New psychoactive substances (NPSs) have become an integral part of the recreational drug market with "new" compounds being reported by the European Monitoring Centre for Drugs and Drug Addiction weekly. Due to the changing nature of NPSs, it is impractical to carry out single analyte or even simple class quantitation. Although several gas chromatography-mass spectrometry (GC-MS) methods have been developed these are typically class specific. We present a validated GC-MS method for the quantitation of 2-DPMP, 3-MeO-PCE, 3-MeO-PCP, 5-APB, 6-APB, benzedrone, butylone, ethylone, flephedrone, methiopropamine, MDPV, mephedrone, methoxetamine, methylone, naphyrone, 25B-NBOME, 25C-NBOME, 25D-NBOMe, 25E-NBOME, 25H-NBOME, 25I-NBOME, Mescaline-NBOME and 25P-NBOME in blood and urine samples. Sample preparation was carried out using solid-phase extraction followed by derivatisation and analysis by GC-MS. Parameters investigated for validation included bias, precision, linear calibration model, carryover, interferences, limit of detection, limit of quantification, and autosampler and freeze/thaw stability. All drugs yielded successful results for each of these parameters as per SWGTOX guidelines. The GC-MS method was used for the reanalysis of 12 blood samples (eight cases) where 25I-NBOMe, 25C-NBOMe, methoxetamine and methylone had previously been detected by NMS laboratories. This GC-MS method was able to quantitatively detect these drugs in 75% of the blood samples, 42% of which contained either 25C-NBOMe or 25I-NBOMe. This method accurately allows for the simultaneous quantification of a wide variety of compounds via GC-MS, in particular NBOMe compounds which are typically analysed by liquid chromatography-tandem mass spectrometry which is not available in all laboratories.

Knowledge of the stability of analytes in solvents and biological matrices is of high importance in the field of forensic toxicology. This is particularly true where quantitative analysis is undertaken; degradation of analytes will result in under‐estimation of concentrations, whereas the production of analytes/metabolites will lead to over‐estimation. Although stability is included as part of method validation, this typically focuses on processed sample stability, and the impact of freeze/thaw cycles upon analytes. Although beneficial for method evaluation, this does not assist laboratory analysts with the short‐ and long‐term storage of samples prior to this step and does not consider the impact the biological matrix may have on the degradation or production of the analyte in question. The timeframe for these studies is also relatively short (typically 72 h), which does not cover the time frame between sample receivership and analysis for many forensic cases. This review collects previously published work on long‐term stability studies, grouping compounds into their associated drug classes and matrices. Research shows that the majority of compounds are more stable at lower storage temperatures, and that analysis should be completed as quickly as possible. It is advised that analyte stability be considered prior to any interpretation of concentrations in a forensic setting.This article is categorized under: Toxicology > Analytical Toxicology > Drug Analysis Toxicology > Drug Stability

Synthetic cannabinoid receptor agonists (SCRAs) remain a major public health concern, with their use implicated in intoxications and drug-related deaths worldwide. Increasing our systematic understanding of SCRA metabolism supports clinical and forensic toxicology casework, facilitating the timely identification of analytical targets for toxicological screening procedures and confirmatory analysis. This is particularly important as new SCRAs continue to emerge on the illicit drug market. In this work, the metabolism of ADB-HEXINACA (ADB-HINACA, N-[1-amino-3,3-dimethyl-1-oxobutan-2-yl]-1-hexyl-1H-indazole-3-carboxamide), which has increased in prevalence in the United Kingdom and other jurisdictions, was investigated using in vitro techniques. The (S)-enantiomer of ADB-HEXINACA was incubated with pooled human hepatocytes over 3 hours to identify unique and abundant metabolites using liquid chromatography–quadrupole time-of-flight mass spectrometry. In total, 16 metabolites were identified, resulting from mono-hydroxylation, di-hydroxylation, ketone formation (mono-hydroxylation then dehydrogenation), carboxylic acid formation, terminal amide hydrolysis, dihydrodiol formation, glucuronidation and combinations thereof. The majority of metabolism took place on the hexyl tail, forming ketone and mono-hydroxylated products. The major metabolite was the 5-oxo-hexyl product (M9), while the most significant mono-hydroxylation product was the 4-hydroxy-hexyl product (M8), both of which were confirmed by comparison to in-house synthesized reference standards. The 5-hydroxy-hexyl (M6) and 6-hydroxy-hexyl (M7) metabolites were not chromatographically resolved, and the 5-hydroxy-hexyl product was the second largest mono-hydroxylated metabolite. The structures of the terminal amide hydrolysis products without (M16, third largest metabolite) and with the 5-positioned ketone (M13) were also confirmed by comparison to synthesized reference standards, along with the 4-oxo-hexyl metabolite (M11). The 5-oxo-hexyl and 4-hydroxy-hexyl metabolites are suggested as biomarkers for ADB-HEXINACA consumption.

Anabolic–androgenic steroids (AASs) and other image and performance enhancing drugs (IPEDs) are controlled by governments and sport institutions such as the World Anti‐doping Agency (WADA). Although elite athletes and professional bodybuilders are the most visible AAS abusers, the introduction of the internet has increased the accessibility of AASs, with use being observed among recreational gym goers at increasing prevalence. Despite reported increase in use, routine analysis for these substances is uncommon, with many forensic laboratories opting to outsource AAS analysis. This review collates information regarding the extraction and analysis of AASs from various biological matrices with the considered purpose of providing a reference for the development of AAS methods to allow for routine detection by forensic laboratories.This article is categorized under: Toxicology > in Sport Toxicology > Analytical

NBOMes are a group of new psychoactive substances derived from phenethylamines. Recreational abuse is thought to have begun in 2010 and they are commonly associated with the “club drug” scene. They are administered in liquid form or as blotters due to their high potency. An LC–MS-MS method was validated using Scientific Working Group for Forensic Toxicology parameters for the detection of 25B-, 25C- and 4-iodo-2,5-dimethoxy-N-[(2-methoxyphenyl)methyl]-benzeneethanamine (25I-NBOMe) using 4-bromo-2,5-dimethoxy-N-[(2-methoxyphenyl)methyl]-benzeneethanamine (25B-NBOMe)-D₃ as internal standard for urine and hair. Calibration graphs with R² values >0.99 were observed for urine and hair for concentrations ranging from 0.1 to 100 ng/mL and 0.025 to 2.5 ng/mg, respectively. Urine LODs ranged from 5 to 25 pg/mL and had an LOQ of 50 pg/mL. Hair LOD and LOQs ranged from 3 to 5 pg/mg and 6.25 to 12.5 pg/mg, respectively. Intra- and inter-day precision was <20% and accuracy was within ±20% for both matrices. The method was shown to be selective for both exogenous and endogenous compounds. No matrix effects were observed for either matrix. LLE recovery ranged from 90 to 103% for urine samples and solid phase extraction recovery ranged from 80 to 107% for hair samples. Long–Evans rats (n = 55) were administered 25B-, 25C- or 25I-NBOMe at doses ranging from 30 to 300 μg/kg over a period of 10 days. Rats were shaved prior to their first dose and re-shaved after the 10-day period. Hair was separated by color (black: n = 55 and white: n = 55) and analyzed using the validated LC–MS-MS method to assess the impact hair color has on the incorporation of these drugs. All drugs were successfully detected in black hair. 25B-NBOMe from rats receiving the highest dose and 25C-NBOMe from rats receiving the medium and high doses were quantified in white hair. 25I-NBOMe was detected but fell below the limit of quantification. A dose-dependent concentration increase was observed in the black hair. All pooled urine samples tested positive for their expected NBOMes.

The emergence of new synthetic cannabinoid receptor agonists (SCRAs) onto the illicit drugs market continues to cause harm, and the overall availability of physicochemical and pharmacokinetic data for new psychoactive substances is lacking. The lipophilicity of 23 SCRAs and the plasma protein binding (PPB) of 11 SCRAs was determined. Lipophilicity was determined using a validated chromatographic hydrophobicity index (CHI) log D method; tested SCRAs showed moderate to high lipophilicity, with experimental log D_7.4 ranging from 2.48 (AB‐FUBINACA) to 4.95 (4F‐ABUTINACA). These results were also compared to in silico predictions generated using seven commercially available software packages and online tools (Canvas; ChemDraw; Gastroplus; MoKa; PreADMET; SwissADME; and XlogP). Licenced, dedicated software packages provided more accurate lipophilicity predictions than those which were free or had prediction as a secondary function; however, the latter still provided competitive estimates in most cases. PPB of tested SCRAs, as determined by equilibrium dialysis, was in the upper range of the lipophilicity scale, ranging from 90.8% (ADB‐BUTINACA) to 99.9% (BZO‐HEXOXIZID). The high PPB of these drugs may contribute to reduced rate of clearance and extended durations of pharmacological effects compared to lesser‐bound SCRAs. The presented data improve understanding of the behaviour of these drugs in the body. Ultimately, similar data and predictions may be used in the prediction of the structure and properties of drugs yet to emerge on the illicit market.