Identification and characterization of urinary prenylamine metabolites by means of liquid chromatography-tandem mass spectrometry.

Prenylamine is a vasodilator of phenylalkylamine structure and was used for the treatment of angina pectoris, until reports of undesirable effects including ventricular tachycardia led to a decreasing use of the drug in the 1980s. Metabolic N-dealkylation of orally ingested prenylamine can liberate amphetamine in humans and cause positive findings for amphetamine in doping and forensic analysis. In 2010, the World Anti-Doping Agency (WADA) classified prenylamine as a non-specified stimulant according to the 2010 Prohibited List, thus banning its use in sports in-competition. Supporting the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) based detection method, a post-administration urine sample following a single oral prenylamine ingestion (Segontin(®) 60 mg) was analyzed for urinary metabolites. The LC-separated analytes were ionized in positive electrospray ionization (ESI) mode and detected as protonated ions using an AB Sciex TripleTOF 5600 quadrupole-time-of-flight hybrid mass spectrometer. Over 40 phase I metabolites were detected, including previously unknown mono- bis-, tris- and tetra-hydroxylated prenylamine, several hydroxylated and methoxylated prenylamine metabolites and (hydroxylated) diphenylpropylamine. Investigation of the collision-induced dissociation behaviours of the metabolites by high resolution/high accuracy mass spectrometry allowed for the assignment of the nature and the site of observed metabolic transformations. The most abundant phase I metabolite was confirmed as p-hydroxy-prenlyamine by chemical synthesis and stable isotope labelling of reference material. An existing routine screening assay based on direct injection and LC-MS/MS analysis of urine was modified and validated according to common guidelines, in order to allow for the detection of p-hydroxy-prenylamine in sports drug testing. The assay demonstrated the ability to detect the target metabolite at 0.1 ng/ml at intra- and inter-day imprecisions below 10%.

Single- and multiple-dose pharmacokinetics of R-(-)-and S-(+)-prenylamine in man.

The pharmacokinetics of S-(+)- and R-(-)-prenylamine was studied in eight healthy volunteers given single and repeated oral doses of the racemic drug. Distinct differences in various pharmacokinetic parameters were found between the S- and R-enantiomer. The maximum plasma concentrations and AUCs of the R-enantiomer exceeded those of the S-enantiomer five-fold; the apparent oral clearance of the S-form was five-times and the renal clearance three-times higher than of the R-form. Acid catalyzed hydrolysis of urine samples released more S-prenylamine, indicating stereoselective glucuronidation of unchanged prenylamine. Plasma protein binding also differed between the two enantiomers, generally with a higher unbound fraction of the S-form, whereas analysis of the bound fractions showed that prenylamine was bound to different plasma proteins with inverse stereoselectivity.

Simultaneous determination of R- and S-prenylamine in plasma and urine by reversed-phase high-performance liquid chromatography.

A high-performance liquid chromatographic method for the determination of R- and S-prenylamine in human plasma and urine is described. It involves a two-step liquid-liquid extraction of prenylamine from biological material and preparation of diastereomeric urea derivatives with R-(-)-naphthylethyl isocyanate, a chiral fluorescence marker. Separation and quantitation of the diastereomeric prenylamine derivatives are carried out by a reversed-phase high-performance liquid chromatographic system with fluorimetric detection. The limit of determination is less than 2 ng of enantiomer per ml of urine and less than 1 ng of enantiomer per ml of plasma. A preliminary kinetic study on one healthy volunteer who had received a single oral dose of racemic prenylamine (100-mg film tablet) showed distinctly higher plasma and urine concentrations of the R-enantiomer.

Metabolism of DL-[14C]prenylamine in man.

Following oral administration of DL-[14C]prenylamine, about 40% of the dose administered was excreted in urine within 10 days. Less than 0.1% of the dose was excreted as unchanged prenylamine. The drug was extensively metabolized to at least 20 to 25 metabolites. The structure of 12 metabolites could be elucidated by means of g.c.m.s. Ring hydroxylation and further methylation of the phenolic metabolites are the main metabolic pathways involved. A substantial part of the drug and/or its metabolites is metabolized via cleavage of the C--N--C bond, giving rise to amphetamine and diphenylpropylamine which are further metabolized by aromatic and sidechain hydroxylation.