Determination of in vitro Metabolic Pathways for Novel Psychoactive Substance Fluorexetamine

Novel psychoactive substances (NPS) are designed by clandestine laboratories to mimic the effects of illegal drugs such as amphetamine, heroin, or ketamine. These NPS that appear on the black market are termed “legal highs” due to the lack of scheduling through the Drug Enforcement Agency. This presents a unique challenge to death investigators and health professionals since the identity and physiological effects of the substances are initially unknown. Identification of the compounds is further complicated by metabolism in the body once the substances are imbibed. Fluorexetamine is an NPS that was recently reported and is a derivative of ketamine. The purpose of this research is to determine the metabolites of fluorexetamine and develop validated methods for detection and quantitation that can be used in toxicology laboratories investigating cause of death. A gas chromatography-mass spectrometry (GC-MS) method for fluorexetamine was developed utilizing an Agilent GCMS 5977C equipped with an Agilent HP-5MS 30m x 0.25 mm x 0.25 um column. Helium was used as the carrier gas at a flow rate of 1.0 mL/minute. The inlet temperature was 200oC and a starting temperature of 70oC with a one-minute hold was ramped 20oC/min to 330oC with a final 15-minute hold. The transfer line was set at 310oC and the mass spectrometer was set to scan from 50-500 m/z with a solvent delay of 3 minutes. HepG2 cell lines show transcripts of multiple CYP enzymes necessary for metabolism. Fluorexetamine was incubated with HepG2 cell lines under various conditions and the cell cultures were extracted and analyzed by GC-MS. Sample extracts showed no appreciable concentrations of metabolites when incubated for up to 8 hours. Our results indicate that while HepG2 cells show presence of the necessary enzymes for metabolism, the expression and/or activity of those enzymes may be too low to generate metabolites at the time points studied.