Human metabolism of AP-238. A novel synthetic opioid spreading in the shadow of “fentalogues”

Abstract

The phenomenon of new psychoactive substances is constantly evolving, with novel synthetic opioids (NSO) representing a particular threat given their sometimes extremely high potencies and their potential to produce respiratory depression. As a consequence of recently implemented legal restrictions on fentanyl analogues, a new “legal” generation of opioids, embodied by the class of cinnamylpiperazines, popped up on the illicit drug market. AP-238 was the latest NSO of this series to be notified to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) and is involved in an increasing number of acute intoxications. Adverse effects associated with the abuse of NSO, their continued evolution, increased popularity and high availability constitute a serious concern and pose an imminent hazard to public health. The identification of specific markers of consumption for these NSO is essential for clinical and forensic casework and represents a new challenge for toxicology laboratories. Referring to this, we investigated the in vivo and in vitro metabolism of AP-238. For tentative identification of the main phase-I metabolites, a pooled human liver microsomes (pHLM) assay was used. Incubation was performed for 2 hours at 37 °C in triplicates, including a blank control (no pHLM) and a zero control (no reference standard). Analyses were carried out using liquid chromatography coupled to time-of-flight mass spectrometry (LC-qToF-MS). Full scan and auto MS/MS data were acquired in one run. In a second run, full scan and broadband collision-induced dissociation (bbCID) data were acquired, checking for characteristic fragment ions (e.g., m/z 117.0699) to detect unexpected metabolites. To compare the relative abundance of the metabolites, a multiple reaction monitoring (MRM) method comprising at least two ion transitions of each metabolite, was applied using liquid chromatography coupled to tandem-mass spectrometry (LC-MS/MS). Chromatographic separation was achieved on a Kinetex® C18 column (100 × 2.1 mm, 2.6 μm). Both instruments were operated in positive ion mode. Anticipated phase-I metabolites detected in the pHLM assay were confirmed in three authentic blood samples collected during postmortem examinations and tested positive for AP-238 in the Institute of Forensic Medicine of Freiburg. In total, ten AP-238 phase-I metabolites were identified by means of LC–qToF–MS in the pHLM assay and confirmed five of these by corresponding signals in the human blood samples. They mainly consisted of products of hydroxylation, oxidation, dealkylation, and combinations of these reactions. A dealkylated/monohydroxylated metabolite was detected with the highest abundance in blood. Suggested biomarkers for AP-238 consumption are generated by monohydroxylation combined with dealkylation (most abundant) and by sole hydroxylation at the cinnamyl substituent, with the latter being highly specific. Although this investigation was carried out with blood samples due to the non-availability of urine, it is likely that these metabolites are suitable markers for urine screening as well. The combination of dealkylation with (multiple) hydroxylation(s) was also observed for further minor metabolites. The exact chemical structures of these metabolites remain unclear and would require isolation and structure elucidation e.g. by nuclear magnetic resonance spectroscopy or synthesis of reference material. However, this does not preclude their use as valid biomarkers. Implementing analytical methods for the detection of NPS metabolites is often crucial to document consumption in clinical and forensic toxicology. In vitro methods like the used pHLM assay proved to be suitable tools for prediction of biomarkers for NSO intake, although they have to be confirmed by authentic samples. If urine is not available, blood can be used for this purpose.