Metabolism of selegiline in human Identification, Excretion, and Stereochemistry of Urine Metabolism เมตาบอล ซมข องซล จลน ในมนษย นำเสนอโดย น. ส. อรทย กฤษณา นวฒน รหสนกศกษา 51312336
Introduction n n 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
n n n Monoamine oxidase, L-dopamin Later, Heinonen et. al Methamphetamine, desmethyl – selegiline urine human p-hydroxyamphetamine, p-hydroxymetamphetamine rat
Material and Method n d-, l-, R-, S-, or(+)- and (-)- systems of naming optical isomers H 2 N H CH 3 l-Amphetamine CH 3 NH H NH 2 CH 3 d-Amphetamine H CH 3 l-Methamphetamine H NHCH 3 d. Methamphetamine enantiomers of amphetamine and methamphetamine
Materials and Methods n Chemical (R)-amphetamine sulfate (S)- methamphetamine (1 R, 2 R)-norpseudoepedrine · HCl sigma
n n n n (1 R, 2 S)-norephedrine·HCl (1 S, 2 R)-ephedrine·HCl (1 S, 2 R)-pseudoephedrine·HCl p- hydroxyamphetamine p- hydroxymethamphetamine p- hydroxynorephedrine p- chlorphetermine Fluka Instd.
n - - 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 n n n Urine 3 ml+ 100 mg 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 15 ml glass centrifuge tube + 0. 4 ml of 0. 06 M HCl
n Mixing, Centrifuge 5 min at 1200 g n Organic layer was aspirate , dried
Isolation of Conjugate Metabolite n n n Urine 3 ml adjusted ph 5. 2 with 0. 2 M Sodium acetate buffer Incubate with 50 µl of arylsulfatase / β glucolonidase 52 °c Solution was neutralized with 5 M KOH and adjusted p. H 9. 6 with 200 mg of sodium bicarbonate : potassium carbonate (2: 1)
Quantification of Selegiline and Its Metabolite n n n 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
n n Two drops of MSTFA added to the reaction mixture 2 µl of the solution was injected into the GC/MS
GC/MS, GC-NPD n n HP 5890/5971 A 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 n n 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 (A 1 -A 6) N-trifluoroacetylation, Otrimethylsilylation showed 10 peaks(B 1 B 10)
Identification of the Stereoisomer of the Selegiline Metabolites n n 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 n Trifluoroacetylated form, Ntrifluoroacetylation-O-trimethlysilation derivative n Detection limit of selegiline was ~ 0. 3 ng/ml other metabolites were ~ 0. 1 ng/ml - Correlation(r) =0. 999 between injection amounts and detection response n
n n 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 n n n 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 n n n 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
ขอบคณ อาจารย ธงชย เตโชวศาล ทปรกษาสมมนา Quesssion
A 2 Abundance 4. 0 E 3. 0 E A 1 2. 0 E 1. 0 E A 3 A 4 A 6 A 5
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