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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

Opioid plasma concentrations during a switch from transdermal fentanyl to methadone.

Opioid switching is often used to improve the opioid response in patients with cancer experiencing poor analgesia or adverse effects. When switching between drugs with delayed effect because of pharmacokinetics or type of delivery, concerns exist about the correct timing of introducing the second drug after stopping the previous one. The aim of this study was to assess plasmatic changes of fentanyl and methadone underlying the clinical events occurring during opioid switching. Eighteen patients with cancer receiving transdermal fentanyl with uncontrolled pain and/or moderate to severe opioid adverse effects, were switched to oral methadone using an initial fixed ratio of 1:20. Fentanyl patches were removed and the first of three daily doses of methadone was started concurrently. Blood samples were obtained at intervals after removing the fentanyl patch, and at 5-hour intervals for the first 25 hours. The intensity of pain and the adverse effects were assessed before switching, the day after, and then daily up to dose stabilization. A successful switch was considered when the intensity of pain and distress score decreased at least of 33% of the basal value recorded before the switch, within a reasonable period of time. Complete blood samples were obtained in 16 patients. Methadone plasma concentration increased from 2 to 245 ng/mL, and fentanyl plasma concentration decreased from 15 to 8 ng/mL, 25 hours after. A successful switch was determined the day after in 7 patients, while 4 patients did not respond favorably (effective switching, 63%). Five patients were considered too terminal for an appropriate evaluation. No differences in plasma concentration pattern of the two opioids were found between patients considered responders and nonresponders. Conversion ratios between opioids at time of stabilization did not significantly change in comparison with the initial conversion ratio chosen. Starting methadone soon after removing fentanyl patches results in a rapid increase of methadone concentration, while the half-life of transdermal fentanyl is reached after 25 hours. As a result, the rapid achievement of a clinical effect is obtained avoiding distressing therapeutic holes in patients with a clinical condition, mainly characterized by poor pain control and severe adverse effects, requiring an immediate intervention.

A case of fatal intoxication from metformin.

A case of fatal intoxication from metformin is presented. The decedent was an obese 58-year-old-woman with type II diabetes, in whom severe lactic acidosis secondary to metformin accumulation was precipitated by acute renal failure. She had been on metformin 500 mg twice a day. Postmortem blood was deproteinated with acetonitrile, washed with dichloromethane, and the resulting supernatant injected into high-performance liquid chromatography system. Separation was performed on a analytical 125 x 4 mm i.d. RP-8 column. The wavelength was set at 235 nm. The mobile phase was acetonitrile (40%), sodium lauryl sulfate, and sodium dihydrogen phosphate adjusted to pH 5.1 (60%) at a flow rate of 1.0 mL/min. The concentration of metformin in postmortem blood was 77.3 microg/mL. The qualitative result was also confirmed by LC/APCI/MS/MS analysis.

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.

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.