21
13
3.2
1.5
–
–
–
1.1
7.2
19
29
99
190
199
131
36
26
1.7
–
–142
–136
–110
–94
–87
63
107
35
–12
–108
–120
–157
–157
3.24
0.17
0.25
0.50
0.77
1.36
2.22
3.18
2.34
1.59
1.70
1.63
1.48
FP 3
0.0
1.8
3.9
6.0
8.0
12.3
23.5
47.2
71.0
95.0
123.2
242.3
335.3
–
0.6
1.8
1.5
1.0
0.9
1.0
–
–
–
–
–
–
–
1.6
5.6
9.8
9.1
240
355
144
74
30
18
0.5
–
–
–
–
–
–
1.7
2.2
1.3
1.0
–
–
–
–
–
–
–
1.6
2.0
45
57
35
266
13
10
–
–
–
0.9
4.5
8.1
9.5
43
36.
15
8.1
2.1
2.1
–
–
–
–
2.8
8.8
9.8
53
102
93
63
24
18
0.9
–
–142
–143
–123
–108
–115
71
96
35
–32
–104
–119
–149
–159
1.41
0.12
0.23
0.50
0.45
1.79
2.44
1.87
1.68
1.23
1.46
1.32
0.78
FP 5
0.0
1.9
3.9
5.9
7.9
11.9
24.2
47.6
74.6
98.0
121.3
241.0
335.3
–
0.8
1.0
0.9
0.8
1.0
0.5
–
–
–
–
–
–
–
4.5
11
21
43
163
130
54
34
17
13
0.8
–
–
–
–
–
–
0.7
–
–
–
–
–
–
–
–
–
0.7
3.0
5.5
23
29
28
19
12
13
1.4
–
–
–
–
1.3
2.0
3.1
2.0
1.2
–
–
–
–
–
–
–
0.5
1.4
3.3
6.6
10
8.4
4.7
4.8
5.4
0.8
–
–137
–133
–121
–98
–81
44
35
–83
–103
–127
–137
–155
–165
2.17
0.34
0.22
0.61
0.99
1.03
1.02
1.12
1.11
1.15
1.35
2.41
1.33
FP 8
0.0
2.3
4.3
6.3
8.3
12.3
24.3
48.8
72.8
96.3
120.3
243.0
336.3
–
1.1
1.0
1.2
0.7
0.8
1.4
0.5
–
–
–
–
–
–
3.9
15
22
21
208
412
161
120
46
21
0.6
–
–
–
–
0.6
–
1.3
3.6
1.4
1.4
0.8
–
–
–
–
–
2.0
3.5
6.0
28
129
76
72
41
29
1.6
0.9
–
–
1.0
2.2
2.0
4.5
7.9
1.9
1.4
0.6
0.3
–
–
–
–
0.9
2.6
3.8
13
57
17
26
12
7.6
0.8
0.4
–137
–130
–111
–80
–90
54
138
51
45
–54
–96
–154
–161
2.99
0.57
0.44
0.74
0.63
1.29
3.23
1.22
2.27
1.66
1.54
1.84
3.65
Subject
* Absorbance rate in comparison to calibrator’s (positive number = above cutoff).
† – = negative result.
498
Journal of Analytical Toxicology, Vol. 33, October 2009
Table VII. (Continued) Results From Each Subject Given 2.0 mg Flunitrazepam
Time
(h)
Flu
(µg/L)
7AFlu
(µg/L)
dmFlu
(µg/L)
7AdmFlu
(µg/L)
7AcFlu
(µg/L)
7AcdmFlu
(µg/L)
CEDIA*
Creatinine
(g/L)
FP 10
0.0
2.0
4.0
6.0
8.0
12.6
24.3
48.3
72.4
96.3
120.3
239.8
336.0
–†
0.7
2.0
0.8
1.2
1.8
0.5
0.7
–
–
–
–
–
–
4.0
14
13
76
262
255
189
73
69
33
1.6
–
–
–
–
–
0.5
1.4
1.0
0.8
–
–
–
–
–
–
–
0.6
1.2
6.1
34
42
54
20
24
9.9
1.2
–
–
–
1.1
1.0
3.7
5.7
8.2
4.7
1.8
1.2
1.0
–
–
–
–
0.6
1.4
3.5
15
23
25
9.4
9.9
4.0
0.6
–
–146
–142
–113
–119
–56
93
72
64
–36
–46
–113
–164
–156
2.39
0.25
0.22
0.24
0.57
1.31
1.68
1.58
1.04
1.68
1.12
1.02
1.69
FP 12
0.0
2.0
4.0
6.3
8.0
12.0
23.6
47.6
71.6
95.6
119.6
240.0
338.0
–
5.5
5.3
1.0
0.8
0.6
–
–
–
–
–
–
–
–
26
80
28
18
46
122
50
35
9.1
3.2
–
–
–
1.3
2.6
0.8
0.6
1.2
–
–
–
–
–
–
–
–
1.1
7.6
11
5.7
30
54
40
34
12
5.4
0.5
–
–
0.6
1.9
2.1
1.0
2.3
2.4
0.6
0.4
–
–
–
–
–
–
1.9
3.9
2.2
10
11
7.0
8.4
2.3
0.8
–
–
–153
–107
14
–76
–97
–45
22
–63
–79
–133
–151
–172
–169
1.11
0.24
0.44
0.51
0.26
1.11
1.08
0.73
1.04
0.65
0.46
0.53
0.81
FP 14
0.0
2.0
4.0
6.0
8.0
12.0
24.0
45.6
70.8
95.0
120.0
247.6
336.0
–
1.1
1.3
1.4
1.3
0.6
1.2
–
–
–
–
–
–
–
5.7
9.7
24
37
29
136
58
20
11
11
1.2
–
–
–
–
–
0.7
–
1.1
–
–
–
–
–
–
–
0.8
2.0
6.4
7.6
10
35
25
13
7.6
4.5
1.0
–
–
1.9
4.8
11
11
5.7
13
6.2
3.4
1.1
0.9
–
–
–
1.1
3.3
11
21
13
60
36
14
6.8
8.5
1.4
–
–174
–151
–139
–104
–79
–101
54
–54
–123
–146
–135
–168
–179
0.36
0.23
0.22
0.62
0.86
0.50
1.37
1.07
0.74
0.45
1.43
2.17
1.30
FP 15
0.0
2.0
4.0
6.2
8.3
12.2
24.2
48.2
72.7
96.6
120.8
240.2
335.4
–
n.a.‡
n.a.
2.5
1.3
0.8
1.0
1.0
0.6
–
–
–
–
–
n.a.
n.a.
95
75
8.0
232
164
95
44
40
1.0
–
–
n.a.
n.a.
2.4
2.2
–
3.2
3.1
1.7
0.7
0.7
–
–
–
n.a.
n.a.
7.2
16
2.6
57
86
63
37
28
2.5
0.6
–
n.a.
n.a.
6.7
10
1.4
12
10
3.7
2.1
1.3
–
–
–
n.a.
n.a.
5.3
17
2.9
51
86
52
33
36
2.1
0.6
–153
n.a.
n.a.
36
39
–126
101
94
28
–58
–65
–161
–167
3.18
n.a.
n.a.
1.16
1.85
0.49
2.05
2.62
1.93
1.48
2.77
1.80
2.49
Subject
* Absorbance rate in comparison to calibrator’s (positive number = above cutoff).
† – = negative result.
‡ n.a. = not available because no sample was obtained.
499
Journal of Analytical Toxicology, Vol. 33, October 2009
2.0 mg were screened positive. These samples were obtained
between 4 and 72 h post-dose. Results for all subjects are presented in Tables VI–VII. Excretion graphs are shown in Figures 3–5. Time on X-axis is the norm time for sampling. Within
one dose, the concentrations varied considerably between the
subjects but followed similar excretion patterns. Figure 6
depict the ratio of 7-aminodesmethylflunitrazepam to 7aminoflunitrazepam showing a steady increase over time.
There was a difference in the slope before and after the time for
the peak concentration.
Discussion
The most important findings from this study were 1. that the
evaluation of metabolite ratios may be used to estimate the time
of intake, 2. that in the first 2–4 h after intake, the metabolite
concentrations were very low and may go undetected, and 3.
with a standard immunoassay screening it was impossible to detect a low dose intake. These three findings all have implications
for DFSA cases. The first because the toxicologist may be able to
corroborate the victim’s story of when the assault happened; the
second because the common direction for samplings are to obtain samples as soon as possible after the assault; and the third
because many laboratories rely on immunoassays for their
screening. Immunoassays with increased performance towards
low dose benzodiazepines are available (7,9), but to what extent
these are used in any but specialized laboratories are not well
known. For chromatographic methods, the Society of Forensic
Toxicologists DFSA committee has suggested a 5 µg/L cutoff (17)
for 7-aminoflunitrazepam that, in the light of our results, seems
adequate for most DFSA cases if samples aren’t obtained very
early after ingestion.
The quantitative data from 7-aminoflunitrazepam from our
study are consistent with other reports in the literature. Peak
concentrations ranged between 11 and 100 µg/L for 7-aminoflunitrazepam, 9 and 22 µg/L for 7-aminodesmethylflunitrazepam,
and 4 and 41 µg/L for 7-acetamidodesmethylflunitrazepam after
the low dose, and between 122 and 412 µg/L, 28 and 95 µg/L, and
10 and 199 µg/L, respectively, for the high dose.
Peak concentrations appeared relatively late after administration as can be seen in Figures 3–5. For 7-aminoflunitrazepam, the concentration usually peaked either after 12 h (n
= 4) or 24 h (n = 4) for the low dose and after 12 h (n = 3) or
24 h (n = 5) for the high dose. Jourdil et al. (12) reported a
positive urine sample from a DFSA victim where the sample
obtained 4–6 h after the incident contained 11.9 µg/L 7aminoflunitrazepam. This is in agreement with our findings
and is also a good example on the low concentrations obtained
shortly after an intake.
The predominant metabolite found in our study was 7aminoflunitrazepam followed by 7-aminodesmethylflunitrazepam and 7-acetamidodesmethylflunitrazepam, which
both had very similar excretion profiles. The metabolite 3-OHflunitrazepam has also been detected after chronic use but was
not included in our study (12). The parent compound and Ndesmethylflunittrazepam were only found in very low concen-
500
trations, which are consistent with previous reported findings
(5,12). We chose to follow the ratio of 7-aminodesmethylflunitrazepam to 7-aminoflunitrazepam, two of the most predominant metabolites, and found that the ratio increased with
time independent of the dose given. Thus, instead of using the
concentration, which may differ because of dose or urine dilution, the ratio seems a better means to estimate the time of
intake. A ratio of less than 0.1 strongly suggests that intake
was less than 4 h earlier and a ratio over 0.6 that more than 72
h have passed since ingestion. In addition, the presence of
other minor metabolites or even the parent compound may
also help in the interpretation of time of intake.
Urinary detection times of flunitrazepam metabolites are,
of course, dependent on method and cutoffs used, and some
researchers have developed very sensitive methods to overcome late sampling after a DFSA (6,12). Negrusz et al. (6),
for example, reported urinary detection times for 7-aminoflunitrazepam of 28 days after administration of 2 mg using a
very low cutoff of 10 pg/mL. Our main objective for this study
was not increased sensitivity, even though we lowered the
threshold considerably in relation to our routine method
that has a 10 µg/L threshold for 7-aminoflunitrazepam. Even
at that cutoff, the samples from this study presented positive
for up to five days (range 3–5 days), probably a long enough
detection time even for samples taken later in the excretion
time after a single dose. After the low dose, the three predominant metabolites were detected together up to five days
after dose; however, in four subjects either 7-aminoflunitrazepam or 7-aminodesmethylflunitrazepam were found
above the 0.5 µg/L cutoff after 10 days. After the high dose,
six of the subjects showed positive samples for all three
predominant metabolites up to 10 days after dose. One subject
was positive for 7-aminodesmethylflunitrazepam and 7acetamidodesmethylflunitrazepam in the very last urine
sample obtained after 14 days.
Conclusions
We conclude that the ratio of 7-aminodesmethylflunitrazepam to 7-aminoflunitrazepam increased with time independently of dose and may be used to estimate the time of intake, and we think that measuring more than one metabolite
adds valuable information for the toxicologist. For some subjects, the metabolite concentrations in the early samples were
below the proposed SOFT DFSA cutoff of 5 µg/L, and a chromatographic method may fail to detect a flunitrazepam intake,
as did a standard immunoassay during screening. We think
laboratories should consider this when advising police and
hospitals about sampling as well as when they set up strategies for analysis.
Acknowledgment
The authors acknowledge Dr. Maria Cherma for taking good
care of the study participants.
Journal of Analytical Toxicology, Vol. 33, October 2009
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