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Articles |
Drug Dependence Research Center, Department of Psychiatry, University of California, San Francisco, San Francisco, CA 94143.
a Address correspondence to this author at: Langley Porter Psychiatric Institute, Box CPR–0984, University of California, San Francisco, 401 Parnassus Ave., San Francisco, CA 94143-0984. Fax 415-476-7690.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-[(S)-1-hydroxy-1,2,2-trimethylpropyl]-6,14-endo-ethano-6,7,8,14-tetrahydrooripa-vine,
is an analgesic with a long duration of action
(1)(2), 25–40 times more potent than morphine
(2)(3)(4), which elicits partial agonist effects at the µ
receptor and antagonist effects at the 
receptor
(5)(6)(7)(8)(9)(10). Buprenorphine has been used for the control of
both acute postoperative pain and the chronic pain associated with
terminal cancer (4)(6)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21).
Because of its relatively low abuse potential
(22)(23) and because it produces less
respiratory depression than methadone
(14)(16)(24), buprenorphine is
currently in clinical trials as a pharmacotherapeutic agent for the
treatment of opiate dependence
(14)(17)(23)(25)(26)(27)(28)(29)(30)(31). Because the analgesic dosage of buprenorphine in humans is low (13)(14)(15)(17)(18)(19)(20)(21)(32), 0.3–1 mg parenterally and 4–8 mg sublingually, and because the parent drug is metabolized by hepatic enzymes to the dealkylated product, norbuprenorphine (33), and conjugated to ß-glucuronic acid (34), concentrations of buprenorphine in plasma often fail to exceed 1 µg/L (32)(35). Analysis of buprenorphine concentrations in plasma is, therefore, challenging (36).
The literature of the analytical chemistry of buprenorphine is extensive, with methods based on HPLC with ultraviolet (37)(38)(39), fluorescence (37)(40), electrochemical (41)(42)(43)(44)(45)(46), and ion spray–mass spectrometry (HPLC/ISP-MS) (47) detection; gas chromatography (GC) interfaced with nitrogen–phosphorus (48), electron-capture (ECD) (33)(48)(49), and MS detectors [electron impact (EI) (34)(46)(50), chemical ionization (51), and tandem negative chemical ionization (52)]; RIA (46)(53); radioreceptor assay (54); and enzyme-linked immunoassay (54) applied to the analysis of buprenorphine in plasma and urine.1 HPLC, with the exception of the recently published HPLC/ISP-MS method (47), appears not to possess the routine sensitivity required for the reliable measurement of buprenorphine in plasma, whereas RIA, potentially the most sensitive analytical technique, has cross-reactions with norbuprenorphine and ß-glucuronide conjugates, which, because of metabolite buildup in the plasma, presents particular problems for the measurement of the drug in chronically dosed subjects (36).
Of the published methods, GC-MS (34) initially appeared to be the most suitable for our needs, but we were unable to achieve adequate sensitivity. The tandem negative chemical ionization method of Kuhlman et al. (52) had not been published at the time of the present work.
Cone et al. (49) reported a GC-ECD method with a detection limit of 10 µg/L for the measurement of buprenorphine, norbuprenorphine, and demethoxybuprenorphine, the acid-catalyzed degradation product of buprenorphine (55), in urine and fecal extracts. The method utilizes etorphine as the internal calibrator, conversion of the analytes to the pentafluoropropionyl esters, and chromatography on a packed column. By refining the extraction and derivatization procedure of Cone et al., using a capillary column and a structural analog of buprenorphine, N-n-propylnorbuprenorphine, as an internal calibrator, we developed a method for the measurement of buprenorphine in plasma with a limit of quantification of 0.1 µg/L. This method was used to obtain pharmacokinetic data from human volunteers given intravenous and sublingual tablet and solution formulations of buprenorphine. This data is being used in support of a new drug application filed by the National Institute on Drug Abuse (NIDA) and Reckitt and Colman Products with the Food and Drug Administration for approval of the use of buprenorphine as a pharmacotherapeutic agent for the treatment of opiate addiction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following analytical reagent-grade materials were used: acetone, t-amyl alcohol, sodium bicarbonate, 10 mol/L sodium hydroxide, and concentrated sulfuric acid (Fisher Scientific). Toluene (Fisher Scientific) and n-butyl acetate (Aldrich) were of HPLC grade. Ethyl acetate and heptane were of ultraresianalyzed grade (J.T. Baker). Heptafluorobutyric anhydride was obtained in 1-mL prescored ampules (Pierce). All extraction tubes and GC microvial inserts were deactivated with water-soluble siliconizing fluid (Aquasil, Pierce).
Synthesis of the internal calibrator,
N-
n-propylnorbuprenorphine. A solution of
norbuprenorphine (28.4 mg, 63.1 µmol) in 11 mL of methanol:acetic
acid (10:1 by vol) was treated with 0.500 mL (403 mg, 6.93 mmol) of
freshly distilled propionaldehyde and the mixture allowed to stir at
room temperature for 30 min. After cooling the mixture to -78 °C in
a dry ice–acetone bath and slow addition of sodium borohydride (923
mg, 24.4 mmol) followed by the addition of another 10 mL of methanol,
the mixture was allowed to stir for 1 h and then warm to room
temperature. Evaporation of the solvent under reduced pressure was
followed by cooling to 0 °C and slow addition of 20 mL of water. The
pH of the aqueous phase was adjusted to 9.1 with 85% phosphoric acid
and the crude product extracted with 3 x 20 mL of ethyl acetate.
The combined extracts were washed with 50 mL of saturated sodium
chloride, dried over anhydrous magnesium sulfate, filtered, and
evaporated under reduced pressure to yield a viscous oil, which was
dissolved in 5 mL of 2-propanol, cooled to 0 °C, and treated with
125 µL of 1 mol/L HCl. Addition of 50 mL of ether caused the
precipitation of the hydrochloride salt, which was separated from
solution, washed with fresh ether, and air-dried to yield 20.3 mg (41.2
µmol, 60%) of white, crystalline material that was used without
further purification.
preparation of plasma calibrator, control, and internal calibrator
solutions
Buprenorphine
. One stock solution of 100 mg/L
buprenorphine was prepared in 0.01 mol/L sulfuric acid and diluted to a
concentration of 10 mg/L. The dilution was used in the preparation of
the plasma calibrators. A second 100 mg/L solution, also diluted to 10
mg/L, was used for the preparation of the plasma control solutions.
Individual calibrators and controls were prepared by diluting the appropriate volume of 10 mg/L stock solution with enough blank plasma to attain a total volume of 100 mL. The solutions were then mixed well and aliquoted into single-run amounts in 1-dram, screw-capped glass vials, which were stored at -20 °C until needed. Calibrators were prepared at concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 5, 10, and 20 µg/L. Plasma controls were prepared at 0.1, 0.2, 0.5, 2, and 10 µg/L.
Internal calibrator.
A stock solution of 100 mg/L
N-n-propylnorbuprenorphine was prepared in 0.01 mol/L
sulfuric acid, and 500 µL was diluted with 99.5 mL of 0.01 mol/L
sulfuric acid to produce a solution at 500 µg/L. This solution was
aliquoted into single-run amounts (about 7.0 mL per vial) in 2-dram,
Teflon-lined, screw-capped vials, which were stored at -20 °C until
needed.
Calibration graphs.
Quantification was achieved by
integration of detector responses and constructing calibration curves
of the response (peak-height) ratios of analyte/internal calibrator vs
amount (concentration) ratios of analyte/internal calibrator by linear
regression. For this assay, the linear range was from at least 0.1 to
20 µg/L.
Sample preparation.
To 1 mL of plasma in a 16 x
100 mm silanized, screw-top culture tube was added 100 µL of 500
µg/L internal calibrator (N-n-propylnorbuprenorphine), 4
mL of ethyl acetate:heptane (4:1 by vol), and 0.5 mL of pH 9.13 sodium
bicarbonate buffer. The tube was then capped, vortex-mixed for ~10
min, centrifuged for 20 min at 1500g, and subsequently
immersed in a dry ice–acetone bath to freeze the bottom aqueous layer.
The top organic layer was decanted into a clean 16 x 100 mm
silanized culture tube containing 0.5 mL of 0.25 mol/L sulfuric acid,
and the tube was recapped, vortex-mixed for ~10 min, centrifuged for
10 min at 1500g, and immersed in a dry ice–acetone bath to
freeze the aqueous layer. The organic layer was discarded. Two
milliliters of toluene:t-amyl alcohol (9:1 by vol) was
added, and after the aqueous layer had thawed, the tube was recapped,
vortex-mixed for ~5 min, centrifuged for 10 min at 1500g,
and immersed in a dry ice–acetone bath to freeze the aqueous layer.
The organic layer was discarded. Four milliliters of ethyl
acetate:heptane (4:1 by vol) and enough pH 9.45 sodium bicarbonate
buffer (usually 0.5–0.9 mL) to adjust the aqueous phase to pH 9.13
were added to the tube, which was recapped, vortex-mixed for ~10 min,
centrifuged for ~10 min at 1500g, and immersed in a dry
ice–acetone bath to freeze the aqueous layer. The organic layer was
decanted into a clean 16 x 100 silanized culture tube and
evaporated to dryness under a stream of dry nitrogen. Two milliliters
of dry heptane was added and evaporated to dryness.
To the residue was added 100 µL of dry toluene and 50 µL of heptafluorobutyric anhydride. The tube was recapped and the mixture was vortex-mixed and allowed to stand at room temperature for at least 1 h and no longer than overnight. After uncapping, the excess reagent was evaporated to dryness at room temperature under a stream of dry nitrogen.
The residue was dissolved in 20 µL of n-butyl acetate and transferred to an autosampler vial in a 200-µL silanized insert. The vial was crimp-capped and loaded onto the autosampler tray, which was water-thermostated to the ambient temperature of the laboratory, and the sample analyzed by ECD GC.
Chromatography.
GC analyses were performed with a
Hewlett-Packard (HP) 5890 GC equipped with an HP 7673A autosampler, an
HP G1223A ECD, and an HP 5895A data collection and processing
system. An HP Ultra-1 fused-silica capillary column (25 m x 0.2 mm
i.d.) was used, with a stationary phase of cross-linked methylsilicone
gum (0.33-µm film thickness). A Merlin Microseal (Merlin Instruments)
was used instead of a standard silicone–rubber septum. Helium was used
as the carrier gas, with a head pressure of 200 kPa, which resulted in
a flow rate of approximately 3 mL/min (37 cm/s) at 150 °C.
The sample (2 µL) was injected in the splitless mode via the autosampler, with a septum purge on-time of 1 min and an injector-port temperature of 285 °C. The column-oven temperature was programmed from 150 °C (after a hold of 1 min) to 325 °C at a rate of 10 °C/min and held for another 6 min. The detector (ECD) temperature was 325 °C. Typical retention times for the heptafluorobutyryl derivatives were as follows: 19.2 min (internal calibrator), 20.4 min (buprenorphine). Before commencement of an analytical run, active sites within the chromatographic system were plugged with 8–10 replicate injections of a derivatized extract of a plasma sample supplemented with 50 ng of both the internal calibrator and buprenorphine.
Repeatability, precision, and accuracy.
The
repeatability of the method was estimated by comparing the linear
regression slopes, intercepts, and correlation coefficients of the
calibrator plots from individual runs. Precision and accuracy were
determined from the analysis of supplemented plasma controls at five
concentrations ranging from 0.1–10 µg/L. The precision of the method
was expressed as the within-run and total (n = 40)
(56) CVs, and the accuracy as the ratio of the average
calculated concentrations to their supplemented values.
Long-term and freeze–thaw stability
. The long-term
stabilities of both plasma controls and clinical samples were
determined. Replicate analyses (n = 7) of aliquots of four
different plasma control concentrations spanning the range 0.2–20
µg/L were performed and the mean calculated concentrations, CVs, and
accuracies determined. After storage at -20 °C for 14 months,
replicate analyses (n = 4) of aliquots of the same four batches of
plasma controls were performed and the results compared.
After storage at -20 °C for 14 months, 20 previously measured and randomly selected clinical samples were reanalyzed. The original and repeat measurements were compared, the differences calculated, and the average change determined.
Freeze–thaw stability was determined for five consecutive cycles of freezing and thawing. Supplemented plasma quality-control (QC) samples were prepared at 0.5 and 10 µg/L, aliquoted, and frozen at -20 °C immediately after preparation. For each freeze–thaw cycle, the samples at each concentration were thawed to room temperature, allowed to stand for 1 h, and then refrozen. After duplicate samples at each concentration had been through 1–5 cycles of freezing, all samples were thawed and analyzed for buprenorphine.
Recovery.
The recovery of buprenorphine from extracted
plasma samples was determined at four concentrations spanning the range
from 0.5 to 50 µg/L. A stock solution of buprenorphine was prepared
at a concentration of 100 mg/L in methanol, and serial dilutions of an
aliquot of this stock solution produced buprenorphine solutions at 10,
1, 0.1, and 0.01 mg/L. An aliquot of the 10 mg/L solution was diluted
with blank plasma to yield a concentration of 50 µg/L. Serial
dilutions of the 50 µg/L plasma with the appropriate volumes of blank
plasma resulted in buprenorphine plasma concentrations of 10, 2, and
0.5 µg/L.
Four 1-mL aliquots at each plasma concentration were extracted by the standard procedure, with the final extracts decanted into tubes previously supplemented with 50 ng of the internal calibrator. The extracts were evaporated to dryness and derivatized by the standard method.
The 0.1 and 0.01 mg/L stock solutions were used to supplement four 16 x 100 mm silanized screw-top culture tubes that had been previously supplemented with 50 ng of internal calibrator, with each of the following amounts of buprenorphine: 50, 10, 2, and 0.5 ng. The solutions were evaporated to dryness and the residues derivatized with heptafluorobutyric anhydride.
GC analysis of the samples was followed by integration and calculation of the peak-height ratios of the buprenorphine/internal calibrator peaks in each chromatogram. The mean peak-height ratio for each concentration was calculated for both the extracted and unextracted samples. The ratio of averages at each concentration was considered to represent the recovery of buprenorphine at each concentration.
Derivative stability.
Stability of the derivatized
extracts during the chromatographic run was established by using two
identical and concurrently prepared sets of calibrator and QC samples.
One set was chromatographed immediately; the second set was injected
after sitting on the temperature-thermostated autosampler tray for
36 h. The first set was then reinjected.
pharmacokinetic studies
This method has been used in human pharmacokinetic studies of the
bioavailability of a sublingual 2-mg, 300 mL/L ethanol solution
(57), and the bioequivalence of an 8-mg tablet compared
with an ethanolic solution formulation.
Bioavailability
. Six healthy subjects, five men and one
woman between the ages of 21 and 36 years, opiate experienced but
non-opiate dependent by DSM-IV criteria (58), were tested.
Written consent was obtained. In a three-session balanced design at
weekly intervals, subjects were tested under the following experimental
conditions: (a) buprenorphine 2 mg in 1 mL of a 300 mL/L
ethanol solution held sublingually for 3 min; (b)
buprenorphine 2 mg in 1 mL of a 300 mL/L ethanol solution held
sublingually for 5 min; (c) buprenorphine 1 mg by
intravenous infusion over 30 min. Sublingual treatments were random in
sequence; the intravenous treatment was always given between the
sublingual treatments.
Sublingual doses were administered with a 1-mL tuberculin syringe. The buprenorphine solution was placed in the right posterior sublingual area at the base of the tongue. Dose exposure was terminated by swallowing, and subjects were instructed not to swallow until told to do so by an observer. After 3 or 5 min elapsed, subjects immediately swallowed once and thereafter were allowed to swallow ad libitum.
The intravenous dose of buprenorphine was infused into a forearm vein at a rate of 1 mL/min under syringe pump control over a 30-min period. Blood samples were obtained through an intravenous catheter placed in the opposite forearm.
Blood samples (10 mL) were collected into iced, heparinized Vacutainer Tubes (Becton Dickinson) before buprenorphine administration and at 5, 20, 30, 40, 60, 90, 120, 180, 240, 300, 360, 480, 600, and 720 min postdose. Samples were centrifuged and the plasma separated and frozen at -20 °C within 30 min of blood collection.
Statistical analyses were performed on areas under the curves (AUCs), peak concentration, and peak time by using the SAS Statistical Analysis System program (59).
Bioequivalence.
Six healthy male subjects between the
ages of 23 and 42 years who met the same eligibility requirements as
the participants in the bioavailability study gave informed written
consent and were tested in a two-session, balanced crossover design
approximately 7 days apart, under the following experimental
conditions: (a) buprenorphine 8 g/L in 1 mL of a 300 mL/L
ethanol solution held sublingually for 5 min; (b) a
prototype buprenorphine 8-mg tablet held sublingually for 5 min.
The liquid formulations were administered in the same manner as for the bioavailability study. The tablet was placed in the midportion of the lateral sublingual space. At 5 min after tablet dosing, the sublingual area was inspected briefly for any residual tablet. If tablet fragments were visible, the subject was instructed to continue sublingual holding and not to swallow until the tablet had subjectively completely dissolved or for an additional 5 min, whichever occurred first. If full dissolution was confirmed by visual examination, the time was recorded, the subject swallowed once, and then swallowed ad libitum.
Blood samples (10 mL) were obtained through an intravenous catheter placed in the forearm of the nondominant hand. Samples were collected into heparinized Vacutainer Tubes immediately before dosing and at 0.25, 0.50, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, 36, and 48 h after administration. Samples were centrifuged and the plasma separated and frozen at -20 °C within 30 min of blood collection.
Statistical analyses were performed by the same methodology as that used for the bioavailability study.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Derivatization.
Derivatization of buprenorphine with
heptafluorobutyric anhydride results in the formation of the
heptafluorobutyryl esters of the parent compound as well as its
acid-catalyzed degradation products,
21-cyclopropylmethyl-7-[2-(3,3-dimethyl-1-butenyl)]-6,14-endo-ethanotetrahydrooripavine
(buprenorphine olefin) and
21-cyclopropylmethyl-6,14-endo-ethano-2',3',4',5',7,8-hexahydro-4',4',5',5'-tetramethylfurano-
[2',3':6,7]-normorphide (demethoxybuprenorphine) (Fig. 1
). Thesedegradation products (55)
result from simple elimination of water and elimination of the elements
of methanol accompanied by cyclization, respectively, from the 7-
side chain of the tetrahydrooripavine nucleus. The internal calibrator,
N-n-propylnorbuprenorphine, undergoes analogous elimination
and cyclization.
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When the derivatization reaction is carried out in silanized culture
tubes, the losses of analytes via this pathway are minimized, typically
averaging <10%. However, when the acylation is performed in
unsilanized tubes, losses may approach 25% or more (Fig. 2
). The side reaction may also be suppressed by pretreatment of
the culture tubes for several hours with dilute aqueous disodium EDTA.
Silanization of microvial inserts was necessary to prevent irreversible
adsorption of the derivatives to the glass walls of the inserts.
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Chromatography.
A typical chromatogram of a derivatized
extract of a 1-mL blank plasma sample supplemented with 50 ng of
internal calibrator and 20 ng of buprenorphine is shown in Fig. 3
. Expanded chromatograms of derivatized extracts of a 1-mL blank
plasma supplemented with 50 ng of the internal calibrator and 0.5 ng of
buprenorphine, and a 1-mL blank plasma sample are displayed in Figs. 4
and
5, respectively. No extraneous peaks that would interfere with
the analysis at concentrations above the quantification limit were
found in either blank plasma or in clinical samples. Additionally, a
study was performed to examine the possibility of interference with the
assay from some commonly abused narcotic analgesics, opiate
antagonists, amphetamines, anticonvulsants, antidepressants,
antipyretics, benzodiazepines, hal-lucinogens, ß-blockers,
trimethylxanthines, and the major metabolite of buprenorphine,
norbuprenorphine. Although most of the drugs examined yielded
derivatives upon reaction with heptafluorobutyric anhydride, none
presented any interference with the assay. The results are summarized
in Table 1
.
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Calibration curves.
Calibration plots were broken into
low (0.1–2.0 µg/L) and high (2.0–20 µg/L) concentration ranges.
The curves were linear in each range, with average coefficients of
determination (r2) of 0.998 and 0.999,
respectively, for the low and high ranges.
Sensitivity.
The limit of detection for accurate
quantification was 0.1 µg/L, defined by a total CV of <20%;
however, concentrations of 0.05 µg/L could still be detected.
Precision and accuracy.
The within-run and total means,
accuracies, SDs, and CVs of the method were determined with blank
plasma samples supplemented with known amounts of buprenorphine at five
different concentrations spanning the analytical range of the method,
and included in single runs performed on 20 different days. For the
study, two replicate samples were analyzed at each concentration. CVs
were <10% at every concentration with the exception of the 0.1 and
0.2 µg/L, where the total CVs were 15.9% and 12.5%, respectively
(Table 2
).
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Long-term and freeze–thaw stability.
Aliquots (n =
7) of supplemented QC samples at four different concentrations were
measured immediately after preparation. The remaining sample at each
concentration was aliquoted into glass vials in single-run amounts and
frozen at -20 °C. Fourteen months later the samples were thawed and
remeasured (n = 4) with no evidence of decomposition (Table 3
).
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Twenty randomly chosen clinical samples were examined for stability in the same manner. However, statistical analysis of the data indicated some decomposition, averaging 10%, over the same time frame.
Supplemented plasma QC samples were prepared at 0.5 and 10.0 µg/L, aliquoted, and frozen at -20 °C immediately after preparation. The samples were put through repetitive cycles of freezing and thawing. For each freeze–thaw cycle, the samples were thawed to room temperature, allowed to stand for 1 h, and then refrozen. When duplicate samples at each concentration had been through 1–5 cycles of freezing, all samples were thawed and analyzed for buprenorphine, with no evidence of decomposition.
Recovery.
The recovery of buprenorphine averaged 77%
over two orders of magnitude of concentration, and ranged from 71% at
10 µg/L to 85% at 0.5 µg/L (Table 4
).
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Stability of derivatives.
Decomposition of the
derivatives was found to be accelerated by the heat from the hot
injector port transferred to the samples on the autosampler tray. To
retard this decomposition, cold water was circulated through the sample
trays throughout the course of the run. Stability was established with
two identical and concurrently prepared sets of calibration and QC
samples. The first set was chromatographed immediately. Twenty-four
hours after completion of the first run, corresponding to 36 h
after initiation of the first run, the second set of samples was
chromatographed. Finally, the first set was rechromatographed and the
data analyzed. No evidence of decomposition was found.
Pharmacokinetic studies.
Fig. 6
shows the mean concentration–time curves for buprenorphine
obtained from six patients after receiving, in three different sessions
approximately 7 days apart: 1 mg of buprenorphine, 30-min intravenous
infusion; 2 mg of buprenorphine in 1 mL of 300 mL/L ethanol, held
sublingually for 3 min before swallowing was allowed; and 2 mg of
buprenorphine in 300 mL/L ethanol, held sublingually for 5 min. Table 5
lists the corresponding pharmacokinetic parameters: AUC, peak
concentration, and peak time. Bioavailability from the 3-min sublingual
administration, based on extrapolated AUC, was 36.5 ± 13.4%
(±SD). The corresponding parameter for the 5-min sublingual
administration was 33.1 ± 13.4% (±SD) with no significant
difference in the extent of bioavailability between the two sublingual
holding times.
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Figure 7
shows the mean plasma concentration–time curves for
buprenorphine obtained from six patients after receiving, in two
different sessions approximately 7 days apart: buprenorphine 8 g/L in 1
mL of 300 mL/L ethanol, held sublingually for 5 min; and a prototype
buprenorphine 8-mg tablet, also held sublingually for 5 min. Table 5
displays the corresponding pharmacokinetic parameters derived from
these curves. On the basis of these parameters, we concluded that the
two products differed in both rate and extent of bioavailability of
buprenorphine. The relative bioavailability of buprenorphine from the
prototype 8-mg tablet, when using the solution as a reference, was
58.4% ± 31.8% for extrapolated AUC.
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These results are discussed in detail in a separate publication (57).
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Discussion |
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The first reported GC method for the analysis of buprenorphine in plasma was the GC-MS method of Lloyd-Jones et al. (50), which involved a complex liquid–liquid extraction of horse plasma, followed by derivatization with heptafluorobutyric anhydride for GC separation and analysis. The reported limit of quantification was 20 µg/L. Ohtani et al. (51) found that selected-ion monitoring (SIM) in the EI mode, even with extensive sample cleanup before derivatization with pentafluoropropionic anhydride, resulted in chromatograms with high background noise, precluding analysis of buprenorphine at subnanogram concentrations in plasma. SIM of the same derivatized extracts in the positive chemical ionization mode resulted in a method with a limit of detection of 0.2 µg/L. A GC method involving solid-phase extraction and ECD of the heptafluorobutyryl ester of buprenorphine, and claiming a limit of detection of 0.5 µg/L, was reported by Martinez et al. (48). This method, however, requires 2–4 mL of plasma and still does not achieve the desired limit of quantification. Debrabandere et al. (46), using an extensive liquid–liquid extraction procedure, silylation of the final extract, and SIM in the EI mode, claimed limit of detection of 0.2 µg/L for buprenorphine in plasma.
We initially attempted to use the GC-MS method of Blom et al.
(34), with a reported limit of detection of 0.15 µg/L of
buprenorphine in human plasma, but abandoned this method when we could
not consistently reproduce the reported acid-catalyzed degradation of
the 7- side chain. Subsequently, it became apparent that this
degradation was neither necessary nor advantageous for the
chromatography, and that the amount of elimination and cyclization that
occurs upon reaction with heptafluorobutyric anhydride could be
controlled by appropriate deactivation of the culture tubes utilized
for the derivatization reaction (Fig. 2
).
To obtain maximum sensitivity we decided to use ECD. The method of Cone et al. (49), developed for the analysis of buprenorphine in urine and feces, and with a reported limit of detection of 10 µg/L in urine, appeared to be a good starting point. Preliminary experiments determined that the extraction procedure described in this report provided cleaner extracts than those obtained with solid-phase extraction.
The method was optimized by using N-n-propylnorbuprenorphine, a closer structural analog of buprenorphine than the etorphine used by Cone et al. The pH was maintained at the isoelectric point during the extraction procedure to maximize recovery, and silanizing the culture tubes used for derivatization minimized decomposition. Heptafluorobutyryl esters were prepared instead of the pentafluoropropionyl esters because the heptafluorobutyryl esters are more stable to hydrolysis. Microvial inserts were deactivated to prevent adsorption of the heptfluorobutyryl derivatives of buprenorphine and the internal calibrator, which increased dramatically with time in the absence of silanization. The autosampler tray was temperature-thermostated to retard thermal degradation of the derivatives. Finally, modern capillary GC was used instead of packed-column chromatography.
Implementation of these modifications resulted in a method with the precision, accuracy, specificity, and sensitivity (0.1 µg/L) required for the measurement of buprenorphine in plasma.
After the completion of this work, a GC tandem (GC/MS-MS) mass-spectrometric method (52) was reported for the simultaneous measurement of buprenorphine and norbuprenorphine in plasma. The method involves a triple-stage quadrupole instrument in the negative chemical ionization mode. The reported limits of quantification are 0.2 µg/L for buprenorphine and 0.03 µg/L for norbuprenorphine. A separate publication (60) reports the use of this method to study the pharmacokinetics of intravenous, sublingual, and buccal buprenorphine.
The method described here has the necessary specificity, sensitivity, precision, and accuracy for pharmacokinetic studies of buprenorphine. The extensive sample cleanup procedure is more labor-intensive than the simple extraction procedures utilized in either the HPLC/ISP-MS (47) or GC/MS-MS (52) methods described above. The advantage of our method is that it can be performed by any laboratory that owns a capillary GC equipped with a data system, autosampler, and an ECD. The instruments required for the HPLC/ISP-MS and GC/MS-MS methods, although state of the art, require the capital investment of several hundred thousand dollars, and are thus beyond the means of most analytical laboratories.
Measurement of the metabolite norbuprenorphine, which has a much lower
analgesic potency than buprenorphine (61), was not
required for our studies. However, norbuprenorphine is extracted and
derivatized by the method (Table 1
). Consequently, the method could be
appropriately modified to measure norbuprenorphine as well.
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Acknowledgments |
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Footnotes |
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References |
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The following articles in journals at HighWire Press have cited this article:
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  D. A. Ciraulo, R. J. Hitzemann, E. Somoza, C. M. Knapp, J. Rotrosen, O. Sarid-Segal, A. M. Ciraulo, D. J. Greenblatt, and C. N. Chiang Pharmacokinetics and Pharmacodynamics of Multiple Sublingual Buprenorphine Tablets in Dose-Escalation Trials J. Clin. Pharmacol., February 1, 2006; 46(2): 179 - 192. [Abstract] [Full Text] [PDF]
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), 2 mg of buprenorphine in
1 mL of 300 mL/L ethanol sublingually for 3 min (
), and 2 mg of
buprenorphine in 1 mL of 300 mL/L ethanol sublingually for 5 min
(
).
)
and an 8-mg buprenorphine tablet sublingually for 5 min (•).