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- Determination of Ketamine and Norketamine in hair samples using MEPS as sample clean-upPublication . Simão, Ana Y.; Oliveira, Patrik; Rosendo, Luana M.; Rosado, Tiago; Gallardo, Eugenia; Andraus, Maristela; Barroso, MárioIntroduction and Aim: The use of hair samples to determine ketamine (K) and its metabolite, norketamine (NK), has been studied by several authors, using different clean-up approaches. However, microextraction by packed sorbent (MEPS), a miniaturized form of the classic solid-phase extraction has not been applied to date for the purpose. Therefore, a method to determine K and NK in hair samples was developed, optimized, and validated using MEPS as sample clean-up. Materials & Methods: Hair samples were scissor-cut into small fragments, and rinsed with methanol to remove dirt and externally deposited material. Following an overnight incubation with methanol at 65 ºC (no stirring), the compounds were analysed by GC-MS/MS without the need of derivatization procedures; MEPS conditions were: conditioning (5 x 250 µL of methanol and 4 x 250 µL of deionized water); load (10 x 150 µL of hair extract); washing (50 µL of 0.1% acetic acid and 50 µL of 10% methanol); and elution (100 µL of 3% ammonium hydroxide in methanol). Results & Discussion: The procedure resulted in acceptable recoveries, 39-61% for K and 32-43% for NK, and allowed reaching limits of quantification (LOQs) of 50 pg/mg for both analytes. The analytical method presented acceptable accuracy and precision with coefficients of variation typically lower than 15% and BIAS within ± 15%, except at the LOQ (20%). The method was successfully applied to 2 authentic samples, and ketamine concentrations were below 0.05 and 0.18 ng/mg. Norketamine was not detected. Conclusions: This work is the first analytical method using MEPS coupled to GC-MS/MS for the determination of K and NK in hair samples. Following a comparison with a SPE-based method using authentic samples, it was considered rapid and suitable for routine analysis. Acknowledgments: FCT (UIDB /00709/2020 and UIDP/00709/2020). A.Y. Simão acknowledges the PhD fellowship from FCT (2020.09070.BD).
- Otimização de uma metodologia por microextração em seringa empacotada e cromatografia gasosa-espetrometria de massa em tandem para a identificação de arilciclohexaminas em amostras de cabeloPublication . Oliveira, Patrik; Simão, Ana Y.; Rosendo, Luana M.; Pedro, Soraia; Rosado, Tiago; Andraus, Maristela; Barroso, Mário; Gallardo, EugeniaAs novas substâncias psicoativas (NPS) têm vindo a tornar-se um problema cada vez mais um problema de saúde pública. Em Portugal, a ketamina (KET) entrou para a lista de NSP com o Decreto-Lei nº 54/2013. Inicialmente sintetizada com o intuito de substituir a fenciclidina (PCP) como anestésico, acabou por ser usada recreativamente devido aos seus efeitos alucinogénios e estimulantes. Recentemente, a esketamina (análogo da KET) foi introduzida com finalidade terapêutica no tratamento de depressão major refractária a outra terapêutica. Para distinguir o consumo terapêutico do recreativo é necessário que existam métodos que para a sua deteção e quantificação. Neste trabalho foi desenvolvido e otimizado um método para a deteção de KET e seu metabolito (norketamina – NK) em amostras de cabelo com recurso à microextração em seringa empacotada (MEPS) e cromatografia de gases acoplada à espetrometria de massa em tandem (GC-MS/MS). (1) Foi desenvolvido um método analítico para a determinação de KET e NK em amostras de cabelo com recurso à MEPS; (2) A GC-MS/MS foi crucial para deteção inequívoca dos analitos, aumentando a sensibilidade e seletividade do método; (3) As recuperações obtidas são adequadas e foram alcançados LODs baixos (alcançando os cut-offs (0,2 ng/mg) propostos pela Society of Hair Testing) (4) Primeiro método analítico que permite a determinação de KET e NK em amostras de cabelo com recurso a MEPS e GC-MS/MS
- Differentiation of selegiline metabolites and illicit methamphetamine and amphetamine use in elderly postmortem cases: Insights from LC-MS/MS and immunoassay techniquesPublication . Costa, Suzel; Rosendo, Luana M.; Barroso, Mário; Franco, João MiguelAbstract: Introduction: In forensic toxicology, distinguishing between pharmaceutical metabolites and illicit substances is crucial for accurate case interpretation. This study explores two post-mortem cases involving elderly individuals where methamphetamine and amphetamine were detected, raising initial concerns about illicit drug use. Aims: To demonstrate the application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoassay techniques in differentiating between selegiline metabolites and illicit methamphetamine and amphetamine. Methods: Blood samples from three deceased elderly individuals (ages 91 and 89) were analyzed using LC-MS/MS for the quantitative detection of selegiline and metabolites. Initial screening revealed methamphetamine and amphetamine levels suggestive of potential illicit use. Immunoassay testing for dextro isomers (those associated with drug abuse) was subsequently performed to differentiate between isomers. Results and Discussion: LC-MS/MS analysis confirmed selegiline in both cases, with methamphetamine and amphetamine detected. Immunoassay results were negative for dextro isomers, indicating that the detected substances were L-isomers, which is consistent with selegiline metabolism rather than illicit use. Conclusion: This study highlights the critical role of combining LC-MS/MS and immunoassay techniques in forensic toxicology. The negative immunoassay results for dextro isomers clarified that the methamphetamine and amphetamine present were metabolic byproducts of selegiline therapy. Accurate differentiation between therapeutic and illicit substances is essential to avoid misinterpretation, particularly in elderly individuals, where illicit drug use is infrequent. These findings emphasize the need for comprehensive analytical approaches in forensic investigations to ensure accurate and reliable results.
- High concentrations of nordiazepam may impair the detection of benzoylecgonine by GC-MS with derivatizationPublication . Costa, Suzel; Rosendo, Luana M.; Barroso, Mário; Gallardo, EugeniaIntroduction: Forensic toxicology plays a pivotal role in legal investigations, particularly in detecting substances in biological samples. However, the simultaneous presence of multiple substances can pose analytical challenges. For instance, nordiazepam, a major metabolite of some benzodiazepines, can interfere with the detection of benzoylecgonine, a primary metabolite of cocaine, when using gas chromatography-mass spectrometry (GC-MS) with N- methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) derivatization. This interference could result in false negatives for benzoylecgonine detection. Aims: This study aims to assess whether N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide (MBDSTFA) derivatization can reduce interference from nordiazepam, thereby improving the detection and quantification of benzoylecgonine in biological samples. Methods: Solid phase extraction (SPE) was carried out on post-mortem blood samples using Oasis® MCX columns. Microwave derivatization with MSTFA or MBDSTFA followed after the extracts were evaporated. Results and discussion: Two derivatization reagents, MSTFA and MBDSTFA, were compared using GC-MS to evaluate their effectiveness. For this assessment, both the limit of detection (LOD) and limit of quantification (LOQ) for benzoylecgonine in this chromatographic method were 10 and 25 ng/mL, respectively. MBDSTFA derivatization significantly reduced analytical interference between nordiazepam and benzoylecgonine. In contrast, MSTFA derivatization resulted in substantial interference at high concentrations of nordiazepam, potentially leading to false negatives for cocaine detection. These findings highlight the superior performance of MBDSTFA in complex toxicological analyses. Conclusion: MBDSTFA derivatization is more effective than MSTFA for minimizing interference in the detection of benzoylecgonine in the presence of nordiazepam. This method enhances the specificity of forensic toxicological analyses and helps prevent false negatives for detecting cocaine consumption through its principal metabolite, thereby supporting more reliable legal and clinical decisions.