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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.
- Suicide by self-administration of pethidine, propofol and anxiolytics : a case reportPublication . Proença, Paula; Castanheira, Alice; Castanheira, Fernando; Monteiro, Carla; Simoes Da Silva, Beatriz; Franco, João Miguel; Corte Real, FranciscoBackground and aim: Recreational use of anesthetic and sedative agents in health care practitioners is becoming an increasing problem due to its easy availability and, therefore, they become vulnerable to misusing or abusing of these drugs. The authors present a fatal case involving a a 50-year-old man, nurse, found dead in a hospital room, in supine position, with intravenous access devices in the forearm that were attached to two packages of saline solutions. Some empty packages were close to the victim. A white viscous fluid found at the death scene was send to analysis. The expert's investigation at the scene indicated possible propofol poisoning. Methods: The blood samples were initially subjected to a qualitative analysis. A systematic toxicological drug screening was carried out in blood with a combination of enzyme immunoassays (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Femoral blood alcohol concentration was measured by gas chromatography with headspace (GC/FID) technique. Due to suspected propofol intoxication, a specific analytical methodology was developed in order to detect and quantify this substance in the postmortem samples. Chromatographic analysis of propofol was preceded by protein precipitation of 0.1 mL of blood samples with acetonitrile. Chromatographic separation was achieved with a reversed-phase column, using a gradient elution of 2 mM aqueous ammonium formate (0.1% formic acid), and 2mM ammonium fluoride in methanol, at a flow rate of 0.4 mL/min, with a run time of 10 minutes. Results and discussion: Toxicological results revealed the presence of the anesthetic (propofol) in femoral blood (33.5 mg/L) and heart blood (113.3 mg/L), and also in the white viscous fluid found at the death scene. The remaining laboratory analyses revealed the presence of nordiazepam, temazepam and metoclopramide in therapeutic concentrations, but toxic blood concentrations of midazolam (1473 μg/L), diazepam (5182 μg/L) and pethidine (8622 μg/L). The blood alcohol analysis result was positive (0.18 g/L) as well as positive for opiates (morfine < 25 μg/L). Pesticides were not present in the analysed samples. Conclusion: The development of the method for confirmation and quantification of the anesthetic propofol was crucial given the suspicion of poisoning by this substance. However, laboratory analysis has also revealed the simultaneous presence of an opioid analgesic agent (pethidine) and anxiolytics in high blood concentrations. These findings have allowed to identify a case where several drugs were abused and whose synergy of effects and intoxication, that were not considered at first, were lately found to have contributed to the medico-legal etiology of suicide.
- 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.
- Autópsias virtuais no âmbito forense: alcances e limitaçõesPublication . Lopes, Matilde Figueiredo; Corte Real, Francisco; Monteiro, Carla PintoA autópsia virtual é um método inovador, que tem vindo a solidificar a sua relevância na área da imagiologia forense. Há várias técnicas post mortem, contudo na área forense evidenciam-se a tomografia computorizada post mortem e a ressonância magnética post mortem. Esta abordagem é capaz de fornecer elementos periciais relevantes para a Patologia Forense, através da análise de imagens recolhidas, de forma não invasiva e preservando a integridade do cadáver, com o intuito de esclarecer a causa e as circunstâncias da morte, a identificação do cadáver e a realização de estudos tanatológicos e antropológicos. O objetivo principal deste trabalho é expor os alcances e as limitações da abordagem da autópsia virtual assim como a sua aplicabilidade no âmbito forense, focando, essencialmente, nas técnicas de tomografia computorizada post mortem e de ressonância magnética post mortem. Mas será que a autópsia virtual poderá vir a substituir a autópsia invasiva, em algum momento? Ou será considerada, apenas, como um complemento? O uso desta abordagem como um método de “screening” será exequível? São estas e outras questões que os autores pretendem esclarecer através de uma revisão narrativa. Para isso foi realizado um levantamento por pesquisa na PubMed e outras bases de dados a partir de 1 de janeiro de 2015 até 31 de novembro de 2023, tendo sido incluído conteúdo que se mostrou pertinente fora desse espaço temporal. Como conclusão, a autópsia virtual pode ser recomendada como uma alternativa prática de diagnóstico quando a autópsia convencional não é possível e pode desempenhar um papel como método de “screening”, sobretudo para mortes traumáticas