Journal of Analytical Toxicology, Vol. 32, October 2008
Urinary Detection Times and Metabolite/Parent
Compound Ratios After a Single Dose of Buprenorphine
Robert Kronstrand1,*, Ingrid Nyström1, Malin Andersson1, Lina Gunnarsson1, Staffan Hägg2,3,
Martin Josefsson1, and Johan Ahlner1
1National
Board of Forensic Medicine, Department of Forensic Genetics and Forensic Toxicology, Linkoping, Sweden;
of Clinical Pharmacology, Sahlgrenska University Hospital, Göteborg, Sweden; 3Division of Clinical
Pharmacology, Linköping University, Linköping, Sweden
2Department
Abstract
The objective was to estimate the detection times and
metabolite/parent compound ratios in urine after a single dose of
buprenorphine. Eighteen healthy volunteers received a single dose
of 0.4 mg buprenorphine sublingually. Urine samples were
collected prior to dosing and at 2, 4, 6, 8 12, 24, 48, 72, and 96 h
post-dose. The samples were screened using cloned enzyme donor
immunoassay (CEDIA) reagent and quantitation was performed
with liquid chromatography–tandem mass spectrometry (LC–MS–
MS) with a cut-off of 0.5 ng/mL for buprenorphine and
norbuprenorphine. The mean time of continuous positive results
was 9 h (range 4 to 24 h) with CEDIA, whereas for an LC–MS–MS
method it was 76 h (range 23–96 h) for buprenorphine, and for
norbuprenorphine all samples were positive at 96 h. Some subjects
had positive CEDIA results after a negative sample, owing to
differences in creatinine concentration. The time when the ratio
norbuprenorphine/buprenorphine exceeded 1 was estimated at
7 h. The metabolite/parent ratio may be used to estimate the time
of intake even though the individual ratios showed an increased
variation the more distant the collection time. We believe that
using this ratio, rather than the actual concentrations, it is possible
to compensate for urine dilution and different doses, and to
improve interpretation.
Introduction
Buprenorphine is an opioid acting as partial agonist and
antagonist on the µ- and κ-opioid receptors. It has been used
for the prevention or treatment of moderate to severe chronic
pain with therapeutic doses of 0.3–0.6 mg in the form of sublingual tablets (Temgesic®). In most European countries including Sweden, buprenorphine is also used in treatment of
heroin addiction as an alternative to methadone (1,2). The
doses used on this indication range from 1 to 32 mg/day. Until
2005, sublingual buprenorphine for treatment of opioid
* 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, Linkoping, Sweden; E-mail [email protected].
586
dependence (Subutex®) was increasingly prescribed in Sweden
and a rapid spread to the black market was noted. Therefore, restrictions on the prescription of Subutex came in 2005. Despite
these restrictions, there has been a steady increase in the request for forensic buprenorphine analysis. During 2007, the
National Board of Forensic Medicine, which serves all of
Sweden, screened approximately 20,000 and confirmed 400
urine samples (2%) as positive for buprenorphine. A majority
of these samples came from either the prison boards or the police (petty drug offences) but an increase in requests from
forensic autopsy cases has also occurred. Recently, a new formulation of buprenorphine for treatment of opioid dependence, Suboxone, was introduced on the Swedish market. This
formulation contains not only buprenorphine but also the µ-receptor antagonist naloxone. In the ratio 4:1, naloxone has no
pharmacological effect after oral or sublingual administration
(3,4). It will only exert its opioid receptor antagonist effect if the
tablet is injected. In this way, the new formulation should reduce the potential for abuse.
Buprenorphine is usually administered sublingually because
it has a very low bioavailability after oral administration owing
to an extensive first-pass metabolism. After sublingual administration the maximum plasma concentration is reached after
40 min to 3.5 h. In humans, buprenorphine is metabolized primarily in the liver by N-dealkylation to norbuprenorphine
(norbuprenorphine) through CYP3A4 (5). Both the metabolite
and the parent drug undergo extensive conjugation to glucuronides that are excreted in the urine (5,6). Norbuprenorphine is an active metabolite but the conjugated metabolites
seem to be inactive (5). Urine concentrations of free buprenorphine and norbuprenorphine have been reported to be low,
with mean percentages of 6% and 35% (7) or 10% and 22%, as
reported by Blom et al. (8), and thus a hydrolysis step is recommended before analysis, unless the conjugates are analyzed
directly (9,10). Even though glucuronide reference materials
are available, many laboratories tend to use a hydrolysis before
analysis and thus measure the total concentration of the compounds (7,11–17). Feng et al. (12) reported the quantitative hydrolysis of buprenorphine-glucuronide using β-glucuronidase
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
Journal of Analytical Toxicology, Vol. 32, October 2008
from E. coli after 2 h of incubation at 37°C and at pH 6.8 (12).
Their study did not include evaluation of the hydrolysis of norbuprenorphine-glucuronide. However, Lisi et al. (13) optimized the hydrolysis of norbuprenorphine-glucuronide in a
urine sample from a volunteer who was administered 0.2 mg
buprenorphine. The hydrolysis required very high concentrations of the enzyme β-glucuronidase from H. pomatia, even at
a norbuprenorphine-glucuronide concentration as low as 6
µg/L. Kronstrand et al. (11) studied the hydrolysis using β-glucuronidase from E. coli and found that the cleavage of the
buprenorphine glucuronides was much easier to break than
that of norbuprenorphine. Differences in glucuronide hydrolysis have also been reported for codeine, morphine, and other
opioids (18,19). To be able to use a ratio between parent compound and metabolite quantitative hydrolysis is of great importance.
Urine concentrations of buprenorphine from both drug addicts and patients have been reported (7,11,14,16,20). George et
al. found concentrations between 28 and 1458 µg/L of buprenorphine and 28 to 1843 µg/L of norbuprenorphine in patients receiving decreasing doses of buprenorphine (16). They also concluded that the ratio between norbuprenorphine and
buprenorphine was unrelated to the dose regimen. Kronstrand
et al. reported buprenorphine concentrations between 31 and
1080 µg/L in patients and concentrations in abusers from 2.9 to
796 µg/L (11). Cirimele et al. (20) found buprenorphine concentrations ranging from only 1 µg/L up to 1052 µg/L in patients
in treatment for heroin addiction, whereas Vincent et al. (14) reported concentrations from 1.0 to 3.3 µg buprenorphine/mg
creatinine and 0.6–7.0 µg norbuprenorphine/mg creatinine in
five buprenorphine abusers. Thus, as expected concentrations in
living humans seem to vary substantially due to the dose intake
and the time of sampling. Also, postmortem concentrations
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Sex
(Male/Female)
Age
(years)
Height
(cm)
Weight
(kg)
BMI*
M
M
F
F
M
F
M
M
M
F
M
F
F
M
F
F
F
F
24
33
22
22
25
23
22
23
27
21
29
22
23
29
21
22
22
27
176
176
168
171
181
170
180
168
178
168
188
170
177
186
181
167
160
168
65
67
60
68
73
69
83
72
72
65
81
64
60
82
72
53
60
67
21.0
21.6
21.3
23.3
22.3
23.9
25.6
25.5
22.7
23.0
22.9
22.1
19.2
23.7
22.0
19.0
23.4
23.7
* Body mass index.
Materials and Methods
Chemicals and reagents
The reference materials buprenorphine, norbuprenorphine,
buprenorphine-d4, and norbuprenorphine-d3 were purchased
from Cerilliant (Round Rock, TX). All organic chemicals were of
gradient or analytical grade and all inorganic solvents were of
analytical grade. Enzymatic hydrolysis was performed using βglucuronidase (E. coli, 200U/mL) from Roche (Mannheim, Germany). Drug-free urine was obtained from laboratory personal
and was tested negative for buprenorphine and norbuprenorphine before use as matrix for controls and calibrators.
Study design
Table I. Demographics of the 18 Subjects
Subject
range extensively (21–25). Tracqui et al. reported postmortem
urine concentrations of 4–1000 µg/L in 20 fatalities involving
buprenorphine (21). We have recently investigated buprenorphine-related overdose deaths and found that in deaths where
buprenorphine was the major (or only) cause of death, the urine
concentrations were low and suggested a recent abstinence before the last dose, whereas other cases had much higher metabolite concentrations than parent compound (25). Thus, analysis
of urine in buprenorphine-related deaths could help to rule out
or confirm recent abstinence.
One of the most frequently asked inquiries from prison
boards regarding urine sample results is when the drug was ingested. The information regarding buprenorphine excretion is
very limited and the aim of this study was to describe the excretion of buprenorphine into urine and to investigate the
norbuprenorphine/buprenorphine ratio changes over time. In
this way, we aimed to establish a method to estimate the time
for buprenorphine use from urine data.
The study was approved by the Regional Research Ethics
Committee in Linköping (#M5-06). Eighteen healthy volunteers were recruited by advertising. There were no specific inclusion criteria. Participation in any other study during the
time for this study was considered an exclusion criterion, as
Figure 1. Mean excretion curves for buprenorphine and norbuprenorphine corrected for creatinine concentration. N = 18. Error bars represent
standard error of the mean (s.e.m.). The two curves cross at approximately
7 h after dose.
587
Journal of Analytical Toxicology, Vol. 32, October 2008
were pregnancy or nursing. Ongoing
medication with opiates, opioids, or benzodiazepines, or a documented abuse of
any of these substances were considered
an exclusion criterion, as were allergies.
Ten females aged 21 to 27 (mean 23) and
eight males aged 22 to 33 (mean 27)
were recruited. Their mean body mass
indexes were 22.1 and 23.2, respectively.
The subject demographics are shown in
Table I. In the morning of the first day,
subjects administered 0.4 mg of
buprenorphine sublingually (Temgesic®). The subjects provided urine
samples prior to dosing and approximately at 2, 4, 6, 8, 12, 24, 48, 72, and 96
h post dose. During the first 10 h of the
study, the subjects remained at the clinic
and were monitored for adverse effects.
Table II. Individual Analytical Results From Each Subject Given
0.4 mg Buprenorphine
Subject
FP 1
FP 2
Samples
The urine samples were collected in
plastic bottles and were transferred into
10-mL tubes within 24 h. One aliquot
was subjected to immunoassay and two
1-mL aliquots and one 2-mL aliquot
were frozen in 10-mL glass tubes
pending LC–MS–MS analysis. The urine
samples were first screened for buprenorphine using CEDIA reagent with a cut-off
of 5 µg/L, and the urine creatinine concentration was determined with the Jaffé
method. These two analyses were performed on an Advia 1650 from Bayer AB
(Gothenburg, Sweden).
FP 3
FP 4
Instrumentation
588
FP 5
BUP*
(µg/L)
NorBUP*
(µg/L)
Ratio
NorBUP/BUP
CEDIA
Creatinine
(g/L)
0.0
2.0
4.0
6.2
8.1
12.0
24.0
48.0
72.0
96.0
NEG
n.a.†
n.a.
17.00
9.55
2.21
1.79
0.94
1.34
0.68
NEG
n.a.
n.a.
11.00
6.26
4.07
5.45
5.90
6.20
4.10
0.65
0.66
1.84
3.04
6.28
4.63
6.06
–28
n.a.
n.a.
73
27
–15
–16
–24
–24
–23
2.27
n.a.
n.a.
1.51
0.86
0.79
1.38
1.46
1.78
1.65
0.0
2.2
4.2
6.3
8.1
12.0
24.0
48.0
72.0
96.0
NEG
52.60
45.50
19.40
20.00
12.20
5.41
3.76
1.74
0.26
NEG
5.68
16.50
14.40
19.00
19.60
13.00
12.40
6.92
1.90
0.11
0.36
0.74
0.95
1.61
2.40
3.30
3.98
7.31
–29
181
166
74
83
37
7
–13
–24
–24
3.55
1.70
1.96
1.76
1.89
2.27
2.61
2.55
1.82
1.23
0.0
2.1
4.0
6.1
8.2
12.0
22.8
47.0
72.0
96.0
NEG
12.90
32.60
26.50
18.90
1.37
3.79
1.80
1.44
0.75
NEG
3.56
16.80
25.70
22.50
3.47
12.30
7.89
5.26
2.66
0.28
0.52
0.97
1.19
2.53
3.25
4.38
3.65
3.55
–29
39
154
114
69
–20
–13
–10
–13
–13
2.2
0.44
1.43
2.24
2.57
0.47
2.37
1.66
1.55
1.52
0.0
2.1
4.1
6.1
8.2
12.0
22.8
46.6
72.0
96.0
NEG
4.78
9.55
5.80
1.29
4.76
3.72
1.25
0.97
0.71
NEG
2.31
7.25
8.48
2.30
10.70
12.80
4.03
4.27
1.57
0.48
0.76
1.46
1.78
2.25
3.44
3.22
4.40
2.21
–28
–1
20
4
–17
–2
–8
–12
–9
–12
2.66
0.18
0.61
0.96
0.32
1.53
2.24
1.11
1.47
1.13
0.0
2.1
4.0
6.1
8.2
12.0
22.4
46.6
72.0
96.0
NEG
9.00
15.60
13.80
4.34
4.65
6.07
3.27
2.92
NEG
NEG
2.68
7.28
12.30
5.06
4.83
14.00
10.50
8.32
0.95
–26
21
46
41
–3
–3
2
–2
–8
–17
1.88
0.58
0.91
1.6
0.77
0.85
2.33
2.72
3.03
0.27
0.30
0.47
0.89
1.17
1.04
2.31
3.21
2.85
Table continues on next page
* BUP = Buprenorphine and NorBUP = norbuprenorphine.
† n.a. = Not available because no sample was obtained.
▲
The LC–MS–MS analysis was performed on a PerkinElmer series 200
chromatography system consisting of
two 200 micro pumps, a “hot-pocket”
column oven, a 200 auto sampler, and a
SCIEX API 2000 MS–MS instrument (Applied Biosystems, Toronto, ON) equipped
with an electrospray interface. Ion spray
voltage was set to 5000 V. Nitrogen was
used as nebulizer gas (25 psi), auxiliary
gas (50 psi heated to 300°C), curtain gas
(30 psi), and as collision-activated dissociation gas (set on 5). A 50 × 2.1-mm
Zorbax phenyl analytical column with
3.5-µm particle size (Agilent Technologies, Kista, Sweden) was used. Mobile
phase A was a 10:10:80 mixture of acetonitrile/methanol/20mM ammonium
formate buffer (pH 3.0), and mobile
phase B was a 35:35:30 mixture. The
system was run in a linear gradient from
Time
(h)
Journal of Analytical Toxicology, Vol. 32, October 2008
75% A-phase to 10% A-phase during 5
min, followed by a 1-min equilibration
with 90% A-phase. The total flow rate
was 0.25 mL/min. The column oven was
set at 30°C. A 10-µL aliquot of the samples was injected.
Table II. Individual Analytical Results From Each Subject Given
0.4 mg Buprenorphine (Continued)
Subject
FP 6
Extraction and quantification of
buprenorphine and norbuprenorphine
FP 7
FP 8
FP 9
FP 10
BUP*
(µg/L)
NorBUP*
(µg/L)
Ratio
NorBUP/BUP
CEDIA
Creatinine
(g/L)
0.0
2.1
4.0
6.2
8.1
12.0
23.0
46.9
70.9
95.5
NEG
6.58
8.90
12.40
2.75
5.53
2.82
1.78
0.81
0.44
NEG
0.81
4.75
9.70
4.24
8.92
8.34
5.84
2.56
1.13
0.12
0.53
0.78
1.54
1.61
2.96
3.28
3.15
2.57
–21
12
26
44
–8
9
–6
–12
–17
–17
2.67
0.37
0.44
1.12
0.56
1.50
1.54
1.48
1.06
0.90
0.0
2.1
4.0
6.0
8.1
12.0
24.0
47.7
71.7
95.7
NEG
5.63
8.24
2.55
1.62
2.36
1.76
1.08
0.60
0.61
NEG
1.89
4.04
2.39
2.26
5.66
5.02
5.02
2.78
2.76
0.34
0.49
0.94
1.40
2.40
2.85
4.65
4.66
4.54
–19
11
23
–3
–6
–7
–12
–17
–25
–23
2.41
0.50
0.70
0.55
0.55
2.35
2.23
2.86
2.77
2.83
0.0
2.2
4.0
6.0
8.2
12.0
22.8
47.7
72.7
96.8
NEG
59.80
n.a.
n.a.
36.10
0.62
1.35
1.67
0.61
1.05
NEG
11.40
n.a.
n.a.
17.40
1.78
5.93
8.27
5.09
5.83
–75
199
n.a.
n.a.
141
–17
–14
–11
–20
–17
5.50
3.64
n.a.
n.a.
3.07
0.23
0.85
2.14
1.56
3.04
0.0
2.1
4.2
6.1
8.1
12.0
22.9
46.8
75.2
98.8
NEG
32.60
28.90
11.00
2.46
4.79
3.12
1.52
0.55
0.68
NEG
9.13
20.00
13.50
3.55
9.86
12.50
7.36
6.12
6.48
0.28
0.69
1.23
1.44
2.06
4.01
4.84
11.13
9.57
–22
136
119
39
–3
6
–2
–16
–21
–17
4.17
1.62
2.00
1.39
0.47
1.28
1.79
1.92
2.74
1.87
0.0
2.2
4.2
6.3
8.2
12.0
22.4
47.5
71.2
95.4
NEG
3.08
4.42
1.37
0.51
0.96
0.71
0.60
0.54
0.28
NEG
3.84
11.80
4.35
2.37
4.63
5.95
3.86
3.52
1.61
1.25
2.67
3.18
4.65
4.84
8.33
6.42
6.52
5.75
–17
–6
8
–9
–13
–12
–19
–21
–22
–21
0.46
0.22
0.62
0.31
0.23
0.71
0.82
1.47
1.77
1.49
0.19
0.48
2.86
4.39
4.95
8.33
5.55
Table continues on next page
▲
Samples were analyzed in batches of
17 samples and three controls at 2 µg/L,
50 µg/L, and a hydrolysis control made
of pooled samples. Quantitation of
buprenorphine and norbuprenorphine
was performed using LC–MS–MS as described by Kronstrand et al. (11). In
brief, to 1.0 mL of urine were added 25
µL of internal standard (buprenorphined4 and norbuprenorphine-d3 2 mg/L),
300 µL 0.5 M BIS-TRIS propane buffer
(pH 6.8), and 40 µL β-glucuronidase.
The tube was capped and incubated in a
water bath (with orbital shaking) at 37°C
for 20 h and the pH was then adjusted to
6.1 with 4 mL of 0.2 M phosphate buffer.
Thereafter, solid-phase extraction was
performed. BondElute Certify columns
were activated and conditioned with 2
mL of methanol followed by 2 mL of 0.2
M phosphate buffer (pH 6.1). The sample
was drawn through the column, which
was then rinsed with 2 mL of deionized
water, 2 mL of 0.1M HCl, and 2 mL of
methanol. Then the column was dried
for 5 min (10 in. Hg), and the analytes
were eluted with 2 mL of a mixture of
dichloromethane/2-propanol (80:20)
containing 2% ammonia (25%). The
eluate was evaporated in a TurboVap at
40ºC and 5 psi nitrogen. The residue was
reconstituted in 100 µL of mobile phase.
The calibration range of the original
method was 2–500 µg/L. In this study,
the concentrations were expected to be
low, and the calibrator concentrations
were set at 0.5, 2, 10, 25, 50, and 100
µg/L. Standard solutions were added to 5
mL batches of negative urine. One
milliliter from each calibration batch
was pipetted and treated as authentic
samples. Calibrations were performed in
duplicates to minimize random errors
as one calibration curve could be used
for several series of samples. To estimate
the imprecision at the lowered calibration level, five controls at 0.5 µg/L were
analyzed within one batch. The coefficients of variation were 7.1% (buprenor-
Time
(h)
* BUP = buprenorphine and NorBUP = norbuprenorphine.
† n.a. = Not available because no sample was obtained.
589
Journal of Analytical Toxicology, Vol. 32, October 2008
phine) and 6.8% (norbuprenorphine).
Samples with concentrations lower than
0.5 µg/L were re-analyzed using 2 mL
urine.
Table II. Individual Analytical Results From Each Subject Given
0.4 mg Buprenorphine (Continued)
Subject
FP 11
FP 12
FP 13
FP 14
FP 15
Time
(h)
BUP*
(µg/L)
NorBUP*
(µg/L)
Ratio
NorBUP/BUP
CEDIA
Creatinine
(g/L)
0.0
2.3
4.1
6.0
8.2
12.0
23.0
47.5
69.5
96.2
NEG
12.20
30.50
13.10
11.50
1.36
1.57
1.44
0.83
0.75
NEG
3.00
13.20
9.71
8.30
1.92
4.29
6.62
4.18
2.97
0.25
0.43
0.74
0.72
1.41
2.73
4.60
5.01
3.98
–23
46
155
58
42
–11
–6
–16
–19
–19
2.28
0.46
2.01
2.07
2.43
0.52
1.52
1.95
1.77
2.01
0.0
2.0
4.0
6.0
8.0
12.0
22.8
47.0
71.2
96.5
NEG
n.a.
n.a.
n.a.
n.a.
n.a.
1.90
0.48
0.39
NEG
NEG
n.a.
n.a.
n.a.
n.a.
n.a.
2.17
1.92
1.65
1.29
–20
n.a.
n.a.
n.a.
n.a.
n.a.
–12
–16
–19
–19
0.5
n.a.
n.a.
n.a.
n.a.
n.a.
0.95
0.52
0.56
0.70
0.0
2.0
4.1
6.3
8.3
12.0
22.9
47.2
72.7
96.7
NEG
7.38
12.00
6.55
6.03
4.30
2.60
0.48
0.45
NEG
NEG
3.81
10.60
13.30
10.60
12.80
12.30
6.09
3.11
1.19
–25
17
43
16
9
3
–9
–21
–20
–15
2.90
0.33
0.75
1.17
1.08
1.43
2.38
1.06
1.60
0.98
0.0
2.2
4.2
6.1
8.2
12.0
23.2
47.2
72.0
96.0
NEG
9.40
28.40
12.70
0.85
1.57
1.81
1.79
0.76
0.67
NEG
1.83
11.20
9.89
1.05
2.54
7.65
11.60
4.80
2.97
0.19
0.39
0.78
1.23
1.62
4.23
6.48
6.30
4.47
–24
27
128
46
–15
–12
–12
–15
–19
–18
2.44
0.44
1.83
1.92
0.23
0.61
1.65
2.99
2.20
2.44
0.0
2.2
4.2
6.1
8.1
12.0
25.7
50.0
73.8
96.8
NEG
3.67
6.06
6.05
3.01
6.17
2.24
0.68
0.46
0.38
NEG
0.92
5.14
8.56
5.23
15.70
8.87
4.92
3.55
2.40
0.25
0.85
1.41
1.74
2.54
3.96
7.22
7.72
6.32
–19
2
14
12
–2
12
–7
–14
–17
–20
2.28
0.32
0.63
1.26
0.93
2.69
1.92
1.13
1.27
1.63
1.14
4.00
4.23
0.52
0.88
2.03
1.76
2.98
4.73
12.69
6.91
* BUP = buprenorphine and NorBUP = norbuprenorphine.
† n.a. = Not available because no sample was obtained.
590
▲
Table continues on next page
Results
In total, the 18 subjects provided 170
urine samples. Due to failures to mictate, 10 urine samples were not possible
to obtain. The concentrations of the analytes varied substantially between the
subjects even though the same dose was
administered. Peak concentrations of
buprenorphine ranged from only 4.4 to
60 µg/L and norbuprenorphine peak
concentrations ranged from 5.7 to 26
µg/L. The excretion profiles were similar between the study subjects with initial higher concentrations of buprenorphine than of norbuprenorphine that
later changed. The time when the ratio
between norbuprenorphine and
buprenorphine turned higher than 1 was
approximately 7 h as derived from
Figure 1 where the mean concentration
profiles over 96 h of all subjects are
shown. Individual data are shown in
Table II. The mean time for the last consecutive positive using CEDIA (> 5
ng/mL) was calculated to 9 h (range 4 to
24 h) for 17 subjects. Subject 12 never
provided a positive sample and no samples were obtained during the first day.
Owing to differences in urine dilution a
negative sample could sometimes be followed by a positive. The corresponding
detection times using the LC–MS–MS
method was 76 h for buprenorphine and
96 h for norbuprenorphine using the 0.5
µg/L cut-off. However, when using 2 mL
urine for the extraction, 14 of the subjects were positive also for buprenorphine in the last sample at 96 h. In
Figure 2, the mean ratios between norbuprenorphine and buprenorphine are
depicted. Figure 3 shows the relationship between the mean ratio and sampling time during the first 24 h. The relationship was linear during the first 24
h after intake and then a significant curvature was observed. After administration of buprenorphine, all subjects suffered from sedation and vertigo, but at
different degrees. In addition, other
common adverse events reported during
Journal of Analytical Toxicology, Vol. 32, October 2008
the first study day were nausea (12 subjects), vomiting (7 subjects), and tremor (7 subjects); however, two or more subjects
also reported sweating, impaired hearing, dry mouth, and
headache.
shown by George et al. (16), independent of buprenorphine
dose. Still, interindividual differences in metabolism will affect
the ratio. Buprenorphine is metabolized through CYP3A4,
which have shown some polymorphism even though it is not
bimodal. As seen in Table II, the individual ratios differed significantly between the subjects. One reason was the different
intervals between urine samples in the beginning of the first
Discussion
day. Several subjects failed to leave urine samples and
buprenorphine and metabolites accumulated in the urine until
We have shown that buprenorphine and its metabolite, norlater times, increasing the range of ratios for each sampling
buprenorphine, can be detected in the urine up to 96 h after a
time. However, this reflects the authentic samples from casesingle dose and that the concentration of norbuprenorphine
work. After 48 h, the ratio showed considerable scatter and the
became higher than that of the parent compound within a few
mean values at 48, 72, and 96 h were in the same order. This inhours after intake. The urine concentrations of a drug and its
dicated that buprenorphine and norbuprenorphine have simmetabolites show a considerable scatter owing to differences in
ilar excretion rates and Vincent et al. (14) came to a similar
dose, uptake, distribution, metabolism, and excretion, but also
conclusion when following the decline in urinary concentradepend on urine dilution. Thus, solely the concentration of a
tions in some buprenorphine addicts in a detoxification center.
drug in urine is not suitable for estimations of time of intake.
Using the ratio, an estimation of the time from intake to samMetabolite/parent compound ratios, on the other hand, may
pling can be made. A ratio less than 0.5 indicated very recent
prove useful as the ratio is unaffected by urine dilution, and as
use, and a ratio of 1 indicated use within 7 to 10 h. A ratio of
about 3 indicated use 1 day before sampling and this correlated well with the
Table II. Individual Analytical Results From Each Subject Given
0.4 mg Buprenorphine (Continued)
study of George et al. (16) where daily
urine samples from patients treated with
Time
BUP*
NorBUP*
Ratio
Creatinine
buprenorphine had ratios of 3, indepenSubject
(h)
(µg/L)
(µg/L)
NorBUP/BUP
CEDIA
(g/L)
dently of the dose given and with that of
Böttcher et al. (15), who found a mean
FP 16
0.0
NEG
NEG
–17
1.42
ratio of 4.3. In the present study, ratios
2.2
13.20
3.22
0.24
52
0.35
higher than 4 indicated use farther back
4.1
18.90
6.97
0.37
89
0.62
in time, but the relationship showed a
6.2
17.50
10.20
0.58
71
1.03
very distinct curvature after 48 h and an
8.2
16.70
10.40
0.62
70
1.08
estimation of the time of intake became
12.0
8.29
8.07
0.97
26
1.19
22.9
4.75
8.72
1.84
5
2.27
difficult.
47.2
2.09
8.07
3.86
–12
1.89
The mean detection time with the
70.2
1.22
9.75
7.99
–12
1.85
CEDIA reagent was 9 h, and considering
96.3
0.36
3.23
8.97
–15
0.98
the cut-off of 5 ng buprenorphine per
mL urine, this was in accordance with
FP 17
0.0
NEG
NEG
–22
1.65
the method specifications and the actual
1.9
9.06
2.21
0.24
32
0.34
4.0
30.30
17.20
0.57
131
2.22
buprenorphine concentrations in the
6.0
23.40
17.80
0.76
114
2.54
samples as measured by LC–MS–MS. An
8.1
13.60
14.10
1.04
57
2.94
earlier study has also shown that the
12.0
7.95
16.30
2.05
–12
3.05
CEDIA reagent performed well at the
21.8
3.89
9.16
2.35
–4
2.89
proposed cut-off (15). However, the
46.2
2.47
9.42
3.81
–9
3.54
metabolite was present in significant
69.5
0.93
3.63
3.92
–17
2.40
concentrations even after 96 h. The
93.8
0.40
1.98
4.95
–18
2.10
specificity of the reagent towards buprFP 18
0.0
NEG
NEG
–19
0.68
enorphine resulted in negative outcome
2.0
5.84
3.37
0.58
19
0.18
even short times after ingestion of
4.0
n.a.
n.a.
n.a.
n.a.
buprenorphine, and thus failed to iden6.2
12.10
9.44
0.78
42
0.87
tify even a recent intake.
8.2
3.07
4.83
1.57
1
0.68
A limitation of the data from our study
12.0
2.47
4.05
1.64
–6
0.66
is
the single intake as compared to
23.4
3.73
13.40
3.59
–2
1.80
multiple
doses that is likely to occur
46.5
1.99
9.08
4.56
–10
1.81
among
abusers.
In chronic users, the
72.2
1.44
6.79
4.72
–12
1.82
norbuprenorphine/buprenorphine ratio
94.3
NEG
2.12
–15
0.63
might be overestimated because of
* BUP = buprenorphine and NorBUP = norbuprenorphine.
residual norbuprenorphine from former
† n.a. = Not available because no sample was obtained.
intakes. However, the similar 24-h ratios
591
Journal of Analytical Toxicology, Vol. 32, October 2008
Board of Forensic Medicine, Sweden. Ingela Jacobsson is acknowledged for excellent clinical assistance.
References
Figure 2. Mean norbuprenorphine/buprenorphine ratios for the 18 subjects
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Figure 3. Mean norbuprenorphine/buprenorphine ratios during the first
24 h. The change in ratio over time could be described by a simple linear
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Sweden, most requests for estimating the time of administration has so far been by the prison boards when a detained
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addition, the approach can be used in suspected buprenorphine overdose deaths to estimate the time since the last dose.
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in acute overdose cases, indicating a short period of survival
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estimate the time of intake. Further studies using different
doses of buprenorphine are warranted to investigate this issue.
Acknowledgments
This study was partly financed by a grant from the National
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