Development and clinical application of a
mass spectrometric method to quantify methadone
in dog plasma
Lena Thunander Sundbom
20 p Master´s Degree Project, Fall semester 2003.
Department of Medicinal Chemistry, Division of Analytical Pharmaceutical Chemistry, Uppsala University.
Performed at Departement of Chemistry, National Veterinary Institute (SVA), Uppsala.
Supervisors: Ulf Bondesson, Professor, and Mikael Hedeland, PhD,
Departement of Chemistry, National Veterinary Institute (SVA),
In cooperation with the Swedish University of Agricultural Sciences (SLU), Uppsala and Astra Zeneca, Lund.
Examiner: Curt Pettersson, Professor,
Departement of Medicinal Chemistry,
Division of Analytical Pharmaceutical Chemistry, Uppsala University.
1
ABSTRACT
3
INTRODUCTION
4-5
MATERIALS AND METHODS
6-8
Chemicals
6
Instrumentation
6
Plasma samples
6
Sample pre-treatment (extraction procedure)
6
Gas chromatographic and mass spectrometric procedure
7
Standard solutions and calibrations
7
Analysis
7
Validation
7-8
RESULTS AND DISCUSSION
9-15
Method development
9-13
Extraction procedure
Gas chromatographic and mass-spectrometric procedure
Validation
9
9-10
11-13
Bioanalysis of dog plasma
13-15
CONCLUSION
15
REFERENCES
16
2
ABSTRACT
A liquid-liquid extraction method and a gas chromatographic method with chemical
ionization tandem mass spectrometric detection was developed and validated to
quantify methadone in dog plasma. Validation parameters evaluated was: limit of
detection, limit of quantification, stability in plasma, stability of extracts, selectivity,
precision and accuracy. The method was adequate to quantify methadone in dog
plasma at concentrations above 2.2ng/ml (LOQ). The application of the assay was
demonstrated in a pharmacokinetic study on four healthy beagle dogs. The dogs
were treated with methadone either with subcutaneous injections or with
intramuscular injections every forth hour during three days. The elimination was
studied 36 hours after the last given dose.
3
INTRODUCTION
The tertiary amine methadone (d,l-dimethylamino-4,4-diphenyl-3-heptanone, Fig.1a)
is a synthetic analgesic opiate closely related with morphine. Since the introduction
into human clinical practice in 1946, methadone has been widely used for cancer
pain relief, in postoperative pain therapy and as a substitute for patients dependent
upon heroin1,2. In Swedish veterinary medicine there are no approved drugs
containing opiates. Therefore, the use of human drugs like methadone are, since
many years, well established for postoperative pain relief in dogs, even though there
is very little knowledge about metabolism, kinetics and bio-availability in the animal3.
Doses and regimens have to be based upon human data and it is uncertain if the
treatment is carried out in the optimal way. The pharmacokinetic parameters of
methadone differ from that of other opiates. In all investigated species, methadone is
described as a drug with a rapid distribution phase and a slow rate of elimination 4.
Most of the pharmacokinetic studies conducted in humans have indicated highly
variable interindividual half-lives1,5. Thus, a potential risk for toxic accumulation exists
with repeated administration. This variability may require dose adjustment on the
basis of individual pharmacokinetic parameters5. For humans, a dosage regimen
recommended for pain relief is a dose every 4-6 hours the first 24 hours and then one
dose every 12 hours to avoid accumulation and unwanted side-effects6.
Recommended doses and regimens for dogs with a dose every 3-4 hours7 do not
take that into consideration, although the risk for accumulation might be as great as
for humans. Both in rats8, goats4 and dogs3,9, studies have shown an wide
interindividual range in the terminal half-lives.
CH3
O
H3C
N
CH3
CH3
O
CH3
D3C
N
CH3
CH3
Fig.1b The structure of 2H3-methadone.
Fig.1a The structure of methadone.
In order to quantify methadone in biological fluids, many analytical methods have
been developed, including gas chromatography with nitrogen-phosphorus detection1,
gas chromatography–mass spectometry10,11,12, high-performance liquid
chromatography with UV-detection2,3,13,14 and radioimmunoassay4,5,8. Previously
reported analytical procedures for measuring methadone in biological matrices have
primarily involved multi-step extraction procedures, which gives a tedious and time
consuming sample pre-treatment. Another problem with some of the published
methods is lack of sensitivity. However, HPLC with UV detection has shown a limit of
quantification (LOQ) of 2.5 ng/ml2,14, and gas chromatography with nitrogenphosphorus detector has shown even better sensitivity, with a LOQ of 0.5 ng/ml1.
Mass spectrometric assays have been shown to provide increased sensitivity and
selectivity over a wide range of chemical classes, and have been successfully
applied to the analysis of several opiates15. Therefore, the use of mass spectrometric
detection should increase the sensitivity even more. Despite that, studies using gas
chromatography with single mass spectrometric detection (MS) has not shown
increased sensitivity, when analysing methadone, compared to other methods (LOQ
5-40ng/ml)10,11,12. The use of tandem mass spectrometry (MS/MS) should,
4
theoretically, increase the sensitivity and selectivity, but no such study has been
published. Mass spectrometric analytical methodologies offer several advantages
over alternative means of quantitative analysis. For example MS enables the use of
deuterated internal standards, which closely compensate for variations in the
analytical procedure15.
Methadone is a frequently used post-operative analgesic drug for dogs and it is
important to be able to determine concentrations of methadone in dog plasma and
thereby be able to decide effective doses and regimens to prevent side-effects. The
aim of this study was to develop and validate a new bio-analytical method for
quantification of methadone in dog plasma and apply it on a pharmacokinetic study.
The intention was to develop a GC-MS/MS method, working in the SRM mode, in
order to obtain increased sensitivity and more selective detection compared to other
methods. Furthermore, simplification of the extraction procedure was also a main
objective.
5
MATERIALS AND METHODS
Chemicals
Methadone (d,l – dimethylamino-4,4-diphenyl-3-heptanone, Fig.1a) hydrochloride
and the analogue labelled with three deuterium atoms (2H3 methadone hydrochloride,
used as internal standard, Fig.1b) was donated from Ulleråker hospital, Uppsala,
Sweden. Other chemicals used, hexane, 2-buthanol, NaOH and toluene were of
analytical grade and used without further purification. The water used was purified in
a Milli-Q water purification system from Millipore (Bedford, MA, USA).
Instrumentation
A Hewlett-Packard 5890 Series II Gas Chromatograph (Waldbronn, Germany)
equipped with a CTC analytics automatic sampler (Zwingen, Switzerland) and a
ThermoFinnigan Mat TSQ 7000 Mass Selective Detector (triple quadrupole MS/MS;
San Jose, CA, USA) was used for the analysis. A HP-5MS capillary column (30m x
0.32 mm ID) with film thickness of 0.25 m (5% phenylmethylsilyl) was used
(Hewlett-Packard, Waldbronn, Germany). For instrument control, a computer with the
software Xcalibur (ThermoFinnigan, San Jose, CA, USA) was used. The shaking
apparatus used was an Edmund Bűhler 7400 Tűbingen SM 25 and the centrifuge
used was a Heraeus Sepatech Megafug 1,0R.
Plasma samples
Four adult healthy beagle dogs (Astra Zeneca R&D animal department, Lund,
Sweden) were given doses of methadone hydrochloride (Metadon ® Pharmacia,
10mg/ml) every fourth hour during two (dog 2 and 4) or three (dog 1 and 3) days.
Blood samples were collected, right before the given doses, i.e. four hours after every
injection, by a permanent catheter in V.jugularis. Blood samples were also collected
10min, 20min, 30min, 45min, 1h, 2h, 4h, 6h, 8h, 12h, 16h, 24h and 36h (48h for dog
2 and 4) after the last given dose. The four dogs were treated either with 0.1 mg/kg or
0.4 mg/kg and either with intramuscular (I.M.) injections or subcutaneous (S.C.)
injections.
Dog 1: methadone 0.1 mg/kg S.C. injection
Dog 2: methadone 0.4 mg/kg S.C. injection
Dog 3: methadone 0.1 mg/kg I.M. injection
Dog 4: methadone 0.4 mg/kg I.M. injection
Blood samples were centrifuged for 10 minutes and plasma were stored at -20C
until analysis. The Ethical Committee for Animal Experiments, Lund, Sweden,
approved the study protocol.
Sample pre-treatment (extraction procedure)
A single step liquid-liquid extraction procedure was used. 100 l of the internal
standard solution (56ng/ml plasma or 14ng/ml plasma), 500 l Milli-Q water, 5.0 ml
hexane:2-buthanol (97:3) and 100 l 1M NaOH was added to 500 l plasma. The
sample was shaken for 15 min and then centrifuged for 10 min at 3500 rpm. The
organic phase was transferred to clean tubes and evaporated to dryness at 50C
under a gentle stream of dry nitrogen. The dry residue was redissolved in 50 l
toluene and transferred to vials before GC-MS/MS analysis.
6
Gas chromatographic and mass spectrometric procedure
Helium was used as the carrier gas at a flow-rate of 1.5 ml/min. Injections of 1.0 l
were performed with the injector working in the split-less mode at 250C. The oven
temperature program used was: 150C the first minute and then the temperature was
gradually increased with 20C per minute for 6.5 minutes to 280C and then 280C
for 1 minute. The MS interface was working at 300C. The mass spectrometer
operated in the positive chemical ionization mode (NH3 (2.8x10-5Torr), 150eV) and
[M+H]+ parent ions of methadone and the internal standard were monitored in the
first quadropole (methadone m/z 310 and internal standard m/z 313). Argon (22.3mTorr) was used as collision gas to create daughter ions. Selected Reaction
Monitoring (SRM) was used and the common daughter ion m/z 105 was chosen for
both methadone and IS.
Standard solutions and calibration
Three different stock solutions from three independently weighed methadone
hydrochloride portions and one of the internal standard were prepared by dissolving
the adequate amount of drug in methanol. Appropriate solutions were then made in
distilled water to prepare series of standard solutions containing approximately 0.0051.4ng methadone/ul (1.0-280ng/ml plasma). The standard solutions for the calibration
curve were prepared from two different stock solutions. Two standard curves were
obtained by adding constant amount of internal standard (14ng/ml in the interval 126ng methadone/ml and 56ng/ml in the interval 26-280ng methadone/ml) and varying
amounts of methadone to drug-free plasma. Extraction was carried out as described
above and the samples were analysed. Methadone/internal standard peak area
ratios were calculated and standard curves were constructed by plotting the
methadone/internal standard peak area ratios as a function of the known
concentrations of methadone. A linear regression analysis was performed and the
correlation coefficient was calculated. Quality control samples for pre-analysis
validation and analytical verification (prepared from the third stock solution) with
concentrations of 2.2, 9.0, 56 and 112ng/ml plasma were analysed together with
standard curves.
Analysis
During the analysis of a set of clinical samples, control samples with the
concentrations of 2.2, 9.0, 56 and 112ng/ml, calibration samples for two standard
curves and plasma blank were extracted and analysed along with the unknowns. In
order to avoid carry-over effects, rinsing with toluene was performed twice between
every analysis.
Validation
Linearity: calibration samples were analysed, a linear regression method was used
for curve fitting and the correlation coefficient was calculated.
Limit of detection (LOD): calibration samples (0.1-26ng/ml plasma) were analysed to
investigate the lowest concentration that could be detected. The LOD was defined as
a peak with a height at least three times as high as the baseline noise level (S/N=3).
The LOD was calculated by extrapolation because the lowest concentration
investigated (0.1ng/ml) had S/N=20.
7
Limit of quantification (LOQ): acceptable limits for LOQ were 80-120% for accuracy
and RSD ≤20% for precision16.
Precision and accuracy: validation samples with the concentrations 2.2, 9.0,
56,112ng/ml plasma were analysed together with calibration samples and the
precision and accuracy were calculated. Acceptable limits for the lowest
concentration were 80-120% for accuracy and RSD ≤20% for precision. Acceptable
limits for higher concentrations were 85-115% for accuracy and RSD ≤15% for
precision16. During three days, samples with the concentration 112ng/ml (five
samples a day) were analysed to get the between-day and within-day variability. To
get the chromatographic precision, each sample was injected ten times. The samples
were analysed together with calibration samples and accuracy and precision were
calculated.
Stability in plasma: a plasma pool with a methadone concentration of 112ng/ml
plasma was prepared and stored in room temperature. During three days the
samples from the pool (five samples a day) were extracted and analysed. The mean
levels of methadone during these three days were then compared.
Stability of extracts: a plasma pool with a methadone concentration of 112ng/ml
plasma was prepared, extracted and stored in the vials in +4C. During five days the
extracts (three extracts a day) were analysed and the mean levels of methadone
during these five days were then compared.
Selectivity: drug free samples from the dogs were analysed to make sure that there
were no disturbing peaks at the retention time of methadone and internal standard.
Plasma samples containing only internal standard were also investigated to control
that no cross-talk between the SRM channels occurred.
8
RESULTS AND DISCUSSION
Method Development
Extraction procedure
The use of a simple extraction procedure, i.e. a single step liquid-liquid extraction
procedure, was possible because of the selective detection technique (MS/MS) that
was used and was preferable to minimize the risk for analyte loss. The use of a
deuterium-labelled internal standard, which has almost identical physio-chemical
properties as methadone and therefore controls sample loss closely, was also
possible when using mass spectrometric detection. Methadone has a tendency to
adsorb to surfaces17. To prevent that, hexane was mixed with 2-buthanol as the
organic phase. 2-buthanol competes with methadone for the surface sites. The
alcohol also increases the degree of extraction by increasing hydrogen bonding to
the analyte. Comparison between saturated carbonate buffer (pH 9.39) and 1M
NaOH for setting of plasma-pH, was made by extracting identical samples with the
two methods. No difference could be seen in recovery and NaOH was chosen
because, theoretically, the higher pH the better when extracting a base. During the
development of this assay it was observed that addition of 0.5 ml water to the plasma
samples (n=5), prior to extraction, gave cleaner chromatograms with less background
noise. We also compared to shake the samples (n=5) horizontally respective
vertically in the extraction tubes and could see that the samples shaken horizontally
gave ten times higher peaks in the chromatograms compared to the samples shaken
vertically. The samples shaken vertically had probably not reached equilibrium after
15 minutes and with extended shaking time the differences would most likely not
have been seen.
Gas chromatographic and mass spectrometric procedure
To start with, a comparison between single-stage gas chromatographic mass
spectometry (GC-MS) operating in electron ionization (EI) mode and MS operating in
chemical ionisation (CI) mode were made. When scanning m/z 40-400 in EI mode,
the fragments m/z 294 for methadone and m/z 297 for the internal standard were
chosen for SIM because those were largest. The same procedure was then made in
CI mode and the protonated molecular ions m/z 310 for methadone and m/z 313 for
the internal standard were chosen. CI was selected because it gave the greatest
signal-to-noise (S/N) ratio when running identical samples: CI/EI (S/N)~100.
Thereafter, a comparison between MS and MS/MS was made. The use of CI, that
gave more intense parent ions compared to EI, was also preferable when using
MS/MS because the parent ions then split again into daughter ions. Selected
Reaction Monitoring (SRM) was used and the common daughter ion m/z 105 was
chosen for methadone with the parent ion m/z 310, and for the internal standard with
the parent ion m/z 313. Fig.2a shows the full scan daughter ion spectrum for
methadone. The retention time was 5.11 minutes (Fig.2b) and the chromatographic
procedure was completed within 8.5 minutes. No interfering peaks were observed.
9
Fig.2a The full scan daughter ion spectrum of methadone (parent ion m/z 310).
Fig.2b. SRM chromatogram of methadone: parent ion m/z 310, daughter ion m/z 105.
MS/MS has advantage over MS due to superior sensitivity and selectivity, which also
was shown in the test. The limit of detection (LOD, signal/noise, S/N=3) when
running CI-MS was approximately 10ng/ml to compare with LOD <0.1ng/ml (S/N=20,
see further section Validation below) when running MS/MS. MS/MS was therefore
selected for quantitative purposes. Fig.3 shows representative chromatogram from
plasma with a spiked concentration of 0.1ng/ml, which was the lowest concentration
that was investigated.
RT: 0.00 - 8.51
100
NL: 1.16E4
RT: 5.14
AA: 23114
TIC F: + c CI SRM
ms2 310.00@25.00
[ 104.50-105.50] MS
ICIS
1117met04_031120
095317
90
Relative Abundance
80
70
60
50
40
30
20
RT: 6.57
AA: 1066
10
0
NL: 9.97E4
RT: 5.12
AA: 186296
TIC F:parent
+ c CI SRM ion m/z 310 and daughter
Fig.3
Plasma sample with the spiked concentration
0.1ng/ml. SRM:
100
ms2 313.00@25.00
ion90 m/z 105.
[ 104.50-105.50] MS
ICIS
1117met04_031120
095317
80
70
60
50
40
30
10
Validation
A linear regression method was used to obtain the best fit of the peak-area ratios of
methadone and the internal standard as a function of methadone concentration. The
calibration curve covered two wide ranges, from approximately 1.0 to 26ng/ml
(IS14ng/ml) and from 26 to 280ng/ml (IS 56ng/ml) with good linearity (Fig.4). The
correlation coefficients (R2) obtained were 0.97-0.99, indicating that a good
proportionality existed between the response and concentration of methadone. The
limit of detection (LOD, S/N= 3) of the assay was calculated to be 15pg/ml by
extrapolation from the results obtained at the lowest concentration investigated
(0.1ng/ml).
ratio metadon/metadon-d3
8,0000
7,0000
6,0000
2
R = 0,9989
5,0000
4,0000
3,0000
2,0000
1,0000
0,0000
0
5
10
15
20
25
30
35
40
45
50
ng/0.5ml
Fig.4 Calibration curve in the interval 0.5-13ng/0.5ml (1.0-26ng/ml).
Table 1 shows that the limit of quantification (LOQ), i.e. the lowest concentration that
could be determined with precision of RSD ≤20% and accuracy of 80-120% was
2.2ng/ml plasma (S/N= 60). For higher concentrations, acceptable limits for precision
was RSD ≤15% and for accuracy 85-115%16. The results were within acceptable
limits when the validation samples, with the concentrations 2.2, 9.0, 56 and 112ng/ml,
were run together with calibration samples. The precision became worse at lower
concentrations of the validation samples, but the accuracy seemed to be
independent of the concentration.
Conc. (ng/ml)
Mean(10samples)
SD
Precision(RSD%)
Accuracy (%)
2.2
2.3
0.2
18.3
105
9.0
9.7
0.6
12.7
108
56
60
1.4
4.7
108
112
119
2.3
3.9
107
Table 1. Accuracy and precision of validation samples with the concentrations 2.2, 9.0, 56 and
112ng/ml plasma.
The between-day and within-day accuracy and precision, where plasma samples
(c=112ng/ml) were analysed three different days, are shown in Table 2. The withinday results are based on the means for five samples a day and the between-day
results are shown as Total in the table below. Acceptable limits for accuracy is 85115% and for precision RSD ≤15%. The accuracy was not within acceptable limits
day two. That may be due to unstable GC-MS/MS conditions (see Table 3, day 2).
11
Day1(mean)
Precision(RSD%) 3.9
Accuracy (%)
110
Day2(mean)
12
117
Day3(mean)
6.6
106
Total
7.5
111
Table 2. Five samples a day were run for three days. Mean day 1-3 gives the within-day accuracy
and precision and Total gives the between-day accuracy and precision.
The chromatographic precision, when the samples above were injected ten times
each, showed great variability. The results were notably worse for three samples day
two. The reason for this variability may come from the fact that the collision-gas
pressure was not stable during the runs. The results for each sample are shown in
Table 3.
Day1
Sample1
Precision(RSD%) 2.7
Accuracy(%)
113
Sample2
4.7
111
Sample3
5.7
107
Sample4
1.6
112
Sample5
2.4
108
Day2
Sample6
Precision(RSD%) 3.8
Accuracy(%)
128
Sample7
7.0
114
Sample8
5.6
109
Sample9
18
121
Sample10
16
111
Day3
Sample12
3.5
97
Sample13
2.9
111
Sample14
3.9
109
Sample15
4.3
112
Sample11
Precision(RSD%) 0.6
Accuracy(%)
101
Table 3. The chromatographic precision when the samples were injected ten times each.
Table 4 shows the results for stability in plasma where the plasma samples
(c=112ng/ml) stored in room temperature were extracted and analysed together with
fresh calibrants for three different days (five samples a day). There seemed to be no
major degradation of methadone during the three days studied. The alterations
shown are probably due to the extraction procedure and/or small differences in the
GC-MS/MS between the three days studied.
Day1(mean of 5 samples)
100%
Day2(mean of 5 samples)
105%
Day3(mean of 5 samples)
93%
Table 4. Plasma samples stored in room temperature showed no major degradation of methadone
during the three days studied.
Stability of the extracts was examined by analysing plasma samples (c=112ng/ml)
that were extracted on the same day and then stored in the vials in +4°C. For five
days, three extracts a day were analysed. There seemed to be no major degradation
of methadone during the three days studied. The alterations shown are probably due
to the extraction procedure and/or small differences in the GC-MS/MS between the
five days studied (Table 5).
12
Day1(mean) Day2(mean) Day3(mean) Day4(mean) Day5(mean)
100%
98%
104%
104%
110%
Table 5. The mean of three samples a day is compared with each other. No major degradation of
methadone could be seen during the five days studied.
The selectivity test, when methadone-free samples from the dogs were analysed,
showed peaks corresponding to 0.4ng/ml, negligible compared to LOQ. When blank
plasma samples containing only internal standard were run, a peak corresponding to
0.2ng/ml in the methadone chromatogram could sometimes be seen. Whether that
was due to crosstalk between the methadone-internal standard SRM-channels or a
carry-over effect was unclear.
The quantification limit achieved by this method was comparable to other methods
used to analyse methadone in plasma2,12. A comparison between LOQ and LOD
according to signal-to-noise ratios gives that it could be possible to improve the
quantification limit because LOQ, 2.2 ng/ml (S/N=60), was much higher than LOD,
15pg/ml (S/N= 3). That may be possible by decreasing the variability in the GCMS/MS procedure by keeping the gas pressure more stable during the runs. A
smaller amount of toluene for redissolution of methadone or an increase in injection
volume might also improve the quantification limit.
Bioanalysis of dog plasma
The application of the developed assay was demonstrated after administration of
methadone (0.1 or 0.4mg/kg, S.C. or I.M.) every fourth hour during two or three days
to four healthy beagle dogs. The doses given were recommended doses for pain
relief in dogs and used in clinical practice7. Plasma samples were collected four
hours after every injection, i.e. right before the next dose. The concentrations were
found to vary over a wide range (Fig.5a,b). For the two dogs that were given
0.1mg/kg, the concentrations varied between 2 to 45ng/ml and for the two dogs given
0.4mg/kg the concentrations varied between 5 to 190ng/ml four hours after the
injections. For the dogs given the higher dose (dog 2 and 4) the study had to be
interrupted after injection eight. The dogs then showed severe side-effects like
vomiting and diarrhoea, hyper ventilation and apathy. The symptoms gradually
vanished and eight hours after the last injection the dogs were completely recovered.
The two dogs given the lower dose did not show any side-effects during the study.
13
50
40
30
S.C.
20
I.M.
10
pl
e1
5
sa
m
pl
e1
3
sa
m
pl
e1
1
sa
m
pl
e9
sa
m
pl
e7
sa
m
pl
e5
sa
m
sa
m
sa
m
pl
e3
0
pl
e1
ng methadone/ml
0.1mg/kg
4h after injection
Fig.5a methadone concentrations four hours after every injection for the dogs treated with 0.1 mg/kg,
either S.C.(dog 1) or I.M.(dog 3).
0.4mg/kg
ng methadone/ml
200
150
S.C.
I.M.
100
50
pl
e8
sa
m
pl
e7
sa
m
pl
e6
sa
m
pl
e5
sa
m
pl
e4
sa
m
pl
e3
sa
m
pl
e2
sa
m
sa
m
pl
e1
0
4h after injection
Fig.5b methadone concentrations four hours after every injection for the dogs treated with 0.4 mg/kg,
either S.C.(dog 2) or I.M. (dog 4).
The elimination was studied by collecting plasma samples up to 36 hours (48 hours
for dog 2 and 4) after the last dose (Fig.6 a,b). Initially, the two dogs treated with
methadone S.C. showed higher concentrations compared to the two dogs given
methadone I.M., but at the end of the study the concentrations had decreased
substantially for all four dogs, for two dogs (3 and 4) even below LOQ.
14
0.1mg/kg
ng methadone/ml
100
80
60
S.C.
40
I.M.
20
36h
24h
16h
12h
8h
6h
4h
2h
1h
45min
30min
20min
10min
0
Elimination
Fig.6a methadone concentrations after the last given dose for the dogs treated with 0.1mg/kg, either
S.C.(dog 1) or I.M.(dog 3).
0.4mg/kg
ng methadone/ml
350
300
250
S.C.
200
150
I.M.
100
50
48h
36h
24h
16h
12h
8h
6h
4h
2h
1h
45min
30min
20min
10min
0
Elimination
Fig.6b methadone concentrations after the last given dose for the dogs treated with 0.4mg/kg, either
S.C.(dog 2) or I.M. (dog 4).
It was unclear if steady state was achieved during the days studied and it had
therefore been interesting to continue the study further days to be able to evaluate if
any accumulation appeared. Because of the limited sample size it was not adequate
to draw any quantitative pharmacokinetic conclusions from the results. Hopefully, a
greater study with several dogs will be performed in the future.
CONCLUSION
The GC-MS/MS assay described in this report had a simple sample preparation and
a sensitivity and selectivity that was sufficient for the purpose to quantify methadone
in dog plasma at the concentrations encountered in clinical practice.
15
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fluid, and Urine by Gas Chromatography and its Application to Routine Drug Monitoring, Pharmaceutical
Research, Vol.10, No.3, 1993
2.
Boulton D.W, Lindsay Devane C, Development and Application of a Chiral High Performance Liquid
Chromatography Assay for Pharmacokinetic Studies of Methadone, Chirality 12:681-687, 2000
3.
Garret E.R, Derendorf h, Mattha A.G, Pharmacokinetics of Morpine and its Surrogates VII: HighPerformance Liquid Chromatographic Analyses and Pharmacokinetics of Methadone and its Derived
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