The analysis and detection of new psychoactive substances in biological matrices

New psychoactive substances (NPSs) have appeared on the recreational drug market at an unprecedented rate in recent years. Many are not new drugs but failed products of the pharmaceutical industry. The speed and variety of drugs entering the market poses a new complex challenge for the forensic toxicology community. The detection of these substances in biological matrices can be difficult as the exact compounds of interest may not be known. Many NPS are sold under the same brand name and therefore users themselves may not know what substances they have ingested. The majority of analytical methods for the detection of NPSs tend to focus on a specific class of compounds rather than a wide variety. In response to this, a robust and sensitive method was developed for the analysis of various NPS by solid phase extraction (SPE) with gas chromatography mass spectrometry (GCMS). Sample preparation and derivatisation were optimised testing a range of SPE cartridges and derivatising agents, as well as derivatisation incubation time and temperature. The final gas chromatography mass spectrometry method was validated in accordance with SWGTOX 2013 guidelines over a wide concentration range for both blood and urine for 23 and 25 analytes respectively. This included the validation of 8 NBOMe compounds in blood and 10 NBOMe compounds in urine. This GC-MS method was then applied to 8 authentic samples with concentrations compared to those originally identified by NMS laboratories. The rapid influx of NPSs has resulted in the re-analysis of samples and thus, the stability of these substances is crucial information. The stability of mephedrone was investigated, examining the effect that storage temperatures and preservatives had on analyte stability daily for 1 week and then weekly for 10 weeks. Several laboratories identified NPSs use through the cross-reactivity of these substances with existing screening protocols such as ELISA. The application of Immunalysis ketamine, methamphetamine and amphetamine ELISA kits for the detection of NPS was evaluated. The aim of this work was to determine if any cross-reactivity from NPS substances was observed, and to determine whether these existing kits would identify NPS use within biological samples. The cross- reactivity of methoxetamine, 3-MeO-PCE and 3-MeO-PCP for different commercially point of care test (POCT) was also assessed for urine. One of the newest groups of compounds to appear on the NPS market is the NBOMe series. These drugs pose a serious threat to public health due to their high potency, with fatalities already reported in the literature. These compounds are falsely marketed as LSD which increases the chance of adverse effects due to the potency differences between these 2 substances. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was validated in accordance with SWGTOX 2013 guidelines for the detection for 25B, 25C and 25I-NBOMe in urine and hair. Long-Evans rats were administered 25B-, 25C- and 25I-NBOMe at doses ranging from 30-300 µg/kg over a period of 10 days. Tail flick tests were then carried out on the rats in order to determine whether any analgesic effects were observed as a result of dosing. Rats were also shaved prior to their first dose and reshaved after the 10-day period. Hair was separated by colour (black and white) and analysed using the validated LC-MS/MS method, assessing the impact hair colour has on the incorporation of these drugs. Urine was collected from the rats, analysed using the validated LC-MS/MS method and screened for potential metabolites using both LC-MS/MS and quadrupole time of flight (QToF) instrumentation

A review of the analytical techniques for the detection of anabolic–androgenic steroids within biological matrices

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

Application of a urine and hair validated LC-MS/MS method to determine the effect of hair colour on the incorporation of 25B-NBOMe, 25C-NBOMe and 25I-NBOMe into hair in the rat.

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 SWGTOX parameters for the detection of 25B-, 25C- and 25I-NBOMe using 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 -100 ng/mL and 0.025-2.5 ng/mg respectively. Urine LODs ranged from 5-25 pg/mL and had an LOQ of 50 pg/mL. Hair LOD and LOQs ranged from 3-5 pg/mg and 6.25-12.5 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 90-103% for urine samples and SPE recovery ranged from 80-107% for hair samples. Long-Evans rats (n=55) were administered 25B-, 25C- or 25I-NBOMe at doses ranging from 30-300 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 colour (black: n=55 and white: n=55) and analysed using the validated LC-MS/MS method to assess the impact hair colour 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

Detection of anabolic-androgenic steroids in ecigarettes seized from prisons:A case study

The administration of new psychoactive substances (NPS), in particular synthetic cannabinoid receptor agonists (SCRAs), via e-cigarettes, within prison settings has been well publicized. This study provides an overview of five e-cigarette case samples seized from Scottish prisons between May 2022 and July 2023 where the anabolic-androgenic steroids (AASs) mestanolone and oxandrolone were identified following gas chromatography-mass spectrometry (GC-MS) analysis. These e-cigarette samples represented 2.9% of all samples containing e-cigarette cartridges (n = 170) and 9.4% of all samples found to contain AASs (n = 53) seized during the same time period. The AASs were detected in combination with other drugs, including cocaine, Δ 9-tetrahydrocannabinol (Δ 9-THC), SCRAs and nicotine. This represents a new and novel route of administration for AASs.</p

Detection of anabolic-androgenic steroids in ecigarettes seized from prisons:A case study

The administration of new psychoactive substances (NPS), in particular synthetic cannabinoid receptor agonists (SCRAs), via e-cigarettes, within prison settings has been well publicized. This study provides an overview of five e-cigarette case samples seized from Scottish prisons between May 2022 and July 2023 where the anabolic-androgenic steroids (AASs) mestanolone and oxandrolone were identified following gas chromatography-mass spectrometry (GC-MS) analysis. These e-cigarette samples represented 2.9% of all samples containing e-cigarette cartridges (n = 170) and 9.4% of all samples found to contain AASs (n = 53) seized during the same time period. The AASs were detected in combination with other drugs, including cocaine, Δ 9-tetrahydrocannabinol (Δ 9-THC), SCRAs and nicotine. This represents a new and novel route of administration for AASs.</p

Log D7.4 and plasma protein binding of synthetic cannabinoid receptor agonists and a comparison of experimental and predicted lipophilicity

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 D7.4 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

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Extended Stability Evaluation of Selected Cathinones

Understanding the stability of drugs in a forensic toxicology setting is critical for the evaluation of drug concentrations. Synthetic cathinones are new psychoactive substances structurally derived from cathinone, the psychoactive component of Catha edulis (“khat”), a shrub that is indigenous to the Middle East and East Africa. Previous research has evaluated the stability of synthetic cathinones in biological matrices, including blood preserved with the combination of NaF and K2C2O4 used in gray-top tubes. However, it does not assess their stability in blood preserved with Na2EDTA, used for some clinical samples. Further, stability in unpreserved urine samples was only studied for two weeks. This research evaluates the stabilities of four Schedule I synthetic cathinones: mephedrone, MDPV (3,4-methylenedioxypyrovalerone), naphyrone, and α-PVP (alpha-pyrrolidinopentiophenone) at 20°C (room temperature), 4°C (refrigerator), and −20°C (freezer). Stability was assessed in methanolic and acetonitrile solutions, as well as in Na2EDTA-preserved blood and unpreserved urine. Solutions (1 mg/L) of each drug in each matrix stored in aliquots (100 μL, solvents; 1.2 mL, biological samples; n = 12) at each of the three temperatures for triplicate analysis on days 3, 7, 14, and 30. On day 0 of each study, three additional aliquots of each solution were analyzed. Biological samples underwent solid-phase extraction before analysis. All samples were analyzed in full-scan by gas chromatography-mass spectrometry (GC-MS). The results of this study show that under room temperature and refrigerator storage conditions, mephedrone, naphyrone, and MDPV will degrade in methanol. This degradation starts are early as day 3. Additionally, all four drugs will degrade in Na2EDTA-preserved human whole blood samples in at least one evaluated storage environment. However, when in acetonitrile-based working solutions and unpreserved urine samples, they proved to be more stable. Methanolic working solutions and samples of Na2EDTA-preserved blood containing these cathinones should be stored in the freezer and used or tested with urgency to ensure that quantitative sample analysis is as accurate as possible in forensic casework