Phase I Metabolism of Novel Phencyclidine Derivative 3‐Cl‐PCP: In Vitro Studies With Pooled Human Liver Microsomes and Investigation of a Post‐Mortem Case

Assessing fatalities linked to new psychoactive substances (NPS) often remains challenging. This study investigates a fatal intoxication involving the novel phencyclidine derivative 3-chloro-phencyclidine (3-Cl-PCP), analyzing its phase I metabolism aided by pooled human liver microsomes (pHLMs) and supported by in silico models. Postmortem samples (bile, urine, cardiac and femoral blood, and gastric contents) and three syringes were collected from a fatal intoxication case. Quantification was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In vitro metabolism studies employed pHLM incubation, with metabolites tentatively identified by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). To evaluate phase II metabolism, urine and bile samples were measured with and without β-glucuronidase treatment. 3-Cl-PCP concentrations ranged from 610 ng/mL in cardiac blood to 830 ng/mL in femoral blood. Higher levels were detected in urine (1200 ng/mL) and bile (4300 ng/mL). Seven phase I metabolites were tentatively identified in vitro and postmortem, primarily involving hydroxylation of the cyclohexyl (M1-M3) and piperidine (M4-M5) rings. Additionally, piperidine ring-opened carboxyl (M6) and ring-opened alcohol (M7) metabolites were tentatively identified. Post-enzyme urine analysis revealed extensive glucuronidation of metabolites M1-M5, with M4 (hydroxyl piperidine) showing the highest abundance. Bile showed elevated abundance of unconjugated metabolites, particularly M6. Metabolites M4-M7 were detected in all investigated matrices. Unchanged 3-Cl-PCP predominantly accumulated in bile. Urine analysis should prioritize the parent compound along with the metabolites hydroxy cyclohexyl (M3), hydroxy piperidine (M4), and piperidine ring-opened carboxyl (M6) and should include a prior β-glucuronidase cleavage step.