Electronic Theses and Dissertations
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Browsing Electronic Theses and Dissertations by Department "Forensic Science"
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Item Alternate DNA extraction and STR profiling strategies for skeletal and other challenging samples(2019-11-15) Harrel, LeAnn Michelle; Hughes-Stamm, Sheree; Houston, RachelSkeletal remains are often submitted for DNA analysis for human identification (HID) purposes, to either supplement or substitute other forensic identification methods, such as anthropological and odontological analyses. However, the identification of skeletal samples via DNA testing is often challenging and alternate sample processing methods may offer some effective solutions. Current protocols for processing skeletal material involve crushing the bone into a fine powder, which requires specialized equipment, reagents, training, and can pose an increased risk of contamination. Although methods that involve powdering bone tissues for extraction often yield sufficient results for statistical comparisons, bypassing this step can eliminate many of these risks, save time and resources, and also make bone extractions easier for forensic DNA laboratories to implement. Several issues, such as PCR inhibition and DNA degradation, can also make identification more difficult. Therefore, products that provide analysts with more information about sample quality at various stages of the HID process could greatly improve the genotyping process and assist with sample triage and workflow decisions. The main aim of this research was to evaluate the effectiveness of various protocols that eliminate the need to powder bone tissue prior to DNA extraction. A secondary aim of this study was to investigate the benefits of internal STR quality controls for assessing sample quality and determining rework strategies when challenging samples fail to produce complete STR profiles. Several non-powdering DNA extraction methods were tested with human skeletonized remains of varying quality to identify the most efficient protocol to achieve the highest genotyping success. In addition, a broader scope of forensically relevant sample types were genotyped with the Investigator® 24plex QS and GO! kits that include quality controls to test their effectiveness in identifying issues that negatively impact STR success, and guide the most efficient sample rework strategies. As a result of this work, an effective powder-free DNA extraction workflow was identified and shown to be successful with a variety of environmentally challenged skeletal samples. Additionally, we have demonstrated that internal STR quality sensors can simplify STR profile interpretation, help reduce the number of sample reworks, and generate more complete STR profiles when samples are reworked based on the information provided by the quality sensors. These alternate methodological approaches can reduce overall processing time and costs for a wide range of challenging samples subjected to STR typing for human identification purposes. This research has also been used as the scientific basis to amend recommended protocols for some commercial HID products, which will directly benefit the forensic community.Item Alternative Sample Processing Techniques for Rootless Hair Shafts and other Challenging Samples(2021-08-01T05:00:00.000Z) Gutierrez, Ryan Matthew; Houston, RachelEvidence recovered for forensic testing often includes challenges that will impede typical genetic analysis using short tandem repeat markers with capillary electrophoresis platforms. This can include degradation, inhibition, and the lack of sufficient template for successful processing. To increase successful analysis of samples, optimization of testing protocols has been implemented with varying success in forensic crime laboratories. Even with these improvement attempts, it is sometimes necessary to perform alternate analysis on forensic samples. For some cases the sequencing of mitochondrial DNA is performed as a secondary option to traditional workflows. One sample type that is commonly considered challenging and relegated to mitochondrial sequencing is rootless hair shafts. Given these challenges, novel strategies that attempt to improve the recovery of genetic information are often proposed to either circumvent or supplement existing analysis options. These include amplification of markers that are not STRs and analysis of any amplified DNA with alternative instrumentation such as massively parallel sequencing (MPS) platforms. In this research several approaches are implemented to improve the recovery of genetic information from challenging sample types, with a focus on rootless hair samples. The first series of experiments focused on the extraction and amplification of DNA. Several variables were tested during this process including hair pre-treatment, extraction procedure, amplification strategy, and instrumentation. Results from this research showed that while hair decontamination did decrease the occurrence of allele drop-in with rootless hair samples, it also drastically decreased the recovery of DNA. Optimized extraction techniques can increase the recovery of DNA from challenging sample types. Lastly, with challenging samples, it is necessary to concentrate amplification strategies on small amplicon fragments to improve success. To improve results with mitochondrial DNA sequencing, a novel MPS workflow was rigorously assessed for use with forensic samples. Testing parameters included limits of detection, concordance with alternate sequencing techniques, mixture detection, repeatability, and success with forensic type samples. Overall, the workflow tested performed similarly to or better than the comparison methods with a high potential for implementation in the immediate future. Another major concern with many of the challenging forensic sample types is the potential for genetic content to be mixed or contaminated from an external source. To better characterize the occurrence of mixtures in MPS, mixture samples were created using known profiles and amplified for MPS. This data was analyzed to identify potential strengths and weaknesses when compared to capillary electrophoresis. The MPS platform included Y-STR results that would need to be generated using a separate STR amplification on CE. The limit of detection for MPS is also an improvement. The information gathered from isoalleles and additional STR markers can also lead to more discriminatory profiles when looking at some DNA mixtures. However, there are limitations, with a noticeably less reproducible peak height ratios being generated on the MPS platform. The implementation of probabilistic genotyping software with MPS data should be considered to ease mixture interpretation.Item APPLICATIONS OF FORENSIC PLANT SCIENCE IN DRUG TRAFFICKING AND ENVIRONMENTAL CRIMES(2020-05-01T05:00:00.000Z) Roman, Madeline G; Houston, Rachel M; LaRue, BobbyForensic plant science deals with the use of plants as evidence in court. Plant genetic techniques, such as DNA barcoding or DNA fingerprinting, can be used to combat trafficking of illicit drugs by providing leads for law enforcement concerning entry points into the country and linking cases. Additionally, they can be used in court as evidence of environmental crimes, including illegal logging, which often go unpunished due to lack of forensic evidence. DNA barcoding is a technique that involves sequencing regions of the genome to identify a species or population of origin (biogeographical origin), and DNA fingerprinting involves individualizing samples based on their unique genetic profile, usually by using short tandem repeat markers (STRs). Cannabis sativa L. is the source of both an illegal drug, marijuana, and a legal crop, hemp. Marijuana is the most commonly used illicit drug in the United States, and due to state-specific legalization of the drug, law enforcement must prevent and investigate trafficking of marijuana between states, as well as from international sources (e.g., at the border with Mexico). Current methods of identifying C. sativa use microscopic features of the plant or quantify delta-9-tetrahydrocannabinol (THC), the psychoactive component of marijuana. A DNA barcoding method could assist investigations by indicating the biogeographical origin and crop type of a sample and providing a means for linking cases from common growers and distributors. In the first phase of this study, seven polymorphic regions in the chloroplast genome of C. sativa were reported and explored as DNA barcodes for determining biogeographical origin and crop type. An MPS assay was then developed to genotype these hotspots in a high throughput manner, which will facilitate the creation of a worldwide haplotype database, similar to the model of human mitochondrial haplotypes. Additionally, single nucleotide polymorphisms (SNPs) in the tetrahydrocannabinolic acid (THCA) synthase gene were evaluated for their ability to distinguish between marijuana and hemp. The majority of marijuana samples and hemp flowers were classified correctly; however, other variables influence cannabinoid content in C. sativa, resulting in incorrect classifications for some sample types (i.e., hemp seeds and cannabigerol strains). Quantification of THC and THCA is the gold standard for distinguishing between marijuana and hemp, but several sample types (including juvenile plants, seeds, roots, and trace residues) may yield inconclusive chemical results. An alternate DNA approach should be taken with these samples, and the chloroplast DNA barcoding regions proposed in this dissertation may offer a viable future approach. Papaver somniferum (opium poppy) is the source of opiates and opioids, a class of narcotic drugs with high abuse potential. Users who become addicted after being prescribed opiates may turn to alternatives, such as heroin or poppy seed tea, once their prescriptions end. There is currently no forensic method for genetically individualizing samples in cases of poppy seed tea overdoses. The Drug Enforcement Agency’s Heroin Signature Program uses chemical analyses to determine the origin of heroin samples; however, addition of a genetic method would supplement the program and be capable of analyzing difficult sample types (such as trace residues found on drug paraphernalia). Three DNA extraction methods were evaluated for poppy seeds, and a novel quantitative real-time PCR assay was developed and validated for future genetic studies involving P. somniferum. STR markers from the literature were evaluated, and a preliminary STR multiplex was used in a proof-of-concept study to show the potential of future STR panels for individualizing or determining biogeographical origin of heroin or poppy seed tea samples. Eucalyptus is a genus of gum trees (eucalypts) planted around the world for use in the production of paper pulp, hardwood, essential oils, and other industrial products. Illegal logging of eucalypts and other trees costs the world economy billions of dollars annually, and cases of wood theft are often dismissed due to a lack of forensic evidence. Over 1,200 STRs have been discovered in eucalypts, but there has been a lack of forensic research testing these markers for evidence of illegal logging. This project evaluated nine STR markers for Eucalyptus and applied them to a case of illegal logging to demonstrate the utility of STR analysis for providing evidence in court.Item Collection, Direct Amplification, and Genotyping Methods for DNA Recovered from Pipe Bombs and Firearms(2019-07-05) Tasker, Esirioghene; Hughes-Stamm, Sheree; LaRue, BobbyImprovised explosive devices (IEDs) and firearms have often been used to cause mass destruction and harm within communities. When a crime has been committed with these weapons, it is critical that a suspect or suspects be identified quickly. There are several approaches that can be used to identify a potential suspect, but few are as discriminatory as DNA analysis. Short tandem repeat (STR) analysis via capillary electrophoresis (CE) has proven to be robust method of human identification (HID); however, DNA remaining on touched or handled items is likely to be poor in quality and quantity, which can make STR profile interpretation extremely difficult or hinder it completely. Therefore, this study explores various collection strategies, novel genetic markers, and alternate analysis methods to increase the likelihood of retrieving usable genetic information from these challenging items. This was accomplished is four separate phases. The first study compared the informativeness of alternate genetic markers and analysis methods to standard STR analysis. PVC pipe bombs and copper wires were constructed and spiked with known quantities of biological material to recover mock low-template DNA from the surface PVC pipes and traces of blood from the ends of copper wires. DNA collected from PVC pipe fragments underwent traditional STR analysis as well as insertion/null (INNULs) analysis via CE and single nucleotide polymorphism (SNP) analysis via massively parallel sequencing (MPS) for HID. Blood from copper wires were analyzed using ancestry informative SNPs (AISNPs) via MPS. The results of this study showed that a complete INNUL profiles had a higher power of discrimination when less than 14 STR alleles were called. SNP analysis via MPS were the most discriminatory of all the methods tested when the same DNA concentration input was used. In addition, AISNPs correctly predicted the ancestry for five of the six blood samples tested. In the second study, three swab types and a swab storage device called the SwabSaver® were evaluated. Traditional cotton swabs, nylon-flocked swabs, and layered cotton paper swabs were used to collect DNA from handled assault rifle magazines and were then extracted or stored at room-temperature in a centrifuge tube or SwabSaver® device for one or two months. While nylon swabs resulted in higher DNA yields overall, swab storage had a greater impact on STR profile completeness, and swabs stored in the SwabSaver® device resulted in profiles comparable to swabs that were not stored. The third study examined two swab substrates and two direct amplification strategies for DNA collected from common pipe bomb substrates. PVC and steel pipes, electrical tape, and copper wires were spiked with known quantities of epithelial cells and swabbed with a conventional cotton swab or a microFLOQ® Direct swab (nylon-flocked swab). Swabs either underwent traditional DNA processing, direct amplification, or a pre-treatment strategy prior to STR amplification. The results of this proof-of-concept study showed that direct amplification using microFLOQ® Direct swabs was the most successful as it had the shortest processing time and resulted in the most complete STR profiles. Lastly, the fourth and final study examined two sub-sampling strategies for direct amplification using microFLOQ® Direct swabs. The first strategy involved DNA collection from firearms using a regular-tipped nylon swab, which was then sub-sampled using a microFLOQ® Direct swab. Rather than collecting from the larger swab head, the second strategy involved sub-sampling directly from post-blast pipe bomb substrates. A microFLOQ® Direct swab was used to collect from the fragments first and was followed with a more thorough collection with a regular-tipped nylon swab. All microFLOQ® Direct swabs underwent direct amplification while regular nylon swabs underwent traditional processing. Traditional DNA processing resulted in higher yields overall; however, sub-sampling from the larger swab head did result in partial and complete profiles. Sub-sampling from the pipe bomb substrates was mostly unsuccessful, which was likely due poor DNA deposition by the handler and high heat and pressure from the explosion.Item DEVELOPMENT AND APPLICATION OF ANALYTICAL METHODS FOR FENTANYL ANALOGS IN TRADITIONAL AND ALTERNATIVE MATRICES(2021-08-01T05:00:00.000Z) Palmquist, Kaitlyn Bethany; Swortwood, Madeleine JOver the last decade, the United States has experienced increasing numbers of overdose deaths attributed to opioid abuse. While the opioid crisis originated with prescription opioids, recent impacts are credited to synthetic novel psychoactive substances (NPS). Synthetic opioids, a category of NPS, can be divided into two categories: fentanyl- and non-fentanyl derivatives. Fentanyl analogs, the focus of this research, not only pose a significant threat to public safety, but also challenges to forensic laboratories due to their high potency, low concentrations, similar molecular structures, and progressive prevalence. To address detection issues faced by forensic toxicologists, it is necessary to develop highly sensitive analytical methods for detecting fentanyl analogs in traditional and alternative biological matrices. The goals of this study were to 1) develop and validate a data-independent screening method for fentanyl analogs in whole blood and oral fluid for application to postmortem specimens and antemortem oral fluid collected from detainees, respectively; 2) develop and validate a quantitative method for furanyl fentanyl and its metabolites in human and rat plasma for future pharmacological assessment; 3) develop and validate a quantitative method for prevalent fentanyl analogs in whole blood and perform a long-term stability study; and 4) develop and validate a quantitative method for fentanyl analogs in oral fluid for application to antemortem oral fluid samples collected from probationers/parolees. A data-independent screening method was developed and validated for fentanyl analogs (n=14) in whole blood and oral fluid using liquid chromatography-quadrupole-time-of-flight-mass spectrometry (LC-QTOF-MS). Data were acquired in time of flight (TOF) and All Ions fragmentation (AIF) modes and low limits of detection were achieved. A personal compound database and library (PCDL) was developed for targeted and exogenous compound identification. Postmortem blood samples (n=30) received from National Medical Services (NMS) Labs and oral fluid samples (n=20) collected from detainees in Texas detention centers were screened for fentanyl analogs. In the blood samples, analogs of furanylfentanyl (n=16), 4-ANPP (n=16), cis-3-methylfentanyl (n=4), fentanyl (n=4), norfentanyl (n=2), and valerylfentanyl (n=1) were detected. No fentanyl analogs were detected in the oral fluid samples. A quantitative method was developed and validated for furanylfentanyl and its metabolites (4-ANPP and furanyl norfentanyl) in human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using American National Standards Institute/American Standards Board (ANSI/ASB) Standard 036: Standard Practices for Method Validation in Forensic Toxicology. Low limits of detection and small sample volumes (100 μL) were achieved. The method was cross validated in rat plasma for potential application to a pre-clinical pharmacodynamic/pharmacokinetic (PD/PK) study. A method was developed and validated for the quantification of prevalent fentanyl analogs (n=13) in blood using targeted data acquisition on an LC-QTOF-MS. The method was validated according to ANSI/ASB Standard 036. The method was applied to a long-term stability study assessing fentanyl analog degradation over 9 months at four temperature conditions (-20⁰C, 4⁰C, 25⁰C, and 35⁰C). Results described minimal instability under room temperature and refrigerated storage, degradation after 4 freeze/thaw cycles, and instability after 1 week of elevated exposure. Acrylfentanyl had a high degree of instability under most temperature conditions and breakdown mechanism remains undetermined. Authentic forensic blood specimens stored under refrigeration were analyzed 6 months apart to assess stability in postmortem samples. Furanylfentanyl (n=4) and 4-ANPP (n=7) were quantifiable and exhibited percent loss of 0.2-26.8% and 16.3-37.4%, respectively. Loss was attributed to sample source, age, and composition. The aforementioned data acquisition was utilized to develop and validate a quantification method for fentanyl analogs (n=13) in oral fluid using LC-QTOF-MS by ANSI/ASB Standard 036. The method was applied to authentic oral fluid samples (n=16) received from Redwood Toxicology obtained from probationers/parolees. Oral fluid samples were positive for fentanyl (n=16) and 4-ANPP (n=3) at concentrations of 1.0-104.5 ng/mL and 1.2-5.7 ng/mL, respectively. No fentanyl analogs were detected. The present work describes sensitive analytical methods for the detection and quantification of fentanyl analogs with proven applicability to forensically relevant samples. In addition, challenges associated with analyte detection, compound differentiation, and drug instability have been addressed. With the constant emergence of novel fentanyl analogs, forensic toxicologists must be proactive with advancement of analytical analyses and sample treatment.Item Development and Evaluation of miRNA and mRNA Panels for Body Fluid Identification(2019-07-05) Mayes, Carrie A.; Hughes-Stamm, Sheree; LaRue, BobbyThe attribution of biological material to a tissue source, also known as body fluid identification (BFID), can aid investigators in corroborating statements and in the reconstruction of events. Chemical tests, microscopy, enzymatic activity, and immunochromatographic tests are widely employed in crime laboratories at present but have a high false-positive rate and interpretations are often subject to conjecture. Forensic testing requires a methodology that can analyze low level and challenging samples with high specificity. While several molecular targets have been considered for BFID, this project explores the use of microRNA (miRNA) and messenger RNA (mRNA) for BFID. The results of this study provide the forensic community with information on the performance of miRNA and mRNA for BFID using capillary electrophoresis (CE), quantitative reverse transcription PCR (RT-qPCR), and massively parallel sequencing (MPS) technologies, particularly for challenging samples. In the first study, an eight-marker miRNA multiplex was developed for capillary electrophoresis using a linear primer system. Markers were chosen to identify venous blood, menstrual blood, semen, saliva, as well as an endogenous reference gene. Reverse transcription and PCR primers were developed for each marker, evaluated in singleplex, and then multiplexed. Each fluid were co-extracted (DNA/RNA) and amplified with STR and miRNA multiplexes, respectively. All DNA profiles were complete and miRNA profiles correctly identified the body fluid using a decision tree-based interpretation strategy. In the second study, the stability and persistence of miRNA and mRNA was evaluated in challenging samples. Custom reverse transcription quantitative PCR (RT-qPCR) assays were performed to detect the presence of miRNA and mRNA in samples. mRNA targets and miRNA targets were chosen for blood, semen, and an endogenous reference gene respectively. For the evaluation of these markers over time, blood and semen samples were placed in a glass enclosed area exposed to natural heat, humidity, and UV light as well as controlled conditions in a lab cabinet (room temperature, low humidity, and darkness) for up to six months. mRNA was undetected in experimental samples after 30 days, while control mRNA and all miRNA transcripts were detected for the duration of the experiment. A persistence study was also performed by laundering blood and semen stained swatches and either machine drying or allowing the samples to air dry. Blood specific markers were detected in all bloodstained samples, while semen specific markers were observed in all but one semen stained sample. Transfer of both miRNA and mRNA was observed by taking an unstained portion of the swatch. In the final study, an early access mRNA panel for BFID was evaluated. Samples of venous blood, menstrual blood, semen, saliva, and vaginal secretions were placed in a variety of challenging conditions including outside with and without exposure to direct precipitation, buried, on a decomposing cadaver, laundered, aged, and post-coital samples. Co-extracted DNA profiles were evaluated, and RNA was successfully extracted and typed in most samples. Reverse transcription negatives, total reads, and composition of reads attributed to body fluid specific markers were used to evaluate the performance of the panel. Based on the results of the study, potential areas for improvement were highlighted; however, the performance of the panel overall is encouraging.Item DEVELOPMENT AND VALIDATION OF A TECHNIQUE FOR ANALYSIS OF TONER PRINTED DOCUMENTS USING MAGNETIC FLUX MEASUREMENTS(2021-05-01T05:00:00.000Z) Polston, Carrie Elizabeth; Buzzini, PatrickToner printed documents are frequently submitted for forensic examination, yet there is no standard methodology for examination of the documents, and laboratories are recommended to implement ‘complementary analyses’ using multiple different types of instrumentation and analytical techniques which provide categorical information about the production of a document until they have enough information to make a determination about its authenticity, for example. Laboratories often face difficulties in implementing the ‘complementary analyses’ approach, because it requires access to multiple types of costly instrumentation, lengthy analyses and data interpretation, and the results are often inconclusive due to the categorical nature of the data provided by most techniques. It has been acknowledged that there is a need for the development of a method for toner analysis that is non-destructive, cost-efficient, and provides quantitative data that can easily be interpreted using statistical methods. The purpose of this dissertation was to develop a method for toner analysis that fulfilled this need using magnetic flux measurements. To achieve this aim, the project objectives were to 1) assess the printing and device factors which affect the repeatability of measurements; 2) determine the critical method and instrument parameters which affect measurement uncertainty as well as to develop a method protocol which considers those parameters and could be implemented in a forensic laboratory setting; 3) conduct a method validation and apply the refined method protocol to a representative population sample of toners. First, an initial assessment of the magneto-optical imaging technology was conducted which assessed the repeatability and reproducibility of magnetic flux measurements of toners, as well as sample stability. Stability was assessed by measuring the magnetic properties of documents produced by five electrophotographic devices which had been analyzed one year prior. A representative population of 150 toner samples was then assessed using the magnetic flux as a function of toner area as a normalizing unit to determine if significant variation could be observed. The results indicated that magnetic flux does remain stable over time and that there is a relationship between toner area and magnetic flux. The relationship between toner area and magnetic flux allowed for the normalization of data and assisted in comparison of samples with variable text and print features, which is necessary for casework application. The variation present in the population distribution was large enough to allow for discrimination of most samples based on magnetic flux measurement. After the initial assessment, method development was completed, which included assessing the instrument and method parameters which affect measurement uncertainty and developing a method protocol which considers them. At this stage, multiple analyses were conducted to analyze the impact of the variables which had been identified as potential contributors to the measurement uncertainty. One study assessed the impact of changing toner area within the field of the sensor and controlling the grey value variation during the area determination process using seven samples from different electrophotographic devices. This study found that the toner area could vary without significantly impacting measurement uncertainty as long as contacts with the sensor periphery were minimized, and that limiting the variation of the mean grey value to ± 0.5 pixels was effective in reducing measurement uncertainty. The next study assessed the impacts of hysteresis effects and biasing induction current spatial effects on measurement uncertainty, using five samples from different devices. This study found that both induction current spatial effects and hysteresis effects significantly increased intra sample variation, but that hysteresis effects were reciprocal in different planes of rotation and their impacts can be mitigated using a sampling methodology which conducts measurements from four different orientations. Induction current spatial effects could not be completely mitigated via sampling methodology, but there was a relationship between the percent of toner surface area incident to the induction current and measurement enhancement, so this relationship could be exploited to reduce the impacts of biasing induction current spatial effects, and potentially a method could be developed with further research to correct for the signal enhancement. The last stage of method development involved assessing factors which impacted the reproducibility of measurements via an inter-laboratory study. A method protocol was designed which controlled the variables known to impact measurement uncertainty, and this method protocol was issued together with three toner samples to laboratories which were recruited to participate in the study. Laboratory conditions including the instrument version, software update version, operator, and laboratory setup were found to have a significant impact on the reproducibility of results, though the implementation of a standard method did reduce the levels of measurement uncertainty from those observed in the initial assessment, where the operator was the only reproducibility variable assessed. Though measurements cannot be compared between laboratories at this time, it is possible that the development and deployment of a known reference material for use with the instrument would further reduce measurement uncertainty and allow for meaningful inter-laboratory comparisons. Finally, the developed method was validated using a published forensic validation guideline in the field of forensic toxicology. The positive quality control sample provided by the instrument manufacturer was used to assess the bias and precision of measurements, and the potential for matrix effects and interferences were also assessed using five matrix samples from different sources and a collection of 25 different media types that may be encountered on a mixed media document. The bias and precision were found to be within acceptable levels, and no matrix effects or interferents were identified. After validation, the method was applied to a representative population sample. The distribution of measurements in the population sample was similar to the distribution in the initial assessment study, and it was determined that toner populations consistently exhibited variation which provides the potential to discriminate between samples of different origin. Magnetic flux measurements of toners were found to provide the potential for a robust, non-destructive, cost-efficient method for toner examination which provides continuous quantitative data which can be easily interpreted using commonly employed statistical testing methodologies. Although there is still a need for the development of a known reference material for use with the instrument to reduce measurement uncertainty to a level which would allow for inter-laboratory data comparisons, magnetic flux measurement methods can be validated to forensic laboratory standards, allowing practitioners to deploy another analytical tool for toner printed documents analysis.Item Development and Validation of Toxicological Methods for Cognitive Stimulants in Traditional and Alternative Matrices(2021-12-01T06:00:00.000Z) Smith, Christina; Swortwood, Madeleine JAttention-deficit/hyperactivity disorder (ADHD) is a neurological disorder that arises from a lack of dopamine in the brain. Patients with this disorder have an increased level of dopamine reuptake transporters in the brain which leads to a lack of dopamine in the synapse. The dopamine deficiency leads to inability to pay attention, lack of focus and boredom. Medications work to combat this disorder by blocking dopamine reuptake transporters thus increasing dopamine and speeding up brain activity. One of the main medications prescribed to treat ADHD is methylphenidate. Methylphenidate has two chiral centers which gives rise to four stereoisomers: the threo- and erythro- configurations of the dextro- and levo-methylphenidate enantiomers. The threo-methylphenidate configuration is known to be responsible for the pharmaceutical effects, specifically the d enantiomer as the l enantiomer has been proven to be toxic. Methylphenidate is typically sold as a racemic mixture of threo-methylphenidate with both the d and l enantiomer present. Due to the differing effects of the enantiomers, it is important to separate the enantiomers to better understand the pharmacodynamic and pharmacokinetics (PD/PK) of these medications. Methylphenidate is metabolized to ritalinic acid and, in the presence of ethanol, can break down to ethylphenidate. There are minimal comprehensive methods that separate the enantiomers of methylphenidate and its metabolites. To bridge the gap in knowledge, this study aims to analyze these cognitive stimulants in traditional and alternative matrices across multiple analytical platforms. Additionally, stability of these analytes needs to be assessed to better understand proper handling conditions of forensic toxicology specimens. To better understand cognitive stimulants, such as methylphenidate, this study sought to develop analytical methods that can be used for the quantification of these analytes. Additionally, proof of applicability was conducted to demonstrate method validity. The main goals of this study were to 1) develop and validate a method for the chiral separation and analysis of d,l-methylphenidate, d,l-ethylphenidate and ritalinic acid in blood using liquid chromatography-tandem mass spectrometry (LC-MS/MS); 2) develop and validate an achiral method for d,l-methylphenidate, d,l-ethylphenidate, lisdexamfetamine, and amphetamine in oral fluid using LC-MS/MS with application of the method to authentic oral fluid samples; 3) develop and validate a method for the chiral separation and analysis of d,l-methylphenidate, d,l-ethylphenidate and d,l-ritalinic acid in blood using supercritical fluid chromatography (SFC) coupled to LC-MS/MS and apply the method to authentic postmortem blood samples; and 4) assess long- and short-term stability of d,l-methylphenidate, d,l-ethylphenidate and ritalinic acid in blood. A method was developed, optimized and validated for quantification of d,l-methylphenidate, d,l-ethylphenidate, and ritalinic acid in blood using LC-MS/MS. Chiral separation of the enantiomers was achieved using an Agilent Chiral-V column and this method was considered acceptable per validation guidelines with the exception of ion suppression/enhancement. However, the deuterated internal standards compensated for this as well as reproducibility of the effects. This method proved to be suitable for chiral separation without the need for hazardous and costly derivatizing agents traditionally used for separating enantiomers. A method was developed, optimized, and validated for quantification of methylphenidate, ethylphenidate, lisdexamfetamine and amphetamine in oral fluid using LC-MS/MS. This method was considered sensitive and acceptable per validation guidelines except for ion suppression/enhancement. Similar to the blood method, the deuterated internal standard compensated for this phenomenon. For proof of applicability, this method was applied to 4 authentic oral fluid samples collected from college students alongside self-reported medication use. Both lisdexamfetamine and amphetamine were detected in the samples, as expected from subject surveys. This method demonstrates that oral fluid can be used as an alternative forensic toxicology matrix for detection of cognitive stimulants. A method was developed and optimized for quantification of d,l-methylphenidate, d,l-ethylphenidate and d,l-ritalinic acid in blood using SFC-MS/MS. Method validation was conducted and was deemed acceptable. This method was applied to 49 authentic postmortem samples in which the enantiomers of the analytes were quantified and compared to results achieved from an achiral assay. Of the 49 samples, d,l-ritalinic acid was detected in all 49 samples, d-methylphenidate was detected in 29 samples, l-methylphenidate was detected in 15 samples, d-ethylphenidate was detected in 5 samples and l-ethylphenidate was detected in 1 sample. This technique offers an alternative way to achieve chiral separation of analytes. Lastly, the stability of d,l-methylphenidate, d,l-ethylphenidate, and ritalinic acid were assessed over a 9 month period. Storage under frozen temperatures (-20ºC) was the only condition in which all analytes remained stable. A follow up study was conducted to assess methylphenidate degradation and determined that methylphenidate degrades to ritalinic acid under non-frozen conditions. This study demonstrates the importance of understanding proper sample handling and storage conditions as well as time of analysis for unstable drugs where quantification may be of important toxicological value. The developed analytical methods herein offer chiral separation and quantification of methylphenidate and other cognitive stimulants in blood and oral fluid through various analytical techniques. As the potential for cognitive stimulant abuse and misuse is rising, it is important to analyze these analytes in forensic toxicology samples. Data from these studies can be useful for laboratories to better understand chiral analysis, alternative matrices and stability of these analytes for proper detection, quantification, and interpretation.Item Development of a Comprehensive Genetic Tool for Identification of Cannabis sativa Samples for Forensic and Intelligence Purposes(2018-04-13) Houston, Rachel Michelle; Gangitano, David; Hughes-Stamm, ShereeCannabis sativa L. (marijuana) is the most commonly used illicit drug in the United States. Due to partial legalization, law enforcement faces a unique challenge in tracking and preventing flow of the legal marijuana to states where it is still prohibited. Moreover, significant illegal C. sativa traffic from Mexico exists at the US border. To date, no DNA method for Cannabis using short tandem repeat (STR) markers following International Society of Forensic Genetics (ISFG) or Scientific Working Group on DNA Analysis Methods (SWGDAM) recommendations (e.g., use of sequenced allelic ladder, use of tetra-nucleotide STR markers, etc.) has been reported. In addition, there is no existing Cannabis STR reference population database that can be used for forensic purposes (e.g., population in Hardy-Weinberg and linkage equilibrium, parameters of forensic interest). There have been very limited chloroplast (cpDNA) and mitochondrial DNA (mtDNA) studies investigating C. sativa haplotypes in the Americas. Lastly, massively parallel sequencing (MPS) technology has not yet been applied to targeted sequencing of C. sativa for forensic purposes. This project explores the use of genetic tools to identify and determine the origin of C. sativa for forensic purposes. Results provide the forensic DNA community a comprehensive genetic tool (STR, cpDNA, mtDNA, and MPS) that allows for the individualization of Cannabis samples, the association of different cases as well as origin determination of samples for forensic and intelligence purposes. First, a previously reported 15-loci STR multiplex was evaluated. Results of the evaluation indicated that this STR system is not suitable for forensic identification due to several issues; namely high heterozygote peak imbalance in some markers, overlapping alleles between two closely located STR markers, high stutter peaks in dinucleotide markers, inter-loci peak imbalance and presence of null alleles in four of the markers. Therefore, a novel 13-loci STR multiplex was developed and optimized for C. sativa identification (3500 Genetic analyzer), according to ISFG and SWGDAM recommendations, using primer and multiplex STR design software, and a gradient PCR approach for optimal annealing temperature determination. This STR multiplex was validated according SWGDAM guidelines. Case-to-case comparisons were performed by phylogenetic analysis using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) method and parsimony analysis with statistically significant differences detected using pair-wise genetic-distance comparisons. Homogeneous subpopulations (low FST) were determined by phylogenetic analysis and confirmed by bootstrap analysis (95% confidence interval). Results revealed a homogeneous subpopulation that could be used as a Cannabis reference STR population database (N=101) with parameters of population genetics (observed heterozygosity, expected heterozygosity, Hardy-Weinberg equilibrium, and linkage disequilibrium) and of forensic interest (allele frequencies and power of discrimination, etc.). Another previously reported multi-loci system was modified and optimized to genotype five chloroplast and two mitochondrial markers. For this purpose, two assays were designed: a homopolymeric STR pentaplex and a SNP triplex with one chloroplast (Cscp001) marker shared by both methods for quality control. For successful mitochondrial and chloroplast typing, a novel real-time PCR quantitation method was developed and validated to accurately estimate the quantity of the chloroplast DNA (cpDNA) using a synthetic DNA standard. Moreover, a sequenced allelic ladder was also designed for accurate genotyping of the homopolymeric STR pentaplex. And finally, as a proof of concept, a custom panel for MPS was designed to interrogate 12 Cannabis-specific STR loci by sequence. A simple workflow was designed to integrate the custom PCR multiplex into a workflow compatible with the Ion Plus Fragment Library Kit, Ion Chef, and Ion S5 system. For data sorting and sequence analysis, a custom configuration file was designed for STRait Razor v3 to parse and extract STR sequence data. The study resulted in a preliminary investigation of sequence variation for 12 autosomal STR loci in 16 Cannabis samples. Results revealed intra-repeat variation in eight loci where the nominal or size-based allele was identical, but variances were discovered by sequence. In addition, full concordance was observed between the MPS and capillary electrophoresis (CE) data. Although the panel was not fully optimized and only a small number of samples were evaluated, this study demonstrated that more informative STR typing of Cannabis samples can successfully be performed on a MPS platform.Item Development of Magnetic Carbon Nanotubes Facilitated Dispersive-Micro Solid Phase Extraction (Mag-CNT/d-µSPE) for Forensic Applications(2021-08-01T05:00:00.000Z) Li, Sun Yi; Yu, Jorn (Chi Chung)In the past decade, magnetic carbon nanotubes (Mag-CNTs) facilitated dispersive-micro solid phase extraction (d-µSPE) (Mag-CNTs/d-µSPE) methods have been applied in a wide range of analytes. Despite the successful applications of Mag-CNTs/d-µSPE in other fields, limited research was invested in the development and application of Mag-CNTs/d-µSPE in forensic toxicology analysis. The goal of this work was to explore the properties of two in-house synthesized Mag-CNTs and to develop Mag-CNTs/d-µSPE methods for forensic applications. Two surface functionalized Mag-CNTs: carboxyl Mag-CNTs (Mag-CNTs-COOH) and sulfonyl Mag-CNTs (Mag-CNTs-SO3H), were synthesized and characterized. Successful Mag-CNTs/d-µSPE were developed using both Mag-CNTs to extract a cyanide metabolite, 2-aminothiazoline-4-carboxylic acid (ATCA) from biological matrices coupled with gas chromatography-mass spectrometry (GC-MS) analysis. Sensitivity of the Mag-CNTs-COOH/d-µSPE was enhanced using a one-step desorption/derivatization approach. The methods of both Mag-CNTs were validated according to the ANSI/ASB guidelines and their performances were compared to a SPE method. The successful results hopefully will accelerate the adoptability of ATCA analysis in routine cyanide analysis in crime laboratories. Other target analytes focused in this work was opiates present in poppy seed tea samples. Tea brewed from “unwashed” poppy seed was used as home remedy for pain relief and was considered as “legal high.” Different recipes were suggested on online forums to prolong the euphoria and analgesic effects after tighter regulations were imposed. A Mag-CNTs-SO3H/d-µSPE workflow was developed and validated to determine the concentrations of morphine, codeine, and thebaine present in poppy seed tea samples brewed using these suggested recipes. Potential lethal opiate concentrations were obtained from some poppy seed tea samples when moderate amount suggested for new users were used. The reusability and the potential carryover issues of the Mag-CNTs were also assessed in this work. The two Mag-CNTs maintained comparable extraction efficiency through at least four regeneration cycles and demonstrated no carryover issues. The successful development, validation, and applications of the renewable Mag-CNTs/d-µSPE methods showed the strong potential as a greener and efficient alternative sample preparation method for forensic analyses.Item Enhanced sample preparation and data interpretation strategies using massively parallel sequencing for human identification in missing persons' and DVI casework(2018-11-26) Elwick, Kyleen; Hughes-Stamm, Sheree; Gangitano, DavidWhen the remains of victims from mass disasters, military conflicts, or missing persons’ cases are recovered, identification is the most important objective. The recovered unidentified remains may be intact, fragmented, comingled, highly decomposed, or skeletonized. The DNA within these tissues is often degraded, damaged, and/or contains inhibitory agents depending on the environment in which the remains were discovered. This project explores the use of traditional genotyping and newer DNA sequencing technologies for the identification of challenging human remains commonly recovered from mass disasters and missing persons’ cases. The results of this study will provide the forensic community with additional information on the comparative performance of massively parallel sequencing (MPS) chemistries and platforms with compromised samples, particularly highly inhibited samples. This study was comprised of four projects. First, two CE-based STR megaplex kits (GlobalFiler® PCR Amplification and Investigator® 24plex QS kits) were evaluated for their tolerance to PCR inhibitors (humic acid, melanin, hematin, collagen, calcium) and overall sensitivity of detection for high and low quantity (1 ng and 0.1 ng) DNA samples. The results suggested that the GlobalFiler® kit was more sensitive down to 7.8 pg of DNA while the Investigator® kit was more tolerant to all PCR inhibitors at both DNA concentrations. The GlobalFiler® kit produced more alleles, higher peak heights, and higher peak height ratios when determining sensitivity. Conversely, the Investigator® kit produced more alleles and balanced profiles for every inhibitor and inhibitor concentration than the GlobalFiler® kit. Second, two MPS chemistries and platforms (Ion AmpliSeq™ kit on the Ion PGM and the ForenSeq™ kit on the MiSeq FGx™) were evaluated side-by-side using the same inhibited DNA samples. The AmpliSeq™ and ForenSeq™ kits were found to be tolerant and susceptible to different common PCR inhibitors. The AmpliSeq™ chemistry demonstrated tolerance to collagen and calcium; however, it was highly susceptible to humic acid and hematin. Conversely, the ForenSeq™ kit showed extreme tolerance to hematin and calcium inhibitors but was greatly affected by melanin. The third study focused on determining the effectiveness of common DNA extraction methods to remove inhibitors from forensically relevant samples and their downstream compatibility with two MPS chemistries. Three substrates (blood, hair, and bone) were spiked with high concentrations of four inhibitors (humic acid, melanin, hematin, and calcium) and extracted using five DNA extraction methods (DNA IQ™, QIAamp® DNA Investigator, PrepFiler®, and two total demineralization protocols (bone only)). The results showed that all extraction methods were able to efficiently remove all PCR inhibitors with no sign of inhibition and provide sufficiently pure DNA extracts for sequencing. Although the amount of DNA recovered using the different extraction methods differed, the sequencing data indicated that none of the extraction methods negatively influenced the downstream sequencing performance on either MPS system. The fourth and final study reports the comparative performance of two MPS systems when sequencing challenging human skeletal remains. Thermally degraded, embalmed, cremated, burned, and decomposed bones and teeth (N = 24) were extracted using a total demineralization protocol and processed with two MPS chemistries and platforms in addition to traditional CE-based STR typing. The results demonstrated that CE-based STR profiling was still a valuable approach by providing at least a partial DNA profile for every sample, whereas MPS did fail to produce a profile in some instances. However, these MPS chemistries are still not fully optimized to tolerate such difficult samples and further optimization is warranted. Conversely, MPS has the capability to analyze more markers and multiple marker systems (STRs, SNPs, etc.) simultaneously. Therefore, even though some CE samples produced more complete profiles, the additional markers within MPS multiplexes may result in higher powers of discrimination for identification, and thereby provide results to assist with solving missing persons’, forensic, and DVI cases.Item Forensic Applications of DNA Sequencing to Combat Drug Trafficking and Biothreats(2022-05) Graham, Kari Ann; Houston, Rachel M; Hughes-Stamm, Sheree R; Kalafut, Timothy S; Primm, Todd PNonhuman forensic genetics encompasses a variety of fields that focus on using the genome to assess variations within species and between species for legal purposes. Technological advances in sequencing technologies and data analysis have allowed for a better understanding of these variations and have helped develop assays that can be used as investigative tools. This dissertation sought to apply sequencing technologies for use in both microbial forensics by assessing the utility of a third-generation sequencing platform, the Oxford Nanopore Technologies (ONT) MinION portable sequencer for taxonomic identification of bacterial pathogens of interest; and in forensic plant science by applying Sanger sequencing to assess the genetic variation of two forensically relevant species, Papaver somniferum (opium poppy) and Mitragyna speciosa (kratom), for the development of comprehensive tools for species identification and biogeographical source attribution. Microbial forensics focuses on identification of pathogenic agents used in bioterrorism and biocrime for the purpose of source attribution to an individual(s). In 2001, the Anthrax letters bioterrorism attack revealed the need for more robust, universal, and rapid microbial forensic analysis on unknown biological evidence. Traditional approaches for bacterial identification include culturing isolates and molecular approaches involving Sanger and next generation sequencing (NGS) of the 16S ribosomal RNA gene (16S rRNA) but these are laborious and laboratory based. The ONT MinION sequencer can generate long read lengths that span the entire 16S rRNA gene and can accurately identify bacteria to the species level. This platform is also portable and allows for real-time analysis of the pathogen on-site, negating the need for transportation and laboratory analysis. However, it requires large quantities of pure high molecular weight (HMW) DNA compared to other sequencing platforms, thus, the bacterial DNA extraction method of choice is important for successful MinION sequencing. Most extraction methods are tailored to a priori knowledge of the taxonomic grouping, which is difficult to know for certain during an attack. Therefore, it is important to identify a universal extraction method that can isolate high-quality DNA for application to MinION sequencing. Forensic plant science can be used for origin identification of seized illegal drugs derived from plants, such as marijuana, opium poppy, and kratom. P. somniferum, also known as the opium poppy, has been used for the opium stored in the flower pods. This opium contains morphine, codine, and thebaine, which have relaxing and pain-relieving properties. However, these alkaloids are highly addictive and are tightly controlled under the Controlled Substances Act (CSA). This control has led to the production of illegal heroin, which is derived from morphine. These substances have led to an epidemic that is responsible for many deaths due to overdoses. Seized plant material and heroin are identified and sourced using morphological and chemical methods, which have been moderately successful but have major drawbacks. However, the use of the genome, especially the chloroplast genome (cpDNA), could aid in strengthening these methods. Two other species, P. setigerum and P. bracteatum, further complicate the analysis because they are morphologically similar and produce opium. However, these two species are not controlled and are used as a common defense in criminal proceedings. Therefore, methods must be developed that can individualize P. somniferum from its nearest neighbors and source the material to producers, suppliers, and distributors. Mitragyna speciosa, also known as Kratom, is a ‘legal’ drug of abuse that is currently gaining popularity in the US. It has been banned in many countries, such as Thailand, but it is not federally controlled in the United States. Instead, each state has its own laws on sales. The effects are unique: in small quantities it acts like a stimulant, but in larger quantities it has opioid-like effects. There have been deaths documented from kratom use, many in polydrug users resulting in the safety of this product being questioned. The sale of this product is uncontrolled, and users often do not know what they are consuming, the total active alkaloid content, or if it has been adulterated. The plant is very diverse morphologically, and seized components are usually powdered, making identification very difficult. Methods are needed to identify M. speciosa and help source and track the kratom coming into the US, especially in areas where it is illegal to possess and sell. This is confounded by the very limited literature and sequencing data available in databases. The first aim of this study was to identify a universal extraction method that can be coupled with MinION sequencing for use in a forensic situation. It also evaluated the cloud-based data analysis software, EPI2ME, for accurate taxonomic identification. This study revealed that there was no true universal method, but the DNeasy PowerSoil kit and a Chelex-100 method described in the literature are compatible for rapid bacterial identification using MinION sequencing. However, the 16S Barcoding Kit 1-24 coupled with the EPI2ME 16S FASTQ data analysis workflow may not be appropriate for taxonomic identification of forensically relevant species. Furthermore, the intrinsic properties of the bacteria may play a greater role in sequencing accuracy rather than extraction methods. The second aim of this study was to identify cpDNA barcoding markers that can be used as a comprehensive tool for the individualization of P. somniferum. Ten poppy seed samples (used as a proxy for heroin) were extracted, and the target regions of interest were sequenced using Sanger sequencing. Data were compared with previously published P. somniferum cpDNA genomes to assess intraspecies variability, as well as previously published genomes of P. setigerum and P. bracteatum for interspecies variation. There is a lack of sequencing data for the cpDNA genome of this species, and therefore, no hotspot regions were identified when comparing published genomes. Therefore, 11 previously described polymorphic barcode regions found in the literature were screened. Two regions, trnH-psbA and petA-psbJ, showed the most promise in their use for individualization of P. somniferum. Lastly, the third aim of this study was to evaluate intraspecies variation of M. speciosa at recently published barcode markers, ITS, rbcL, matK, and trnH-psbA, for their utility in biogeographical source location. Commercially purchased kratom samples were extracted and sequenced using Sanger sequencing. The intraspecies variability was determined by aligning and comparing our data with the data available in public databases. A novel homopolymer T region was found in trnH-psbA, and two novel SNPs were found in the nuclear ITS region. However, our data appeared to group in two distinct haplotypes with no observable biogeographical trend. These novel applications of sequencing to two distinct forensic fields can help aid investigations by providing intelligence, investigative leads, and for exclusionary purposes.The potential for bioterrorism or biocrime is not misplaced, and robust analytical methods for microbial forensics must be developed and tested. Drug trafficking has major economic and health impacts; therefore, identifying the source and trafficking routes is important to apprehending the individuals involved and stopping the import of illegal drugs. This work has also laid the foundation for future studies using sequencing data in microbial forensics and to aid in combating of drug trafficking.Item Identification and metabolism of suvorexant: Implications for forensic toxicology(2019-11-13) Skillman, Britni Nicole; Kerrigan, SarahSuvorexant (Belsomra®), a novel dual orexin receptor antagonist for the treatment of insomnia, was recently introduced to the pharmaceutical market in 2015. Insomnia affects up to one-third of the American population, which could make suvorexant a popular option for treating these patients. However, due to its recent introduction to the market, few methods have been developed for the detection of suvorexant and limited case reports have been published that examine suvorexant in forensic toxicology casework. Since a limited number of studies exist detailing the analysis of suvorexant, little is known regarding its role in human performance toxicology and postmortem investigations. This study aimed to further the understanding related to its analytical detection, the identification of metabolites, and the drug’s physicochemical properties. In broader terms, the potential for drug-mediated interferences using liquid chromatography-mass spectrometry (LC-MS) is also addressed. Methods for the detection of suvorexant in blood at forensically relevant concentrations were developed and validated using liquid chromatography-quadrupole/time-of-flight-mass spectrometry (LC-Q/TOF-MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Ion suppression and matrix effects using electrospray (ESI) techniques were evaluated and strategies for mitigating interferences in quantitative targeted assays were assessed. The importance of using stable isotope labeled internal standards (SIL-IS) was highlighted using a statistical comparative approach with a structurally similar analog. Suvorexant was quantitated in forensic case specimens and its lipophilicity was determined experimentally and theoretically to evaluate its potential to undergo postmortem redistribution (PMR). In the absence of commercially available metabolite standards, major metabolites for suvorexant were produced in vitro using recombinant cytochrome P450 enzyme systems and were subsequently identified in authentic case specimens.Item Identification and stability of synthetic cathinones in biological samples(2017-11-15) Glicksberg, Lindsay C.; Kerrigan, SarahSynthetic cathinones (“bath salts”) are a class of novel psychoactive substances abused for their psychostimulant and euphoric effects. However, these drugs have received international and national attention due to severe and life threatening adverse effects. In order to properly associate pharmacological, impairing, or toxic effects with synthetic cathinone use, toxicologists must be able to detect and reliably interpret results. The detection of these synthetic phenethylamines relies on validated analytical techniques. Quantitative assays determine the concentration of drug present in biological samples at the time of analysis, which may be significantly different from the concentration at the time of collection or time of death. Drug stability must be understood in order to determine the extent to which these changes influence analytical results. This research provides the forensic toxicology community with a comprehensive understanding of the stability of these compounds in biological matrices. A method for the detection of twenty-two synthetic cathinones, isolated from blood and urine using liquid chromatography quadrupole/time of flight mass spectrometry (LC-Q/TOF MS) was developed and validated. This method was used to assess synthetic cathinone stability in blood (pH 7) and urine (pH 4 and 8) stored at 32°C, 20°C, 4°C, and -20°C for six months. The selected synthetic cathinones were representative of the various structural analogs, including unsubstituted secondary amines (methcathinone, ethcathinone, buphedrone, and pentedrone); ring substituted secondary amines (3-FMC, 4-FMC, 4-MEC, 4-EMC, 3,4-DMMC, mephedrone, and methedrone); methylenedioxy-substituted secondary amines (methylone, ethylone, butylone, pentylone, eutylone); and tertiary amines (α-PVP, naphyrone, pyrovalerone, MPBP, MDPBP, and MDPV). The significance of analyte, storage temperature, storage time, concentration, and matrix pH were systematically assessed. Stability was influenced by structure, matrix pH, and storage temperature. Halogenated cathinones (3-FMC, 4-FMC) were the least stable and the tertiary cathinones bearing the methylenedioxy group (MDPBP, MDPV) were the most stable. The analysis of authentic urine samples from cathinone users supported these experimental findings. Matrix pH and cathinone structure had a more profound influence than prolonged storage. In addition to detecting synthetic cathinones from antemortem specimens to support experimental stability findings, synthetic cathinones were also identified in a series of fifty fatalities to determine postmortem distribution and redistribution. Drugs were identified in central and peripheral blood, urine, liver, vitreous humor, and stomach contents. Central to peripheral blood (C/P) and liver to peripheral blood (L/P) ratios were determined for seven synthetic cathinones to assess postmortem redistribution (PMR). While synthetic cathinones appear to exhibit low to moderate PMR, the highest C/P ratios were observed for cathinones bearing a secondary amine and a methylenedioxy group.Item Identifying the challenges of emerging novel psychoactive substances(2019-11-11) Truver, Michael Thomas; Swortwood, MadeleineNovel psychoactive substances (NPS) are compounds similar to common drugs of abuse with slight modifications to their chemical structure. These modifications can be dangerous and sometimes fatal due the lack of knowledge and/or studies of their adverse effects. NPS are sometimes marketed as “legal highs” or “research chemicals” in order to mimic effects of illicit drugs while simultaneously bypassing legislation. Although there are many drugs that are classified as NPS, novel synthetic opioids and synthetic cannabinoids are the focus of this research. Since little is known about these novel psychoactive substances difficulties may arise when analyzing toxicological samples suspected to contain NPS. To address the emergence of novel psychoactive substances, it is necessary to properly identify issues that could hinder analyses by assessing prevalence, examining instability, determining pharmacology, and identifying potential metabolites. The goals of this study were to 1) develop and validate a method to quantify novel synthetic opioids (NSO), buprenorphine, and heroin markers in oral fluid and apply the method to the analysis of oral fluid collected from detainees; 2) using the acquisition method from the oral fluid method, validate a blood method for the NSO and perform a long term stability study; 3) develop and validate a method to detect a NSO (U-47700) and its metabolites in plasma using a small sample volume (100 µL) and cross validate in rat plasma for a pharmacokinetic study in rats; and 4) identify metabolites for two prominent synthetic cannabinoids in vitro verify metabolism with analysis of authentic urine samples. A method was validated for the quantification of morphine, 6-acetylmorphine, buprenorphine, U-47700, U-49900, U-50488, AH-7921, MT-45, W-18, and W-15 in oral fluid and was deemed acceptable according to Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines. This method was applied to analysis of oral fluid collected from detainees (n=20) in Texas detention centers participating in a drug recognition evaluatoion (DRE) certification program. Although NSO were not detected, valuable data were collected that reinforced oral fluid as viable matrix when compared to presumptive urine results and impairment observations. A blood method was validated for seven NSO and then applied to assess the stability of these analytes over a 36-week study at four temperature conditions (-20°C,4°C, 25°C , and 35°C). The results showed minimal effect on stability at the elevated temperature during the first two weeks, indicating that these analytes would be stable in the event of improper transport/handling within this timeframe. A method was validated for the quantification of U-47700, N-desmethyl-U-47700, and N,N-didesmethyl-U-47700 in human and rat plasma. This method was applied to a pharmacokinetic study where rats were injected with 0 (saline), 0.3, 1.0 or 3.0 mg/kg U-47700. Blood samples were collected at 15, 30, 60, 120, 240, 480 min after injection for quantification of U-47700 and its metabolites. Pharmacodynamic effects were also assessed at the same time points. It was determined that doses of U-47700 had a positive correlation with the behaviors observed which further demonstrates the analgesic effects of this novel synthetic opioid. Finally, a metabolic study utilizing human liver microsomes was conducted to investigate 5F-MDMB-PICA and 5F-MDMB-PINACA in vitro metabolism. In vitro metabolites were verified in vivo by analyzing authentic case specimens. Additionally, the potency and efficacy of 5F-MDMB-PICA and 5F-MDMB-PINACA were identified by examining activity at the CB1 receptor. The EC50 at the CB1 receptor for 5F-MDMB-PICA and 5F-MDMB-PINACA were found to be comparable to each other and JWH-018. There were 22 metabolites identified for 5F-MDMB-PICA and 21 metabolites identified for 5F-MDMB-PINACA. These studies have sought to identify toxicological issues that could arise when detecting a new NPS while providing the necessary data to the forensic toxicology community to further understand the activity and prevalence of such compounds. The challenges that arise when faced with a newly emerged NPS may be detrimental for forensic analysis and thorough characterization of new compounds is necessary for proper identification and detection.Item Improved Detection of Kratom Alkaloids in Forensic Toxicology(2019-11-13) Basiliere, Stephanie Pauline; Kerrigan, SarahKratom is a botanical drug with psychoactive properties that produces both stimulant and opiate effects depending on the dosage. Its major psychoactive components are mitragynine and 7-hydroxymitragynine. The drug may not be included as part of routine toxicological screening and as such, its use may be underreported. This research seeks to improve the analysis of kratom alkaloids in toxicological specimens, and increase overall understanding related to their properties and drug metabolism. Five Mitragyna alkaloids including speciociliatine (SC), mitragynine (MG), paynantheine (PY), speciogynine (SG), and 7-hydroxymitragynine (7-MG-OH) were analyzed in blood and urine using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q/TOF-MS). Each method was validated in accordance with published guidelines for forensic use. LC-Q/TOF-MS was used throughout the study due to its selectivity and sensitivity. Recombinant human cytochrome P450 isoenzymes (rCYPs) were used to investigate the biotransformational pathways involved during drug metabolism. Phase I metabolism was attributed to four rCYPS (CYP3A4, CYP2D6, CYP2C19, and CYP2C18) producing a total of four metabolites (9-O-demethylmitragynine, 16-carboxymitragynine, 9-O-demethyl-16-carboxymitragynine, and 7-hydroxymitragynine). The pH and temperature dependent stability of MG, SC, SG, PY, and 7-MG-OH were also investigated. All five alkaloids were acid labile, with 7-MG-OH being the most unstable. However, SC, SG, and PY were more stable than MG and 7-MG-OH. In addition, two degradation products of mitragynine were identified. Chemical and enzymatic hydrolysis of conjugated metabolites of mitragynine were investigated using postmortem urine specimens. Acid and base hydrolysis, in addition to nine enzyme systems (β-glucuronidase: Escherichia coli, Patella vulgata, Helix pomatia; sulfatase: abalone entrails, Aerobacter aerogenes. Patella vulgata, Helix pomatia; recombinant systems; BGTurbo™, BGS™, ASPC™, IMCSzyme) were evaluated. Ultimately, hydrolysis did not improve the analysis of 9-O-demethylmitragynine and 7-hydroxymitragynine, but did improve the detection of 16-carboxymitragynine. Postmortem blood (n=40), urine (n=16), and tissue (n=20) specimens from 20 fatalities were also evaluated. Speciociliatine and speciogynine were shown to be biomarkers of kratom use, with concentrations higher than mitragynine in the majority of specimens. In addition, the majority of blood and urine specimens contained several metabolites of mitragynine, with 9-O-demethylmitragynine and 7-hydroxymitragynine being the most prominent.Item Influence of genetic variation on social behaviors and frontal cortex differences in generalized anxiety disorder(2019-07-12) Chesna, Elizabeth Ann; Boisvert, Danielle; LaRue, BobbyCertain behaviors have a major impact on the criminal justice system and medical field. The research presented here focuses on antisocial behaviors and generalized anxiety disorder (GAD). Antisocial behaviors such as aggression, criminal behavior, and drug abuse contribute to violent crime. In developed countries, the majority of violent crime is committed by a reduced group of antisocial recidivistic offenders. Currently, the United States has the largest incarceration rate in the world. Identification of genetic variants that influence these behaviors is crucial for the prevention of crime, reduction in recidivism, and the understanding of the etiology of criminal behavior in general. In the first part of this study, a custom primer panel for massively parallel sequencing (MPS) was designed to include 48 single nucleotide polymorphisms (SNPs) potentially associated with social behaviors. Traditional methods, such as single base extension (SBE), are limited in multiplexing capability and time consuming. MPS is more cost effective and allows for a large number of SNPs to be analyzed simultaneously. A preliminary sample set of 100 Caucasian male students were used to assess the validity and concordance of this custom MPS panel. Eight SNPs were genotyped using both SBE and MPS techniques, with all successful profiles being 100% concordant. Participants also completed a survey assessing multiple behaviors and psychological traits. While no significant associations were found in this preliminary sample pool, some trends were observed in behavioral traits. The findings of this study suggest that this panel can be used to simultaneously assess a large number of behavioral and psychological markers. To further explore these results, genetic variants observed in the preliminary control population were compared to a set of high risk individuals. Therefore, in the second part of this study, 19 markers associated with dopamine (DA) turnover and oxytocin (OXT) were compared between an inmate (N=100) and control (N=100) population. Two SNPs (rs909525 and rs1799836) associated with monoamine oxidase had significantly different major allele frequencies between control and inmate populations (p=0.00002 and p=0.00004 respectively). Moreover, haplotype analysis revealed strong linkage disequilibrium in markers associated with monoamine oxidase A (MAOA), catechol-O-methyl transferase (COMT), and OXT. Two haplotypes associated with MAOA had differences in frequency between controls and inmates. Haplotype GAT was observed more often in inmates than controls (p=0.0012) and GGT was not observed in the inmate population (p=0.000004). Multifactor dimensionality reduction was used to test for gene-gene interaction. Epistasis between markers was not found; however, strong redundancies between rs4680 and rs11476, and rs1799836 and rs740603 were observed. These results provide evidence that marker variation occurs between inmate and control samples and this variation may contribute to behaviors associated with delinquency. Anxiety disorders also have a major impact on society, as they are the most common type of psychiatric disorder. Among these, generalized anxiety disorder (GAD) is one of the most prevelant. GAD involves persistent anxiety and may worsen over time if left untreated. As a result, an individual’s daily life is impaired. Furthermore, there is an economic burden on society and the healthcare system. Imaging techniques, including functional magnetic resonance imaging (fMRI), have allowed for better understanding of structural and functional changes involved in GAD. In the third part of this study, fMRI was used to assess thickness and surface area differences in GAD patients. Moreover, eleven bilateral frontal regions defined in the Desikan-Kiliany Atlas were compared. A total of 300 participants were included in this study within three groups: GAD patients (N=100), psychiatric controls (N=100), and healthy controls (N=100). Groups were matched for demographic characteristics and other psychiatric conditions. No significant differences were observed for surface area in the left or right hemisphere; however, significant differences were found for thickness in both hemispheres. In the left hemisphere, lower thickness was observed in GAD patients verses healthy controls (p=0.0001) for the pars triangularis and superior frontal region (p=0.0000). Also, significantly lower thickness was observed in psychiatric controls compared to healthy controls (p=0.0000) for the superior frontal region. In the right hemisphere, lower thickness was observed in GAD patients versus healthy controls (p=0.0006) for the caudal middle frontal region and superior frontal region in GAD (p=0.0000). These findings provide evidence that these structures may be involved in GAD. Furthermore, they also suggest GAD may be due to damage from chronic stress as it suppresses neurogenesis, dendritic growth, and synaptic strength.Item Metabolism and Analysis of Desomorphine(2018-11-20) Winborn, Jessica; Kerrigan, Sarah; Haines, DonovanDesomorphine is a semi-synthetic opioid that is ten times more potent than morphine, with a faster onset but shorter duration of action. It is a major component of the drug referred to as “Krokodil”, which is used as a heroin substitute. Its prevalence is difficult to estimate due to a lack of analytically confirmed cases, which may in part be due to the limited studies regarding its pharmacology or methodology to detect the drug in biological specimens. This research seeks to further the understanding of both desomorphine’s metabolism and its detection in biological specimens, to facilitate its identification in clinical and forensic toxicology laboratories. Six commercially available enzyme-linked immunosorbent assays were evaluated to determine their effectiveness with respect to desomorphine detection. Cross-reactivities were highly variable between assays, ranging from <2.5-77%. Recombinant human cytochrome P450 enzymes (rCYPs) and recombinant uridine 5'-diphospho-glucuronosyltransferases (rUGTs) were used to investigate the biotransformational pathways involved in desomorphine metabolism. Phase I metabolism could be attributed to seven rCYPs (rCYP2B6, rCYP2C8, rCYP2C9, rCYP2C18, rCYP2C19, rCYP2D6 and rCYP3A4), producing a total of nine phase I metabolites (nordesomorphine, desomorphine-N-oxide, two norhydroxydesomorphine isomers, and five hydroxylated isomers). During phase II metabolism, desomorphine-glucuronide was produced by nine rUGTs (rUGT1A1, rUGT1A3, rUGT1A8, rUGT1A9, rUGT1A10, rUGT2B4, rUGT2B7, rUGT2B15, and rUGT2B17). Chemical and enzymatic hydrolysis of conjugated metabolites were investigated using desomorphine-glucuronide generated in situ using rUGT enzyme. Acid hydrolysis and five β-glucuronidase sources (BGTurbo™, IMCSzyme™, Escherichia coli, Helix pomatia and Patella vulgata) were evaluated. Acid hydrolysis produced complete hydrolysis of desomorphine-glucuronide, and under optimal conditions, each enzyme produced complete or near complete hydrolysis (≥96%), with BGTurbo™ and IMCSzyme™ offering the shortest incubation times. Under simulated challenging conditions, P. vulgata was the most effective enzyme evaluated. Desomorphine was analyzed in blood and urine samples using gas chromatography-mass spectrometry (GC-MS), and urine samples were additionally analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-quadrupole/time of flight-mass spectrometry (LC-Q/TOF-MS). Each method was validated in accordance with published guidelines for forensic use. Additionally, LC-Q/TOF-MS was used to analyze desomorphine metabolites, which in the absence of commercially available reference material or authentic urine specimens, were generated in-vitro.Item The collection, preservation, and processing of DNA samples from decomposing human remains for more direct disaster victim identification (DVI)(2018-04-13) Holmes, Amy; Hughes-Stamm, ShereeForensic DNA analysis plays a vital role in forensic casework and mass fatality incidents to identify victims. In these situations, human remains are often subjected to extreme heat, humidity, possible mutilation or fragmentation, microbial activity, decomposition and putrefaction, which can all complicate disaster victim identification (DVI). In many cases, the disaster event results in damage to the local infrastructure causing a loss of electricity and lack of facilities with refrigeration to house large numbers of human remains. Without refrigeration, the DNA in tissues become more degraded and damaged making traditional short tandem repeat (STR) typing more difficult. Therefore, immediate and simple in-field collection and stabilization of DNA samples from decomposing human remains without the need for refrigeration would be of great benefit to the forensic community. In addition, quick victim identification is also a goal for DVI operational teams as they attempt to bring closure to the victim’s families, and assist government agencies that must account for the deceased. As a result, the demand for a rapid turnaround time is often stressed; however, due to the overwhelmingly large number of samples requiring processing, this may not be feasible. Significant rate-limiting steps in the STR typing workflow are DNA extraction (approx. 3-4 hours), DNA quantification (1-2 hours), and standard PCR amplification (approx. 2-3 hours). Digestion of dense connective tissues such as skin and muscle prior to DNA purification may add another 8-12 hours, or hard tissues such as bone or teeth another 24-48 hours to the overall time required for generating an STR profile. If some of these procedures could be reduced (or avoided) during a mass fatality incident involving thousands of victims, sample throughput could be substantially increased. This doctoral research focuses on testing various in-field DNA collection and room temperature preservation methods for decomposing human remains as mock DVI samples. We compared rapid DNA purification protocols or direct amplification approaches that will eliminate unnecessary steps in the DNA analysis workflow, increasing the throughput and reducing the costs of analysis. Overall, results indicate that sufficient DNA can be collected and preserved at ambient temperature using some of these methods, provided that DNA is not already severely degraded before collection. In-field sample collection from human remains using biopsy punches or simply making an incision in the skin and swabbing the underlying muscle with cotton or foam swabs proved to be the most successful and easiest methods of DNA collection. Biopsy punches should be immediately stored in a modified TENT preservative, and swabs allowed to dry for transport to the laboratory for refrigeration, or stored until DNA analysis is possible. TENT and swab samples were processed with both traditional DNA analysis workflows and a direct PCR approach. In all cases, complete profiles were obtained from fresh tissues using all methods tested in this study. However, severely decomposed tissues were more challenging, with most samples yielding partial (or no) STR profiles. We proposed a method for triaging swab samples based on the quantification results of samples prepared for direct PCR in order to increase the first-pass success rate. Results indicate that foam swabs used to collect from muscle tissue may generate the most complete STR profiles for the majority of decomposed tissues, with cotton swabs yielding similar results. In addition, aliquots of TENT containing DNA leached from tissues were successfully diluted and directly added to the PCR reaction, thereby skipping DNA extraction and quantification all together. This protocol is the quickest of all methods tested, generating STR profiles in a fraction of the time it takes for traditional DNA processing. If this first-pass approach fails due to insufficient amounts of DNA, then the tissue itself stored in the TENT buffer can be quickly extracted in under 20 minutes with the PDQeX DNA extraction system. We found that this method generated the most complete STR profiles from severely decomposed tissues. Overall, we have demonstrated that tissues preserved in a modified TENT buffer or collected and stored using cotton and foam swabs show potential as alternate methods for the immediate in-field collection and preservation of DNA at room temperature for human identification purposes. However, these methods warrant further investigation to optimize protocols to achieve more efficient DNA preservation and higher STR success rates from severely decomposed human tissues.