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