Gamma-hydroxybutyric acid endogenous production and post-mortem behaviour – The importance of different biological matrices, cut-off reference values, sample collection and storage conditions

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.info:eu-repo/semantics/acceptedVersio

Simultaneous analysis of some club drugs in whole blood using solid phase extraction and gas chromatography–mass spectrometry

The use of psychoactive substances to improve social relations and increase body energy, in Rave Culture, has raised many legal and health public concerns, both for illicit trade and consumption. Therefore, forensic toxicology plays an important role in this area, mainly linked to the detection and quantitation of these substances, both in vivo and in post-mortem samples. In fact, at the moment, forensic sciences have been under public authorities’ scrutiny and critical look, due to the increasing attention of the media and public opinion, always applying for the use of scientific knowledge to help solving forensic cases. However, forensic toxicology results are only reliable to solve legal cases if all the analytical methodologies used are appropriately validated. In this work, a methodology for the extraction and analysis of 7-aminoflunitrazepam, buprenorphine, flunitrazepam, ketamine, methadone, phencyclidine (PCP) and d-propoxyphene was developed for whole blood samples, with Solid-Phase Extraction (SPE), using OASIS MCX SPE columns, and gas chromatography coupled to mass spectrometry. The procedure presented here proved to be reliable, specific, selective and sensitive, with good LODs and LOQs and good precision.The adoption of a SPE procedure with an automatic SPE extraction device, allowed an increased level of automation in sample treatment, being contemporarily less time-consuming, increasing productiveness, and allowing good recovery and appropriate selectivity being, also, simple and reproducible. The simultaneous detection and quantitation of all compounds by the same extraction and detection methodology is crucial and has a great potential for forensic toxicology and clinical analysis.info:eu-repo/semantics/acceptedVersio

Quantificação de Club Drugs em amostras biológicas em toxicologia forense

Mestrado em Métodos Biomoleculares AvançadosAs ciências forenses, incluindo a toxicologia forense, apresentam, na actualidade, uma importância acrescida e uma exposição mediática nunca antes atingidas. Os seus resultados são cada vez mais valorizados pelas autoridades judiciais e a sua abrangência é cada vez mais vasta. Tendo como objectivo a aplicação de conhecimentos e métodos científicos na resolução de questões judiciais, a credibilidade e validação das metodologias utilizadas é fulcral para a aceitação de um resultado que terá sempre consequências judiciais. O consumo de substâncias psicoactivas usadas para intensificar fenómenos individuais e colectivos de socialização, associadas a um tipo de sub-cultura organizado ou não em “Clubes de Dança” (dance clubs), denominada Rave Subculture, levanta questões prementes de saúde pública e de envolvimento das autoridades judiciais, quer na prevenção do tráfico, quer na minimização e controlo do seu consumo. As consequências sanitárias e judiciais trazem também as ciências forenses neste plano, justificando a implementação de métodos de detecção e quantificação deste grupo de compostos, quer para avaliação de estado de influenciado, quer para avaliação e determinação de possíveis causas de morte associadas ao seu consumo. Neste trabalho, foi desenvolvido um método de determinação analítica de GHB, GBL, cetamina, propoxifeno, PCP, metadona, flunitrazepam, 7- aminoflunitrazepam e buprenorfina em líquidos biológicos (urina e sangue total) por GC-MS em modo SIM, precedido de extracção por SPE com colunas OASIS® MCX. Os resultados obtidos permitaram a validação da metodologia desenvolvida, a partir de níveis terapêuticos, para a cetamina, propoxifeno, PCP, metadona, flunitrazepam, 7-aminoflunitrazepam e buprenorfina em amostras de urina e sangue total. O método apenas não se mostrou eficaz a concentrações terapêuticas para o flunitrazepam em amostras de urina, com uma taxa de recuperação de 41%. Os restantes compostos apresentaram taxas de recuperação superiores a 68%. Finalmente, o método de extracção não se mostrou eficaz para o GHB e a GBL. Com este método o Serviço de Toxicologia Forense adoptou um procedimento generalista para a detecção deste grupo de substâncias, resultando numa maior rentabilização de recursos físicos e humanos e consequente redução de custos. ABSTRACT: Presently, forensic sciences have been under public authorities’ scrutiny and critical look, due to the increasing attention of the media and public opinion, always applying its aim, the use of scientific knowledge to help solving forensic cases. However, to use forensic toxicology results to solve legal cases, it is crucial to have all the analytical methodologies appropriately validated. The use of psychoactive substances in order to improve social relations and increase body energy in Rave Culture has raised many legal and health public concerns, both for illicit trade and consumption. Forensic toxicology plays an important role in this area, mainly linked to the detection and quantitation of these substances, both in vivo as in post-mortem samples. In this work, we developed a methodology for the extraction and analysis of GHB, GBL, ketamine, propoxyphene, PCP, methadone, flunitrazepam, 7- aminoflunitrazepam and buprenorphine from urine and whole blood samples by SPE (using OASIS® MCX SPE columns) and GC-MS in SIM mode. The results obtained allowed to validate the analytical method for ketamine, propoxyphene, PCP, methadone, flunitrazepam, 7-aminoflunitrazepam and buprenorphine, both for urine and whole blood samples, starting in therapeutic values (except for Flunitrazepam in urine samples, with an extraction efficiency of 41%); all the others compounds shown extraction efficiencies above 68%. Finally the extractive procedure wasn’t efficient both for GHB and GBL. This method constitutes a systematic procedure for club drugs detection able to be routinely applied with evident advantages in terms of a more rational and economic use of equipments and human resources

Endogenous GHB concentrations in whole blood postmortem samples as a biomarker for post mortem interval estimation – A set of real cases analysis

Poster apresentado no 54th Annual Meeting of The International Association of Forensic Toxicologists, Brisbane (Austrália), 2016Gamma-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.N/

Comparison of endogenous GHB concentrations in blood and hair in death cases with emphasis on the post mortem interval

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.info:eu-repo/semantics/acceptedVersio

A fast and reliable method for GHB quantitation in whole blood by GC–MS/MS (TQD) for forensic purposes

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.info:eu-repo/semantics/acceptedVersio

Determinação de GHB em Amostras Biológicas

Poster apresentado no I Encontro de Doutorandos da Faculdade de Medicina da Universidade do Porto, 2011Apresentação dos parâmetros de Validação Analítica já estudados no desenvolvimento da técnica de determinação de GHB em amostras biológicas (sangue, urina, cabelo). Serão apresentados os estudos preliminares realizados para o desenvolvimento do método analítico e os resultados de validação obtidos até ao momento no estudo e desenvolvimento da técnica analítica, designadamente, extracção em fase sólida (SPE) e Cromatografia Gasosa acoplada a Espectrometria de Massa em tandem (GC/MS-MS).info:eu-repo/semantics/publishedVersio

GC-MS – Still standing for clinical and forensic analysis: validation of a multidrug method to detect and quantify illicit drugs

An SPE-GC-MS analytical method using whole blood samples has been developed and validated to detect and quantify nineteen compounds belonging to the Drugs of Abuse (DA) groups of cocaine and metabolites, opiates and new psychoactive substances (NPS). The method detailed here is necessary because the recreational consumption of these DA has increased considerably in recent years and poly-drug-consumption is now very common. The method developed was both specific and selective. Three different working ranges have been defined due to the differences between therapeutic, toxic and lethal concentrations of DA. Linearity was confirmed for the defined working ranges of all DA, except pseudoephedrine, ephedrine, norephedrine, and TFMPP. Since the remaining nineteen substances showed heteroscedasticity, six ponderation factors were studied to find the best fit for each compound. Limits of Detection and Lower Limits of Quantification have been studied and defined. No carryover was noted, with acceptable extraction recoveries. The achievement of all validation criteria according to international guidelines allows the application of the proposed method in routine forensic analysis. Using this method can also significantly reduce response times and GC-MS analysis costs.info:eu-repo/semantics/acceptedVersio

Analysis of organophosphorus pesticides in whole blood by GC-MS-μECD with forensic purposes

In the present work, two multi-residue methods for the determination of ten organophosphorus pesticides (OPs), namely chlorfenvinphos, chlorpyrifos, diazinon, dimethoate, fenthion, malathion, parathion, phosalone, pirimiphos-methyl and quinalphos, in post-mortem whole blood samples are presented. The adopted procedure uses GC-MS for screening and quantitation, and GC-µECD (electron capture detector) for compound confirmation. Three different Solid Phase Extraction (SPE) procedures for OPs with Oasis® hydrophilic lipophilic balanced (HLB) and Sep-Pak® C18 cartridges were tested, and followed by GC-µECD and GC-MS analysis. The Sep-Pak® C18 cartridges extraction procedure was selected since it generated analytical signals 5 times higher than those obtained with the two different Oasis® HLB cartridges extraction procedures. The method has shown to be selective for the isolation of selected OPs as well as to the chosen internal standard (ethion) in postmortem blood samples. Calibration curves between 50 and 5000 ng/mL were prepared using weighted linear regression models (1/x2). It was not possible to establish a working range for fenthion by GC-µECD due to the lower sensitivity of the detector to this compound, whereas for pirimiphos-methyl it was set between 500 and 5000 ng/mL. The limit of quantitation was established at 50 ng/mL for all analytes, except for pirimiphos-methyl by GC-µECD analysis (500 ng/mL). The average extraction efficiency ranged from 72 to 102%. The developed methods were considered robust and fit for the purpose, and had already been adopted in the laboratory routine analysis.info:eu-repo/semantics/acceptedVersio