Polydrug fatal intoxication involving MDPHP: Detection and in silico investigation of multiple 3,4-methylenedioxy-derived designer drugs and their metabolites

Abstract

A drug-related fatality involving 3,4-methylenedioxy-α-pyrrolidinohexanophenone (MDPHP) is here reported. Belonging to the class of synthetic cathinones (SCs), MDPHP is a 3,4-methylenedioxy-derived designer (MDDs) drug with a pyrrolidine moiety and an alkyl portion with six carbon atoms. Other MDD pyrrolidine derivatives belong to the alkyl homologous series (C3–C5) and are known as 3,4-methylenedioxy-α-pyrrolidinopropiophenone (MDPPP), 3,4-methylenedioxy-α-pyrrolidinobutyrophenone (MDPBP) and 3,4-methylenedioxypyrovalerone (MDPV). MDDs are psychostimulant drugs of abuse that primarily act on monoamine transporters; little is known about their off-target liability. Recently, MDPHP has gained attention due to increasing seizures and involvement in human intoxications, but currently there is a lack of data about its pharmaco-toxicological effects. In the case reported here, a 58-year-old man with a history of MDPV addiction was found dead in a waterway. While no evidence of natural disease or trauma was found to account for the death, toxicological analysis revealed the presence of MDPHP in addition to MDPPP, MDPV, MDPBP, clonazepam, and citalopram. Since no standards of MDPPP and MDPBP were available at the time of the analysis, LC–QTOF analysis of the drugs and their metabolites were performed. The following concentrations of MDPHP were reported: 350 ng/mL in femoral blood (FB), 110 ng/mL in cardiac blood (CB), 1900 ng/mL in urine, 3000 ng/mL in bile, 490 ng/g in kidney, 80 ng/g in liver, 480 ng/g in lung, 98 ng/g in brain, 700 ng/mL in gastric content and 8 ng/mg in pubic hair. Other MDDs concentrations in biological fluids and tissue were significantly lower than MDPHP suggesting their presence as synthetic impurities. Finally, to better understand the binding properties of the abovementioned MDDs to several documented transporters and receptors, an in silico evaluation was performed. The medical examiner reported that the cause of death was an acute multidrug intoxication by MDPHP and clonazepam in presence of MDPPP, MDPV, MDPBP and citalopram.

Introduction

The dynamic nature of the European drug market is highlighted by increased reports of the production and use of synthetic cathinones (SCs), a class of drugs relatively new to Europe [1]. SCs first appeared on the European illicit drug market in 2005; in 2022, 162 out of 930 monitored new psychoactive substances (NPS) by the European Union Drug Agency (EUDA) belonged to the class of SCs [1]. These substances are central nervous system (CNS) stimulants, and they are derivatives of 2-amino-1-phenylpropan-1-one, or cathinone, which is an alkaloid agent present in the Khat plant (Catha edulis). Their main effects are paranoia, hallucinations, increased friendliness (entactogen effect), panic, and agitation. Moreover, these substances can induce tachycardia, hypertension, chest pain, and rhabdomyolysis. Nonhuman animal studies and human reports suggest they have high abuse and dependence liability [2–4]. SCs are commonly encountered as white or brown crystal powder. Oral use and snorting are the main modes of consumption, but modalities of intake include also rectal (“boofing”), and intravenous (“slamming”) use. Injection of SCs has been linked to long-term abstinent ex-opiate users, young people beginning their injecting career with cathinones as well as chemsex practices and resulting in a highly increased risk of the spread of blood-borne and sexually transmitted diseases like Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV) [5]. 3,4-Methylenedioxy-α-pyrrolidinohexanophenone or MD-αP-hexanophenone (MDPHP or MD-αP-HP, IUPAC name 1-(2H-1,3-Benzodioxol-5-yl)-2-(pyrrolidin-1-yl)hexan-1-one) (Fig. 1) belongs to the class of α-pyrrolidinophenones with close structural similarity to 3,4-methylenedioxypyrovalerone or MD-αP-valerophenone (MDPV or MD-αP-VP) (Fig. 1), the first pyrovalerone derivative that occurred as an NPS [6]. These derivatives possess a pyrrolidine moiety as an amino substituent. Other known α-pyrrolidinophenone derivatives closely correlated to MDPHP and MDPV are 3,4-methylenedioxy-α-pyrrolidinopropiophenone or MD-αP-propanophenone (MDPPP or MD-αP-PrP), and 3,4-methylenedioxy-α-pyrrolidinobutyrophenone or MD-αP-butanophenone (MDPBP or MD-αP-BP) [7] (Fig. 1). MDPV, MDPPP, MDPBP, and MDPHP were formally notified to the European Early Warning System for the first time in 2008, 2009, 2010, and 2014, respectively [7]. MDPV has been detected in up to 107 non-fatal intoxications and 99 deaths during 2008–2013, and in the same period, over 200 kg of the drug were seized in Europe [8]. No MDPPP-related intoxication or death has been reported in the literature, while MDPBP has been reported in three fatal accidents between 2011 and 2015 [9–11]. Recently, Arillotta et al. described 17 cases of intoxication by concomitant consumption of MDPHP and other psychoactive substances, except for one case where MDPHP was the only detected substance [12], and Grapp et al. have reported 9 polydrugs intoxication cases involving MDPHP [13]. Other cases have previously been reported by Beck et al. within the frame of the STRIDA project [14]. Moreover, only three cases of death involving MDPHP have been reported so far: (1) a fetal death in Poland in 2019 was associated with the mother's use of MDPHP together with α- pyrrolidinohexanophenone (α-PHP) as both substances could be detected in the fetus’ blood as well as in the mother's blood and urine [15]; (2,3) two fatal acute intoxications caused by MDPHP in a 48- and 30-year-old were described in Italy [16, 17] (2021 and 2022, respectively). The EUDA annual reports refer to 149 kg of MDPHP seized in 2021 [8] and 180 kg in 2022 [1].

Figure 1.

MDD pyrrolidine derivative structures.

MDPHP structure is closely related to the notorious MDPV and therefore causes a similar mechanism of action such as increment the dopamine, norepinephrine and serotonin release and inhibit their reuptake [18–22]. However, the longer aliphatic chain than MDPV (4C vs 3C), seems to slightly increase the potency in the inhibition of dopamine transporters (DAT) [23] as well as the muscarinic receptors (MR) [24]. Little is known about the pharmacokinetic features of MDPHP because no studies are available; even less is known about postmortem (PM) alterations [17].

To our knowledge, this paper describes the first polydrug-­related fatal intoxication involving MDPHP and other 3,4-methylenedioxy-derived designers (MDDs) (MDPV, MDPBP, and MDPPP) combining analytical and computational characterization.

On-site medicolegal investigation and autopsy examination

The body of a 58-year-old Caucasian man was found floating in prone position in a waterway in the Milan area. The man was last seen alive by his partner 7 days previously; moreover, the partner reported that the deceased suffered from HIV (for which he was undergoing drug treatment) and regularly used MDPV. At the on-site inspection, rigor was present at major joints, the postmortem lividity was distributed accordingly with the body’s position, the environmental temperature and the rectal one were isothermic (air: 8.1°C; water: 7.8°C; rectal: 7.8°C), both hands were macerated, and no greenish staining of the skin was present. At autopsy, the heart was slightly enlarged (weight: 480 g) with mild ventricular hypertrophy, and the lungs and the brain were congested but not edematous. No traumatic injuries were present. A diagnosis of drowning was ruled out due to inconsistent pathological findings. The estimation of time since death rounded between 24 and 72 hours before the on-site investigation. The public prosecutor authorized toxicological analyses due to insufficient morphological findings to determine the cause of death. Samples of cardiac and femoral blood, bile, urine, brain, liver, gastric content, kidney, lung, and pubic hair were collected and maintained at –20°C until toxicological analysis.

Materials and methods

Toxicological analysis of postmortem specimens

A comprehensive systematic toxicological analysis was performed on the postmortem tissue specimens to investigate alcohol, volatile substances, and illegal and medical drugs. Firstly, peripheral postmortem blood was screened for ethanol and volatile compounds by headspace gas chromatography with flame-ionization detection (GC–FID). Liquid chromatography–tandem mass spectrometry (LC–MS/MS) screening was performed on cardiac blood and urinary samples for the research of drugs of abuse (amphetamines, buprenorphine, cannabinoids, cocaine, methadone and opiates), benzodiazepines, antipsychotics and NPS (cathinones, cannabinoids, opioids and benzodiazepines). In addition, general unknown screening in blood, urine and gastric content was conducted by gas chromatography–mass spectrometry (GC–MS) in order to identify additional drugs or metabolites after acidic and basic extraction and derivatization according to Maurer et al. [25]. These procedures were also used to identify MDPHP and MDPV and to hypothesize the presence of MDPBP and MDPPP in the biological samples as described in the next section. Drugs, drugs of abuse and NPS were also investigated in pubic hair specimens.

MDDs confirmation analysis

Since no commercial MDPPP and MDPBP standards were available in the beginning for analysis, a prior identification was performed by an X500R liquid chromatograph with tandem quadrupole time-of-flight mass spectrometry (LC–QTOF/MS) (Sciex, Darmstadt, Germany) analysis of the drugs and their metabolites in the urinary sample. The fragmentation pattern was hypothesized based on the literature and illustrated using ChemDraw software as a support. Once reference compounds were available, an ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was set-up and validated for their confirmation and quantitation, according to Peters et al. [26].

Chemicals and standards

Ultrapure water and organic solvents were of analytical grade and purchased from Carlo Erba (Milan, Italy). The certified reference MDPHP was obtained from Cayman Chemical (Ann Arbor, MI, USA), MDPV from Istituto Superiore di Sanità (Italy), MDPPP and MDPBP from LGC (Teddington, UK). MDMA-d₅ as internal standard (100 μg/mL solution in methanol) were purchased from Cerilliant (Milan, Italy). Formic acid (98–100%) was from Sigma–Aldrich (Milan, Italy).

Instrumental conditions

UHPLC–MS/MS analyses were performed on a 1290 Infinity ultra-high-performance liquid chromatography system (Agilent Technologies, Palo Alto, CA, USA) coupled to a Q Trap 5500 linear ion trap triple-quadrupole mass spectrometer (Sciex, Darmstadt, Germany) and equipped with an electrospray ionization (ESI) source. Chromatographic separation was carried out on a a Kinetex UHPLC XB-C18 column (100 mm × 2.1-mm i.d, 2.6 particle size, Phenomenex, CA, USA) at 35°C using a linear gradient elution with two solvents: 0.1% formic acid (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Solvent A and B were 95% and 5% at 1.00 min, respectively. Solvent B was increased to 35% from 1.00 to 6.00 min, then increased to 95% from 6.00 to 7.10 min, held at 95% from 7.10 to 9.00 min, and then decreased back to 5% from 9.00 to 9.10 min and held at 5% from 9.10 to 11.00 min for re-equilibration. The flow rate was kept constant at 0.45 mL/min during the analysis. The separated analytes were detected with a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode via positive ESI using the precursor ion and product ion transitions shown in Supplementary Table S1. The instrumental conditions were optimized by direct infusion (flow rate 7 μL/min) of a mix standard solution (100 ng/ml) and were as follows: entrance potential 10 eV, curtain gas 25 psi, ion spray voltage 5500 eV, ion source temperature 500°C, ion source gas 1 45 psi and ion source gas 2 40 psi. Data acquisition and processing was performed using Analyst^®1.7.1 and MultiQuant^®2.1.1 software (Sciex, Darmstadt, Germany), respectively.

Validation procedure

UHPLC–MS/MS procedure for MDDs determination was performed in accordance with international recommendations for the validation of new analytical methods in single case studies according to Peters et al. [26]. Selectivity was calculated by the analysis of six sources of blank plasma and the analysis of two zero samples (blank matrix + internal standard). Calibration standards (0.01, 0.025, 0.05, 0.1, 0.25, 0.5 ng/mL for MDPV; 0.1, 0.25, 0.5, 1, 2.5, 5 ng/mL for MDPBP and MDPPP, 0.25, 0.5, 1, 2.5, 5, 10 ng/mL for MDPHP) were obtained by spiking 100-μl blank plasma aliquots with appropriate amounts of standard working solutions. Seven points calibration curves for each compound were generated based on the peak area ratios of the analytes to the IS against nominal analyte concentration using a y = mx + b linear regression. The correlation was tested over the whole range of concentration to ensure linear regression. Linearity was considered satisfactory if R² ≥ 0.990 and CV ≤ 15%. Sensitivity was expressed in terms of limit of detection (LOD) and limit of quantification (LOQ). The LOQ was determined as the lowest concentration with values for precision and accuracy within ±20% and a signal-to-noise (S/N) ratio of the peak areas ≥10, the LOD as the lowest concentration with S/N of the peak areas ≥3. Precision and accuracy of the method were determined through the analysis of six independent replicates of quality control (QC) materials at low and high concentrations levels for each compound according to their calibration ranges. Precision and accuracy were determined by calculating the coefficient of variation (CV%) and the Bias (BIAS%) of the QC samples under the following conditions: intraday, interday, and spiking by different analysts. The analytes recovery was determined by comparing the mass spectrometric response of a first set of standards-free plasma samples (n = 3) fortified with MDDs prior to extraction at low, intermediate and high concentrations level, and a second set of MDDs-free plasma samples (n = 3) fortified with analytes at the same final concentration after extraction. Absolute recovery was determined by comparing the peak areas of the two sets of samples and expressed as percentage. The matrix effect (%) was determined by comparing the peak areas of a first set of extracted aqueous samples (n = 3) in the low, intermediate and high concentration range with the peak areas of a second set of MDDs-free plasma samples (n = 3), both fortified with MDDs after to extraction. Additionally, the analytes recovery and the matrix effects were studied also in MDDs-free urine.

Specimens preparation and extraction

Biological specimens such as kidneys, liver, lungs and brain were homogenized in H₂O (5 mL for 1 g), ultrasonicated for 20 minutes and incubated at 4°C overnight. Accordingly, with the calibration ranges, an appropriate aliquot of diluted tissues (10 or 100 μL) and liquid specimens such as cardiac and femoral blood (10 and 1000 µL), urine (2 and 500 µL) and bile (10 µL) were added by 10 μL internal standard (MDMA-d₅ 1 μg/mL) and 2 mL of borate buffer solution at pH 9 (Avantor, Inc., Radnor, PA, USA). Samples were added with 4 mL of a mixture of chloroform/n-heptane/2-propanol (50/33/17; v/v/v) and extracted. After centrifugation, the organic layer was separated and evaporated to dryness with a gentle stream of nitrogen. The residue was dissolved with methanol (50 μL), and a 1-μL aliquot was then injected into the UHPLC–MS/MS system and analyzed as described previously.

Molecular modelling

Molecular structures

The structures of monoamine transporters and MR have been obtained either from the Protein Data Bank (https://www.rcsb.org/), when the experimental structure was accessible, or via deep learning modelling as reported in the AlphaFold Protein Structure Database [27] and accessible via Uniprot web service (https://www.uniprot.org): Dopamine uptake transporter (DAT): PDB ID AF-Q01959-F1; Norepinephrine uptake transporter (NET): PDB ID AF-P23975-F1; Serotonin uptake transporter (SERT): PDB ID 7LI9 complexed with serotonin; Muscarinic receptor 1 (MR1): PDB ID 6ZFZ complexed with 77-LH-28-1; Muscarinic receptor 2 (MR2): PDB ID 5KZC complexed with NMS; Muscarinic receptor 3 (MR3): PDB ID 8EA0 complexed with iperoxo; Muscarinic receptor 4 (MR4): PDB ID 5DSG complexed with tiotropium; and Muscarinic receptor 5 (MR5): PDB ID 6OL9 complexed with tiotropium.

The structures of MDPV, MDPPP, MDPHP, and MDPBP were obtained from the NIH PubChem repository (https://pubchem.ncbi.nlm.nih.gov/).

Ligands docking

The experimentally determined starting coordinates files have been manually processed in order to remove the ligands complexed with the receptors and transporters. The docking calculations were performed using AutoDock 4.2.6, a widely used software suite for predicting interactions between large biomolecules and small molecules like ligands [28]. Ligands were processed using Meeko (https://meeko.readthedocs.io/en/release/), a Python package used to prepare ligands directly from SMILES or other molecule formats for docking to AutoDock. Both transporter and receptor structures were manually processed via AutoDock tools [28] graphical user interface to properly detect atomic charges, geometric torsional degrees of freedom and protonation state, which have been then stored in PDBQT format. The ligand binding site was determined based on the ligand’s position from the original PDB file, whereas for the AlphaFold models of the DAT and NET transporters pocket residues were selected by comparing them to the SERT structure. A consistent docking center was used for all ligands in each transporter model. The grid dimensions were set to 40 × 40 × 40 Å in the x, y, and z directions, with spacing between grid points of 0.375 Å to ensure that ligands could fully explore the binding pocket. Docking calculation was performed using a genetic algorithm. Each configuration file was used for one docking run, producing up to 100 potential binding poses, eventually statistically analyzed according to their predicting affinity for the protein (Ki, expressed in terms of ligand concentration).

Results

As the first step, GC–MS screening was applied to postmortem specimens revealing the presence of MDPHP by means of a good fit of the obtained mass spectra with the SWGDRUG library version 3.13 installed on the Agilent Chemstation. Moreover, citalopram (FB 530 ng/mL), clonazepam (FB 0.20 ng/mL), 7-aminoclonazepam (FB 350 ng/mL), MDPV and MDPHP were identified and quantified in biological specimens by LC–MS/MS (as reported in Table 1), while the presence of MDPBP and MDPPP was postulated according to m/z of molecular mass ion and fragment ions, but no reference standards were available (Fig. 2).

Table 1.

Distribution of MDDs in postmortem biological samples in the present case and two previous cases

Matrix	Present case				Di Candia et al. [16]	Croce et al. [17]
	MDPHP	MDPV	MDPPP	MDPBP	MDPHP	MDPHP
FB ng/mL	350	0.08	0.93	0.61	399	1601.90
CB ng/mL	110	0.02	0.38	0.04	–	1639.99
Urine ng/mL	1900	0.64	10.8	9.1	222	3028.54
Bile ng/mL	3000	0.48	3.5	10	–	–
Brain ng/g	98	0.03	< LOQ	0.06	–	–
Liver ng/g	80	0.01	0.13	0.19	–	–
Kidneys ng/g	490	0.04	0.20	0.20	–	–
Lungs ng/g	480	0.03	0.20	0.13	–	–
Gastric Content ng/g	700	0.08	0.30	0.14	50	1846.45
Hair ng/mg	8 (0–1.5 cm pubic hair)	< LOQ	< LOQ	< LOQ	–	152.38 (0–1.5 cm) 451.33 (1.5–3 cm)

Figure 2.

LC–QTOF chromatograms, molecular mass and isotopic abundance and proposed fragmentation at CE = 35 eV for MDPPP (A, B, C), MDPBP (D, E, F), MDPV (G, H, I), and MDPHP (L, M, N), respectively.

Therefore, our approach was to investigate MDPBP and MDPPP, as well as their metabolic pathways, using urine sample analyzed by LC–QTOF/MS. An aliquot of 0.5 mL of urine was deconjugated overnight at 37°C by adding 100 µL of β-glucuronidasi from Escherichia coli (Type VII-A, Sigma-Aldrich) previously prepared by dissolving the powder in 5 mL of ultrapure water. Then we extracted urine as described in the text at paragraph 3.2.4 and performed an HRMS-LC–MS analysis. The exact mass and the proposed fragmentation of MDPBP and MDPPP are reported in Fig. 1.

Furthermore, it can be assumed that MDPBP and MDPPP may follow a similar metabolic pathway as structural analogues MDPHP and MDPV [13, 29, 30]. First, we confirm five phase I metabolites for MDPHP according to Kavanagh et al. [31], moreover we were able to suppose the presence of the demethylenyl-methyl-metabolites of MDPBP and MDPPP (MDPBP-M1 and MDPPP-M1) (Table 2). These metabolites are the structural homologues of the most abundant MDPHP metabolite.

Table 2.

Calculated and measured accurate masses of supposed MDPHP (M1–M5) metabolites, the most abundant MDPPP (M1) and MDPBP (M1) metabolites (red) and their fragmentation patterns observed in the urine sample of our case by using LC–QTOF.

MDPHP metabolites are represented according to their relative decreasing abundances.

As soon as MDPBP and MDPPP reference compounds were available, both extraction procedure and UHPLC–MS/MS method were developed and validated for their identification and quantitation, as described here previously. Regarding the selectivity, no interferent peaks have been detected in proximity of the analytes and IS retention times. All calibration curves showed good linearity (R² > 0.9976) over the entire investigated range when using linear correlation. The mean % CV was found to be lower than 15%, whereas LOD and LOQ obtained were 0.4 and 1.4 pg/mL for MDPHP, 0.1 and 0.3 pg/mL for MDPV, 1.7 and 5.6 pg/mL for MDPBP, 0.4 and 1.3 pg/mL for MDPPP respectively. An overview of the assessed validation data is shown in Supplementary Table S2. Regarding the extracted stability, the deviation of MDDs in vial at –20°C after 7 days ranged between –33.3% and +16.3%, whereas after 21 days between –44.4 and +25.0.

The analyte recoveries and the matrix effects were calculated as reported in both MDDs-free blood and urine samples. As shown in Supplementary Table S3, the absolute recovery from blood ranged between 90.5% and 118.8%, whereas the matrix effect was between 88.7% and 126.3%. For urine, the recovery was lower than blood (from 61.7% to 87.5%) and the matrix effect was higher, up to 236.5%.

The validated method was then applied to obtain a distribution of MDDs in the postmortem fluids (urine, bile, cardiac and femoral blood), tissue specimens (kidneys, liver, lungs, and brain), gastric content and pubic hair. MDDs were quantified in all postmortem specimens, at different levels, as reported in Table 1.

In order to model on molecular basis, the MDDs pharmacodynamic properties, a molecular docking protocol has been used for understanding the binding properties of MDDs to several documented transporters and receptors, that is, the dopamine, norepinephrine and serotonin uptake transporters and the muscarinic receptors. The binding affinity was assessed using the inhibition constant (Ki) distribution sampled over a family of poses, which constitutes a measure of how strongly an inhibitor binds to its target. A lower Ki value signifies a more potent inhibitor, reflecting stronger binding, hence being a key parameter in evaluating the effectiveness of potential drugs and in gaining insights into the mechanisms of enzyme regulation. For both monoamine transporters (Fig. 3, top) and MRs (Fig. 3, bottom), the Ki values display an improvement trend as the lipophilic chain of the synthetic cathinones is extended to butyl. This suggests that MDPHP has a higher affinity for the dopamine/norepinephrine/serotonin transporters and MRs.

Figure 3.

Probability distribution function (PDF) of inhibition constant (Ki) computed by AutoDock between MDPPP/MDPBP/MDPV/MDPHP and (a) dopamine uptake transporter (DAT), (b) norepinephrine uptake transporter (NET) and (c) serotonin uptake transporter (SERT) (top panel) and (a) muscarinic receptor 1, (b) 2 (c) 3, (d) 4 and (e) 5 (bottom panel).

Discussion and conclusion

This paper reports a case of fatal polydrug intoxication involving MDPHP. The cause of death was based on a forensic multidisciplinary approach which included a medicolegal on-site investigation and autopsy examination with full and specific toxicological analyses. The cause of death was identified in an acute multidrug intoxication produced by MDPHP and clonazepam in presence of MDPPP, MDPV, MDPBP, and citalopram. The victim had a positive history of “MDPV” or “PV” addiction when alive, especially in the context of the chemsex. However, it is not clear if the decedent was aware of the real composition of MDPHP or he thought he was taking MDPV. Thus, there are reported cases in which MDPHP was purchased as MDPV [12, 17]. Noteworthy, the postmortem toxicological investigations that were performed on the biological specimens showed the presence of MDPHP in both femoral and cardiac blood, urine, bile, brain, liver, lungs and kidneys, together with MDPV, MDPBP, MDPPP, citalopram (femoral blood 530 ng/mL), clonazepam (femoral blood 0.2 ng/mL) and 7-aminoclonazepam (femoral blood 350 ng/mL). The MDDs concentrations in the different postmortem specimens are summarized in Table 1.

The forensic literature reports that concentrations of MDPHP in nonfatal acute intoxication among polydrug assumptions range between 3 and 136 ng/mL (median 8 ng/mL, in serum) [14], 1.26 and 73.3 ng/mL (median: 12.8 ng/mL) [12], 3.3 and 140 ng/mL (median: 16.0 ng/mL, in serum) [13]. On the other hand, serum concentration of MDPHP taken alone in acute nonfatal intoxications varies between <1 ng/mL and 14.3 ng/mL [14].

Analytical data about MDPHP fatal intoxications were compared with those reported by Di Candia et al. [16] and Croce et al. [17] (Table 1). In this case, the MDPHP concentrations were similar to Di Candia et al., but lower than the ones reported by Croce et al. [17] in all specimens, although both papers describe fatal intoxication caused by MDPHP. This statement could be supported by a different interindividual tolerance rate to SCs. Therefore, in our case, the 1.5 cm-pubic hair analysis revealed the presence of MDPHP at concentration of 8.3 ng/mg, whereas Croce et al. [17] found 152.38 ng/mg in the proximal segment and 451.33 in the distal segment (1.5–3 cm). Croce et al. evaluate the postmortem distribution of MDPHP by comparing its concentration in CB and PB: specifically, CB (1639.99 ng/mL) and PB (1601.90 ng/mL) were described as similar, and thus postmortem redistribution was considered not relevant. In our case, the MDPHP concentration in CB (110 ng/mL) was lower than PB (350 ng/mL) suggesting a potential water dilution of the cardiac sample during the autopsy dissection. However, we excluded the onset of the postmortem redistribution from central to peripheral districts. Finally, the MDPHP gastric content (700 ng/mL) concentration may suggest an oral intake of the NPS.

MDPV, MDPPP, and MDPBP concentrations in biological fluids and tissue (Table 1) were significantly lower than MDPHP suggesting their presence as synthetic impurities. Additionally, their main metabolites were identified by LC–QTOF in urine sample, as reported in Table 2.

All these MDDs, which possess a pyrrolidine moiety at the nitrogen position of the cathinone and a methylenedioxy moiety, were formally notified to the European Early Warning System from 2008 (MDPV) to 2014 (MDPHP), respectively [7]. Substituents play a key role in the pharmacokinetics and pharmacodynamics profiles. In particular, pyrrolidine moieties and the length of the aliphatic chain increase lipophilicity thus increasing the potency. Indeed, MDPV (C3), closely related to MDPHP (C4) (Fig. 1), readily permeates the blood-brain barrier as determined in vitro study [32].

The in silico analysis of the binding of MDDs to a documented series of transporters and receptors support the idea that the last generation of cathinones has evolved following a route towards ubiquitous binding improvement despite the increase of its lipophilicity. Interestingly, our data reveal for MDPPP a wider range of affinities for all the considered monoamine transporters with respect to the next generations of molecules, suggesting that affinity and specificity of cathinone-transporters synergically led the chemical evolution of this class of molecules. The same trend is detectable for the affinities of MDDs for the MR, with the notable exception of MR3, for which an optimum had been already reached for MDPPP.

In literature, the use of pyrrolidines is reported, not coincidentally, among autoerotic fatalities for which these substances could enhance sexual pleasure [33]. However, Pelletti et al. stated that the use of such toxics could be seriously underestimated due to a systematic lack of autopsies and postmortem toxicological analyses [34]. Moreover, the analytical definition of such chemical molecules is difficult due to their continuous and rapid evolution as well as the absence of available standards. From a medicolegal point of view, this issue is challenging since forensic practitioners may not have scientific evidence to define the cause of death which could be dramatic in a judicial setting. Similar considerations can be expanded to other circumstances of forensic interest such as the evaluation of psychophysical alterations in the event of accidental deaths or suicides.

In conclusion, to our knowledge, this is the first case reporting a comprehensive MDPHP distribution in a MDPHP-related acute intoxication in presence of other MDD compounds and their metabolites. Moreover, the in silico evaluation of the MDD binding properties suggests that MDPHP has a higher affinity for the transporters of dopamine, norepinephrine and serotonin as well as MRs.

Supplementary data

Supplementary data are available at Journal of Analytical Toxicology online.

Funding

This study does not receive funding from any institution.

Data availability

The data underlying this article are available in the article and in its online supplementary material.

Ethical permission

This article does not contain any studies with living human participants or animals performed by any of the authors (clinical trial number: not applicable). The subject involved in this study underwent a judicial autopsy at the Institute of Forensic Medicine of Milan in order to identify the cause of death. Data collecting, sampling and subsequent forensic analysis were authorized by the public prosecutor. Therefore, data were acquired as part of a forensic judicial investigation and in accordance with Italian Police Mortuary Regulation. Publication of data is allowed when the cases have been closed, but the anonymity of the subject must be guaranteed. All the study was also conducted in accordance with the Declaration of Helsinki.