Parent Synthetic Cannabinoids Research Articles

Synthesis, characterization and differentiation of the structural isomers of valine and tert-leucine derived synthetic cannabinoids.

The identification of the synthetic cannabinoids receptor agonists (SCRAs) has always posed a great challenge to drug testing laboratories with slight structural modifications aimed at evading drug legislation. In addition, the most prevalent synthetic cannabinoids have valine and tert-leucine amino acid moieties where re-arrangement of the carbon chains can result in structural isomers that are very similar to the parent synthetic cannabinoids. This makes their analysis and identification challenging, and the problem is compounded with the difficulty in purchasing reference standards quickly and a lack of literature for comparison. Therefore, in this investigation, four series of synthetic cannabinoids (AB-PINACA, AB-CHMINACA, MMB-FUBINACA and 5-fluoro-MDMB-PINACA) and their alkyl chain structural isomers at the amino acid moieties were synthesized and characterized using various analytical techniques-gas chromatography-mass spectrometry (GC-MS), gas chromatography-infrared detection (GC-IRD) and nuclear magnetic resonance (NMR) spectroscopy to evaluate the ability of each analytical technique to differentiate the respective isomers for their identification. A total of 12 isomers were synthesized and analysed together with the four parent synthetic cannabinoids. NMR was able to differentiate between all the compounds, whereas GC-IRD was able to discern between most of the synthetic cannabinoids and their isomers. GC-MS had the least discriminating power and was not able to differentiate some of the compounds that has very similar mass spectra. The results from this work will be useful to other drug testing laboratories that are facing the identification of related synthetic cannabinoids.

CB1 activity of three classes of synthetic cannabinoids and their metabolites – The tail needs to know what the head is doing

Synthetic cannabinoids (SCs) are amongst the most prevalent of the new psychoactive substances. They encompass various structural classes with a wide range of CB1 activity. Most SCs are extensively bio-transformed in vivo and in vitro. The available data on the CB1 activity of SC metabolites are limited but such knowledge is needed to evaluate SC toxicology, duration of effects and to develop bioassays for screening purposes. To evaluate any structure-activity relationships, we determined the human CB1 receptor efficacy and potency of three SC classes and their metabolites. An activity assay incorporating AequoScreen recombinant CHO-K1 cells expressing the human CB1 receptor was used to study SCs and their metabolites. Dose-response curves were prepared in triplicate in three independent experiments and GraphPad Prism was used to calculate substance efficacy and potency. Eight concentrations, 29 nM–60 μM, were used for each drug and JWH-018 was used as a reference. The three SC classes investigated were (i) JWH-018, AM2201, THJ-018 and THJ-2201 and their corresponding 4-OH-pentyl and 5OH-pentyl metabolites; (ii) MMB-4en-PICA (MMB022) and MDMB-4en-PINACA and their corresponding ester-hydrolysis and dihydrodiol metabolites; and (iii) 5F-MDMB-PINACA, 5F-MDMB-PICA, 4F-MDMB-BINACA, and 4F-MDMB-BICA and their oxidative-defluorination and ester-hydrolysis metabolites. Parent SC potency values ranged from 7-200 nM. The 4-OH-pentyl metabolites of JWH-018, AM2201, THJ-018 and THJ-2201 had similar potency to their respective parent SC. The 5-OH-pentyl-JWH-018 and 5-OH-pentyl-THJ-018 metabolites were 5–10 times less potent than their parent SCs. No CB1 activity was observed for MMB-4en-PICA ester-hydrolysis and dihydrodiol metabolites, possibly due to the lower activity of the parent and a subsequent large loss of activity, whilst the dihydrodiol and ester-hydrolysis MDMB-4en-PINACA metabolites were 53 times and 130 times less potent respectively than MDMB-4en-PINACA. 5F-MDMB-PINACA and 5F-MDMB-PICA oxidative-defluorinated metabolites were 7 and 27 times less potent than the parent SCs. The 4OH-butyl metabolites of 4F-MDMB-BINACA, and 4F-MDMB-BICA were 6 and 11 times less potent than the parent drug, respectively, and their ester-hydrolysis metabolites were 21-47 times less potent. Where hydroxylations occur at the non-terminal (4) position of the pentyl tail of JWH-018 and THJ -2201 there is no loss of CB1 activity. The CB1 potency of the JWH-018 and THJ -2201 5OH-pentyl metabolites is reduced but in the same potency range as those reported for other parent SCs. Dihydrodiol metabolites of 4en-compounds show a large reduction in CB1 activity (> 53 times) compared to the parent, as do their ester hydrolysis metabolites (> 130-times). The oxidative defluorinated metabolites of the 4F-MDMB and 5F-MDMB compounds retain some CB1 activity (7–27 times reduction) but a more significant loss of activity is observed when ester hydrolysis occurs in the head group (21–417 times reduction). The head and core moieties of SCs are most influential in determining CB1 potency. The tail, when fulfilling certain steric requirements, acts as an anchor point in the CB1 receptor, influencing the overall position and rigidity of the SC in the CB1 receptor. From a urinary bioassay perspective, where metabolites can be 10 times more abundant than parent SCs, the activity of the metabolites might be important for SC detection. We have established a structure-activity relationship for three SC classes and their metabolites. The relative change in CB1 activity caused by a biotransformation on the tail of the SC depends on the head structure present and biotransformation of the head group leads to a greater reduction in potency than single hydroxylations on the tail. So, “the tail of the SC metabolite needs to know what the head is doing” when activating the CB1 receptor. The study is part of Eurostars-2 Joint Programme NPS-REFORM.

Transesterification of Indazole-3-carboxamide Synthetic Cannabinoids: Identification of Metabolite Biomarkers for Diagnosing Co-abuse of 5F-MDMB-PINACA and Alcohol.

Concurrent use of alcohol with synthetic cannabinoids (SCs) has been widely recorded among drug abusers. The susceptibilities of three indazole-3-carboxamide type SCs with methyl ester moiety, 5F-MDMB-PINACA, 5F-MMB-PINACA, and MMB-FUBINACA, to transesterification in the presence of ethanol warranted further investigation in view of probable augmented toxicity. In vitro metabolite identification experiments were first performed using human liver microsomes (HLMs) to characterize the novel metabolites of the three parent SCs in the presence of ethanol. Formation of transesterified metabolite, hydrolyzed metabolite, and several oxidative metabolites in HLM in the presence of alcohol was further determined for each parent SC and the respective ethyl ester analog, 5F-EDMB-PINACA, 5F-EMB-PINACA, and EMB-FUBINACA, to quantitatively elucidate transesterification and hydrolysis activities. Our results suggested that all three SCs undergo carboxylesterase-mediated transesterification to their respective ethyl ester analog in the presence of ethanol, which was incubation time- and ethanol concentration-dependent. Each ethyl ester metabolite was sequentially and readily metabolized to novel oxidative metabolites with the intact ethyl ester moiety and the same hydrolyzed metabolite as derived from its parent SC. A smaller extent of transesterification was non-enzymatically driven. Notably, we proposed 5F-EDMB-PINACA oxidative defluorination metabolite as the biomarker for diagnosing the potential co-abuse of 5F-MDMB-PINACA and alcohol. Due to the comparable pharmacological activities between each SC and its ethyl ester metabolite, augmented toxicity associated with co-abuse of SCs and alcohol is probable and deserves further investigation.

UHPLC-HRMS and GC-MS Screening of a Selection of Synthetic Cannabinoids and Metabolites in Urine of Consumers.

Background and Objectives: The use of synthetic cannabinoids has increased around the world. As a result, the implementation of accurate analysis in human biological matrices is relevant and fundamental. Two different analytical technologies, ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) and high-sensitivity gas chromatography-mass spectrometry (GC-MS) were used for the determination of three synthetic cannabinoids JWH-122, JWH 210, UR-144 and their metabolites in urine of consumers. Materials and Methods: Sample preparation included an initial hydrolysis with β-glucuronidase and liquid-liquid extraction. The UHPLC-HRMS method included a Kinetex 2.6 u Biphenyl 100A (100 × 2.1 mm, 2.6 μm) (Phenomenex, Italy) column with a gradient mobile phase consisting of mobile phase A (ammonium formate 2mM in water, 0.1% formic acid) and mobile phase B (ammonium formate 2mM in methanol/acetonitrile 50:50 (v/v), 0.1% formic acid) and a full-scan data-dependent MS2 (ddMS2) mode was used (mass range 100–1000 m/z). The GC-MS method employed an ultra-Inert Intuvo GC column (HP-5MS UI, 30 m × 250 µm i.d, film thickness 0.25 µm; Agilent Technologies, Santa Clara, CA, USA) and electron-impact (EI) mass spectra were recorded in total ion monitoring mode (scan range 40–550 m/z). Results: Both methods have been successfully used for screening of parent synthetic cannabinoids and their metabolites in urine samples of consumers. Conclusions: The screening method applied JWH-122, JWH-210, UR-144 and their metabolites in urine of consumers can be applied to other compounds of the JWH family.

Sensitive identification and quantitation of parent forms of six synthetic cannabinoids in urine samples of human cadavers by liquid chromatography–tandem mass spectrometry

Human urine samples are easier to obtain than human blood samples due to noninvasiveness. The urine levels of synthetic cannabinoids (SCs) in unchanged forms, however, are usually much lower than their blood and tissue levels and cannot be detected in most cases. Therefore, in the present work a sensitive analytical method was devised for the determination of urine levels of six SCs in unchanged forms such as N-(1-amino-3-methy-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA), N-(1-amino-3-methy-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (AB-FUBINACA), N-[(1S)-1-(1-aminocarbonyl)-2-methyl-propyl]-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide (AB-CHMINACA), N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide (MAB-CHMINACA), methyl-2-[1-(5-fluoropentyl)-1H-indazole-3-carboxamido]-3-methylbutanoate (5F-AMB) and methyl-2-[1-(5-fluoropentyl)-1H-indazole-3-carboxamido]-3,3-dimethylbutanoate (5F-ADB). These SCs were extracted from urine via liquid–liquid extraction. The identification and quantitation were performed by a relatively new type of an instrument for liquid chromatography–tandem mass spectrometry. The limits of detection were as low as 3–8 pg/mL, and the quantitation range was 10–1000 pg/mL using 400 μL of urine. The urine levels of AB-PINACA and AB-FUBINACA of victim 1 were 23 and 10 pg/mL, those of AB-CHMINACA and 5F-AMB of victim 2 were 239 and 19 pg/mL, and those of MAB-CHMINACA and 5F-ADB of victim 3 were 229 and 19 pg/mL, respectively. To our knowledge, this is the first report dealing with successful analysis of low levels of parent synthetic cannabinoids in authentic human urine specimens.

Analysis of Parent Synthetic Cannabinoids in Blood and Urinary Metabolites by Liquid Chromatography Tandem Mass Spectrometry

Synthetic cannabinoids emerged on the designer drug market in recent years due to their ability to produce cannabis-like effects without the risk of detection by traditional drug testing techniques such as immunoassay and gas chromatography-mass spectrometry. As government agencies work to schedule existing synthetic cannabinoids, new, unregulated and structurally diverse compounds continue to be developed and sold. Synthetic cannabinoids undergo extensive metabolic conversion. Consequently, both blood and urine specimens may play an important role in the forensic analysis of synthetic cannabinoids. It has been observed that structurally similar synthetic cannabinoids follow common metabolic pathways, which often produce metabolites with similar metabolic transformations. Presented are two validated quantitative methods for extracting and identifying 15 parent synthetic cannabinoids in blood, 17 synthetic cannabinoid metabolites in urine and the qualitative identification of 2 additional parent compounds. The linear range for most synthetic cannabinoid compounds monitored was 0.1-10 ng/mL with the limit of detection between 0.01 and 0.5 ng/mL. Selectivity, specificity, accuracy, precision, recovery and matrix effect were also examined and determined to be acceptable for each compound. The validated methods were used to analyze a compilation of synthetic cannabinoid investigative cases where both blood and urine specimens were submitted. The study suggests a strong correlation between the metabolites detected in urine and the parent compounds found in blood.

Determination of selected synthetic cannabinoids and their metabolites by micellar electrokinetic chromatography – mass spectrometry employing perfluoroheptanoic acid-based micellar phase

Perfluoroheptanoic acid was employed as a volatile micellar phase in background electrolyte for micellar electrokinetic chromatography–tandem mass spectrometry separation and determination of 15 selected naphthoyl- and phenylacetylindole- synthetic cannabinoids and main metabolites derived from JWH-018, JWH-019, JWH-073, JWH-200 and JWH-250. The influence of concentration of perfluoroheptanoic acid in background electrolytes on the separation was studied as well as the influence of perfluoroheptanoic acid on mass spectrometry detection. The background electrolyte consisted of 75mM perfluoroheptanoic acid, 150mM ammonium hydroxide pH 9.2 with 10% (v/v) propane-2-ol allowed micellar electrokinetic chromatography separation together with mass spectrometry identification of the studied parent synthetic cannabinoids and their metabolites. The limits of detection of studied synthetic cannabinoids and metabolites were in the range from 0.9ng/mL for JWH-073 to 3.0ng/mL for JWH-200 employing liquid–liquid extraction. The developed method was applied on the separation and identification of studied analytes after liquid–liquid extraction of spiked urine and serum samples to demonstrate the potential of the method applicability for forensic and toxicological purposes.

Quantitative urine confirmatory testing for synthetic cannabinoids in randomly collected urine specimens.

Synthetic cannabinoid intake is an ongoing health issue worldwide, with new compounds continually emerging, making drug testing complex. Parent synthetic cannabinoids are rarely detected in urine, the most common matrix employed in workplace drug testing. Optimal identification of synthetic cannabinoid markers in authentic urine specimens and correlation of metabolite concentrations and toxicities would improve synthetic cannabinoid result interpretation. We screened 20 017 randomly collected US military urine specimens between July 2011 and June 2012 with a synthetic cannabinoid immunoassay yielding 1432 presumptive positive specimens. We analyzed all presumptive positive and 1069 negative specimens with our qualitative synthetic cannabinoid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which confirmed 290 positive specimens. All 290 positive and 487 randomly selected negative specimens were quantified with the most comprehensive urine quantitative LC-MS/MS method published to date; 290 specimens confirmed positive for 22 metabolites from 11 parent synthetic cannabinoids. The five most predominant metabolites were JWH-018 pentanoic acid (93%), JWH-N-hydroxypentyl (84%), AM2201 N-hydroxypentyl (69%), JWH-073 butanoic acid (69%), and JWH-122 N-hydroxypentyl (45%) with 11.1 (0.1-2,434), 5.1 (0.1-1,239), 2.0 (0.1-321), 1.1 (0.1-48.6), and 1.1 (0.1-250) µg/L median (range) concentrations, respectively. Alkyl hydroxy and carboxy metabolites provided suitable biomarkers for 11 parent synthetic cannabinoids; although hydroxyindoles were also observed. This is by far the largest data set of synthetic cannabinoid metabolites urine concentrations from randomly collected workplace drug testing specimens rather than acute intoxications or driving under the influence of drugs. These data improve the interpretation of synthetic cannabinoid urine test results and suggest suitable urine markers of synthetic cannabinoid intake.

O22: Quantification of 22 synthetic cannabinoids and 10 metabolites in human hair

Introduction Synthetic cannabinoids are the most common drugs among an expanding array of compounds that mimic the effects of traditional illicit psychoactive substances. A few methods have been developed for the determination of the parent synthetic cannabinoids in the keratin matrix, opening the scientific debate about how to interpret the quantitative results. In particular, the discrimination between (i) passive or active exposure, and (ii) chronic consumption and occasional use, appears to be essential within the forensic context. Just as occurred for the traditional drugs of abuse, the detection of drug metabolites is likely to represent the key factor to support the decision procedure. Therefore, we updated our existing method [ A. Salomone et al., Drug Testing and Analysis, 2014, 6, 126–134 ] in order to complement the determination of 22 synthetic cannabinoids in human hair with 10 of their metabolites. Afterwards, we applied the new method to 15 real hair samples which previously tested positive for at least one synthetic cannabinoid [ A. Salomone et al., cited ], in order to verify the presence of metabolites. Methods The analytical method was validated in accordance with the criteria and recommendations of national and international guidelines. The following parameters were investigated: selectivity, specificity, linearity range, detection and quantification limits (LOD and LOQ), intra-assay and inter-assay precision and accuracy. Carry-over effect, recovery and matrix effects were also investigated. Two groups of subjects, namely driving re-licensing and drug abuse/withdrawal control subjects, provided hair samples that had previously resulted positive to at least one common drug of abuse and one synthetic cannabinoid. In this procedure, the samples were added with 1 mL NaOH 1N and subsequently incubated at 95 °C for 10 min. Immediately afterwards, the samples were extracted with 5 mL of n-hexane/ethylacetate 90:10 (v/v). The aqueous phase was newly added with glacial CH 3 COOH to reach a pH of 3.5–4.0 and again extracted with n-hexane/ethylacetate 90:10. The two organic phases were unified, dried under a nitrogen flow at 70 °C and reconstituted with 50 μL of methanol. An aliquot of 1 μL methanol solution was directly injected into the UHPLC-MS/MS system. Results The optimized UHPLC – MS/MS method allowed the simultaneous determination of 22 synthetic cannabinoids, 10 metabolites and 6 internal standards. The whole chromatographic run, comprehensive of the time required for column re-equilibration before the following injection, was completed in 8.0 min. Retention times ranged between 2.2 min (WIN 48,098) and 5.5 min (JWH- 020). Two samples out of 15 resulted positive to one metabolite, namely the N-(5-hydroxypentyl) metabolite of JWH-122. The calculated concentrations were 2.5 pg/mg and 0.72 pg/mg. In the same samples, the drug parent concentrations were, respectively, 2800 pg/mg and 760 pg/mg. Conclusion The present preliminary study describes for the first time an extensive search of metabolites of synthetic cannabinoids in human hair. Undoubtedly, the detection of metabolites in hair may represent the decisive factor to support the interpretation of hair analyses, particularly with respect to potential sources of external contamination. More samples will be analyzed in order to (i) verify to what extent the presence of metabolites can prove the active consumption, (ii) evaluate the concentration ratios between parent drugs and metabolites, and (iii) elaborate tentative cut-offs for parent drug and metabolites.

Metabolite profiling of RCS-4, a novel synthetic cannabinoid designer drug, using human hepatocyte metabolism and TOF-MS.

Since 2009, scheduling legislation of synthetic cannabinoids prompted new compound emergence to circumvent legal restrictions. 2-(4-methoxyphenyl)-1-(1-pentyl-indol-3-yl)methanone (RCS-4) is a potent cannabinoid receptor agonist sold in herbal smoking blends. Absence of parent synthetic cannabinoids in urine suggests the importance of metabolite identification for detecting RCS-4 consumption in clinical and forensic investigations. Materials & methods & Results: With 1 h human hepatocyte incubation and TOF high-resolution MS, we identified 18 RCS-4 metabolites, many not yet reported. Most metabolites were hydroxylated with or without demethylation, carboxylation and dealkylation followed by glucuronidation. One additional sulfated metabolite was also observed. O-demethylation was the most common biotransformation and generated the major metabolite. For the first time, we present a metabolic scheme of RCS-4 obtained from human hepatocytes, including PhaseI and II metabolites. Metabolite structural information and associated high-resolution mass spectra can be employed for developing clinical and forensic laboratory RCS-4 urine screening methods.

First Metabolic Profile of XLR-11, a Novel Synthetic Cannabinoid, Obtained by Using Human Hepatocytes and High-Resolution Mass Spectrometry

Since the mid-2000s synthetic cannabinoids have been abused as recreational drugs, prompting scheduling of these substances in many countries. To circumvent legislation, manufacturers constantly market new compounds; [1-(5-fluoropentyl)indol-3-yl]-(2,2,3,3-tetramethylcyclopropyl)methanone (XLR-11), the fluorinated UR-144 analog, is one of the most recent and widely abused drugs, and its use is now linked with acute kidney injury. Our goal was to investigate XLR-11 metabolism for identification of major urinary targets in analytical methods and to clarify the origin of metabolites when one or more parent synthetic cannabinoids can be the source. We incubated 10 μmol/L XLR-11 with pooled human hepatocytes and sampled after 1 and 3 h. Samples were analyzed by high-resolution mass spectrometry with a TOF scan followed by information-dependent acquisition triggered product ion scans with dynamic background subtraction and mass defect filters. Scans were thoroughly data mined with different data processing algorithms (Metabolite Pilot 1.5). XLR-11 underwent phase I and II metabolism, producing more than 25 metabolites resulting from hydroxylation, carboxylation, hemiketal and hemiacetal formation, internal dehydration, and further glucuronidation of some oxidative metabolites. No sulfate or glutathione conjugation was observed. XLR-11 also was defluorinated, forming UR-144 metabolites. On the basis of mass spectrometry peak areas, we determined that the major metabolites were 2'-carboxy-XLR-11, UR-144 pentanoic acid, 5-hydroxy-UR-144, hydroxy-XLR-11 glucuronides, and 2'-carboxy-UR-144 pentanoic acid. Minor metabolites were combinations of the biotransformations mentioned above, often glucuronidated. These are the first data defining major urinary targets of XLR-11 metabolism that could document XLR-11 intake in forensic and clinical investigations.