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.

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.

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.

Identification of 1-butyl-3-(1-(4-methyl)naphtoyl)indole detected for the first time in "herbal high" products on the Italian market.

The results of the analysis of some herbal products and powders adulterated with alkylindoles recovered on the Italian market between 2010 and 2011 are reported. Besides the well-known alkyindoles JWH-018 and JWH-073, other derivatives such as JWH-250 and AM-694 have been detected and for the first time in Italy 1-butyl-3-(1-(4-methyl)naphthoyl)indole (compound 1), the 4-methylnaphthoyl analogue of JWH-073. This compound as well as the other alkylindoles has been synthesized and characterized by (1)H NMR, (13)C NMR, DSC, GC/MS, and elemental analysis. The quantitative analyses of the samples have been carried out by means of the GC/FID method developed in our laboratory for the analysis of herbal high products containing naphthoylindoles; the quantity of the cannabimimetic substances ranged from 6 mg/g to 47 mg/g.

Analysis of fluid extracts obtained from Papaver rhoeas petals contaminated with Papaver bracteatum petals.

In this paper, we report a case of misidentification of medicinal plants involving dried petals of Papaver rhoeas (red poppy) contaminated with Papaver bracteatum (scarlet poppy) petals. Preliminary TLC analysis indicated the presence of thebaine either in the fluid extracts or in the petals. It was therefore necessary to carry out an accurate botanic examination of the plant material, which revealed contamination of the red poppy petals with scarlet poppy petals. Moreover, to confirm the adulteration, we developed and validated an efficient, reversed-phase ion pair HPLC method for determination of the alkaloids specific for the Papaver species. Six petal batches and five commercial fluid extracts were analyzed. Only one petal batch from Iran contained thebaine and its analogue oripavine while the alkaloids typical for the Papaver bracteatum species were identified in all fluid extracts, meaning that they were all prepared with contaminated petals.

The effects of excipients for topical preparations on the human skin permeability of terpinen-4-ol contained in Tea tree oil: infrared spectroscopic investigations.

This work aimed to evaluate the effect induced by excipients conventionally used for topical dosage forms, namely isopropyl myristate (IPM) or oleic acid (OA) or polyethylene glycol 400 (PEG400) or Transcutol (TR), on the human skin permeability of terpinen-4-ol (T4OL) contained in the pure Tea tree oil. The effect of such excipients was determined by evaluating the absorption of T4OL using human epidermis and the perturbation of the organization of stratum corneum by ATR-FTIR. Among the tested excipients OA enhanced the absorption of T4OL by perturbing the stratum corneum lipid barrier. Other excipients caused a weak enhancement effect and their use should be carefully monitored.

Formulation study of tea tree oil patches.

The antimicrobial, antifungal and anti-inflammatory properties of tea tree oil (TTO), the essential oil of Melaleuca alternifolia are well documented. In order to optimize its therapeutic activity, TTO patches were designed. The aim of this work was the formulation of monolayer patches containing TTO. Moreover, the performance of oleic acid (OA) as a skin penetration enhancer in patches was evaluated. Terpinen-4-ol (T4OL), the main component of TTO, was the marker used to evaluate TTO skin permeability. The permeation study was performed through human epidermis by using Franz diffusion cells. Patches were prepared by using methacrylic copolymers, Eudragit E100 (EuE100) or Eudragit NE (EuNE), and a silicone resin, BioPSA7-4602 (Bio-PSA). TTO and OA contents were fixed at 10% w/w and 3% w/w, respectively. The patches were prepared by a casting method and characterised in terms of T4OL content and skin permeability. All the selected polymers were suitable as the main component of the patch matrix. Since the main critical issue in the use of TTO is related to its toxicity after absorption, the local administration of TTO can take advantage of the use of patches based on EuE100 because of the high retained amount and the low permeation of T4OL. In this matrix, OA slightly increased the T4OL retained amount, improving the efficacy and safety of TTO patches.

Blood cyanide determination in two cases of fatal intoxication: comparison between headspace gas chromatography and a spectrophotometric method.

Blood samples of two cases were analyzed preliminarily by a classical spectrophotometric method (VIS) and by an automated headspace gas chromatographic method with nitrogen-phosphorus detector (HS-GC/NPD). In the former, hydrogen cyanide (HCN) was quantitatively determined by measuring the absorbance of chromophores forming as a result of interaction with chloramine T. In the automated HS-GC/NPD method, blood was placed in a headspace vial, internal standard (acetonitrile) and acetic acid were then added. This resulted in cyanide being liberated as HCN. The spectrophotometric (VIS) and HS-GC/NPD methods were validated on postmortem blood samples fortified with potassium cyanide in the ranges 0.5-10 and 0.05-5 mug/mL, respectively. Detection limits were 0.2 mug/mL for VIS and 0.05 mug/mL for HS-GC/NPD. This work shows that results obtained by means of the two procedures were insignificantly different and that they compared favorably. They are suitable for rapid diagnosis of cyanide in postmortem cases.