Determination of Selegiline and its Metabolites in Human Urine by GC/MS

Summary

This experiment is mainly used for doping control.

Operation method

GC, GC/MS method

Principle

In this experiment, the urinary metabolites of orally administered Selegiline Hydrochloride, a monoamine oxidase inhibitor, were analyzed and determined by GC and GC/MS techniques, and 10 metabolites were confirmed to be detected, three of which were newly detected hydroxylated metabolites.

Materials and Instruments

Urine
Selegiline5 tablets selegiline-HCl amphetamine-HCl methylamphetamine-HCl p-OH-methylamphetamine
Gas Chromatograph Nitrogen and Phosphorus Detector Gas Chromatography Mass Spectrometer

Move

1. Urine sample collection

A single dose of 10 mg Selegiline 5 tablets was administered orally to healthy males (weight 65 Kg). All of the urine from each of the urine sessions performed and the blank urine prior to the administration of the drug were collected separately at -20°C.

2. Sample preparation

Take 5 ml of urine sample, add 100 mg L-cysteine, 0.5 ml of concentrated HCl, hydrolyzed at 100 ℃ for 30 min. after cooling, extracted with 3 ml of tert-butyl methyl ether, separated, and discarded the ether layer. To the aqueous phase was added 0.5 ml 12M NaOH, 2 g of solid buffer ( _NaHCO3: _K2CO3 = 3:2, pH 9.6). A solution of tert-butyl methyl ether/isopropanol (9:1) containing diphenylamine as an internal standard was prepared with 3 ml of tert-butyl methyl ether/isopropanol (9:1) and shaken. The extract was separated, blown dry under N2 gas, and the residue was dissolved with 1 ml of ethyl acetate and injected with 2 μL of GC/NPD, and then concentrated to about 50 μL under N2 gas, and injected with 2 μL of GC/MSD, and then blown dry, and the reaction was carried out with the addition of 100 μL of ethyl acetate and 100 μL of trifluoroacetic anhydride (TFAA) to the residue for 20 min at 70 °C. The residue was blown dry under N2 gas, and then dissolved with 100 μL of ethyl acetate and 100 μL of trifluoroacetic anhydride (TFAA) for 20 min. The reaction was dried under N2 gas, then dissolved in 100 μL of ethyl acetate, and 2 μL of GC/MSD was injected into the sample.In the urinary excretion trend test of selegiline metabolites amphetamine, methylamphetamine and norSelegiline, the procedure of urine sample extraction was as follows: 5 ml of each of the urine samples collected at 0-72 h were taken separately, and 0.5 ml of 5M KOH, 3 g NaCl, 2 ml of 5 ppm diphenylamine (internal standard) in tert-butyl methyl ether solution, shaking, centrifugation, separation of the ether layer, and injection of 2 μL into the GC.

Caveat

Urine samples taken from 0 to 72 hours should be handled with care to avoid introducing contamination.

Common Problems

Source: Determination of Selegiline and its Metabolites in Human Urine by GC, GC/MS Methods.

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