Metabolism of selegiline in human
Identification, Excretion, and Stereochemistry of
Urine Metabolism
เมตาบอลิซึ่มของซิลจิ ีลนี ในมนุษย์
นาเสนอโดย
น.ส. อรทัย กฤษณานุวฒ
ั น์ รหัสนักศึกษา 51312336
Introduction
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The chemical name is (R)-(-)-N,2dimethyl-N-2-propynylphenethylamine
hydrochloride
It is a white to near white crystalline
powder, soluble in water, chloroform
and methanol
Molecular weight of 223.75
Boling point 80 °c
The structural formula of
selegiline
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Monoamine oxidase, L-dopamin
Later, Heinonen et.al
Methamphetamine, amphetamine,
desmethyl – selegiline
urine
human
p-hydroxyamphetamine,
p-hydroxymetamphetamine
rat
Material and Method
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d-, l-,R-,S-, or(+)- and (-)- systems of
naming optical isomers
H2N
H
CH3
l-Amphetamine
CH3NH
CH3
l-Methamphetamine
H
NH2
CH3
d-Amphetamine
H
H
NHCH3
CH3
d-Methamphetamine
enantiomers of amphetamine and methamphetamine
Materials and Methods
Chemical
(R)-amphetamine sulfate
(S)-amphetamine sulfate
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(S)- methamphetamine
(1R,2R)-norpseudoepedrine · HCl
sigma
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(1R,2S)-norephedrine·HCl
(1S,2R)-norephedrine·HCl
(1S,2R)-ephedrine·HCl
(1S,2R)-pseudoephedrine·HCl
p- hydroxyamphetamine
p- hydroxymethamphetamine
p- hydroxynorephedrine
p- chlorphetermine
Fluka
Instd.
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Drug administration and sample
collection
10, 5, and 2.5 mg of selegiline.HCl
Urine sample collection at various times
over 72 hr and stored at 4 °c
Isolation of Unconjugate Metabolism
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Urine 3 ml+ 100mg sodium
bicarbonate: potassium carbonate (2:1)
+150 ng instd. (p- chlorphentamine)
8 ml of diethylether-tert butanol (7:1)
for metabolite extracted
Organic layer was transfer into a 15ml
glass centrifuge tube + 0.4ml of 0.06M
HCl
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Mixing, Centrifuge 5 min at 1200 g
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Organic layer was aspirate , dried
Isolation of Conjugate
Metabolite
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Urine 3 ml adjusted ph 5.2 with 0.2M
Sodium acetate buffer
Incubate with 50 µl of arylsulfatase / β glucolonidase 52 °c
Solution was neutralized with 5 M KOH
and adjusted pH 9.6 with 200mg of
sodium bicarbonate : potassium carbonate
(2:1)
Quantification of Selegiline and Its
Metabolite
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Dry residue was dissolved in 50 µl of
ACN:TFA (60:40)
Titrated with MSTFA until color reaction
changes red to yellow
Sample was heated for 10 min at 60°c
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Two drops of MSTFA added to the
reaction mixture
2 µl of the solution was injected into
the GC/MS
GC/MS, GC-NPD
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HP 5890/5971A instrument, HP 9144
disk drive,HP Think Jet Printer
HP fused-silica capillary column, 5%
phenylmethylsilicon(SE-54)
17 min length, with 0.2 mm diameter,
0.33 µl film thickness
Temperature and column condition
identical for GC/MS
Results and Discussion
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Identification of Metabolites by GC-NPD
and GC/MS
Comparison of the EI mass spectra and
gas chromatographic retention times
GC-NPD gave six peaks (A1-A6)
N-trifluoroacetylation, Otrimethylsilylation showed 10 peaks(B1B10)
Identification of the Stereoisomer
of the Selegiline Metabolites
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Applicable to the quantification of trace
optical isomer containing more than one
reactive functional gr.
The stereochemical identities of the
metabolites confirmed by comparison of
the GC RT of derivative of the extractd
metabolites with of the authentic std.
Urinary excretion of
metabolites
Summarized in table 1
 Trifluoroacetylated form, Ntrifluoroacetylation-O-trimethlysilation
derivative
 Detection limit of selegiline was ~
0.3ng/ml
other metabolites were
were ~
0.1ng/ml
- Correlation(r) =0.999 between injection
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The major metabolite was (R)methamphetamine for ~ 37% of dose
β-hydroxylation and aromatic
hydroxylation were minor metabolite
Dealkylation, β-hydroxylation of
selegiline lead to unconjugate
Most of the p-hydroxylated of selegiline
excreted conjugate (57.3-77.3%)
The sum of the amount of
urinary metabolite in 2 days
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44-58% dose-independent amount of
selegiline
48.67-48.8% dose of dealkylated
metabolites (Asia)
30.78-34.09% (Europeans)
4.84-8.77% dose of aromatic
hydroxylate metabolites (Asia)
12.42-17.54% (Europeans)
Kinetic Studies
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Quantification of urine concentration of
selegiline and the metabolite
The excretion rate for selegiline and
metabolite was calculated by division of
the total amount via the urine collection
interval
Readily absorbed, rapidly distributed
ขอบคุณ
อาจารย์ ธงชัย เตโชวิศาล
ที่ปรึ กษาสัมมนา
Quesssion
A2
Abundance
4.0E
3.0E
A1
2.0E
1.0E
A3
A4
A6
A5