Phase I metabolism of the methadone analog methiodone (IC-26) in pooled human liver microsomes and its detection in seven authentic human cases

The recent detection of methiodone (IC-26; 4-(Ethanesulfonyl)-N, N-dimethyl-4,4-diphenylbutan-2-amine) in Europe, a new synthetic opioid associated with a growing number of fatal intoxications, highlights the need for reliable toxicological markers to support its detection in routine screening. As a structural analog of methadone, methiodone exemplifies the structural diversity of new emerging synthetic opioids. In this study, the metabolism of methiodone was investigated using an integrated approach combining in silico prediction (GLORYx, EAWAG-PPS, BioTransformer 3.0), in vitro incubation with pooled human liver microsomes (pHLM), and liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QTOF/MS) analysis of authentic biological samples from one non-fatal intoxication and six post-mortem cases. Two major phase I metabolites were identified, N-desmethyl methiodone (M1) and N, N-didesmethyl methiodone (M2), and both were consistently detected across all fatal cases by LC-QTOF/MS. Seven additional minor metabolites, likely formed via hydroxylation or combined N-demethylation and hydroxylation, were tentatively identified exclusively in authentic biological samples by LC-MS/MS. In the non-fatal case, M1 was the sole detectable analyte in serum, suggesting its particular value as a long-term exposure marker. The detection of trace M1 in microsome-free control incubations indicates a minor non-enzymatic degradation pathway, with implications for the cautious interpretation of low-level M1 findings, e.g. in hair analysis. Blood methiodone concentrations in fatal cases ranged from 25 to 930 ng/mL, and in urine from 220 to 37,700 ng/mL. The integration of methiodone, M1, and M2 into routine opioid screening methods is strongly recommended to ensure reliable detection across all stages of drug elimination.