Journal of Analytical Toxicology,Vol. 21, October 1997
Determination of Buprenorphinein Human Plasmaby
Gas Chromatography-PositiveIon Chemical Ionization
Mass Spectrometryand Liquid ChromatographyTandem Mass Spectrometry
David E. Moody*, John D. Laycock, Alan C. Spanbauer, Dennis J. Crouch, and Rodger L. Foltz
Center for Human Toxicology, University of Utah, Salt Lake City, Utah 84112
Jonathan L. Josephs~
Finnigan MAT, SanJos~, California 95134
Leslie Amass* and Warren K. Bickel
Behavioral Pharmacology Laboratory, Department of Psychiatry, University of Vermont, Burlington, Vermont05401
Abstract I
Buprenorphine is used for the management of pain and has been
advocated for the treatment of opioid addiction. Therapeutic doses
result in low plasma concentrations of buprenorphine. In order to
assessthe safety and efficacy of buprenorphine, sensitive
analytical methods are needed. Until recently, gas
chromatography-positive ion chemical ionization mass
spectrometry (GC-PCI-MS) offered the most sensitive method to
selectively quantitate buprenorphine. We have developed and
validated a sensitive liquid chromatography-electrospray
ionization-tandem mass spectrometry (LC-ESI-MS-MS) method for
buprenorphine. The method is described and compared with a
GC-PCI-MS method validated in this laboratory. One-milliliter
aliquots of plasma are required for the LC-ESI-MS-MS method and
2-mL aliquots for the GC-PCI-MS method. Buprenorphine-d4 is
used as internal standard for both methods. Derivatization with
pentafluoropropionic acid anhydride is used for the GC-PCI-MS
method, in which the derivatized protonated molecular ions after
loss of water are monitored at m/z596 and 600. For LC-ESIMS-MS, the parent protonated molecule ions are monitored at
m/z468 and 472. A single-step extraction of basic plasma with
n-butyl chloride provided recoveries of 70-87%. Although a limit
of quantitation (LOQ) of 0.1 ng/mL could be established for
LC-ESI-MS-MS, we could only achieve an LOQ of 0.5 ng/mL with
the GC-PCI-MS assay. The GC-PCI-MS method has a linear range
of 0.5 to 40 ng/mL (mean r 2 = 0.998, n = 7). For quality control
samples at 1.0, 2.5, and 12.5 ng/mL, the intra- and interassay
coefficients of variation (CV) did not exceed 14%, and percent of
targets were within 16%. The LC-ESI-MS-MS method had a linear
range of 0.1 to 10 ng/mL (mean r 2 = 0.999, n = 7).
* Address correspondence to David E. Moody, Center for Human Toxicology, 8PRB, Rm 490,
University of Utah, Salt Lake City, UT 84112. E-mail [email protected].
f Current address: Pharmaceutical Research Institute, Bristol-Meyers Squibb, New Brunswick,
NJ, 089O3.
r Current address: Department of Psychiatry, University of Colorado School of Medicine,
Denver, CO 80262.
406
For quality control samples at 0.25, 2.5 and 7.5 ng/mL, the intraand interassay CVs did not exceed 4%, and percent of targets were
within 12%. Stability studies demonstrated buprenorphine was
stable for up to 24 h, 125 days, and 55 days when stored at room
temperature, 4~ and -20~ respectively. The utility of the lower
LOQ was demonstrated in 40 plasma samples collected up to 96 h
after a sublingual dose of buprenorphine; 10 were quantitatable
using GC-PCI-MS and 38 using LC-ESI-MS-MS.
Introduction
Buprenorphine is an oripavine derivative having partial
agonist and antagonist opioid activity (1-3). For the treatment
of moderate to severe pain, buprenorphine has been used successfullyby intramuscular, intravenous, or sublingual routes at
doses ranging from 0.3 to 0.6 mg (4). Clinical studies have
shown that buprenorphine, like methadone, can also be used
for the treatment of opioid addiction and may be safelyused to
withdraw patients from heroin (5-7) (For a review, see Bickel
and Amass [8]). When used for treatment of opioid dependence, buprenorphine is usually administered sublingually in
doses of 2-32 rag.
Clinical pharmacokinetic, safety, and efficacystudies examining the utility of buprenorphine as a treatment of opioid
dependence require an accurate and sensitive analytical
method. Immunoassay methods have been used to detect
buprenorphine and its metabolites in urine and plasma (9-13).
Although some of these assays reported good sensitivity (e.g.,
10 pg/mL [12]), they were not specific for buprenorphine
because they cross-reacted with norbuprenorphine and/or conjugated buprenorphine metabolites. Reversed-phase highperformance liquid chromatographic (HPLC) methods with
UV (14), fluorescence (15,16), and electrochemical (17,18)
Reproduction(photocopyin8)of editorialcontentof thisjourna[is prohibitedwithoutpublisher'spermission.
Journal of Analytical Toxicology, Vol. 21, October 1997
detection have been reported for analysis of buprenorphine in
plasma and urine. These methods lacked the sensitivity to
detect buprenorphine in plasma at concentrations less than
1 ng/mL. Other published methods for the analysis of
buprenorphine in biological fluids include gas chromatography (GC)with electron capture detection (19) and quantitative thin-layer chromatography (20). These methods were
limited to analysis of buprenorphine in urine.
Quantitation of buprenorphine has also been examined by
mass spectrometric methods. Biota et al. (21) used gas chromatography-mass spectrometry (GC-MS) with electron ionization to measure buprenorphine in human plasma and urine.
The method consisted of extraction at pH 9.4, back extraction
into dilute sulfuric acid, and heating at 110~ which caused
the buprenorphine to undergo cyclization with the loss of
methanol. The cyclizedproduct was then extracted and derivatized with pentafluoropropionicanhydride (PFPA).The limit of
detection of this method was 0.15 ng/mL. A gas chromatography-positive ion chemical ionization-mass spectrometry
(GC-PCI-MS) method was reported by Ohtani et al. (22). In
that method, samples were made acidic and extracted with an
organic solvent to remove potential interferences. The pH was
adjusted to 10.5, and the samples were extractedwith a second
organic solvent and derivatized with PFPA. These authors
reported a limit of quantitation (LOQ)of 0.2 ng/mL. Recently,
Kuhlman et al. (23) used solid-phase extraction and derivatization with heptafluorobutyric anhydride to prepare samples
for GC coupled with negative ion chemical ionization and
tandem mass spectrometry. Selected reaction monitoring of a
prominent product ion at m/z 464 formed by collision-induced
dissociation of the molecular anion permitted an LOQ of 0.2
ng/mL.Twoother recent publications have employedthe combination of HPLC with single-stage MS for determination of
buprenorphine in biologicalsamples. Tracqui et al. (24) used a
single-stepliquid-liquid extraction of biologicalfluids and hair
samples, whereas Hoja et al. (25) used acetonitrile to deproteinize whole blood then solid-phase extraction and liquidliquid backextraction.Both of these research groups monitored
the protonatedmoleculesfor buprenorphine(m/z 468) formedby
electrosprayionization(ESI).The more extensiveextractionprocedure by Hoja et al. (25) permitted a lower LOQ of 0.1 ng/mL.
We reporttwo validatedMS methods for the determination of
buprenorphine in human plasma. One method employs a
single-step liquid-liquid extraction followedby derivatizatioff
with PFPAand GC-PCI-MSanalysiswith an LOQof 0.5 ng/mL.
The other method consists of a verysimilar liquid-liquidextraction followeddirectly by liquid chromatography-electrospray
ionization-tandem mass spectrometry (LC-ESI-MS-MS)with
an LOQ of 0.1 ng/mL. Both methods use a deuterium-labeled
isotopomer of buprenorphine as the internal standard.
Experimental
Materials
Drug reference standards of buprenorphine (100 lag/mLin
methanol) and buprenorphine-d4 (100 l~g/mLin methanol)
were obtained from Radian (Austin,TX). Sodium hydroxidewas
purchased from Sigma (St. Louis, MO). High-purity grade
n-butyl chloride, acetonitrile, and toluene were obtained from
Baxter Diagnostics (McGrawPark, IL), and PFPAwas obtained
from Regis (Morton Grove, IL). Formic acid (88%) was purchased from J.T. Baker (Phillipsburg, NJ). The GC column was
a DB-1 fused-silica capillary column (15 m x 0.32-mm i.d.,
1-1~m film thickness, J&W Scientific, Folsom, CA). The LC
column was an Alltech (Deerfield,IL) Solvent Miser| C8 150 x
2.l-ram column with a 5-1~mparticle size.
Specimens
Plasma specimens were collected as part of a larger clinical
trial of buprenorphine using dosing schedules previously
described in a preliminary communication (26). Three dosing
schedules were employed: an 8-mg/70-kg dose was administered every 24 h, a 16-mg/70-kgdose was administered every
48 h, and a 24-mg/70-kg dose was administered every 72 h.
Four opioid-dependent outpatients abstinent from illicit opioids who had been maintained on 8 mg/70 kg sublingual
buprenorphine were exposed to the dosing schedules in a
random sequence. A dosing schedule lasted 17 days and consisted of dailyadministration of a sublingual dose of buprenorphine (8, 16, or 24 mg/70 kg) or saline placebo. During each
dosing schedule, laboratorysessions (each separated by at least
72 h) were randomly scheduled to occur 24 and 48 h after an
8-mg/70-kg dose; 24, 48, and 72 h after a 16-mg/70-kg dose;
and 24, 48, 72, and 96 h after a 24-mg/70-kg dose. Therefore,
an additional placebo was interposed before the laboratory
session with the longest time since dosing. A control session
was interposed that consistedof three consecutive24-h dosings
with 2 mg/70 kg buprenorphine. During laboratory sessions,
subjects were also administered 0 (saline), 6, and 12 rag/70 kg
of subcutaneous hydromorphone at 90-rain intervals using a
cumulative-dosing paradigm. Blood was collected in 10-mL
heparinized Venoject| tubes (Becton Dickinson, Franklin
Lakes, NJ) at the beginning of a laboratory session (30 rain
before first subcutaneous injection).The blood was centrifuged
immediately,and the plasma was stored at-20~ until analysis.
Calibrator and control preparation
All buprenorphine stock solutions used for calibrator and
quality control (QC) sample preparation were prepared by
diluting the purchased reference solutions in methanol
(1 to 10). Stock solutions were diluted (1 to 10) in distilled
water to obtain concentrations of 1.0 pg/mL for analysis by
GC-PCI-MS and 0.1 pg/mL for analysis by LC-ESI-MS--MS.
Stock solutions for calibratorsand QC sampleswere preparedby
different individuals.Internal standard spiking solutions were
prepared by successive 1 to 10 dilutions of methanolic stock
solutions of buprenorphine-d4to obtain concentrations of 1.0
I~g/mL for GC-PCI-MS analysis and 0.1 pg/mL for LC-ESIMS-MS analysis.
GC-PCI-MSanalytical method
Extraction. Two-milliliteraliquotsof calibrators (0, 0.5,1.0, 2.5,
5,10, 20, 30, and 40 ng/mL),QCsamples(1.0,2.5 and 12.5ng/mL)
and subject plasma samples were pipetted into 16 x 100-ram
407
Journal of Analytical Toxicology, Vol. 21, October 1997
tubes. Fifty microliters of internal standard (buprenorphine-d4
silanized tubes. Twenty microliters of internal standard
at 0.1 ng/pL) was added to each tube; the tubes were mixed
(buprenorphine-d4 at 1.0 ng/pL) was added to each tube; the
brieflyand allowedto equilibrate for approximately 1 h. The pH
tubes were mixed briefly, allowed to equilibrate for approximately 1 h, and the pH adjusted to approximately 10.5 with
of each sample was adjusted to approximately 10.5 with 25 pL
of 2N sodium hydroxide. Four milliliters of n-butyl chlo40 pL of 2N sodium hydroxide.Four milliliters of n-butyl chloride/acetonitrile (4:1, v/v) was added to each tube. The tubes
ride/acetonitrile (4:1, v/v) was added to each tube. The tubes
were mixed on a rocker for 30 rain and centrifuged for 10 rain
were mixed on a rocker for 30 rain and centrifuged for 10 rain
at 2250 rpm. The organic layer was transferred to clean 13 x
at 2250 rpm. The organic layer was transferred to clean 13 x
100-ram silanized tubes and evaporatedat 40~ under a stream
100-ram silanized tubes, and the solvent was evaporated at
of air. Seventy-fivemicroliters of H20/acetonitrile (2:1) con40~ under a stream of air. One-hundred microliters of PFPA
taining 0.1% formic acid was added to each tube. The tubes
and 100 pL of toluene were then added to each tube, and the
tubes were allowed to sit at room temperature for 30 rain. The
were vortex mixed, centrifuged at 2250 rpm for 5 rain, and the
samples were evaporated under a stream of air at room temliquid transferred to labeled autosampler vials for injection
perature until completely dry (i.e., no odor of PFPAremained).
into the LC-ESI-MS-MS.
Forty microliters of n-butyl chloride was added to each tube,
LC-ESI-MS-MS conditions. The analysis was performed
using a Finnigan-MATTSQ 7000 triple-stage quadrupole MS
the tubes vortex mixed, and the liquid transferred to labeled
with an ESI interface. A CTC A200 LC autosampler was used to
autosampler vials for injection into the GC-MS.
GC-PCI-MS conditions. The analysis was performed using a
inject 20 pL of the extracts onto the Alltech Cs LC column. A
LDC Analytical(RivieraBeach, FL) constaMetric| 4100 MS LC
Finnigan-MAT4500 MS equipped with a 9610 GC, INCOSsoftpump was operated isocratically to deliver a flow rate of
ware, and a CTC A200S autosampler (San Jos~, CA). A DB-1
fused-silica capillary column was used with
hydrogen as the carrier gas. A typical temperature program began at 160~ for 0.1
A
(MH*-H20)
100 "
rain, and the temperature was increased at a
CF3CF2C-O"~
rn/z = 596
rate of 20~
to 310~ The final tern=
perature was held for 0.1 min. All injections
"~
=
were made in the splitless mode of opera~ so CH3 7, ~
N'cH
(MH*)
tion. The GC to MS interface temperature
ndz [] 614
HOG-CH3
m/z = 4 6 7
was maintained at 285~ and the injector
C(CH3)3
temperature was held at 270~ The MS was
I I
l
I
I
I
I I /
I
operated in the positive ion detection mode.
o 2O0
600
300
400
500
A reagent-gas mixture of methane and
m/z
ammonia at a ratio of approximately4:1 was
,,.=B
introduced into the MS until an ion source
m/z
=
596
pressure of approximately 1 torr was
reached. The ion source was maintained at
140~ The electron multiplier was operated
at 1600 volts and the conversion dynode at
-3 kV. Selected ion monitoring centroid data
were collected at m/z 596 and 600 for
I
I
I
.=_
Z211416.
buprenorphine and buprenorphine-d4,
C
.~.~
I~,0"
respectively. Peak-height ratios (d0/d4) of
the calibrators were used to generate a calm/z = 600
c~
ibration curve, and a least-square equation
was calculated and applied to the peak
height ratios of QC and subject samples to
determine their buprenorphine concentration.
2"~4MH+_CF3CF2CO
)
t==
LC-ESI-MS-MS analytical method
Extraction. The extraction was performed
as described here previously,except that the
method was modified to use 1-mL samples.
One-milliliter calibrator samples (0, 0.10,
0.25, 0.50, 0.75, 1.0, 2.5, 5.0, 7.5, and 10
ng/mL), QC samples (0.25, 2.5, and 7.5
ng/mL), and subject plasma samples were
pipetted into separate 16 x 100-ramsilanized
408
4121
,=e
4143
~,&
5ll~
l=e
5l2",e
,~
gl48
r&
61|0
Scan time (min)
Figure 1. Fragmentation and chromatography of buprenorphine during GC-PCI-MS. (A) Spectraof
buprenorphine derivatized with PFPA and ionized with methane-ammonia under positive ion
chemical ionization conditions. The full molecular structure of derivatized buprenorphine is
shown. Ion current profile of plasma that was fortified with buprenorphine at 0.5 ng/mL (limit of
quantitation) (B) and buprenorphine-d4 at 10 ng/mL (C), extracted, derivatized with PFPA,and analyzed by GC-PCI-MS.
Journal of Analytical Toxicology, Vol. 21, October 1997
0.25 mL/min. The solvent was H20/MeOH/acetonitrile
(25:30:45, v/v/v) containing 0.1% formic acid. The tube lens
and capillaryvoltages were optimized for maximum buprenorphine signal. The ESI spray voltage was 5 kV,and the capillary
temperature was 225~ The sheath gas pressure was 80 psi
nitrogen, and the auxiliary flow was 10 units. The MS was
operated in the MS-MS mode with a collision energy of-25 eVand 2.5 retort argon
collision gas pressure. Both Q1 and Q3
were set to monitor ions at m/z 468 and
lOO U
472. Peak-area ratios (do/d4) of the calibrators were used to generate a calibration
curve; a least-square equation was calculated and applied to the peak-area ratios of
50.>_
QC samples and subjects to determine
107
buprenorphine concentration.
nr
i
0
Results and Discussion
396 and 414 were monitored over a range of collision energies.
The plot in Figure 2B shows that the buprenorphine MH§ ion
intensity (m/z 468) remains relativelyconstant up to a collision
energy of -20 eV and then drops sharply above -30 eV. The
intensities for the two most intense product ions (m/z 396
and 414) remain small throughout the range of collision ener-
A
m/z =
152 187
/ ,.t II
'
100
267
I
I
312
I II
,
200
I
396
414
\/I
I
I
300
468
,
I
I
400
m/z
B
Chromatography and spectrometry
A positive ion chemical ionization mass
spectrum of the pentafluoropropionyl
derivative of buprenorphine is shown in
Figure 1A.The relativelylow intensity protonate molecule [MH+] at m/z 614 undergoes loss of H20 to give the intense peak at
m/z 596. The only other significant peak
above m/z 200 is the low intensity peak at
m/z 467 that is formedby loss of CF3CF~CO
from the MH+. The corresponding ions of
derivatizedbuprenorphine-d4 occur at m/z
618, 600, and 471. For sample analysis, the
ion currents at m/z 596 and 600 are monitored. Figures 1B and C show the ion current profiles resulting from analysis of a
plasma sample containing 0.5 ng/mL
(LOQ) of buprenorphine and 10 ng/mL of
the deuterated internal standard.
Triple quadrupole MS are commonly
used in pharmaceutical analysis for the
measurement of trace drugs and metabolites. Most often, the technique of selected
reaction monitoring is used in which an
abundant analyte ion is selectively transmitted through Q1, collisionally dissociated to product ions in Q2, and only
selected product ions are transmitted
through Q3. This process greatly reduces
the chemical noise reaching the detector
and, therefore, results in a higher signalto-noise ratio than that achievable by
single-stage mass analysis. However, the
buprenorphine MH+ ion formed by ESI
proved difficult to fragment, even under
rigorous collision cell conditions (Figure
2A). The most intense product ions at m/z
I
500
2e+6
I e+ 6
Oe+O
i
0
10
20
30
40
50
60
-eV
Figure2. Collision-induced dissociation of buprenorphine after LC separation and ESI.(A) Spectra
of fragments formed in Q2 at a collision energy of 30 eV after selection of m/z 468 by Q1. (B)
Changes in intensity of m/z 468 ( 9 ), 414 ( 9 and 396 (o) with changes in collision energy.
Source
91
92
03
n d z = 468
Buprenorphlne
m / z = 468
lm
~
D
Background
Figure3. Illustration of the MS-MS mode of operation. The MS-MS has three quadrupoles, Qt, Q2,
and Q3. The parent ion, along with some background material, is selected by Q1 set at m/z 468.
The collision reactions (i.e., fragmentation) in Q2 do not produce significant fragment ions of the
parent but do fragment background ions. Selection of parent ion (m/z 468) by Q3 increasesthe
signal-to-noise ratio.
409
Journal of Analytical Toxicology,Vol. 21, October 1997
gies. At collision energies above -40 eV the buprenorphine
MH§ is shattered and forms many low intensity product ions.
Consequently, the best signal-to-noise was achieved when the
MH§ ions for buprenorphine (m/z 468) and buprenorphine-d4
(m/z 472) were selected and transmitted through both Q1 and
Q3. In this case, the improvement in signal-to-noise is due to
the fact that ions at m/z 468 and 472 from chemical background undergo collision-induced fragmentation in Q2 and are
Table I. Recovery of Burpenorphine from Human Plasma
after Single-Step Liquid-Liquid Extractions
GC-PCI-MS
LC-ESI-MS-MS
QC
(ng/mt)
Recovery
QC
Recovery
(%)
(rig/rot)
(%)
5.00
25.0
75.0
87.2
81.2
97.2
0.25
2.50
7.50
82.9
74.2
69.5
3.0 E3
~-~ ~
m/z:468
not transmitted through Q3 as a result. The process is depicted
schematically in Figure 3. Buprenorphine was monitored as
the surviving parent ion, which provided symmetrical ion current profiles with a minimum of interference even at the LOQ
of 0.1 ng/mL (Figure 4).
Recovery
Recovery of buprenorphine was determined by comparing
the peak-area ratios calculated when buprenorphine-d0 and
buprenorphine-d4 were extracted together (Batch A) to the
peak-area ratios obtained when buprenorphine-d0 was
extracted and buprenorphine-d4 added just before derivatization (GC-PCI-MS) or before reconstitution (LC-ESI-MS-MS)
(Batch B). Recovery was determined at three concentrations
with five replicates per batch at each concentration. Recovery
was expressed as (mean peak-area ratio for Batch A) / (mean
peak-area ratio for Batch B) x 100% (TableI). Recoveryvaried
from 81 to 97% for the GC-PCI-MS method at concentrations
ranging from 5 to 75 ng/mL and from 70 to 83% for the
LC-ESI-MS-MS method at concentrations ranging from 0.25
to 7.5 ng/mL. Although the extraction
methods were fairly similar, the greater
volume of organic or the 2-mL sample
A
volume used in the GC-PCI-MS method
resulted in greater extraction efficiency.
tinearity
2.0 E3
1.0 E3
B
rn/z = 472
1.0 E5
0.6 E5
0.2 E5
.
.
.
,
.
.
6:00
.
.
.
.
.
.
.
,
_
.
6:50
,
.
.
.
.
,
.
7:40
.
.
.
.
.
.
8:30
9:20
Scan time (rain)
Figure 4. Ion current profiles of (A) 0.10 ng/mL buprenorphine monitored at m/z 468 and
(B) 5 nglml_buprenorphine-d4monitored at m/z 472 after LC-ESI-MS-MS.
410
During the GC-PCI-MS method development, a linear range of 0.5 to 40 ng/mL
was established. The calibration curves
were reproducible and linear through the
entire concentration range. The average
correlation coefficients (r 2) were 0.998
(Table II). The LC-ESI-MS-MS calibration
curve was reproducible and linear from 0.1
to 10 ng/mL with a mean correlation coefficient of 0.999 (TableII). During analysis
of clinical specimens, we found that buprenorphine concentrations rarely exceeded
10 ng/mL. Therefore, during LC-ESIMS-MS method development, we limited
the upper range of the calibration curve to
10 ng/mL. The practicality of this upper
limit is consistent with other studies that
have specifically measured buprenorphine.
With the exception of the first 10-15 rain
following intravenous injection of buprenorphine, no other plasma concentrations
have been reported that exceeded 10 ng/mL
(16,21-23,27).
Accuracy and precision
The accuracy and precision of the
methods were determined from replicate
analysis of the LOQ calibrator and the QC
samples at three concentrations. Preliminary experiments demonstrated that accurate quantitation could not be achieved at
Journal of Analytical Toxicology, Vol, 2 I, October 1997
concentrations below 0.5 and 0.1 ng/mL for the GC-PCI-MS
and LC-ESI-MS-MS methods, respectively.These concentrations were then confirmed as the LOQs from the subsequently
described precision and accuracy experiments. Accuracy was
expressed as the percent of measured concentration relative to
fortified (target) concentration. Precision was expressed as the
percent coefficient of variance (%CV). Intra-assay accuracy
and precision were determined from the analysis of five replicates at each concentration within a single analyticalbatch. For
the GC-PCI-MSmethod, the LOQ accuracy was within 20% of
target concentration, and the three QC samples were all within
14% of their targets. These determinations were precise with
%CVs ranging from 4 to 6% (Table III). The LC-ESI-MS-MS
method demonstrated even better intra-assay accuracy (within
12% of target) and similar precision with %CVsranging from
4 to 10%. Interassay values were determined from seven analytical runs performed on separate days. The average value for
3-5 replicates of the LOQ and QC samples within the run were
used. Interassay accuracy for the GC-PCI-MS method at the
LOQ was within 20% of target and within 8% of target for the
QC samples. The interassay %CVs ranged from 7 to 14%. The
LC-ESI-MS-MS interassay accuracy for the LOQ and the QC
samples was within 8% of target, while the interassay %CVfor
the LOQwas 10%, those for the QC samples did not exceed4%
(Table III). Both methods demonstrated accuracy and precision
that was acceptablefor clinical pharmacokinetic, safety and efficacy studies (28).
An LOQ of 0.1 ng/mL of buprenorphine was established for
the LC-ESI-MS-MS method. Although other methods have
reported LOQs or limits of detection that approach 0.1 ng/mL
(21-24), only Hoja et al. (25) have reported accuracy and precision at this concentration. The lowest concentrations at
which accuracy and/or precision were reported by others were
0.5 (22), 0.5 (23), 1.0 (24), and 5.0 ng/mL (21).
Stability
Buprenorphine undergoes a chemical rearrangement when
heated under acidic conditions. This has been described in
detail for studies performed in aqueous solutions (15,29). The
stability of buprenorphine in biological specimens, however,
has only been addressed in a single study (16). That study
demonstrated that buprenorphine was stable in frozen plasma
for up to 28 days. The bench-top and freeze-thaw stability of
buprenorphine was determined for both of the methods pre-
sented here. At concentrations ranging from 0.25 to 40 ng/mL,
we found that buprenorphine was stable in plasma stored at
room temperature for up to 24 h. Two freeze-thaw cycles did
not result in any appreciable loss of buprenorphine at concentrations ranging from 0.25 to 12.5 ng/mL (Figure 5). In
addition, QC samples routinely analyzed by the GC-PCI-MS
method were stored at 4~ These QC samples were analyzed
over a 124-day period without any loss of buprenorphine
(Figure 6A). QC samples routinely used in the LC-ESI-MS-MS
method were aliquotted and stored at-20~ These QC samples
were followed for 55 days without any noticeable loss of
buprenorphine (Figure 6B). These data set lower limits for
refrigerated and frozen storage of plasma samples that extend
beyond stability limits of 28 days of frozen storage as reported
by Ho (16).
Clinical applications
Forty specimens were collected from four subjects who
received sublingual doses of buprenorphine (2, 8, 16, and 24
mg/70 kg) as described in Experimental. Collection times were
not successive; one collection followed one individual dose.
The results of LC-ESI-MS-MS analysis of these specimens are
presented in Table IV.Averageplasma buprenorphine concentrations were dose and postdose time-dependent. The utility of
the method was demonstrated because buprenorphine could be
detected in the plasma of all four subjects 24-h after administration of a 2-mg/70-kg sublingual dose. A previous study using
a method with an LOQ of 0.2 ng/mL could only follow plasma
buprenorphine in all subjects for 7 h after a 4-mg sublingual
dose. At 24 h after their 4-mg dose, buprenorphine was not
quantitatable in two of the six subjects (27). This difference
between the two studies may be explained by the lower LOQ of
our method. Another possibility is the interindividual differences in buprenorphine pharmacokinetics.
There were interindividual differences in buprenorphine
concentrations between individuals in our study. For example,
subject V008 had dose-dependent concentrations at 24 h postdose of 0.4, 0.5, 1.0 and 1.9 ng/mL following 2, 8, 16, and 24mg/70-kg doses, whereas subject V001 had 24-h postdose
concentrations of 0.2, 0.4, 0.5, and 0.3 ng/mL following the
same respective doses (Table IV). This individual difference
ranged from 1.5-fold in the samples taken 24 h after the
8-mg/70-kg dose to 10-foldin the samples taken 48 h following
the 24-mg/70-kg dose (Table IV). This individual variation is
Table II. Characteristics of Calibration Curves*
FullCurve
Linear range
Intercept
Method
n
(ng/mL)
(ng/mL)
GC-PCI-MS
LC-ESI-MS-MS
7
7
0.5 - 40
0.1 - I 0
-0.108 • 0.155
0.001 • 0.003
Slope
height ratio)]
[(ng/mC)/(peak
11.48 • 1.42
4.82 • 0.32
LowCurve
Correlation coefficient
Intercept
(r z)
(ng/mC)
0.998 • 0.001
0.999 • 0.001
0.026 • 0.033
0.001 • 0.000
* Note: Calibration curves were calculated with concentration on the y-axis and peak height ratios on the x-axis. Split low/high curves were
used. The characteristics of the full curve are shown to demonstrate overall linearity. The intercept for the low curves is also shown to
demonstratethat averageoffset of the curve at the low end did not exceed 5 and 1% of the GC-PCI-MS and LC-ESI-MS-MS LOQs,
respectively. The slopesdiffer since the GC-PCI-MS method employed twice the concentration of internal standard.
411
Journal of Analytical Toxicology,Vol. 21, October 1997
consistent with that reported by Kuhlman et al. (27) who found
up to 21.6-, 8.6-, and 4.8-fold differences among six subjects
after buccal, sublingual, and intravenous administration of
buprenorphine, respectively.
Comparison of methods
The forty clinical specimens were also analyzed by the
GC-PCI-MS method. The utility of decreasing the LOQ from
0.5 to 0.1 ng/mL was demonstrated by these comparative analyses. With the LOQ of 0.5 ng/mL, only 10 of the 40 (25%) specimens had quantitatable buprenorphine, which was also true
for 38 (95%) of the specimens when the LOQ was 0.1 ng/mL.
As only 10 specimens were quantitatable by both methods,
only a limited comparison of quantitation could be made.
Regression analysis gave an r 2 of 0.96. This provides limited
evidence that the two methods provide agreeable results.
Because of the long postdose collection times used in this
study, it provides a relatively rigorous test of the sensitivity of
the methods. It should be noted that the GC-PCI-MS method
was used for a large multi-center study that collected 24-h
postdose specimens, and buprenorphine was quantitatable in
approximately two-thirds of these specimens.
administration is 27.7 h (5.2 to 49.1 h) (27). Therefore, detection of buprenorphine out to 96-h postdose, or longer, is
important for accurate determination of pharmacokinetic
parameters, such as areas under the curve. Reducing the LOQ
from 0.5 to 0.1 ng/mL is also important to follow the pharmacodynamic effects of buprenorphine. For the subjects described
in Table I~ Amass et al. (26) performed additional experiments
to assess the opioid antagonist effects of buprenorphine. They
found that buprenorphine blocked the effects of hydromorphone on observer- and subject-related measures of opioids for
up to 48 h. As shown in Table IV,this antagonism occurred in
several subjects with buprenorphine plasma concentrations
of less than 0.5 ng/mL. This study has also presented long-term
1201008060"
40-
Conclusion
20C~
Of the two assays described here, the LC-MS-MS method
provides the best sensitivity. It also requires less time to perform; there is no derivatization, and the cycle time between
injections is approximately two-thirds that of the GC-PCI-MS
method. The major disadvantage of the LC-MS-MS method is
the high cost of the instrumentation.
We have found that LC-MS-MS can achieve a precise and
accurate LOQ of 0.1 ng/mL for buprenorphine. Although a
similar LOQ was achieved with LC-MS, a more rigorous extraction was required to reach this sensitivity with the single-stage
MS (25). With this LOQ, we were able to demonstrate plasma
buprenorphine concentrations out to 96-h postdose. The
reported mean half-life of buprenorphine after sublingual
Table III. Intra- and Interassay Accuracy and Precision*
Method
GC-PCI-MS
LC-ESI-MS-MS
Target
concentration
(ng/mL)
0.5 (LOQ)
1.0
2.5
12.5
0.1 (LOQ)
0.25
2.5
7.5
O-
0.25
2.5
7.5
12.5
40
2.5
7.5
12.5
40
120
~
100
0
~
80
e~
60
40
20
Accuracy and Precision
Intra-assay Interassay
(% target _+% CV)
120 + 4
116+5
112+6
114__.4
100_+10
108_+4
88 _+4
99 _+4
120 + 10
101+12
102+14
108_+7
100+10
104_+4
92 _+4
96 _+1
* Intra-assay values are from replicates of 5 within a single run. Interassay values
are from means of seven analytical runs performed on separate days with each
samples at an n of 3-5.
412
0
0.25
Sample concentration (ng/mL)
Figure 5. Bench-top and freeze-thaw stability of buprenorphine in
human plasma. Blank plasmas were fortified with buprenorphine at the
concentrations noted. (A) One set of plasma at each concentration
was thawed and stored at room temperature 24 h before controls;
samples were extracted and analyzed once the controls thawed. (B)
One set of plasma at each concentration was thawed and then refrozen
at -20~ These and control sampleswere then thawed a day later and
analyzed. Values are the mean plus or minus standard error of the
mean of three replicatesfor each concentration.Solid bars: analysisperformed by LC-ESI-MS-MS; hatched bars: analysis performed by
GC-PCI-MS.
Journal of Analytical Toxicology, Vol. 21, October 1997
stability data on refrigerated and frozen plasma samples containing buprenorphine.
Recently, we were asked to modify the LC-MS-MS assay in
order to permit simultaneous measurement of buprenorphine
and naloxone. This is because naloxone is being evaluated in a
coformulation with buprenorphine for use in treatment of
opioid addiction. It is anticipated that the dosage of naloxone
can be adjusted in the buprenorphine-naloxone formulation
such that the naloxone will have no antagonistic effect when
administered sublingually, where it undergoes extensive firstpass metabolism, but will act as an opiate-receptor antagonist
when the medication is injected intravenously. This coformulation would thereby discourage diversion of the medication for
purposes of drug abuse.
The modified LC-MS-MS assay has been validated and has a
LOQ of 0.1 ng/mL for both buprenorphine and naloxone. A
preliminary communication describing the assay has been
accepted (30).
Acknowledgment
This work was supported in part by NIDA
contract N01DA-1-9205and NIDAgrant DA
06969.
Table IV. Plasma Buprednorphine Concentrations Versus Doses of Sublingual
Buprenorphine
Plasmabuprendorphine(ng/mL)
Dose
(mg/70 kg)
Subject
Postdose
(h)
V001
V002
V006
V008
2
24
0.2
0.2
0.3
0.4
0.3•
8
24
48
0.4
0.2
0.6
0.3
0.4
0.5
0.5
0.6
0.5•
0.4•
16
24
48
72
0.5
0.2
0.2
1.2
<0.2*
0.5
0.5
0.4
0.3
1.0
0.6
0.2
0.8•
0.3•
0.3•
24
24
48
72
96
0.3
0.2
0.2
<0.1'
1.1
0.4
0.3
0.1
0.5
0.3
0.1
0.1
1.9
2.1
0.1
0.2
1.0•
0.8•
0.2•
0.1•
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9 9
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O l i i i
~:
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Journal of Analytical Toxicology,Vol. 21, October 1997
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