Analytical characterisation of hashish samples

Summary This report describes the analytical characterisation of two hashish samples to establish if they belong to a common lot, although their physical appearance is quite different. The samples were extracted and subjected to HPLC and GC for the separation and quantitation of the main cannabinoids. GC-MS enabled the selective identification of minor cannabinoids and terpenes. Hashish samples were also mineralised and analysed for metal traces by atomic absorption spectrometry (AA).     

Cannabielsoic acids : Isolation and synthesis by a novel oxidative cyclization

Two tricyclic dihydrobenzofuran cannabinoids, cannabielsoic acids A (4a) and B (5a) were isolated from hashish. Their structures were elucidated by chemical transformations and from spectral data. Cannabielsoic acid A was synthesized from cannabidiolic acid by an oxidative cyclization in the presence of air under irradiation, or with manganese dioxide.     

Application of ultrasound‐assisted liquid–liquid microextraction coupled with gas chromatography and mass spectrometry for the rapid determination of synthetic cannabinoids and metabolites in biological samples

The constant emergence of new psychoactive substances is a challenge to clinical and forensic toxicologists who need to constantly update analytical techniques to detect them. A large portion of these substances are synthetic cannabinoids. The aim of this study was to develop a rapid and simple method for the determination of synthetic cannabinoids and their metabolites in urine and blood using gas chromatography–mass spectrometry. The method involves an ultrasound‐assisted dispersive liquid–liquid microextraction that implies a rapid procedure, giving excellent extraction efficiencies with minimal use of toxic solvents. This is followed by silylation and analysis with gas chromatography–mass spectrometry. The chromatographic method allows for the separation and identification of 29 selected synthetic cannabinoids and some metabolites. The method was validated on urine and blood samples with the ability to detect and quantify all analytes with satisfactory limits of detection (from 1 to 5 ng/mL), limits of quantification (5 ng/mL), and selectivity and linearity (in the range of 5–200 ng/mL). The developed assay is highly applicable to laboratories with limited instrumental availability, due to the use of efficient and low‐cost sample preparation and instrumental equipment. The latter may contribute to enhance the detection of new psychoactive substances in clinical and forensic toxicology laboratories.     

Development of Gas-Chromatographic Method for Simultaneous Determination of Cannabinoids and Terpenes in Hemp

An original gas chromatographic method has been developed for simultaneous determination of major terpenes and cannabinoids in plant samples and their extracts. The main issues to be addressed were the large differences in polarity and volatility between both groups of analytes, but also the need for an exhaustive decarboxylation of cannabinoid acidic forms. Sample preparation was minimised, also by avoiding any analyte derivatisation. Acetone was found to be the most appropriate extraction solvent. Successful chromatographic separation was achieved by using a medium polarity column. Limits of detection ranged from 120 to 260 ng/mL for terpenes and from 660 to 860 ng/mL for cannabinoids. Parallel testing proved the results for cannabinoids are comparable to those obtained from established HPLC methods. Despite very large differences in concentrations between both analyte groups, a linear range between 1 and 100 µg/mL for terpenes and between 10 and 1500 µg/mL for cannabinoids was determined.     

Determination of cannabinoids from a surrogate hops matrix using multiple reaction monitoring gas chromatography with triple quadrupole mass spectrometry

Cannabinoids are the primary bioactive constituents of Cannabis sativa and Cannabis indica plants. In this work, gas chromatography in conjunction with triple quadrupole mass spectrometry in multiple reaction monitoring mode was explored for determination of cannabinoids from a surrogate hops matrix. Gas chromatography with mass spectrometry is a reasonable choice for the analysis of these compounds; however, such methods are susceptible to false positives for Δ9‐tetrahydrocannabinol, due to decarboxylation of Δ9‐tetrahydrocannabinolic acid, its acid precursor, in the hot injection port. To avoid this transformation, the carboxyl group of Δ9‐tetrahydrocannabinolic acid was protected through a silylation reaction. Multiple reaction monitoring transitions for both unmodified and silylated cannabinoids were developed and the fragmentation pathways for the different species were assigned. Precision and accuracy were evaluated for cannabinoids spiked into hops at different levels. The developed methods provided good linearity (R² > 0.99) for all the cannabinoids with a linear range from 0.15 to 20 mg/L, and with limits of detection in the orders of low‐ to mid‐picogram on column. The recoveries for the cannabinoids were generally between 75 and 120%. Precisions (<6% coefficient of variation) were within acceptable ranges.     

MEKC–MS/MS method using a volatile surfactant for the simultaneous determination of 12 synthetic cannabinoids

This study describes a method for the simultaneous determination of 12 synthetic cannabinoids by MEKC–MS/MS using a volatile surfactant (ammonium perfluorooctanoate) as a constituent of the micellar pseudostationary phase. Although most synthetic cannabinoids comigrated by a CZE method, sufficient separation could be achieved by the proposed method. The best separation was made possible by 50 mM ammonium perfluorooctanoate in 20% v/v acetonitrile/water (apparent pH* 9.0) as the BGE, followed by MS detection using a sheath liquid composed of 5 mM ammonium formate in 50% v/v methanol/water mixed hydro‐organic solvent. The standard calibration curve for all analytes showed good linearity (r > 0.99). Satisfactory recoveries, ranging from 89.5 to 101.7%, were obtained. The LODs were 6.5–76.5 μg/g for the target analytes. This method appears to be a useful tool for the identification of synthetic cannabinoids in illegal herbal incense blends.     

Detection of intake of hashish in biological material

In order to detect hashish intake, urine, blood and serum were analysed for the main components of hashish, i.e., tetrahydrocannabinol (THC), cannabidiol, cannabinol and the decomposition product of THC, THC-carboxylic acid. After extraction and silylation, the samples were analysed by gas chromatography-mass spectrometry with multiple ion detection. The Emit-st-system is used as a pretest for urine.     

Screening of cannabinoids in industrial‐grade hemp using two‐dimensional liquid chromatography coupled with acidic potassium permanganate chemiluminescence detection

Widely known for its recreational use, the cannabis plant also has the potential to act as an antibacterial agent in the medicinal field. The analysis of cannabis plants/products in both pharmacological and forensic studies often requires the separation of compounds of interest and/or accurate identification of the whole cannabinoid profile. In order to provide a complete separation and detection of cannabinoids, a new two‐dimensional liquid chromatography method has been developed using acidic potassium permanganate chemiluminescence detection, which has been shown to be selective for cannabinoids. This was carried out using a Luna 100 Å CN column and a Poroshell 120 EC‐C₁₈ column in the first and second dimensions, respectively. The method has utilized a large amount of the available separation space with a spreading angle of 48.4° and a correlation of 0.66 allowing the determination of more than 120 constituents and mass spectral identification of ten cannabinoids in a single analytical run. The method has the potential to improve research involved in the characterization of sensitive, complex matrices.     

A Rapid Nano-Liquid Chromatographic Method for the Analysis of Cannabinoids in Cannabis sativa L. Extracts

Cannabis sativa L. is an herbaceous plant belonging to the family of Cannabaceae. It is classified into three different chemotypes based on the different cannabinoids profile. In particular, fiber-type cannabis (hemp) is rich in cannabidiol (CBD) content. In the present work, a rapid nano liquid chromatographic method (nano-LC) was proposed for the determination of the main cannabinoids in Cannabis sativa L. (hemp) inflorescences belonging to different varieties. The nano-LC experiments were carried out in a 100 µm internal diameter capillary column packed with a C18 stationary phase for 15 cm with a mobile phase composed of ACN/H₂O/formic acid, 80/19/1% (v/v/v). The reverse-phase nano-LC method allowed the complete separation of four standard cannabinoids in less than 12 min under isocratic elution mode. The nano-LC method coupled to ultraviolet (UV) detection was validated and applied to the quantification of the target analytes in cannabis extracts. The nano-LC system was also coupled to an electrospray ionization–mass spectrometry (ESI-MS) detector to confirm the identity of the cannabinoids present in hemp samples. For the extraction of the cannabinoids, three different approaches, including dynamic maceration (DM), ultrasound-assisted extraction (UAE), and an extraction procedure adapted from the French Pharmacopeia’s protocol on medicinal plants, were carried out, and the results achieved were compared.     

Analysis of cannabinoids by liquid chromatography— Thermospray mass spectrometry and liquid chromatography— Tandem mass spectrometry

Summary A rapid method based on liquid chromatography and thermospray mass spectrometry without any derivatization or pre-purification steps has been developed for the identification and quantification of cannabinoids in drugs from cannabis plants. The extracts were separated on a C₁₈ reversed-phase column with an acidic acetonitrile-water gradient. Liquid chromatographymass spectrometry was performed with a thermospray interface and protonated molecular ions were obtained from the cannabinoids of interest. Liquid chromatography-tandem mass spectrometry experiments on the molecular ions gave additional structural information online. The sensitivity and selectivity of the method was sufficient to enable the detection of 100 pg of the cannabinoids.     

Quadrupole‐time‐of‐flight mass spectrometry screening for synthetic cannabinoids in herbal blends

‘Legal highs’ are novel substances which are intended to elicit a psychoactive response. They are sold from ‘head shops’, the internet and from street suppliers and may be possessed without legal restriction. Several months ago, a 19‐year‐old woman came searching for medical treatment as she had health problems caused by smoking legal highs. The substances were sold as herbal blends in plastic bags under four different labels. In this work, samples of these herbal blends have been analysed to investigate the presence of psychoactive substances without any reference standard being available at the laboratory. A screening strategy for a large number of synthetic and natural cannabinoids has been applied based on the use of ultra‐high pressure liquid chromatography coupled to quadrupole‐time of flight mass spectrometry (UHPLC‐QTOF MS) under MS^E mode. A customized home‐made database containing literature‐based exact masses for parent and product ions of around 200 synthetic and natural cannabinoids was compiled. The presence of the (de)protonated molecule measured at its accurate mass was evaluated in the samples. When a peak was detected, collision‐induced dissociation fragments and characteristic isotopic ions were also evaluated and used for tentative identification. After this tentative identification, four synthetic cannabinoids (JWH‐081, JWH‐250, JWH‐203 and JWH‐019) were unequivocally confirmed by subsequent acquisition of reference standards. The presence in the herbal blends of these synthetic cannabinoids might explain the psychotic and catatonic symptoms observed in the patient, as JWH compounds could act as potent agonists of CB₁ and CB₂ receptors located in the Limbic System and Basal ganglia of the human brain. Copyright © 2013 John Wiley & Sons, Ltd.     

Elucidation of the Chemical Structure of a Gas Chromatographic Artifact of Synthetic Cannabinoid <Emphasis Type="Italic">N</Emphasis>-(1-Carbamoyl-2-Methylpropyl)-1-(Cyclohexylmethyl)-1<Emphasis Type="Italic">H</Emphasis>-Indazol-3Ccarboxamide by High-Resolution Mass Spectrometry

Synthetic cannabinoids are the most popular psychoactive compounds on the illegal market. In the gas chromatographic determination, some synthetic cannabinoids undergo chemical transformations because of their thermal interaction with the chromatographic system. This paper is devoted to the elucidation of the structure of a gas chromatographic artifact formed from synthetic cannabinoid N-(1-carbamoyl-2-methylpropyl)-1-(cyclohexylmethyl)-1H-indazol-3-carboxamide as a result of dehydration of its terminal carbamoyl moiety. The chemical structure of the artifact is determined by high-resolution mass spectrometry.     

Simultaneous analysis of several synthetic cannabinoids,THC, CBD and CBN,in hair by ultra‐high performance liquid chromatography tandem mass spectrometry. Method validation and application to real samples

A simple procedure for the quantitative detection of JWH‐018, JWH‐073, JWH 200, JWH‐250, HU‐210, Δ⁹‐tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in hair has been developed and fully validated. After digestion with NaOH and liquid–liquid extraction, the separation was performed with an ultra‐high performance liquid chromatography system coupled to a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode. The absence of matrix interferents, together with excellent repeatability of both retention times and relative abundances of diagnostic transitions, allowed the correct identification of all analytes tested. The method was linear in two different intervals at low and high concentration, with correlation coefficient values between 0.9933 and 0.9991. Quantitation limits ranged from 0.07 pg/mg for JWH‐200 up to 18 pg/mg for CBD The present method for the determination of several cannabinoids in hair proved to be simple, fast, specific and sensitive. The method was successfully applied to the analysis of 179 real samples collected from proven consumers of Cannabis, among which 14 were found positive to at least one synthetic cannabinoid. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination and identification of synthetic cannabinoids and their metabolites in different matrices by modern analytical techniques – a review

Synthetic cannabinoids have gained popularity due to their easy accessibility and psychoactive effects. Furthermore, they cannot be detected in urine by routine drug monitoring. The wide range of active ingredients in analyzed matrices hinders the development of a standard analytical method for their determination. Moreover, their possible side effects are not well known which increases the danger.     

Simple approach for evaluation of matrix effect in the mass spectrometry of synthetic cannabinoids

A simple approach for studying and identifying matrix effect is described. This method for the determination of matrix effect combines the advantages of two most popular traditional methods while eliminating their disadvantages. A postcolumn infusion system was used to observe the MS signal alterations of synthetic cannabinoids: UR-144, XLR-11 and STS-135. Protein precipitation, liquid–liquid extraction and solid phase extraction sample preparation methods were tested. The results of the experiments showed that the discussed method of matrix effect estimation can have practical application in the development of analytical methods. The comparison of the normalized matrix effect profiles can be done even for data obtained over time. Obtained results also indicated that matrix effect was highly dependent on sample preparation. Although similar structure, significant differences were observed for different synthetic cannabinoids.     

Simultaneous determination of Delta9-tetrahydrocannabinol, 11-hydroxy-Delta9-tetrahydrocannabinol and 11-nor-9-carboxy- Delta9-tetrahydrocannabinol in human plasma by high-performance liquid chromatography/tandem mass spectrometry

A rapid and sensitive method for the simultaneous confirmatory analysis of three forensic most relevant cannabinoids, Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH), by means of high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) in human plasma was developed and fully validated. Sample clean-up was performed by automated silica-based solid-phase extraction and the separation was carried out using a PhenylHexyl column (50 x 2 mm i.d., 3 micro m) and acetonitrile-5 mM ammonium acetate gradient elution. Data were acquired with an API 3000 LC/MS/MS system equipped with a turboionspray interface and triple quadrupole mass analyzer using positive electrospray ionization and multiple reaction monitoring. Two MS/MS transitions for each substance were monitored and deuterated analogues of analytes were used as internal standards for quantitation. The limit of quantitation was 0.8 ng ml(-1) for THC, 0.8 ng ml(-1) for 11-OH-THC and 4.3 ng ml(-1) for THC-COOH and linearity with a correlation coefficient r(2) = 0.999 was achieved up to 100 ng ml(-1) for THC and 11-OH-THC and 500 ng ml(-1) for THC-COOH. The limits of detection were 0.2 ng ml(-1) for THC, 0.2 ng ml(-1) for 11-OH-THC and 1.6 ng ml(-1) for THC-COOH. The developed LC/MS/MS method was also successfully used for the determination of THC-COOH-glucuronide, the phase II metabolite of THC-COOH.     

Fragmentation pathways of drugs of abuse and their metabolites based on QTOF MS/MS and MS(E) accurate-mass spectra

A study of the fragmentation pathways of several classes of drugs of abuse (cannabinoids, ketamine, amphetamine and amphetamine-type stimulants (ATS), cocaine and opiates) and their related substances has been made. The knowledge of the fragmentation is highly useful for specific fragment selection or for recognition of related compounds when developing MS-based analytical methods for the trace-level determination of these compounds in complex matrices. In this work, accurate-mass spectra of selected compounds were obtained using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry, performing both MS/MS and MS(E) experiments. As regards fragmentation behavior, the mass spectra of both approaches were quite similar and were useful to study the fragmentation of the drugs investigated. Accurate-mass spectra of 37 drugs of abuse and related compounds, including metabolites and deuterated analogues, were studied in this work, and structures of fragment ions were proposed. The accurate-mass data obtained allowed to confirm structures and fragmentation pathways previously proposed based on nominal mass measurements, although new insights and structure proposals were achieved in some particular cases, especially for amphetamine and ATS, 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THC-COOH) and opiates.     

LC/ESI-MS/MS method for quantification of 28 synthetic cannabinoids in neat oral fluid and its application to preliminary studies on their detection windows

Serum and urine samples are commonly used for the analysis of synthetic cannabinoids in biofluids; however, their utilization as analytical matrices for drug abstinence control features some substantial drawbacks. While for blood collection invasive sampling is inevitable, the urinary analysis of synthetic cannabinoids is limited by the lack of available reference standards of the respective major metabolites. Moreover, the long detectability of synthetic cannabinoids in both matrices hampers the identification of a recent synthetic cannabinoid use. This article describes the development, validation and application of an LC/ESI-MS/MS method for the quantification of 28 synthetic cannabinoids in neat oral fluid (OF) samples. OF samples were prepared by protein precipitation using ice-cold acetonitrile. Chromatographic separation was achieved by gradient elution on a Luna Phenyl Hexyl column (50?×?2 mm, 5 μm), while detection was carried out on a QTrap 4000 instrument in positive ionization mode. The limits of detection ranged from 0.02 to 0.40 ng/mL, whereas the lower limits of quantification ranged from 0.2 to 4.0 ng/mL. The method was applied to authentic samples collected during two preliminary studies in order to obtain insights into the general detectability and detection windows of synthetic cannabinoids in this matrix. The results indicate that synthetic cannabinoids are transferred from the blood stream into OF and vice versa only at a very low rate. Therefore, positive OF samples are due to contamination of the oral cavity during smoking. As these drug-contaminations could be detected up to approximately 2 days, neat oral fluid appears to be well suited for detection of a recent synthetic cannabinoid use.

Analysis of abuse drugs in urine using surfactant-assisted dispersive liquid-liquid microextraction

The process of surfactant-assisted dispersive liquid-liquid microextraction (SA-DLLME) followed by high-performance liquid chromatography-UV detection was successfully applied for the extraction and determination of selected cannabinoids (cannabidiol, Δ(9)-tetrahydrocannabinol, and cannabinol) in urine samples. The effective parameters on the extraction efficiency were studied and optimized utilizing two different optimization methods: one variable at a time (OVAT) and face center design (FCD). Under the optimum conditions (extraction solvent and its volume, toluene, 85 μL; disperser agent and its concentration, 1.0 mL of ultra-pure water containing 0.5 mmol/L tetradecyl tremethyl ammonium bromide (TTAB); sample pH, 2.0 and salt concentration, 11% w/v NaCl), the limits of detection of the method were in the range of 0.1-0.5 μg/L and the repeatability and reproducibility of the proposed method, expressed as relative deviation, varied between 4.1 and 8.5% and 6.7 and 11.6%, respectively. Linearity was found to be in the range of 1.0-200 μg/L and under the optimum conditions, the preconcentration factors (PFs) were between 190 and 292. This proposed method was successfully applied in the analysis of three male advocate urine samples and good recoveries were obtained.     

Determination of cannabinoids in plasma using salting-out-assisted liquid–liquid extraction followed by LC–MS/MS analysis

The detection of the markers of Cannabis consumption in biological specimens is an important task for drug testing laboratories in varous contexts. A simple assay combining salting-out assisted liquid–liquid extraction sample preparation and LC–MS/MS analysis was applied to the measurement of Δ⁹-tetrahydrocannabinol, 11-nor-9-carboxy-Δ⁹-tetrahydrocannabinol (THC-COOH), 11-hydroxy-Δ⁹-tetrahydrocannabinol, cannabinol and cannabidiol concentrations in 100 μl plasma specimens. The assay had linearity of 1–100 ng ml⁻¹ for THC-COOH and 0.5–50 ng ml⁻¹ for the other tested cannabinoids. Assay validation criteria were fulfilled. Extraction yields (88.7–97.3%) and internal-standard correct matrix effects (−9.6 to +5.4%) were acceptable. The assay was applied to 238 clinical specimens from trauma patients, with 19 samples presenting quantifiable concentrations of at least one of the target compounds. The developed assay is a simple and efficient strategy for simultaneous measurement of Δ⁹-tetrahydrocannabinol, THC-COOH, 11-hydroxy-Δ⁹-tetrahydrocannabinol, cannabinol and cannabidiol concentrations in plasma specimens.