Studies on the metabolism of five model drugs by fungi colonizing cadavers using LC-ESI-MS/MS and GC-MS analysis

Abstract

It is well-known that cadavers may be colonized by microorganisms, but there is limited information if or to what extent these microbes are capable of metabolizing drugs or poisons, changing the concentrations and metabolic pattern of such compounds in postmortem samples. The aim of the present study was to develop a fungal biotransformation system as an in vitro model to investigate potential postmortem metabolism by fungi. Five model drugs (amitriptyline, metoprolol, mirtazapine, promethazine, and zolpidem) were each incubated with five model fungi known to colonize cadavers (Absidia repens, Aspergillus repens, Aspergillus terreus, Gliocladium viride, and Mortierella polycephala) and with Cunninghamella elegans (positive control). Incubations were performed in Sabouraud medium at 25 °C for 5 days. After centrifugation, a part of the supernatants was analyzed by liquid chromatography-tandem mass spectrometry with product ion scanning. Another part was analyzed by full scan gas chromatography-mass spectrometry after extraction and derivatization. All model drugs were metabolized by the control fungus resulting in two (metoprolol) to ten (amitriptyline) metabolites. Of the model fungi, only Abs. repens and M. polycephala metabolized the model drugs: amitriptyline was metabolized to six and five, metoprolol to two and two, mirtazapine to five and three, promethazine to six and nine, and zolpidem to three and four metabolites, respectively. The main metabolic reactions were demethylation, oxidation, and hydroxylation. The presented in vitro model is applicable to studying drug metabolism by fungi colonizing cadavers.

Studies on drug metabolism by fungi colonizing decomposing human cadavers. Part II: biotransformation of five model drugs by fungi isolated from post‐mortem material

“…MRT and its NFM show a cleavage through the piperazine moiety leaving fragments with m/z 195 and 209 (Figures a, d, and e) or m/z 211 and 225 (Figure b) representing the unchanged or hydroxylated tricyclic ring system, respectively. The hydroxy group in the latter metabolite (Figure b) is most likely attached to the aliphatic carbon atom of the methylene group between the two aromatic rings because the retention time of this metabolite differs from the hydroxyaryl metabolites previously reported by Martínez et al . The metabolites with pseudomolecular ions m/z 268 and unchanged tricyclic ring systems (Figures d and e) can be interpreted as combinations of N ‐demethylation and hydroxylation at the piperazine moiety because they have a prominent neutral loss of water in their EPI spectra.…”

“…The metabolites with pseudomolecular ions m/z 268 and unchanged tricyclic ring systems (Figures d and e) can be interpreted as combinations of N ‐demethylation and hydroxylation at the piperazine moiety because they have a prominent neutral loss of water in their EPI spectra. Martínez et al . previously reported a human metabolite with the same pseudomolecular ion and fragmentation pattern to the latter two, but with different retention time.…”

“…The system was controlled by Analyst 1.5.1. software which was also used for data analysis. For the chromatographic separation, data acquisition and mass spectrometric evaluation was performed as described by Martínez et al 29 with minor modifications. An Eclipse XDB C18 5 μm (4.1 × 150 mm) column was used with the following linear gradient of the mobile phases A (aqueous ammonium formate, 50 mM, pH 3.0) and B (0.1 % formic acid in acetonitrile): 10 % B to 60 % B within 10 min.…”

“…The aim of this second part was to evaluate the capability of these strains to metabolize drugs in vitro . The experiments were performed employing our previously described approach for studying in vitro fungal metabolism using incubation experiments with five model drugs followed by gas chromatography‐mass spectrometry (GC‐MS) or liquid chromatography‐tandem mass spectrometry (LC‐MS/MS)‐based analysis and interpretation of mass spectral data …”

Experimental Study on the Postmortem Redistribution of the Substituted Phenethylamine, 25B‐NBOMe

“…Enteric bacteria and invading bacteria and fungi in cadavers can metabolize drugs, causing postmortem changes of blood drug concentrations . In particular, an in vitro model showed that fungi known to colonize cadavers (e.g., Absidia repens and Mortierella polycephala) could metabolize drugs via demethylation, oxidation, and hydroxylation . As the primary route of 25B‐NBOMe metabolism is 5′‐demethylation, followed by conjugation to glucuronic acid , the redistributed 25B‐NBOMe from the lung to the blood might be degraded by colonizing microorganisms in the blood.…”