Download Determination of Buprenorphine

Journal of AnalyticalToxi(olog~,Vol. 23, July/August1999
Determination of Buprenorphineand
Norbuprenorphinein Urine and Hair by
Gas Chromatography-MassSpectrometry
Fram;oise Vincent, Janine Bessard, J~r6me Vacheron, Michel Mallaret, and Germain Bessard"
Laboratoire de Pharmacologie et Analyses Toxicologiques, Centre Hospitalier Universitaire de Grenoble B.P. 217,
38043 Grenoble Cedex 9, France
Abstract
Buprenorphine, which is used in France as a substitution drug for
opioid addiction, is widely abused, and several fatal cases have been
reported. In order to confirm a recent intoxication or to establish
retrospectively chronic abuse, a simple and reliable gas
chromatographic-mass spectrometric method was developed and
validated for quantitation of buprenorphine and its active metabolite
norbuprenorphine in urine and hair. Two milliliters of urine or
50 mg of pulverized hair was submitted to a pretreatment
(enzymatic hydrolysis for urine and decontamination with
dichloromethane followed by incubation in 0.1M HCI for hair).
Buprenorphine-d4 was chosen as the internal standard. Selective
solid-phase extraction with Bond Elut Certify ~columns provided
recoveries higher than 85% for urine and 43% for hair. By using a
mixture of MSTFA/TMSIM/TMCS (100:2:5), buprenorphine and
norbuprenorphine produced stable silylated derivatives. The
detection was carried out with a quadrupole mass detector working
in El selected ion monitoring mode. Ions at m/z450 and 468 were
chosen for the quantitation of buprenorphine and
norbuprenorphine, respectively (m/z454 was used for the internal
standard). Limits of quantitation were 0.25 and 0.20 ng/mL,
respectively, for buprenorphine and norbuprenorphine in urine and
0.005 ng/mg for the two compounds in hair. Calibration curves were
linear from 0 to 50 ng/mL in urine and from 0 to 0.4 ng/mg in hair.
Between-day and within-day precisions were lessthan 8.4% in hair
and 6.1% in urine for both molecules in all cases.This method was
applied to urine and hair samples collected from patients in a
withdrawal treatment program and demonstrated its good
applicability in routine analysisand its benefit for clinicians. This
technique, which requires instruments already available to many
toxicology laboratories, offers an attractive alternative to more
sophisticated techniques.
Introduction
Buprenorphine, a semisynthetic derivative of thebaine is a
narcotic analgesic. It binds to the p opioid receptors with a
270
high affinity as a partial agonist and to the ~r opioid receptors
as an antagonist. Consequently, it develops a ceiling effect on
respiratory depression (1).
Since 1996, buprenorphine has been used in France as a
substitution drug for opioids and is available more easily than
methadone. Buprenorphine (high dosage tablets, Subutex |
0.4, 2, and 8 rag) may be initially prescribed by general practitioners, but methadone must be initially prescribed by a psychiatrist in a "methadone clinic" ("centre de substitution").
More than 40,000 patients are maintained with buprenorphine,
whereas only 6000 are maintained with methadone. It has
been shown that buprenorphine is widely abused, and 20 fatal
cases have been reported recently (2). Moreover, lethal intoxications with buprenorphine are probably underestimated, as
few French laboratories have the ability to quantitate
buprenorphine and its metabolite in biological fluids (3).
In this paper, a method for screening populations of addicts,
for toxicological monitoring of withdrawal therapeutics, or for
forensic applications is described9
A poor correlation between blood levels (a few nanograms
per milliliter) and clinical effects or toxicity has been described
for buprenorphine (2,3). Taking repetitive blood samples is
not convenient with drug addicts undergoing withdrawal treatment. Therefore, urine, which is the most common biological
sample analyzed in the case of drug addicts, was selected for
this study. Buprenorphine was determined together with norbuprenorphine, its N-dealkylated metabolite, which is also present in urine, usually at even higher concentrations9
Information about recent exposure to a drug (2-3 days)
acquired by urine analysis can be complemented by hair analysis, which can provide a retrospective view of drug intake
over several weeks or months depending on its length (4,5). In
order to benefit from this important information, hair analysis
was included in this study.
Literature reports several analytical methods for the determination of buprenorphine and norbuprenorphine in biological samples.
Radioimmunoassay methods (RIA) were developed for urine
and plasma analysis (6-8). They offer a rapid and very sensitive
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Journal of Analytical Toxicology, Vol. 23, July/August 1999
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identification of buprenorphine and are well
adapted to general screening situations. Unfortunately, two main drawbacks limit their
application in toxicology: the legislation in
course about the use of radioactive isotopes
and the lack of specificity due to cross-reactivity with metabolites. An alternative
assay to RIA, substituting a fluorescent
marker for the classical radioactive one, led
to a less sensitive detection (9).
Up to now, no EMIT or FPIA immunoassay has ever been developed to analyze
buprenorphine and its metabolites.
Various modes of detection have been reported for gas chromatographic (GC)
methods. The nitrogen phosphorus detector was not sensitive enough to detect
buprenorphine concentrations lower than
50 ng/mL (10). The use of an electron capture detector enabled a 10-fold increase in
sensitivity (11,12) or even a 100-fold increase after derivatization with heptafluorobutyric anhydride (10). Thermal instability was reported if no derivatization was
performed before analysis (8,12,13).
Reversed-phase high-performance liquid
chromatographic (HPLC) methods require
no derivatization and are less time consuming. Among the classical modes of detection, fluorimetric detection was reported
(14). This last method is simple and easy to
perform, but it lacks sensitivity and specificity, for the detection of buprenorphine in
the nanogram-per-milliliter range. HPLC
with electrochemical detection (15-17) offers better sensitivity, allowing a limit of detection (LOD) of 0.02 ng/mg in hair (8,18).
Nevertheless, this detection mode remains
delicate to operate and is not well suited for
routine screening.
Independently of the choice of the chromatographic separation method, mass spectrometric (MS) detection always provides
better specificity.
LODs in the range of 0.1 to 0.2 ng/mL
could be reached using either the GC-MS
in electron impact (El) mode (6,13), or negative (NCI) or positive (PCI) chemical ionization mode (11,19,20).
The use of HPLC combined with MS was
reported for the analysis of buprenorphine
in hair (8,21) with an LOD of 0.02 ng/mg, in
blood (21-23) with an LOD of 0.05 ng/mL,
and in homogenates of viscerae (liver, brain,
kidney, myocardium) with an LOD of 2 ng/g
(3).
Recently, a GC-NCI method (24) and a
271
Journal of Analytical Toxicology, Vol. 23, July/August 1999
liquid chromatography-electrospray ionization (LC-ESI)
method (19), both coupled with tandem mass spectrometry
(MS-MS), were proposed for buprenorphine blood determination with limits of quantitation (LOQ) of 0.2 and 0.1 ng/mL,
respectively.
These last methods (GC-MS-MS, LC-MS, and LC-MS-MS)
provide a very good specificityand sensitivity,but the necessary
equipment is rarely available in standard toxicologylaboratories.
The procedure described in this paper enables the quantitation of buprenorphine and its active metabolite in urine and
hair samples. We used the selective properties of the solidphase extraction (SPE) and produced stable silylatedderivatives
for both compounds. Good sensitivity and specificity were
reached with a GC coupled with a classical benchtop mass
spectrometric detector operated in EI selected ion monitoring
(SIM) mode.
(St. Louis, MO).N-Methyl-N-trimethylsilyltrifluoroacetamide
(MSTFA)was provided by Pierce ChemicalCompany (Rockford,
IL). Trimethylchlorosilane (TMCS)and 1-(trimethylsilyl) imidazole (TMSIM)were from Fluka (Buchs, Switzerland). The
GC column was a fused-silica capillary column (HP-5MS
5% pheny[ methyl siloxane, 30 m x 0.25-ram i.d., 0.25-pm
film thickness) from Hewlett-Packard (Wilmington, DE). SPE
was performed with Bond Elut Certify| cartridges for rapid extraction of drugs of abuse (type LRC, size 10 mL/130 mg of sorbent phase, Varian Sample Preparation Products, Harbor City,
CA). SPE columns were positioned on a VacElut VacuumManifold (Varian). Urine and hair were spiked with standards using
a Hamilton Microliter| Syringe 7105 (Hamilton Bonaduz AG,
Bonaduz, Switzerland). Creatinine urinary dosages were conducted on an Hitachi 917 | automatic analyzer (Boehringer
Mannheim, Mannheim, Germany).
Working solutions of the drugswere prepared by diluting the
standard solutions in methanol to obtain concentrations of
10 and 1 mg/L for buprenorphine and norbuprenorphine and
of 10 mg/L for the internal standard. The solutions were kept
in the dark at -18~ for one month. HCI (0.1M) and 1M KOH
were prepared by diluting concentrated solutions with deionized water obtained on a MilliQwater purification system (Millipore, Bedford, MA). Phosphate buffer (0.1M, pH 6.0) was
made up of potassium dihydrogen phosphate (13.61 g) dissolved in deionized water and was adjusted to pH 6.0 with 1M
KOH. The total volume was brought up to 1 L. Acetate buffer
(2M, pH 5.0) was obtained by mixing 67.8 mL ofa 2M sodium
acetate solution (16.4 g sodium acetate/100 mL deionized
water) with 32.2 mL of a 2M acetic acid solution (28.6 mL
glacial acetic acid/250 mL deionizedwater). Bufferswere stored
at 4~ for one month. The working solution of ff-glucuronidase
was prepared daily with 15 mg of [3-glucuronidasein 1 mL of
2M acetate buffer (pH 5.0). The silylating reagent was prepared by mixing 1000 IJL of MSTFA,50 IJLof TMCS, and 20 pL
of TMSIM; it was stored at 4~ for two weeks.
Experimental
Samples
Drug-free urine and hair samples spiked with different concentrations of buprenorphine and norbuprenorphine were
used for method validation. Positive urine and hair samples
were collected from patients who had just been included in a
methadone program or who were hospitalized for a withdrawal
treatment. All subjects had a long history of buprenorphine
abuse. Their age ranged from 24 to 46 years.
Chemicals and materials
Drug reference standards of buprenorphine, norbuprenorphine, and buprenorphine-d4 (100 mg/L in methanol) were
obtained from Radian (Austin,TX). Methanol (for pesticides
analysis), dichloromethane (for trace analysis), and isopropyl
alcohol (HPLC grade) were purchased from SDS (ValdonnePeypin, France). Glacial acetic acid 100%, potassium hydroxide,
potassium dihydrogen phosphate (Pro analysis), and ammonia
solution 25% (Suprapur) were purchased from Merck (Darmstadt, Germany). Sodium acetate and hydrochloric acid 36%
(RP Normapur) were obtained from Prolabo (Paris, France).
~3-Glucuronidase from Helix pomatia was supplied by Sigma
GC-MS method
Sample pretreatment. To prepare urinary calibration, seven
aliquots of drug-free urine were spiked with buprenorphine and
norbuprenorphine at concentrations of 0, 1, 2, 5, 10, 20, and
50 ng/rnL. After addition of the internal standard (2 IJL of
buprenorphine-d4 at 10 rag/L) and 250 IJL of [g-glucuronidase
Table I. Characteristics of Calibration Curves*
Biological
sample
Compound
Concentration
range
No. of
curves
Slopet
Interceptt
Correlation
coefficient(r2) t
Urine
buprenorphine
norbuprenorphine
0-50 ng/mL
0-50 ng/mL
7
7
1.06 _+0.05
2.8 _+0.1
-0.02 _+0.03
-0.07 _+0.07
0.999 _+0.001
0.998 _+0.001
Hair
buprenorphine
norbuprenorphine
0 0.4 ng/mg
0 0.4 ng/mg
10
10
0.84 _+0.08
1.95 + 0.07
0.08 + 0.05
0.07 _+0.09
0.998 _+0.001
0.998 + 0.001
9 C a l i b r a l i ( ) n ( ur~.('~ are ux[;r(~ss(2d as y = a x + / ) . x = a l l l ( ) u n l ratio : c o n c e n t r a t i o n
v = area rati~l: p e a k area o l a n a l y t e / p e a k area of i n t e r n a l s t a n d a r d .
' M e a n + SD
272
of
ana[yte/concentration r
internal standard.
Journal of Analytical Toxicology, Vol. 23, July/August 1999
working solution, the calibration points and patient samples
(2 mL) were incubated at 37~ for 20 h. Finally,4 mL of 0.1M
phosphate buffer (pH 6.0) were added before extraction.
Hair strands were cut into sections (usually I cm). Decontamination was achieved with two dichloromethane washes:
hair was mechanically shaken by inversion in a 20-mL stoppered glass tube with 5 mL of dichloromethane for 5 min.
After drying,hair sampleswere pulverizedin a ball mill (Retsch
MM 2000) for 15 rain. Samples for calibration (50 rag) were
prepared by spiking pulverized drug-free hair with buprenorphine and norbuprenorphine at concentrations of 0, 0.02, 0.04,
0.1, 0.2, 0.3, and 0.4 ng/mg. Afteraddition of the internal standard (1 ~L of buprenorphine-d4 at 10 mg/L), calibration samples were incubated at 56~ overnight in 1 mL of 0.1M HCI.
Patient samples were spiked with internal standard and incubated similarly. After neutralization with 1 mL of 0.1M KOH
and addition of 4 mL of 0.1M phosphate buffer (pH 6.0), samples were centrifugedfor 10 min at 3500 rpm before extraction.
SPE and derivatization procedures. This extraction procedure was closely related to the one recommended by the manufacturer for the extraction of cocaine and benzoylecgonine.
After being positioned on the Vac Elut Vacuummanifold,the
Bond Elut Certify LRC columns were conditionedwith 2 mL of
methanol followedby 2 mL of 0.1M phosphate buffer (pH 6.0).
Then the samples were poured into the cartridge reservoirsand
passed through the column at a flow rate of 0.5 mL/min. After
that, the columns were washed with 6 mL of deionized water,
dried under vacuum for 5 min, rinsed with 3 mL of 0.1M HC1,
dried again under vacuum for 5 min, and washed with 5 mL of
methanol. After dryingunder vacuum for 5 min, all of the compounds of interest were eluted successivelyby 2 mL and I mL of
dichloromethane/isopropyl alcohol (80:20, v/v) with 2% am-
monia solution (prepareddaily)at a flowrate of 0.5 mL/min. The
eluates were collected in a borosilycated glass tube. Samples
were evaporated under nitrogen flow at room temperature.
Residueswere reconstitutedin 0.5 mL of dichloromethane,transferred to a vial, and again evaporated under nitrogen at room
temperature.To the dried extracts, 301JLof silylatingreagent was
added, and the vials were heated at 65~ for 30 min. A 1-~L
aliquot was injected into the chromatographic system.
GC-MS conditions. The quantitative analysiswas performed
using a Hewlett-Packardbenchtop GC-MS system consisting of
an HP 5973 mass selective detector (MSD), an HP 6890 series
GC, and an HP 6890 series automatic liquid sampler. HP ChemStation software was used for data acquisition and processing.
The initial oven temperature of 100~ was maintained for 1 min
and then increased at a rate of 20~
to reach a maximum
temperature of 300~ which was held for 9 rain. There was a
final isotherm at 310~ for 2 min to purge the column. The injector system mode was splitless (45 s). The carrier gas was helium at a constant flowrate of 1 mL/min. GC-MStemperatures
were as follows:injector 250~ interface 300~ source 220~
and quadrupole 100~ The MS was operated in EI mode at
70 eV. The electron multiplier voltage was set at +250 V above
the autotune voltage. The MSD was autotuned daily with
PFTBA. Ions at m/z 450, 482, and 506 served for buprenorphine identification; m/z 468, 500, and 524 served for norbuprenorphine identification;and m/z 454, 486, and 510 served
for buprenorphine-d4 identification. Ions at m/z 450, 468, and
454 were selected for quantitation. The dwell time for the different ions was set at 30 ms. Identification was established by
taking into account both retention times and relative abundance of qualifier ions. Concentrationswere evaluatedin patient
samples with calibration curves calculated using peak-area ratios (analyte/internal standard) plotted
versus concentration ratios.
Table II. Within-Day Precision
Biological
sample
Urine
Hair
Concentration
2 ng/mL
10 ng/mL
50 ng/mL
0.04 ng/mg
0.2 ng/mg
0.4 ng/mg
n
Coefficientof variation(%)
Buprenorphine Norbuprenorphine
10
10
10
2.9
3.7
2.5
3.3
5.3
2.1
10
10
10
8.4
4.1
3.4
7.5
6.8
7.9
Table Ill. Between-Day Precision
Coefficient of variation (%)
Biological
sample
Urine
Hair
Buprenorphine Norbuprenorphine
Concentration
2 ng/mL
10 ng/mL
5,0 ng/mL
0.02 ng/mg
0.4 ng/mg
10
10
10
3.8
1.8
3.2
5.3
6.1
6.1
10
10
4.1
5.4
7.6
4.5
Results
Chromatogram and mass spectra of buprenorphine-lTMS and norbuprenorphine2TMS are presented in Figure 1. The two
compounds appear well separated: their
mean retention times were 17.70 and 15.38
rain, respectively (Figure 1A).
Linearity
Linear correlation was found for buprenorphine and norbuprenorphine in the
ranges 0 to 50 ng/mL for urine and 0 to 0.4
ng/mg for hair. The slopes, the intercepts
and the average linear correlation coefficients (r2) for urine and hair are presented
in Table I.
Limit of linearity
The limit of linearity was determined by
extracting drug-free urine and hair samples
273
Journal of Analytical Toxicology, Vol. 23, July/August 1999
spiked with increasing concentrations of buprenorphine and
norbuprenorphine. The calibration curves of buprenorphine
and norbuprenorphine were found to be linear over the range
0-500 ng/mL for urine and 0-10 ng/mg for hair.
Within-day and between-day precision
samples spiked with decreasing concentrations of buprenorphine and norbuprenorphine until a response equivalent to
three times the mean background noise registered on blank
samples at the retention time of drugs tested was obtained.
LOD was 0.10 ng/mL in urine and 0.002 ng/mg in hair for the
two compounds.
Within-day precision was calculated from repeated analysis
of urine and hair, during one working day, by the same operator. It was calculated for three concentrations of buprenorphine and norbuprenorphine. Results are given in Table II.
Between-day precision was calculated from the analysis of
samples at the same concentrations of buprenorphine and norbuprenorphine. One analysis was performed per day; results are
given in Table III.
LOQ
Similarly, the LOQ was estimated by testing decreasing concentrations of buprenorphine and norbuprenorphine until a response equivalent to 10 times the mean background noise was
obtained. In urine, LOQ was 0.25 and 0.20 ng/mL for buprenorphine and norbuprenorphine, respectively. LOQ was 0.005
ng/mg in hair for the two compounds.
LOD
Figure 2 shows typical selected ion monitoring chromatograms, recorded from a urine extract, spiked with 2
ng/mL of buprenorphine and 2 ng/mL of norbuprenorphine.
The LOD was estimated by extracting drug-free urine or hair
Im ,154.00(453.70 to 454.70)
ion 4M.CO(467.70 to 4 M . ~
1105
Extraction recovery
Extraction recovery, expressed as a percentage, was defined as the ratio of calibration curve slope of extracted analyte to calibration curve slope of non-extracted analytes. In all
cases, buprenorphine-d4 was added just before derivatization. The recoveries were 88% and 43%
for buprenorphine in urine and hair; for
norbuprenorphine, they were 85% and
51%, respectively.
e~O0
eO00,
Discussion
5B~O
Choice of the analyzed molecules
Although norbuprenorphine shows a notably reduced analgesic effect compared
with its parent compound (25), its analysis
provides useful information. Norbuprenorphine respiratory depressant activity is estimated to be 10 times higher than that of
buprenorphine (26). The study of parent
compound/metabolite ratio can also give
interesting information about the time of
drug intake and patient biotransformation
capacity. Presence of norbuprenorphine in
biological samples brings the proof of drug
intake. This positive result excludes an external addition of the drug to urine or an
environmental contamination of hair.
45OO
4OOO
3OOO
15.37
25O0
2OOO
No
1500
Sample collection and
pretreatment methods
1000
50O
15.(30
15.50
18.00
1650
17.00
17.~D
18.00
18.50
Time (mini
Figure 2. Chromatogram obtained after the injection of 1 pL of an extracted drug-free urine spiked with
2 ng/mL of each compound to dose and 10 ng/mL of internal standard. Data are recorded in the SIM
mode at m/z 468 for norbuprenorphine, m/z 450 for buprenorphine, and m/z 454 for buprenorphined.+.
274
As patients were hospitalized, urine was
easily collected. In order to limit errors
linked to daily variation in urinary dilution,
especially when daily quantitative analysis
had to be done, sampling was standardized
by collecting the first morning urine. When
this was not possible, creatinine dosages
were achieved and concentrations were expressed as nanograms of buprenorphine per
milligrams of creatinine.
Journal of Analytical Toxicology, Vol. 23, July/August 1999
Buprenorphine is metabolized to norbuprenorphine by Ndealkylation. Both molecules appear in urine mainly in the
form of glucuronide conjugates (11). Before analysis, urine
samples must be hydrolyzed to determine the total amount of
buprenorphine and norbuprenorphine. A classical enzymatic
method (27) that has already been used for other opiate derivatives was performed.
Collection of hair samples was standardized by cutting them
in the region of the vertex posterior. This area presents less
variability in hair growth rate (4,28-30). Hair analysis first requires a washing to remove external contamination, then an
extraction of the drugs from the hair matrix. Various methods
were used to liberate drugs of abuse; the most commonly
used were HCI extractions, enzyme digestions, methanol extractions, and NaOH extractions. According to the results of
Welch et al. (31,32), all techniques gave comparable results.
The preparation method used here was described by Kintz et
al. (8,33). It involves two washes with dichloromethane, pulverization in a ball mill, and incubation at 56~ overnight in
I mL of 0.1M HCI.
Choice of internal standard
guprenorphine-d4 has been recently used for the determination of both buprenorphine and its metabolite, as norbuprenorphine-d4 was not yet available (19,21-23). Norcodeine has been used as the internal standard for the
determination of norbuprenorphine (24). Norcodeine was
tested but not selected because of the lack of stability of its
1! ~r
2TMS derivative. Therefore, buprenorphine-d4 was the only
internal standard.
Choice of extraction method
Both liquid-liquid extraction (LLE) and SPE methods are
reported in literature.
Cleanliness of extracts is a determining factor in overall
sensitivity. The important background noise observed after a
single-step liquid extraction can be overcome by using a
MS-MS detection method (19), but this technology is not yet
widely used. Most authors proceed to multiple step LLE to
limit the interfering peaks (11,13,21,34). These methods are
delicate, lengthy, and lead to decreased recoveries.
In 1996, Kuhlman et al. (24), investigating different extraction methods, concluded that SPE was more rapid, reproducible, and efficient than LLE. They described an SPE method
using Clean Screen (ZCDAU020)| columns; the columns' phase
combines both hydrophobic and cation exchange functional
groups. Similar columns (Bond Elut Certify columns) had already been found satisfactory in our laboratory for the analysis
of other morphinic drugs and dextropropoxyphene (35); their
use was extended to buprenorphine analysis. Chromatograms
of urine and hair extracts from two drug addicts are presented
in Figure 3.
Recoveries obtained in urine were at least as satisfactory as
those obtained with a multiple-step LLE method for buprenorphine and even higher for norbuprenorphine (21,22). The extraction with hair proved to be less efficient, which is consis-
[on 454.C0 (453.79 rr 4S4.10):
Ion 4~O.GO (44D.70 ~ 4 ~ 0 . ; ~ :
~ n 454,00 (4~.79 to 484.70)
~J
5e~oJ
s4om]
mooo]
;,r
~oco
/
4e~o!
Norbuprenorphine-2TMS
Norbuprenorphine-2TMS
~ooo
,a~ot
,c~o!
~ec~J
eQoo
3~oo!
j~m:oJ
::~eoJ
:~cm!
4~o
2(~o
17.87
Bup:,eno~hine-d4-1TMS ~7~
110 ng/mL)
le~OJ
x I[I
te~oJ
14~o]
l~COJ
' i
ICOOo]
e~o.]
8ooo~
a~o
9mo
Ill""Bupren~
Buprenorphi
ne-d4-1TMS
(0.2 ng/mL)
16.g4
17.a3
Buprenorphine-lTMS
~ -Z-Z~? ,:
9 ' "15111o '
"
Time (rain)
Time (min)
Figure 3. Chromatograms of extracts from patients under buprenorphine therapy. Data are recorded in the SIM mode at m/z468 for norbuprenorphine, m/z450
for buprenorphine, and m/z 454 for buprenorphine-d4. A, urine extract; B, hair extract.
275
Journal of Analytical Ioxicotogy, Vol. 23, July/August 1999
tent with similar observations made with LLE (18). This result
seems to be better explained by difficulties in releasing drugs
from the hair matrix due to a holding-back phenomenon (31)
rather than by real difficulties with the SPE method, which
gives good results with other biological samples.
Choice of detection and derivatization methods
GC-MS was chosen for its specificity and sensitivity.
When GC is used, it is necessary to derivatize polar functions
of buprenorphine and norbuprenorphine in order to improve
their chromatographic behavior. Several derivatizing agents
have been proposed; many authors have used fluorinated
reagents such as pentafluoropropionic anhydride (PFPA) and
heptafluorobutyric anhydride (HFBA) (12,13,19,20,24); some
were working in PCI mode (20) or in NCI mode (24).
Few authors have studied silylating reagents. Lloyd-Jones
et al. (34) prepared monosilyl derivative with BSA after heating
at 40~ for 15 rain for GC-MS analysis. In 1993, Debrabandere
et al. (6) chose MSTFA at 70~ for 15 rain to measure
buprenorphine in horse urine. As opiates are usually analyzed
as their silylated derivatives, a first series of trials was conducted with bis(trimethylsilyl)trifluoroacetamide (BSTFA).
Buprenorphine could be easily silylated on its hydroxy phenolic
function, resulting in buprenorphine-lTMS. However, norbuprenorphine-2TMS was not produced in a reproducible
manner because of the difficulty of silylating the amine function. Both norbuprenorphine-]TMS and -2TMS were present in
an unpredictable ratio regardless of the temperature and the
duration of the reaction. No improvement was observed with
the addition of a catalyst such as TMCS.N-MethyI-N-trimethylsilyltrifluoroacetamide (MSTFA) possesses a high silylating
power which can be increased by adding a catalyst, such as
TMCS and/or l-(trimethylsilyl)imidazole (TMSIM). A mixture
of MSTFA, TMCS, and TMSIM constitutes a powerful silylating
reagent used to determine free steroids (36) or to silylate indolic amine functions (36). To detect nalbuphine, another
morphinic compound structurally related to buprenorphine, a
mixture of MSTFA/TMSIM/TMCS(100:2:5) allowed 100% conversion to nalbuphine-3TMS, whereas the use of BSTFA even
associated with TMCS led to both nalbuphine-2TMS and nalbuphine-3TMS (37). Using the same mixture, norbuprenorphine was quantitatively converted into norbuprenorphine2TMS. After testing the influence of temperature and reaction
time, optimal parameters were found to be 65~ and 30 rain.
Although thermal instability of buprenorphine has been re-
ported in literature (8,12,13), we have never experimented any
problem after derivatization of this molecule. Moreover the
derivatives of both molecules remained stable during several
days. Nevertheless, in the case of norbuprenorphine, deactivated glass liners were very important to preserve the integrity
of the derivative-2TMS.
Quantitation method
Convenient performances in terms of both sensitivity and
specificity were achieved with the use of the SIM method.
In Figure 1B, the peak at m/z 539 corresponds to the molecular ion of buprenorphine-lTMS (C32H49NO4Si);other significant ion peaks are at m/z 450 (M-89; loss of CH3OH and C4H9)
and m/z 482 (M-57; loss of C4H9) (6). The base peak (m/z 450)
was chosen for the quantitation of buprenorphine, and m/z 482
and 506 were selected as qualifier ions.
Figure 1C shows the full scan mass spectrum of norbuprenorphine-2TMS. The molecular ion peak is at rn/z 557
(C31H51NO4Si2).Buprenorphine-lTMS and norbuprenorphine2TMS undergo a similar fragmentation. The base peak at m/z
468 was selected for quantitation and ions at m/z 500 and 524
as qualifier ions.
Validation parameters
Calibration curves were always linear through the examined concentration range. Given that concentrations can be
very high in the urine of addicts, it was often necessary to dilute the patient samples. In the case of hair, limit of linearity
was much higher than concentrations usually found in human
samples.
Between-day and within-day precisions were systematically
less than ]0%, which is appropriate in terms of quality.
Because SPE provides clean extracts, detection and quantiration limits were almost equivalent to values obtained through
GC-MS-MS (24), LC-MS (21,23), or LC-MS-MS (19) analysis. In the case of hair, it should be noted that optimal instrument conditions were needed to achieve these values.
Clinical applications
Results presented in Table IV refer to urine and hair samples
collected at the same time from five different patients. All of
them had a long history of buprenorphine use at high doses
and were candidate for a withdrawal treatment.
Urinary concentrations of total buprenorphine and norbuprenorphine reach several hundreds or even thousands of
Table IV. Buprenorphine and Norbuprenorphine Concentrations in Hair and Urine of Drug Addicts
Urine
Patient
Buprenorphine
(ng/mgcreatinine)
A
B
C
D
E
1041
2366
3~I 6
1431
] 007
276
Hair
Norbuprenorphine Buprenorphine/
(ng/mgcreatinine) norbuprenorphine
789
984
6990
2053
636
1.32
2.40
0.47
0.70
1.58
Buprenorphine
Norbuprenorphine
(ng/mg)
(ng/mg)
0.361
0.266
0.060
0.265
0.305
0.546
0.785
0.029
0.772
0.470
Buprenorphine/
norbuprenorphine
0.66
0.34
2.03
0.34
0.65
Journal of Analytical Toxicology, Vol. 23, July/August1999
nanograms per milliliter or nanograms per milligram of creatinine. The buprenorphine/norbuprenorphine concentration
ratio showed a great interindividual variability.
Buprenorphine and norbuprenorphine were found in the
hair of the fivesubjects. Buprenorphine concentrations ranged
from 0.060 to 0.360 ng/mg; norbuprenorphine was often found
in even more abundant quantity, ranging from 0.029 to 0.785
ng/mg. Few reports are related to buprenorphine in human
hair. Kintz et al. (8) determined buprenorphine concentrations in the range 0.020 to 0.590 ng/mg in the hair of 14 young
drug addicts admitted to a withdrawal program; norbuprenorphine concentrations ranged from not detected to 0.150 ng/mg
in the same subjects. For Tracqui et al. (21), concentrations
measured in the hair of six addicts under substitutive therapy
ranged from 0.004 to 0.140 ng/mg and from undetectable to
0.067 ng/mg for buprenorphine and norbuprenorphine, respectively. Morerecently, Valdezet al. (38) presented hair analysis of samples from four subjects admitted to a buprenorphine-treatment program. The authors observed a gradual
trend of increasing hair concentrations over time and noted
9 Buprenorphlne
[] Norbuprenorphine I
A
600
SO0
400
300
200
100
0
,
2
3
4
5
6
7
8
9
10
11
Days after admission to hospital
I
9 Buprenorphine
r~Norbupmnorphlne I
that in all cases norbuprenorphine metabolite hair concentrations were greater than those of the parent compound, as we
obtained in many cases. It should be noted that this result is
somewhat unusual as drugs are generally incorporated into
hair according to their lipophilicity; this fact is well known for
cocaine and 6-monoacetylmorphine (5).
Figure 4A displays the daily urinary elimination of buprenorphine and of its major metabolite concerning a drug addict
hospitalized for voluntary withdrawal treatment. This patient
had been consuming buprenorphine for one year at an average daily intake of 8 mg by the sublingual route. During his
8-day stay in the hospital, this patient suffered two relapses in
buprenorphine intake that caused his eviction from the program. In this case, the buprenorphine/norbuprenorphineconcentration ratio was systematically lower than 0.1. Variations
in the metabolite concentrations were more significant than
those in the parent compound and brought the proof of the patient moral contract breach.
Figure 4B shows the toxicological monitoring of another
drug addict, taking 16 mg of buprenorphine per day by the intravenous route. He was hospitalized as the previous subject,
but he did behave as agreed during the withdrawal treatment.
His buprenorphine and norbuprenorphine urinary concentrations regularly decreased, reaching very low levels (nanograms
per milliliter) after about 8 days, as generally observed after
stopping the intake. This patient benefited, of course, from
the whole withdrawal treatment.
In this case, as in other cases of withdrawal treatment
without relapse, even if norbuprenorphine concentrations were
initially higher than buprenorphine, norbuprenorphine could
not be detected any longer than buprenorphine.
Figure 5 presents the toxicological hair monitoring of a
third patient who had received 16 mg of buprenorphine per day
by the intravenous route for 3 years, then was admitted to a
withdrawal treatment. Concentrations of buprenorphine and
norbuprenorphine were measured in 1-cm hair segments.
During the period of chronic buprenorphine intake, hair concentrations were relatively constant. The buprenorphine/norbuprenorphine concentration ratio was close to 0.2, indicating
B
4:T
I 9
i
360
BNorbuprenorphlne I
:i
1
300 ~
0,9
0,8
0,7
0,6
=~ o,5
0,4
0,3
0,2
0,1
0
0',-- I
1
2
3
4
5
6
7
8
9
10
11
Days after admission to hospital
Figure4. Urineanalysisof buprenorphineand norbuprenorphinefor daily
toxicologicol monitoring in the caseof patientshospitalizedfor a voluntary
withdrawal treatment. A, Caseof a patient who relapsed into buprenorphine abuse.B,Caseof a patientwho complied with abstinencefrom drug.
11
10
9
8
7
6
5
4
3
2
1
Centimeters from root
Figure 5. Determination of buprenorphine and norbuprenorphine in a
11-cm hair lock from a young drug addict. The patientwas monitored after
having undergonea withdrawal cure. Measurementswere carried out on
1-cm hair segmentsbeginning from the root.
277
Journal of Analytical Toxi(ology, Vol. 23, July/August 1999
that norbuprenorphine was more incorporated than buprenorphine as previously observed. Then, the withdrawal treatment
induced a decrease in hair concentrations until the levels were
undetectable. Segmental hair analysis requires a lot of care in
result interpretation until a precise knowledge about the dynamics of appearance and disappearance is acquired, especially
for buprenorphine. A delay occurs between drug intake and
drug incorporation into the hair (4,5,28-30). Valdez et al. (38)
showed that there was a delay of several weeks after beginning
buprenorphine treatment before buprenorphine and norbuprenorphine could be detected in hair. Hair is also known to
grow approximately by 1 cm per month, but to define precisely the time of a particular drug exposure, it may be necessary to establish an accurate growth rate of hair for each
patient (5,28). Hair pigmentation also appears to be an important factor in drug incorporation.
After the withdrawal period, only four random urinary controis were conducted once each month. All were negative, but
they could reflect the abstinence over the last few days only.
Hair analysis, adjunct to urinalysis, brought a reliable proof of
nonrecidivism.
Conclusions
An analytical procedure combining SPE and GC-MS analysis
was developed to demonstrate a buprenorphine use or a drug
abstinence. This method is sensitive enough for essaying
buprenorphine and norbuprenorphine in urine and hair samples. A wide interindividual variability is observed in urine and
hair samples. Norbuprenorphine appeared as a marker of
buprenorphine intake in both matrices: however, the precise
buprenorphine/norbuprenorphine ratio could not be defined.
Norbuprenorphine appeared to be more incorporated in hair
than its parent compound. Historical records constituted
through hair segmentation coupled with another technique
such as urinalysis can be very useful to monitor recidivism or
abstinence in withdrawal treatments. This method, even if
somewhat time consuming, can be applied to a series of samples and can be wholly automated. Its practice does not require
too sophisticated or expensive equipment. As no immunoassay
is yet available, it can be used as a "routine" method in clinical
toxicology and forensic medicine laboratories.
References
1. J.W. Lewis. Buprenorphine. DrugAIcohol Depend. 14:363-372
(1985).
2. A. Tracqui, C. Tournoud, F. Flesch, J. Kopferschmitt, R Kintz,
M. Deveaux, M.H. Ghysel, R Marquet, G. P@in, G. Petit, A.
Jaeger, and B. Ludes. Intoxications aigiJes par traitement substitutif
~.~base de bupr~norphine haut dosage. PresseM6d. 27:557-561
(1998).
3. A. Tracqui, G. Petit, D. Potard, F. Levy, P. Kintz, and B. Ludes. Intoxications mortelles par bupr~norphine (Subutex| et benzodiaz6pines: 4 cas. J. M~d. L6[4. DroitM6d. 40:213-223 (1997).
278
4. T. Mieczkowski. The use of hair analysis for the detection of
drugs: an overview. J. Clin. Forensic Med. 3:59-71 (1996).
5. M.R. Moeller. Drug detection in hair by chromatographic procedures. J. Chromato~,r. 580:125-134 (1992).
6. U Debrabandere, M. Van Boven, L. Laruelle, and P. Daenens.
Routine detection of buprenorphine in horse urine: possibilities
and limitations of the combined use of radioimmunoassay, liquid
chromatography and gas chromatography-mass spectrometry.
Anal. Chim. Acta. 275:295-303 (t993).
7. C.W. Hand, K.E. Ryan, S.K. Dutt, R.A. Moore, J. O'Connor,
D. Talbot, and H.J. McQuay. Radioimmunoassay of buprenorphine in urine: studies in patients in a drug clinic. J. Anal. Toxicol.
13:100-104 (1989).
8. P. Kintz, V. Cirimele, Y. Edel, C. Jamey, and P. Mangin. Hair analysis for buprenorphine and its dealkylated metabolite by
radioimmunoassay and confirmation by LC-ECD. J. Forensic Sci.
39:1497-1503 (1994).
9. L. Debrabandere, M. Van goven, and P. Daenens. Development
of a fluoroimmunoassay for the detection of buprenorphine in
urine. J. Forensic Sci. 40:250-253 (1995).
10. D. Martinez, M.C. Jurado, and M. Repetto. Analysis of buprenorphine in plasma and urine by gas chromatography. J. Chromatogr.
528:459-463 (1990).
11. E.J. Cone, C.W. Gorodetzky, D. Yousefnejad, W.F. Buchwald,
and R.E. Johnson. The metabolism and excretion of buprenorphine in humans. Drug Metab. and Dispos. 12:577-581 (1984).
12. E.J.Cone, C.W. Gorodetzky, D. Yousefnejad, and W.D. Darwin.
63Ni electron-capture gas chromatographic assay for buprenorphine and metabolites in human urine and feces. J. Chromatogr.
337:291-300 (1985).
13. Y. Biota, U. Bondesson, and E. Anggard. Analysis of buprenorphine and its N-dealkylated metabolite in plasma and urine by selected-ion monitoring. J. Chromatogr. 338:89-98 (1985).
14. S.T. Ho, J.J. Wang, W. Ho, and O.Y.P. Hu. Determination of
buprenorphine by high-performance liquid chromatography with
fluorescence detection: application to human and rabbit pharmacokinetic studies. J. Chromatog, r. 570:339 350 (1991).
15. L. Debral)andere, M. Van Boven, and P. Daenens. Analysis of
buprenorphine in urine specimens. J. Forensic Soi. 37:82-89
(199] ).
16. L. Debrabandere, M. Van Boven, and P. Daenens. High-performance liquid chrornut()graphy with electrochemical detection
of buprenorphine and its major metaholite in urine. J. Chromato~r. 564:557-566 (1991).
17. E. Schleyer, R. Lohmann, C. Roll, A. Gralow, C.C. Kaufmann,
M. Unterhalt, and W. Hiddemann. Column-switching solid-phase
trace-enrichment high-performance liquid chromatographic
method for measurement of buprenorphine and norbuprenorphine in human plasma and urine by electrochemical detection.
J. Chromatogr. 614:275-283 (199~).
18. R Kintz. Determination of buprenorphine and its dealkylated
metabolite in human hair. J. Anal. Toxkol. 17:443-444 (1993).
19. D.E. Moody, i.D. Laycock, A.C. Spanbauer, D.J. Crouch,
R.L. Foltz, J.L. Josephs, L. Amass, and W.K. Bickel. Determination
of buprenorphine in human plasma by gas chromatography-positive ion chemical ionization mass spectrometry and liquid chromatography-tandem mass spectrometry. J. Anal. Toxicol. 21:
406-414 (1997).
20. M. Ohtani, F. Shibuya, H. Kotaki, K. Uchuno, Y. Saitoh, and
F. Makagawa. Quantitative determination of buprenorphine and
its active metabolite, norbuprenorphine, in human plasma by
gas chromatography-chemical ionization mass spectrometry. J.
Chromato~r. 487:469-475 (1989).
21. A. Tracqui, P. Kintz, and P. Mangin. High-performance liquid
chromatographic mass spectrometry determination of buprenorphine and norbuprenorphine in biological fluids and hair samples.
J. Forensic Sci. 42:111-114 (I 997).
22. H. Hoja, R Marquet, B. Verneuil, H. Lotfi, J.L. Dupuy, M.F. Dreyfuss, and G. Lach~tre. Dosage de bupr6norphine et de
Journal of Analytical Toxicology,Vol. 23, July/August1999
23.
24.
25.
26.
27.
28.
29.
30.
norbupr~norphine dans I'urine et le s~rum par chromatographie
liquide coupl~e ,~ la spectrom~trie de masse avec ionisation de
type ~lectrospray. Analusis 24:104-107 (1996).
H. Hoja, P. Marquet, B. Verneuil, H. Lotfi, J.L. Dupuy, and
G. Lach~tre. Determination of buprenorphine and norbuprenorphine in whole blood by liquid chromatography-mass spectrometry. J. Anal. Toxicol. 21:160-165 (1997).
J.J. Kuhlman, J. Magluilo, E. Cone, and B. Levine. Simultaneous
assay of buprenorphine and norbuprenorphine by negative chemical ionization tandem mass spectrometry. J. Anal. Toxicol. 20:
229-235 (1996).
M. Ohtani, H. Kotaki, Y. Sawada, and T. Iga. Comparative analysis of buprenorphine and norbuprenorphine-induced analgesic
effects based on pharmacokinetic-pharmacodynamic modeling.
J. Pharmacol. Exp. Ther. 272:505-510 (1995).
M. Ohtani, H. Kotaki, K. Nishitateno, Y. Sawada, and T. Iga. Kinetics of respiratory depression in rats induced by buprenorphine
and its metabolite, norbuprenorphine. J. Pharmacol. Exp. Ther.
281:428-433 (1997).
R.W. Romberg and L. Lee. Comparison of the hydrolysis rates of
morphine-3-glucuronide and morphine-6-glucuronide with acid
and 13-glucuronidase. J. Anal. Toxicol. 19:157-162 (1995).
W.A. Baumgartner, V.A. Hill, and W.H. Blahd. Hair analysis for
drugs of abuse. ]. Forensic Sci. 34:1433-1453 (1989).
R Kintz and R Mangin. What constitutes a positive result in hair
analysis: proposal for the establishment of cut-off values. Forensic
Sci. Int. 70:3-11 (1995).
Society of Hair Testing. Forensic Sci. Int. 84:3-6 (1997}.
31. M. Welch, L. Sniegoski, and C. Allgood. Interlaboratory comparison studies on the analysis of hair for drugs of abuse. Forensic $ci.
InL 63:295-303 (1993).
32. U Sniegoski and M. Welch. Interlaboratory studies on the analysis
of hair for drugs of abuse: results from the fourth exercise. J.
Anal. Toxicol. 20:242-247 (I 996).
33. P. Kintz. Interlaboratory comparison of quantitative determinations
of drugs in hair samples. Forensic Sci. InL 70:105-109 (1995).
34. JiG. Lloyd-Jones, P. Robinson, R. Henson, S.R. Biggs, and
T. Taylor. Plasma concentration and disposition of buprenorphine
after intravenous and intramuscular doses to baboons. Eur. J.
Drug Metab. Pharmacokinet. 5:233-239 (1980).
35. G. Amalfitano, J. Bessard, F. Vincent, H. Eysseric, and G. Bessard.
Gas chromatographic quantification of dextropropoxyphene and
norpropoxyphene in urine after solid-phase extraction. J. Anal.
Toxicol. 20:547-554 (I 996).
36. G. Van Look, G. Simchen, and J. Heberle. SilylatingAgents, 2nd
ed. Fluka Chemie A.G. Buchs, Switzerland, 1995, pp 13-53.
37. Y.C. Yoo, H.S. Chung, I.S. Kim, W.T. Jin, and M.K. Kim. Determination of nalbuphine in drug abusers' urine. ]. Anal. To•
19:120-123 (1995).
38. A.S. Valdez, D.G. Wilkins, M.H. Slawson, C. Sison, W. Ling, and
D.E. Rollins. Buprenorphine and norbuprenorphine in human
hair of substance abuse treatment subjects. Poster presented in
College on Problems of Drug Dependence, 60th annual scientific
meeting, Scottsdale, AZ, 1998.
Manuscript received May 26, 1998;
revision received September 8, 1998.
279