As an improvement in efficiency and in Human Discrimination Power, the new six dye multiplex kit PowerPlex® Fusion 6C System, by Promega, available for human identification can co-amplify 27 loci, in a single reaction, have been introduced in the last years with great success. This kit allows the amplification and detection of autosomal loci included in the expanded Combined DNA Index System CODIS, plus the loci Penta D, PENTA E and SE33 as well as Amelogenin for gender determination. Furthermore, this kit includes three Y –STRs (DYS391, DYS576 and DYS570), allowing allelic attribution in a total of 27 loci. This genetic markers extension satisfies not only CODIS but also European Standard Set recommendations. Thinking about continuous human migration movements, especially in a very cosmopolitan region like Lisbon and south of Portugal, and also, in keeping population studies and actualized STR databases we decided to update our previous studies. Our sample is composed of 600 unrelated individuals, from paternity testing with laboratory identity anonymised. DNA was extracted by Prep-n-go BufferTM(Thermo-Fisher Scientific). PCR amplification was performed with PowerPlex® Fusion 6C System, according to manufacturer’s guidelines. Fragment analysis was carried out in an Applied Biosystems® 3500 Genetic Analyser. Electrophoresis results were analysed with GeneMapper® ID-X v1.4. Allele frequencies and population statistics, including Hardy-Weinberg equilibrium p-values from exact test probabilities and forensic parameters were calculated with adequate software. In conclusion, our population information was updated in order to apply most recent data in our casework weight of evidence.

Ayahuasca is a beverage consumed at shamanic ceremonies and currently has gained popularity on recreational scenarios. It contains beta-carboline alkaloids and N,N-dimethyltryptamine, which possesses hallucinogenic effects. Only a few studies have elicited the psychoactive effects and the dose of such compounds on neurological dopaminergic cells or animals. In this work, we aimed to study the cytotoxic effects of these compounds present in ayahuasca beverages and on five different teas (Banisteriopsis caapi, Psychotria viridis, Peganum harmala, Mimosa tenuiflora and Dc Ab (commercial name)) preparations on dopaminergic immortalized cell lines. Moreover, a characterization of the derivative alkaloids was also performed. All the extracts were characterized by chromatographic systems and the effect of those compounds in cell viability and total protein levels were analyzed in N27 dopaminergic neurons cell line. This is the first article where cytotoxicity of ayahuasca tea is studied on neurological dopaminergic cells. Overall, results showed that both cell viability and protein contents decreased when cells were exposed to the individual compounds, as well as to the teas and to the two mixtures based on the traditional ayahuasca beverages. View Full-Tex

The importance of studying non-conventional biological matrices such as oral fluid (OF) is increasingly being recognized. This sample presents several advantages, mainly related to its collection procedure: it is non-invasive, easy to perform by non-medical personnel, can be performed under supervision to prevent adulteration, and provides low biohazard risk. OF samples are more likely to contain parent drugs, reflecting recent drug use – a major advantage of this matrix1. A fast and robust analytical methodology was developed in OF samples for the determination of tetrahydrocannabinol (THC), 11-hydroxy-tetrahydrocannabinol (THC-OH), 11-carboxytetrahydrocannabinol (THC-COOH), cannabinol (CBN) and cannabidiol (CBD) by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), aiming at documenting cannabis consumption. Briefly, 200-μL aliquots of OF were subjected to protein precipitation with a refrigerated methanol/acetonitrile mixture (80:20, v/v). After centrifugation, the extracts were evaporated to dryness, reconstituted in methanol, and 5-μL aliquots were injected into the UPLC-QTRAP-MS 6500+ (SCIEX®) system (in a 14-minute run). The analysis was carried out in MRM mode with two transitions for each compound and one transition for each internal standard. The method was validated according to the guidelines of ANSI/ASB 0362. Parameters such as ion suppression/enhancement, interferents, linearity, precision and accuracy, limits of detection and quantification, dilution integrity and stability were studied and showcased satisfactory results. The 2 ng/mL cut-off for THC3 was achieved, and the method was successfully applied to real samples (57.95- 898.28 ng/mL for THC; 0.17-4.09 ng/mL for THC-COOH; 1.26-44.57 ng/mL for CBN; 0.42-1007.86 ng/mL).

STRs are the standard genetic markers mainly used in forensic cases. In routine casework it is important to establish a population genetic database for further reliable statistical analyses. AmpF1STRIdentifiler (Applied Biosystems) and Geneprint Powerplex 16 (Promega Corporation, Madison WI, USA) are multiplex kits wich co-amplifie 17 STR - loci including the segment of X-Y homologous gene Amelogenina routinely used in our laboratory. 13 core short tandem repeat loci standardized under the combined DNA Index System (Codis): CSF1PO, D3S1358, D5S818, D13S317, D16S539, D18S51, D21S11, vWA, FGA, TH01, TPOX, two additional tetranucleotide loci - D2S1338 and D19S433 – and two additional pentanucleotides – Penta E and Penta D. The purpose of this study is to determine the allele distribution data of the 17 STR loci in 2445 caucasian unrelated individuals from the south of Portugal, 176 unrelated individuals from Cabo Verde and 102 unrelated individuals from Angola and compare it with the values of the all the population resident in the same area. Allele frequencies for each locus observed heterozygosity, expected heterozygosity, power of exclusion, power of discrimination, and p values of chi-square test for departures from Hardy-Weinberg expectations were calculated.

Introduction and aims: Different extraction and clean-up protocols have been used for amphetamine-related compounds determination in hair. Microextraction by packed sorbent (MEPS) is a miniaturized version of solidphase extraction (SPE) which has been applied to determine a number of drugs in hair (e.g., opiates, cocaine, ketamine, etc.) [1-4]. However, concerning amphetamine-related drugs, MEPS has only been used in hair for the determination of amphetamine (AMP) and methamphetamine (MAMP). The aim of this work was the development and validation of a method to determine AMP, MAMP, MDA, MDMA, MDE and MBDB in hair using MEPS for sample clean-up. Materials and methods: Hair (50 mg) was incubated with NaOH 1M at 45 °C overnight [5], after which HCl 10M was added for neutralization and the solution was centrifuged. The MEPS procedure for extract clean-up was optimized using the Design of Experiments (DoE) approach, and the final steps were: conditioning (1 x 250 μL of methanol and 1 x 250 μL of deionized water); loading (18 x 100 μL); and elution (7 x 100 μL of 2% NH4OH in acetonitrile). To the eluted extract, 30 μL of MBTFA was added (to avoid loss of analytes), and extracts were evaporated to dryness, following microwave-assisted derivatization with 50 μL of MBTFA. A gas chromatography coupled to mass spectrometry (GC-MS) system was used for determination. Results and discussion: The MEPS procedure resulted in recoveries of 8-14% for AMP, 14-20% for MAMP, 10-15% for MDA, 18-28% for MDMA, 25-43% for MDE and 34-52% for MBDB, in the linear range of 0.2-5.0 ng/mg. Precision and accuracy of the developed method were in accordance with the statements of international guidelines for method validation. Conclusions: This is the first analytical method using MEPS coupled to GC-MS to determine the selected amphetamines in hair samples, proving to be a great alternative to the classic procedures, being rapid, eco-friendly and less expensive.

O consumo de drogas de abuso pertencentes aos grupos das anfetaminas e metanfetaminas, opiáceos e cocaína e derivados, constitui uma ameaça à saúde pública e estabilidade social. Segundo o Relatório Europeu sobre Drogas de 2022, o consumo de drogas atingiu números elevados em toda a União Europeia. O objetivo deste estudo foi a deteção e confirmação de anfetamina, metanfetamina, benzoilecgonina e morfina em amostras de fluído oral e verificar se houve alguma degradação destas substâncias durante o período de armazenamento das amostras estudadas. Para o efeito foram usadas 53 amostras, previamente fortificadas com concentrações conhecidas das substâncias em estudo (pertencentes aos kits da OraSure Technologies, Inc. e destinados à análise de triagem de drogas de abuso pela técnica imunoenzimática ELISA do sistema Evolis Twin Plus, Bio Rad). As amostras apresentavam prazos de validade situados entre os anos 2021 e 2024. Foram usados volumes de 200 μL de fluido oral, para a extração das substâncias estudadas, usando o procedimento de precipitação proteica com acetonitrilo gelado. Seguidamente os extratos foram analisados por cromatografia de gases associada à espectrometria de massas. A identificação das substâncias foi realizada com um cromatógrafo de gases 7890B equipado com uma coluna cromatográfica HP-5MS (5% de fenilmetilpolisiloxano, 30m x 0,32mm; 0,25μm d.i.) e acoplado a um espetrómetro de massa de quadrupolo simples com uma fonte de impacto eletrónico de alta eficiência 5977A (GC-MS-EI, Agilent Technologies). Os dados foram inicialmente adquiridos em full SCAN e posteriormente em modo selected ion monitoring (SIM) usando o software de aquisição MassHunter WorkStation. As substâncias foram quimicamente derivatizadas por aquecimento induzido por microondas durante 90s (soluções de MBTFA para a anfetamina e metanfetamina e de MSTFA com 5% de TMCS para a morfina e benzoilecgonina) e estudados os seus padrões de fragmentação. Os iões foram escolhidos com base na melhor seletividade e abundância, de modo a maximizar as relações sinal-ruído. O método apresenta um limite de deteção de 20 ng/mL para todas as substâncias,tendo sido possível confirmar a presença de anfetamina, metanfetamina,benzoilecgonina e morfina, de acordo com os critérios de aceitação da World Anti-Doping Agency (WADA). Não se verificaram alterações significativas associadas ao tempo de armazenamento das amostras de fluido oral.

According to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), approximately 1.3 million individuals have used opiates, both for medical and illicit purposes1, presenting a significant public health challenge2,3. To address this issue, methods for quantifying these substances are needed. Urine is a commonly employed matrix in clinical and forensic toxicological analyses due to its ease of collection and ample availability. Its short detection window is particularly effective for monitoring recent drug exposure4,5. This study aimed to optimize a method for determining tramadol, codeine, morphine, 6-acetylmorphine, 6- acetylcodeine, and fentanyl in urine samples (250 μL). The process involved centrifugation, acid hydrolysis, and extraction using microextraction by packed sorbent (MEPS). MEPS offered a rapid, environmentally friendly, and reusable extraction technique6. All parameters that influence the extraction were previously optimized. The method was validated following international guidelines, demonstrating excellent linearity [1 to 1000 ng/mL for all compounds, except for fentanyl (10 to 1000 ng/mL), with coefficients of determination of at least 0.99], and presenting coefficients of variation and bias ≤ 15% for precision and accuracy, except for the lowest calibrator (≤ 20%). Recoveries obtained ranged from 17 to 107%, with lowest percentages for morphine (12 to 17%). Despite the low extraction efficiency obtained for morphine, it was possible to detect concentrations as low as 1 ng/mL for all compounds, except for fentanyl (10 ng/mL). The method was successfully applied to real samples from consumers of these substances. This is the first method to use MEPS and GC-MS/MS for the simultaneous determination of these six opioids in urine samples.