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- LC-MS/MS-MS3 for determination and quantification of ∆9-tetrahydrocannabinol and metabolites in blood samplesPublication . Proença, Paula; Martinho, Beatriz; Teixeira, Helena; Monteiro, Carla; Simões, Susana; Franco, João; Corte Real, F.Due to the high prevalence of cannabinoids in forensic toxicology, it is crucial to have an efficient method that allows the use of a small sample amount and that requires a minimal sample preparation, for the determination and quantification of low concentrations. A simple, highly selective and high throughput liquid chromatography tandem mass spectrometry method (LC-MS/MS-MS3) was developed for the determination and quantification of ∆9-tetrahydrocannabinol (THC), 11-hydroxy-∆9-THC (THC-OH) and 11-nor-∆9-THC-9-carboxylic acid (THC-COOH) in blood samples. Chromatographic analysis was preceded by a protein precipitation of 0.1 mL of blood samples with acetonitrile, then THC, THC-OH, THC-COOH and deuterated internal standards were separated on an Acquity UPLC® HSS T3 (100 mm x 2.1 mm i.d., 1.8 mm) reversed-phase column using a gradient elution with 2 mM aqueous ammonium formate (0.1% formic acid) and methanol, at a flow rate of 0.4 mL/min, and with a run time of 10 min. For MS/MS-MS3 analysis, a SCIEX QTRAP® 6500+ linear ion trap triple quadrupole mass spectrometer was used via electrospray ionization (ESI), operated in multiple reaction mode (MRM) and linear ion trap mode (MS3). The method was validated in accordance with international accepted criteria and guidelines. The method was selective and linear between 0.5-100 ng/mL (r2>0.995). The lower limits of quantification (LLOQ) corresponded to the lowest concentrations used for the calibration curves. The coefficients of variation obtained for accuracy and precision were less than 15%. The mean recoveries were between 88.0-101.4% for the studied concentration levels (1 ng/mL, 5 ng/mL and 50 ng/mL). No significant interfering compounds, matrix effects or carryover were observed. The validated method provides a sensitive, efficient and robust procedure for the quantitation of cannabinoids in blood using LC–MS/MS-MS3 and a sample volume of 0.1 mL. This work is also a proof of concept for using LC-MS3 technique to determine drugs in biological samples.
- LC–MS-MS-MS3 for the determination and quantification of ∆9-tetrahydrocannabinol and metabolites in blood samplesPublication . Proença, Paula; Teixeira, Helena; Martinho, Beatriz; Monteiro, Carla; Franco, João; Corte-Real, FranciscoDue to the high prevalence of cannabinoids in forensic toxicology analysis, it is crucial to have an efficient method that allows the use of a small sample amount and that requires a minimal sample preparation for the determination and quantification of low concentrations. A simple, highly selective and high throughput liquid chromatography–tandem mass spectrometry methodology (LC–MS-MS-MS3) was developed for the determination and quantification of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9- tetrahydrocannabinol (THCOH) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) in blood samples. Chromatographic analysis of THC, THC-OH and THC-COOH and their deuterated internal standards was preceded by protein precipitation (PPT) of 0.1 mL of blood samples with acetonitrile. Chromatographic separation was achieved by use of an Acquity UPLC® HHS T3 (100 mm × 2.1 mm i.d., 1.8 μm) reversed-phase column, using a gradient elution of 2 mM aqueous ammonium formate, 0.1% formic acid and methanol at a flow rate of 0.4 mL/min, with a run time of 10 min. For the MS-MS-MS3 analysis, a SCIEX QTRAP® 6500+ triple quadrupole linear ion trap mass spectrometer was used via electrospray ionization (ESI), operated in multiple reaction monitoring (MRM) and linear ion trap mode (MS3). The method was validated in accordance with internationally accepted criteria and guidelines, and proved to be selective and linear between 0.5 and 100 ng/mL (r2 > 0.995). The lower limits of quantification (LLOQ) corresponded to the lowest concentrations used for the calibration curves. The coefficients of variation obtained for accuracy and precision were <15%. The mean recoveries were between 88.0% and 117.2% for the studied concentration levels (1 ng/mL, 5 ng/mL and 50 ng/mL). No significant interfering compounds, matrix effects or carryover were observed. The validated method provides a sensitive, efficient and robust procedure for the quantification of cannabinoids in blood, using LC–MS-MS-MS3 and a sample volume of 0.1 mL. This work is also a proof of concept for using LC–MS3 technique to determine drugs in biological samples.
- A retrospectÍve analysis of data from cannabinoid forensic cases in the Centre of Portugal between 2020 and 2023Publication . Proença, Paula; Teixeira, Helena M.; Monteiro, Carla; Monsanto, Paula; Coelho, Maria; Franco, João Miguel; Corte Real, FranciscoBackground and aim: Marijuana, hashish and other psychoactive products obtained from Cannabis are the most produced and trafficked illicit drugs around the world. Methods: The authors studied all the positive cases for cannabinoids in the Centre of Portugal; from autopsies at the Forensic Clinical and Pathology Service of Coimbra’s Delegation of the National Institute of Legal Medicine and Forensic Sciences (NILMFS), from other autopsies in the Centre of Portugal, and road traffic and labour accidents from June 2020 until March 2023. Results were from blood samples routinely tested for the presence of cannabinoids by enzyme immunoassay (ELISA) and confirmed by LC-MS/MS. When requested, other substances were analysed. Results and discussion: During the study period, a total of 4599 toxicological requests for cannabinoids were received at the Forensic Toxicology Laboratory. 461 cases (10.0%) tested positive in screening tests, all confirmed by LC-MS/MS. Among positive cases, 77% were road traffic surveillance cases , and 24.9% related to other antemortem and postmortem cases.We observed that there was an accentuated consumption of cannabinoids between ages 21 and 30 years old, average age 33 years, and a higher prevalence in men (92.4%). THC was confirmed and quantified in 76.5% of the cases, with an average blood concentration of 13.5 ng/mL; the mean and percent for the active metabolites, 11-OH-THC, was 5.9 ng/mL in 44.5% and for THCCOOH was 20.1 ng/mL in 56.2% of the positive cases. It is important to highlight that in the 75.1% positive road traffic surveillance cases the achieved THC average was 11.6 ng/mL and in postmortem cases related to road traffic accidents this value increased to 16.0 ng/mL. 25.4% of the cases (n=117) had cannabinoids together with other psychoactive substances and ethanol was present in 162 of the cases, with 33.9% presenting a BAC ≥0.5 <1.2 g/L and 50.6% a BAC ≥1.2 g/L. 20.9% of the Forensic Pathology and Clinic cases were road accidents with 79.2% drivers. Conclusion: Cannabis is frequently used before and during driving with high concentrations of the active compounds demonstrating impairment increasing theof a crash. It is also important to be aware of the results showing simultaneous use of THC and alcohol increasing accident risk.
- Phytocanabinoids and synthetic cannabinoids: from recreational consumption to potential therapeutic use – a reviewPublication . Teixeira, Helena M.Cannabinoids are part of the most popular group of illicit substances in the Western world. The word “cannabinoid” refers to any chemical substance, regardless of structure or origin, that binds to the body’s cannabinoid receptors and that has effects similar to those produced by the Cannabis plant. Regarding their origin, cannabinoids can be classified into endocannabinoids, phytocannabinoids and synthetic cannabinoids. The behavioral and physiological effects of cannabinoids have received particular attention over the last few decades, including sensations of euphoria, relaxation and loss of concentration, with their repeated use being associated with short and long-term side effects, including respiratory and cardiovascular disorders, cognitive changes, psychoses, schizophrenia and mood disorders. On the other hand, recent investigations have proposed a promising therapeutic potential of cannabinoid-based drugs for a wide range of medical situations, including neurological and psychiatric disorders, among other indications. The growing popularity in the use of cannabinoid-based compounds, both for recreational and therapeutic purposes, has been accompanied by an equally continuous and growing evolution of knowledge regarding their potential harmful and beneficial effects. However, there are several open questions and challenges to be answered, which require more and better investigations. This article’s main objectives are: i) to understand the importance of the action of cannabinoids in humans; ii) identify the different types of cannabinoids that exist and understand the differences in their action; iii) distinguish the legislative framework for cannabinoid consumption; iv) identify the possible adverse effects of cannabinoid consumption, as well as their potential benefits; v) know the existing medical-scientific evidence in terms of therapeutic potential, particularly in relation to aspects of safety and efficacy; vi) encourage critical thinking about the recreational consumption and therapeutic use of cannabinoids, based both on currently available evidence and gaps in knowledge.