New challenges and innovation in forensic toxicology: Focus on the “New Psychoactive Substances”

Abstract

In the recent years, new molecules have appeared in the illicit market, claimed to contain “non-illegal” compounds, although exhibiting important psychoactive effects; this heterogeneous and rapidly evolving class of compounds are commonly known as “New Psychoactive Substances” or, less properly, “Smart Drugs” and are easily distributed through the e-commerce or in the so-called “Smart Shops”. They include, among other, synthetic cannabinoids, cathinones and tryptamine analogs of psylocin. Whereas cases of intoxication and death have been reported, the phenomenon appears to be largely underestimated and is a matter of concern for Public Health. One of the major points of concern depends on the substantial ineffectiveness of the current methods of toxicological screening of biological samples to identify the new compounds entering the market. These limitations emphasize an urgent need to increase the screening capabilities of the toxicology laboratories, and to develop rapid, versatile yet specific assays able to identify new molecules. The most recent advances in mass spectrometry technology, introducing instruments capable of detecting hundreds of compounds at nanomolar concentrations, are expected to give a fundamental contribution to broaden the diagnostic spectrum of the toxicological screening to include not only all these continuously changing molecules but also their metabolites. In the present paper a critical overview of the opportunities, strengths and limitations of some of the newest analytical approaches is provided, with a particular attention to liquid phase separation techniques coupled to high accuracy, high resolution mass spectrometry.

Introduction

“A spectre is haunting Europe – the spectre of Communism. All the powers of old Europe have entered into a holy alliance to exorcise this spectre …” (Manifesto of the Communist Party, 1848).

With these inspired words, Karl Marx (1818–1883) described the spreading of the communist faith in the society of the 19th century and the related concerns of all the European powers.

In the modern times, different specters haunt Europe, among which some are patent, such as the economical crisis, the increasing unemployment, and the global warming, whereas other specters, fairly unapparent but not less dangerous, do exist, e.g., loneliness, depression, unhappiness, suicides, deaths caused by occupational and traffic incidents… Among these subtle dangers, the hidden penetration inside the western societies of new psychotropic compounds is not the least in importance.

Indeed, in the recent years, inside the European Union, and in the USA, new psychoactive molecules have appeared, often under “innocent” appearances (house scents, bath salts, incenses, etc.), finding a wide and efficient distribution through the “e-commerce” or specialized shops. These products are claimed to contain only natural “non-illegal” compounds and consequently have no limitations in their commercial distribution, although exhibiting important psychoactive effects. Hence, this heterogeneous class of products is also named “Smart Drugs”, or more properly “New Psychoactive Substances” (NPSS), and the shops where they are available are known as “Smart Shops”.

The vast class of NPSs includes primarily synthetic cannabinoids and synthetic cathinones. The first group refers to a number of synthetic molecules developed as potential drugs acting on the THC receptors CB1 and CB2, originally designed with a therapeutical intent [1], [2], [3], [4]. In Fig. 1 are reported the first synthetic THC analogs, HU-210 and CP-47,497, and WIN 55,212-2, the precursor of aminoalkyl-indole cannabimimetics acting on both CB1 and CB2. When John W. Huffman, at Clemson University, determined that a simple alkyl chain could replace the aminoalkyl group, the investigation of hundreds of related “JWH compounds”, characterized by their binding affinities for CB receptors, ensued. JWH 018 (see Fig. 2) was the first synthetic cannabinoid identified in herbal blends [1], [5], followed by JWH-019 [6], JWH-122 [7], JWH-398 [8], HU-210 [9], CP47,497 [10], JWH-073 [10], JWH-398 [8], JWH-200 [11] and JWH-250 [8] as the most “popular”, although many other have been reported. Thereafter, JWH compounds have been classified into 5 groups with the aim of developing generic legislation to control all synthetic CBs. The Group 1 naphthoylindoles is typified by JWH 018 and includes 73 other compounds. The related Group 2 naphthylmethylindoles contains 9 compounds. Several of the 32 known naphthoylpyrroles (Group 3) are potent CB receptor agonists (e.g., JWH 147) and therefore have a high abuse potential. The Group 4 napthylmethylindenes has 3 members, like JWH 176. Finally, the Group 5 phenylacetylindoles covers 28 synthetic CBs, like JWH 203, some of which have been detected in blends. In order to circumvent the legislation controlling the drugs of abuse they are usually added to “incenses” or “house scents”, often with the warning “not for human consumption”. However, in reality they must be smoked in order to produce strong THC-like effects. In Fig. 3 are reported molecular structures of non-JWH-like compounds, also exhibiting selective activity at either receptor CB1 or CB2.

Other typical NPSs are synthetic cathinones, derivatives of a naturally occurring amphetamine-like molecule (Cathinone) present in the well known psychoactive plant Catha edulis. This group of compounds includes buthylone, dimethylcathinone, etcathinone, ethylone, mephedrone, methylone, pyrovalerone, methylenedioxypyrovalerone (MDPV), etc. which are most often sold under the “innocent” form of bath salts [12], [13].

Moreover, tryptamine analogs of the hallucinogenic psylocin and phenylpiperazines have also been synthesized and released into the market [14].

Also, several herbal products can be included in the NPSs, particularly those traditionally used in other cultures for their psychotropic effects, whose active principles, even if known, are not well characterized, not available as pure standard and not scheduled as illicit drugs. Among these herbal products, just to mention a few, Areca catechu, Ipomoea violacea, Mitragyna speciosa, Hawaiian baby woodrose and Voacanga Africana [15], [16].

Eventually, a few “energetic” dietary supplements labeled as “totally natural without side effects” and available in herbalist's shops, were reported to contain sildenafil (the active principle of Viagra) or its unregistered analogs [17], [18].

Because of their innocent appearance and the lack of any warnings on possible side effects the above-mentioned products look particularly attractive for the young generations. However it is widely reported that the use of NPSs may cause dissociate mental states and even psychedelic sensations. Some may also induce amphetamine-like effects on both perception of fatigue and mood, as well as enhancement of the “sexual performance.”

In the recent years, numerous cases of acute intoxications and a few deaths associated with NPS intake have been reported [19], [20], [21], [22].

However, at the present state of the art there are few analytical methods for NPSs and their metabolites in biological samples, and the great majority of intoxications were reported on the mere basis of anamnestic and clinical data, that often suffer of low diagnostic sensitivity and specificity. For the same reasons, the potential association between NPSs use and traffic or occupational accidents is so far totally unknown.

Thus, the diffusion of the NPSs at international level has become a problem of the highest concern, also because these psychoactive substances, although on the basis of a still limited body of literature, might induce tolerance and dependence [23], [24], [25], [26]. This international concern led to the creation of the “Early-warning System” (EWS) in the European Union. The main task of the EWS is to spread information on the New Psychoactive Substances to the countries included in this network, particularly concerning those compounds that can cause social and public health problems. Another important task of EWS is to activate a risk evaluation process in order to control the identified compounds at European level [27]. Updated information on the penetration of NPSs in the EU can be found also in the most recent annual reports of the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) [20].

On this basis, the diffusion of the use of NPSs in the population may affect the traditional approaches adopted by forensic toxicology to monitor the drug abuse in the population.

The aim of the present contribution is not to present a review of the rapidly increasing analytical methods for the determination of the NPSs in illicit preparations and in body fluids, which can be the subject of specific, more documented and detailed papers, but just to discuss critically how the modern analytical toxicology may face this new and multiform phenomenon, which at present is silently expanding all over the “developed countries”, still in the absence of efficient tools for its monitoring, control and contrast.