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Metabolism of selegiline in human Identification, Excretion, and Stereochemistry of Urine Metabolism

Metabolism of selegiline in human Identification, Excretion, and Stereochemistry of Urine Metabolism. เมตาบอลิซึ่มของซิลิจีลีนในมนุษย์ นำเสนอโดย น.ส. อรทัย กฤษณานุวัฒน์ รหัสนักศึกษา 51312336. Introduction. The chemical name is (R)-(-)-N,2-dimethyl-N-2-propynylphenethylamine hydrochloride

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Metabolism of selegiline in human Identification, Excretion, and Stereochemistry of Urine Metabolism

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  1. Metabolism of selegiline in humanIdentification, Excretion, and Stereochemistry of Urine Metabolism เมตาบอลิซึ่มของซิลิจีลีนในมนุษย์ นำเสนอโดย น.ส. อรทัย กฤษณานุวัฒน์ รหัสนักศึกษา 51312336

  2. 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

  3. The structural formula of selegiline

  4. Monoamine oxidase, L-dopamin Later, Heinonen et.al Methamphetamine, amphetamine, desmethyl – selegiline urine human p-hydroxyamphetamine, p-hydroxymetamphetamine rat

  5. H2N H H NH2 CH3 CH3 l-Amphetamine d-Amphetamine CH3NH H H NHCH3 CH3 CH3 l-Methamphetamine d-Methamphetamine Material and Method • d-, l-,R-,S-, or(+)- and (-)- systems of naming optical isomers enantiomers of amphetamine and methamphetamine

  6. Materials and Methods Chemical (R)-amphetamine sulfate (S)-amphetamine sulfate sigma (S)- methamphetamine (1R,2R)-norpseudoepedrine · HCl

  7. (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

  8. 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

  9. 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

  10. Mixing, Centrifuge 5 min at 1200 g • Organic layer was aspirate , dried

  11. 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)

  12. 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

  13. Two drops of MSTFA added to the reaction mixture • 2 µl of the solution was injected into the GC/MS

  14. GC/MS, GC-NPD • HP 5890/5971Ainstrument, 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

  15. 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)

  16. 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.

  17. 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

  18. 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%)

  19. 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)

  20. 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

  21. ขอบคุณ อาจารย์ ธงชัย เตโชวิศาล ที่ปรึกษาสัมมนา Quesssion

  22. A2 4.0E Abundance A1 3.0E 2.0E A6 A4 A3 A5 1.0E

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