Confirmation of metabolites of the neuroleptic drug prothipendyl using human liver microsomes, specific CYP enzymes and authentic forensic samples-Benefit for routine drug testing

Abstract

Metabolism of the tricyclic azaphenothiazine neuroleptic drug prothipendyl was investigated with in vitro studies using human liver microsomes but also specific isoforms of cytochrome P450 (CYP) enzymes. Identification and analysis of metabolites was done by liquid chromatography (LC) coupled with quadrupole time of flight mass spectrometry (LC-QTOF-MS) as well as triple quadrupole mass spectrometry (LC-QQQ-MS). Results of the herein presented study revealed the proof of various demethylated and oxidized metabolites (-CH₂, -C₂H₄, four derivatives of prothipendyl +O and three derivatives of prothipendyl -CH₂+O). Metabolic reactions of prothipendyl were mainly catalyzed by CYP enzymes CYP1A2, CYP2D6, CYP2C19 and CYP3A4. N-demethyl-prothipendyl was predominantly formed by isoforms CYP2C19 and CYP1A2, while particularly the CYP isoenzyme 3A4 was responsible for the formation of prothipendyl sulfoxide. To confirm the formation of previously identified metabolites in vivo, cardiac blood samples that were tested positive for prothipendyl during routine drug testing and serum and urine samples, collected after a voluntary intake of prothipendyl, were analyzed by LC-QQQ-MS. All metabolites of prothipendyl were proven in these authentic specimens. Neither in serum samples nor in urine samples, a prolonged detectability of metabolites in comparison to prothipendyl could be demonstrated.

Keywords: Cytochrome P450; LC-QQQ-MS; LC-QTOF-MS; Metabolism; Prothipendyl.