Toxicological interpretation of N-ethylpentedrone and its metabolites in twelve fatal and non-fatal forensic cases.

N-Ethylpentedrone (NEP) is a synthetic cathinone that remained legally available in the Netherlands until its inclusion under the Opium Act as of 1 July 2025. Although NEP emerged in the mid-2000s, toxicological data, especially regarding its metabolism and concentration in real-world cases, are scarce. The aim of this study is to demonstrate analytical methods for the detection and quantification of NEP and its metabolites in antemortem and postmortem biological matrices and to provide reference values and guidance for interpretation. Cases where NEP was detected between May 2024 and September 2025 were selected. All cases have undergone Systematic Toxicological Analyses, which consists of a selection of screening procedures (HS-GC-FID and LC-QToF-MS) and a targeted semi-quantitative LC-MS/MS method. Additional quantitative LC-MS/MS analyses were performed if needed. NEP metabolites were investigated by LC-Q-Orbitrap-MS. NEP was detected in 12 cases (all male, age range 18-56 years). The median antemortem (n = 8) and postmortem (n = 5) NEP (femoral) blood concentrations were 0.062 mg/L (range <0.005-0.45 mg/L) and 0.63 mg/L (range 0.26-0.93 mg/L), respectively. In four cases, a NEP (multidrug) intoxication was concluded as a possible cause of death. One case indicated a half-life of around 2-3 hours, a blood/plasma ratio of around 1.1 and an up to 5-fold postmortem increase of NEP within 48 hours after death. Several phase-1 metabolites of NEP were detected, but only NEP itself was identified in all investigated blood, plasma, urine, and vitreous humor samples. NEP recently resurfaced in the Netherlands, with four potentially fatal cases. For the analysis and interpretation of NEP blood concentrations, degradation of NEP in blood stored at room temperature, (survival) time since intake and a potential significant postmortem increase should be taken into account. NEP itself remains the target compound of choice, with dihydro-NEP and N-desethyl-NEP serving as complementary analytes depending on case circumstances.