Development and validation of a color spot test method for the presumptive detection of 25-NBOMe compounds.

The great increase of new psychoactive substances over the past decade has substantially transformed the illicit drug industry to an ever-changing dynamic market. 25-NBOMe compounds are just one of these new substance groups that pose a public health risk in many countries around the world. These highly potent, hallucinogenic phenethylamines have previously been sold as "legal highs" or "synthetic LSD" and the necessity to rapidly identify their presence is crucial. While there are many laboratory-based analytical methods capable of identifying these compounds, the lack of presumptive test methods indicates the need for a specific and timely test that could be used in the field. Herein we outline the developed chemical spot test that can selectively identify the presence of 25-NBOMe compounds and related analogs through the reaction with a substituted benzoquinone reagent under basic conditions. This test method has been comprehensively validated showing a high level of selectivity, specificity, and precision with only two other illicit substances producing similar positive results as 25-NBOMe and few false-negative results seen. The working limit of detection was determined to be 225 µg and there was no cross-reactivity from potential adulterants of significance. This test has also been shown to work directly with blotter papers containing 25-NBOMe compounds, indicating no interference from this common matrix and the ability to differentiate these compounds from LSD. This method shows a high potential to be translated to a field compatible test that is simple, rapid, and selective for 25-NBOMe compounds.

Identification of Unique 4-Methylmethcathinone (4-MMC) Degradation Markers in Putrefied Matrices†.

Drug degradation as a consequence of putrefactive bacterial activity is a well-known factor that affects the identification and quantitation of certain substances of forensic interest. Current knowledge on putrefaction-mediated degradation of drugs is, however, significantly lacking. This study aimed to investigate the degradation of 4-methylmethcathinone (4-MMC or mephedrone) and to detect its degradation products in putrefied biological matrices containing 4-MMC. The bacteria species Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Proteus vulgaris were grown in brain-heart infusion broth, spiked with 4-MMC and incubated at 37°C for 24 h. Postmortem human blood and fresh porcine liver macerate were also left to putrefy in sample tubes at room temperature for 1 week. Structural elucidation was based on modern spectroscopic analyses including the use of high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. All four putrefactive bacteria were capable of degrading 4-MMC extensively under the experimental conditions explored. Of particular interest was the discovery of a novel degradation product common to all four bacterial species, which was assigned as 2-hydroxy-1-(4-methylphenyl)propan-1-one (HMP) based on the spectroscopic data. This degradation product was detectable in both postmortem human blood and porcine liver samples. The stability of the identified degradation products, especially HMP, should be further investigated to assess their validity of serving as marker analytes for monitoring 4-MMC in postmortem toxicology.

Synthetic Cathinones Induce Cell Death in Dopaminergic SH-SY5Y Cells via Stimulating Mitochondrial Dysfunction.

Increasing reports of neurological and psychiatric complications due to psychostimulant synthetic cathinones (SCs) have recently raised public concern. However, the precise mechanism of SC toxicity is unclear. This paucity of understanding highlights the need to investigate the in-vitro toxicity and mechanistic pathways of three SCs: butylone, pentylone, and 3,4-Methylenedioxypyrovalerone (MDPV). Human neuronal cells of SH-SY5Y were cultured in supplemented DMEM/F12 media and differentiated to a neuronal phenotype using retinoic acid (10 µM) and 12-O-tetradecanoylphorbol-13-acetate (81 nM). Trypan blue and lactate dehydrogenase assays were utilized to assess the neurotoxicity potential and potency of these three SCs. To investigate the underlying neurotoxicity mechanisms, measurements included markers of oxidative stress, mitochondrial bioenergetics, and intracellular calcium (Ca2+), and cell death pathways were evaluated at two doses (EC15 and EC40), for each drug tested. Following 24 h of treatment, all three SCs exhibited a dose-dependent neurotoxicity, characterized by a significant (p < 0.0001 vs. control) production of reactive oxygen species, decreased mitochondrial bioenergetics, and increased intracellular Ca2+ concentrations. The activation of caspases 3 and 7 implicated the orchestration of mitochondrial-mediated neurotoxicity mechanisms for these SCs. Identifying novel therapeutic agents to enhance an altered mitochondrial function may help in the treatment of acute-neurological complications arising from the illicit use of these SCs.

Color Tests for the Preliminary Identification of New Psychoactive Substances.

Color tests are a key tool for the rapid and simple identification of seized illicit drugs. This chapter outlines a series of color tests that can be used for the preliminary identification of new psychoactive substances such as cathinones, piperazines, tryptamines, and amphetamine-type stimulants.

Color Spot Test As a Presumptive Tool for the Rapid Detection of Synthetic Cathinones.

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and other border protection agencies globally. Color testing is a presumptive identification technique indicating the presence or absence of a particular drug class using rapid and uncomplicated chemical methods. Owing to their relatively recent emergence, a color test for the specific identification of synthetic cathinones is not currently available. In this study, we introduce a protocol for the presumptive identification of synthetic cathinones, employing three aqueous reagent solutions: copper(II) nitrate, 2,9-dimethyl-1,10-phenanthroline (neocuproine) and sodium acetate. Small pin-head sized amounts (approximately 0.1-0.2 mg) of the suspected drugs are added to the wells of a porcelain spot plate, and each reagent is then added dropwise sequentially before heating on a hotplate. A color change from very light blue to yellow-orange after 10 min indicates the likely presence of synthetic cathinones. The highly stable and specific test reagent has the potential for use in the presumptive screening of unknown samples for synthetic cathinones in a forensic laboratory. However, the nuisance of an added heating step for the color change result limits the test to laboratory application and decreases the likelihood of an easy translation to field testing.

A review of chemical 'spot' tests: A presumptive illicit drug identification technique.

Chemical 'spot' tests are a presumptive illicit drug identification technique commonly used by law enforcement, border security personnel, and forensic laboratories. The simplicity, low cost, and rapid results afforded by these tests make them particularly attractive for presumptive identification globally. In this paper, we review the development of these long-established methods and discuss color test recommendations and guidelines. A search of the scientific literature revealed the chemical reactions occurring in many color tests are either not actively investigated or reported as unknown. Today, color tests face many challenges, from the appearance of new psychoactive substances to concerns regarding selectivity, sensitivity, and safety. Advances in technology have seen color test reagents used in digital image color analysis, solid sensors, and microfluidic devices for illicit drug detection. This summarizes current research and suggests the future of presumptive color testing.

A study to model the post-mortem stability of 4-MMC, MDMA and BZP in putrefying remains.

There is currently limited data available on the stabilities of the three stimulants 4-methylmethcathinone (4-MMC), 3,4-methylenedioxymethamphetamine (MDMA) and N-benzylpiperazine (BZP) in a putrefying matrix. A Gas Chromatography Mass Spectrometry (GC-MS) method to determine the concentration of the three drugs in putrefying porcine liver over a three month period was developed and validated. Both 4-MMC and BZP were found to be unstable, becoming undetectable and having an average recovery of 52% respectively after one month at ambient room temperature (20°C). MDMA was found to be moderately stable, with an average recovery of 74% after three months at room temperature. This study indicated that the putrefaction process could have a significant impact on concentrations of 4-MMC and BZP in post-mortem cases involving putrefied remains.