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- Drogas Clássicas versus Novas Drogas - reflexão sobre a situação atualPublication . Tarelho, Sónia; Castro, André Lobo; Dias, Mário; Franco, João
- GHB – amostragem e armazenamento de amostras biológicas de interesse com fins forensesPublication . Castro, André Lobo; Dias, Mário; Reis, Flávio; Teixeira, HelenaO Ácido gama-hidroxibutírico (GHB) é um composto endógeno com um historial de utilização clínica desde os anos 1960’s. No entanto, devido aos seus efeitos secundários, foi classificado como uma substância controlada. É uma substância associada ao consumo ilícito para fins recreativos, para aumento forçado de massa muscular por parte de praticantes de culturismo e ainda ao abuso sexual facilitado por substâncias. A interpretação medico-legal de um resultado positivo para GHB está dependente do seu contexto endógeno e do comportamento post-mortem do composto. Neste pressuposto, serão discutidos os valores endógenos de GHB, quer in-vivo, quer post-mortem, e serão sugeridos alguns cuidados no que diz respeito à amostragem e armazenamento das diferentes amostras biológicas passíveis de ser utilizadas em contexto clínico e forense.
- Quantificação de D9-THC, 11-OH-THC E THC-COOH por SPE e GC/MS-MS em sangue totalPublication . Castro, André Lobo; Tarelho, Sónia; Quintas, Maria José; Costa, Pedro; Melo, Paula; Franco, João Miguel; Dias, MárioIntrodução: Os canabinóides, obtidos a partir de plantas da espécie Cannabis sativa, continuam a ser as substâncias ilícitas com maior prevalência de consumo a nível mundial. O seu abuso representa um problema importante de saúde pública não só devido aos efeitos nefastos para o organismo do indivíduo consumidor mas, também, devido ao facto de o seu consumo afetar negativamente diversas características psicomotoras relevantes para o desempenho de tarefas como a condução (e.g. capacidade de reação). Geralmente os canabinóides encontram-se presentes no organismo em baixas concentrações, nomeadamente o D9-THC e o 11-OH-THC, pelo que o laboratório de toxicologia forense deve dispor de métodos analíticos dotados de uma capacidade de deteção adequada a esta realidade. Acresce ainda o facto de a legislação em vigor para a avaliação do estado de influenciado por substâncias psicotrópicas, ao nível da fiscalização rodoviária, não definir um valor como critério de positividade, algo que torna ainda mais importante a adoção de métodos de elevada sensibilidade a fim de conferir uma representatividade adequada aos resultados obtidos. O objetivo deste trabalho incluiu a validação analítica de um procedimento para a deteção e quantificação, com fins forenses, de D9-THC, 11-OH-THC e THC-COOH em amostras de sangue total obtidas in vivo e post-mortem, bem como a avaliação da prevalência de casos positivos após a aplicação do respetivo procedimento analítico a casos reais. Material e Métodos: O procedimento analítico inclui a extração em fase sólida (SPE) das substâncias de interesse com recurso a cartuchos HLB OASIS® (Waters), a derivatização com BSTFA:TMCS 99:1 (Supelco) e a utilização de uma técnica instrumental hifenada com recurso a um cromatógrafo de gases GC-450 acoplado a um espectrómetro de massa do tipo triplo quadrupolo MS-300 (Bruker). Resultados: Este trabalho conduziu ao desenvolvimento de um método cromatográfico capaz de permitir a separação total dos três compostos e a deteção por MS-MS através da monitorização de dois iões (m/z 289 e 305) obtidos a partir do ião-percursor m/z 371, para cada composto. O ião m/z 289 é utilizado para efeitos de quantificação. Como padrões internos utilizaram-se os análogos deuterados. A validação analítica incluiu o estudo, entre outros, dos seguintes parâmetros: especificidade e seletividade (0% de falsos resultados positivos ou negativos), limiares analíticos de deteção (LoD) e quantificação (LoQ) e a linearidade na gama de trabalho considerada (LoQ de 1 ng/mL e desempenho linear entre 1-100 ng/mL para todos os compostos avaliados), rendimento do procedimento extrativo, arrastamento entre análises e repetibilidade (inferior a 15%). Discussão: O método desenvolvido evidenciou ser adequado para a deteção e quantificação dos canabinóides selecionados tendo sido obtidos bons resultados ao nível do LoD e LoQ. Estes limiares analíticos são adequados para a deteção e quantificação destas substâncias em contexto forense, nomeadamente no caso das amostras biológicas colhidas âmbito da aplicação do Código da Estrada. Este procedimento analítico foi aplicado à rotina laboratorial representando uma melhoria efetiva na qualidade da resposta do laboratório, evidenciada através do aumento no número de casos positivos nas amostras analisadas relativamente ao procedimento analítico anteriormente em vigor.
- Endogenous GHB concentrations in whole blood postmortem samples as a biomarker for post mortem interval estimation – A set of real cases analysisPublication . Castro, André Lobo; Tarelho, Sónia; Dias, Mário; Reis, Flávio; Teixeira, HelenaGamma-hydroxybutyric acid (GHB) is an endogenous compound which has a story of clinical use and illicit abuse since the 1960’s. Its postmortem behaviour, namely regarding degradation and metabolism, has been increasingly studied to be used as a putative biomarker for post-mortem interval (PMI) estimation. Thus, whole blood post-mortem GHB levels were obtained in thirty two real cases with previous information on death and autopsy data. The samples were treated through sample methanolic precipitation followed by GC-MS/MS analysis (LLOQ=0.1 mg/L). No differences were encountered for the other parameters evaluated, including age [under 44 years-old - 7.87 2.06 mg GHB/L (n=7), 45 to 60 years-old - 6.80 3.67 mg GHB/L (n=13) and over 61 years-old - 5.72 2.39 mg GHB/L (n=12), p0.05], gender [men - 7.79 5.04 mg GHB/L (n=23), women - 6.72 2.60 mg GHB/L (n=9), p=0.273], cause of death [accident - 7.96 ± 2.26 mg GHB/L (n=8), suicide - 6.75 ± 3.22 mg GHB/L (n=7) and unknown/natural death - 5.14 ± 2.96 mg GHB/L (n=17), p0.05] and presence or absence of substances [absence - 6.37 2.61 mg GHB/L, presence - 6.96 3.38 mg GHB/L, p=0.405]. On the other hand, the results obtained suggest that the PMI (until 5 days between death and sampling) influences GHB whole blood concentration, noticed namely between 48 and 72 hours (24 - 48 hours (p=0.893), 48 - 72 hours (p<0.05); 72 - 96 hours (p=0.123). This study brings additional data regarding the usefulness of GHB levels in forensic toxicology, which might be further strengthened with larger, but comparable, studies from other laboratories and institutions in the forensic toxicology context.
- Comparison of endogenous GHB concentrations in blood and hair in death cases with emphasis on the post mortem intervalPublication . Castro, André Lobo; Tarelho, Sónia; Dias, Mário; Reis, Flávio; Teixeira, Helena M.Gamma-hydroxybutyric acid (GHB) is an endogenous compound which has a story of clinical use and illicit abuse since the 1960’s. The possibility to use a multi-sample approach of GHB evaluation, including whole blood and hair, to better characterize a forensic toxicology case and evaluate a possible causal association with the death, is an exciting up-to-date issue. In addition, its post-mortem behaviour, namely regarding degradation and metabolism, has been increasingly investigated as a putative biomarker for post-mortem interval (PMI) estimation. Thus, In order to contribute to clarify this specific aspect, whole blood and hair post-mortem GHB levels were evaluated in thirty two real cases with previous information on death and autopsy data. The results obtained suggest that the PMI (until 5 days between death and sampling) influences GHB whole blood concentration, but not GHB levels in hair samples. No differences were encountered for the other parameters evaluated, including age, gender, cause of death and presence or absence of substances. This study brings new insights regarding the usefulness of GHB levels in forensic toxicology, which might be further strengthened with larger, but comparable, studies from other laboratories and institutions in the context of legal medicine.
- Quantification of Δ9-THC, 11-OH-THC and THCCOOH by SPE and GC-MS-MS in whole blood samples for forensic purposesPublication . Castro, André Lobo; Tarelho, Sónia; Quintas, Maria José; Costa, Pedro; Melo, Paula; Castañera, Antonio; Dias, MárioIntroduction: Cannabis sativa represents the most frequently abused illicit drug around the world. Its abuse represents a major issue in terms of public health, not only due to its physical and psychological effects in terms of individual behaviour, but also due to consequences in terms of impairment, mainly as to driving capabilities is concerned. Detection and accurate quantification at low levels, namely for 9-THC and 11-OH-THC, seems crucial, in terms of response of a forensic toxicology laboratory to answer to legal requirements. The aim of this work considers the analytical validation for detection and quantification of 9-THC, 11-OH-THC and THCCOOH in whole blood samples, in vivo and post-mortem, for forensic purposes. Material and methods: The method included an SPE procedure with HLB OASIS® (WATERS) cartridges, derivatization with BSTFA:TMCS 99:1 (SUPELCO) and an hyphenated instrumental technique based in a GC-450 gas chromatograph coupled to a MS-300 triple quadrupole mass detector (BRUKER). Results: The procedure included a chromatographic run, with full separation of the three compounds, and the MS-MS detection was characterized by two product-ions (289 m/z ; 305 m/z), obtained from the parent-ion 371 m/z. The ion 289 was used for quantification. The respective tri-deuterated substances were used as internal standards. The validation included several parameters, as specificity/selectivity (with 0% of False Positives and False Negatives), LOD and LOQ (1 ng/mL to all compounds), linearity (1 – 100 ng/mL to all compounds), extraction recovery, carryover, inter and intra-day run precision (both under 15%), among others. Discussion: The developed method has shown to be fit to purpose, with good results in terms of increased sensibility and detection limits, due to an optimal response from the analytical equipment, based on tandem MS technology. The LOD and LOQ are compatible with reference values for positive real samples, namely applied to traffic roadside testing. This method has been applied to the laboratory routine, representing an effective improvement in terms of the lab response to legal requirements.
- Quantification of GHB by SPE and GC-MS-MS in whole blood samples for forensic purposesPublication . Castro, André Lobo; Dias, Mário; Reis, Flávio; Teixeira, HelenaIntroduction: Gamma-Hydroxybutyrate (sodium hydroxybutyrate; sodium oxybutyrate; GHB) is known to be an endogenous, naturally occurring, short-chained fatty acid found in mammalian tissues, with wide distribution and action in several brain areas (hipothalamus, basal ganglia). Although it was first synthesised in 1960, it soon was noticed that it is no more than an endogenous compound. With more than 30 years of clinical use, both in Europe and the U.S.A, its illicit use includes recreational use, muscle building effects in bodybuilders and drug-facilitated sexual abuse. Used as a club drug, alone or mixed with other substances, it´s symptoms mimetize MDMA, ketamine and ehtanol. On the ohter hand, it is also used for drug-facilitated sexual abuse (DFSA) purposes. Aim: In this work, the authors developed and validated an analytical procedure for GHB detection in whole blood (in vivo and post-mortem), for forensic purposes. Material and Methods: The analytical method was developed preparing the samples by a SPE procedure with MCX OASIS® cartridges, followed by derivatization with BSTFA-TMCS (99:1) and instrumental analysis developed by GC-MS-MS in a Triple Quadrupole apparatus (BRUKER). Results and Discussion: The described method shows good fitness for purpose for whole blood samples. The obtained LOD and LOQ were 200 ng/mL, for 100 uL of sample. This increase in sensitivity was obtained due to an optimized SPE procedure and an instrumental technique state-of-the-art. The work range started at 200 ng/mL, far below the suggested cut-off for whole blood samples (5-10 mg/L). These results will allow the possibility to distinguish post-mortem production, endogenous values and external consumption, whenever this diagnosis should be determined, being applicable to forensic purposes.
- A fast and reliable method for GHB quantitation in whole blood by GC–MS/MS (TQD) for forensic purposesPublication . Castro, André Lobo; Tarelho, Sónia; Dias, Mário; Reis, Flávio; Teixeira, Helena M.Gamma-Hydroxybutyric Acid (GHB) is an endogenous compound with a story of clinical use since the 1960’s. However, due to its secondary effects, it has become a controlled substance, entering the illicit market. A fully validated, sensitive and reproducible method for the quantification of GHB by methanolic precipitation and GC-MS/MS (TQD) in whole blood is presented. Using 100 µL of whole blood, obtained results included a LOD and LLOQ of 0.1 mg/L and a recovery of 86% in a working range between 0.1 and 100 mg/L. This method is sensitive and specific to detect the presence of GHB in small amounts of whole blood (both ante-mortem or post-mortem), and is, to the authors’ knowledge, the first GC-MS-MS TQD method that uses different precursor ions and product ions for the identification of GHB and GHB-D6 (internal standard). Hence, this method may be especially useful for the study of endogenous values in this biological sample.
- Gamma-hydroxybutyric acid endogenous production and post-mortem behaviour – The importance of different biological matrices, cut-off reference values, sample collection and storage conditionsPublication . Castro, André Lobo; Dias, Mário; Reis, Flávio; Teixeira, Helena M.Gamma-Hydroxybutyric Acid (GHB) is an endogenous compound with a story of clinical use, since the 1960's. However, due to its secondary effects, it has become a controlled substance, entering the illicit market for recreational and “dance club scene” use, muscle enhancement purposes and drug-facilitated sexual assaults. Its endogenous context can bring some difficulties when interpreting, in a forensic context, the analytical values achieved in biological samples. This manuscript reviewed several crucial aspects related to GHB forensic toxicology evaluation, such as its post-mortem behaviour in biological samples; endogenous production values, whether in in vivo and in post-mortem samples; sampling and storage conditions (including stability tests); and cut-off reference values evaluation for different biological samples, such as whole blood, plasma, serum, urine, saliva, bile, vitreous humour and hair. This revision highlights the need of specific sampling care, storage conditions, and cut-off reference values interpretation in different biological samples, essential for proper practical application in forensic toxicology.