Ce travail avait pour objectif d'étudier l'intérêt et les limites de l'analyse de deux matrices alternatives que sont la bile et l'humeur vitrée (HV), en toxicologie médicolégale. Pour chacune des deux matrices, une revue de la littérature visait à investiguer les connaissances utiles à leur application en toxicologie médicolégale. Une place importante de ces revues est réservée à l'anatomie et la physiologie de l'HV et du système biliaire ainsi qu'aux mécanismes de distribution des xénobiotiques dans ces matrices. La partie expérimentale décrit trois études: deux menées sur populations autopsiques et une associant expérimentations animales et études de populations autopsiques. Les deux premières ont permis de proposer des outils statistiques d'interprétation des concentrations de méprobamate mesurées dans ces matrices. Ils peuvent être utilisés dans diverses situations à la place ou en complément de l'interprétation des concentrations sanguines: cadavre exsangue, putréfaction avancée du corps, redistribution post mortem des xénobiotiques… La troisième étude concernait six molécules (diazépam, citalopram, cyamémazine, morphine, caféine et méprobamate). Les molécules détectées dans le sang l'étaient systématiquement dans l'HV et la bile aussi bien dans les prélèvements des populations autopsiques que ceux issus des expérimentations animales. Les concentrations vitréennes chez l'animal et chez l'homme étaient systématiquement corrélées aux concentrations sanguines, exceptées celles de cyamémazine et de citalopram chez l'homme. Pour la bile, une corrélation significative était observée pour le méprobamate et la caféine chez l'homme et l'animal. Il ressort de ces résultats, que l'analyse de l'HV et de la bile permettent de disposer d'informations relatives à la nature des molécules absorbées et à leur rôle dans la survenue du décès / The present study sought to assess the interest and limitations of analyzing two alternative matrices, bile and vitreous humor (VH), in forensic toxicology. For each matrix, a literature review established the state of knowledge relating to their forensic application. The review placed special focus on the anatomy and physiology of VH and the biliary system and the mechanisms of xenobiotic distribution within the specific matrix. The experimental sections describe three studies: two performed on autopsy populations, and one associating autopsy populations to an animal model. The first two studies resulted in statistical tools for interpreting meprobamate concentrations in these matrices, which can be used as alternatives or complements to blood concentrations in various situations: exsanguination, advanced putrefaction, postmortem xenobiotic redistribution, etc. The third study focused on 6 molecules: diazepam, citalopram, cyamemazine, morphine, caffeine and meprobamate. Molecules detected in blood were also systematically detected in VH and bile samples from both the autopsy and animal populations. Animal VH and blood levels showed systematic correlation. In autopsy samples, cyamemazine and citalopram showed no such correlation. In bile, significant correlations with blood concentrations were found for meprobamate and caffeine in both the autopsy and animal populations. This study confirmed the interest of postmortem analysis of bile and VH. Results show that analyzing bile and VH sheds light on drugs intake and on their implication in cause of death

Orientadores: Nelci Fenalti Höehr, Marcos Nogueira Eberlin / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T10:40:41Z (GMT). No. of bitstreams: 1 SantosJunior_JulioCesar_M.pdf: 4092105 bytes, checksum: 8809a688f7411f1a87fd956c6674fe41 (MD5) Previous issue date: 2014 / Resumo: Embora as anfetaminas estejam proibidas no Brasil, elas continuam sendo adquiridas ilegalmente assim como os canabinóides e cocaína, que representam um dos principais problemas de saúde pública a serem enfrentados no nosso país. Um dos grandes desafios desta área é a dificuldade de obtenção de material para exames periciais. O humor vítreo por encontrar-se isolado em um compartimento relativamente protegido de contaminação externa, invasão de microorganismos e traumatismos em geral, bem como por sua simplicidade/estabilidade analítica e esterilidade durante um longo período após a morte constitui-se como uma excelente amostra para a determinação de xenobióticos em corpos politraumatizados, carbonizados ou em decomposição, auxiliando na delegação da causa mortis. O uso da espectrometria de massas e o advento de novas metodologias de ionização são ferramentas essenciais à toxicologia forense, a V-EASI (venture easy ambient sonic-spray), é uma fonte de ionização de fácil aplicação e instalação, que não requer fluxo de eluente e os demais fatores utilizados nas fontes comerciais. Além disso, o uso da espectrometria de massas de ressonância ciclotrônica de íons por transformada de Fourier (FT-ICR-MS) de ultra-alta resolução e exatidão (valores de m/z exatos) leva a exata composição molecular, alcançando erros abaixo de 1 ppm (partes-por-milhão). Perante isso a avaliação da fórmula molecular normalmente é inequívoca. Quando acoplada a ionização por eletrospray (ESI) espécies moleculares suaves são formadas reduzindo a complexidade do espectro e produzindo informação composicional livre de fragmentos em misturas complexas facilitando sua compreensão. Portanto este trabalho visa o desenvolvimento de metodologias analíticas para análise de drogas de abuso presentes em humor vítreo, empregando o uso de técnicas modernas de espectrometria de massas (FT-ICR-MS e V-EASI-MS) / Abstract: Although amphetamines are banned in Brazil, they remain illegally acquired as cannabinoids and cocaine, which account for a public health task to be faced in this country. The major challenge is to obtain material for investigation exams. The vitreous humor constitutes a good alternative for these exams, since it occurs isolated in a protected space, free of external contamination and of microorganisms and traumatisms, and also due to its analytical stability and sterility preserving it for a long period after death. Moreover, the vitreous humor constitutes an excellent sample for the determination of xenobiotics even in polytraumatized bodies, carbonized or in decomposition, involved in the causa mortis. The use of mass spectrometry and the advent of new ionization methods are essential tools for forensic toxicology, the V-EASI (venture easy ambient sonic-spray), is a source of ionization easy to use and install, not requiring nitrogen flow, eluent flow and other factors used in commercial sources. Furthermore, the use of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) provides ultra high resolution and accuracy in mass analysis and its accurate m/z values lead to the exact molecular composition, reaching errors below 1 ppm (parts-per-million) on normal operational conditions, the assigned molecular formula are normally unequivocal. When electrospray ionization (ESI) is used, soft molecular species are formed reducing spectra complexity and providing fragment-free compositional information about complex mixtures facilitating comprehension. Therefore, this work aims at the development of analytical methodologies for the analysis of drugs of abuse present in the vitreous humor, employing the use of modern techniques of mass spectrometry (FT-ICR-MS and V-EASI-MS / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Novel psychoactive substances (NPS) represent a great challenge to toxicologists due to the ability of illicit drug manufacturers to alter NPS chemical structures quickly and with ease to circumvent legislation regulating their use. Each time a new structure is introduced, there is a possibility that it has not been previously recorded in law enforcement or scientific databases. Many toxicology laboratories use targeted analytical methods that rely on libraries of known compounds to identify drugs in samples. However, these libraries do not include large numbers of NPS which could result in non-identification or detection. High-resolution mass spectrometry (HRMS) has been suggested as a method for screening a wide variety of analytes due to its higher sensitivity and mass accuracy as compared to some other forms of mass spectrometry. This technique can generate characteristic MS/MS spectral data for use in compound identification. The main goal of this research was to create a high-resolution mass spectrometry (HRMS) library of NPS and metabolites, as well as validate a method for screening and confirmation of these substances. The study consisted of three main tasks which included; the development of a large high-resolution MS/MS spectral library and database, validation of a method for screening and confirmation of over 800 NPS and metabolites, and screening of blind-spiked and authentic urine specimens to determine real-world applicability of the HRMS library and method. During validation, several isomeric and structurally related NPS were observed which could not be adequately separated using traditional LC methods. A fourth task was therefore added to investigate improved separation using two-dimensional liquid chromatography (2D-LC). Increased resolving power is achieved in 2D-LC through the coupling of multiple orthogonal separation systems. Ultimately, an on-line, comprehensive method was developed using orthogonal reversed-phase columns in each dimension (RP x RP) for improved separation of co-eluting and isomeric synthetic cannabinoids. This work can aid laboratories in the identification of NPS through the use of a validated LC-QTOF-MS method for screening and confirmation and HRMS spectral library. In instances where isomeric and structurally related NPS are not sufficiently separated, RP x RP methods can be explored.

This research study successfully completed three objectives: 1) validate liquid-liquid, supported-liquid, and solid-phase extractions for the quantitation of three barbiturates (amobarbital, butalbital, and phenobarbital) in blood and urine using liquid chromatography-tandem mass spectrometry; 2) to compare the efficiency and effectiveness among methods in accomplishing extraction of barbiturates under the laboratory setting at Boston University School of Medicine; and 3) to report all the analytical data to RTI International for interlaboratory comparison. For the validation study, a six-point linear calibration model (20-2000 ng/mL) with inversely weighted concentration (1/x) was reproducible in all three sample preparation methods for both blood and urine with r2 greater than or equal to 0.994. Bias and precision evaluated from three controls throughout the range of the curve were within ±20% and ±20%CV, respectively. Neither carryover nor interference was observed. Detection limits were evaluated down to 5 ng/mL depending on the extraction procedure. Samples were able to be diluted up to 50 times prior to instrumental analysis. Samples were stable on autosampler at room temperature up to 72 hours after their initial analysis. Recovery of barbiturates from blood and urine all ranged from 45% to 86%. The effect of ionization suppression or enhancement was found to have minimal impact on the validation. For choosing the most suitable method quantifying barbiturates, efficiency and effectiveness were studied. Efficiency evaluates the time and ease of sample preparation required to prepare a sample for analysis. Supported-liquid extraction was found to be the most efficient method for extracting barbiturates as it required the least amount of time to perform and could be easily automated with minimal training. Effectiveness is an assessment of one’s ability to selectively recover target analyte at a reasonably low concentration. By considering a method’s recovery, extract cleanliness, detection limits, and reproducibility, liquid-liquid extraction was the best at quantifying barbiturates in blood and supported-liquid extraction was the most suitable method for extracting barbiturates from urine. For interlaboratory comparison, all the data collected has been reported to RTI International. These findings can be used for examining the overall reliability and reproducibility of the validated methods. Results obtained can also be used to explore the possibility for streamlining sample preparation in the forensic laboratory, and hence reducing the case backlog.

In forensic toxicology, analysis of drugs and metabolites in biological fluids is performed to determine cause of death, suspected drug use, drug facilitated sexual assaults, or whether someone was driving under the influence. Analyte identification and concentration determination can be determined in a variety of matrices (e.g., blood, urine, or oral fluid) and can be complex. It is therefore necessary to have optimal sample preparation and instrumental conditions that work for all matrices of interests. Determining the best approach can be challenging due to the amount of time and resources to perform expansive evaluations of sample preparation, stationary/mobile phases, liquid chromatography (LC) conditions and mass spectrometry (MS) operating parameters. In this study three different sample preparation methods were validated for blood and urine. The three sample preparation methods were solid-phase extraction (SPE), supported liquid extraction (SLE), and liquid-liquid extraction (LLE). Six different drug groups were used as the analytes being tested by the methods. These drug groups were amphetamines, local anesthetics, opioids, hallucinogens, antidepressants, and novel psychoactive substances (NPS). A total of twenty-three drugs were used: amphetamine, methamphetamine, (3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), and 3,4-methylenedioxymethamphetamine (MDMA), benzoylecgonine (BZE), cocaine, lidocaine, codeine, methadone, morphine, 6-monoacetylmorphine (6-MAM), fentanyl, oxycodone, lysergic acid diethylamide (LSD), phencyclidine (PCP), amitriptyline, citalopram, fluoxetine, trazodone, ethylone, α-pyrrolidinopentiophenone (α-PVP), and 25I-NBOMe. The methods were validated according to guidelines set forth by the Scientific Working Group for Forensic Toxicology (SWGTOX) Standard Practices for Method Validation in Forensic Toxicology and the American Academy of Forensic Science (AAFS) Standards Board (ASB) draft of Standard Practices for Method Validation in Forensic Toxicology. Parameters of calibration model, bias, precision, limit of detection (LOD), limit of quantitation (LOQ), dilution integrity, ion suppression/enhancement, interference studies, and stability were evaluated. Recovery was also assessed to determine the efficiency of the extraction. Calibration models met the 0.98 R2 minimum requirement. For all sample preparations the compounds evaluated in each were found to be stable for at least 72 hours. Interferences were found to be similar across all three sample preparation methods. Parameters of bias, precision, and dilution integrity were largely comparable between all three methods. Overall for LOD, SLE resulted in lower values for blood and urine ranging for 0.1 to 5 ng/mL. Overall for LOQ, SLE resulted in lower values for blood and LLE resulted in lower values for urine in the range of 0.5-10 ng/mL. SLE resulted in the highest recovery for all twenty-three analytes, due to LLE failing to extract consistently or completely for benzoylecgonine, morphine, and 6-monoacetylmorphine. Overall, SLE resulted in the lowest percent values for ion suppression and enhancement for both blood and urine. Overall, blood resulted in high ion suppression (exceeding -20%) for SPE and LLE. Final determination overall was that SLE was the best sample preparation method for all twenty-three analytes. This was determined based on the evaluation of recovery, ion suppression/enhancement, and LOD, as well as sample preparation time. Sample preparation time for SLE was approximately 1 hour, while SPE took 2.5 hours and LLE 2 hours.

Despite efforts to control synthetic cannabinoids, clandestine manufacturers continue to modify their structures to avoid legal consequences, creating an ever-changing analytical target for forensic laboratories (1). Forensic toxicology laboratories often lack the needed resources or do not have the capabilities to test for these compounds and metabolites, requiring specimens to be submitted to reference laboratories (2). Drug stability can be affected by long storage times, temperature and preservatives (3). Although these factors can be controlled, systematic research is necessary to identify their impacts on the stability of these new synthetic cannabinoids that are continually emerging. The purpose of this research is to assess the stability of 17 synthetic cannabinoids in human whole blood and 10 synthetic cannabinoid metabolites in human urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) over thirty-five weeks. The analysis methods were validated in accordance to the Academy Standards Board (ASB) method validation guidelines for quantitative analysis and stability evaluation of the following analytes in blood: 4-cyano CUMYL-BUTINACA, ADB-PINACA, EMB-FUBINACA, JWH-250, MO-CHMINACA, 5-fluoro-3,5-ABPFUPPYCA, 5-fluoro ADB-PINACA, APP-PICA, CUMYL-THPINACA, PB-22, XLR11, 5-fluoro PY-PINACA, MDMB-FUBICA, MEP-CHMICA, NM2201, RCS-8, and UR144. The stability analysis in urine includes the following metabolites: 5-fluoro MDMB-PICA metabolite 7, 5-fluoro PB-22 3-carboxyindole, AB-FUBINACA metabolite 3, ADB-PINACA N-(4-hydroxypentyl), ADB-PINACA pentanoic acid, UR-144 Degradant N-pentanoic acid, PB-22 N-(5-hydroxypentyl), MDMB-FUBICA metabolite 3, UR-144 N-(5-hydroxypentyl), and JWH-250 N-pentanoic acid. Research samples were prepared by spiking with certified reference standards (Cayman Chemical, Ann Arbor, MI, USA) of each select synthetic cannabinoid in certified drug-free human whole blood (Boston Medical Center, Boston, MA, USA; Biological Specialty Corporation, Colmar, PA) and drug-free urine that was received as donations following the approved Institutional Review Board guidelines (Boston University School of Medicine, Boston, MA, USA). Blood samples were aliquoted into 6 mL BD Vacutainer Plastic Collection Tubes (Fisher Scientific, Waltham, MA, USA) and urine samples were stored in 15 mL Falcon Conical Centrifuge Tubes (Fisher Scientific, Waltham, MA, USA). Stability under room temperature (20ºC), refrigerator (4ºC), and freezer (-20ºC) at low and high concentrations were evaluated at select time points. A 5% solution of potassium oxalate and sodium fluoride or ethylenediaminetetraacetic acid (EDTA) was added to the preserved blood samples by the manufacturer prior to storage. The anticoagulant, potassium oxalate, was only added in solution to the preserved samples whereas none was added to the nonpreserved samples. Short-term urine samples were preserved with 1% of sodium fluoride prior to storage. Extraction of analytes was conducted using supported-liquid extraction (SLE) ISOLUTE 1 mL cartridges (Biotage, Charlotte, NC, USA) and reconstituted in 100 μL of 50:50 mixture of 0.1% formic acid in millipore deionized water and 0.1% formic acid in acetonitrile (Fisher Scientific, Waltham, MA, USA). Analysis was performed in triplicate using a reverse-phase C18 column (Waters XBridge C18 3.5 μM, 2.1 x 50 mm, Milford, MA, USA) on the Shimadzu Prominence Ultra-Fast Liquid Chromatography (UFLC, Kyoto, Japan) with SCIEX 4000 Q-Trap Electrospray Ionization Tandem Mass Spectrometry (ESI/MS/MS, Waltham, MA, USA) in positive ionization mode. The total run time was 8 minutes with a flow rate of 0.6 mL/min and injection volume of 10 μL. Linear calibration curves for each analyte with the exception of a quadratic regression for PB-22, all had acceptable R2 values > 0.99 using a weighting factor of 1/x. A linear dynamic range of 0.5 – 25 ng/mL was used for all analytes in blood except for NM2201 and APP-PICA with a limit of quantitation (LOQ) of 0.1 ng/mL and MO-CHMINACA with a working range of 0.5 – 15 ng/mL. A linear working range of 5 – 40 ng/mL was utilized for all metabolites in urine. No signs of carryover were observed. In general, analytes were considered stable if the average area ratio between the analyte and internal standard at the time point was within ± 20% of the average area ratio response at time point zero. In some cases, it was necessary to evaluate the complete picture of the stability data by reviewing analyte area, concentration, and overall stability data trend between timepoints at the low and high concentrations. In certain situations, an analyte was considered stable even if specific timepoints for a concentration were outside the ±20% range. For example, in cases where one concentration at a timepoint was within the ±20% range and the other concentration fell within ±30% range the analyte was considered stable overall. Long-term stability results revealed that all synthetic cannabinoids were stable at 21 to 35 weeks in frozen blood preserved with sodium fluoride except for APP-PICA. The preservatives are recommended to be added to blood to reduce the possibility of matrix inferences and minimize detrimental impacts on the stability of synthetic cannabinoids. Analytes experienced lower degradation in the order of samples that were kept frozen, refrigerated, and then at room temperature. Blood analytes that were stable up to 35 weeks in freezer generally had a core structure of a carbonyl substituent on a pyrazole or pyrrole with surrounding nonpolar groups; whereas compounds with two polar carbonyl functional groups present were found to experience degradation much earlier at 1 week or less in room temperature and refrigerator storage conditions. 5-fluoropentyl analogs, like XLR11 and 5-fluoro ADB-PINACA, in comparison to their counterpart analyte, UR144 and ADB-PINACA, were unstable at earlier time points under all storage conditions. Instability in the majority of the urine metabolites was not observed until after 9 weeks and was generally consistent across all storage conditions. The validated methods demonstrate a sensitive and reliable way to positively identify 17 different synthetic cannabinoids in human whole blood and 10 synthetic cannabinoid metabolites in urine for rapid time stability analysis at various storage conditions. The use of SLE improved sample preparation efficiency by decreasing the extraction time from 1 hour to 30 minutes compared to traditional extraction methods, such as solid-phase extraction (SPE) and liquid-liquid extraction (LLE). Further studies into additional matrices, such as oral fluid, longer storage times, and other emerging synthetic cannabinoid analytes would expand the scope of this research.

Per- and polyfluoroalkyl substances (PFAS) encompass a large group of manufactured compounds that have been used in various production processes such as food packaging, commercial products, workplaces, homes, water supplies, and food. PFAS are persistent, resistant to degradation, and can bioaccumulate. Although an exposure limit that predicts adverse health effects has yet to be determined, the Center for Disease Control and Prevention’s 2015-16 health survey found average blood levels of 4.72 ng/ml for PFOS and 1.56 ng/ml for PFOA. The objective of this research was to evaluate the use of laminar flow tandem mass spectrometry following solid phase extraction (SPE) using weak anion exchange (WAX) properties on the detection and quantitation of PFAS compounds. Seven-point calibration standards applied to this research were prepared using certified reference materials (Wellington Laboratories, Ontario, CA), and calibrators were run without sample extraction. The concentrations varied slightly based on the PFAS analyte of interest. All samples and quality controls were prepared by spiking certified reference material (Wellington Laboratories) into pooled human serum (BioIVT, Westbury, NY, USA). A laminar flow QSight®220 ultra-high pressure liquid chromatography-tandem mass spectrometer (LC-MS/MS, PerkinElmer, Waltham, MA, USA) was equipped with a Selectra C18 100 x 2.1mm x 3μm (UCT, Bristol, PA, USA) column with a Brownlee C18 delay column (PerkinElmer) and followed the LC-MS/MS parameters developed for the method. Extraction was accomplished using a WAX SPE column (UCT, ECWAX053) by first conditioning the columns with 1 mL of methanol (Fisher Scientific, Fair Lawn, NJ, USA) followed by 1 mL of 100 mM pH 7 phosphate buffer (Acros Organics, Geel, Belgium, EU). Samples were loaded onto the column at a rate of 1-2 mL/min. The SPE cartridges were washed with 1 mL of 100 mM pH 7 phosphate buffer and 1 mL of millipore water (Millipore Milli- Q Ultrapure Type 1 water system, Millipore Sigma, Burlington, MA, USA), then dried under full flow for 5 minutes. Elution was carried out with 2.5mL of a 98:2 methanol: OptimaTM grade ammonium hydroxide (Fisher Scientific) solution. The eluted samples were then evaporated to dryness using a MULTIVAP® Nitrogen Evaporator (Organomation,Berlin,MA,USA) at 55°C and 5psi. All samples were reconstituted in 100 μL of a 96:4 methanol:water solution. The parameters assessed followed Academy Standards Board Standard 036: Standard Practices for Method Validation in Forensic Toxicology, including matrix interferences, limit of detection (LOD), limit of quantitation (LOQ), a recovery study, and a calibration model. The results of the study were gathered from the following eleven analytes: PFBA, PFBS, PFHxA, PFHpA, PFHxS, PFOA, PFOS, PFNA, PFDA, PFUnA, and PFDoA. Depending on the analyte, a lower LOQ was established at 0.16 – 1.75ng/mL and an upper LOQ at 43.75 – 51.41 ng/mL. Based on the established linear calibration model an LOD in the range of 0.11 - 0.51 ng/mL was achieved. All eleven PFAS analytes showed an acceptable bias of ±20%. All analytes showed a between-run precision (%CV) in an acceptable range of ±20%. No matrix interferences were detected. The average recovery for SPE ranges from 77.64- 104.73% with recovery of 77.64% for PFBS, 83.89% for PFBA, and 95.64-104.73% for PFHxA, PFHpA, PFHxS, PFOA, PFOS, PFNA, PFDA, PFUnA, and PFDoA. Utilizing the UCT WAX SPE column, good recovery for the PFAS compounds was demonstrated. Further, the extraction technique was efficient for high throughput analysis with the extraction time comparable to other traditional SPE methods. The total analytical run time of 11 minutes using the QSight®220 coupled with the UCT Selectra C18 100 x 2.1mm x 3μm column allowed for adequate re-equilibration and system washes to prevent carryover and contamination of these persistent pollutants with excellent chromatography. Having the ability to efficiently and accurately quantify PFAS compounds in biological matrices will allow for better understanding of prevalence, bioaccumulation in biological matrices, and will aid in understanding how these concentrations relate to various health outcomes.

In postmortem toxicology, certain cases require the examination of embalmed biological specimens to investigate the presence and potential role drugs may have played in a person’s death. Key factors, like postmortem distribution, which can be greatly affected by temperature, may alter drug concentrations in different areas of the body. In the United States, the involvement of cocaine in overdose deaths has significantly increased between 2012 and 2019 (1). The purpose of this project was to examine the stability of cocaine and its primary metabolite, benzoylecgonine, in perfused postmortem rat tissues stored at different temperatures over a one month. Twelve frozen cocaine positive rat specimens, intracardially perfused with a saline and formaldehyde solution, were received from a chronic cocaine rat study at Boston University Department of Psychological and Brain Sciences (Dr. Kathleen Kantak, Boston, MA, USA). The specimens were dissected, and the spleen, one kidney, and one lung were removed from each specimen. A fine-needle aspiration biopsy was performed on each organ to collect a time zero (T0) sample. One set of four rat specimens were stored at room temperature (20-22°C), another four were stored at refrigerator temperature (4°C), and another four were stored at freezer temperature (-20°C). A section of each organ was collected for analysis at two weeks (T1) and one month (T2). Samples underwent solid-phase extraction before liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis using a QSight 220 CR Laminar Flow Triple Quadrupole Mass Spectrometer with electrospray ionization, operated in positive ion mode (PerkinElmer, Shelton, CT, USA). Simplicity 3QTM software (PerkinElmer) was used for all data collection, analysis, and quantification. All calibration curves generated for each analyte had acceptable R2 values greater than 0.98 using a weighted linear regression model (1/x). Between T1 and T2, eight samples demonstrated a 15-873% increase in cocaine concentration and four samples had a 13-45% decrease in cocaine concentration. For benzoylecgonine, nine samples demonstrated an 18-289% increase in concentration between T1 and T2 and six samples had a 3-57% decrease in concentration. In samples collected at one month, concentration values for cocaine were highest in samples stored at freezer temperature (-20°C) and lowest in samples stored at refrigerator temperature (4°C). The highest benzoylecgonine values were found in samples stored at freezer temperature (-20°C) as well, and the lowest concentrations were in samples stored at room temperature (20-22°C). Due to the variability in analyte concentration in the organs of the intracardially perfused specimens, the impact storage conditions had on analyte stability could not be determined.

Novel psychoactive substances (NPS) are synthetic drugs that pose serious public health and safety concerns as their ingestion by recreational drug users continues to cause adverse events and death. A multitude of NPS have been implicated in forensic investigations in the United States, but the identification of these emerging substances is challenging and complex, requiring advanced analytical capabilities and novel analysis workflows. The most common and effective manner for identifying NPS is by the use of mass spectrometry, while the true utility of this technology lies within non-targeted acquisition techniques. This research sought to utilize novel drug screening technologies and customized methodologies to characterize current NPS use in high risk populations through the analysis of biological sample extracts discarded from a partnering forensic toxicology reference laboratory. Specifically, NPS detection, identification, and characterization were the primary foci to produce increased awareness and education on a national level. To accomplish these goals, two novel workflows were developed: sample mining and data mining. A liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) assay was developed, validated, and implemented for forensic toxicology analytical testing. A SCIEX TripleTOF™ 5600+ QTOF-MS with SWATH® acquisition coupled to a Shimadzu Nexera XR UHPLC was used. Resulting data were compared against an extensive in-house library database containing more than 800 analytes. The LC-QTOF-MS assay was applied to the re-analysis of biological sample extracts to discover emergent NPS, their metabolites, and trends in use patterns. In total, 3,543 biological sample extracts were analyzed during this research and 21 emerging NPS were detected, some for the first time, through sample mining. Among these emerging substances were the NPS opioids: isopropyl-U-47700, 3,4-methylenedioxy-U-47700, and fluorofuranylfentanyl; the NPS opioid precursors: N-methyl norfentanyl and benzylfuranylfentanyl; the NPS hallucinogens: 2F-deschloroketamine, methoxy-PCP, and hydroxy-PCP; the NPS stimulants: 3,4-methylenedioxy-alpha-PHP, eutylone, and N-ethyl hexedrone; and the NPS benzodiazepine: flualprazolam. With respect to trends, NPS opioid positivity declined over time during this research; however, fentanyl positivity was persistent. Heroin and 3,4-methylenedioxymethamphetamine (MDMA) positivity appeared to decline slightly, but further temporal evaluation is necessary. NPS were less likely to be found in combination with other NPS; only one NPS substance was found in 82.5% of NPS-positive samples. Fentanyl poly-drug use was common, including concurrent or proximate use with traditional opioids (42.8%), NPS opioids (27.3%), cocaine (26.4%), methamphetamine (13.1%), NPS stimulants (4.2%), and other substances. The evaluation of in vitro metabolism for five emerging NPS detected for the first time during this research (3,4-methylenedioxy-U-47700, ortho-fluorofuranylfentanyl, 2F-deschloroketamine, eutylone, and N-ethyl hexedrone) resulted in the characterization of major metabolic pathways and the identification of metabolites presence in vivo by data mining of extract datafiles. These major metabolites provide utility for forensic laboratories to prolong detection windows for NPS. The primary metabolite identified for 3,4-methylenedioxy-U-47700 was N-demethyl-3,4-methylenedioxy-U-47700; the primary metabolite identified for ortho-fluorofuranylfentanyl was fluoro-4-ANPP; the primary metabolite identified for 2F-deschloroketamine was 2F-deschloro-norketamine; and the primary metabolites identified for eutylone and N-ethyl hexedrone were products of hydrogenation to the beta-ketone. As shown through this research, NPS continue to appear in forensic toxicology casework and novel assays for their detection and characterization are critical to remaining at the forefront of emerging drug trends and recreational drug use. LC-QTOF-MS was a vital piece of the analytical puzzle for discovering and characterizing emerging NPS and their metabolites. Analytical chemists must continue research involving NPS to broaden our understanding of synthetic drugs and their public health and safety impacts. / Chemistry

L'objectif de cette thèse était de faire le point sur la place de l'analyse de la moelle osseuse (MO) en tant que matrice alternative au sang en toxicologie médicolégale. Une méthode analytique a été développée et validée pour la quantification du citalopram, du diazepam et ses métabolites (nordazepam, temazepam, oxazepam) dans la MO et 10 autres matrices d'intérêt médicolégal. Cette procédure a été appliquée avec succès dans des cas réels pour l'analyse de matrices dégradées et a permis l'établissement d'une cinétique tissulaire chez l'animal au cours d'une étude pharmacocinétique. Cette cinétique animale a été intégrée dans une modélisation PBPK afin de prédire chez l'homme la distribution tissulaire du citalopram, du diazepam et son métabolite principal, le nordazepam, après administration orale thérapeutique. Ces simulations donnent des clefs intéressantes pour l'interprétation quantitative des concentrations tissulaires en toxicologie médicolégale. Une étude a été conduite pour déterminer l'influence du site de prélèvement sur la détermination des concentrations médullaires de caféine et sur la corrélation de ces concentrations avec les dosages sanguins. Elle montre que le site de prélèvement de MO est un paramètre important à prendre en considération dans l'interprétation quantitative des analyses de MO. L'ensemble de ce travail confirme l'intérêt de la MO en toxicologie médicolégale. Des études expérimentales ont permis d'approfondir les connaissances de cette matrice autour des problématiques du prélèvement, de l'analyse et de la distribution ante mortem afin de contribuer à l'interprétation qualitative et quantitative des analyses réalisées sur la MO / The aim of this work was to evaluate the interest of bone marrow (BM) analysis in forensic toxicology, as an alternative matrix to blood. An analytical method was developed and validated for the quantification of citalopram, diazepam, and its main metabolites (nordazepam, temazepam, oxazepam) in BM and 10 others matrices of forensic interest. This procedure was successfully applied to real cases for putrified sample analyses and to establish a tissue kinetic in rabbit samples for a pharmacokinetic study. These animals kinetics were implemented in PBPK modeling to predict in human tissue distribution of citalopram, diazepam, and its metabolite, nordazepam, after oral therapeutic administration. These predictions gave some clues to interpret quantitatively tissue concentrations in forensic toxicology. A study was also performed to examine whether BM sample location may influence post mortem BM quantification and correlation between BM and blood concentrations. Caffeine was used as test compound. Sample location was found to be an important parameter to consider in quantitative interpretation of BM analyses. This work confirmed the interest of BM in forensic toxicology. Experimental studies improved our knowledge on this matrix about the problematic of sample location, analytical procedure and ante mortem distribution to contribute to qualitative and quantitative interpretation of BM analyses