Alpha-pyrrolidinoisohexanophenone (α-PHiP/α-PiHP) is a synthetic drug structurally related to cathine, a natural psychoactive alkaloid isolated from khat plant. α-PiHP is a structural isomer of α-PHP. α-PHP and α-PiHP may be considered as an analogue of α-PVP, a Schedule I drug under the Convention on Psychotropic Substances by the United Nations. This α-pyrrolidinophenone was first reported to EMCDDA by Slovenia in December 2016. In Hungary it appeared in August 2016 but before 2021 it had been detected in seizures only two times and it had never been identified in biological samples. However, in 2021 its consumption became prevalent in Hungary. α-PiHP was detected in one blood sample and twenty-one urine samples collected during the last two months of 2021 and submitted to our laboratory for forensic toxicological analysis. It means that about 5% of our cases were confirmed positive for α-PiHP during these months. Until February 2022 α-PiHP has been identified in one fatal intoxication case in Poland. This study aims to investigate and rank the in vivo urinary metabolites of α-PiHP.

Urinary metabolites of α-PiHP have been investigated by analysing pooled human liver microsome (pHLM) and pooled S9 fraction (pS9) samples in vitro, blank urine samples as negative controls and in vivo urine samples from users (n=4) as positive controls using a Waters Xevo G2-XS UHPLC-MS-QToF. The analysis was performed in MSe mode and Unify software was applied to search for metabolites and localize the metabolic transformation according to the fragment ions of the spectra resulted from high energy collisions. Phase I and Phase II metabolites were investigated and ranked according to their relative abundance.

In this metabolism study ten in vivo urinary metabolites of α-PiHP were tentatively identified. The identifications were confirmed by use of in vitro metabolites detected in pHLM and pS9 samples. Eight of them were Phase I metabolites and two of them were Phase II metabolites. According to the metabolite ranking study five metabolites were more abundant in urine than the parent compound. For α-PiHP, the two major metabolites via reduction of the keto moiety (M01) and via hydroxylation of the pyrrolidine ring combined with the oxidation of the isoalkyl chain (M06) were identified. The metabolites via the combination of keto reduction and oxidation of the isoalkyl chain (M04), via a ring-opening followed by a caboxylation (M09) and via the glucuronidation of the keto reduced metabolite (M07) were also dominant. The minor metabolites were one Phase II metabolite (M08), two metabolites via oxidation of the isoalkyl chain (M02, M03), one metabolite via the combination of keto reduction and hydroxylation of the pyrrolidine ring (M05) and one metabolite via oxidation of the pyrrolidine ring (M10).

As a result of this present study for the urinary metabolites of α-PiHP, its proposed metabolic pathways in humans include reduction of the keto group, oxidation of the isoalkyl chain, oxidation of the pyrrolidine ring to the oxo moiety or ring opening followed by carboxylation, and the combination of these pathways. Finally, it should be pointed out that the detection of relevant metabolites in addition to the parent drug is important for indisputably proving ingestion of the drug. It is especially important in differentiating similar compounds having identical ESI+ fragmentation pattern and very similar retention time but being concerned by different legislation control. Besides, according to the present study the relative abundance for the major metabolites of α-PiHP is much higher than for the parent compound. To sum up, the relevant metabolites are key components in screening and confirmatory methods developed for sensitively and selectively detecting α-PiHP consumption.