To demonstrate analytical methods for detection and quantification of N-ethylpentedrone (NEP) and its metabolites in biological samples and to provide guidance for interpretation of blood concentrations.

The post-mortem cases have undergone Systematic Toxicological Analyses (STA), which consists of screening procedures (HS-GC-FID, LC-ToF-MS and LC-QToF-MS) and a targeted semi-quantitative LC-MSMS method. These were supplemented with targeted quantitative LC-MSMS analyses, depending on the outcome of the screening results. For Driving Under the Influence of Drugs (DUID) cases, screening was performed with LC-QToF-MS and supplemented with similar targeted quantitative methods. NEP metabolites were investigated by LC-QOrbitrap-MS.

Although first user reports of NEP were already described in the mid 2000's, post-mortem toxicological data on this cathinone-type NPS are scarce. It recently resurfaced in the Netherlands and surrounding countries. NEP was found in two post-mortem cases from a secured forensic psychiatric clinic. Before their death, both patients had symptoms of hyperactivity. NEP also appeared in three DUID cases.

Besides unknown concentrations of NEP, initial toxicological investigations in femoral blood revealed the presence of low to therapeutic concentrations of aripiprazole, metformin, valproic acid and zuclopenthixol in post-mortem case 1 (PM1) and promethazine and aripiprazole in post-mortem case 2 (PM2), respectively. For the three DUID cases, NEP was found in blood either as a single drug of abuse (DUID1), in combination with a single other drug (DUID2, presence of ritalinic acid) or in combination with four other NPSs (DUID3, presence of bromazolam, bromonordiazepam, alpha-PHiP and 4-fluoro-3-methyl-alpha-PVP).

An LC-MSMS method was validated for determination of NEP in whole blood, stabilized with sodium fluoride and potassium oxalate. Following protein precipitation with acidified acetonitrile, NEP was determined in MRM mode (m/z 206.1–>146.1 and 206.1–>130.0) using 4-MMC-D3 as internal standard. No interferences from the matrix or the internal standard occurred. A calibration model (quadratic fit) was assessed from 0.005 to 1mg/L using a 1/x² weighting factor. Within-run precision, between-run precision and between-run accuracy (n=6) at LLOQ/low/mid/high concentrations (0.005/0.015/0.4/0.8mg/L) were 5.4/3.2/3.9/5.3%, 13.1/8.0/5.4/3.5% and 88.2/99.6/99.5/114.4%, respectively. No carry-over was noticed and absolute and relative matrix effects (n=6) at low/high concentrations fulfilled validation criteria, both for antemortem as well as post-mortem blood samples. Freeze-thaw and autosampler stability were within acceptance criteria (85–115%). However, bench-top stability at 4hours was sometimes outside acceptance criteria (maximum loss of 18%).

For PM1 and PM2 NEP was measured in femoral blood at concentrations of 0.83 and 0.93mg/L, respectively. This is in line with recent literature about fatal intoxications involving NEP. For DUID cases, lower blood concentrations of 0.076, 0.17 and 0.035mg/L were measured in DUID1, DUID2 and DUID3, respectively.

Several phase-1 metabolites of NEP (N-desethyl-NEP, dihydro-NEP and dihydro-N-desethyl-NEP) were identified in blood and urine. NEP itself was present in all investigated blood and urine samples.

NEP recently resurfaced. Two NEP-related deaths had femoral blood concentrations of about 0.9mg/L, while three DUID cases had lower blood concentrations (all below 0.2mg/L). Based on the outcomes of the pathological and toxicological investigations, and the obtained medical information, the cause of death for both post-mortem cases was attributed to a fatal NEP intoxication. Although several metabolites are present, NEP itself is the target compound of choice – even in urine – if samples are collected in tubes preserved with sodium fluoride.