Simple and straightforward analysis of cannabinoids in medicinal products by fast-GC–FID

Forensic Toxicology -     

Analysis of 62 synthetic cannabinoids by gas chromatography–mass spectrometry with photoionization

Gas chromatography–mass spectrometry (GC–MS) in electron ionization (EI) mode is one of the most commonly used techniques for analysis of synthetic cannabinoids, because the GC–EI-MS spectra contain characteristic fragment ions for identification of a compound; however, the information on its molecular ions is frequently lacking. To obtain such molecular ion information, GC–MS in chemical ionization (CI) mode is frequently used. However, GC–CI-MS requires a relatively tedious process using reagent gas such as methane or isobutane. In this study, we show that GC–MS in photoionization (PI) mode provided molecular ions in all spectra of 62 synthetic cannabinoids, and 35 of the 62 compounds showed only the molecular radical cations. Except for the 35 compounds, the PI spectra showed very simple patterns with the molecular peak plus only a few fragment peak(s). An advantage is that the ion source for GC–PI-MS can easily be used for GC–EI-MS as well. Therefore, GC–EI/PI-MS will be a useful tool for the identification of synthetic cannabinoids contained in a dubious product. To the best of our knowledge, this is the first report to use GC–PI-MS for analysis of synthetic cannabinoids.     

Driving under the influence of synthetic cannabinoids (“Spice”): a case series

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.     

Application of high-performance liquid chromatography with charged aerosol detection (LC–CAD) for unified quantification of synthetic cannabinoids in herbal blends and comparison with quantitative NMR results

Purpose

The analysis of products which contain synthetic cannabinoids (SCs) is very challenging due to their diversity and rapidly changing SC structures, variable herbal matrices and, above all, inaccessibility of reference standards. Therefore, the aim of this study was to develop a method which allows quantification of SCs’ contents in herbal blends without their reference standards.

Methods

Identification of SCs was performed using liquid chromatography–high-resolution tandem mass spectrometry with a quadrupole time-of-flight analyser (LC–QTOF-MS/MS). A liquid chromatography–charged aerosol detection (LC–CAD) method with unified calibration for the quantification of SCs was developed and validated. Two available reference standards were used as universal standards. Quantitative analysis using a nuclear magnetic resonance spectroscopy method was also performed to externally validate the developed LC–CAD method.

Results

All peaks of SCs observed in LC–CAD chromatograms were identified by LC–QTOF-MS/MS. Validation data and results from a CAD response evaluation indicated that the elaborated quantitative method was sufficiently accurate for the determination of SCs belonging to various chemical families. The LC–CAD method turned out to be very flexible, because it was successfully applied for the analysis of 19 herbal products.

Conclusions

In this study, methods which enable identification and quantification of currently known SCs as well as novel unknown derivatives without their reference standards were developed. These methods can be applied to the control of suspect SC products and may support the risk assessment of SC presence on the market. To our knowledge, this is the first trial to use LC–CAD for unified quantification of SCs without their reference standards.

     

DART – LTQ ORBITRAP as an expedient tool for the identification of synthetic cannabinoids

Synthetic cannabinoids as designer drugs constitute a major problem due to their rapid increase in number and the difficulties connected with their identification in complex mixtures. DART (Direct Analysis in Real Time) has emerged as an advantageous tool for the direct and rapid analysis of complex samples by mass spectrometry. Here we report on the identification of six synthetic cannabinoids originating from seized material in various matrices, employing the combination of ambient pressure ion source DART and hybrid ion trap - LTQ ORBITRAP mass spectrometer. This report also describes the sampling techniques for the provided herbal material containing the cannabinoids, either directly as plant parts or as an extract in methanol and their influence on the outcome of the analysis. The high resolution mass spectra supplied by the LTQ ORBITRAP instrument allowed for an unambiguous assignment of target compounds. The utilized instrumental coupling proved to be a convenient way for the identification of synthetic cannabinoids in real-world samples.     

Comments on the paper entitled “Rapid tentative identification of synthetic cathinones in seized products taking advantage of the full capabilities of triple quadrupole analyzer”

Forensic Toxicology -     

Pharmacological evaluation of new constituents of “Spice”: synthetic cannabinoids based on indole,indazole, benzimidazole and carbazole scaffolds

Purpose

In the present study we characterized a series of synthetic cannabinoids containing various heterocyclic scaffolds that had been identified as constituents of “Spice”, a preparation sold on the illicit drug market. All compounds were further investigated as potential ligands of the orphan receptors GPR18 and GPR55 that interact with some cannabinoids.

Methods

The compounds were studied in radioligand binding assays to determine their affinity for human cannabinoid CB₁ and CB₂ receptors expressed in CHO cells, and in cAMP accumulation assays to study their functionality.

Results

Structure-activity relationships were analyzed. The most potent CB₁ receptor agonist of the present series MDMB-FUBINACA (12) (K_i?=?98.5 pM) was docked into the human CB₁ receptor structure, and a plausible binding mode was identified showing high similarity with that of the co-crystallized THC derivatives. MDMB-CHMCZCA (41) displayed a unique profile acting as a full agonist at the CB₁ receptor subtype, but blocking the CB₂ receptor completely. Only a few weakly potent antagonists of GPR18 and GPR55 were identified, and thus all compounds showed high CB receptor selectivity, mostly interacting with both subtypes, CB₁ and CB₂.

Conclusions

These results will be useful to assess the compounds’ toxicological risks and to guide legislation. Further studies on 41 are warranted.

     

Analysis of cannabinoids in oral fluid by liquid chromatography–tandem mass spectrometry

A sensitive method was developed for quantifying a wide range of cannabinoids in oral fluid (OF) by liquid chromatography–tandem mass spectrometry (LC–MS/MS). These cannabinoids include ?⁹-tetrahydrocannabinol (THC), 11-hydroxy-?⁹-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-?⁹-tetrahydrocannabinol (THCCOOH), cannabinol (CBN), cannabidiol (CBD), ?⁹-tetrahydrocannabinolic acid A (THC-A), 11-nor-9-carboxy-?⁹-tetrahydrocannabinol glucuronide (THCCOOH-gluc), and ?⁹-tetrahydrocannabinol glucuronide (THC-gluc). Samples were collected using a Quantisal? device. The advantages of performing a liquid–liquid extraction (LLE) of KCl-saturated OF using heptane/ethyl acetate versus a solid-phase extraction (SPE) using HLB copolymer columns were determined. Chromatographic separation was achieved in 11.5 min on a Kinetex? column packed with 2.6-μm core–shell particles. Both positive (THC, 11-OH-THC, CBN, and CBD) and negative (THCCOOH, THC-gluc, THCCOOH-gluc, and THC-A) electrospray ionization modes were employed with multiple reaction monitoring using a high-end AB Sciex API 5000? triple quadrupole LC–MS/MS system. Unlike SPE, LLE failed to extract THC-gluc and THCCOOH-gluc. However, the LLE method was more sensitive for the detection of THCCOOH than the SPE method, wherein the limit of detection (LOD) and limit of quantification (LOQ) decreased from 100 to 50 pg/ml and from 500 to 80 pg/ml, respectively. The two extraction methods were successfully applied to OF samples collected from volunteers before and after they smoked a homemade cannabis joint. High levels of THC were measured soon after smoking, in addition to significant amounts of THC-A. Other cannabinoids were found in low concentrations. Glucuronide conjugate levels were lower than the method’s LOD for most samples. Incubation studies suggest that glucuronides could be enzymatically degraded by glucuronidase prior to OF collection.     

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.     

Fast and reliable profiling of cannabinoids in seized samples using the method of HPLC–DAD followed by chemometrics

Forensic Toxicology -     

What is the message of a meeting? Analysis and comparison of the 2458 abstracts of the EANM/WCNMB congress in Berlin and the SNM meeting in Toronto in 1998

The abstracts of the joint congress of EANM/ WCNMB in Berlin 1998 and of the 45th Annual Meeting of the Society of Nuclear Medicine in Toronto 1998 have been analysed and compared in terms of comprehensibility, composition, questions at issue, methods, patient/subject number, type of conclusion and duplication of information between the meetings. All 1362 and 1096 abstracts, respectively, were analysed from the abstract books with regard to ten ”hard” and four ”soft” variables. The dominant topics were new radiopharmaceuticals, methods of synthesis, examination methods, evaluation of examinations, investigation algorithms, technical devicesand novel use of radiopharmaceuticals. In addition to these topics, there were numerous reports about established radiopharmaceuticals and techniques, often without a specific merit mentioned. There were also many abstracts with questions outside nuclear medicine, but using such techniques. Few papers reported negative findings or dealt with quality assurance, dosimetry, and cost-effectiveness. Many of the conclusions contained hyperbole. Some abstracts were very extensive and detailed. Sixty-seven contributions conveyed identical information at both meetings. Structured and/or paragraphed abstracts promote clarity and reduce the number of lines that need to be read in order to comprehend the background and aim of the abstract. Such contributions were more frequent at the EANM/WCNMB congress while the SNM meeting covered a wider field with a greater representation of radiophysics, instrumentation, and computer evaluations.     

Myxofibrosarcoma: prevalence and diagnostic value of the “tail sign” on magnetic resonance imaging

Objective

Myxofibrosarcoma frequently shows curvilinear extensions of high T2 signal that also enhance on magnetic resonance imaging; these “tails” represent fascial extension of tumor at histopathological examination. This study was performed to determine whether the tail sign is helpful in distinguishing myxofibrosarcoma from other myxoid-containing neoplasms.

Materials and methods

The study group consisted of 44 patients with pathologically proven myxofibrosarcoma; the control group consisted of 52 patients with a variety of other myxoid-predominant tumors. Three musculoskeletal radiologists independently evaluated T2-weighted (and/or short-tau inversion recovery) and post-contrast MR images for the presence of one or more enhancing, high-signal intensity, curvilinear projections from the primary mass. Sensitivity and specificity for the diagnosis of myxofibrosarcoma were calculated for each reader. Interobserver variability was assessed with kappa statistic and percentage agreement.

Results

A tail sign was deemed present in 28, 30, and 34 cases of myxofibrosarcoma and in 11, 9, and 5 of the controls for the three readers respectively, yielding a sensitivity of 64–77 % and a specificity of 79–90 %. The interobserver agreement was moderate-to-substantial (kappa?=?0.626).

Conclusion

The tail sign at MRI is a moderately specific and sensitive sign for the diagnosis of myxofibrosarcoma relative to other myxoid-containing tumors.     

Ossification and pseudoepiphysis formation in the “nonepiphyseal” end of bones of the hands and feet

Metacarpals, metatarsals, and phalanges were studied to assess the developmental morphology of secondary ossification in the nonepiphyseal ends of these bones as well as the formation of the pseudoepiphysis as an epiphyseal ossification variant. Both direct ossification extension from the metaphysis into the epiphysis and pseudoepiphysis formation preceded, and continued to be more mature than, formation and expansion of the classic epiphyseal (secondary) ossification center at the opposite end of each specific bone. Direct metaphyseal to epiphyseal ossification usually started centrally and expanded hemispherically, replacing both physeal and epiphyseal cartilage simultaneously. In contrast, when remnants of physis were retained, while juxtaposed epiphyseal cartilage was replaced, a pseudoepiphysis formed. There were three basic patterns of pseudoepiphysis formation. First, a central osseous bridge extended from the metaphysis across the physis into the epiphysis and subsequently expanded to create a mushroom-like osseous structure. In the second pattern a peripheral osseous bridge formed, creating either an osseous ring or an eccentric bridge between the metaphysis and the epiphysis. In the third pattern, multiple bridging occurred. In each situation the associated remnant physis lacked typical cell columns and was incapable of significantly contributing to the postnatal longitudinal growth of the involved bone. Pseudoepiphyses were well formed by 4–5 years and coalesced with the rest of the bone months of years before skeletal maturation was attained at the opposite epiphyseal end, which ossified in the typical pattern (i.e., formation of a secondary center de novo completely within the cartilaginous epiphysis). This process may also affect the development and appearance of ossification within the longitudinal epiphyseal bracket (delta phalanx).     

Detection of pyrovalerone as a possible synthetic by-product of 4′-methyl-α-pyrrolidinohexanophenone and 4-methyl-α-ethylaminopentiophenone in illicit drug products

Forensic Toxicology - Impurity profiling is an important intelligence-gathering tool that can be used to link batches of drugs, and it provides valuable insights into manufacturing and supply...     

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 unregulated “herbal” products circulated in the Tokyo area

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)-1H-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)-1H-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.     

Location of the “zero zone” and its role in the pathogenesis of cervical spondylosis

Summary Significant correlation between the location of the zero zone and spondylosis was found in the cervical spine. The possible role of the zero zone in the development of spondylosis is suggested and the deleterious effect of counteracting forces as compared with a unidirectional traction is stressed.

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Die Stelle der Nullzone und ihre Rolle in der Pathogenese der zervikalen Spondylose

Zusammenfassung Die Ergebnisse der dynamischen Studien scheinen darauf hinzuweisen, daß ein Zusammenhang zwischen den häufigsten Stellen der Nullzone und der Spondylose besteht. Es scheint, daß das Bindegewebssystem der Halswirbelsäule auf die Überlastung durch entgegengesetzte Zugkomponenten sehr empfindlich ist und durch diese stärker beschädigt wird als durch eine Traktion, die nur in einer Richtung ausgeübt wird.

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Localisation de la zone zero et son rôle dans la pathogenie de la spondylose cervicale

Résumé Il semble y avoir une corrélation entre la zone zéro et le développement d'une spondylose cervicale et que la détérioration discale se fasse spécialement au niveau des mouvements intervertébraux bidirectionnels opposés.

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From the annual congress of the European Society of Neuroradiology (Geilo, September 1975)