Trajectory reconstruction of shooting incidents can help investigators determine critical case information regarding the number of shooters involved, their location(s), and intent. The examination of trace amounts of intermediate target materials collected on the surface of spent bullets provides crucial information needed for trajectory reconstruction. Determining the origin of an unknown material adhered to a spent bullet allows for the identification of intermediate targets the bullet either contacted or penetrated during flight. Although significant information can be obtained from examination of these trace materials adhered to spent bullets, this aspect of trajectory reconstruction is often ignored. The ability of different bullet types to collect trace materials from intermediate targets and the ability to associate these trace materials to their origin was examined using microscopy and Direct Analysis in Real Time (DART). Full metal jacket (FMJ), jacketed hollow point (JHP), and lead round nose (LRN) bullets were fired into sheets of five different commonly used building materials (oriented strand board, sanded plywood, white melamine board, synthetic PVC board, and medium density fiberboard) to produce a total of 45 spent bullets to be examined. All spent bullets were examined and photographed using a DSLR camera paired with a stereomicroscope. The spent bullets were then examined using DART/MS to determine if any ion profiles generated from the trace materials could be associated with those of the intermediate target building materials which the bullets were fired through. The collection of trace material from all five types used was highly dependent on the type of bullet. Very minimal amounts of trace materials were observed on the majority of LRN bullets, which failed to produce an identifiable ion signature. The FMJ bullets that were fired through PVC material collected trace material that produced an ion profile, while all other building materials failed to transfer to the FMJ bullets. All JHP bullets collected significant amounts of the five building materials tested inside the hollow point cavity and along the nose of the bullet. In every spent JHP bullet sample, the trace material collected produced a unique ion profile. Additionally, MS data from four of the five building materials tested matched the MS data generated from trace material collected on JHP bullets from the respective target materials.

Direct Analysis in Real Time (DART) ionization/mass spectrometry allows for the high throughput analysis of a wide range of materials including but not limited to: solids, liquids, powders, tablets, and plant materials. The ability to detect cocaine was established in a reproducible manner with the use of a DART ionization source (IonSense Inc., Saugus, MA) interfaced to a modified single quadrupole mass spectrometer. Development of a methodology for the detection of cocaine within contrived street quality drug mixtures involved the optimization of the ionization source, sample introduction mechanism, ion guide, and mass analysis parameters. An analytical method was created that utilized ionized helium carrier gas heated to 300°C and an automated sample introduction apparatus consisting of a Linear Rail Enclosure that holds consumable QuickStrip^TM sample cards. Ionized molecules were then fragmented by manipulation of voltage levels within the ion guide to gain more structural information prior to detection by a single quadrupole mass spectrometer. Cocaine was detected by the modified DART/MS analytical platform and gave two peaks within the mass spectrum at m/z 304 and 182. Optimization of in-source fragmentation by manual adjustment of the skimmer focus voltage allowed for the reproducible fragmentation of cocaine and the ability to increase or decrease the amount of fragmentation seen between the two peaks detected for cocaine. With the use of fragmentation, this analytical platform can be classified as a Category A technique as defined by the Scientific Working Group for the Analysis of Seized Drugs. The robust detection of cocaine was demonstrated for reference samples at concentrations as low as 10 ng/μL (50 ng) with high signal abundance greater than ten times the signal to noise ratio. Furthermore, the detection of cocaine at 10 ng/μL was demonstrated for multi component mixtures of up to 14 additional components containing common adulterants and diluents found within street quality samples. In total, 25 common excipients were tested using the same method parameters as optimized for cocaine analysis. Of these 25 excipients tested, five were not detected in positive ion mode (one could be detected in negative ion mode). Of the twenty excipients that could be detected by mass spectrometry, two pairs of excipients (levamisole/tetramisole and creatine/creatinine) could not be differentiated from each other. There were no excipients tested that had equivalent m/z values as those of cocaine. Experimentation into the effects of various excipients at multiple concentrations on the abundance of the two cocaine peaks was performed. Regardless of excipient amount (up to 10 times more concentrated than cocaine) and the number of components (up to 15 total components) the ratio of abundance between the m/z 304 to 182 peaks did not vary greater than 22% relative standard deviation. A match criteria protocol was developed for the ability of an analyst to confirm the presence of cocaine within unknown forensic case samples that have previously tested positive for the presumptive identification of cocaine. The identification of cocaine was based on various factors such as the signal to noise ratio at m/z 304 and 182, the ratio of abundance between those two peaks as well as positive and negative controls. This match criteria protocol was utilized for 25 double blind mock forensic casework samples was performed. Determination for the presence of cocaine within these unknown samples gave an analyst error rate of 0%, with no false positives or false negatives predicted. To further aid human interpretation and identification of compounds within mixtures, the advanced chemometric software, Analyze IQ, was utilized. Development of predictive classification models using a combination of pre-processing steps, principle component analysis and machine learning techniques was achieved. Models were built using 381 unique samples for the purposes of identifying the presence of cocaine within unknown samples. Of all methods available within the Analyze IQ software, the optimization of a model using principle component analysis with support vector machine regression with a radial basis function kernel yielded an initial error rate of 0% for 72 samples tested. Furthermore, of the samples tested against the model, 20 samples were comprised of excipients that were not incorporated into the initial model development process. The inclusion of these samples (10 spiked with cocaine, 10 absent of cocaine), shows that predictive modeling based software can provide an accurate, robust, and evolving approach to the identification of cocaine within sample compositions that have not previously been tested and stored in a database of known reference samples. Predictive modeling has advantages over current mass spectral libraries, which are limited to the identification of pure compounds. To further test the abilities of predictive models, optimized machine learning models were applied to 25 double blind mock forensic casework samples. The predictive modeling error rate was identical to the human interpretation rate for the double blind mock casework samples with a 0% error rate. Using the DART/MS analytical platform, 25 mock forensic casework samples along with positive and negative controls were analyzed and identified for the presence of cocaine within 30 minutes. On the order of 15 to 30 times faster than modern GC/MS and LC/MS methods, the ability to analyze and identify samples faster would allow for an increase in samples being processed on a daily basis and allow for the reduction of case backlogs that currently plague controlled substances sections of forensic science laboratories throughout the United States.

Canines have been employed in matters of law enforcement because of their keen sense of smell. Presently, law enforcement officials are utilizing trained canines in routine traffic stops to assess if the vehicle contains any illicit substances. Many believe that this is an infringement on an individual’s fourth amendment rights, which has garnered the attention of both the media and the courts. Many questions have been raised with respect to canines alerting to locations where illicit substances were no longer present. Thus, the purpose of this dissertation research is to evaluate the manner in which active odorants transport and persist onto various substrates. Direct Analysis in Real Time (DART) coupled to an accurate-mass time-of-flight (AccuTOF™) mass spectrometer was used to rapidly analyze the volatile organic compounds (VOC’s) from a variety of narcotic and explosive substances. The DART ion source is a soft ionization technique used in ambient conditions to sample liquids, solids or gases in real time. Thermal desorption of the VOC’s could thus be conducted in seconds. The present study found that the VOC’s from illicit substances transport from one location to another, in a short amount of time, through a process known as advection, which may contribute to canines producing unconfirmed alerts during their training and certifications. Three of the four odorants used in this study produced positive results, with the exception being 2-ethyl-1-hexanol, as it was not detected at any time when held at distances between 0.5 and 3 meters. In addition, the amount of time needed for an active odorant to contaminate an object in its immediate vicinity was explored and the results were determined to be inconclusive. Although they were observed following longer exposure times, it was still deemed to be inconclusive since it was still possible for these odorants to be present, albeit not in detectable amounts, at lower exposure times. Controlled odor mimic permeating systems (COMPS), patented technology by IFRI were tested to determine the possibility of cross-contamination between the training aids, and the results conclude that there was not any evidence of cross-contamination observed during any of the trials.

Molecular ionization is owed much of its development from the early implementation of electron ionization (EI). Although dramatically increasing the library of compounds discovered, an inherent problem with EI was the low abundance of molecular ions detected due to high fragmentation leading to the difficult task of the correct chemical identification after mass spectrometry (MS). These problems stimulated the research into new ionization methods which sought to "soften" the ionization process. In the late 1980s the advancements of ionization techniques was thought to have reached its pinnacle with both electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). Both ionization techniques allowed for "soft" ionization of large molecular weight and/or labile compounds for intact characterization by MS. Albeit pervasive, neither ESI nor MALDI can be viewed as "magic bullet" ionization techniques. Both techniques require sample preparation which often included native sample destruction, and operation of these techniques took place in sealed enclosures and often, reduced pressure conditions. New open-air ionization techniques termed "ambient MS" enable direct analysis of samples of various physical states, sizes and shapes. One particular technique named Direct Analysis In Real Time (DART) has been steadily growing as one of the ambient tools of choice to ionize small molecular weight (< 1000 Da) molecules with a wide range of polarities. Although there is a large list of reported applications using DART as an ionization source, there have not been many studies investigating the fundamental properties of DART desorption and ionization mechanisms. The work presented in this thesis is aimed to provide in depth findings on the physicochemical phenomena during open-air DART desorption and ionization MS and current application developments. A review of recent ambient plasma-based desorption/ionization techniques for analytical MS is presented in Chapter 1. Chapter 2 presents the first investigations into the atmospheric pressure ion transport phenomena during DART analysis. Chapter 3 provides a comparison on the internal energy deposition processes during DART and pneumatically assisted-ESI. Chapter 4 investigates the complex spatially-dependent sampling sensitivity, dynamic range and ion suppression effects present in most DART experiments. New implementations and applications with DART are shown in Chapters 5 and 6. In Chapter 5, DART is coupled to multiplexed drift tube ion mobility spectrometry as a potential fieldable platform for the detection of toxic industrial chemicals and chemical warfare agents simulants. In Chapter 6, transmission-mode DART is shown to be an effective method for reproducible sampling from materials which allow for gas to flow through it. Also, Chapter 6 provides a description of a MS imaging platform coupling infrared laser ablation and DART-like phenomena. Finally, in Chapter 7 I will provide perspective on the work completed with DART and the tasks and goals that future studies should focus on.

Ambient mass spectrometry (MS) is a new and growing sub-field in MS which has opened new research avenues, particularly for applications relating to the analysis of solid samples. Results on the implementation and application of ambient MS techniques including: desorption electrospray ionization (DESI) and direct analysis in real time (DART) indicated that these techniques could serve as complementary tools for the rapid qualitative screening of pharmaceuticals, allowing up to two orders of magnitude improvement in throughput compared to traditional methods such as liquid chromatography MS. The selectivity of DESI could be enhanced by performing the experiment in the reactive mode. In this mode, complexation reactions between reagents added to the spray solvent and analytes on the sample surface resulted in analyte stabilization, inhibiting fragmentation. They also resulted in a concomitant enhancement in the analyte surface activity, facilitating their evaporation from secondary droplets culminating in an improvement in sensitivity. Also for drug tablets analysis, the analyte signal dependency on DESI geometrical set-up variables could be mitigated following the careful and controlled addition of an isotopically labeled internal standard (IS) to the sample or by spraying samples with a pair of reagents with different affinities for the analyte. Either of these approaches resulted in an analyte-to-IS signal ratio (in the former) or an analyte complex ratio (in the later), which was largely independent of DESI experimental variables allowing quantitative analysis using this technique. DESI MS was also observed to be a very powerful tool for determining the 2-D distribution of various pharmaceutically important compounds on tablet and tissue surfaces. The ability to map the distribution of molecules of interest by DESI MS has very great implications in drug tablet quality control and in determining the role of chemical signals presented on tissue surfaces. DESI was observed to be limited to ionizing molecules of medium to high polarities without much limitation in terms of mass range, whereas DART was better suited for the analysis of molecules within a broader range of polarities, but within a more limited mass range (up to 800 Da approximately). These limitations were circumvented by implementing a novel multimode ambient ion source, desorption electrospray/metastable-induced ionization (DEMI), which combines various aspects of DESI and DART. Initial experiments with the DEMI ion source demonstrated its ability to enable the simultaneous analysis of molecules within a broader range of polarities and masses than DESI and DART alone.

The aim of this study was to investigate the utility of direct analysis in real time quadrupole time-of-flight mass spectrometry and gas chromatography quadrupole time-of-flight mass spectrometry in the analysis of 162 different synthetic cannabinoids. Direct analysis in real time quadrupole time-of-flight mass spectrometry is shown to be a rapid and accurate analytical method for synthetic cannabinoids. Spectra can be generated with less than 1.5 ng of the drug in under a minute and be successfully searched against previously generated ESI-QTOF libraries in most cases (118/130 drugs tested) as well as can also be applied to the identification of synthetic cannabinoids in a mixture. Gas chromatography quadrupole time-of-flight mass spectrometry, while requiring a much longer analysis time, is shown to accurately distinguish all but 19 compounds (140/159). These two instruments have proven to be viable alternatives in synthetic cannabinoid analysis and will greatly benefit forensic laboratories.

The presence of inhibitory substances in biological forensic samples has, and continues to affect the quality of the data generated following DNA typing processes. Although the chemistries used during the procedures have been enhanced to mitigate the effects of these deleterious compounds, some challenges remain. Inhibitors can be components of the samples, the substrate where samples were deposited or chemical(s) associated to the DNA purification step. Therefore, a thorough understanding of the extraction processes and their ability to handle the various types of inhibitory substances can help define the best analytical processing for any given sample. A series of experiments were conducted to establish the inhibition tolerance of quantification and amplification kits using common inhibitory substances in order to determine if current laboratory practices are optimal for identifying potential problems associated with inhibition. DART mass spectrometry was used to determine the amount of inhibitor carryover after sample purification, its correlation to the initial inhibitor input in the sample and the overall effect in the results. Finally, a novel alternative at gathering investigative leads from samples that would otherwise be ineffective for DNA typing due to the large amounts of inhibitory substances and/or environmental degradation was tested. This included generating data associated with microbial peak signatures to identify locations of clandestine human graves. Results demonstrate that the current methods for assessing inhibition are not necessarily accurate, as samples that appear inhibited in the quantification process can yield full DNA profiles, while those that do not indicate inhibition may suffer from lowered amplification efficiency or PCR artifacts. The extraction methods tested were able to remove >90% of the inhibitors from all samples with the exception of phenol, which was present in variable amounts whenever the organic extraction approach was utilized. Although the results attained suggested that most inhibitors produce minimal effect on downstream applications, analysts should practice caution when selecting the best extraction method for particular samples, as casework DNA samples are often present in small quantities and can contain an overwhelming amount of inhibitory substances.

The DART ion source was introduced in 2005 at the ASMS Sanibel Conference and immediately afterward Professor Sparkman was contemplating of a way to get our lab this revolutionary mass spectrometry ionization technique. It did not take long because it was delivered to the Pacific Mass Spectrometry Facility in August 2006 and I was able to being using and learning the technique. The ion source creates excited state helium metastables (2 3 S) with an ionization potential of 19.8 eV are created by a glow discharge at atmospheric pressure. The metastables are sent through an optional heater, to aid in desorption, enter the open-air to directly ionize your sample or ionize reagent species to react with the analyte molecules. The most observed ionization mechanism is the formation of protonated molecules from a proton-transfer reaction between the analyte and protonated water clusters. The limited to no sample preparation with the "soft" ionization provide very quick identification of intact organic ions in or on various types of matrices. When the DART is coupled to a high resolving power instrument, such as the JEOL AccuTOF, accurate masses and accurate isotope ratios are assigned to aid in the determination of unknown elemental compositions. This research discusses the formation of the metastable species and how they are used to produce analyte and reagent ions within the open-air sample gap of the DART-mass spectrometer interface. A description of the fundamentals on the operation including real time visualization of the fluid dynamics and confirmation of the formation of a hydroxyl radical in the proposed formation of the protonated water clusters, along with applications developed in the Pacific Mass Spectrometry Facility will also be discussed. These include cleavage, desorption, and ionization of solid-phase peptides, desorption of aqueous metal ions using a heated wire filament and the increased ion transmission with the Vapur interface using metal coated glass tube for the transfer tube.

Metabolomics is the methodical scientific study of biochemical processes associated with the metabolome—which comprises the entire collection of metabolites in any biological entity. Metabolome changes occur as a result of modifications in the genome and proteome, and are, therefore, directly related to cellular phenotype. Thus, metabolomic analysis is capable of providing a snapshot of cellular physiology. Untargeted metabolomics is an impartial, all-inclusive approach for detecting as many metabolites as possible without a priori knowledge of their identity. Hence, it is a valuable exploratory tool capable of providing extensive chemical information for discovery and hypothesis-generation regarding biochemical processes. A history of metabolomics and advances in the field corresponding to improved analytical technologies are described in Chapter 1 of this dissertation. Additionally, Chapter 1 introduces the analytical workflows involved in untargeted metabolomics research to provide a foundation for Chapters 2 – 5. Part I of this dissertation which encompasses Chapters 2 – 3 describes the utilization of mass spectrometry (MS)-based untargeted metabolomic analysis to acquire new insight into cancer detection. There is a knowledge deficit regarding the biochemical processes of the origin and proliferative molecular mechanisms of many types of cancer which has also led to a shortage of sensitive and specific biomarkers. Chapter 2 describes the development of an in vitro diagnostic multivariate index assay (IVDMIA) for prostate cancer (PCa) prediction based on ultra performance liquid chromatography-mass spectrometry (UPLC-MS) metabolic profiling of blood serum samples from 64 PCa patients and 50 healthy individuals. A panel of 40 metabolic spectral features was found to be differential with 92.1% sensitivity, 94.3% specificity, and 93.0% accuracy. The performance of the IVDMIA was higher than the prevalent prostate-specific antigen blood test, thus, highlighting that a combination of multiple discriminant features yields higher predictive power for PCa detection than the univariate analysis of a single marker. Chapter 3 describes two approaches that were taken to investigate metabolic patterns for early detection of ovarian cancer (OC). First, Dicer-Pten double knockout (DKO) mice that phenocopy many of the features of metastatic high-grade serous carcinoma (HGSC) observed in women were studied. Using UPLC-MS, serum samples from 14 early-stage tumor DKO mice and 11 controls were analyzed. Iterative multivariate classification selected 18 metabolites that, when considered as a panel, yielded 100% accuracy, sensitivity, and specificity for early-stage HGSC detection. In the second approach, serum metabolic phenotypes of an early-stage OC pilot patient cohort were characterized. Serum samples were collected from 24 early-stage OC patients and 40 healthy women, and subsequently analyzed using UPLC-MS. Multivariate statistical analysis employing support vector machine learning methods and recursive feature elimination selected a panel of metabolites that differentiated between age-matched samples with 100% cross-validated accuracy, sensitivity, and specificity. This small pilot study demonstrated that metabolic phenotypes may be useful for detecting early-stage OC and, thus, supports conducting larger, more comprehensive studies. Many challenges exist in the field of untargeted metabolomics. Part II of this dissertation which encompasses Chapters 4 – 5 focuses on two specific challenges. While metabolomic data may be used to generate hypothesis concerning biological processes, determining causal relationships within metabolic networks with only metabolomic data is impractical. Proteins play major roles in these networks; therefore, pairing metabolomic information with that acquired from proteomics gives a more comprehensive snapshot of perturbations to metabolic pathways. Chapter 4 describes the integration of MS- and NMR-based metabolomics with proteomics analyses to investigate the role of chemically mediated ecological interactions between Karenia brevis and two diatom competitors, Asterionellopsis glacialis and Thalassiosira pseudonana. This integrated systems biology approach showed that K. brevis allelopathy distinctively perturbed the metabolisms of these two competitors. A. glacialis had a more robust metabolic response to K. brevis allelopathy which may be a result of its repeated exposure to K. brevis blooms in the Gulf of Mexico. However, K. brevis allelopathy disrupted energy metabolism and obstructed cellular protection mechanisms including altering cell membrane components, inhibiting osmoregulation, and increasing oxidative stress in T. pseudonana. This work represents the first instance of metabolites and proteins measured simultaneously to understand the effects of allelopathy or in fact any form of competition. Chromatography is traditionally coupled to MS for untargeted metabolomics studies. While coupling chromatography to MS greatly enhances metabolome analysis due to the orthogonality of the techniques, the lengthy analysis times pose challenges for large metabolomics studies. Consequently, there is still a need for developing higher throughput MS approaches. A rapid metabolic fingerprinting method that utilizes a new transmission mode direct analysis in real time (TM-DART) ambient sampling technique is presented in Chapter 5. The optimization of TM-DART parameters directly affecting metabolite desorption and ionization, such as sample position and ionizing gas desorption temperature, was critical in achieving high sensitivity and detecting a broad mass range of metabolites. In terms of reproducibility, TM-DART compared favorably with traditional probe mode DART analysis, with coefficients of variation as low as 16%. TM-DART MS proved to be a powerful analytical technique for rapid metabolome analysis of human blood sera and was adapted for exhaled breath condensate (EBC) analysis. To determine the feasibility of utilizing TM-DART for metabolomics investigations, TM-DART was interfaced with traveling wave ion mobility spectrometry (TWIMS) time-of-flight (TOF) MS for the analysis of EBC samples from cystic fibrosis patients and healthy controls. TM-DART-TWIMS-TOF MS was able to successfully detect cystic fibrosis in this small sample cohort, thereby, demonstrating it can be employed for probing metabolome changes. Finally, in Chapter 6, a perspective on the presented work is provided along with goals on which future studies may focus.