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Volker Auwärter - One of the best experts on this subject based on the ideXlab platform.
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Hair analysis for JWH-018, JWH-122, and JWH-210 after passive in vivo exposure to synthetic cannabinoid smoke
Forensic Toxicology, 2015Co-Authors: Melanie Hutter, Bjoern Moosmann, Volker Auwärter, Merja A. NeukammAbstract:Hair analysis is often used to confirm abstinence from drug use. However, interpretation of hair analysis results can be challenging, particularly with regard to smoked substances like synthetic cannabinoids, because hair can be contaminated by side-stream smoke. In this study, we measured the concentrations of synthetic cannabinoids in scalp hair after exposure to side-stream smoke from a cigarette containing the synthetic cannabinoids JWH-018, JWH-122, and JWH-210. Three participants exposed their hair to the side-stream smoke once each working day for 3 weeks to mimic realistic conditions experienced by consumers of these drugs. Two other participants exposed their hair once to the side-stream smoke of one cigarette. Scuba regulators with external air supply were used to avoid inhalation of smoke. Hair segments and wash solutions were analyzed by liquid chromatography–tandem mass spectrometry. The highest measured concentrations were 70 pg/mg of JWH-018, 260 pg/mg of JWH-122, and 950 pg/mg of JWH-210 in distal hair segments collected at the end of the exposure period. At 2–3 weeks after the end of the repeated exposure, all three synthetic cannabinoids were detected in the hair samples of both participants with longer hair. In these samples, the ratio of cannabinoid amount in acetone wash to that in hair was below 0.5 for all synthetic cannabinoids, which could be interpreted as evidence of consumption. However, with the nonconsumption of synthetic cannabinoids by our study participants being confirmed by urine testing, it is apparent that even high substance concentrations in hair samples do not prove consumption and can be explained by external contamination after contact with synthetic cannabinoids alone.
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O23: Hair analysis for synthetic cannabinoids: How does handling of herbal mixtures during forensic analysis affect the analyst's hair concentrations?
Toxicologie Analytique et Clinique, 2014Co-Authors: Volker Auwärter, Melanie Hutter, Merja A. Neukamm, Bjoern MoosmannAbstract:Introduction If narcotics police officers or other persons handling drug material at work are suspected of consuming drugs, hair analysis may be used to prove or refute such suspicion. However, it is known for many drugs that differentiation of actual drug use from external contamination can be challenging or sometimes impossible. The aim of this study was to evaluate the extent of external contamination caused by handling of synthetic cannabinoid containing drug material under realistic conditions in a forensic laboratory. Methods Hair of laboratory workers was systematically analyzed for synthetic cannabinoids with a validated LC-MS/MS method after a big seizure of legal high products had to be analyzed in our laboratory. Hair samples were taken two days after the last exposure and again one week later. In addition, hair samples of laboratory staff not directly in contact with the drug material and close relatives of exposed subjects were analyzed to check for cross contamination. Results All samples of persons who were in direct contact with drug material were tested positive for at least one of the synthetic cannabinoids (JWH-018, JWH-073, JWH-081, JWH-122, JWH-210, JWH-307, JWH-368, AM-2201, AM-2201 indazole derivative, AM- 2232, MAM-2201, RCS-4, XLR-11, 5F-PB-22, RCS-4 ortho isomer). Concentrations ranged from trace amounts up to a maximum of 170 pg/mg (JWH-210) and roughly reflected duration and intensity of exposition. There was no significant decline in concentrations from sample 1 to sample 2 (one week later). Unexpectedly, also subjects without direct contact to drug material showed measurable hair concentrations. In one case, a hair sample (21 cm) was taken 10 weeks after the last exposition with plant material. In this case, relevant concentrations of 5F-PB-22 were detected with an increase of concentrations from distal to proximal segments (7.9 – 20 pg/mg). Conclusion Depending on duration and intensity of exposition, relevant concentrations of synthetic cannabinoids may be found in hair samples of persons exposed to these drugs at work. Unexpectedly, even cross contamination from an exposed person to a close relative may occur and lead to (false) positive hair findings. Concentrations caused by contamination are in the typical range found in known users of these drugs and could lead to wrong conclusions. In contrast, detection of metabolites could strongly suggest an actual consumption. However, we did not detect such metabolites so far even in samples of known consumers of synthetic cannabinoids showing extremely high concentrations of the unchanged compounds. Therefore, body fluids have to be analyzed to unambiguously prove use of these drugs.
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Investigation of the in vitro toxicological properties of the synthetic cannabimimetic drug CP-47,497-C8
Toxicology and Applied Pharmacology, 2014Co-Authors: Verena J. Koller, Bjoern Moosmann, Volker Auwärter, Tamara Grummt, Miroslav Mišík, Siegfried KnasmüllerAbstract:Abstract Cannabicyclohexanol (CP-47,497-C8) is a representative of a group of cannabimimetic cyclohexylphenols which is added to herbal mixtures as a cannabis substitute since 2008. Although in the beginning CP-47,497-C8 was the main ingredient of “Spice” and similar products, it was partly replaced by aminoalkylindole-type cannabinoid receptor agonists like JWH-018, JWH-073 or JWH-250, but never completely disappeared from the market. Since information on its toxicological properties is scarce, we investigated the effects of the drug in human derived cell lines. The cytotoxic effects were studied in a panel of assays (SRB, XTT, LDHe and NR tests) in a buccal derived (TR146) and a liver derived (HepG2) cell line. The strongest effects were seen in the two former assays at levels ≥ 7.5 μM indicating that the compound interferes with protein synthesis and causes membrane damage. In additional comet assays, DNA damage was detected at levels ≥ 10 μM. Experiments with lesion specific enzymes showed that these effects are not due to oxidative damage of DNA bases. The negative findings obtained in Salmonella/microsome assays and the positive results of micronucleus tests with the cell lines indicate that the compound does not cause gene mutations but acts on the chromosomal level. In contrast to other synthetic cannabinoids, no indication for estrogenic/antiestrogenic properties was seen in a luciferase assay with bone marrow derived U2-OS cells. In conclusion, our findings show that the drug has only weak cytotoxic properties. However, the induction of chromosomal damage indicates that it may cause adverse effects in users due to its impact on the stability of the genetic material.
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Driving under the influence of synthetic cannabinoids (“Spice”): a case series
International Journal of Legal Medicine, 2014Co-Authors: Frank Musshoff, Melanie Hutter, Stefan Kneisel, Gerhard Kernbach-wighton, Burkhard Madea, Wolfgang Bicker, Volker AuwärterAbstract:Recreational use of synthetic cannabinoid receptor agonists—so-called “Spice” products—became very popular during the last few years. Several reports on clinical symptoms and poisonings were published. Unfortunately, most of these reports do not contain any analytical data on synthetic cannabinoids in body fluids, and no or only a limited number of cases were reported concerning driving under the influence (DUI) of this kind of drugs. In this article, several cases of DUI of synthetic cannabinoids (AM-2201, JWH-018, JWH-019, JWH-122, JWH-210, JWH-307, MAM-2201 (JWH-122 5-fluoropentyl derivative), and UR-144) are presented, focusing on analytical results and signs of impairment documented by the police or the physicians who had taken the blood sample from the suspects. Consumption of synthetic cannabinoids can lead to impairment similar to typical performance deficits caused by cannabis use which are not compatible with safe driving. These deficits include centrally sedating effects and impairment of fine motor skills necessary for keeping the vehicle on track. Police as well as forensic toxicologists and other groups should become familiar with the effects of synthetic cannabinoid use, and be aware of the fact that drug users may shift to these “legal” alternatives due to their nondetectability by commonly used drug screening tests based on antibodies. Sophisticated screening procedures covering the complete range of available compounds or their metabolites have to be developed for both blood/serum and urine testing.
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Toxicological profiles of selected synthetic cannabinoids showing high binding affinities to the cannabinoid receptor subtype CB_1
Archives of Toxicology, 2013Co-Authors: Verena J. Koller, Gerhard J. Zlabinger, Sabine Fuchs, Volker Auwärter, Siegfried KnasmuellerAbstract:Products containing synthetic cannabinoids are consumed as a surrogate for marihuana due to their non-detectability with commonly used drug tests and their strong cannabimimetic effects. Because data concerning their toxicological properties are scarce, the cytotoxic, genotoxic, immunomodulatory, and hormonal activities of four naphthoylindole compounds (JWH-018, JWH-073, JWH-122 and JWH-210) and of one benzoylindole (AM-694) were studied in human cell lines and primary cells; tetrahydrocannabinol was included as the classical non-endogenous cannabinoid receptor ligand. All compounds induced damage to the cell membranes of buccal (TR146) and breast (MCF-7) derived cells at concentrations of ≥75–100 μM. No cytotoxic responses were seen in other assays which reflect mitochondrial damage, protein synthesis, and lysosomal activities. JWH-073 and JWH-122 induced DNA migration in buccal and liver cells (HepG2) in single cell gel electrophoresis assays, while JWH-210 was only in the latter cell line active. No estrogenic activities were detected in bone marrow cells (U2-OS), but all compounds caused anti-estrogenic effects at levels between 2.1 and 23.0 μM. Furthermore, no impact on cytokine release (i.e., on IL-10, IL-6, IL-12/23p40 and TNFα levels) was seen in LPS-stimulated human PBMCs, except with JWH-210 and JWH-122 which caused a decrease of TNFα and IL-12/23p40. All toxic effects were observed with concentrations higher than those expected in body fluids of users. Since genotoxic effects are in general linear over a wide concentration range and the exposure levels may be higher in epithelial cells or in serum, further experimental work is required to find out if DNA damage takes place in drug users.
Stefan Kneisel - One of the best experts on this subject based on the ideXlab platform.
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Driving under the influence of synthetic cannabinoids (“Spice”): a case series
International Journal of Legal Medicine, 2014Co-Authors: Frank Musshoff, Melanie Hutter, Stefan Kneisel, Gerhard Kernbach-wighton, Burkhard Madea, Wolfgang Bicker, Volker AuwärterAbstract:Recreational use of synthetic cannabinoid receptor agonists—so-called “Spice” products—became very popular during the last few years. Several reports on clinical symptoms and poisonings were published. Unfortunately, most of these reports do not contain any analytical data on synthetic cannabinoids in body fluids, and no or only a limited number of cases were reported concerning driving under the influence (DUI) of this kind of drugs. In this article, several cases of DUI of synthetic cannabinoids (AM-2201, JWH-018, JWH-019, JWH-122, JWH-210, JWH-307, MAM-2201 (JWH-122 5-fluoropentyl derivative), and UR-144) are presented, focusing on analytical results and signs of impairment documented by the police or the physicians who had taken the blood sample from the suspects. Consumption of synthetic cannabinoids can lead to impairment similar to typical performance deficits caused by cannabis use which are not compatible with safe driving. These deficits include centrally sedating effects and impairment of fine motor skills necessary for keeping the vehicle on track. Police as well as forensic toxicologists and other groups should become familiar with the effects of synthetic cannabinoid use, and be aware of the fact that drug users may shift to these “legal” alternatives due to their nondetectability by commonly used drug screening tests based on antibodies. Sophisticated screening procedures covering the complete range of available compounds or their metabolites have to be developed for both blood/serum and urine testing.
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acute toxicity due to the confirmed consumption of synthetic cannabinoids clinical and laboratory findings
Addiction, 2013Co-Authors: Maren Hermannsclausen, Stefan Kneisel, Bela Szabo, Volker AuwärterAbstract:Aims Recently, several synthetic cannabinoids were identified in herbal mixtures consumed as recreational drugs alternative to cannabis products. The aim was to characterize the acute toxicity of synthetic cannabinoids as experienced by emergency patients. Design This was a retrospective study targeting patients seeking emergency treatment after recreational use of synthetic cannabinoids. Setting and participants Patients were selected from the database of the Poisons Information Center Freiburg between September 2008 and February 2011. The inclusion criteria were: hospitalization, available clinical reports and analytical verification of synthetic cannabinoid uptake. In total, 29 patients were included (age 14–30 years, median 19; 25 males, four females). Measurements Clinical reports were evaluated and synthetic cannabinoids and other drugs were determined analytically. Findings CP-47,497-C8 (one), JWH-015 (one), JWH-018 (eight), JWH-073 (one), JWH-081 (seven), JWH-122 (11), JWH-210 (11), JWH-250 (four) and AM 694 (one) were quantified in blood samples. JWH-018 was most common in 2008–9, JWH-122 in 2010, and JWH-210 in 2011. Tachycardia, agitation, hallucination, hypertension, minor elevation of blood glucose, hypokalaemia and vomiting were reported most frequently. Chest pain, seizures, myoclonia and acute psychosis were also noted. Conclusions There appears to have been an increase in use of the extremely potent synthetic cannabinoids JWH-122 and JWH-210. Acute toxic symptoms associated with their use are also reported after intake of high doses of cannabis, but agitation, seizures, hypertension, emesis and hypokalaemia seem to be characteristic to the synthetic cannabinoids, which are high-affinity and high-efficacy agonists of the CB1 receptor. Thus, these effects are due probably to a strong CB1 receptor stimulation.
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A fatal case involving several synthetic cannabinoids
2013Co-Authors: Nadine Schaefer, Stefan Kneisel, Dietmar Bregel, Benjamin Peters, Peter H Schmidt, Volker Auwärter, Andreas H. EwaldAbstract:A 36-year-old man collapsed at home right after he had smoked an herbal blend named "Mary Joy Annihilation". After arrival of the ambulance, the man already suffered seizures and died after admission to hospital despite continued attempts of resuscitation. In the decedent's apartment, the police found the residual of a joint, which was seized for analysis. The aim was to determine whether a synthetic cannabinoid intoxication could be considered as a contributing factor to the cause of death. METHODS: Femoral blood, bile fluid, gastric content, hair, brain, lung, kidney, liver, and adi- pose tissue samples were obtained during autopsy. Blood, bile fluid and gastric content sam- ples, homogenized tissues and pulverized hair were extracted liquid-liquidly and analyzed by LC-MS/MS (MRM/ESI+). The residual of the joint was macerated in ethanol and analyzed by GC-MS in scan mode. The femoral blood sample was also analyzed by GC-MS in scan and SIM mode. RESULTS: In femoral blood 0.1 ng/mL JWH-018, 0.3 ng/mL JWH-122, 1.4 ng/mL AM-2201, 1.5 ng/mL MAM-2201, approximately 6 ng/mL UR-144, and 250 ng/mL amphetamine could be detected. The synthetic cannabinoids were also found in other tissues. In addition, JWH- 210 could be detected in hair and adipose tissue samples. The joint contained the same syn- thetic cannabinoids except for JWH-018 and JWH-210. CONCLUSION: An acute influence of several synthetic cannabinoids and amphetamine can be assumed. Detection of JWH-210 in adipose tissue and hair and lack of it in blood and joint indicates that a previous consumption of other substances had occurred. Language: en
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Analysis of 30 synthetic cannabinoids in oral fluid using liquid chromatography‐electrospray ionization tandem mass spectrometry
Drug Testing and Analysis, 2012Co-Authors: Stefan Kneisel, Volker Auwärter, Jürgen KempfAbstract:In recent years, the analysis of synthetic cannabinoids in human specimens has gained enormous importance in the broad field of drug testing. Nevertheless, the considerable structural diversity among synthetic cannabinoids already identified in 'herbal mixtures' hampers the development of comprehensive analytical methods. As the identification of the main metabolites of newly appearing substances is very laborious and time-consuming, the detection of the parent compounds in blood samples is the current approach of choice for drug abstinence testing. Whenever blood sampling is not possible however, the need for alternative matrices arises. In this article, we present a fully validated liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for the analysis of 30 synthetic cannabinoids in oral fluid samples collected with the Drager DCD 5000 collection device. The method proved to be suitable for the quantification of 28 substances. The limits of detection were in the range from 0.015 to 0.9 ng/ml, while the lower limits of quantification ranged from 0.15 to 3.0 ng/ml. The method was successfully applied to 264 authentic samples during routine analysis. A total of 31 samples (12%) was tested positive for at least one of the following synthetic cannabinoids: AM-694, AM-2201, JWH-018, JWH-019, JWH-081, JWH-122, JWH-203, JWH-210, JWH-250, JWH-307, MAM-2201, and RCS-4. Given that stabilization of the collection pads after sampling is warranted, the collection device provides satisfactory sensitivity. Hence, whenever blood sampling is not possible, the Drager DCD 5000 collection device offers a good tool for the analysis of synthetic cannabinoids in oral fluid in the broad field of drug testing. Language: en
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Determination of 22 synthetic cannabinoids in human hair by liquid chromatography-tandem mass spectrometry.
Journal of Chromatography B, 2012Co-Authors: Melanie Hutter, Stefan Kneisel, Volker Auwärter, Merja A. NeukammAbstract:Abstract Herbal mixtures of the “Spice“-type contain a variety of synthetic cannabinoids. To prove the contact of a person with synthetic cannabinoids in a previous period of up to several months, hair testing is ideally suited. A rapid, simple and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay was developed to determine 22 synthetic cannabinoids in human hair. The synthetic cannabinoids JWH-007, JWH-015, JWH-018, JWH-019, JWH-020, JWH-073, JWH-081, JWH-122, JWH-200, JWH-203, JWH-210, JWH-250, JWH-251, JWH-398, AM-694, AM–2201, methanandamide, RCS-4, RCS-4 ortho isomer, RCS-8, WIN 48,098 and WIN 55,212-2 were extracted from 50 mg hair by 3-h ultrasonification in ethanol. The extracts were analysed on a triple-quadrupole linear ion trap mass-spectrometer in scheduled multiple reaction monitoring mode (sMRM). The method was fully validated and proved to be accurate, precise, selective and specific with satisfactory linearity within the calibrated range and a lower limit of quantification of 0.5 pg/mg for 20 compounds. Authentic hair samples from chronic consumers showed the presence of two to six synthetic cannabinoids in the same segment. In the first segment, concentrations of up to 78 pg/mg JWH-081 were present. In segmented hair, the concentrations of most substances increased from the first (proximal) to the third segment. The highest concentration was ca. 1100 pg/mg JWH-081. The results of segmental hair analysis in chronic users suggest incorporation of the drugs in head hair via side-stream smoke condensation as a major route. In summary, the method can be used to prove the contact with herbal mixtures containing synthetic cannabinoids and thus contributes to an efficient abstinence control.
Tomoko Hamano - One of the best experts on this subject based on the ideXlab platform.
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Identification and quantitation of two new naphthoylindole drugs-of-abuse, (1-(5-hydroxypentyl)-1H-indol-3-yl)(naphthalen-1-yl)methanone (AM-2202) and (1-(4-pentenyl)-1H-indol-3-yl)(naphthalen-1-yl)methanone, with other synthetic cannabinoids in unre
Forensic Toxicology, 2012Co-Authors: Jun’ichi Nakajima, Jin Suzuki, Misako Takahashi, Masao Yoshida, Takako Seto, Chieko Kanai, Tomoko HamanoAbstract:During our continual surveillance of unregulated drugs in May–June 2011, we found two new compounds as adulterants in herbal products obtained at shops in the Tokyo area. These compounds were identified by liquid chromatography–mass spectrometry, gas chromatography–mass spectrometry, accurate mass spectrometry, and nuclear magnetic resonance spectroscopy. The first compound identified was a naphthoylindole (1-(5-hydroxypentyl)-1 H -indol-3-yl)(naphthalen-1-yl)methanone (AM-2202, 1 ), which is a side-chain hydroxyl analogue of JWH-018. The second compound was (1-(4-pentenyl)-1 H -indol-3-yl)(naphthalen-1-yl)methanone ( 2 ), which is side-chain double bond analogue of JWH-018. This is the first report to identify 1 and 2 in a commercial “herbal” product to our knowledge. For quantitation of the above compounds 1 and 2 , and chemical analysis for previously reported compounds (AM-2201, 3 ; JWH-203, 4 ; JWH-019, 7 ; JWH-210, 8 ; mitragynine, 9 ), each product was extracted with methanol under ultrasonication to prepare solutions for analysis by liquid chromatography with ultraviolet detection. For the sake of identifying JWH-203 ( 4 ) and its positional isomers [JWH-203-3-chloroisomer ( 5 ) and 4-chloroisomer ( 6 )] correctly, simultaneous liquid chromatography analysis on fluorocarbon-bonded silica gel column was performed. And a case report of commercially available products containing synthetic cannabinoids 7 and 8 , and a natural occurring alkaloid 9 , was also shown. Each of 6 commercially circulated products contained compounds 1 – 4 and 7 – 9 ; the amounts of the compounds ranged from 4.1 to 222 mg per pack.
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Identification and quantitation of two benzoylindoles AM-694 and (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone, and three cannabimimetic naphthoylindoles JWH-210, JWH-122, and JWH-019 as adulterants in illegal products obtained via the Internet
Forensic Toxicology, 2011Co-Authors: Jun’ichi Nakajima, Jin Suzuki, Misako Takahashi, Masao Yoshida, Takako Seto, Chieko Kanai, Tomoko HamanoAbstract:During our careful surveillance of unregulated drugs, we found five new compounds used as adulterants in herbal and drug-like products obtained via the Internet. These compounds were identified by liquid chromatography–mass spectrometry, gas chromatography–mass spectrometry, accurate mass spectrometry, and nuclear magnetic resonance spectroscopy. The first compound identified was a benzoylindole AM-694, which is 1-[(5-fluoropentyl)-1 H -indol-3-yl]-(2-iodophenyl)methanone ( 1 ). The second compound was (4-methoxyphenyl)(1-pentyl-1 H -indol-3-yl)methanone ( 2 ), which was also classified as a benzoylindole. The three other compounds were identified as naphthoylindoles JWH-210 (4-ethylnaphthalen-1-yl-(1-pentylindol-3-yl)methanone; 3 ), JWH-122 (4-methylnaphthalen-1-yl-(1-pentylindol-3-yl)methanone; 4 ), and JWH-019 (1-hexyl-3-(naphthalen-1-oyl)indole; 5 ). All compounds except compound 2 had been reported to be cannabinoid receptor agonists. For quantitation of the five compounds and previously reported compounds, each product was extracted with methanol under ultrasonication to prepare a test solution for analysis by liquid chromatography with ultraviolet detection. Each compound detected in 43 commercial products showed large variation in content ranging from 4.0 to 359 mg per pack.
Marilyn A Huestis - One of the best experts on this subject based on the ideXlab platform.
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Urinary prevalence, metabolite detection rates, temporal patterns and evaluation of suitable LC-MS/MS targets to document synthetic cannabinoid intake in US military urine specimens.
Clinical Chemistry and Laboratory Medicine, 2015Co-Authors: Ariane Wohlfarth, Marisol S. Castaneto, Adarsh Gandhi, Nathalie A. Desrosiers, Thomas M. Martin, Kevin L. Klette, Karl B. Scheidweiler, Marilyn A HuestisAbstract:Identifying synthetic cannabinoid designer drug abuse challenges toxicologists and drug testing programs. The best analytical approach for reliably documenting intake of emerging synthetic cannabinoids is unknown. Primarily metabolites are found in urine, but optimal metabolite targets remain unknown, and definitive identification is complicated by converging metabolic pathways. We screened 20,017 US military urine specimens collected from service members worldwide for synthetic cannabinoids between July 2011 and June 2012. We confirmed 1432 presumptive positive and 1069 presumptive negative specimens by qualitative liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis including 29 biomarkers for JWH-018, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210, JWH-250, RCS-4, AM2201 and MAM2201. Specimen preparation included enzyme hydrolysis and acetonitrile precipitation prior to LC-MS/MS analysis. We evaluated individual synthetic cannabinoid metabolite detection rates, prevalence, temporal patterns and suitable targets for analytical procedures. Prevalence was 1.4% with 290 confirmed positive specimens, 92% JWH-018, 54% AM2201 and 39% JWH-122 metabolites. JWH-073, JWH-210 and JWH-250 also were identified in 37%, 4% and 8% of specimens, respectively. The United States Army Criminal Investigation Command seizure pattern for synthetic cannabinoid compounds matched our urine specimen results over the time frame of the study. Apart from one exception (AM2201), no parent compounds were observed. Hydroxyalkyl metabolites accounted for most confirmed positive tests, and in many cases, two metabolites were identified, increasing confidence in the results, and improving detection rates. These data also emphasize the need for new designer drug metabolism studies to provide relevant targets for synthetic cannabinoid identification.
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nontargeted swath acquisition for identifying 47 synthetic cannabinoid metabolites in human urine by liquid chromatography high resolution tandem mass spectrometry
Analytical and Bioanalytical Chemistry, 2015Co-Authors: Karl B. Scheidweiler, Michael Jarvis, Marilyn A HuestisAbstract:Clandestine laboratories constantly produce new synthetic cannabinoids to circumvent legislative scheduling efforts, challenging and complicating toxicological analysis. Sundstrom et al. (Anal Bioanal Chem 405(26):8463–8474, [9]) and Kronstrand et al. (Anal Bioanal Chem 406(15):3599–3609, [10]) published nontargeted liquid chromatography, high-resolution, quadrupole/time-of-flight mass spectrometric (LC-QTOF) assays with validated detection of 18 and 38 urinary synthetic cannabinoid metabolites, respectively. We developed and validated a LC-QTOF urine method for simultaneously identifying the most current 47 synthetic cannabinoid metabolites from 21 synthetic cannabinoid families (5-fluoro AB-PINACA, 5-fluoro-AKB48, 5-fluoro PB-22, AB-PINACA, ADB-PINACA, AKB48, AM2201, JWH-018, JWH-019, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210, JWH-250, JWH-398, MAM2201, PB-22, RCS-4, UR-144, and XLR11). β-Glucuronidase-hydrolyzed urine was extracted with 1-mL Biotage SLE+ columns. Specimens were reconstituted in 150-μL mobile phase consisting of 80 % A (0.1 % formic acid in water) and 20 % B (0.1 % formic acid in acetonitrile). Fifty microliters was injected, and SWATH™ MS data were acquired in positive electrospray mode. The LC-QTOF instrument consisted of a Shimadzu UFLCxr system and an ABSciex 5600+ TripleTOF® mass spectrometer. Gradient chromatographic separation was achieved with a Restek Ultra Biphenyl column with a 0.5-mL/min flow rate and an overall run time of 15 min. Identification criteria included molecular ion mass error, isotopic profiles, retention time, and library fit criteria. Limits of detection were 0.25–5 μg/L (N = 10 unique fortified urine samples), except for two PB-22 metabolites with limits of 10 and 20 μg/L. Extraction efficiencies and matrix effects (N = 10) were 55–104 and −65–107 %, respectively. We present a highly useful novel LC-QTOF method for simultaneously confirming 47 synthetic cannabinoid metabolites in human urine.
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Nontargeted SWATH acquisition for identifying 47 synthetic cannabinoid metabolites in human urine by liquid chromatography-high-resolution tandem mass spectrometry
Analytical and Bioanalytical Chemistry, 2015Co-Authors: Karl B. Scheidweiler, Michael J. Y. Jarvis, Marilyn A HuestisAbstract:Clandestine laboratories constantly produce new synthetic cannabinoids to circumvent legislative scheduling efforts, challenging and complicating toxicological analysis. Sundstrom et al. (Anal Bioanal Chem 405(26):8463–8474, [ 9 ]) and Kronstrand et al. (Anal Bioanal Chem 406(15):3599–3609, [ 10 ]) published nontargeted liquid chromatography, high-resolution, quadrupole/time-of-flight mass spectrometric (LC-QTOF) assays with validated detection of 18 and 38 urinary synthetic cannabinoid metabolites, respectively. We developed and validated a LC-QTOF urine method for simultaneously identifying the most current 47 synthetic cannabinoid metabolites from 21 synthetic cannabinoid families (5-fluoro AB-PINACA, 5-fluoro-AKB48, 5-fluoro PB-22, AB-PINACA, ADB-PINACA, AKB48, AM2201, JWH-018, JWH-019, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210, JWH-250, JWH-398, MAM2201, PB-22, RCS-4, UR-144, and XLR11). β-Glucuronidase-hydrolyzed urine was extracted with 1-mL Biotage SLE+ columns. Specimens were reconstituted in 150-μL mobile phase consisting of 80 % A (0.1 % formic acid in water) and 20 % B (0.1 % formic acid in acetonitrile). Fifty microliters was injected, and SWATH™ MS data were acquired in positive electrospray mode. The LC-QTOF instrument consisted of a Shimadzu UFLCxr system and an ABSciex 5600+ TripleTOF® mass spectrometer. Gradient chromatographic separation was achieved with a Restek Ultra Biphenyl column with a 0.5-mL/min flow rate and an overall run time of 15 min. Identification criteria included molecular ion mass error, isotopic profiles, retention time, and library fit criteria. Limits of detection were 0.25–5 μg/L ( N = 10 unique fortified urine samples), except for two PB-22 metabolites with limits of 10 and 20 μg/L. Extraction efficiencies and matrix effects ( N = 10) were 55–104 and −65–107 %, respectively. We present a highly useful novel LC-QTOF method for simultaneously confirming 47 synthetic cannabinoid metabolites in human urine. Graphical Abstract SWATH acquisition MS experiment
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Simultaneous quantification of 20 synthetic cannabinoids and 21 metabolites, and semi-quantification of 12 alkyl hydroxy metabolites in human urine by liquid chromatography-tandem mass spectrometry.
Journal of Chromatography A, 2013Co-Authors: Karl B. Scheidweiler, Marilyn A HuestisAbstract:Clandestine laboratories constantly produce new synthetic cannabinoids to circumvent legislative efforts, complicating toxicological analysis. No extensive synthetic cannabinoid quantitative urinary methods are reported in the literature. We developed and validated a liquid chromatography tandem mass spectrometric (LC-MS/MS) method for simultaneously quantifying JWH-018, JWH-019, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210, JWH-250, JWH-398, RCS-4, AM-2201, MAM-2201, UR-144, CP 47,497-C7, CP 47,497-C8 and their metabolites, and JWH-203, AM-694, RCS-8, XLR-11 and HU-210 parent compounds in urine. Non-chromatographically resolved alkyl hydroxy metabolite isomers were considered semi-quantitative. β-glucuronidase hydrolyzed urine was extracted with 1 ml Biotage SLE+ columns. Specimens were reconstituted in 150 µL mobile phase consisting of 50% A (0.01% formic acid in water) and 50% B (0.01% formic acid in 50:50 methanol:acetonitrile). 4 and 25 µL injections were performed to acquire data in positive and negative ionization modes, respectively. The LC-MS/MS instrument consisted of a Shimadzu UFLCxr system and an ABSciex 5500 Qtrap mass spectrometer with an electrospray source. Gradient chromatographic separation was achieved utilizing a Restek Ultra Biphenyl column with a 0.5 ml/min flow rate and an overall run time of 19.5 and 11.4 min for positive and negative mode methods, respectively. Quantification was by multiple reaction monitoring with CP 47,497 compounds and HU-210 ionized via negative polarity; all other analytes were acquired in positive mode. Lower and upper limits of linearity were 0.1–1.0 and 50–100 µg/l (r2 > 0.994). Validation parameters were evaluated at three concentrations spanning linear dynamic ranges. Inter-day analytical recovery (bias) and imprecision (N=20) were 88.3–112.2% and 4.3–13.5% coefficient of variation, respectively. Extraction efficiencies and matrix effect (N=10) were 44–110 and −73 to 52%, respectively. We present a novel LC-MS/MS method for simultaneously quantifying 20 synthetic cannabinoids and 21 metabolites, and semi-quantifying 12 alkyl hydroxy metabolites in urine.
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Automated and semi-automated screening, LCMSMS confirmation and human hepatocyte high-resolution metabolism studies of synthetic cannabinoids
2013Co-Authors: Ariane Wohlfarth, Marisol S. Castaneto, Adarsh Gandhi, Nathalie A. Desrosiers, Eliani Spinelli, Sheena Young, Allan J. Barnes, Karl B. Scheidweiler, Marilyn A HuestisAbstract:Designer drug availability changes rapidly. Assays are needed to identify synthetic cannabinoids in human urine, but metabolites are frequently unknown. This research evaluates available screen performance, develops sensitive and specific LCMSMS confirmation assays, and utilizes human hepatocyte cultures and high-resolution (HR) mass spectrometry to identify unique urinary targets. Homogeneous (Immunalysis), and heterogeneous (Randox; National Medical Services (NMS)) screening assays were evaluated with authentic urine specimens. An LCMSMS qualitative confirmation method for synthetic cannabinoids (JWH-018, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210, JWH-250, AM 2201 and RCS-4) and 20 metabolites in human urine was developed. Specimens were hydrolyzed and protein precipitated, followed by single MRM transition monitoring or survey scan that triggered an enhanced product ion scan at 3 different collision energies on the ABSciex 5500 Qtrap. 2500 specimens were screened by Randox, Immunalysis, NMS, and LCMSMS assays for 19 hydroxyalkyl, hydroxyindole and carboxy metabolites of 9 synthetic cannabinoids. Positive criteria included retention time, purity of fit, and presence of 3 ions, including the molecular ion. Confirmation based on full spectra increased identification confidence. 285 samples were LCMSMS positive for synthetic cannabinoids. Immunalysis and NMS assays had high sensitivities (87.7 and 84.9 per cent) and specificities (98.7 and 97.7 per cent) at a 5ng/mL cutoff; Randox identified almost all positive specimens (97.9 per cent sensitivity), but had 1149 presumptive positive unconfirmed specimens (48.1 per cent specificity). Possible reasons for low specificity, and advantages and disadvantages of approaches are discussed. Human hepatocyte cultures and HR mass spectrometry identified new synthetic cannabinoids metabolites to improve emerging drug identification. Identification of new synthetic cannabinoids is a difficult and critical laboratory problem.
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Identification and quantitation of two new naphthoylindole drugs-of-abuse, (1-(5-hydroxypentyl)-1H-indol-3-yl)(naphthalen-1-yl)methanone (AM-2202) and (1-(4-pentenyl)-1H-indol-3-yl)(naphthalen-1-yl)methanone, with other synthetic cannabinoids in unre
Forensic Toxicology, 2012Co-Authors: Jun’ichi Nakajima, Jin Suzuki, Misako Takahashi, Masao Yoshida, Takako Seto, Chieko Kanai, Tomoko HamanoAbstract:During our continual surveillance of unregulated drugs in May–June 2011, we found two new compounds as adulterants in herbal products obtained at shops in the Tokyo area. These compounds were identified by liquid chromatography–mass spectrometry, gas chromatography–mass spectrometry, accurate mass spectrometry, and nuclear magnetic resonance spectroscopy. The first compound identified was a naphthoylindole (1-(5-hydroxypentyl)-1 H -indol-3-yl)(naphthalen-1-yl)methanone (AM-2202, 1 ), which is a side-chain hydroxyl analogue of JWH-018. The second compound was (1-(4-pentenyl)-1 H -indol-3-yl)(naphthalen-1-yl)methanone ( 2 ), which is side-chain double bond analogue of JWH-018. This is the first report to identify 1 and 2 in a commercial “herbal” product to our knowledge. For quantitation of the above compounds 1 and 2 , and chemical analysis for previously reported compounds (AM-2201, 3 ; JWH-203, 4 ; JWH-019, 7 ; JWH-210, 8 ; mitragynine, 9 ), each product was extracted with methanol under ultrasonication to prepare solutions for analysis by liquid chromatography with ultraviolet detection. For the sake of identifying JWH-203 ( 4 ) and its positional isomers [JWH-203-3-chloroisomer ( 5 ) and 4-chloroisomer ( 6 )] correctly, simultaneous liquid chromatography analysis on fluorocarbon-bonded silica gel column was performed. And a case report of commercially available products containing synthetic cannabinoids 7 and 8 , and a natural occurring alkaloid 9 , was also shown. Each of 6 commercially circulated products contained compounds 1 – 4 and 7 – 9 ; the amounts of the compounds ranged from 4.1 to 222 mg per pack.
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Identification and quantitation of two benzoylindoles AM-694 and (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone, and three cannabimimetic naphthoylindoles JWH-210, JWH-122, and JWH-019 as adulterants in illegal products obtained via the Internet
Forensic Toxicology, 2011Co-Authors: Jun’ichi Nakajima, Jin Suzuki, Misako Takahashi, Masao Yoshida, Takako Seto, Chieko Kanai, Tomoko HamanoAbstract:During our careful surveillance of unregulated drugs, we found five new compounds used as adulterants in herbal and drug-like products obtained via the Internet. These compounds were identified by liquid chromatography–mass spectrometry, gas chromatography–mass spectrometry, accurate mass spectrometry, and nuclear magnetic resonance spectroscopy. The first compound identified was a benzoylindole AM-694, which is 1-[(5-fluoropentyl)-1 H -indol-3-yl]-(2-iodophenyl)methanone ( 1 ). The second compound was (4-methoxyphenyl)(1-pentyl-1 H -indol-3-yl)methanone ( 2 ), which was also classified as a benzoylindole. The three other compounds were identified as naphthoylindoles JWH-210 (4-ethylnaphthalen-1-yl-(1-pentylindol-3-yl)methanone; 3 ), JWH-122 (4-methylnaphthalen-1-yl-(1-pentylindol-3-yl)methanone; 4 ), and JWH-019 (1-hexyl-3-(naphthalen-1-oyl)indole; 5 ). All compounds except compound 2 had been reported to be cannabinoid receptor agonists. For quantitation of the five compounds and previously reported compounds, each product was extracted with methanol under ultrasonication to prepare a test solution for analysis by liquid chromatography with ultraviolet detection. Each compound detected in 43 commercial products showed large variation in content ranging from 4.0 to 359 mg per pack.
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Identification and quantitation of cannabimimetic compound JWH-250 as an adulterant in products obtained via the Internet
Forensic Toxicology, 2011Co-Authors: Jun’ichi Nakajima, Misako Takahashi, Takako Seto, Jin SuzukiAbstract:During our careful survey of unregulated drugs in Tokyo, a new compound was disclosed as an adulterant in herbal and powder products. This compound was found to have a molecular weight of 335 by liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry, and the accurate mass measurement suggested an elementary composition of C_22H_26NO_2. Using these mass data together with those obtained by nuclear magnetic resonance analysis, the compound was identified as 1-pentyl-3-(2-methoxyphenylacetyl)indole (JWH-250), which had been reported by Huffman and coworkers in 2005. This compound was classified as a phenylacetylindole and a cannabinoid receptor agonist. For quantitation of the compound in herbal and powder products, each product was extracted with methanol under ultrasonication to prepare the solution for analysis by liquid chromatography with ultraviolet detection. The contents of JWH-250 in five products ranged from 77.4 to 165 mg per pack.
