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

• Published in: Drug testing and analysis 2012-09, Vol.4 (9), p.701-716
• Main Authors: Beuck, S., Sigmund, G., Koch, A., Schänzer, W., Pokrywka, A., Kwiatkowska, D., Thevis, M.
• Format: Article
• Language: English
• Subjects: Adrenergic Agents - metabolism; Adrenergic Agents - urine; Chromatography, Liquid - methods; doping; Doping in Sports; Humans; Limit of Detection; Male; Middle Aged; Prenylamine - metabolism; Prenylamine - urine; stimulants; Substance Abuse Detection - methods; Tandem Mass Spectrometry - methods; TripleTOF; urine analysis; Vasodilator Agents - metabolism; Vasodilator Agents - urine
• ISSN: 1942-7603; 1942-7611
• DOI: 10.1002/dta.1388

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%. Copyright © 2012 John Wiley & Sons, Ltd. The human urinary metabolism of the phenylalkylamine prenylamine was elucidated by liquid chromatography coupled to quadrupole‐time‐of‐flight hybrid mass spectrometry by electrospray ionization. The structures of three hydroxy‐metabolites, which were identified together with over 40 additional phase I metabolites, were confirmed by chemical synthesis of reference material. An existing urine analysis method used for routine doping controls was extende