Forensic biotechnology

EW SYCHOACTIVE UBSTANCES

They represent a very large group of molecules mainly of a synthetic nature, characterized by

pharmacological and toxicological properties that are particularly dangerous for the health of consumers: the

emergence of NPS in recent years represents a phenomenon of increasingly impressive dimensions and a

worldwide problem. NPS can be very different, and they can be divided in different groups:

Phencyclidine-

Aminoindanes phenethylamines

type substances

Plant-based Synthetic

Piperazines substances cannabinoids

Synthetic Tryptamines Others

cathinones

The most important problem is that these compounds are sold in the illicit market very easily and we don’t

know anything about them (internal standard, cut-off, pharmacokinetic and dynamic, half-life, etc).

We can classify NPS on the base of their pharmacological aspect:

1. Stimulants: cathinones, phenethylamine

2. Substances with entactogenic activity: phenylpiperazine and new synthetic hallucinogens (es. 2,5-

dimethoxy4-bromoamphetamine (DOB))

3. Dissociative anaesthetics: ketamine, methoxetamine

4. Synthetic cannabinoids (serie JWH, HU, CP)

5. Synthetic opioids (FENTANYL DERIVATES, NITAZENES)

6. Designer BDZ University of Verona Bacci Anna

Forensic Biotechnology 2023/2024

14.1 S

TIMULANTS

14.1.1 Cathinones

The identification of cathinone as the main psychoactive compound of khat leaves, resulted in the synthesis

of several derivatives. The effect of cathinones is similar to those of amphetamine: they act through the

inhibition of dopamine and noradrenaline reuptake, they induce effects similar to those of cocaine and

amphetamines (increased sociability, energy, sexual libido and work capacity).

Some adverse effects are: panic attacks, tremors, agitation, insomnia, nausea, headaches, tinnitus,

dizziness, muscle twitching, increased heart rate, altered vision, short term mental confusion, memory

difficulties, suicidal thoughts, psychosis, tolerance and dependence. Also neurotoxicity and lethality.

The first cathinone that appears in the market was mephedrone. It’s illegal in Italy from 2010.

Methylone (beta-keto-MDMA) is similar to MDMA but less potent and with a shorter duration of effect, it’s

used alone or with mephedrone and it could be sold in liquid, powder or tablet form.

As soon as methcathinone was considered illegal, the illicit market started to synthesise new molecules that

only had a different added group, so they were not actually illegal, such as NRG (mixture of cathinones,

etylone, methedrone, nafirone, flufedrone, 3-fluoromethcathinone, pentylone, buphedrone, ...

14.1.2 Phenethylamine

It’s a stimulant similar to amphetamines, with mainly hallucinogenic effects. There are so many NPS that are

called by simplicity as compounds of series 2C (n>27), compounds of series NBOMe (N> 33) extremely potent

μg, aminoindanes and compounds of series D (DOM, DOB, DOI,..).

→

5-APB it’s phenethylamine, structurally analogue of MDA (sold as Benzo Fury), also positive at

immunoassays for amphetamine/MDMA. It’s used for dissociative anaesthetics.

→

5-MAPB (N-metil-5(2aminopropil)benzofurane) N-methyl derivative of 5-APB, it’s metabolized by CYP450

to 5-APB. Positivity at immunoassays for amphetamine/MDMA.

University of Verona Bacci Anna

Forensic Biotechnology 2023/2024

14.2 D

ISSOCIATIVE ANAESTHETICS

Effects: visual-auditory hallucinations defined as near-death, with the perception of disembodied entities,

apparent visions of the future and views of one’s body from outside.

Adverse effects: dizziness, state of confusion and psychomotor agitation, time distortion, aphasia, dynes

thesis and temporary cognitive alterations with memory loss, tachycardia, hypertension, stupor, ataxia,

mydriasis. Induction of an abstinence syndrome characterised by mood changes and/or depressive thoughts.

14.2.1 Methoxetamine

It’s available in internet as powder since 2010. It’s known as special M, MXE, M-ket. Similar to ketamine.

Act as an NMDA receptor antagonist, similarly to other arylcyclohexylamines.

14.3 S

YNTHETIC CANNABINOIDS

- 2006: Herbal mixtures marketed as environmental perfumers or 'hemp-flavoured' herbal incense

- Sold in 'smart shops' or via the internet as legal alternatives to cannabis with a low perception of

toxicity

- End of 2008: 'Spice' phenomenon in Europe

- Start of alert system monitoring

- December 2008: JWH-018 detected in Austria

They can interact with the same receptor as cannabinoids, but THE STRUCTURE IS DIFFERENT, so there’s no

possibility for our system to detect synthetic cannabinoids if we are detecting for natural ones. There are still

some portions that are able to stimulate the same receptor of THC. These compounds have the same effects

of cannabinoids, but they are chemically synthesised.

They started from the ‘90s:

- Discovery of cannabinoids receptors (CB1: CNS, peripheral neurons; CB2: cells of the immune system)

- Identification of the endogenous ligand (anandamide)

- Synthesis of new molecules capable of mimicking the effects of cannabinoids with an

aminoalkylindole structure

- The aim was to identify new active molecules, selective for peripheral CB1 receptors, for the

treatment of chronic pain without effects on the CNS.

In 2009/2010 they appeared on the illicit market, and they were developed as soon as the first molecules

were scheduled.

Effects on humans overlapping those of THC: tachycardia, altered perception and mood, dry mouth,

reddening of the eyes.

Affecting: CNS and cardiovascular system, endocrine, respirator and immune system.

Psycotoxic effects described after the use of N-joy: hallucinations, altered perception of one’s body, mental

confusion, panic attacks, paresthesias, tachycardia and nausea.

The intoxication is more severe than THC and fatalities are also reported. Because synthetic cannabinoids

react more strongly with the brain's cannabis receptors they're more potent than natural cannabis.

he effects of taking synthetic cannabinoids with other drugs − including over-the-counter or prescribed

medications − can be unpredictable and dangerous.

• Synthetic cannabinoids + antidepressants: mixing with antidepressant drugs such as SSRIs (selective

serotonin reuptake inhibitors) is dangerous and can lead to fever, fast heartbeat, convulsions, organ

failure, coma and death.

• Synthetic cannabinoids + stimulants such as crystal methamphetamine (ice) or cocaine: effects can

be particularly dangerous and increase likelihood of experiencing anxiety.

University of Verona Bacci Anna

Forensic Biotechnology 2023/2024

14.4 S

YNTHETIC OPIOIDS

Synthetic opioids are substances that are synthesised in laboratory (2012 and in particular 2016) and that act

on the same targets in the brain as natural opioids to produce analgesic effects.

Some common illicitly produced synthetic opioids are acetyl fentanyl, butyryl fentanyl, beta-

hydroxythiofentanyl, carfentanyl, furanyl fentanyl, 4- fluoroisobutyryl fentanyl, acryl fentanyl, and U-47700.

They can be sold as synthetic heroin, or mixed with heroin or other illicit drugs, and even counterfeit or fake

medicines. One of the most important aspects of the synthetic opioids crisis is about their increasing potency

and toxicity.

MORPHINE (natural opioid product)<HEROINE(semisynthetic opioid)<FENTANYL (synthetic)< CARFENTANYL

Also, we have to consider the fact that clandestinely manufactured pills present different doses in tablets

(tablet matrix+active substances).

Some effects of clandestinely produced synthetic opioids, similar to other commonly used opioid

analgesics (e.g., morphine), may include relaxation, euphoria, pain relief, sedation, confusion, drowsiness,

dizziness, nausea, vomiting, urinary retention, pupillary constriction, and respiratory depression (=DEATH).

USE NALOXONE AS ANTIDOT. University of Verona Bacci Anna

Forensic Biotechnology 2023/2024

14.5 D

ESIGNER BENZODIAZEPINES The rising use of designer benzodiazepines

(DBZD) is a cat-and-mouse game between

organized crime and law enforcement. Non-

prohibited benzodiazepines are introduced

onto the global drug market and scheduled as

rapidly as possible by international authorities.

In response, DBZD are continuously modified to

avoid legal sanctions and drug seizures and

generally to increase the abuse potential of the

DBZD.

Similar to BDZ but not used in therapy (21

compounds).

Potency scale:

2-metizolam, 1-pyrazolam, 3-

deschloroetizolam, 3-nifoxipam, 4-

meclonazepam, 4-etizolam, 5-diclazepam, 7-

flubromazepam, 6-clonazepam, 6-

flubromazolam.

14.6 T I

HE TALIAN LEGISLATION

➢ Ordinanza 6 aprile 2010: Prohibition of the manufacture, import, placing on the market, trade and

use of products called 'n-Joy' and 'Spice'. (G.U. Serie Generale n. 87 del 15 aprile 2010)

➢ Decree 16 June 2010: (G.U. Serie Generale n. 146 del 25 giugno 2010) Update of Table I D.P.R. 309/90:

Insertion of JWH-018, JWH-073 and Mephedrone

➢ Minister of Health. Ordinanza 3 december 2010 Precautionary measures to protect health, on the

use of products called Forest Green, Jamaican Spirit, Star of Fire, Amazonas, Amazonas Vanilla, B-52

e Jamaican Gold. (G.U. Serie Generale n. 12 del 17 gennaio 2011).

➢ Minister of Health. Ordinanza 30 decembre 2010 Prohibition of manufacturing and marketing the

product «Jungle Mistic Incense». (G.U. Serie Generale n. 44 del 23 febbraio 2011)

➢ DECREE 11 may 2011 (G.U. Serie Generale n. 112 del 16 maggio 2011) Updating and completion of

tables listing narcotic and psychotropic substances: insertion in Table I of: 3,4-

Methylenedioxypyrovalerone (MDPV), JWH-250, JWH-122 and structural analogues derived from 3-

phenylacetylindole and 3-(1- naphthoyl) indole.

THEY BECOME ILLEGAL ONLY WHEN THEY ARE SCHEDULE (IN THE LIST).

The analysis of non biological samples:

- Herbal blends (43.6 %) - Beverages (3.9 %)

- Capsule, tablets (10.2 %) - Syringes (containing mushroom

- Powders (3.9 %) spores) (7.7 %)

- Sweets (2.5 %) - Fertilizers (26.9 %)

- scentes incences - Bath salt

- Mushroom (1.3 %) University of Verona Bacci Anna

Forensic Biotechnology 2023/2024

14.7 A :

NALYTICAL APPROACH

❖ Untargeted screening by using HRMS (ESI-TOF MS)

❖ n

Confirmation of the structures of the unknown molecules’ specific fragmentations (ESI-ion trap MS )

[ION TRAP is the only system that can perform SPECIFIC multi-stage FRAGMENTATION to deconstruct

the structure of the molecule, we got the MW and neutral ion from ion HRMS (High Resolution MS)

and then we have the structure]

❖ n

Identification of the compounds with LC-ESI-Ion trap MS and LC-ESI-QTOF

❖ Quali-quantitative confirmation by GC-MS

➔ The great variety of active substances makes the identification of NPS extremely difficult, often

lacking both reference standards and information on chemical physical charact