Metabolism of selegiline in human
Identification, Excretion, and Stereochemistry of Urine Metabolism
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Introduction
The chemical name is (R)-(-)-N,2-dimethyl-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
Monoamine oxidase, L-dopamin Later, Heinonen et.al Methamphetamine, amphetamine, desmethyl – selegiline urine human
p-hydroxyamphetamine, p-hydroxymetamphetamine rat
Material and Method
d-, l-,R-,S-, or(+)- and (-)- systems of naming optical isomers
l-Methamphetamine
HCH3NH
CH3
l-Amphetamine
H2N
CH3
H
d-Amphetamine
NH2
CH3
H
d-Methamphetamine
HNHCH3
CH3
enantiomers of amphetamine and methamphetamine
Materials and Methods
Chemical (R)-amphetamine sulfate (S)-amphetamine sulfate
sigma
(S)- methamphetamine (1R,2R)-norpseudoepedrine · HCl
(1R,2S)-norephedrine·HCl (1S,2R)-norephedrine·HCl Fluka (1S,2R)-ephedrine·HCl (1S,2R)-pseudoephedrine·HCl p- hydroxyamphetamine p- hydroxymethamphetamine
Instd. p- hydroxynorephedrine p- chlorphetermine
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
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
Mixing, Centrifuge 5 min at 1200 g
Organic layer was aspirate , dried
Isolation of Conjugate Metabolite
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
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
Two drops of MSTFA added to the reaction mixture
2 µl of the solution was injected into the GC/MS
GC/MS, GC-NPD
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
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, O-
trimethylsilylation showed 10 peaks(B1-B10)
Identification of the Stereoisomer of the Selegiline Metabolites
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, N-
trifluoroacetylation-O-trimethlysilation derivative
Detection limit of selegiline was ~ 0.3ng/ml other metabolites were were ~ 0.1ng/ml - Correlation(r) =0.999 between injection
amounts and detection response
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
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
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
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Quesssion
Abundance
1.0E
2.0E
3.0E
4.0E
A1
A2
A3A4
A5A6