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%.
Institute of Biochemistry, German Sport University Cologne, Cologne, Germany.
S
S
Beuck
G
G
Sigmund
A
A
Koch
W
W
Schänzer
A
A
Pokrywka
D
D
Kwiatkowska
M
M
Thevis
D018663:Adrenergic Agents / Q000378:metabolism* / Q000652:urine*; D002853:Chromatography, Liquid / Q000379:methods; D004300:Doping in Sports; D006801:Humans; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D011299:Prenylamine / Q000378:metabolism* / Q000652:urine*; D015813:Substance Abuse Detection / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014665:Vasodilator Agents / Q000378:metabolism / Q000652:urine
D018663:Adrenergic Agents; D014665:Vasodilator Agents; D011299:Prenylamine