Chemical Characterization and Evaluation of the Antibacterial Activity of Essential Oils from Fibre-Type Cannabis sativa L. (Hemp).

Volatile terpenes represent the largest group of Cannabis sativa L. components and they are responsible for its aromatic properties. Even if many studies on C. sativa have been focused on cannabinoids, which are terpenophenolics, little research has been carried out on its volatile terpenic compounds. In the light of all the above, the present work was aimed at the chemical characterization of seventeen essential oils from different fibre-type varieties of C. sativa (industrial hemp or hemp) by means of GC-MS and GC-FID techniques. In total, 71 compounds were identified, and the semi-quantitative analysis revealed that α- and ß-pinene, ß-myrcene and ß-caryophyllene are the major components in all the essential oils analysed. In addition, a GC-MS method was developed here for the first time, and it was applied to quantify cannabinoids in the essential oils. The antibacterial activity of hemp essential oils against some pathogenic and spoilage microorganisms isolated from food and food processing environment was also determined. The inhibitory effects of the essential oils were evaluated by both the agar well diffusion assay and the minimum inhibitory concentration (MIC) evaluation. By using the agar diffusion method and considering the zone of inhibition, it was possible to preliminarily verify the inhibitory activity on most of the examined strains. The results showed a good antibacterial activity of six hemp essential oils against the Gram-positive bacteria, thus suggesting that hemp essential oil can inhibit or reduce bacterial proliferation and it can be a valid support to reduce microorganism contamination, especially in the food processing field.

Pharmacokinetics and tolerability of oral cannabis preparations in patients with medication overuse headache (MOH)-a pilot study.

PURPOSE: The recent release of a medical cannabis strain has given a new impulse for the study of cannabis in Italy. The National Health Service advises to consume medical cannabis by vaporizing, in decoction or oil form. This is the first study that explores the pharmacokinetics and tolerability of a single oral dose of cannabis as decoction (200 ml) or in olive oil (1 ml), as a first step to improve the prescriptive recommendations. METHODS: This is a single-center, open-label, two-period crossover study designed to assess the pharmacokinetics and tolerability of oral cannabis administered to 13 patients with medication overuse headache (MOH). A liquid chromatography tandem-mass spectrometry (LC-MS/MS) method was conducted for the quantification of THC, CBD, 11-OH-THC, THC-COOH, THC-COOH-glucuronide, THCA-A, and CBDA. Blood pressure, heart rate, and a short list of symptoms by numerical rating scale (NRS) were assessed. RESULTS: Decoctions of cannabis showed high variability in cannabinoids content, compared to cannabis oil. For both preparations, THCA-A and CBDA were the most widely absorbed cannabinoids, while THC and CBD were less absorbed. The most important differences concern the bioavailability of THC, higher in oil (AUC0-24 7.44, 95% CI 5.19, 9.68) than in decoction (AUC0-24 3.34, 95% CI 2.07, 4.60), and the bioavailability of CBDA. No serious adverse events were reported. CONCLUSIONS: Cannabis decoction and cannabis oil showed different pharmacokinetic properties, as well as distinct consequences on patients. This study was performed in a limited number of patients; future studies should be performed to investigate the clinical efficacy in larger populations.

Appearance/Image- and Performance-Enhancing Drug Users: A Forensic Approach.

Image- and performance-enhancing drugs include a wide range of substances used to promote physical changes to enhance appearance. Anabolic androgen steroids are the most widely used image- and performance-enhancing drugs along with a wide variety of additional substances. The aim of this study was to identify the pathological changes pertaining to the cardiovascular system possibly involved in the death and characterize the substances associated with steroid use and their possible role in the death. A series of deaths involving image- and performance-enhancing drug users were selected. Nandrolone and testosterone were the most frequently identified steroids. The most commonly illegal drugs found were tetrahydrocannabinol, cocaine, and methadone. Antidepressants, opioids, benzodiazepines, and barbiturates were also found. These findings highlight that results obtained from postmortem investigations should be meticulously evaluated to understand the weight that each identified substance may have played in the death process and exclude any alternative causes of death.

Monitoring of adherence to headache treatments by means of hair analysis.

PURPOSE: The aim of this study was to evaluate the potential of hair analysis to monitor medication adherence in headache patients undergoing chronic therapy. For this purpose, the following parameters were analyzed: the detection rate of 23 therapeutic drugs in headache patients' hair, the degree of agreement between the self-reported drug and the drug found in hair, and whether the levels found in hair reflected the drug intake reported by the patients. METHODS: The study included 93 patients suffering from primary headaches declaring their daily intake of at least one of the following drugs during the 3 months before the hair sampling: alprazolam, amitriptyline, citalopram, clomipramine, clonazepam, delorazepam, diazepam, duloxetine, fluoxetine, flurazepam, levomepromazine, levosulpiride, lorazepam, lormetazepam, mirtazapine, paroxetine, quetiapine, sertraline, topiramate, trazodone, triazolam, venlafaxine, and zolpidem. A detailed pharmacological history and a sample of hair were collected for each patient. Hair samples were analyzed by liquid chromatography-electrospray tandem mass spectrometry, using a previously developed method. RESULTS: All 23 drugs were detected in the examined hair samples. The agreement between the self-reported drug and the drug found in hair was excellent for most analytes (P < 0.001, Cohen's kappa); a statistically significant relationship (P < 0.05, linear regression analysis) between dose and hair level was found for amitriptyline, citalopram, delorazepam, duloxetine, lorazepam, and venlafaxine. CONCLUSIONS: Hair analysis proved to be a unique matrix to document chronic drug use in headache patients, and the level found for each individual drug can represent a reliable marker of adherence to pharmacological treatments.

Hair analysis for detection of triptans occasionally used or overused by migraine patients-a pilot study.

PURPOSE: The aim of this study is to evaluate the detection rate of almotriptan, eletriptan, frovatriptan, sumatriptan, rizatriptan, and zolmitriptan in the hair of migraineurs taking these drugs; the degree of agreement between type of self-reported triptan and triptan found in hair; if the concentrations in hair were related to the reported cumulative doses of triptans; and whether hair analysis was able to distinguish occasional use from the overuse of these drugs. METHODS: Out of 300 headache patients consecutively enrolled, we included 147 migraine patients who reported to have taken at least one dose of one triptan in the previous 3 months; 51 % of the patients overused triptans. A detailed pharmacological history and a sample of hair were collected for each patient. Hair samples were analyzed by liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) by a method that we developed. RESULTS: All the triptans could be detected in the hair of the patients. The agreement between type of self-reported triptan and type of triptan found in hair was from fair to good for frovatriptan and zolmitriptan and excellent for almotriptan, eletriptan, sumatriptan, and rizatriptan (P < 0.01, Cohen's kappa). The correlation between the reported quantities of triptan and hair concentrations was statistically significant for almotriptan, eletriptan, rizatriptan, and sumatriptan (P < 0.01, Spearman's rank correlation coefficient). The accuracy of hair analysis in distinguishing occasionally users from overusers was high for almotriptan (ROC AUC = 0.9092), eletriptan (ROC AUC = 0.8721), rizatriptan (ROC AUC = 0.9724), and sumatriptan (ROC AUC = 0.9583). CONCLUSIONS: Hair analysis can be a valuable system to discriminate occasional use from triptan overuse.

Cardiac Toxicity in Selective Serotonin Reuptake Inhibitor Users.

Several classes of recreational and prescription drugs have been associated with an increased risk of cardiovascular disease and the occurrence of arrhythmias, which may be involved in sudden deaths in chronic users even at therapeutic doses. The study presented herein focuses on pathological changes involving the heart, which may be caused by selective serotonin reuptake inhibitor use and their possible role in the occurrence of sudden cardiac death. A total of 40 cases were included in the study and were divided evenly into 2 groups: 20 cases of patients treated with selective serotonin reuptake inhibitors and 20 cases of sudden deaths involving patients void of any drug treatment. The first group included 16 patients treated with citalopram and 4 with sertraline. Autopsies, histology, biochemistry, and toxicology were performed in all cases. Pathological changes in selective serotonin reuptake inhibitor users consisted of various degrees of interstitial and perivascular fibrosis as well as a small degree of perineural fibrosis within the myocardium of the left ventricle. Within the limits of the small number of investigated cases, the results of this study seem to confirm former observations on this topic, suggesting that selective serotonin reuptake inhibitors may play a potential, causative role in the pathogenesis of sudden deaths in chronic users even at therapeutic concentrations.

Tramadol abuse in a binge pattern in a young depressed woman.

BACKGROUND: Tramadol is a central analgesic with a unique pharmacological profile in that it is an opioid agonist and an inhibitor of serotonin and norepinephrine reuptake. We describe a case of abuse of tramadol in a binge pattern in a young woman who had initially received the prescription of tramadol as an analgesic as needed. CASE DESCRIPTION: The patient had no history of drug or alcohol abuse, but suffered from depression. Over time, she had increased the doses up to 30 ml of tramadol 100 mg/ml oral solution a week. She took the drug in consecutive 'pinches', from afternoon to evening. Tramadol improved her mood, gave her euphoria, but also caused constipation and urinary retention. Detoxification was carried out by partial replacement with tramadol 100 mg extended release and gradual reduction of the number of pinches. The patient found it hard to follow the program because of withdrawal symptoms of an opioid type and especially because of the worsening of depression. CONCLUSION: The action of tramadol on the monoaminergic system is believed to be a factor that limits abuse liability and gives advantageous antidepressant-like properties, but it also involves the risk of inducing abuse of the analgesic to improve mood as if it were a stimulant.

Innovative methods for the preparation of medical Cannabis oils with a high content of both cannabinoids and terpenes.

Cannabis-based medications are being increasingly used for the treatment of different clinical conditions. Among all galenic formulations, olive oil extracts from medical Cannabis are the most prescribed ones for their easy preparation and usage. A great variety of methods have been described so far for the extraction of medical Cannabis oils to reach a high yield of Δ9-tetrahydrocannabinol (Δ9-THC), but poor attention has been paid to the preservation of the terpene fraction from the plant, which may contribute to the overall bioactivity of the extracts. In this context, the present study was aimed at the chemical characterization of different medical Cannabis oils prepared by following both innovative and existing extraction protocols, with particular attention to cannabinoids and terpenes, in order to set up a suitable method to obtain an extract rich in these chemical classes. In particular, six different extraction procedures were followed, based on different techniques, of which all but one included a decarboxylation of the plant material. The profile of cannabinoids was studied in detail by means of HPLC-ESI-MS/MS, while terpenes were characterized by means both GC-MS and GC-FID techniques coupled with solid-phase microextraction operated in the head-space mode (HS-SPME). An innovative method that is based on the extraction of the oil by dynamic maceration at room temperature from plant inflorescences, which were partially decarboxylated in a closed system at a moderate temperature and partially pre-extracted with ethanol, produced similar yields of bioactive compounds as that obtained by using a microwave-assisted distillation of the essential oil from the plant material, in combination with a maceration extraction of the oil from the residue. Both these new methods provided a higher efficiency over already existing extraction procedures of medical Cannabis oils and they can be applied to obtain a product with a high therapeutic value.

Development of a new method for the analysis of cannabinoids in honey by means of high-performance liquid chromatography coupled with electrospray ionisation-tandem mass spectrometry detection.

Fibre-type Cannabis sativa L. (hemp) represents a valuable resource in many different fields, including both the pharmaceutical and food ones. This plant contains non-psychoactive cannabinoids, a class of bioactive compounds biosynthesized in both female and male inflorescences. Among them, cannabidiol (CBD) is the most interesting compound from a medicinal point of view. Indeed, several scientific studies have proved its therapeutic potential in a large number of pathologies, in addition to its biological effects attributable to its antioxidant, neuroprotective and anti-inflammatory properties. The analysis of the amount of cannabinoids in food and food supplements represents a critical issue in the ambit of both the quality assurance and the dietary intake control of these biologically active compounds. In this ambit, a particular attention is necessary for apiary products, since they are widely consumed and they can be produced by bees starting from different floral sources. In the light of all the above, the aim of this study was to develop for the first time a new analytical method based on RP-HPLC with ESI-MS/MS detection for the determination of CBD and related cannabinoids in honey. A quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction procedure with an un-buffered method was selected and optimised as the more suitable protocol. As regards detection, it was carried out by using a linear ion trap quadrupole (QTRAP) mass analyser, operated in the multiple reaction monitoring (MRM) mode. Hemp male inflorescences and pollen were analysed in parallel by means of HPLC-UV/DAD, since bees can transfer pollen into their hives and, consequently, into beehive products. The method developed and validated for the first time in this work was finally applied to the analysis of cannabinoids in honey samples, thus demonstrating to be a useful tool for both quality control and safety assurance.

Intra-patient variability of the hair levels of pain medications in chronic migraine patients - a pilot study.

Monitoring of prescription pain medications is strongly recommended, considering their abuse liability. We used hair analysis for monitoring pain medications in 12 women with chronic migraine and drug overuse. Hair samples were collected at baseline and after 4 and 8 months and analyzed by liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS). Intra-patient variability of the hair-level-to-dose ratio was <20% in most cases. The hair analysis detected changes in the dose taken by the same patient over time, both occasionally and chronically. The agreement between the changes in hair drug levels and those of taken doses was excellent. Therefore, hair analysis appeared advantageous for long-term monitoring of pain medications, which is otherwise difficult to determine in conventional matrices. In the same sample, multiple medications could be simultaneously determined, and with three samples, nine months of therapy were objectively documented.

Seizures of illicit substances for personal use in two Italian provinces: analysis of trends by type and purity from 2008 to 2017.

BACKGROUND: The use of illicit substances represents one of the most difficult problems to confront in the health system. Drug use is a global problem but is not uniform throughout the world, within the same country and changes over time. Therefore, knowing the illicit substances that are used in a territory is essential to better organize health services in that specific geographical area. To this aim, we analysed 4200 samples confiscated from individuals who held them for personal use by police forces in the Italian provinces of Modena and Reggio Emilia from 2008 to 2017. METHODS: The suspected samples were screened by gas-chromatography-mass spectrometry (GC-MS) and by liquid chromatography-tandem mass spectrometry (LC-MS/MS); all samples were subsequently analysed by gas chromatography-flame ionization detector (GC-FID) for quantitative analyses. RESULTS: Cannabis was the most seized illicit substance (70.7%). Over the study period, the number of seizures of herb with a high content of Δ9-THC increased. The number of cocaine seizures remained stable (total 16.1%), but the median purity of seized cocaine increased to 75% in 2017. Heroin seizures decreased over time, but the median purity of seized heroin reached 16.8% in 2017. In almost all the years, heroin samples with a purity exceeding the 97.5 percentile were found. Especially from 2014, the range of seized substances increased and started to include synthetic cathinones, phenylethylamines, UR-144, LSD, psilocybe, prescription opioid and hypnotics. In two cases, tramadol together with tropicamide was seized. Most of the seizures involved male subjects and 82% of the seizures were from individuals younger than 35 years of age. CONCLUSIONS: The persistence of old illicit drugs and the rapid emergence of new psychoactive substances represented a serious challenge for public health in the studied Italian area. Some useful interventions might be: informing mainly young people about the possible complications of cannabis use; implementing standardized procedures to diagnose and treat cocaine-related emergencies in hospitals; increasing the distribution of naloxone to antagonize possible heroin overdoses; equipping laboratories to be able to identify the new psychoactive substances.

Polypharmacy Among Headache Patients: A Cross-Sectional Study.

BACKGROUND: Polypharmacy can appropriately treat multiple chronic conditions, but it can also increase potential harm. Polypharmacy information for primary headaches is minimal, despite drugs being the main tools to manage headaches. OBJECTIVE: The aim was to evaluate the prevalence, characteristics and risk factors of polypharmacy in patients with primary headaches and examine whether these variables differ between episodic and chronic headache patients. METHODS: We analysed polypharmacy (simultaneous use of five or more medications), medication type, comorbidity, and risk factors in 300 patients (mean age 42.81 ± 13.21 years) with primary headaches, divided into episodic and chronic, afferent to a headache centre. RESULTS: Patients took an average of 4.37 medications. Polypharmacy was common in 40.7% of patients, and among chronic patients, it reached 58.8%. Most patients used medications (mainly nonsteroidal anti-inflammatory drugs; 73.5%) to treat acute headaches, and 30.4% of episodic and 64.7% of chronic sufferers underwent prophylactic treatment (P < 0.0001), mostly using antidepressants (77.3%). Up to 76.7% of the cohort was taking other medications, primarily for acid-related disorders (21.7%). Comorbidities were present in 59.7% of the cohort. Variables significantly associated with polypharmacy were comorbidities, prophylactic treatment, and triptans (P < 0.001). CONCLUSIONS: Patients with primary headaches, mainly young adults, are exposed to high polypharmacy, comparable to that of the elderly. Because increased numbers of drugs increase the risk of adverse reactions, the many medications concomitantly taken by primary headache sufferers should be frequently reviewed.

Development of a simple and sensitive liquid chromatography triple quadrupole mass spectrometry (LC-MS/MS) method for the determination of cannabidiol (CBD), Δ9-tetrahydrocannabinol (THC) and its metabolites in rat whole blood after oral administration of a single high dose of CBD.

The investigation of the possible conversion of cannabidiol (CBD) into Δ9-tetrahydrocannabinol (THC) in vivo after oral administration of CBD is reported herein since recent publications suggested a rapid conversion in simulated gastric fluid. To this end, single high dose of CBD (50mg/kg) was administered orally to rats and their blood was collected after 3 and 6h. A highly sensitive and selective LC-MS/MS method was developed and fully validated in compliance with the Scientific Working Group of Forensic Toxicology (SWGTOX) standard practices for method validation in forensic toxicology. This method also involved the optimization of cannabinoids and their metabolites extraction in order to remove co-eluting phospholipids and increase the sensitivity of the MS detection. Neither THC nor its metabolites were detected in rat whole blood after 3 or 6h from CBD administration. After oral administration, the amount of CBD dissolved in olive oil was higher than that absorbed from an ethanolic solution. This could be explained by the protection of lipid excipients towards CBD from acidic gastric juice.

The role of ethyl glucuronide in supporting medico-legal investigations: Analysis of this biomarker in different postmortem specimens from 21 selected autopsy cases.

Ethanol determination in postmortem blood is one of the most frequently requested analyses in legal medicine and forensic toxicology. Ethyl glucuronide is a non-oxidative ethanol metabolite. It is also a useful marker of ante-mortem alcohol ingestion when ethanol itself has been completely eliminated from the body and could be considered in autopsy cases to obtain more reliable indications. The aim of the present study was to investigate the ethyl glucuronide distribution in postmortem specimens from autopsy cases found to be positive for ethanol. We presented 21 autopsy cases in which central blood, peripheral blood and liver samples were available. Specimens were analyzed for ethyl glucuronide by liquid chromatography tandem mass spectrometry; we also recorded postmortem interval, case history, cause of death, use of drugs, metabolic disorders if present, putrefaction if present, history of ethanol abuse and information about ethanol intake before death. Our aim was to evaluate and to compare the ethyl glucuronide levels in different matrices taken from the same subject in order to provide a better understanding of the interpretation of postmortem ethyl glucuronide concentrations.

Untargeted rat brain metabolomics after oral administration of a single high dose of cannabidiol.

Cannabidiol (CBD), for long time considered as a minor cannabinoid of Cannabis sativa, has recently gained much attention due to its antioxidant, anti-inflammatory, analgesic and anticonvulsant properties. A liquid chromatography coupled to mass spectrometry based method was developed for the quantitative determination of CBD and other cannabinoids (Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC and 11-nor-9-carboxy-THC) in rat brain samples after oral administration of a single high dose (50 mg/kg) of CBD. The main challenge of the present work was to study CBD pharmacokinetics in rat cortex: the identification of its metabolites and pharmacodynamics through the study of variations in endogenous compounds' concentrations following CBD administration. An untargeted metabolomics approach revealed the formation of some CBD metabolites that are not commonly found in other body tissues or fluids. Lastly, the changes in some endogenous compounds' concentrations were correlated with some of the pharmacological properties of this cannabinoid.

Therapeutic and recreational methadone cardiotoxicity.

Several classes of drugs have been associated with an increased risk of cardiovascular disease and occurrence of arrhythmias potentially involved in sudden deaths in chronic users even at therapeutic doses. The study presented herein focuses on pathological changes involving the heart possibly due to methadone use. 60 cases were included in the study in total and were divided into three groups (therapeutic methadone users: 20 cases, recreational methadone users: 20 cases, and sudden death group in subjects who had never taken methadone: 20 cases). Autopsies, histology, biochemistry and toxicology were performed in all cases. Macroscopic and microscopic investigation results in therapeutic methadone users were similar to those observed in sudden, unexpected deaths in non-methadone users. In recreational methadone consumers, macroscopic and microscopic examination of the heart failed to provide results consistent with acute or chronic myocardial or coronary damage, thereby corroborating the hypothesis of death most likely following respiratory depression.

Development and validation of a liquid chromatography-tandem mass spectrometric assay for quantitative analyses of triptans in hair.

Triptans are specific drugs widely used for acute treatment of migraine, being selective 5HT1B/1D receptor agonists. A proper assumption of triptans is very important for an effective treatment; nevertheless patients often underuse, misuse, overuse or use triptans inconsistently, i.e., not following the prescribed therapy. Drug analysis in hair can represent a powerful tool for monitoring the compliance of the patient to the therapy, since it can greatly increase the time-window of detection compared to analyses in biological fluids, such as plasma or urine. In the present study, a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has been developed and validated for the quantitative analysis in human hair of five triptans commonly prescribed in Italy: almotriptan (AL), eletriptan (EP), rizatriptan (RIZ), sumatriptan (SUM) and zolmitriptan (ZP). Hair samples were decontaminated and incubated overnight in diluted hydrochloric acid; the extracts were purified by mixed-mode SPE cartridges and analyzed by LC-MS/MS under gradient elution in positive multiple reaction monitoring (MRM) mode. The procedure was fully validated in terms of selectivity, linearity, limit of detection (LOD) and lower limit of quantitation (LLOQ), accuracy, precision, carry-over, recovery, matrix effect and dilution integrity. The method was linear in the range 10-1000pg/mg hair, with R(2) values of at least 0.990; the validated LLOQ values were in the range 5-7pg/mg hair. The method offered satisfactory precision (RSD <10%), accuracy (90-110%) and recovery (>85%) values. The validated procedure was applied on 147 authentic hair samples from subjects being treated in the Headache Centre of Modena University Hospital in order to verify the possibility of monitoring the corresponding hair levels for the taken triptans.

Hair testing in clinical setting: Simultaneous determination of 50 psychoactive drugs and metabolites in headache patients by LC tandem MS.

Headache patients suffering from recurrent attacks are a population at risk of overuse and abuse of analgesic medications. Associated with triptans, the first-line drugs recommended for the acute treatment, these patients usually take other medications such as opioids analgesics for the attack treatment, antidepressants and antiepileptics for prophylaxis treatment and benzodiazepines, non-benzodiazepine hypnotics and antipsychotics for the treatment of comorbidities. Regular and frequent use of triptans, like of any other symptomatic analgesic, can cause chronic headache and medication-overuse headache (MOH). In these circumstances, a detoxification treatment is necessary and therefore the monitoring and follow-up of the patients are crucial to the success of the treatment. In the present study, a LC tandem MS method has been developed for the identification of 50 psychoactive drugs in human hair, including triptans, benzodiazepines and metabolites, analgesics, antiepileptic, antidepressants and metabolites, a non-benzodiazepine hypnotic (z-drug), antipsychotics and metabolites. Hair samples were decontaminated, pulverized and incubated overnight in methanol; the extracts were then purified by a new and rapid QuEChERS procedure and analyzed by LC-MS/MS under gradient elution with positive ionization MRM mode. The procedure was fully validated in terms of selectivity, linearity, limit of detection and lower limit of quantitation, precision and accuracy, carry-over, matrix effect, recovery and dilution integrity. The validated procedure has been applied to 234 real hair samples collected from headache patients with known type and dosage of the taken drugs; the obtained data could be of interest to evaluate the xenobiotic concentrations in patients with known therapy.

Delta9 THC content in illicit cannabis products over the period 1997-2004 (first four months).

The aim of this study is to determine delta 9 tetrahydrocannabinol (delta9 THC) content in 5227 seizures of cannabis products over the period 1997-2004 (first four months). The products were seized in Modena country (Italy). The samples were classified as marijuana and hashish and divided into subgroups. The following results show an increase in the potency (concentration of delta9 THC) of these products.