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Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede Policlinico

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2022 - Cannabinoid Stability in Postmortem Brain Samples Stored at Different Temperatures [Articolo su rivista]
Santunione, A L; Palazzoli, F; Verri, P; Vandelli, D; Chiapelli, F; Silingardi, E

Drug stability is an important concern of forensic toxicological testing, particularly postmortem samples that may be stored for an extensive period of time before analysis. In postmortem 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 Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH), and 11-nor-Δ9-tetrahydrocannabinol-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 a valuable specimen in the interpretation of postmortem 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, 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 authentical 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.

2021 - Post-mortem distribution of mephedrone and its metabolites in body fluids and organ tissues of an intoxication case [Articolo su rivista]
Palazzoli, F.; Santunione, A. L.; Verri, P.; Vandelli, D.; Silingardi, E.

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.

2020 - Cannabinoids Determination in Brain: A Supplemental Helpful in Postmortem Evaluation [Articolo su rivista]
Palazzoli, F; Santunione, A L; Marchesi, F; Verri, P; Vandelli, D; Licata, M; Silingardi, E

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 LC-MS-MS technique, designed to simultaneous determination of Delta9-tetrahydrocannabinol (THC), cannabidiol (CBD), 11-hydroxy-Delta9-tetrahydrocannabinol (11-OH-THC), 11-nor-Delta9-tetrahydrocannabinol-carboxylic acid (THC-COOH) and 11-nor-Delta9-tetrahydrocannabinol-carboxylic acid-glucuronated (THC-COOH-glucuronide) 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 in a helpful supplement in the investigation of the role of cannabinoids as crucial or contributory factor in leading to death.

2019 - Intrapatient variability of the hair levels of pain medications in chronic migraine patients - a pilot study [Articolo su rivista]
Ferrari, Anna; Rustichelli, Cecilia; Baraldi, Carlo; Vandelli, Daniele; Verri, Patrizia; Marchesi, Filippo; Licata, Manuela

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‐MS/MS). Intrapatient 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.

2019 - Seizures of illicit substances for personal use in two Italian provinces: analysis of trends by type and purity from 2008 to 2017 [Articolo su rivista]
Verri, Patrizia; Rustichelli, Cecilia; Ferrari, Anna; Marchesi, Filippo; Baraldi, Carlo; Licata, Manuela; Vandelli, Daniele; Palazzoli, Federica; Potì, Francesco; Silingardi, Enrico

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.

2018 - Pharmacokinetics and tolerability of oral cannabis preparations in patients with medication overuse headache (MOH)—a pilot study [Articolo su rivista]
Pellesi, L.; Licata, M.; Verri, P.; Vandelli, D.; Palazzoli, F.; Marchesi, F.; Cainazzo, M. M.; Pini, L. A.; Guerzoni, S.

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.

2018 - The role of ethyl glucuronide in supporting medico-legal investigations: Analysis of this biomarker in different postmortem specimens from 21 selected autopsy cases [Articolo su rivista]
Santunione, Anna Laura; Verri, Patrizia; Marchesi, Filippo; Rustichelli, Cecilia; Palazzoli, Federica; Vandelli, Daniele; Licata, Manuela; Silingardi, Enrico

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.

2017 - Monitoring of adherence to headache treatments by means of hair analysis [Articolo su rivista]
Ferrari, Anna; Licata, Manuela; Rustichelli, Cecilia; Baraldi, Carlo; Vandelli, Daniele; Marchesi, Filippo; Palazzoli, Federica; Verri, Patrizia; Silingardi, Enrico

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.

2016 - Development and validation of a liquid chromatography-tandem mass spectrometric assay for quantitative analyses of triptans in hair [Articolo su rivista]
Vandelli, Daniele; Palazzoli, Federica; Verri, Patrizia; Rustichelli, Cecilia; Marchesi, Filippo; Ferrari, Anna; Baraldi, Carlo; Giuliani, Enrico; Licata, Manuela; Silingardi, Enrico

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 10–1000 pg/mg hair, with R2 values of at least 0.990; the validated LLOQ values were in the range 5–7 pg/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.

2016 - Hair analysis for detection of triptans occasionally used or overused by migraine patients-a pilot study [Articolo su rivista]
Ferrari, Anna; Baraldi, Carlo; Licata, Manuela; Vandelli, Daniele; Marchesi, Filippo; Palazzoli, Federica; Verri, Patrizia; Rustichelli, Cecilia; Giuliani, Enrico; Silingardi, Enrico

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.

2016 - Hair testing in clinical setting: simultaneous determination of 50 psychoactive drugs and metabolites in headache patients by LC tandem MS [Articolo su rivista]
Licata, Manuela; Rustichelli, Cecilia; Palazzoli, Federica; Ferrari, Anna; Baraldi, Carlo; Vandelli, Daniele; Verri, Patrizia; Marchesi, Filippo; Silingardi, Enrico

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.

2015 - Tramadol chronic abuse: An evidence from hair analysis by LC tandem MS [Articolo su rivista]
Verri, Patrizia; Rustichelli, Cecilia; Palazzoli, Federica; Vandelli, Daniele; Marchesi, Filippo; Ferrari, Anna; Licata, Manuela

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 R2 values of at least 0.995. The method offered satisfactory precision (RSD < 10%), 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 x 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.

2012 - L'integrazione dell'analisi del capello nel trattamento terapeutico multidisciplinare di un soggetto con dipendenza combinata da alcol e cocaina [Articolo su rivista]
Palazzoli, Federica; Santunione, Anna Laura; Vandelli, Daniele; S., Malak; C., Ferretti; Licata, Manuela

objective toxicology tests combined with self report data, are resulted an appropriate outcome measure. Furthermore comparing self reports and cocaine hair test, hair test result highly sensitive and specific in identifying past cocaine use.

2010 - Mefedrone: una nuova designer drug identificata nel mercato clandestino [Articolo su rivista]
Licata, Manuela; Verri, Patrizia; Vandelli, Daniele; Palazzoli, Federica

recentemente il mercato delle droghe ha subito un considerevole cambiamento: accanto alle sostanze d'abuso tradizionalmente segnalate si è delineato l'incremento delle 'designer drug', le droghe sintetiche, rappresentate da numerose sottoclassi. la finalità del contribbuto è rivolta da un lato ala caratterizzazione chimico-tossicologica del mefedrone, dall'altro alla sua determinaszione in matrici biologiche con l'obiettivo di controllare nuove abitudini assuntive