OILVEQ: an Italian external quality control scheme for cannabinoids analysis in galenic preparations of cannabis oil.

OBJECTIVES: Italy legalized cannabis oil for specific medical conditions (neuropathic pain, refractory epilepsy and other established pathologies) in 2015, but mandates titration of principal cannabinoids before marketing each batch using iphenated techniques coupled with mass spectrometry. To assess reliability of laboratories from the Italian National Health Service in charge of titrating the batches, the Italian National Institute of Health set up an quality control program on determination of Δ9-tetrahydrocannabinol l (THC), cannabidiol (CBD), Δ9-tetrahydrocannabinolic acid A (THCA-A) and cannabidiolic acid (CBDA) in cannabis oil preparations. METHODS: Two rounds of exercises have been carried out since 2019, involving sixteen Italian laboratories. Five different cannabis oil samples (19-1A and 19-1B for the first round and 22-1A, 22-1B and 22-1C for the second one were prepared and 1 mL amount of each sample was sent to the laboratories. The quantitative performance of each laboratory was assessed calculating the z-score value, a statistical measurement for value's relationship to the mean of a group of values. RESULTS: In the first round, eight out of fourteen laboratories employed an LC-MS while the remaining six used GC-MS. Differently, in the second round, six out of eleven laboratories employed a GC-MS while the remaining five used LC-MS. In the first round, only 28.6 % laboratories achieved an acceptable performance (z-score±2), and all of them used LC-MS as analytical method. In the second round, none of the laboratories achieved an acceptable performance. Satisfactory results, based on z-scores, were generally low (0.0-75.0 %), with only one exception of 100 % for THCA-A determination in sample 22-1B. In the second round, three false negatives (two THC and one CBD by GC-MS determination) were reported while no false positives were described in the blank sample. The two rounds yielded a mean ERR% of 42 % approximately and a mean CV% around 70 % in GC-MS determination. When applying LC-MS determination, the two rounds yielded a mean ERR% of 36 % approximately and a mean CV% around 33 %. CONCLUSIONS: The obtained results underline the need for a clear and consistent protocol to be adopted by all laboratories intending to include the titration of oily cannabis-based products into their routinely analytical techniques. This emphasis on methodology standardization and participation to quality control schemes is essential for ensuring reliable and accurate measurements, ultimately enhancing the overall effectiveness and reliability of medical cannabis treatments.

Technical and health governance aspects of the external quality assessment system for classical and new psychoactive substances analysis testing in blood.

New psychoactive substances (NPS) are uncontrolled analogues of existing drugs or newly synthesized chemicals that exhibit psychopharmacological effects. Due to their diverse nature, composition, and increasing prevalence, they present significant challenges to the healthcare system and drug control policies. In response, healthcare system laboratories have developed analytical methods to detect NPS in biological samples. As a Regional Reference Centre, the Sicilian CRQ Laboratory (Regional Laboratory for Quality Control) developed and conducted an External Quality Assessment (EQA) study to assess, in collaboration with the Istituto Superiore di Sanità (ISS), the ability of different Italian laboratories to identify NPS and traditional drugs of abuse (DOA) in biological matrices. Two blood samples were spiked with substances from various drug classes, including synthetic cannabinoids, cathinones, synthetic opiates, and benzodiazepines, at concentrations ranging from 2 to 10 ng/mL. The blood samples were freeze-dried to ensure the stability of DOA and NPS. Twenty-two laboratories from the Italian healthcare system participated in this assessment. The information provided by the laboratories during the registration in an in-house platform included a general description of the laboratory, analytical technique, and the chosen panels of analytes. The same platform was employed to collect and statistically analyze the data and record laboratory feedback and comments. The evaluation of the results revealed that the participating laboratories employed three different techniques for analyzing the samples: GC-MS, LC-MS, and immunoenzymatic methods. Approximately 90 % of the laboratories utilized LC-MS techniques. Around 40 % of false negative results were obtained, with the worst results in the identification of 5-chloro AB PINACA. The results showed that laboratories that used LC-MS methods obtained better specificity and sensitivity compared to the laboratories using other techniques. The results obtained from this first assessment underscore the importance of external quality control schemes in identifying the most effective analytical techniques for detecting trace molecules in biological matrices. Since the judicial authorities have not yet established cut-off values for NPS, this EQA will enable participating laboratories to share their analytical methods and expertise, aiming to establish common criteria for NPS identification.

NPS-EQA PART I: Four years' experience in external quality assessment program in Italy for classical and new psychoactive substances analysis in oral fluid.

In 2019, Italian National Institute of Health established an external quality assessment program (EQA) to evaluate the performance of oral fluid testing for classical and new psychoactive substances by laboratories participating in the National Early Warning System collaborative centres. This report presents the results of four rounds between 2019 and 2023. Eleven oral fluid specimens, including 3 blank samples, were prepared by adding different classes of and new psychoactive drugs at known concentrations to pre-screened drug-free oral fluid. False-negative and false-positive results were calculated for the qualitative data evaluation. The quantitative evaluation measured the imprecision and accuracy of the results, in terms of coefficient of variation (CV%) and percent error (ERR%), respectively, with respect to a mean value obtained by reference laboratories. Z-score values were then calculated. Over the years, there has been a significant improvement in false-negative results (from 42.7% in the first year to 19.4% in the last year), but not in false-positive results (from 33.3% in the first year to 22.2% in the last one). In addition to the classic drugs of abuse (e.g. cocaine, amphetamine, methadone), the substances found in false positive samples belonged to the class of synthetic cannabinoids (e.g 5-fluoro CUMYL-PINACA and 5-fluoro-EDMB-PICA), synthetic opioids (e.g butyrylfentanyl) and tryptamines (e.g. 5-methoxy-N-methyl-N-isopropyltryptamine). The four rounds yielded a mean ERR% of approximately 22.1% and a mean CV% of around 41.5%. The participating laboratories demonstrated variable performances in relation to the class of analysed psychoactive substances, as evidenced by the calculated Z-scores. Between 25% and 60% of the reported results in all rounds should be considered satisfactory. EQA is a crucial element of laboratory quality management systems. It promotes continuous improvement and maintains high standards in the field of forensic and clinical drug testing.

NPS-EQA PART II: Four years' experience in external quality assessment program in Italy for classical and new psychoactive substances analysis in hair.

In 2019, the Italian National Institute of Health established an external quality assessment (EQA) program to evaluate the performance of laboratories of collaborative centres participating in the National Early Warning System in hair testing for classical and new psychoactive substances (NPS). The results obtained in the four rounds (2019-2023) and the evolution in hair testing performance for classic drugs of abuse and new psychoactive substances are presented. A total of 11 hair specimens, including 3 blank samples, were prepared by adding different classes of classical and NPS at known concentrations to pre-screened drug-free hair. False negative and false positive results were calculated for the qualitative data evaluation. The quantitative evaluation included the imprecision (as % coefficient of variation, CV%) and the accuracy (as % error, ERR%) of the results with respect to a mean value obtained by reference laboratories and Z-score values were assessed. Over the years, an improvement in false negative results (from 52.4% in the first year to 34.3% in the last one) and false positive results (from 55.0% in the first year to 30.8.% in the last one) was observed. In the first round, the mean ERR% ranged from 6.2% to 112.8% due to NPS determination. However, in the subsequent three rounds, the mean ERR% ranged from 10.4% to 22.4%, The mean CV% in the four rounds was approximately 41.5% (ranging from 44.3% to 53.3%). Between 12.0% and 56.6% of the reported results in all rounds should be considered satisfactory. EQA programs help laboratories to identify and correct problems within their processes by highlighting errors and variations. This ensures that the results produced are accurate and reproducible.

Spontaneous Pneumothorax Secondary to Bullous Lung Emphysema Positive for Cannabinoids upon Toxicological Examination.

Cannabis can be related to respiratory diseases, but the relationship between smoking marijuana and the development of a pneumothorax has scarcely been investigated. We aimed to analyze, in patients with a history of cannabis smoking abuse submitted to lung apicectomy for a primary spontaneous pneumothorax (PSP), the correlation between the presence of cannabinoids in the resected lung and the detection of bullous emphysema within the same tissue. Patients undergoing lung apicectomy for a PSP were prospectively enrolled, and the correlation between the presence of cannabinoids in the resected lung tissue and histological finding of bullous emphysema was investigated with Fisher's exact test. There were 21 male patients, with a median age of 27 years. The cannabinoids found by the toxicological examination in surgical specimens were mainly delta-9-tetrahydrocannabinol (THC), cannabinol (CBN), and cannabidiol (CBD). In 14/21 patients, cannabinoids were detected in the resected lung tissue, and bullous emphysema was present in 13/14 of these (93%), while bullous emphysema was found in only 1/7 (14%) of the remaining patients who were negative for cannabinoids in the lung tissue, and the difference was found to be statistically significant (p < 0.0009). Our study demonstrated the presence of bullous emphysema in most cannabinoid-positive patients and its absence in most of those who were cannabinoid-negative, supporting the correlation between cannabinoids in the lung tissue and bullous emphysema with the development of a "secondary" spontaneous pneumothorax.

GC-MS/MS Determination of Synthetic Cathinones: 4-chloromethcathinone, N-ethyl Pentedrone, and N-ethyl Hexedrone in Oral Fluid and Sweat of Consumers under Controlled Administration: Pilot Study.

This study presents a validated GC-MS/MS method for the detection and quantification of 4-chloromethcathinone or clephedrone (4-CMC), N-ethyl Pentedrone (NEP), and N-ethyl Hexedrone (NEH, also named HEXEN) in oral fluid and sweat and verifies its feasibility in determining human oral fluid concentrations and pharmacokinetics following the administration of 100 mg of 4-CMC orally and 30 mg of NEP and NEH intranasally. A total of 48 oral fluid and 12 sweat samples were collected from six consumers. After the addition of 5 µL of methylone-d3 and 200 µL of 0.5 M ammonium hydrogen carbonate, an L/L extraction was carried out using ethyl acetate. The samples, dried under a nitrogen flow, were then derivatized with pentafluoropropionic anhydride and dried again. One microliter of the sample reconstituted in 50 µL of ethyl acetate was injected into GC-MS/MS. The method was fully validated according to international guidelines. Our results showed how, in oral fluid, the two cathinones taken intranasally were absorbed very rapidly, within the first hour, when compared with the 4-CMC which reached its maximum concentration peak in the first three hours. We observed that these cathinones were excreted in sweat in an amount equivalent to approximately 0.3% of the administered dose for 4-CMC and NEP. The total NEH excreted in sweat 4 h after administration was approximately 0.2% of the administered dose. Our results provide, for the first time, preliminary information about the disposition of these synthetic cathinones in the consumers' oral fluid and sweat after controlled administration.

Synthetic cannabinoids use in a sample of opioid-use disorder patients.

Cannabis is the most widely consumed illegal drug in the world and synthetic cannabinoids are increasingly gaining popularity and replacing traditional cannabis. These substances are a type of new psychoactive substance that mimics the cannabis effects but often are more severe. Since, people with opioids use disorder use widely cannabis, they are a population vulnerable to use synthetic cannabinoids. In addition, these substances are not detected by the standard test used in the clinical practice and drug-checking is more common in recreational settings. A cross-sectional study with samples of 301 opioid use disorder individuals was carried out at the addiction care services from Barcelona and Badalona. Urinalysis was performed by high-sensitivity gas chromatography-mass spectrometry (GC-MS) and ultra-high-performance liquid chromatography-high -resolution mass spectrometry (UHPLC-HRMS). Any synthetic cannabinoid was detected in 4.3% of the individuals and in 23% of these samples two or more synthetic cannabinoids were detected. Among the 8 different synthetic cannabinoids detected, most common were JWH-032 and JWH-122. Natural cannabis was detected in the 18.6% of the samples and only in the 0.7% of them THC was identified. Several different synthetic cannabinoids were detected and a non-negligible percentage of natural cannabis was detected among our sample. Our results suggest that the use of synthetic cannabinoids may be related to the avoidance of detection. In the absence of methods for the detection of these substances in clinical practice, there are insufficient data and knowledge making difficult to understand about this phenomenon among opioid use disorder population.

Carbamazepine Overdose after Psychiatric Conditions: A Case Study for Postmortem Analysis in Human Bone.

Carbamazepine is the main option used as a preventive medication to treat bipolar disorder when there is no response to lithium. Carbamazepine toxicity is defined as serum levels greater than 12 µg/mL, with severe toxicity occurring over 40 µg/mL, reduced to 30 µg/mL when combined with pharmacological treatment, i.e., benzodiazepines or antidepressants. For these reasons, it is necessary to find a validated tool to determine carbamazepine levels in an autopsy to rule out suicide or to know if the death was a consequence of an adverse drug reaction (ADR), especially when only bones can be accessed. We have validated a tool to detect and quantify drug concentration in bone. Our results showed a peak for carbamazepine at minute 12 and a mass fragment of 193 m/z. This case study is the first time in the literature that carbamazepine has been detected and quantified in bone. These results demonstrate that carbamazepine can be detected in bone tissue from forensic cases, but almost more importantly, that the method proposed is valid, reliable, and trustworthy.

New Psychoactive Substances Consumption in Opioid-Use Disorder Patients.

(1) Background: Since the beginning of the 21st century, the large number and wide chemical variety of new psychoactive substances (NPS) that enter the market every year has become a public health problem. Given the rapidity with which the drug market is changing, many NPS are not clinically investigated and their effects and health risks are unknown. Drug testing is a very useful tool for this purpose, but, unfortunately, it is not very widespread in individuals with opioid-use disorder under detoxification treatment. The aim of this study is to investigate the use of illicit drugs and NPS in opioid-use disorder (OUD) patients on opioid agonist treatment. (2) Methods: A multicenter, descriptive, cross-sectional study was conducted at two addiction care services in Barcelona and Badalona, Spain. Urine samples were collected from OUD individuals attending these two centers, who anonymously donated a urine sample at the time of a periodical visit. Samples were analyzed by high-sensitivity gas chromatography-mass spectrometry (GC-MS) and ultra-high-performance liquid chromatography-high -resolution mass spectrometry (UHPLC-HRMS). (3) Results: Out of the 187 collected and analyzed urine samples, 27.3% were positive for any type of NPS and 8.6% were positive for new synthetic opioids, including fentanyl and its derivatives (NSO). Other frequently detected substances were benzodiazepines in 46.0% of samples, antipsychotics in 27.8% of samples, or cocaine and cannabis in 23.5% of samples. (4) Conclusion: A wide number of NPS, including NSO, have been detected in urine samples from an OUD population. A lack of NPS detection in standard drug screening among drug users can hide the identification of a potential public health problem.

Prevalence of Licit and Illicit Drugs Use during Pregnancy in Mexican Women.

For the first time, the present study employed hair testing to investigate the prevalence of classical drugs of abuse and new psychoactive substances use during gestation in a cohort of 300 Mexican pregnant women. An interview was conducted to collect data on sociodemographic aspects of the patients, and a 9 cm-long hair strand was taken from the back of the head of each mother one month after delivery. A validated ultra-high-performance liquid chromatography−high-resolution mass spectrometry method was used for the screening of classic drugs, new psychoactive substances, and medications in maternal hair. Out of 300 examined hair samples from pregnant women, 127 (42.3%) resulted positive for psychoactive substances: 45 (35.4%) for cannabis only, 24 (18.9%) for methamphetamine only, 13 (10.2%) for cocaine only, 1 (0.3%) for heroin, 1 for N-N-dimethyltryptamine (0.3%), 1 for ketamine (0.8%), and 35 (16.3%) for more than one psychoactive substance. Furthermore, seven samples (2.3%) resulted positive for new psychoactive substances (NPS): two samples for synthetic cannabinoids, two for synthetic cathinones, and three for nor-fentanyl, and 3.3% of women hair resulted positive for anticonvulsant, antidepressant, and antipsychotic medications. Finally, 83 women hair samples (27.7%) tested positive for nicotine. Nonsteroidal anti-inflammatory drugs (NSAIDs) and other painkillers (60.0%), medications for the treatment of nausea and vomiting (12.3%), antihistamines (8.7%) and nasal/sinus decongestants (6.7%), cough suppressants (5.0%), and bronchodilator agents (5.0%) were also detected in pregnant women hair. The gestational use of psychoactive substances and exposure to tobacco smoke, assessed by hair testing, were associated with a significantly younger age and with a low education grade of the mothers (p < 0.005). This study provides a significant preliminary indication of the under-reported gestational consumption of licit and illicit psychoactive and pharmacologically active drugs in a Mexican environment, showing the value of toxicological and forensic analyses in the global effort to determine the health risks caused by classic drugs and new psychoactive substances during pregnancy.

Assessment of licit and illicit drugs consumption during pregnancy by Ultra-High Performance Liquid Chromatography-High Resolution Mass Spectrometry (UHPLC-HRMS) target screening in Mexican women hair.

Substance use in pregnancy is a global public health problem, both in developed and developing countries. Whereas information is available for major western countries, scarce data are present for the second ones. The objective assessment of pregnancy consumption of xenobiotic is provided by analysis of maternal hair, which can account for gestational consumption, given the possibility to analyze 9 cm hair corresponding to the pregnancy months. Here, we describe an ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) method used as screening analysis of classic drugs, new psychoactive substances and medications in hair from a cohort of pregnant Mexican women. The UHPLC-HRMS method included Accucore™ phenyl Hexyl (100 × 2.1 mm, 2.6 µm, Thermo, USA) column with a gradient mobile phase and a full-scan data-dependent MS2 (ddMS2) mode for substances identification (mass range 100-750 m/z). Results from the first 100 samples disclosed the presence of several undeclared and declared psychoactive substances and medications, being methamphetamine and paracetamol the most prevalent ones found in 20% and 43% cases, respectively. In addition, biomarkers of cannabis and tobacco use as well as those of antihistamines and antiemetic drugs were also prevalent. Albeit preliminary, these data confirm the feasibility of hair screening by UHPLC-HRMS to objectively assess xenobiotic consumption in pregnant women with consequent risk of fetal exposure to toxic substances.

Hair Testing for Classic Drugs of Abuse to Monitor Cocaine Use Disorder in Patients Following Transcranial Magnetic Stimulation Protocol Treatment.

In recent years, hair has become an alternative biological specimen for drug testing in the fields of forensic and clinical toxicology. The advantages of hair testing include larger detection windows (months/years), depending on the length of the hair shaft, compared to those of urine/blood (hours to 2-4 days for most drugs). Segmental hair analysis can disclose a month-to-month (considering 1 cm segment cuts) information of drug exposure (single or repeated) and potentially identify patterns of drug use/administration. Repetitive transcranial magnetic stimulation (rTMS) was recently proposed as a valid tool for therapeutic purposes in addictions, including cocaine use disorder (CocUD). Here, we proposed hair testing analyses of classic drugs of abuse in a clinical setting to monitor the clinical changes in treatment-seeker CocUD patients undergoing protocol treatments with rTMS stimulating the left dorsolateral prefrontal cortex (l-DLPFC). We collected hair samples from nine CocUD patients at different stages from the beginning of treatments. Hair sample analyses revealed significant changes in the patterns of cocaine use, according to the negativity of urine screening tests and the clinical reductions of craving. These data, albeit preliminary, suggest that hair testing analysis of classic drugs of abuse could be extended to clinical settings to monitor the clinical efficacy of innovative therapeutic interventions, such as rTMS.

Postmortem Analysis of Benzodiazepines in Human Bone by Gas Chromatography-Mass Spectrometry.

A procedure based on gas chromatography-mass spectrometry was developed for the analysis of benzodiazepines (nordiazepam, oxazepam, lormetazepam, lorazepam, clonazepam, bromazepam and alprazolam) in postmortem human ribs. Powdered bone samples, including marrow remains inside, with the internal standard diazepam-d5 were subjected to enzymatic hydrolysis with 100 µL of ß-glucoronidase and were incubated in sodium hydroxide for 1 h in a 70°C oven. Samples underwent liquid phase extraction and ethyl acetate was used as eluent. Chromatography was performed on a fused silica capillary column and the selected-ion-monitoring mode was used for analytes determination. The method was validated in the range 0.1-0.5 ng/mg (depending on the benzodiazepine) to 100 ng/mg with average values of recovery, matrix effect and process efficiency ranged from 83.2 to 94.3%, from 97.3 to 102.1% and from 80.5 to 91.2%, respectively. The intra- and inter-day accuracy was <15%. The procedure was tested in rib specimens obtained during routine autopsies from 20 cases where these benzodiazepines were found in blood. Benzodiazepines were detected in the combined bone and marrow samples in 60% of cases. Lorazepam was detected in bone in the range of 0.3-0.7 ng/mg, nordiazepam at 1.3-4.2 ng/mg and oxazepam at 1.1-1.2 ng/mg. To our knowledge, this protocol for the simultaneous analysis of these benzodiazepines is the first performed and validated using human ribs.

Turmeric (Curcuma longa L.) food supplements and hepatotoxicity: an integrated evaluation approach.

INTRODUCTION: Turmeric is the common name for the rhizome of Curcuma longa L. In the recent years, food supplements containing turmeric have been marketed and widely used by an increasing number of consumers. Spontaneous reports of suspected adverse reactions to food supplements are collected within the Phytovigilance system. METHODS: An ad hoc multidisciplinary group investigated the suspected cases of hepatotoxicity reported to the Italian Phytovigilance system associated with the assumption of turmeric food supplements with the methodology specific to pharmacovigilance as well as for the evaluation of the quality and safety of food supplements. RESULTS: A cluster of 28 spontaneous reports of acute hepatitis, mostly with cholestasis, associated with turmeric products were sent to the Italian Phytovigilance system in the first six months of 2019. In all cases, except one, the causality assessment was at least possible. The suspected products were collected and analysed for the presence of drugs, heavy metals, aflatoxins, pesticides, synthetic dyes and pyrrolizidine alkaloids. CONCLUSION: On the basis of the results of all the activities performed by multidisciplinary group, regulatory intervention was taken. This study highlights the importance of developing an integrated evaluation approach for the evaluation of the adverse effects associated with the use of food supplements.

Postmortem analysis of quetiapine and pregabalin in human bone.

In this study quetiapine and pregabalin were analyzed in human bones. A method previously developed for the determination of antidepressants in human bone was tested for the analysis of these two substances. Bones were pulverized and subjected to the extraction protocol, and after undergoing solid-phase extraction, samples were analyzed using gas chromatography-mass spectrometry. The assay was validated in the range 0.3-500 ng/mg, mean analytical recovery was 76.9% for quetiapine and 90.9% for pregabalin, matrix effect was 83% for quetiapine and 91% for pregabalin and process efficiency was 63.8% for quetiapine and 82.7% for pregabalin. The intra- and inter-day precision was below 3% in all cases and the intra- and inter-assay accuracy values were in almost all cases better than 12%. The validated method was then applied to bone samples from forensic cases. Drugs were detected in bone in 2 of the 3 blood positive cases. The approximate concentrations in bone were 40 ng/mg for pregabalin and 7 ng/mg for quetiapine. To our knowledge, this is the first time these substances were detected in bones. With this study the number of substances with a validated protocol to be used in human bones in case of necessity is expanded.

THC and CBD concentrations in blood, oral fluid and urine following a single and repeated administration of "light cannabis".

Background "Light cannabis" is a product legally sold in Europe with Δ9-tetrahydrocannabinol (THC) concentration lower than 0.2% and variable cannabidiol (CBD) content. We studied THC and CBD excretion profiles in blood, oral fluid (OF) and urine after smoking one or four light cannabis cigarettes. Methods Blood, OF and urine samples were obtained from six healthy light cannabis consumers after smoking one 1 g cigarette containing 0.16% THC and 5.8% CBD and from six others after smoking four 1 g cigarettes within 4 h. Sample collection began 0.5 and 4.5 h after smoking one or four cigarettes, respectively. Cannabinoid concentrations were quantified by gas chromatography-mass spectrometry (GC-MS). Results At the first collection, the highest THC and CBD concentrations occurred in blood (THC 7.0-10.8 ng/mL; CBD 30.2-56.1 ng/mL) and OF (THC 5.1-15.5 ng/mL; CBD 14.2-28.1 ng/mL); similar results occurred 0.5 h after the last of four cigarettes in blood (THC 14.1-18.2 ng/mL, and CBD 25.6-45.4 ng/mL) and OF (THC 11.2-24.3 ng/mL; CBD 14.4-37.0 ng/mL). The mean OF to blood ratio ranged from 0.6 to 1.2 after one and 0.6 to 1.9 after four light cannabis cigarettes. THC/CBD ratios in blood and OF were never greater than 2. Urinary 11-nor-9-carboxy-THC concentrations peaked 8 h after one and four cigarettes. Conclusions OF was a valuable alternative to blood in monitoring consumption of light cannabis. Blood and OF THC/CBD concentration ratios, never exceeded 2, possibly providing a useful biomarker to identify light cannabis vs illegal higher THC cannabis use, where THC/CBD ratios are generally greater than 10.

Cannabinoids Identification in Lung Tissues of Young Cannabis Smokers Operated for Primary Spontaneous Pneumothorax and Correlation with Pathologic Findings.

BACKGROUND: Several studies suggested the association between tobacco and cannabis smoking and the risk of primary spontaneous pneumothorax (PSP), but none demonstrated cannabinoids in human lung tissues. OBJECTIVES: The aim of this study was to identify cannabinoids in lung specimens of young cannabis smokers, operated for PSP, and investigate on their pathologic findings, to determine the role of cannabis in PSP pathogenesis. METHOD: A prospective, multicenter study was conducted, enrolling patients admitted for PSP. Inclusion criteria were PSP requiring surgical treatment and history of cannabis smoking, associated or not to tobacco. Control cases were nonsmokers, and tobacco only smokers operated for PSP. Lung apex wedge resection by video-assisted thoracic surgery was performed. Two lung specimens, for pathological and toxicological examination, were taken from each patient. RESULTS: Twenty-nine male patients were enrolled: 21 (72.4%) tobacco and cannabis smokers, 2 (7%) cannabis only smokers, 3 (10.3%) tobacco only smokers, 3 (10.3%) nonsmokers; all underwent lung apicectomy, 4 bilateral surgery, for a total of 33 procedures. Typical PSP pathologic findings were mainly detected in control cases, other alterations in cannabis users. Lung specimens resulted positive for cannabinoids on 22/33 cases (19/22 reported being, 3/22 not being cannabis smokers), negative on 11/33 (3/11 reported not being, 7/11 having been cannabis smokers, 1/11 cannabis smoker). CONCLUSIONS: Our study demonstrated the presence of cannabinoids and particular pathologic alterations in lung tissues of young cannabis smokers with PSP, supporting the correlation between this disease and marijuana abuse and suggesting spontaneous pneumothorax "secondary to marijuana" as a new nosological entity.

Development and Validation of a Method for the Analysis of Bisoprolol and Atenolol in Human Bone.

A method based on gas chromatography-mass spectrometry (GC-MS) is described for the determination of bisoprolol and atenolol in human bone. After the addition of lobivolol as internal standard, pulverized samples were incubated in acetonitrile for 1 h under ultrasounds. After adjusting the pH of the samples to 6, they were centrifuged, and the supernatants were subjected to solid phase extraction. Elution was achieved by using 3 mL of 2% ammonium hydroxide in 80:20 dichloromethane:isopropanol solution. Eluted samples were evaporated and derivatized. Chromatography was performed on a fused silica capillary column and analytes were determined in the selected-ion-monitoring (SIM) mode. The assay was validated in the range 0.1-0.3 ng/mg (depending on the drug) to 150 ng/mg, the mean absolute recoveries were 60% for bisoprolol and 106% for atenolol, the matrix effect was 69% for bisoprolol and 70% for atenolol and process efficiency was 41% for bisoprolol and 80% for atenolol. The intra- and inter-assay accuracy values were always better than 12%. The validated method was then applied to bone samples from two real forensic cases in which toxicological analysis in blood were positive for atenolol in the first case (0.65 µg/mL) and bisoprolol in the second case (0.06 µg/mL). Atenolol was found in bone samples from the corresponding case at the approximate concentration of 148 ng/mg and bisoprolol was found at 8 ng/mg.

Development and validation of a method for analysing of duloxetine, venlafaxine and amitriptyline in human bone.

A method based on gas chromatography-mass spectrometry (GC-MS) is described for the determination of venlafaxine, amitriptyline and duloxetine in human bone. Pulverized samples were incubated in methanol for 1 h under ultrasonication, after the addition of sertraline as internal standard. The samples were centrifuged, and the supernatants were evaporated. Samples were then resuspended in 0.1 M phosphate buffer pH 6 and subjected to solid phase extraction. Chromatography was performed on a fused silica capillary column and analytes were determined in the selected-ion-monitoring (SIM) mode. The assay was validated in the range 0.3-1 ng/mg (depending on the drug) to 500 ng/mg. The mean absolute recoveries ranged from 92.6% to 96.2%, the matrix effect from 76.9% to 103.3% and process efficiency from 74% to 95.9% depending on the analyte. The intra- and inter-assay accuracy values were always better than 20%. The validated method was then successfully applied to real bone samples from forensic cases in which toxicological analysis for these drugs in blood had been positive. Drugs were detected in bone in all blood positive results, the approximate concentrations being 36.4 ng/mg for amitriptyline, 19.3-3 ng/mg for duloxetine and 4.6-2 ng/mg for venlafaxine.