Forensic biotechnology

New psychoactive substances

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.

Types of NPS

• Phencyclidine-type substances
• Aminoindanes
• Phenethylamines
• Plant-based synthetic substances
• Piperazines
• Synthetic cannabinoids
• Tryptamines
• Others like 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).

Pharmacological classification of NPS

• Stimulants: cathinones, phenethylamine
• Substances with entactogenic activity: phenylpiperazine and new synthetic hallucinogens (e.g., 2,5-dimethoxy-4-bromoamphetamine (DOB))
• Dissociative anaesthetics: ketamine, methoxetamine
• Synthetic cannabinoids (series JWH, HU, CP)
• Synthetic opioids (fentanyl derivates, nitazenes)
• Designer BDZ

University of Verona, Bacci Anna, Forensic Biotechnology 2023/2024

Stimulants

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, inducing 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, which has been illegal in Italy since 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 could be sold in liquid, powder, or tablet form. As soon as methcathinone was considered illegal, the illicit market started synthesizing new molecules that had only a different added group, so they were not actually illegal, such as NRG (mixture of cathinones, ethylone, methedrone, nafirone, flufedrone, 3-fluoromethcathinone, pentylone, buphedrone, ...).

Phenethylamine

It’s a stimulant similar to amphetamines, with mainly hallucinogenic effects. There are 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 is phenethylamine, structurally analogous to MDA (sold as Benzo Fury), also positive at immunoassays for amphetamine/MDMA. It’s used for dissociative anaesthetics.
• 5-MAPB (N-methyl-5(2-aminopropyl)benzofurane) is the N-methyl derivative of 5-APB, and it’s metabolized by CYP450 to 5-APB. It shows positivity at immunoassays for amphetamine/MDMA.

Dissociative anaesthetics

Effects include 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 include dizziness, state of confusion and psychomotor agitation, time distortion, aphasia, dynesthesia, and temporary cognitive alterations with memory loss, tachycardia, hypertension, stupor, ataxia, mydriasis. Induction of an abstinence syndrome characterized by mood changes and/or depressive thoughts.

Methoxetamine

It’s available on the internet as a powder since 2010, known as special M, MXE, M-ket. It acts as an NMDA receptor antagonist, similarly to other arylcyclohexylamines.

Synthetic cannabinoids

In 2006, herbal mixtures were 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. By the end of 2008, the 'Spice' phenomenon emerged in Europe, leading to the start of alert system monitoring. In December 2008, JWH-018 was 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 synthesized.

They started from the ‘90s with the discovery of cannabinoid receptors (CB1: CNS, peripheral neurons; CB2: cells of the immune system) and the identification of the endogenous ligand (anandamide), leading to the 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 overlap those of THC: tachycardia, altered perception and mood, dry mouth, reddening of the eyes. They affect the CNS and cardiovascular system, endocrine, respiratory, and immune systems. Psychotoxic effects described after the use of N-joy include 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.

The 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 the likelihood of experiencing anxiety.

University of Verona, Bacci Anna, Forensic Biotechnology 2023/2024

Synthetic opioids

Synthetic opioids are substances that are synthesized in laboratories (2012 and in particular 2016) and 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 their increasing potency and toxicity.

Morphine (natural opioid product) < Heroin (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 an antidote.

University of Verona, Bacci Anna, Forensic Biotechnology 2023/2024

Designer 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.

Italian 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 December 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 scheduled (in the list).

Analytical approach

• Untargeted screening by using HRMS (ESI-TOF MS)
• 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]
• 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 characteristics.