<p>Mass spectrometry, the science and technology of ions, owes much of its current popularity to the development of electrospray ionization. The development of electrospray ionization, along with its low flow-rate analog nanoelectrospray ionization, has increased the chemical space that can be investigated using mass spectrometers by orders of magnitude. While the interfacial chemistry of charged microdroplets that are generated by nanoelectrospray has been studied in detail, the physics of their motion, particularly in the presence of an applied field at ambient pressures, remains relatively unexplored. In this dissertation, an increase in ion currents detected by a commercial triple quadrupole mass spectrometer is used to demonstrate that: (i) the orthogonal injection of counterions into an electrode assembly can compensate for space charge effects and enhance the sampling of charged microdroplets from a nanoelectrospray focused electrostatically under ambient conditions into the mass spectrometer; and (ii) the ease of ion evaporation from charged microdroplets may be elucidated for small molecules based on their relative transmission through an electrode assembly for the simultaneous ambient electrostatic focusing of two nanoelectrosprays. In each case, the development is characterized by using ion trajectory calculations in conjunction with experiments, using homebuilt devices designed and fabricated in-house as rapid prototypes via 3D printing. In the open air, charged microdroplets have low kinetic energies with a narrow energy spread. Despite these limitations, this dissertation demonstrates, through the electrostatic manipulation of charged microdroplets produced via nanoelectrospray ionization, that a better understanding of the physics of moving charges in the open air can be used to increase the sensitivity of atmospheric pressure ionization.</p>

<p dir="ltr">Originating from J. J. Thomsons original work and the development of electrospray ionization (ESI) by John B. Fenn, mass spectrometry offers a versatile analytical tool to measure beyond an ion’s m/z, especially for biomolecules. Gas-phase ion/ion reactions within a mass spectrometer offers an attractive approach to study biomolecules as they take place on the millisecond and sub millisecond time scale, have high efficiency, allow oppositely charged ions to interact with each other in a controlled manner, and a allows for selection of each reactant prior to the reaction via ion isolation. This can be used to probe gas-phase chemistry that can reflect reactions in solution, however gas-phase reactions have no solvent effects and happen faster, making it a simpler experiment. Here, a variety of gas-phase ion/ion reactions and ion trap methodologies are described to study mostly lipids with a minor amount of transmetalation at the beginning.</p><p dir="ltr">First, a series of multivalent metals complexed to neutral ligands are demonstrated to form ion-pairs with tetraphenylborate anions via ion/ion reactions. The resulting products were subjected to collision induced activation (CID) to observe their involvement in transmetalation, complementary density functional theory (DFT) calculations are provided as well. Next, sequential ion/ion reactions were performed to convert isomeric phosphoinositol phosphates dianions to monocations to reveal structural characterization and isomeric differentiation utilizing tandem MS and dissociation kinetics. The following two chapters after, reports on complementary efforts to separate lipids in the gas-phase of different mass and charge but similar mass-to-charge (m/z) resulting in overlapping m/z signals. The first report demonstrates a physical approach where singly and double charged lipids are separated in space from each other, trapped simultaneously such that no information is lost. The second utilizes a lanthanide, Yb3+ trication complex that underwent ion/ion reactions with singly and doubly charged lipid anions of similar m/z that result in different m/z products for each singly and doubly charged lipids. Lastly, a sequential ion/ion approach utilizing hexa(ethylene glycol) dithiol as a novel reagent to charge invert structurally uninformative lipid cations to structurally informative anions with subsequent carbon-carbon double bond localization.</p>

<p>The thesis consists of two main parts of nonlinear optical instrumentation development. </p> <p>Fluorescence-detected mid-infrared photothermal (F-PTIR) microscopy is demonstrated for sub-diffraction limited mid-infrared microspectroscopy of model systems and applied to probe phase transformations in amorphous solid dispersions. To overcome the diffraction limit in infrared imaging, a highly localized temperature-dependent photothermal effect is an attractive alternative indicator to infrared absorption. Photothermal atomic force microscopy infrared spectroscopy (AFM-IR) achieves nanometer resolution by monitoring heat caused expansion but only restricted on the surface. For 3D imaging, optically detected photothermal infrared (O-PTIR) combines an infrared laser with a visible probe source with to transduce photothermal refractive index changes (e.g., from changes in beam divergence or scattering). The sensitivity of O-PTIR is ultimately limited by the relatively weak dependence of refractive index with temperature, exhibiting changes of ~0.01% per oC. Fluorescence-detected photothermal mid-infrared (F-PTIR) spectroscopy (Fig. 1) is demonstrated herein to support 3D imaging with improved photothermal sensitivity. In F-FTIR, the sensitivity of fluorescence quantum efficiency to temperature change (~1-2% per oC) is used to transduce transient heat flux from localized IR absorption. The infrared spatial resolution of F-FTIR is defined by fluorescence microscopy and the thermal diffusivity of the sample instead of infrared wavelength. Initial F-PTIR proof of concept studies are described for microparticle assemblies of silica gel and polyethylene glycol, followed by applications of F-PTIR for analysis of localized composition within phase-separated domains induced by water vapor exposure of an amorphous solid dispersion (ritonavir in copovidone).</p> <p>Fluorescence recovery while photobleaching (FRWP) is demonstrated as a method for quantitative measurements of rapid diffusion mapping over the microsecond to millisecond time scale. Diffusion measurements are critical for molecular mobility assessment in cell biology, materials science and pharmacology. Fluorescence recovery after photobleaching (FRAP) is a well-known noninvasive optical microscopy method for measuring diffusion coefficients of macromolecules, such as proteins in cells and viscous solutions. However, conventional point-bleach FRAP is challenging to implement with multi-photon excitation and typically only supports diffusion analysis over millisecond time scales due to camera frame rate limitations. FRWP with patterned illumination addresses these limitations of FRAP by probing the fluorescence intensity changes while bleaching a comb pattern within a field of view (FoV). Fast-scanning of an ultrafast excitation beam distributes heat rapidly over multiple adjacent pixels, minimizing local heating effects that could complicate analogous diffusion measurements by point-bleach FRAP with multiphoton excitation. In FRWP, time-scales of the probed diffusion events are defined by a single line-pass time of a resonant scanning-mirror with a period of 125  s. In FRWP, the bleach pattern spans locations across the whole FoV, enabling diffusion mapping through image segmentation. More than a hundred bleaching and recovery events can be recorded during a single 10s measurement. Normal and anomalous diffusion of rhodamine-labeled bovine serum albumin (BSA) molecules was studied as a model system, with applications targeting rapid assessment of therapeutic macromolecule mobility within heterogeneous biological environments.</p>

<p>Mass spectrometry (MS) can provide rapid, sensitive, and specific analysis, making it a valuable tool to characterize biomolecules, especially their dynamic changes when involved in significant processes.  Compared to other analytical techniques, which mostly focus on solution-phase or solid-phase characterization, MS enjoys a more general and efficient detection of gas-phase analytes since it ultimately measures abundances of bare ions in vacuum. This unique detection capability of MS has been demonstrated, in this dissertation, by characterizing the neutral serine octamer, a gas-phase amino acid cluster that has been detected by MS only so far. Besides its existence, the progress of chiral enrichment has also been monitored and quantified by MS during octamer formation. The acquired MS data is crucial to interpreting the mechanism of chiral enrichment achieved by serine octamer and might suggest its involvement in the prebiotic world to eventually achieve biohomochirality. The work also showcases the capability of detecting neutral compounds by MS, which breaks the stereotype that MS is exclusively an ion-based technique. </p> <p><br></p> <p>Besides process monitoring in the open air, MS also monitors the highly complicated metabolism processes inside biosamples, primarily benefiting from its excellent sensitivity, specificity, and throughput of ion detection. Since altered cellular metabolism is being recognized as a hallmark of cancer, MS is suitable for cancer diagnostics, whose performance of diagnosing glioma, a common brain cancer, has been tested.  Desorption electrospray ionization(DESI) has been used as it avoids sample preparation and allows direct characterization of raw tissue, therefore well suited for on-site analysis such as in the operating room. In short, we have applied intraoperative DESI-MS analysis on raw brain biopsies to provide glioma diagnostics within 5 min. Specifically, the molecular features revealed by MS are translated into pathological information of analyzed tissue, like genetic mutations and tumor concentrations, which is highly desired during surgeries to guide tumor resection and improve patient management. </p> <p><br></p> <p>Knowledge of diagnostic biomarkers is essential to the translation from MS data to pathology, which can be obtained by metabolic profiling using MS. Despite the tradeoff between comprehensive characterization and analysis time, we have extensively explored endogenous metabolites by using tandem MS and expedited analysis by avoiding the use of chromatography. After fast profiling, statistical analysis of all MS features has been applied to discover diagnostic markers to distinguish healthy brain tissue from cancerous tissue. DESI-MS methods have been developed to facilitate a simple and rapid characterization of these biomarkers in tissue for a smooth clinical transition. </p> <p><br></p> <p>However, the complete characterization of endogenous metabolites in a complicated biomixture, like tissue, is challenging, especially without the orthogonal separation provided by chromatography. This unmet demand calls for the development of novel MS scans to improve the metabolite coverage. For lipidomics by direct infusion MS, the MS scans used for lipid profiling have not been greatly expanded since its introduction. These conventionalMS scans only target one structural moiety of lipids and leave the rest unresolved, which limits the structure elucidation and biological interpretation of diagnostic lipids. We have introduced additional lipid scans that target both the lipid headgroup and one fatty acyl chain, leaving the other fatty acyl chain flexible. These scans with higher specificity can further alleviate the matrix effect by uncovering fewer ions in each scan and provide more structural information to support lipid identification. As a proof-of-concept, we have used them to profile both common phospholipids and the rarer ether lipids that display significant variations between healthy mice tissue and those with metabolic syndrome. The additional structural information provided by these scans ensures a clear message expressed by the disease metabolism and potentially indicates invention points and therapeutic candidates.</p>

<p dir="ltr">Metal ions are typically considered a nuisance for mass spectrometry, as they can introduce chemical noise and distribute an analyte’s signal into multiple peaks. In some cases however, metal ions in biological solutions are either necessary for biomolecular structures, or so ubiquitous in a sample’s native solution conditions that they are difficult to fully remove. In this work, the role of metal ions in biological analytes is explored. For analytes that require metal ions to maintain higher order structures, a mass spectrometry method was developed to determine whether a stable structure is formed from metal ion adducts, or if the metal ion adducts are nonspecifically bound. Electron transfer of these structures reveals complementary fragmentation information, with the added discovery of new radical fragmentation pathways. With mass spectrometry, specific ligand and metal ion affinities can even be determined for analytes at low enough concentrations. In addition to analytes that require metals, an exploration on unwanted metal ion adduction during the electrospray ionization process is shown via gas-phase ion/ion reactions. Observing how specific anionic ligands exchange metals with protons from proteins on a small and controlled scale gives a greater understanding of what solutions can lead to the cleanest results. In addition, this work shows the possibility of finding anionic ligands that will instead exchange protons with metal ions found on proteins. In the gas-phase, these experiments have a high degree of control, leading to a much greater understanding of how metal ions influence mass spectrometry samples.</p>

<p dir="ltr">The field of nanoclusters includes a broad range of sizes and structures that influence both their physical and chemical properties. Scientists use several techniques, such as atom-by-atom substitution, to synthesize atomically precise nanoclusters, and ligand shell mixing to protect nanoclusters from unwanted side reactions, while controlling their reactivity and solubility. These combined techniques can provide stable products, but isomers and structural analogs often remain in the product mixture, complicating the structural characterization of individual nanoclusters. Leading structural characterization techniques in nanocluster research are often limited in their ability to examine both the structure of the metal core and ligand shell in sufficient detail. The primary aim of this research is to systematically characterize the structures and chemical properties of several types of transition metal oxide nanoclusters of interest to applications in energy production, catalysis, and magnetic resonance imaging, without requiring purification. Specifically, this work focuses on 1) Polyoxovanadates (POV) with a mixture of methoxy, ethoxy, and ether ligands, 2) Fe- and W-substituted POV alkoxides, and 3) Octanuclear iron oxide clusters substituted with In atoms. Mass spectrometry techniques enable the structural characterization of individual clusters from multicomponent mixtures without interference. Specifically, we use ion mobility spectrometry to explore how surface ligands affect the metal core in mixed-ligand POV alkoxide species. We examine structure-specific fragments to identify the positions of ligands and heteroatoms within the metal core of mixed-ligand species and W and Fe-substituted POV methoxides. Additionally, we use ion soft-landing to purify W-substituted POV methoxide anions on surfaces for characterization using cyclic voltammetry and infrared spectroscopy. We discovered unique characteristics of each nanocluster including the position of heteroatoms, ligands shell mobilities, structures and collisional cross sections, and provided first insights into the redox properties of W-substituted POV alkoxide. These results highlight the growing influence of mass spectrometry in the field of nanocluster characterization and design.</p>

<p dir="ltr">The detection of explosives and explosive devices based upon the volatile compounds they emit is a long-standing tool for law enforcement and physical security. Towards that end, solid-phase microextraction (SPME) combined with gas chromatography-mass spectrometry (GC-MS) has become a crucial analytical tool for the identification of volatiles emitted by explosives. Previous SPME studies have identified many volatile compounds emitted by common explosive formulations that serve as the main charge in explosive devices. However, limited research has been conducted on initiators like fuses, detonating cords, and boosters. In this study, a variety of SPME fiber coatings (i.e., polydimethylsiloxane (PDMS), polydimethylsiloxane/divinylbenzene (PDMS/DVB), divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS), carboxen/polydimethylsiloxane (CAR/PDMS), and polyacrylate (PA)) were employed for the extraction and analysis of volatiles from Composition C-4 (cyclohexanone, 2-ethyl-1-hexanol, and 2,3-dimethyl-2,3-dinitrobutane (DMNB)) and Red Dot double-base smokeless powder (nitroglycerine, phenylamine). The results revealed that a PDMS/DVB fiber was optimal. Then, an assortment of explosive items (i.e., detonation cord, safety fuse, slip-on booster, and shape charge) were analyzed with a PDMS/DVB fiber. A variety of volatile compounds were identified, including plasticizers (tributyl acetyl citrate, N-butylbenzenesulfonamide), taggants (DMNB), and degradation products (2-ethyl-1-hexanol). </p><p dir="ltr">Taggants, like DMNB, are one of the pivotal components added to explosives. These distinctive chemical markers, deliberately introduced during manufacturing to facilitate the identification of explosives, are commonly detected using SPME GC- MS, but their quantitation remains underexplored. To address this, we investigated total vaporization headspace (TV- HS) GC- MS for quantifying taggants in the headspace of Composition C4. Factors effecting the extraction of DMNB, such as shape and age of the sample, and surface depletion, were also examined. The results revealed that the shape of the sample did not affect the amount of DMNB in the headspace but the older the sample, the more DMNB was detected in the headspace. Surface depletion was also seen in samples that were exposed to air for more than one week. Then calibration curves with calibrants of DMNB in acetone were established for quantitation. The average concentration of DMNB in the headspace was determined to be 125 parts per million (ppm).</p><p><br></p>

<p dir="ltr">Multiply-charged ions of intact biomolecules generated from electrospray ionization (ESI) have drawn researchers' interest in the field of native mass spectrometry (MS) for decades because these ions carry mass and charge information of the intact molecules and interactions among different units. However, the confinement of multiple charge states in a narrow range of <i>m/z</i> makes mass and charge assignments challenging, especially for analytes with a mass greater than 100 kDa. Gas-phase ion/ion reactions have proven to be powerful techniques that facilitate the interpretation of mass spectra of natively sprayed macromolecular analytes by manipulating the masses and charges of ions detected.</p><p dir="ltr">The proton-transfer reaction (PTR) is the most used gas-phase ion/ion reaction method. It utilizes perfluorinated PTR reagents to "grab" protons away from the analyte ions, thereby reducing their charges. A novel charge state manipulation technique called "ion parking," based on PTR, has been developed. In this method, ion signals are accumulated to one or a range of charge states by selectively inhibiting reactions between the target charge state and the PTR reagents via resonance excitation.</p><p dir="ltr">The multiply-charged ion attachment (MIA) reaction is another gas-phase ion/ion reaction approach. It utilizes the significant <i>m/z</i> displacement caused by the attachment of multiply-charged reagent ions, and it has been proven useful for mass analysis of heterogeneous macromolecular analytes with a mass greater than 1 MDa.</p><p dir="ltr">All gas-phase ion/ion reaction techniques require mutual storage of ions in opposite polarities within an electrodynamic quadrupole ion trap, such as a 3D quadrupole ion trap (QIT) or a linear quadrupole ion trap (LIT). Electrodynamic ion traps use high-voltage (HV) drive radio frequencies (RF) to trap ions in a quadrupolar field, typically employing a sinusoidal waveform (sine wave). A digital quadrupole ion trap (DIT) is an unconventional electrodynamic ion trap that uses a digital waveform (square wave) as the drive RF. The high agility of square waves makes DIT an ideal mass analyzer for studying high <i>m/z</i> ions resulting from gas-phase ion/ion reactions. This dissertation describes the development of a novel home-built digital dual-trap mass spectrometer and ion/ion chemistry studies of large biomolecules within the instrument.</p>

<p dir="ltr">This thesis focusses on developing novel data driven oral drug formulation analysis methods by employing technologies such as Fourier transform analysis and generative adversarial learning. Data driven measurements have been addressing challenges in advanced manufacturing and analysis for pharmaceutical development for the last two decade. Data science combined with analytical chemistry holds the future to solving key problems in the next wave of industrial research and development. Data acquisition is expensive in the realm of pharmaceutical development, and how to leverage the capability of data science to extract information in data deprived circumstances is a key aspect for improving such data driven measurements. Among multiple measurement techniques, chemical imaging is an informative tool for analyzing oral drug formulations. However, chemical imaging can often fall into data deprived situations, where data could be limited from the time-consuming sample preparation or related chemical synthesis. An integrated imaging approach, which folds data science techniques into chemical measurements, could lead to a future of informative and cost-effective data driven measurements. In this thesis, the development of data driven chemical imaging techniques for the analysis of oral drug formulations via Fourier transformation and generative adversarial learning are elaborated. Chapter 1 begins with a brief introduction of current techniques commonly implemented within the pharmaceutical industry, their limitations, and how the limitations are being addressed. Chapter 2 discusses how Fourier transform fluorescence recovery after photobleaching (FT-FRAP) technique can be used for monitoring the phase separated drug-polymer aggregation. Chapter 3 follows the innovation presented in Chapter 1 and illustrates how analysis can be improved by incorporating diffractive optical elements in the patterned illumination. While previous chapters discuss dynamic analysis aspects of drug product formulation, Chapter 4 elaborates on the innovation in composition analysis of oral drug products via use of novel generative adversarial learning methods for linear analyses.</p>

The combination of analytical sensitivity and selectivity provided by time-of-flight secondary ion mass spectrometry (ToF-SIMS), with advanced statistical interrogation by principal component analysis (PCA), has allowed a significant advancement in the forensic discrimination of pen, pencil and glass materials based on trace characterisation.