Microbial biotransformation of five pyrrolidinophenone‐type psychoactive substances in wastewater and a wastewater isolated Pseudomonas putida strain

Wastewater‐based epidemiology (WBE) employs the analysis of wastewater to detect and quantify drug use and discharge within a community. In this work, transformation products (TP) by microbes in the environment were identified after incubations in wastewater and an isolated microbial strain. The microbial strain was isolated from an enrichment culture of wastewater supplemented with 3,4‐methylenedioxy‐pyrovalerone, and identified by matrix assisted laser desorption – time of flight mass spectrometry as Pseudomonas putida (P. putida ). Five pyrrolidinophenone‐type psychoactive substances (PPPS) were then incubated in wastewater and in P. putida tryptic soy broth (TSB) growth cultures. TPs were identified using liquid chromatography coupled to mass spectrometry techniques. All TPs observed in P. putida TSB growth cultures were also identified in wastewater incubations. The main TP for all PPPSs in P. putida TSB growth cultures, and two PPPSs incubated in wastewater, were the N ‐desalkyl‐carboxy‐TPs. The study showed P. putida TSB growth cultures used for identification of TPs in wastewater, represent parts of the microbial community. With data provided in this type of experiments more information will be available to select targets for monitoring drug use by WBE. Copyright © 2017 John Wiley & Sons, Ltd.     

Metabolism studies of 4′Cl-CUMYL-PINACA, 4′F-CUMYL-5F-PINACA and 4′F-CUMYL-5F-PICA using human hepatocytes and LC-QTOF-MS analysis

4′Cl-cumyl-PINACA (SGT-157), 4′F-cumyl-5F-PINACA (4F-cumyl-5F-PINACA, SGT-65) and 4′F-cumyl-5F-PICA (4F-cumyl-5F-PICA, SGT-64) are a series of new halogenated cumyl synthetic cannabinoid receptor agonists (SCRAs). Due to rapid metabolism, monitoring and screening for SCRAs in biological matrices requires identification of their metabolites. It is an essential tool for estimating their spread and fluctuations in the global illicit market. The purpose of this study was to identify human biotransformations of 4′Cl-cumyl-PINACA, 4′F-cumyl-5F-PINACA and 4′F-cumyl-5F-PICA in vitro and characterize for the first time the metabolic pathways of halogenated cumyl SCRAs. 4′Cl-cumyl-PINACA, 4′F-cumyl-5F-PINACA and 4′F-cumyl-5F-PICA were incubated with human hepatocytes in duplicates for 0, 1, 3 and 5 h. The supernatants were analysed in data-dependent acquisition on a UHPLC-QToF-MS, and the potential metabolites were tentatively identified. A total of 11 metabolites were detected for 4′Cl-cumyl-PINACA, 21 for 4′F-cumyl-5F-PINACA and 10 for 4′F-cumyl-5F-PICA. The main biotransformations were oxidative defluorination, followed by hydroxylation with dehydrogenation, N-dealkylation, dihydrodiol formation and glucuronidation. Hydroxylations were most common at the tail moieties with higher abundancy for indole than indazole compounds. N-dealkylations were more common for fluorinated tail chain compounds than the non-fluorinated 4′Cl-cumyl-PINACA. In conclusion, many metabolites retained halogen groups at the cumyl moieties which, in various combinations, may be suitable as analytical biomarkers.     

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.     

Pharmacology,prevalence in Germany,and analytical data of cyclobutylmethyl- and norbornylmethyl-type synthetic cannabinoid receptor agonists

Synthetic cannabinoid receptor agonists (SCRAs) are distributed on the drug market to produce THC-like effects while evading routine drug testing and legislation. The cyclobutylmethyl (CBM) and norbornylmethyl (NBM) side chain specifically circumvented the German legislation and led to the emergence of exploratory SCRAs in 2019–2021. The NBM SCRAs were detected post-amendment of the new psychoactive substances act in 2020, which scheduled all CBM SCRAs. All six SCRAs are full agonists at the cannabinoid receptor 1 compared with Δ⁹-tetrahydrocannabinol and CP-55,940. The CBM SCRAs showed binding affinities of K_i: 29.4–0.65 nm and potencies of EC₅₀: 483–40.1 nm (CBMICA << CBMINACA < CBMeGaClone). The norbornyl derivatives exhibited high affinities (K_i: 1.87–0.25 nm ), with indazole being the most affine. Functional activity data confirmed that the indazole derivative tends to be the most potent of all three NBM SCRAs (EC₅₀: 169–1.78 nm ). The sterically demanding NBM side chain increased the affinity and activity of almost all core structures. Future studies should be conducted on similarly voluminous side chain moieties. The ‘life cycle’ of all SCRAs on the drug market was less than a year. Notably, Cumyl-CBMICA was the most prevalent while also having the weakest cannabimimetic properties. Quantification of Cumyl-CBMICA during peak consumption in late 2019 and early 2020 revealed an increase in the concentration on the herbal material, which, together with forum entries and blog posts, corroborates the low in vitro cannabimimetic properties. Seizure prevalence data indicate that almost all SCRAs continue to be identified in 2022, potentially due to remaining stocks.     

Functional evaluation of carboxy metabolites of synthetic cannabinoid receptor agonists featuring scaffolds based on L‐valine or L‐tert‐leucine

Indole‐ and indazole‐based synthetic cannabinoid receptor agonists (SCRAs), featuring valine or tert‐leucine substituents, are commonly abused new psychoactive substances (NPS). A major metabolic pathway for these SCRAs is hydrolysis of the terminal amide or methylester functionalities. Although these hydrolysis products were already detected as main ingredients in some “legal highs,” these metabolites are often poorly characterized. Here, we report a systematic investigation of the activity of 7 common hydrolysis metabolites of 15 SCRAs featuring scaffolds based on L‐valine or L‐tert‐leucine in direct comparison to their parent compounds. An activity‐based cannabinoid receptor 1 (CB₁) bio‐assay was used for activity profiling of SCRAs and their metabolites in a stable HEK293T cell system. The recruitment of β‐arrestin2 to the activated CB₁ (each fused to one part of a split Nanoluciferase) was provoked by adding the (putative) SCRAs. Luminescence of the functionally complemented luciferase was monitored by a 96‐well plate‐reader. The major hydrolysis metabolites of 5F‐AB‐PINACA, ADB‐CHMICA, ADB‐CHMINACA, ADB‐FUBICA, and their methyl‐ and ethylester derivatives showed no detectable CB₁ activation at concentrations up to 1 μM. On the other hand, metabolites of 5F‐ADB‐PINACA, AB‐CHMINACA, and ADB‐FUBINACA did retain activity, although significantly reduced as compared to the parent compounds (EC₅₀ values >100 nM). Activity‐based characterization of SCRAs and their metabolites at CB₁ may not only allow a better insight into the complex interplay between SCRAs and their metabolites in intoxications, but may also allow application of the concept of “activity equivalents” present in biological fluids or, alternatively, in confiscated materials.     

Factors affecting the stability of drugs and drug metabolites in biological matrices

Evaluation of the stability of drugs and drug metabolites in a biological matrix is a critical element to bioanalytical method validation. It is critical to understand the most common factors that affect the stability of such analytes in order to properly develop methods for their detection and measurement. The degradation of drugs and drug metabolites in samples can occur through either reversible or irreversible processes. Common factors that affect this stability include temperature, light, pH, oxidation and enzymatic degradation. Special considerations are also required when dealing with chiral molecules, deuterated internal standards and large biomolecules. Relevant examples of these degradation effects and approaches for dealing with them are presented is this review as taken from the fields of pharmaceutical testing, clinical research and forensic analysis. It is demonstrated through these examples how an understanding of the chemical and physical factors that affect sample stability can be used to avoid stability problems and to create robust and accurate methods for the analysis of drugs and related compounds.     

Large-scale evaluation of ion mobility spectrometry for the rapid detection of synthetic cannabinoid receptor agonists in infused papers in prisons

Synthetic cannabinoid receptor agonists (SCRAs), colloquially known as “spice,” are commonly used in prisons and enter establishments via the mail in the form of infused papers. Many prisons use benchtop ion mobility spectroscopy (IMS) instruments to screen mail and seized materials for the presence of SCRAs and other controlled substances. The selectivity and sensitivity of Rapiscan Itemiser^® 3E and Itemiser^® 4DN Ion Trap Mobility Spectroscopy™ (ITMS™) systems were evaluated using 21 SCRA reference standards. Some differences in the SCRA reduced mobility (K₀) values were observed between this study and those reported previously using IMS detection systems, particularly for cumyl and quinolinyl SCRAs (e.g., 5F-PB-22, Cumyl-4CN-BINACA, and 5F-Cumyl-PEGACLONE), although this was found to have little effect at an operational level. Operational reliability of the systems was evaluated by analyzing 392 paper and card samples with known drug content. ITMS™ system results (e.g., detect or nondetect) were in agreement with gas chromatography–mass spectrometry (GC–MS) analysis in up to 95% of samples tested. Overall, this study found the ITMS™ systems tested to be effective instruments when deployed for the rapid detection of SCRA-infused papers. Used effectively and with up-to-date substance libraries, they will help reduce the supply of SCRAs into prisons and identify emerging threats as they arise. Several emerging SCRAs (5F-MPP-PICA, 5F-EMB-PICA, and 4F-MDMB-BICA) were detected for the first time in Scottish prisons between May and August 2020 as a result of routine monitoring, and all were detected using the ITMS™ systems tested.     

“Walking the nitrogen around the ring”: Chemical synthesis and spectroscopic characterization of novel 4-, 5-, 6-, and 7-azaindazole analogs of the synthetic cannabinoid receptor agonist MDMB-PINACA

Over the past decade, synthetic cannabinoid receptor agonists (SCRAs) have rapidly evolved to encompass a wide range of structurally diverse new psychoactive substances (NPS), including derivatives that incorporate indole, indazole, 7-azaindole, γ-carbolinone, or carbazole heterocyclic scaffolds. The introduction of legislative measures seeking to control the availability of NPS on the recreational drug scene has likely contributed to the continued emergence of novel SCRA analogs, which often evade regulatory control. However, the detection and/or identification of azaindazole-type SCRAs in seized material has not yet been reported (September, 2021). It is plausible that SCRAs bearing a 1,3-disubstituted azaindazole scaffold may possess cannabimimetic activity, given their structural similarity with known indole, indazole, and azaindole SCRAs. In view of these antecedents, a set of four novel isomeric 4-, 5-, 6-, and 7-azaindazole analogs of the known potent indazole SCRA, MDMB-PINACA, were synthesized using a Pd-catalyzed aminocarbonylation strategy. The complementary use of ultraviolet (UV) and infrared (IR) spectroscopy, gas chromatography-mass spectrometry (GC-MS), high resolution mass spectrometry (HRMS), 1D- and 2D-nuclear magnetic resonance (NMR) spectroscopy, and high performance liquid chromatography (HPLC) has permitted the spectroscopic differentiation, unambiguous structural assignment, and rapid separation of novel isomeric 4-, 5-, 6-, and 7-azaindazole analogs of the indazole SCRA, MDMB-PINACA.     

Optimization,validation and application of a high-throughput 96-well elution protocol for the quantification of psychoactive substances in influent wastewater

Wastewater-based epidemiology (WBE) is based on the analysis of human metabolic excretion products (biomarkers) of xenobiotics in wastewater, to gain information about various lifestyles and health aspects of a population in an evidence-based manner. Due to the complex wastewater matrix and trace level occurrence of human biomarkers in the sewage network, it is crucial to have sensitive analytical procedures available. Additionally, to improve the value of WBE as a complementary epidemiological source, there is increasing pressure on the analysis of more compounds, more locations and more samples. A high-throughput method based on 96-well Oasis MCX solid-phase extraction (SPE), requiring less influent wastewater (2 mL), was developed in accordance with the European Medicines Agency guidelines. Validation was successful for 28 parent drugs and metabolites of antidepressants, opioids and drugs of abuse. The selection of biomarkers and quantification limit was chosen to be relevant for WBE and was predominantly 10 ng/L or below. The final method was successfully applied to 24-h composite samples of October 2019 (n = 27), obtained from an urban wastewater treatment plant in Leuven (Belgium).     

Stability studies of vorinostat and its two metabolites in human plasma, serum and urine

Effects of storage time and freeze-thaw procedure on the stability of vorinostat (suberoylanilide hydroxamic acid) and its two metabolites, vorinostat O-glucuronide (M1) and 4-anilino-4-oxobutanoic acid (M2), in human plasma, serum and urine have been examined using high turbulence liquid chromatography (HTLC) online extraction and tandem mass spectrometry (MS/MS) [L. Du, D.G. Musson, A.Q. Wang, Rapid Commun. Mass Spectrom. 19 (2005) 1779–1787]. Vorinostat was demonstrated not to be stable in human plasma during the process of sample processing and storage. Acidifying the plasma sample to prevent possible enzymatic hydrolysis and using plasma with different anticoagulants were evaluated to increase the stability of vorinostat, but neither of these approaches improved stability. Human serum was then used as an alternative to plasma to monitor drug concentration, and vorinostat and its two metabolites maintained consistent concentrations in human serum after 3 freeze-thaw cycles and more than 1 year storage at −70 °C. By comparing the stability results of serum, EDTA plasma and heparin plasma, it was deduced that clotting proteins of plasma might be a major cause of vorinostat degradation. The stability of the three analytes during the process of serum sample collection was verified indicating that prolonged sample collection (up to 180 min) has no effect on the integrity of these analytes.     

In vitro metabolic fate of the synthetic cannabinoid receptor agonists (quinolin-8-yl 4-methyl-3-(morpholine-4-sulfonyl)benzoate [QMMSB]) and (quinolin-8-yl 4-methyl-3-((propan-2-yl)sulfamoyl)benzoate [QMiPSB]) including isozyme mapping and carboxylesterases activity testing

The synthetic cannabinoid receptor agonists (SCRAs) (quinolin-8-yl 4-methyl-3-(morpholine-4-sulfonyl)benzoate [QMMSB]) and (quinolin-8-yl 4-methyl-3-((propan-2-yl)sulfamoyl)benzoate [QMiPSB], also known as SGT-46) are based on the structure of quinolin-8-yl 4-methyl-3-(piperidine-1-sulfonyl)benzoate (QMPSB) that has been identified on seized plant material in 2011. In clinical toxicology, knowledge of the metabolic fate is important for their identification in biosamples. Therefore, the aim of this study was the identification of in vitro Phase I and II metabolites of QMMSB and QMiPSB in pooled human liver S9 fraction (pHLS9) incubations for use as screening targets. In addition, the involvement of human monooxygenases and human carboxylesterases (hCES) was examined. Analyses were performed by liquid chromatography coupled with high-resolution tandem mass spectrometry. Ester hydrolysis was found to be an important step in the Phase I metabolism of both SCRAs, with the carboxylic acid product being found only in negative ionization mode. Monohydroxy and N-dealkyl metabolites of the ester hydrolysis products were detected as well as glucuronides. CYP2C8, CYP2C9, CYP3A4, and CYP3A5 were involved in hydroxylation. Whereas enzymatic ester hydrolysis of QMiPSB was mainly catalyzed by hCES1 isoforms, nonenzymatic ester hydrolysis was also observed. The results suggest that ester hydrolysis products of QMMSB and QMiPSB and their glucuronides are suitable targets for toxicological screenings. The additional use of the negative ionization mode is recommended to increase detectability of analytes. Different cytochrome P450 (CYP) isozymes were involved in the metabolism; thus, the probability of drug–drug interactions due to CYP inhibition can be assessed as low.     

Phase I metabolism of the recently emerged synthetic cannabinoid CUMYL‐PEGACLONE and detection in human urine samples

Indole‐, indazole‐, or azaindole‐based synthetic cannabinoids (SCs), bearing a cumyl substituent are a widespread, recreationally used subgroup of new psychoactive substances (NPS). The latest cumyl‐derivative, CUMYL‐PEGACLONE, emerged in December 2016 on the German drug market. The substance features a novel γ‐carboline core structure, which is most likely synthesized to bypass generic legislative approaches to control SCs by prohibiting distinct core structures. Using liquid chromatography–tandem mass spectrometry and liquid chromatography–high resolution mass spectrometry techniques, the main in vivo phase I metabolites of this new substance were detected. A pooled human liver microsome assay was applied to generate in vitro reference spectra of CUMYL‐PEGACLONE phase I metabolites. Additionally, 30 urine samples were investigated leading to 22 in vivo metabolites. A metabolite mono‐hydroxylated at the γ‐carbolinone core system and a metabolite with an additional carbonyl group at the pentyl side chain were evaluated as highly specific and sensitive markers to proof CUMYL‐PEGACLONE uptake. Moreover, 3 immunochemical assays commonly used for SC screening in urine were tested for their capability of detecting the new drug but failed due to insufficient cross‐reactivity.     

Sewage‐based epidemiology in monitoring the use of new psychoactive substances: Validation and application of an analytical method using LC‐MS/MS

Sewage‐based epidemiology (SBE) employs the analysis of sewage to detect and quantify drug use within a community. While SBE has been applied repeatedly for the estimation of classical illicit drugs, only few studies investigated new psychoactive substances (NPS). These compounds mimic effects of illicit drugs by introducing slight modifications to chemical structures of controlled illicit drugs. We describe the optimization, validation, and application of an analytical method using liquid chromatography coupled to positive electrospray tandem mass spectrometry (LC‐ESI‐MS/MS) for the determination of seven NPS in sewage: methoxetamine (MXE), butylone, ethylone, methylone, methiopropamine (MPA), 4‐methoxymethamphetamine (PMMA), and 4‐methoxyamphetamine (PMA). Sample preparation was performed using solid‐phase extraction (SPE) with Oasis MCX cartridges. The LC separation was done with a HILIC (150 x 3 mm, 5 µm) column which ensured good resolution of the analytes with a total run time of 19 min. The lower limit of quantification (LLOQ) was between 0.5 and 5 ng/L for all compounds. The method was validated by evaluating the following parameters: sensitivity, selectivity, linearity, accuracy, precision, recoveries and matrix effects. The method was applied on sewage samples collected from sewage treatment plants in Belgium and Switzerland in which all investigated compounds were detected, except MPA and PMA. Furthermore, a consistent presence of MXE has been observed in most of the sewage samples at levels higher than LLOQ. Copyright © 2015 John Wiley & Sons, Ltd.     

A transnational perspective on the evolution of the synthetic cannabinoid receptor agonists market: Comparing prison and general populations

The synthetic cannabinoid receptor agonist (SCRA) market is transnational, and the availability of individual SCRAs changes regularly in response to national and international legislative controls. This generates a cyclic pattern and near constant evolution of SCRA compounds. This study reports toxicology-based and/or seized sample-based prevalence data relating to SCRA use in prisons from Germany, the United Kingdom (UK; Scotland and Wales), and the United States (US), representing 4427 individual test results. The study examines SCRA detections in prisons from July 2018 to September 2020, and where possible, prison-based data are compared with SCRA prevalence data in the wider population. The relative influence of Chinese, other international, and national drug legislation on the prevalence of individual SCRAs in prisons is also considered. tert-Leucinate- and valinate-indole- and indazole-3-carboxamides were the most common SCRA detections, and MDMB-4en-PINACA was one of the most commonly detected SCRAs in all jurisdictions by September 2020. However, despite there being a global production and supply market, there were notable regional differences. Analog controls in German and US legislation may have led to increased compound diversity that is not reflected in the UK which has both analog controls and a blanket ban on psychoactive substances. While there were regional differences, SCRA prevalence in prisons closely aligned with the SCRAs detected on the local market, demonstrating that SCRA (and possibly other NPS) monitoring programs in prisons can act as early warning systems for the wider population in that given jurisdiction.     

Synthetic cannabinoid receptor agonists: Analytical profiles and development of QMPSB,QMMSB, QMPCB, 2F-QMPSB,QMiPSB, and SGT-233

A diverse assortment of molecules designed to explore the cannabinoid receptor system and considered new psychoactive substances (NPS) have become known as synthetic cannabinoid receptor agonists (SCRAs). One group of SCRAs that has received little attention involves those exhibiting sulfamoyl benzoate, sulfamoyl benzamide, and N-benzoylpiperidine based structures. In this study, quinolin-8-yl 4-methyl-3-(piperidine-1-sulfonyl)benzoate (QMPSB), quinolin-8-yl 4-methyl-3-(morpholine-4-sulfonyl)benzoate (QMMSB), quinolin-8-yl 4-methyl-3-(piperidine-1-carbonyl)benzoate (QMPCB, SGT-11), quinolin-8-yl 3-(4,4-difluoropiperidine-1-sulfonyl)-4-methylbenzoate (2F-QMPSB, QMDFPSB, SGT-13), quinolin-8-yl 4-methyl-3-[(propan-2-yl)sulfamoyl]benzoate (QMiPSB, SGT-46), and 3-(4,4-difluoropiperidine-1-sulfonyl)-4-methyl-N-(2-phenylpropan-2-yl)benzamide (SGT-233) were extensively characterized (including data on impurities). The analytical profiles may be useful to researchers and scientists who deal with the emergence of NPS during forensic and clinical investigations. The detection of QMPSB was first published in 2016 but it is worth noting that Stargate International, a company originally formed to develop harm reduction solutions, were involved in the investigation and development of these six compounds for potential release between 2011 and early 2014. Whilst information on the prevalence of use of these particular compounds at the present time is limited, one of the key outcomes of the research performed by Stargate International reviewed here was to set the stage for the quinolin-8-yl ester head group that ultimately led to hybridization with an N-alkyl-1H-indole core to give SGT-21 and SGT-32, which became later known as PB-22 (QMPSB/JWH-018 hybrid) and BB-22, respectively, thus, opening the door to a range of SCRAs carrying the quinolin-8-yl head group from about 2012 onwards.     

Tentative identification of the phase I and II metabolites of two synthetic cathinones,MDPHP and α‐PBP,in human urine

Cathinone derivatives are one of the more prominent groups of new psychoactive substances in terms of the number of forensic case reports and the variety of chemical structures available. These substances often sold as “bath salts” are classified as psychostimulants. Using liquid chromatography‐high resolution mass spectrometry, the metabolites of two pyrrolidine cathinone derivatives, α‐PBP and the less common MDPHP, were tentatively identified in urine samples collected from patients admitted to hospital following drug intoxications. The major metabolic pathways for α‐PBP and MDPHP were similar to those of their more common analogs (α‐PVP and MDPV). Metabolites arising from hydroxylation, reduction of the carbonyl group to an alcohol, oxidation to form a lactam and subsequent ring‐opening, and a combination of these processes were identified. In addition, biotransformations of the benzodioxole moiety in MDPHP included demethylenation with subsequent methylation and carboxylation of the butyl group. The majority of the hydroxylated metabolites of α‐PBP and MDPHP were found to be glucuronidated. Both α‐PBP and MDPHP undergo extensive metabolism and the chromatographic peak areas of the metabolites were found to be comparable to or exceeded those of the parent substances. Metabolites resulting from demethylenation and subsequent methylation (MDPHP), reduction of carbonyl group (α‐PBP), and oxidation to form a lactam combined with ring‐opening (α‐PBP and MDPHP) were found to be the most useful target analytes for the confirmation of ingestion.     

Systematic evaluation of a panel of 30 synthetic cannabinoid receptor agonists structurally related to MMB-4en-PICA,MDMB-4en-PINACA,ADB-4en-PINACA,and MMB-4CN-BUTINACA using a combination of binding and different CB1 receptor activation assays—Part II: Structure activity relationship assessment via a β-arrestin recruitment assay

Synthetic cannabinoid receptor agonists (SCRAs) are the second largest class of new psychoactive substances (NPS) and are associated with serious adverse effects and even death. Despite this, little pharmacological data are available for many of the most recent SCRAs. This study consists of three different parts, aiming to systematically evaluate a panel of 30 SCRAs using binding and different in vitro human cannabinoid 1 receptor (CB₁) activation assays. The present Part II investigated the SCRA analogs for their CB₁ activation via a β-arrestin recruitment assay. The panel was systematically designed to include key structural sub-features of recent SCRAs. Thus, the 4-pentenyl tail of MMB-4en-PICA and MDMB-4en-PINACA was retained while incorporating varying head groups from other prevalent SCRAs, including amides and esters of L-valine, L-tert-leucine, and L-phenylalanine, and adamantyl and cumyl moieties. All 30 SCRAs activated CB₁, with indazoles generally showing the greatest potency (EC₅₀ = 1.88–281 nM), followed by indoles (EC₅₀ = 11.5–2293 nM), and the corresponding 7-azaindoles (EC₅₀ = 62.4–9251 nM). Several subunit-linked structure–activity relationships were identified: (i) tert-leucine-functionalized SCRAs were more potent than the corresponding valine derivatives; (ii) no major difference in potency or efficacy was observed between tert-leucine/valine-derived amides and the corresponding methyl esters; however, phenylalanine analogs were affected by this change; and (iii) minor structural changes to the 4-pentenyl substituent had little influence on activity. These findings elucidate structural features that modulate the CB₁ activation potential of currently prevalent SCRAs and a systematic panel of analogs, some of which may appear in NPS markets in future.     

Screening for illicit drugs in pooled human urine and urinated soil samples and studies on the stability of urinary excretion products of cocaine,MDMA, and MDEA in wastewater by hyphenated mass spectrometry techniques

Monitoring population drug use through wastewater‐based epidemiology (WBE) is a useful method to quantitatively follow trends and estimate total drug consumption in communities. Concentrations of drug biomarkers might be low in wastewater due to dilution; and therefore analysis of pooled urine (PU) is useful to detect consumed drugs and identify targets of illicit drugs use. The aims of the study were (1) to screen PU and urinated soil (US) samples collected at festivals for illicit drug excretion products using hyphenated techniques; (2) to develop and validate a hydrophilic interaction liquid chromatography – mass spectrometry / mass spectrometry (HILIC‐MS/MS) method of quantifying urinary targets of identified drugs in wastewater; and (3) to conduct a 24 h stability study, using PU and US to better reflect the chemical environment for targets in wastewater. Cocaine (COC) and ecstasy‐like compounds were the most frequently detected illicit drugs; an analytical method was developed to quantify their excretion products. Hydroxymethoxymethamphetamine (HMMA), 3,4‐methylenedioxymethamphetamine (MDMA), 3,4‐methylenedioxyamphetamine (MDA), HMMA sulfate (HMMA‐S), benzoylecgonine (BE), and cocaethylene (CE) had 85–102% of initial concentration after 8 h of incubation, whereas COC and ecgonine methyl ester (EME) had 74 and 67% after 8 h, respectively. HMMA showed a net increase during 24 h of incubation (107% ± 27, n = 8), possibly due to the cleavage of HMMA conjugates, and biotransformation of MDMA. The results suggest HMMA as analytical target for MDMA consumption in WBE, due to its stability in wastewater and its excretion as the main phase I metabolite of MDMA. Copyright © 2016 John Wiley & Sons, Ltd.     

Application of direct urine LC-MS-MS analysis for screening of novel substances in drug abusers

A newly developed liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was used to study 3000 human urine samples from 3 different populations for 23 analytes covering phenylethylamines, benzylpiperazine, and non-benzodiazepine hypnotics. Direct injection of urine and LC-MS-MS with rapid chromatography and atmospheric pressure chemical ionization was used in the screening step. The cutoff levels were chosen to be at the limit of detection for most analytes to identify as many positive samples as possible. Typically one ion transition was monitored from the pseudo-molecular ions in the multiple reaction monitoring mode. Of the 797 positive screening findings, 518 (65%) were confirmed by a second LC-MS-MS analysis including solid-phase extraction. Confirmed analytical findings included 22 cases positive for N-benzylpiperazine, 88 for 3,4-methylenedioxy-N-methylamphetamine and metabolites, 4 for 1-phenyl-2-butylamine, 24 for zolpidem and metabolites, 118 for zopiclone and metabolites, and 1 for zaleplon. In conclusion, LC-MS-MS was found to be a robust alternative for drugs of abuse screening, offering high sensitivity compared with immunochemical screening methodology.