Comparative study of the analysis of seized samples by GC-MS, 1H NMR and FT-IR spectroscopy within a Night-Time Economy (NTE) setting.

Rapid analysis of surrendered or seized drug samples provides important intelligence for health (e.g. treatment or harm reduction), and custodial services. Herein, three in-situ techniques, GC-MS, 1H NMR and FT-IR spectroscopy, with searchable libraries, are used to analyse 318 samples qualitatively, using technique specific library-based searches, obtained over the period 24th - 29th August 2019. 259 samples were identified as consisting of a single component, of which cocaine was the most prevalent (n = 158). Median match scores for all three techniques were ≥ 0.84 and showed agreement except for metformin (n = 1), oxandrolone (identified as vitamin K by IR (n = 4)), diazepam (identified as zolpidem by FT-IR (n = 2)) and 2-Br-4,5-DMPEA (n = 1), a structural isomer of 2C-B identified as a polymer of cellulose (cardboard) by FT-IR. 51 samples were found to consist of two or more components, of which 49 were adulterated cocaine samples (45 binary and 4 tertiary samples). GC-MS identified all components present in the 49 adulterated cocaine samples, whereas IR identified only cocaine in 88 % of cases (adulterant only = 12 %). The breakdown for 1H NMR spectroscopy was all components identified (51 %), cocaine only (33 %), adulterant only (10 %), cocaine and one adulterant (tertiary mixtures only, 6 %).

A Systematic Study of the In Vitro Pharmacokinetics and Estimated Human In Vivo Clearance of Indole and Indazole-3-Carboxamide Synthetic Cannabinoid Receptor Agonists Detected on the Illicit Drug Market.

In vitro pharmacokinetic studies were conducted on enantiomer pairs of twelve valinate or tert-leucinate indole and indazole-3-carboxamide synthetic cannabinoid receptor agonists (SCRAs) detected on the illicit drug market to investigate their physicochemical parameters and structure-metabolism relationships (SMRs). Experimentally derived Log D7.4 ranged from 2.81 (AB-FUBINACA) to 4.95 (MDMB-4en-PINACA) and all SCRAs tested were highly protein bound, ranging from 88.9 ± 0.49% ((R)-4F-MDMB-BINACA) to 99.5 ± 0.08% ((S)-MDMB-FUBINACA). Most tested SCRAs were cleared rapidly in vitro in pooled human liver microsomes (pHLM) and pooled cryopreserved human hepatocytes (pHHeps). Intrinsic clearance (CLint) ranged from 13.7 ± 4.06 ((R)-AB-FUBINACA) to 2944 ± 95.9 mL min-1 kg-1 ((S)-AMB-FUBINACA) in pHLM, and from 110 ± 34.5 ((S)-AB-FUBINACA) to 3216 ± 607 mL min-1 kg-1 ((S)-AMB-FUBINACA) in pHHeps. Predicted Human in vivo hepatic clearance (CLH) ranged from 0.34 ± 0.09 ((S)-AB-FUBINACA) to 17.79 ± 0.20 mL min-1 kg-1 ((S)-5F-AMB-PINACA) in pHLM and 1.39 ± 0.27 ((S)-MDMB-FUBINACA) to 18.25 ± 0.12 mL min-1 kg-1 ((S)-5F-AMB-PINACA) in pHHeps. Valinate and tert-leucinate indole and indazole-3-carboxamide SCRAs are often rapidly metabolised in vitro but are highly protein bound in vivo and therefore predicted in vivo CLH is much slower than CLint. This is likely to give rise to longer detection windows of these substances and their metabolites in urine, possibly as a result of accumulation of parent drug in lipid-rich tissues, with redistribution into the circulatory system and subsequent metabolism.

Shape matters: The application of activity-based in vitro bioassays and chiral profiling to the pharmacological evaluation of synthetic cannabinoid receptor agonists in drug-infused papers seized in prisons.

Synthetic cannabinoid receptor agonists (SCRAs) elicit many of their psychoactive effects via type-1 human cannabinoid (CB1 ) receptors. Enantiomer pairs of eight tert-leucinate or valinate indole- and indazole-3-carboxamide SCRAs were synthesized and their CB1 potency and efficacy assessed using an in vitro ß-arrestin recruitment assay in a HEK239T stable cell system. A chiral high-performance liquid chromatography method with photodiode array and/or quadrupole time-of-flight-mass spectrometry detection (HPLC-PDA and HPLC-PDA-QToF-MS) was applied to 177 SCRA-infused paper samples seized in Scottish prisons between 2018 and 2020. In most samples, SCRAs were almost enantiopure (S)-enantiomer (>98% of total chromatographic peak area), although in some (n = 18), 2% to 16% of the (R)-enantiomer was detected. (S)-enantiomers are consistently more potent than (R)-enantiomers and often more efficacious. The importance of SCRA-CB1 receptor interactions in the "head" or "linked group" moiety is demonstrated, with the conformation of the "bulky" tert-leucinate group greatly affecting potency (by up to a factor of 374), significantly greater than the difference observed between valinate SCRA enantiomers. (S)-MDMB-4en-PINACA, (S)-4F-MDMB-BINACA, and (S)-5F-MDMB-PICA are currently the most prevalent SCRAs in Scottish prisons, and all have similar high potency (EC50 , 1-5 nM) and efficacy. Infused paper samples were compared using estimated intrinsic efficacy at the CB1 receptor (EIECB1 ) to evaluate samples with variable SCRA content. Given their similar potency and efficacy, any variation in CB1 receptor-mediated psychoactive effects are likely to derive from variation in dose, mode of use, pharmacokinetic differences, and individual factors affecting the user, rather than differences in the specific SCRA present.

Activation of µ-opioid receptors by MT-45 (1-cyclohexyl-4-(1,2-diphenylethyl)piperazine) and its fluorinated derivatives.

BACKGROUND AND PURPOSE: A fluorinated derivative (2F-MT-45) of the synthetic µ-opioid receptor agonist MT-45 (1-cyclohexyl-4-(1,2-diphenylethyl)piperazine) was recently identified in a seized illicit tablet. While MT-45 is a Class A drug, banned in a number of countries, nothing is known about the pharmacology of 2F-MT-45. This study compares the pharmacology of MT-45, its fluorinated derivatives and two of its metabolites. EXPERIMENTAL APPROACH: We used a ß-arrestin2 recruitment assay in CHO cells stably expressing µ receptors to quantify the apparent potencies and efficacies of known (MT-45, morphine, fentanyl and DAMGO) and potential agonists. In addition, the GloSensor protein was transiently expressed to quantify changes in cAMP levels. We measured Ca2+ to investigate whether MT-45 and its metabolites have effects on GluN1/N2A NMDA receptors stably expressed in Ltk- cells. KEY RESULTS: The fluorinated MT-45 derivatives have higher apparent potencies (2F-MT-45: 42 nM) than MT-45 (1.3 µM) for inhibition of cAMP accumulation and ß-arrestin2 recruitment (2F-MT-45: 196 nM; MT-45: 23.1 µM). While MT-45 and 2F-MT-45 are poor recruiters of ß-arrestin2, they have similar efficacies for reducing cAMP levels as DAMGO. Two MT-45 metabolites displayed negligible potencies as µ receptor agonists, but one, 1,2-diphenylethylpiperazine, inhibited the NMDA receptor with an IC50 of 29 µM. CONCLUSION AND IMPLICATIONS: Fluorinated derivatives of MT-45 are potent µ receptor agonists and this may pose a danger to illicit opioid users. Inhibition of NMDA receptors by a metabolite of MT-45 may contribute to the reported dissociative effects.

Hitting the Jackpot - development of gas chromatography-mass spectrometry (GC-MS) and other rapid screening methods for the analysis of 18 fentanyl-derived synthetic opioids.

In recent years, the occurrence of synthetic opioid fentanyl and its derivatives has grown significantly in forensic casework. This study presents the synthesis and analysis of 18 fentalogs, selected based on information received from local law enforcement. This study provides colorimetric tests, thin-layer chromatography (TLC) which can potentially be utilized for presumptive screening of the target compounds, as bulk powders or as trace-level adulterants. The fully validated confirmatory GC-MS method (employing SIM mode) allows the identification of the 18 derivatives, five commonly encountered controlled substances and four adulterants, within 20 minutes. The cross-validated method described herein provides a sensitive screening and quantitation method for the illicit (and potentially harmful) components at trace levels (LOD = 0.007-0.822 µg/mL and LOQ = 0.023-2.742 µg/mL respectively). Spectral data [1 H-NMR, 13 C-NMR, 19 F-NMR, FT-IR, and HRMS] and assignments for the synthesized reference materials are also provided in the Supplementary Information for laboratories engaged in the routine analysis of fentanyl and its derivatives.

Detection and quantitation of synthetic cannabinoid receptor agonists in infused papers from prisons in a constantly evolving illicit market.

Drug misuse in prisons contributes to increased disruption and violence and negatively impacts prisoner safety, rehabilitation, and recovery. Synthetic cannabinoid receptor agonists (SCRAs), colloquially known as "spice", are infused into papers and are of particular concern in a prison setting where they are commonly vaped. Methods for the qualitative and quantitative analysis of SCRA infused papers, including impurity profiling, were developed using gas chromatography-mass spectrometry (GC-MS) with qualitative confirmation by ultra high pressure liquid chromatography with photodiode array and quadrupole time of flight mass spectrometry detection (UPLC-PDA-QToF-MS) and applied to 354 individual seized paper samples originating from 168 seizures from three Scottish prisons. Of these samples, 41% (146 samples from 101 seizures) contained at least one SCRA and multiple SCRAs were detected on 23% of these papers. Concentrations ranged from < 0.05-1.17 mg/cm2 paper, representing the first reported quantitative data for SCRA infused papers. An evolution in the SCRAs detected was demonstrated; 5F-MDMB-PINACA (5F-ADB) predominated until late 2018, after which time 5F-MDMB-PICA and 4F-MDMB-BINACA became increasingly more prevalent, followed by the arrival of MDMB-4en-PINACA in June 2019. Concentration mapping data from two seized paper samples demonstrated that SCRA concentrations across larger papers were highly variable (0.47-2.38 mg/cm2 paper) making consistent dosing by users, and representative sampling by laboratory analysts, difficult. Near real-time qualitative and quantitative information on SCRAs circulating in prisons acts as an early warning system for SCRAs emerging on the wider illicit market, inform the methods used to detect them and limit supply, and provide information to support harm reduction measures.

Hyperpolarization of Pyridyl Fentalogues by Signal Amplification By Reversible Exchange (SABRE).

Fentanyl, also known as 'jackpot', is a synthetic opiate that is 50-100 times more potent than morphine. Clandestine laboratories produce analogues of fentanyl, known as fentalogues to circumvent legislation regarding its production. Three pyridyl fentalogues were synthesized and then hyperpolarized by signal amplification by reversible exchange (SABRE) to appraise the forensic potential of the technique. A maximum enhancement of -168-fold at 1.4 T was recorded for the ortho pyridyl 1H nuclei. Studies of the activation parameters for the three fentalogues revealed that the ratio of ligand loss trans to hydride and hydride loss in the complex [Ir(IMes)(L)3(H)2]+ (IMes=1,3-bis(2,4,6-trimethylphenyl)imidazole-2-ylidene) ranged from 0.52 to 1.83. The fentalogue possessing the ratio closest to unity produced the largest enhancement subsequent to performing SABRE at earth's magnetic field. It was possible to hyperpolarize a pyridyl fentalogue selectively from a matrix that consisted largely of heroin (97 : 3 heroin:fentalogue) to validate the use of SABRE as a forensic tool.

Rapid Identification of Novel Psychoactive and Other Controlled Substances Using Low-Field 1H NMR Spectroscopy.

An automated approach to the collection of 1H NMR (nuclear magnetic resonance) spectra using a benchtop NMR spectrometer and the subsequent analysis, processing, and elucidation of components present in seized drug samples are reported. An algorithm is developed to compare spectral data to a reference library of over 300 1H NMR spectra, ranking matches by a correlation-based score. A threshold for identification was set at 0.838, below which identification of the component present was deemed unreliable. Using this system, 432 samples were surveyed and validated against contemporaneously acquired GC-MS (gas chromatography-mass spectrometry) data. Following removal of samples which possessed no peaks in the GC-MS trace or in both the 1H NMR spectrum and GC-MS trace, the remaining 416 samples matched in 93% of cases. Thirteen of these samples were binary mixtures. A partial match (one component not identified) was obtained for 6% of samples surveyed whilst only 1% of samples did not match at all.

Enantiospecific Synthesis, Chiral Separation, and Biological Activity of Four Indazole-3-Carboxamide-Type Synthetic Cannabinoid Receptor Agonists and Their Detection in Seized Drug Samples.

Synthetic cannabinoid receptor agonists (SCRAs) have been the largest group of illicit psychoactive substances reported to international monitoring and early warning systems for many years. Carboxamide-type SCRAs are amongst the most prevalent and potent. Enantiospecific synthesis and characterization of four indazole-3-carboxamides, AMB-FUBINACA, AB-FUBINACA, 5F-MDMB-PINACA (5F-ADB), and AB-CHMINACA is reported. The interactions of the compounds with CB1 and CB2 receptors were investigated using a G-protein coupled receptor (GPCR) activation assay based on functional complementation of a split NanoLuc luciferase and EC50 (a measure of potency) and Emax (a measure of efficacy) values determined. All compounds demonstrated higher potency at the CB2 receptor than at the CB1 receptor and (S)-enantiomers had an enhanced potency to both receptors over the (R)-enantiomers. The relative potency of the enantiomers to the CB2 receptor is affected by structural features. The difference was more pronounced for compounds with an amine moiety (AB-FUBINACA and AB-CHMINACA) than those with an ester moiety (AMB-FUBINACA and 5F-MDMB-PINACA). An HPLC method was developed to determine the prevalence of (R)-enantiomers in seized samples. Lux® Amylose-1 [Amylose tris(3,5-dimethylphenylcarbamate)] has the greatest selectivity for the SCRAs with a terminal methyl ester moiety and a Lux® i-Cellulose-5 column for SCRAs with a terminal amide moiety. Optimized isocratic separation methods yielded enantiomer resolution values (Rs) ≥ 1.99. Achiral GC-MS analysis of seized herbal materials (n = 16), found 5F-MDMB-PINACA (<1.0-91.5 mg/g herbal material) and AMB-FUBINACA (15.5-58.5 mg/g herbal material), respectively. EMB-FUBINACA, AMB-CHMICA, 5F-ADB-PINACA isomer 2, and ADB-CHMINACA were also tentatively identified. Analysis using chiral chromatography coupled to photodiode array and quadrupole time of flight mass spectrometry (chiral HPLC-PDA-QToF-MS/MS) confirmed that the (S)-enantiomer predominated in all samples (93.6-99.3% (S)-enantiomer). Small but significant differences in synthesis precursor enantiopurity may provide significant differences between synthesis batches or suppliers and warrants further study. A method to compare potency between samples containing different SCRAs at varying concentrations was developed and applied in this small preliminary study. A 10-fold difference in the "intrinsic" potency of samples in the study was noted. With the known heterogeneity of SCRA infused materials, the approach provides a simplified method for assessing and communicating the risk of their use.

Chemical synthesis, characterisation and in vitro and in vivo metabolism of the synthetic opioid MT-45 and its newly identified fluorinated analogue 2F-MT-45 with metabolite confirmation in urine samples from known drug users.

PURPOSE: The detection of a novel psychoactive substance, 2F-MT-45, a fluorinated analogue of the synthetic opioid MT-45, was reported in a single seized tablet. MT-45, 2F-, 3F- and 4F-MT-45 were synthesised and reference analytical data were reported. The in vitro and in vivo metabolisms of MT-45 and 2F-MT-45 were investigated. METHOD: The reference standards and seized sample were characterised using nuclear magnetic resonance spectroscopy, ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry, gas chromatography-mass spectrometry, attenuated total reflectance-Fourier transform infrared spectroscopy and Raman spectroscopy. Presumptive tests were performed and physicochemical properties of the compounds determined. Metabolite identification studies using human liver microsomes, human hepatocytes, mouse hepatocytes and in vivo testing using mice were performed and identified MT-45 metabolites were confirmed in authentic human urine samples. RESULTS: Metabolic pathways identified for MT-45 and 2F-MT-45 were N-dealkylation, hydroxylation and subsequent glucuronidation. The major MT-45 metabolites identified in human in vitro studies and in authenticated human urine were phase I metabolites and should be incorporated as analytical targets to existing toxicological screening methods. Phase II glucuronidated metabolites were present in much lower proportions. CONCLUSIONS: 2F-MT-45 has been detected in a seized tablet for the first time. The metabolite identification data provide useful urinary metabolite targets for forensic and clinical testing for MT-45 and allows screening of urine for 2F-MT-45 and its major metabolites to determine its prevalence in case work.