Development and Early Identification of Cannabis Chemotypes during the Plant Growth: Current Analytical and Chemometric Approaches.

The identification of cannabis chemotypes at an early stage of a plant's growth, which is long before anthesis, has been intensively pursued in order to control the on-target selection of the cultivar type at the beginning of cultivation, so as to avoid economic and legal drawbacks. However, this issue has been systematically addressed by only few and relatively recent studies of analytical chemistry, possibly because result validations require long-term monitoring of the content and ratio of cannabinoids and terpenes in a great number of plant specimens suitably selected and grown. Here, we review the procedures, the chromatographic techniques and the statistics used in topical investigations during the past thirteen years. Through heterogeneous and not easily comparable approaches, they prove the feasibility of chemotypes safe determination within the first month of a plant's life.

Characterization of chemotype-dependent terpenoids profile in cannabis by headspace gas-chromatography coupled to time-of-flight mass spectrometry.

A headspace method called full evaporation technique (FET) coupled to capillary gas chromatography with a mass detector operating in time-of-flight mode (HS-GC/MS-TOF) was developed to characterize the volatile components, especially the terpene fraction, in Cannabis sativa L. inflorescences. This analytical approach allows to reach a high equilibration temperature, that was able to obtain a complete quantification of the volatile components, providing simple sample preparation, specific qualitative detection, high sensitivity, a precise and accurate quantitative determination. The method was applied to 20 cannabis THC-dominant (I) and 13 CBD-dominant (III) chemotypes. The obtained results were then compared with a series of standard solutions consisting of 35 terpenoids and the mass spectra present in a computer library (NIST). The method has an accuracy of more than 90 % and a limit of detection of 5 ppm for all analytes. The main terpenoids in cannabis are namely (% Chemotypes III vs. I of the total terpene content): ß-Caryophyllene (25 vs. 19.3), ß-Mircene (18.2 vs. 20.0), Terpinolene (12.1 vs. 7.0), α-Humulene (6.5 vs. 8.5), D-Limonene (6.2 vs. 7.2), α-Pinene (5.8 vs. 4.9), ß-Pinene (5.0 vs. 5.8) and cis-ß-Ocimene (4.3 vs. 5.2), whose presence is confirmed in both plant chemotypes and account for more than 80 % of the total terpenoids amount. The terpenoids which can clearly distinguish the chemotype are α-Terpineol, Linalool, DL-Menthol, α-Cedrene, and Borneol. This application provides important data on the secondary volatile components of the plant, which may be useful for a better understanding of the therapeutic properties of the cannabis phyto-complex. It gives the possibility of establishing the aroma profile of different Cannabis batches, allowing possible similarities between samples and identifying any artificial adulteration such as hexyl butyrate ester and it provides the opportunity to highlight some target compounds characteristic of the different chemotypes.

Phytocannabinoids Profile in Medicinal Cannabis Oils: The Impact of Plant Varieties and Preparation Methods.

Cannabis (Cannabis sativa L.) is a highly promising medicinal plant with well-documented effectiveness and growing use in the treatment of various medical conditions. Cannabis oils are mostly used in galenic preparations, due to their easy adjustment of the administration dose, together with the enhanced bioavailability of its active compounds. As stated by the Italian Law (9/11/2015, 279 Official Gazette), "to ensure the quality of the oil-based cannabis preparation, the titration of the active substance(s) should be carried out." This study aims to represent the Italian panorama of cannabis oils, which were analyzed (8,201) to determine their cannabinoids content from 2017 to 2019. After application of the exclusion criteria, 4,774 standardized cannabis oils were included, which belong to different medicinal cannabis varieties and prepared according to different extraction methods. The concentration of the principal cannabinoids was taken into account dividing samples on the basis of the main extraction procedures and cannabis varieties. According to this analysis, the most substantial variations should be attributed to different cannabis varieties rather than to their extraction protocols. This study may be the starting point of preparatory pharmacists to assess the correct implementation of the preparation procedures and the quality of the extracts.

A straightforward LC-MS/MS analysis to study serum profile of short and medium chain fatty acids.

Short and medium fatty acids derived from either dietary sources, gut microbiota, and liver production might play a role in the modulation of metabolism and inflammation. The outcome of different autoimmune or inflammatory diseases could be related to microbiota composition and consequently fatty acids production. Their analytical detection, historically completed by GC, was herein investigated using a sensitive approach of LC-MS/MS with straightforward chemical derivatization, using 3-NPH, to the respective acylhydrazines. An isopropanol protein precipitation coupled to LC-MS/MS analysis allowed to separate and quantify butyric, valeric, hexanoic acid and their branched forms. The serum physiological ranges of short and medium chain fatty acids were determined in a heterogeneous healthy population (n = 54) from 18 to 85 years finding a concentration of 935.6 ±â€¯246.5 (butyric), 698.8 ±â€¯204.7 (isobutyric), 62.9 ±â€¯15.3 (valeric), 1155.0 ±â€¯490.4 (isovaleric) and 468.7 ±â€¯377.5 (hexanoic) ng/mL respectively (mean ±â€¯SD). As expected, the biological levels in human serum are reasonably wide-ranging depending on several factors such as body-weight, gut microbiome dysbiosis, gut permeability, cardiometabolic dysregulation, and diet.

The Italian panorama of cannabis light preparation: Determination of cannabinoids by LC-UV.

Cannabis light preparations are products derived or containing dried female inflorescences of Cannabis sativa belonging to Chemotype III (THC/CBD ratio <<1); the total THC (THC+THCA) content in the crop must not exceed 0.2 % in accordance with the EU regulation. In Italy the most recent law for industrial hemp (242/2016) states that only for farmers this limit is extended to 0.6 %. On the other hand, the Ministry of the Interior published a note stating that the sale or the presence in the markets of products (inflorescences, concentrates, essences and resins) or plants with concentrations higher than 0.5 % constitutes a crime. In this confusing legislation framework, it is very important to assess the legality of hemp, determining the total amount of THC. To this end a reliable LC-UV analytical method was developed and validated taking into account parameters such as precision, accuracy, linearity, repeatability of peak area and retention time, limit of detection (LOD= 0.002 % for all cannabinoids) and limit of quantification (LOQ= 0.005 % for all cannabinoids). Accuracy was expressed as the relative error (Er%), while precision was measured as the coefficient of variation (CV%). A CV% below 3 % and Er% between ± 6 % were obtained. The linearity was proven in the concentration range 0.005-1 % for THC, THCA and CBN and 0.005 %-50 % for CBD and CBDA. The analytical method was applied to more than nine hundred cannabis light samples. Based on the law 242/2016, only 18 % of the crops are to be considered legal for the market (total THC<0.2 %). If the circular of the Ministry of the Interior should be converted as a proper law, a substantial amount of cannabis light preparations (24 %) would be considered illegal (total THC>0.5 %). On the other hand, the most of the inflorescences (58 %) have a total THC content comprised between 0.2 % and 0.5 %, and it is not clear whether these products could be sold or not. Moreover, Cannabis light products are not authorized for human consumption, even if everybody knows that this is their primary use. In conclusion, the cannabis light panorama in Italy is quite confused and more specific and clear legislation should be proposed.

Comparison of Different Analytical Methods for the Determination of Carbon Monoxide in Postmortem Blood.

The determination of carbon monoxide (CO) and carboxyhemoglobin (COHb) is of utmost importance in forensic toxicology to determine the cause of death in cases of CO poisoning, fire, and explosions. To this end, reliable and updated analytical methods are required. In this paper, four different methods for the determination of carbon monoxide in postmortem blood samples were compared: (i) the spectrophotometric determination of COHb applying the method proposed by Rodkey and modified by Beutler-West, (ii) the spectrophotometric determination of CO using a micro-diffusion-based method, (iii) the determination of CO by gas chromatography coupled to a TCD detector, and (iv) the determination of COHb by blood gas analysis. Three postmortem blood samples were analyzed with all methods, and the results were comparable. The applied methodologies showed different features depending on the sensitivity, sample preparation, and volume. The HS-GC/TCD method in our hand was the most appropriate, on postmortem samples, and versatile to apply. Unfortunately, only a limited number of postmortem blood samples were available for this study due to the rarity of that kind of intoxication in our jurisdiction.

Urinary TMAO Levels Are Associated with the Taxonomic Composition of the Gut Microbiota and with the Choline TMA-Lyase Gene (cutC) Harbored by Enterobacteriaceae.

Gut microbiota metabolization of dietary choline may promote atherosclerosis through trimethylamine (TMA), which is rapidly absorbed and converted in the liver to proatherogenic trimethylamine-N-oxide (TMAO). The aim of this study was to verify whether TMAO urinary levels may be associated with the fecal relative abundance of specific bacterial taxa and the bacterial choline TMA-lyase gene cutC. The analysis of sequences available in GenBank grouped the cutC gene into two main clusters, cut-Dd and cut-Kp. A quantitative real-time polymerase chain reaction (qPCR) protocol was developed to quantify cutC and was used with DNA isolated from three fecal samples collected weekly over the course of three consecutive weeks from 16 healthy adults. The same DNA was used for 16S rRNA gene profiling. Concomitantly, urine was used to quantify TMAO by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). All samples were positive for cutC and TMAO. Correlation analysis showed that the cut-Kp gene cluster was significantly associated with Enterobacteriaceae. Linear mixed models revealed that urinary TMAO levels may be predicted by fecal cut-Kp and by 23 operational taxonomic units (OTUs). Most of the OTUs significantly associated with TMAO were also significantly associated with cut-Kp, confirming the possible relationship between these two factors. In conclusion, this preliminary method-development study suggests the existence of a relationship between TMAO excreted in urine, specific fecal bacterial OTUs, and a cutC subgroup ascribable to the choline-TMA conversion enzymes of Enterobacteriaceae.

Determination of daptomycin in human plasma and breast milk by UPLC/MS-MS.

During the lactation, the choice of a proper antibiotic is crucial since the drug can cross into breast milk causing toxicity to the infant. Therefore, an extraction protocol and LC/MS-MS method for the determination of daptomycin in human milk and plasma were developed, validated and applied to a case of a breastfeeding mother affected by a purulent acute soft skin infection treated with daptomycin. Because of daptomycin high protein binding and its high molecular weight, the optimisation of the extraction protocol and analytical conditions were deeply investigated, and several parameters were taken into account: in particular the type of extraction, internal standard, the type of organic modifier, pH of the aqueous solution, and gradient. The use of a protein precipitation protocol coupled to a C8-reverse phase LC-MS/MS allows for a reliable quantification of daptomycin in both plasma (in the range of 19-199 µg/mL) and breast milk (in the range of 0.12-0.32 µg/mL). The determination of milk/plasma (M/P) ratio, which ranged from 0.002 to 0.006, allowed to assess that daptomycin, effective for the mother, was contemporarily safe for the breastfed newborn.

Screening of new psychoactive substances (NPS) by gas-chromatography/time of flight mass spectrometry (GC/MS-TOF) and application to 63 cases of judicial seizure.

A screening method for the separation and identification of more than fifty NPS is proposed. The method is based on fast gas-chromatography/time of flight mass spectrometry (FAST-GC/MS-TOF). Thanks to the shorter and narrower capillary column and to the rapid acquisition of the TOF detector a huge number of compounds are separated in a very short time of analysis (10 min). Only a few peaks were overlapped. The possibility to apply deconvolution by the software of the GC/MS-TOF instrument allowed the unequivocal identification also for the superimposed peaks. Linearity and LOD was studied and the method was applied to 63 cases of powders seized by the judicial authority at the airport of Milano Malpensa in Northern Italy in the period 2014-2017.

Determination of Cyanide by Microdiffusion Technique Coupled to Spectrophotometry and GC/NPD and Propofol by Fast GC/MS-TOF in a Case of Poisoning.

A man was found dead in a hotel located near Rome (Italy). The man was still holding a syringe attached to a butterfly needle inserted in his left forearm vein. The syringe contained a cloudy pinkish fluid. In the hotel room the Police found a broken propofol glass vial plus four sealed ones, an opened NaCl plastic vial and six more still sealed, and a number of packed smaller disposable syringes and needles. An opened plastic bottle containing a white crystalline powder labeled as potassium cyanide was also found. Systematic toxicological analysis (STA), carried out on blood, urine and bile, evidenced only the presence of propofol in blood and bile. So the validated L-L extraction protocol and the GC/MS-TOF method for the confirmation of propofol in the biological fluids optimized in our laboratory was applied to blood, urine and bile. The concentration of propofol resulted to be 0.432 µg/mL in blood and 0.786 µg/mL in bile. The quantitative determination of cyanide in blood was carried out by microdiffusion technique coupled to spectrophotometric detection obtaining a cyanide concentration of 5.3 µg/mL. The quantitative determination was then confirmed by GC/NPD and the concentration of cyanide resulted to be 5.5 µg/mL in blood and 1.7 µg/mL in bile. Data emerging from autopsy findings, histopathological exams and the concentrations of cyanide suggested that death might be due to poisoning caused by cyanide, however, respiratory depression caused by propofol could not be excluded.

Extraction Method and Analysis of Cannabinoids in Cannabis Olive Oil Preparations.

Recently, an increasing number of pharmacists had to supply medicinal products based on Cannabis sativa L. (Cannabaceae), prescribed by physicians to individual patients. Cannabis olive oil preparation is the first choice as a concentrated extract of cannabinoids, even though standardized operative conditions for obtaining it are still not available. In this work, the impact of temperature and extraction time on the concentration of active principles was studied to harmonize the different compounding methods, optimize the extraction process, and reduce the variability among preparations. Moreover, starting from the cannabis inflorescence, the effect of temperature on tetrahydrocannabinolic acid decarboxylation was evaluated. For the analysis, a GC/MS method, as suggested by the Italian Ministry of Health, and a GC/flame ionization detection method were developed, validated, and compared.

Determination of Propofol by GC/MS and Fast GC/MS-TOF in Two Cases of Poisoning.

Two cases of suspected acute and lethal intoxication caused by propofol were delivered by the judicial authority to the Department of Sciences for Health Promotion and Mother-Child Care in Palermo, Sicily. In the first case a female nurse was found in a hotel room, where she lived with her mother; four 10 mg/mL vials and two 20 mg/mL vials of propofol were found near the decedent along with syringes and needles. In the second case a male nurse was found in the operating room of a hospital, along with a used syringe. In both cases a preliminary systematic and toxicological analysis indicated the presence of propofol in the blood and urine. As a result, a method for the quantitative determination of propofol in biological fluids was optimized and validated using a liquid-liquid extraction protocol followed by GC/MS and fast GC/MS-TOF. In the first case, the concentration of propofol in blood was determined to be 8.1 µg/mL while the concentration of propofol in the second case was calculated at 1.2 µg/mL. Additionally, the tissue distribution of propofol was determined for both cases. Brain and liver concentrations of propofol were, respectively, 31.1 and 52.2 µg/g in Case 1 and 4.7 and 49.1 µg/g in Case 2. Data emerging from the autopsy findings, histopathological exams as well as the toxicological results aided in establishing that the deaths were due to poisoning, however, the manner of death in each were different: homicide in Case 1 and suicide in Case 2.

A possible biomarker for methadone related deaths.

Methadone (MTH) concentrations in those dying of MTH toxicity totally overlap concentrations where the presence of MTH is only an incidental finding, making it very difficult to make distinctions in actual cases. A biomarker, be it anatomical or biochemical for MTH toxicity is badly needed, particularly if that markers were known to disrupt effective ventilation. Because the brainstem houses the regulatory centers for cardiorespiratory-control enters, it would seem to be the most likely anatomical site to seek abnormalities in cardiorespiratory control. OBJECTIVE: To locate and describe the cells of nucleus of the solitary tract (TS)(NTS) in human brainstem and determine if neuronal cell death, either necrotic or apoptotic, within the TS of humans is more common in deaths due directly to MTH toxicity than with in the solitary tract itself. DESIGN, SETTING, PARTICIPANTS: This was a single cohort study of MTH related decedents autopsied at a large university hospital. Each decedent had a recent history of non medical/illicit MTH use and had been pronounced dead in the field, prior to ever reaching the hospital. Complete autopsy and complete toxicology testing were performed on the formalin fixed brains of each individual. Multiple blocks were prepared of the area of interest, namely the tissue lying immediately between the inferior and the super colliculi. This volume, by definition, would have included the area of the Rostral Ventrolateral Medulla (RVLM), the location of the TS. Immunohistochemistry studies utilizing caspase-9 reaction (a protease enzyme involved in the process of preprogrammed death) were performed in order to estimate the degree and proportion of neuronal apoptosis, and also access the degree of classical necrosis within the NTS. MAIN OUTCOMES AND MEASURES: The primary outcome measure was the presence or absence of neuronal apoptosis and/or necrosis within the NTS. RESULTS: Cells displaying evidence of early apoptosis and advanced apoptosis, consisting primarily of nuclear fragmentation, admixed with other neurons displaying the features of classic necrosis were found. Evidence of classic necrosis was identifiable in most of the controls, though minor degrees of apoptosis were identifiable with Caspase staining and quantitative image analysis of immunohistochemical stains. CONCLUSIONS: and Relevance: Our study shows that neurons, primarily along the TS, but occasionally in other cell nuclei (even controls) are vulnerable, both to direct MTH toxicity (via apoptosis) and indirectly (via hypoxia leading to classical cell necrosis). When MTH is found to be present in significant concentrations, but apoptotic lesions are absent, it would be reasonable to assume that MTH was not primarily the cause of cardiorespiratory arrest.

DNA-based taxonomic identification of basidiospores in hallucinogenic mushrooms cultivated in "grow-kits" seized by the police: LC-UV quali-quantitative determination of psilocybin and psilocin.

The taxonomic identification of the biological material contained in the hallucinogenic mushrooms culture media, was carried out using a DNA-based approach, thus highlighting the usefulness of this approach in the forensic identification of illegal samples also when they are present as basidiospores mixed in culture media and spore-bearing fruiting body are not present. This approach is very useful as it allows the unequivocal identification of potentially illicit material before the cultivation and it enables to stop the material to the Customs and to destroy it due to its dangerousness without cultivating the "grow-kits" and without instructing a criminal case. In fact, even if psilocin and psilocybin and the whole mushrooms are illegal in many countries, there is no specific indication in the law about the so called "grow-kits", containing the spores. To confirm the data obtained by the taxonomic identification, a simple, reliable, efficient LC-UV method, using tryptamine as internal standard, suitable for the forensic quali-quantitative determination of psilocin and psilocybin in hallucinogenic mushroom was optimized, validated and applied to the mushrooms grown after the cultivation of the grow-kits seized by the judicial authority, with the authorization of the Ministry of Health. A cation exchange column was used in a gradient elution mode (Phase A: 50mMK2HPO4; 100mM NaCl pH=3 Phase B: methanol). The developed method was linear over the calibration range with a R(2)>0.9992 for both the analytes. The detection and quantification limits were respectively 0.01 and 0.1µg/mL for psilocybin and 0.05µg/mL and 0.1µg/mL for psilocin and the intra- and inter-day precision was satisfactory (coefficients of variation <2.0% for both the analytes). The content of psilocybin in the mushrooms grown from the seized "grow-kits" ranged from 1.02 to 7.60mg/g of dry vegetable material, while the content of psilocin from 0.415 to 8.36mg/g.

Behavioural and pharmacological characterization of a novel cannabinomimetic adamantane-derived indole, APICA, and considerations on the possible misuse as a psychotropic spice abuse, in C57bl/6J mice.

The novel adamantane derivative APICA (N-(adamantan-1-yl)-1-pentyl-1H-indole-3-carboxamide) was recently identified as a cannabinomimetic indole of abuse. Despite its novel structure, APICA recalls cannabinomimetic indoles, such as representative member JWH-018. In present study, the effects of APICA (1-3mg/kg, i.p.) were tested in C57BL/6J mice, in the Tetrad task which includes the assessment of: body temperature; locomotor activity and behavioural reactivity; nociception; motor coordination; declarative memory. Furthermore, pre-treatment with the CB1 antagonist AM251 (3mg/kg, i.p.) or the CB2 antagonist AM630 (3mg/kg, i.p.) was carried out to characterize APICA activity. Our results show that APICA was able to dose-dependently decrease locomotor activity and behavioural reactivity in the open field, whereas only the highest dose was able to induce hypothermia, analgesia, motor incoordination and recognition memory impairment, with respect to vehicle (p<0.01; p<0.001). The pretreatment with the CB1 antagonist AM251 elicited an increase in body temperature, total distance travelled in the open field, latency to fall down in the Rotarod, and a decrease in tail flick latency (p<0.05; p<0.01). On the other hand, pretreatment with AM630 did not induced significant differences on APICA effects. This study supports preliminary reports on APICA cannabinomimetic properties, extending its detrimental effects on cognitive function. Moreover, these properties can be attributed to the CB1 receptor activity, indicating APICA as a selective CB1 receptor agonist.

Unexpected variation of the codeine/morphine ratio following fatal heroin overdose.

Postmortem samples from 14 cases of suspected heroin overdose were subjected to a preliminary systematic toxicological analysis in order to highlight the presence of unknown exogenous compounds (e.g., drugs of abuse, alcohol) that may have played a role in the mechanism of death. This analysis unveiled histories of poly-drug use in seven of the cases under investigation. Moreover, the concentrations of morphine and codeine in the brain were also investigated, and the results were compared with the data obtained from the blood specimens. The concentration of morphine in blood ranged from 33 to 688 ng/mL, while the concentration of codeine ranged from 0 to 193 ng/mL. However, in the brain, the concentration of morphine was found to be between 85 and 396 ng/g, while the levels of codeine ranged from 11 to 160 ng/g. The codeine/morphine ratio in the blood ranged from 0.043 to 0.619; however, in the brain, the same ratio was found to be between 0.129 and 0.552. In most cases, a significantly higher codeine/morphine ratio was found in the brain, suggesting the accumulation of codeine in brain tissue due its high lipophilicity as compared with morphine.

Characterization of in vitro metabolites of JWH-018, JWH-073 and their 4-methyl derivatives, markers of the abuse of these synthetic cannabinoids.

In vitro incubation with human liver microsomes of JWH-018, JWH-073, JWH-122 and for the first time 1-butyl-3-(1-(4-methyl)naphthoyl)indole (the 4-methylnaphthoyl analogue of JWH-073) was investigated to identify the principal metabolites of alkylindole synthetic cannabinoids, thus helping the discovering of synthetic cannabinoids abusers. The results obtained showed that the most abundant metabolites were mono-hydroxylated derivatives either on the alkyl chain (ω or ω-1 position) or on the indole (presumably in position 5 or 6) and naphthalene moieties. Moreover the extraction conditions of these derivatives from biological fluids, mainly plasma and urine spiked with commercially available metabolite standards, and the incubation procedure were investigated to obtain a fast, reliable and suitable extraction protocol to detect either the parent drugs or their metabolites by means of GC/MS.

Improved GC method for the determination of the active principles of Catha edulis.

The GC method previously reported by our research group for the analysis of the active principles of Catha edulis, i.e. cathine, cathinone and phenylpropanolamine, was considerably improved. N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) as derivatizing agent was employed, thus allowing an accurate determination of the analytes and a suitable internal standard for quantitative analyses (nicotinamide) was introduced. Moreover the chromatographic conditions were carefully studied to improve the separation of the alkaloids and sensitivity. To this end different chromatographic capillary columns and temperature gradients were investigated. The optimized GC method was validated and resulted adequate for the application in forensic analysis. Finally on behalf of the Tribunal, C. edulis vegetable material seized by the police in northern Italy was analyzed, the quantity of cathine ranging from 0.095 to 0.29%, the quantity of PPA from 0.010 to 0.21% and the quantity of cathinone from 0.025 to 0.374% of the weight of the vegetable material.

Capillary electrophoretic and extraction conditions for the analysis of Catha edulis FORKS active principles.

A capillary electrophoretic method, which allowed the detection and separation of the active principles of Catha edulis, i.e. cathinone, cathine and phenylpropanolamine, was developed. A suitable internal standard (nicotinamide), which permitted the quantification of the analytes reducing the variability of the migration times due to EOF changes, was identified. The analytical method was validated, assessing linearity, sensitivity and repeatability, showing optimal features for the analysis of the vegetable material. Moreover extraction conditions were investigated to achieve the exhaustion of the plant material in the fastest and most efficient way to meet the requirements of the Court.