The State of the Art in Post-Mortem Redistribution and Stability of New Psychoactive Substances in Fatal Cases: A Review of the Literature

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review reports a deep overview of the NPS PMR providing many data on it, however, before the publication the manuscript's structure needs to be improved for a better understanding and to avoid repetitions.

1)Methodology:

-the authors should report inclusion and/or exclusion criteria for the literary research (e.g. the selection of a specific publication time range, types of publication…). Did the authors follow the PRISMA flow diagram for literary research?

-the following part should be reported in a descriptive way and not as a list in bold: “A comprehensive bibliographical review was conducted for each NPS family, evaluating

the following parameters:

Type of NPS: Name and comparison with other drugs

Circumstances: Age, sex, cause, and number of deaths…”

-this part should be moved into the discussion section: “The most reliable blood sample for reflecting drug concentrations at the time of death is femoral blood. It is advisable to collect both femoral and cardiac blood samples, as well as liver wedges, to determine C/P and L/P ratios, respectively [13]. Some reviewed publications have used blood from subclavian, iliac, and carotid sources in comparison to cardiac blood for establishing C/P ratios.”

2) The results section should not report the same data in the table.

-Please revise it, ensuring that what is discussed is not yet reported in the table, e.g., data about pKa, concentrations...Post-mortem concentrations are already reported in Table 1 and need to be critically discussed. What need to be highlighted is the PRM.

3)Table 1 is interesting, but its structure is confusing and is too big. In general, it should be harmonized. Tables usually need to be self-explaining. There is a lot of data inside, and the formatting style makes it hard to read. The authors can also consider dividing Table 1 into two or three tables based on the classification reported in the Methodology section.

-The table needs also a list of the spelled abbreviations for the substances as footnotes.

 

4) Please revise all titles of the different sections because some are in capital letters and others are not, please harmonize them.

Author Response

Reviewer 1

 

1)Methodology:

-the authors should report inclusion and/or exclusion criteria for the literary research (e.g. the selection of a specific publication time range, types of publication…). Did the authors follow the PRISMA flow diagram for literary research?

-the following part should be reported in a descriptive way and not as a list in bold: “A comprehensive bibliographical review was conducted for each NPS family, evaluating

the following parameters:

Type of NPS: Name and comparison with other drugs

Circumstances: Age, sex, cause, and number of deaths…”

 

We appreciate your comment and have updated Section 2 to clarify the methodology used to prepare the review. A workflow diagram has been included, as illustrated in the figure below. We outline the initial query used to search bibliographic databases such as PubMed, Wiley, and Google Scholar, and we explain how we refined the search using specific keywords like “blood” and “level,” while limiting the search to the years 2000 to 2024. This search resulted in 300 entries, but we selected only those studies that provided quantitative data on NPS levels in peripheral and cardiac blood, as well as liver concentrations, allowing us to calculate the C/P and L/P ratios. More publications were added to this previous 43 papers using specific key words corresponding to the specific drug classes—such as "designer benzodiazepines," "tryptamines," "synthetic cathinones," "synthetic opioids", etc. Finally, 94 papers were selected to investigate PMR.

Workflown diagram included in section 2.

 

Each of the selected 94 papers has been thoroughly reviewed, and summaries are provided in a series of tables (Tables 1–3). These tables categorize NPS by chemical structure based on the UNODC and EUDA groups and outline the details of each case, including age, sex, cause of death, and postmortem interval (PMI). Additionally, they include samples collected during autopsy, pretreatment methods, analytical techniques, detected postmortem levels, C/P and L/P calculated ratios, co-occurring substances and metabolites, as well as reference numbers and publication years, all organized in columns for easy reference.

 

The remaining articles identified in the initial search were also reviewed, as they provide valuable insights into the physicochemical properties, stability, analytical methods, metabolism, and postmortem levels in a single blood type (typically femoral) for various NPS. These findings will be discussed throughout the document.

 

-this part should be moved into the discussion section: “The most reliable blood sample for reflecting drug concentrations at the time of death is femoral blood. It is advisable to collect both femoral and cardiac blood samples, as well as liver wedges, to determine C/P and L/P ratios, respectively [13]. Some reviewed publications have used blood from subclavian, iliac, and carotid sources in comparison to cardiac blood for establishing C/P ratios.”

 

It has been moved to section 3. “Resuls and discussion”.

 

2) The results section should not report the same data in the table.

-Please revise it, ensuring that what is discussed is not yet reported in the table, e.g., data about pKa, concentrations...Post-mortem concentrations are already reported in Table 1 and need to be critically discussed. What need to be highlighted is the PRM.

We appreciate the reviewer’s suggestion and have removed any duplicity. Section 3, titled “Results and Discussion,” is organized into subsections corresponding to the 11 groups of NPS studied. Each family of NPS includes a brief monograph detailing their physical and chemical properties, comparing them to PMR studies related to their classical counterparts, and assessing their stability across various studies. Additionally, we reference postmortem levels in fatal intoxications (under the epigraph “Postmortem Levels in Fatal Case Reports”) from studies not included in Tables 1-3 due to the absence of cardiac blood samples, as we believe this information is also valuable, that is the reason why we explain some postmortem levels in the text. Finally, we will discuss the C/P and L/P ratios presented in the relevant tables (under the epigraph “Tissue Distribution and PMR”), which are derived from the 94 selected studies, and propose certain phenomena, such as stability, to explain the differences in C/P ratios compared to classical drug analogs.

 

3) Table 1 is interesting, but its structure is confusing and is too big. In general, it should be harmonized. Tables usually need to be self-explaining. There is a lot of data inside, and the formatting style makes it hard to read. The authors can also consider dividing Table 1 into two or three tables based on the classification reported in the Methodology section.

We appreciate the reviewer’s comments and have divided Table 1 into three separate tables (Tables 1-3). The first table provides information on phenylethylamines, aminoindanes, and cathinones; the second focuses on phenmetrazines, phenidates, arylcyclohexylamines, tryptamines, and designer benzodiazepines; while the third table contains case reports related to synthetic opioids, nitazenes, and synthetic cannabinoids.

The tables categorize NPS according to their chemical structure across 11 specified groups, detailing each case report by age, sex, cause of death, and postmortem interval (PMI). They also include data on autopsy samples, pretreatment methods, analytical techniques, detected postmortem levels, calculated C/P and L/P ratios, co-occurring substances and metabolites, along with reference numbers and publication years. All information is organized in a clear, column-based format for easy access. We have renamed the columns, changing “circumstances” to “age and sex” and “matrix” to “samples collected.”

This structure provides readers with a quick overview of each case, while allowing for further in-depth information through bibliographic research. Classifying NPS by chemical structure offers a global view of reported cases, particularly in relation to C/P ratios, and emphasizes certain trends concerning PMR.

-The table needs also a list of the spelled abbreviations for the substances as footnotes.

A glossary has been added at the end of the manuscript, covering all abbreviations and substance names. Additionally, each table includes abbreviations as footnotes for easy reference.

 

4) Please revise all titles of the different sections because some are in capital letters and others are not, please harmonize them.

 

Once again, we appreciate the reviewer’s comments, and we have made the necessary corrections throughout the text.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript reviews post-mortem redistribution and stability of NPS in fatal cases. The authors well examined them. This should be praised. However, some weaknesses are observed. After the correction, this manuscript should be accepted. The comments are indicated below.

 

1)      Some blood concentrations are suspected to be wrong. For example, units of 3-FPM are indicated as mg/L and ng/mL. Please re-confirm all concentration units.

 

2)      3.7. Tryptamines

Psilocybin is a pro-drug of psilocin. Psilocybin itself is not detected in the body. Please re-confirm it.

 

3)      3.8. Designer benzodiazepine

The reviewer does not like this term “designer benzodiazepine” because phenazepam, etizolam, and flutazolam are approved and clinically used in some countries. This term is unilateral one in some western countries. If the authors want to use it, they should define it.

 

4)      3.9. Synthetic opioids

Considering tolerance, it is difficult to discuss the lethal concentration. How did the authors of original papers decide it? Please confirm it.

 

Author Response

1) Some blood concentrations are suspected to be wrong. For example, units of 3-FPM are indicated as mg/L and ng/mL. Please re-confirm all concentration units.

 

We appreciate the reviewer’s attention to this error; it was indeed a discrepancy in concentrations between the table and the text. The correct concentrations are FB: 2400 ng/mL and CB: 2600 ng/mL. We have also reviewed and corrected the concentration units throughout the document.

2) Tryptamines

Psilocybin is a pro-drug of psilocin. Psilocybin itself is not detected in the body. Please re-confirm it.

We appreciate the reviewer’s comments and will clarify this in the document text. Research on metabolism shows that, following oral administration, psilocybin undergoes rapid dephosphorylation due to the acidic environment of the stomach or through alkaline phosphatase (as well as other non-specific esterases) in the intestine, kidneys, and potentially in the blood, resulting in the formation of psilocin that easily crosses the blood-brain barrier. Inhibition of alkaline phosphatase by competitive substrates, such as β-glycerophosphate, can prevent intoxication symptoms. Thus, psilocybin may be regarded as a prodrug, with its in vivo effects being attributable to psilocin. Psilocybin has not been detected in biological fluids in any of the reported cases of poisoning after ingestion of hallucinogenic mushrooms.

To support this claim, a clinical study with eight volunteers administered psychoactive oral doses of psilocybin at 212 ± 25 μg/kg body weight found that, within 24 hours, 3.4 ± 0.9% of the administered dose was excreted as free psilocin. In reported cases of poisoning following the ingestion of hallucinogenic mushrooms, psilocybin itself has not been detected in biological fluids.

 

We add the next text in the manuscript and two new references: “Due to the acidic environment of the stomach and enzymatic action from alkaline phosphatase and other esterases in the intestine, kidney, and possibly blood, psilocybin is dephosphorylated into psilocin. Psilocin, which can easily cross the blood-brain barrier, is the primary agent responsible for psychoactive effects and can be seen as a pro-drug. Psilocin is them metabolized by monoamine oxidase (MAO) and probably aldehyde dehydrogenase (ALDH) to 4-hydroxyindoles-3-acetic acid and 4-hydroxytryptohole. Notably, psilocybin itself has not been detected in biological fluids in reported cases of poisoning following the ingestion of hallucinogenic mushrooms. [173,174]”.

 

References:

[173] Dinis-Oliveira RJ. Metabolism of psilocybin and psilocin: clinical and forensic toxicological relevance. Drug Metab Rev. 2017 Feb;49(1):84-91. doi: 10.1080/03602532.2016.1278228. Epub 2017 Jan 31. PMID: 28074670.

 

[174] Hasler F, Bourquin D, Brenneisen R, Vollenweider FX. Renal excretion profiles of psilocin following oral administration of psilocybin: a controlled study in man. J Pharm Biomed Anal. 2002 Sep 5;30(2):331-9. doi: 10.1016/s0731-7085(02)00278-9. PMID: 12191719.

3) 3.8. Designer benzodiazepine

The reviewer does not like this term “designer benzodiazepine” because phenazepam, etizolam, and flutazolam are approved and clinically used in some countries. This term is unilateral one in some western countries. If the authors want to use it, they should define it.

We appreciate the reviewer’s comments. In general, the studies refer to these substances as “new benzodiazepines” or “designer benzodiazepines.” However, these terms pose a challenge because phenazepam, etizolam, and flutazolam are legally available benzodiazepines in countries like Japan, Russia, and India, yet they are trafficked illegally in the U.S. and Europe, where they are monitored by the EUDA. We will incorporate a definition of these terms to address the concerns raised by the reviewer.

 

We have included: ‘designer benzodiazepines (BZDs)’ refer to NPS that feature a benzodiazepine core and are not regulated by the international drug control system. We also include three benzodiazepines—phenazepam, etizolam, and flutazolam—which were previously classified as NPS but have recently come under international regulation. Etizolam was initially developed and clinically used in Japan and is now licensed in India and Italy; phenazepam originated in the Soviet Union and remains authorized in Russia and neighboring countries; flutazolam was developed primarily for use in Japan. While these drugs are not technically classified as designer BZs, they are monitored by the EUDA and are often categorized with them.

 

4) 3.9. Synthetic opioids

Considering tolerance, it is difficult to discuss the lethal concentration. How did the authors of original papers decide it? Please confirm it.

This commentary is highly valuable and adds significantly to the paper, prompting us to clarify it further. For opioids, tolerance development often causes levels found in regular users to overlap with toxic levels or those observed in postmortem cases. As a result, we refer specifically to concentrations identified in postmortem cases rather than discussing lethal levels directly. Furthermore, aside from potency and tolerance, it is important to highlight that in the majority of reported cases, synthetic opioids were frequently detected in combination with other substances, particularly CNS depressants such as benzodiazepines, antidepressants, antihistamines, ethanol, and additional opioids.

 

In the cases reviewed in Table 3, there were only five reports where a fentanyl analog was the only substance detected: acetylfentanyl (McIntyre IM et al. [243]), 4-fluorobutyryl fentanyl (Rojkiewicz M et al. [250]), p-fluoroisobutyryl fentanyl (Roosendaal J et al. [254]), octfentanil (Coopman V et al. [259]), and U-47700 (Rohrig TP et al. [264]). In these cases, based on circumstantial evidence, autopsy findings, and toxicological results, the medical examiner concluded that acute intoxication with fentanyl derivatives was the cause of death.

 

We proceed to include in the text the following: “Opioid tolerance plays a significant role in assessing possible causes of death, which is why "post-mortem levels" are discussed rather than "lethal concentrations"; levels may overlap depending on whether or not the individual was habituated to opioids. In most of the reported cases, including those displayed in table 3, synthetic opioids were found in combination with other substances, particularly central nervous system (CNS) depressants like benzodiazepines, antidepressants, antipsychotics, antihistaminic drugs, ethanol, and other opioids. Such combinations can lead to pharmacodynamic interactions, raising the risk of respiratory depression. Mono-intoxication by a specific synthetic opioid is discussed only in certain cases where no other compounds are found in the toxicological results or pathological findings to account for the cause of death”.

References:

[243] McIntyre IM, Trochta A, Gary RD, Malamatos M, Lucas JR. An Acute Acetyl Fentanyl Fatality: A Case Report With Postmortem Concentrations. J Anal Toxicol. 2015 Jul-Aug;39(6):490-4. doi: 10.1093/jat/bkv043. Epub 2015 Apr 26.

 

[250] Rojkiewicz M, Majchrzak M, Celiński R, Kuś P, Sajewicz M. Identification and physicochemical characterization of 4-fluorobutyrfentanyl (1-((4-fluorophenyl)(1-phenethylpiperidin-4-yl)amino)butan-1-one, 4-FBF) in seized materials and post-mortem biological samples. Drug Test Anal. 2017 Mar;9(3):405-414. doi: 10.1002/dta.2135. Epub 2016 Dec 29. PMID: 27863134.

 

[254] Roosendaal J, Oosting R, Kloos DP, de Boer HH, van den Berg JDJ, Oldenhof S, Bosman IJ. A fatal mono-intoxication with 4-fluoroisobutyrylfentanyl: Case report with postmortem concentrations. J Anal Toxicol. 2023 Jul 22;47(6):541-546. doi: 10.1093/jat/bkad034. PMID: 37315188.

 

[259] Coopman V, Cordonnier J, De Leeuw M, Cirimele V. Ocfentanil overdose fatality in the recreational drug scene. Forensic Sci Int. 2016 Sep;266:469-473. doi: 10.1016/j.forsciint.2016.07.005. Epub 2016 Jul 17. PMID: 27471990.

 

[264] Rohrig TP, Miller SA, Baird TR. U-47700: A Not So New Opioid. J Anal Toxicol. 2018 Jan 1;42(1):e12-e14. doi: 10.1093/jat/bkx081. PMID: 29040568.

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

As requested, the authors revised the manuscript. The organization has improved and tables are now more understandable. Now in my opinion can be accepted.