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Drug stability is an important concern of forensic toxicological testing, particularly postmortem (PM) samples that may be stored for an extensive period of time before analysis. In PM toxicology, the complex assessment of analyte stability in biological matrices can profoundly impact the interpretation of toxicological results and the outcome of forensic casework. The aim of this work is to assess the stability of ∆9-tetrahydrocannabinol (THC), cannabidiol, 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-∆9-THC (THCCOOH) and 11-nor-∆9-THC-9-carboxylic acid glucuronide (THCCOOH-glucuronide) in brain stored at three different temperatures (4°C, −20°C and −70°C) up to over 12 months (390 days) in order to establish the best storage condition for preventing potential drug degradation during the storage period. Brain is suitable and useful for xenobiotic concentrations and is a valuable specimen in the interpretation of PM toxicological results. In our study, pooled brain specimens were spiked at low and high concentrations to evaluate the change in concentration over time. Stable compounds were quantified within ±20% of the target concentration (the mean concentration resulting from the initial analysis). According to stability criteria, our preliminary findings revealed that all the cannabinoids studied are stable in frozen brain samples (−20°C and −70°C) for over 12 months: all the analytes’ concentrations remained unaffected during storage over time, with the analytical variation staying within ±20%. On the contrary, under refrigeration conditions (4°C), 11-OH-THC, THCCOOH and THCCOOH-glucuronide were instable. Authentic brain samples, collected from eight cases during the autopsy, were analyzed, and the stability was evaluated. This study provided new data on cannabinoid stability in brain. The stability of the brain samples, both in spiked samples and in authentic caseworks, highlights the importance of the brain as a valid testing matrix when retesting is required after a long period of time or when laboratories are faced with backlog.

Cannabis sativa L. is a plant known all over the world, due to its history, bioactivity and also social impact. It is chemically complex with an astonishing ability in the biosynthesis of many secondary metabolites belonging to different chemical classes. Among them, cannabinoids are the most investigated ones, given their pharmacological relevance. In order to monitor the composition of the plant material and ensure the efficacy and safety of its derived products, extraction and analysis of cannabinoids play a crucial role. In this context, in addition to a conventional separation method based on HPLC with UV/DAD detection, a new strategy based on a non-separation procedure, such as 13C-qNMR, may offer several advantages, such as reduced solvent consumption and simultaneous acquisition of the quali/quantitative data related to many analytes. In the light of all the above, the aim of this work is to compare the efficiency of the above-mentioned analytical techniques for the study of the main cannabinoids in different samples of cannabis inflorescences, belonging to fibre-type, recreational and medical varieties. The 13C-qNMR method here proposed for the first time for the quantification of both psychoactive and non-psychoactive cannabinoids in different cannabis varieties provided reliable results in comparison to the more common and consolidated HPLC technique.

The scientific interest in cannabis has been documented by a wide literature, but postmortem studies and interpretations of autopsy findings are lacking or limited to few cases, few matrices analyzed or a small number of analytes. The present study describes the development and full in-house validation of a sensitive and simple method based on an optimized rapid clean-up procedure combined with a robust and highly sensitive liquid chromatography coupled with tandem mass spectrometry (LC–MS-MS) technique, designed to simultaneous determination of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), 11-nor-Δ9-tetrahydrocannabinol-carboxylic acid (THC-COOH) and 11-nor-Δ9-tetrahydrocannabinol-carboxylic acid glucuronated (THC-COOH gluc.) in postmortem samples: central blood (CB), femoral blood (FB) and brain tissue (BR). The developed method was validated and applied to 24 postmortem cases involving cannabinoids. In this study, we presented a full optimization and validation of target analyses for each matrix. The procedure had proven to be reliable and accurate. This study adds new data, particularly about the cannabinoids concentrations in BR samples. Combined pattern (CB, FB, BR) can be used in the interpretation of postmortem cases, proving and strengthening the assessments made on blood data. BR matrix is a helpful supplement in the investigation of the role of cannabinoids as crucial or contributory factor in leading to death.

New psychoactive substances (NPS) are substances that continue to appear on the drug market to bypass controlled substance legislation. Mephedrone or 4-methylmethcathinone is becoming the most popular new psychoactive substance among youth as a recreational drug. The present study describes the optimization and validation of a sensitive method that combined clean up procedure and LC–MS/MS technique designed to simultaneously determine the presence of Mephedrone and its two metabolites (normephedrone as active metabolite and dyhidromephedrone) in post-mortem specimens (body fluids and organ tissues). To date, this is the first determination of Mephedrone metabolites in post-mortem specimens. The validated method was applied to a fatal Mephedrone intoxication case. The distribution of the three analytes in different post-mortem matrices was presented. The toxicological results of the studied case are discussed, along with autopsy, histopathological evidence and crime-scene information. The toxicological results presented in the study provide new data relative to mephedrone and the distribution of its metabolites in post-mortem specimens. In our opinion, the metabolite concentration database must be developed because the metabolites may be linked to toxicity. The pattern of parent drug and its metabolites can be helpful in the interpretation of fatal cases involving mephedrone, which will contribute to the currently limited knowledge about mephedrone and metabolites concentrations.

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

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.

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

Butalbital, a barbiturate, is present in analgesic combinations used by headache sufferers. Overuse/abuse of these combinations may cause dependence, chronic migraine, and medication-overuse headache (MOH). MOH is difficult to manage: it improves interrupting analgesic overuse, but requires monitoring, because relapses are frequent. A gas chromatography-mass spectrometry (GC-MS) method for hair analysis has been developed and validated to document abuse of an analgesic combination containing butalbital and propyphenazone by a patient with MOH. For over ten years the patient managed her headache using eight suppositories/day of an analgesic combination containing butalbital 150mg, caffeine 75mg, and propyphenazone 375mg per suppository. An outpatient detoxification treatment was carried out. After three weeks, the patient reduced the consumption to one suppository/day. At the first control visit, after three months from the beginning of detoxification, the patient increased the use of the combination to four suppositories/day and at the second control visit, after seven months from the beginning of detoxification, she was back to eight suppositories/day. At the two control visits, a hair sample was taken for determination of butalbital and propyphenazone. Moreover blood and urine samples for determination of butalbital were drawn at the beginning of detoxification treatment and at the two control visits. With the segmental analysis of two hair samples the medication history of ten months could be estimated. In the first hair sample, collected at the first control visit, in the distal segment, butalbital and propyphenazone concentrations were, respectively, 17.5ng/mg and 56.0ng/mg, confirming the prolonged abuse; in the proximal segment, concurrently with the detoxification treatment, butalbital and propyphenazone concentrations had reduced respectively to 5.45ng/mg and 11.1ng/mg. The second hair sample, collected at the second control visit, proved the fair course of the detoxification treatment in the distal segment and signalled relapse in the abuse of the analgesic combination in the proximal segment. In the clinical context, hair analysis can be advantageously used to monitor the abuse of analgesic combinations with butalbital, common among headache patients. The validation data showed that GC-MS method developed for determination of butalbital and propyphenazone was rapid, highly sensitive, specific and selective.

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. 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. 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. 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.

Hair analysis, as complementary matrix, has expanded across the spectrum of toxicological investigations for misuse drug monitoring. Hair has become an important matrix for drug analysis, owing to the possibility to detect target analytes for long time periods, depending on hair length. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the quantitation of tramadol, a widely used centrally acting analgesic, and its main metabolites in hair (ODMT, NDMT, NOT). Hair samples were decontaminated and incubated overnight in diluted hydrochloric acid; the extracts were purified by mixed-mode solid phase cartridges and analyzed by LC-MS/MS in positive ionization mode monitoring two transitions per analyte. The procedure was fully validated in terms of linearity, limit of detection and lower limit of quantitation (LLOQ), accuracy, precision, recovery, matrix effect and selectivity. The linear regression analysis was calibrated by deuterated internal standards; for all analytes, responses were linear over the range 0.04-40.00 ng/mg hair, with R(2) values of at least 0.995. The method offered satisfactory precision (RSD10%), accuracy (90-110%) and recovery (90%) values. The found LLOQ values for tramadol and metabolites were in the range 0.010-0.030 ng/mg hair. The proposed procedure was successfully applied to quantify tramadol and metabolites in real hair samples submitted to our laboratory: three cases of tramadol assumption within the therapeutic dosage (3 × 2 segments) and one case of tramadol abuse in a binge pattern (8 segments). The ranges found for TRAM, ODMT, NDMT and NOT were markedly higher in the abuse case (63.42-107.30, 3.76-6.26, 24.88-45.66, 0.22-1.18 ng/mg hair, respectively) compared to the other case reports (3.29-20.12, 0.28-1.87, 0.45-4.32, 0.07-0.80 ng/mg, respectively); also the values of NMDT/ODMT ratio differed significantly. According to the obtained data, we hypothesized that the binge pattern may influence the metabolites' to parent drug concentration ratios; therefore this parameter could represent a target assessment tool to monitor abuse 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.

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.

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.

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