Mass spectrometry is a versatile analytical tool for chemical and biomolecule identification, quantitation, and structural analysis. Tandem mass spectrometry further expands the applications of mass spectrometry, making it more than a mere detector. With tandem mass spectrometry, the mass spectrometer is capable of probing reaction mechanisms, monitoring reaction processes, and performing fast analysis on complex samples. In tandem mass spectrometry, after activation the precursor ions fragment into small fragment ions through one or more pathways, which are affected by the ion’s inherit property, the ion type, and the activation method. To obtain complementary information, one can alter the fragmentation pathway by changing the ion via ion charge manipulation and covalent modification to the ion. Gas-phase ion/ion reactions provide an easy approach to changing ion type and facile modification to the analyte ions. It has been extensively used for spectrum simplification and analyte structural studies. In this dissertation, ion/ion reaction facilitated mass spectrometry methods are studied, and explorations into the method development involving front-end mass spectrometer are discussed.<br>The first work demonstrates a special rearrangement reaction for gas-phase Schiff-base-modified peptides. Gas-phase Schiff-base modification of peptides has been applied to facilitate the primary structural characterization via tandem mass spectrometry. A major or minor fragment pathway related to the novel rearrangement reaction was observed upon in-trap collisional activation of the gas-phase Schiff-base-modified peptides. The rearrangement reaction involves the imine of the Schiff base and a nucleophile present in the polypeptide. The occurrence of the rearrangement reaction is affected by several factors, such as ion polarity, identity of the nucleophile in the peptide (e.g., side chains of lysine, histidine, and arginine), and the position of the nucleophile relative to the imine. The rearrangement reaction does not affect the amount of structural information that can be obtained by collisional activation of the Schiff-base-modified peptide, but when the rearrangement reaction is dominant, it can siphon away signal from the structurally diagnostic processes.<br>Efforts have also been put into the method development of peptide and protein aggregation detection via electrospray ionization mass spectrometry (ESI-MS). People have studied peptide and protein aggregation processes to understand the mechanism of amyloid-related diseases and to control the quality of the peptide and protein pharmaceuticals. ESI-MS is suitable for solution aggregation studies because of its compatibility with solution samples and the straightforward result of the analyte’s oligomeric state on the mass spectrum. However, peak overlap issue and nonspecific aggregation in the ESI process can obscure the result. Here, the application of proton transfer ion/ion reaction to the analyte has been found useful to reduce or eliminate the peak overlap issue. A statistical model based on Poisson statistics has been proposed to deal with the ESI-induced nonspecific aggregation in the droplet and to differentiate the solution-phase aggregation from the droplet-induced aggregation. Factors that affect the accuracy of the statistical model have been discussed with MATLAB simulations.<br>In the era of biological system studies, sample complexity is a challenge every analytical chemist has to face. The analysis of complex sample can be facilitated by the combination of separation techniques outside the mass spectrometer (such as differential mobility spectrometry (DMS)) and ion structure probing techniques inside the mass spectrometer (such as tandem mass spectrometry and gas-phase ion/ion reactions). Here the coupling method between DMS and ion/ion reaction is developed and tested with model peptide systems to demonstrate its possible application in complex sample characterization such as isomer identification.<br>

<p>   </p> <p>Ambient Mass Spectrometry Imaging (MSI) is a powerful analytical tool in biomedical research that enables simultaneous label-free spatial mapping of hundreds of molecules in biological samples under native conditions. Nanospray desorption electrospray ionization (nano-DESI) is an emergent ambient MSI technique developed in 2010 that uses localized liquid extraction of molecules directly from surfaces. Like other liquid-extraction based techniques, nano-DESI relies on gentle removal of molecules from surfaces and soft ionization. High sensitivity and spatial resolution, versatility of the solvent composition, which may be used to tailor the extraction and ionization of selected molecules, quantification capabilities at the single-pixel level as well as compensation for matrix effects by adding a known standard to the solvent, and online derivatization are key features of nano-DESI MSI that position it as a unique analytical tool for studying biological systems. </p> <p>Despite the advantages that nano-DESI provides, there are still challenges associated with the structural characterization, extraction, and detection of certain molecular classes. Therefore, my dissertation research has focused on addressing these analytical challenges by developing innovative approaches that substantially enhance the performance of the nano-DESI technique in the study of complex biological systems. </p> <p>In this thesis, a systematic study of the solvent composition is carried out to aid in the detection of neutral lipids such as triglycerides thereby expanding the molecular coverage of nano-DESI experiments. Taking advantage of the versatility of the solvent composition, I developed an approach for the online derivatization of unsaturated lipids into lipid hydroperoxides using the reaction of singlet oxygen with C=C bonds. This method further expands the specificity of nano-DESI MSI by enabling the detection and imaging of positional lipid isomers. To aid in the analysis of complex mixtures and provide additional structural information in the form of collision cross sections, coupling of nano-DESI with a drift-tube ion mobility spectrometry is also reported along with examples of the powerful capabilities of this platform. Lastly, nano-DESI MSI is used to address the complexity in the analysis of individual skeletal muscle fibers. This collaborative project involves the development of a robust image registration approach of immunofluorescence imaging and high-spatial resolution nano-DESI MSI to obtain accurate chemical maps specific to each fiber type. The developments described in this thesis are key to understanding the dynamic metabolic processes on a molecular level with an unprecedented specificity and sensitivity.</p> <p>  </p>

Mass spectrometry of negative ions is a convenient method for generating, isolating, and analyzing reactive intermediates that would otherwise be too short lived to detect. This ion approach is especially useful for studying the chemical properties of radicals. In this work, a negative charge-carrying group was attached to lignin model compounds and combined with collision-induced dissociation (CID) to generate and characterize radical species involved in the primary pyrolysis of lignin. The charge-tag served to increase the sensitivity of the model compounds using electrospray ionization mass spectrometry (ESI-MS) and promoted charge-remote fragmentations (CRF) upon being collisionally activated. The resulting product ions were comparable to the primary pyrolysis products of lignin; thus, CID-CRF proved to be an effective way of identifying the mechanisms by which lignin decomposes in the gas phase. <br><div><br></div><div>Additionally, this dissertation includes a review of nitrene anions. Nitrene anions are another class of reactive intermediates protected by an electron that provide a means for studying the corresponding neutral molecules via electron photodetachment spectroscopy and photoelectron spectroscopy. The added electron makes it possible for protected nitrene anions to be manipulated by external electric and magnetic fields of a mass spectrometer. Nitrene anions also display their own unique reactivities as reagents, which have been investigated using ion/molecule reactions. Mass spectrometry of negative ions has thereby provided information on the electronic states, reactivities, and thermochemical properties of nitrene intermediates.</div>

<div>Mass spectrometry (MS) is a powerful and versatile analytical tool that is extensively used for the identification and analysis of complex mixtures. The ability to couple MS to atmospheric pressure ionization techniques and high-performance liquid chromatography (HPLC) or gas chromatography (GC) provides a high degree of experimental flexibility. MS is based on the analysis of gas-phase ions. Gas-phase ions are manipulated within the mass spectrometer and separated for detection based on their mass-to-charge (m/z) ratio.</div><div>One of the most commonly used techniques for complex mixture analysis is tandem mass spectrometry (MSⁿ). MSⁿ involves the isolation of the desired ion and allowing it to undergo reactions, such as collision-activated dissociation (CAD) or ion-molecule reactions. Based on the generated product ions, structural information can be obtained for unknown analytes in complex mixtures. In addition, MSⁿ methods based on diagnostic gas-phase ion-molecule reactions have been demonstrated to provide a general and predictable tool to identify specific functional groups in unknown ionized analytes and to classify unknown analytes into different compound classes depending on their functionalities.</div><div>The research described in this dissertation mainly focuses on the development of tandem mass spectrometric methods based on gas-phase ion-molecule reactions and/or CAD for the identification of the <i>N</i>-nitroso functionality, which is present in some potentially mutagenic drug impurities. Furthermore, the dissertation discusses combining machine learning and MSⁿexperiments based on diagnostic ion-molecule reactions of 2-methoxypropene to predict reaction outcomes in a semiautomated fashion for protonated analytes containing specific functional groups. Lastly, chemical characterization and gas-phase reactivity of the boron-centered radical dianion [B₁₂I₁₁]^2-• toward some organic molecules are discussed.</div>

Positively charged aromatic carbon-centered σ-type mono-and biradicals have been studied previously in the gas phase. However, very little is known about the properties of related polyradicals. In this dissertation, the reactions of series of quinolinium-and acridinium-based bi-, tri-, and tetraradicals were studied with cyclohexane and allyl iodide in the gas phase by using tandem mass spectrometry. I atom abstraction and allyl group abstraction were observed as dominant reactions for all the studied radicals upon reactions with allyl iodide. Sequential H atom abstractions were observed as the major reactions for the studied bi-and tetraradicals upon reactions with cyclohexane. Surprisingly, triradicals appeared to undergo addition followed by elimination of a H atom as one of the major reactions upon interactions with cyclohexane. Vertical electron affinity and spin-spin coupling between radical sites were found to control the radical reactivities.<div><br></div><div>The radical site(s) which react first with cyclohexane were experimentally determined. For the studied biradicals, the first reacting radical sites were found to be the ones that are predicted to be more reactive based on the reactivities of related monoradicals. For the studied triradicals, the first reacting radical sites are the ones that are least strongly coupled to the other radical sites. For tetraradicals, the first two sites reacting with cyclohexane are more weakly coupled than the other two radical sites.<br></div><div><br></div><div>The mechanisms for the reactions of the triradicals with cyclohexane were proposed based on tandem mass spectrometry experiments and supported by quantum chemical calculations. Briefly, the least strongly coupled radical site of a triradical reacts with cyclohexane first by abstracting a H atom. The more reactive radical site insome of the produced biradicals will then abstract a H atom from the cyclohexyl radical within the product collision complex to generate a monoradical and cyclohexene. Some of these monoradicals undergo addition to cyclohexene within this product complex,followed by elimination of a H atom. When allowed to react with allyl iodide, all of the monoradicals and most of the biradicals demonstrated predominant I atom abstraction. The quinolinium-based meta-and para-benzynes exhibited allyl group abstraction as the major reaction. The triradicals with a meta-benzyne moiety in the pyridinium ring demonstrated dominant allyl group abstraction, which is likely to occur at the pyridinium moiety. The reaction efficienciesof these triradicals toward allyl iodide are correlated with their calculated vertical electron affinities. The other triradicals showed I atom abstraction as the major reaction. These triradicals react with allyl iodide through different mechanisms compared to those mainly abstract an allyl group. Therefore, their reactivities are not directly related to their calculated vertical electron affinities.<br></div><div><br></div><div>In the tetraradicals, spin-spin coupling between all the radical sites affects their reactivities. The coupling of the radicals in a benzyne moiety is weakened by the couplings of radical sites between two benzyne moieties. This interaction results in higher reaction efficiencies for the tetraradicals than the related benzynes. Particularly, the 2,4,7,8-tetradehydroquinolinium cation was found to have much higher reactivity than the related meta-benzyne, the 2,4-didehydroquinolinium cation. This was rationalized based on the low distortion energy of the meta-benzyne moiety in the tetraradical.<br></div><div><br></div><div>Spin-spin coupling between the radical sites in bi-, tri-, and tetraradicals significantly affect their reactivity. To better understand the relation between the effects of spin-spin coupling and the spatial distance between two radical sites, a series of acridinium-based mono-and biradicals were studied in the gas phase. The acridinium-based monoradicals are less reactive than the related quinolinium-based monoradicals, which is possibly because of the steric hindrance of the additional benzene ring. Unlike quinolinium-based biradicals, which are less reactive than the related monoradicals, acridinium-based biradicals showed higher reactivities than the monoradicals with similar vertical electron affinities. In order to better illustrate the coupling strength in the studied biradicals, the natural logarithm of their total reaction efficiencies toward cyclohexane was plotted as a function of their calculated vertical electron affinities. The plots indicate that the coupling of quinolinium-based biradicals hinders the radical reactivity, while for acridinium-based biradicals, the coupling is negligibly weak and the biradicals react as two individual monoradicals.<br></div>

<p> Illicit drug abuse is a widespread issue in the United States and worldwide. Many methods  seek to ease the analytical workload required to collect, analyze, and identify these drugs. Paper  spray-mass spectrometry (PS-MS) is one response to this analytical workload as it offers a rapid,  affordable, and simple means for drug identification by mass spectrometry. This work centers on  the use of pressure-sensitive adhesive (PSA) lined paper as a PS-MS substrate for drug recovery  and detection. The use of PSA paper as a sampling and analysis substrate has been previously  established but is expanded herein with new capabilities and applications. Chapter 2 introduces  the combination of color tests followed by PS-MS for presumptive and confirmatory drug  identification. Three color tests (cobalt thiocyanate, Simon, or Marquis) were performed on the  PSA paper with subsequent drug confirmation occurring by PS-MS. Chapter 3 examines the use  of PSA paper and PS-MS for the recovery and detection of fentanyl, fentanyl precursors, and  analogs from shipping-related surfaces and in the presence of high amounts of cutting agents. The  use of a cartridge that accommodates a full-sized PSA paper ticket was also explored for drug  detection. Chapter 4 assesses PS-MS with PSA paper on portable MS instrumentation. Analyte  recovery and carryover as well as instrument robustness were evaluated. The color test and PS-MS protocol examined in Chapter 2 was also successfully applied to a portable MS instrument.  Application of PS-MS to the portable system highlights the potential fieldability of the technique. </p>

<p> Mass spectrometry (MS) has become one of the most powerful and versatile tools for chemical analysis due to its ultra-high sensitivity, high throughput, ease of automation, and the large amount of information obtained. Nowadays, MS is extensively used in many tasks, such as identification and quantitation of drug metabolites, analysis of the products of biomass pyrolysis, and study of reactive intermediates, to name a few. However, these mass spectrometric analyses are not without challenges. For example, the requirement for quantifying trace amounts of substances in a complex mixture constantly pushes the detection limit of mass spectrometers, and the increased sample complexity demands higher and higher mass resolution. Therefore, MS is constantly evolving to address more difficult analytical challenges. A variety of MS techniques have been developed over the years, including soft ionization methods that facilitate mass spectrometric analysis of macromolecules, such as proteins and antibodies that enables the development of new therapeutic agents, benchtop high-resolution mass spectrometers, such as the orbitraps that can be used to analyze some of the most complex mixtures, and portable mass spectrometers which can be used in the home and garden and even in cancer surgery. Besides its applications in chemical analysis, MS can serve as a unique tool for the fundamental study of gas-phase ion/molecule reactions, these gas-phase reactions can be used to better understand the reactivities of many reactive intermediates and to obtain structural information for unknown analytes.</p><p></p><p> This thesis is aimed at addressing challenges involved in mass spectrometric analyses of isomeric drug metabolites (Chapter 4), quantitation of drug metabolites by using tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS) (Chapter 5), delineating cellulose depolymerization mechanisms upon fast pyrolysis by using pyrolysis-tandem mass spectrometry (py-MS/MS) (Chapter 6), and studying the reactivities of vinyl cation intermediates (Chapter 7). An overview of the dissertation research is given in Chapter 1, the instrumentation and principles of linear quadrupole ion trap (LQIT) mass spectrometer are discussed in Chapter 2, and the organic synthesis performed for several studies is detailed in Chapter 3.</p>

<div>Mass spectrometry (MS) is a powerful and versatile analytical tool, especially for identification and analysis of complex mixtures. Coupling to high-performance liquid chromatography (HPLC) or gas chromatography (GC) provides additional dimension for mixture analysis. MS manipulates ionized analytes and separates them based on their mass-to-charge (<i>m/z</i>) ratios. MS is capable of providing molecular weight (MW) information by generating pseudo-molecular ions of the analytes. Detailed elemental compositions can be also obtained if high resolution MS is used. MS can also provide extensive structural information of the analyte ions. One of the most commonly used technique is tandem mass spectrometry (MSⁿ). Ions of interest are isolated and subject to sequential reactions (reactions with other molecules or dissociation reactions) to generate product ions that can provide structural information. MS is also a powerful tool for generating and studying highly reactive reaction intermediates, such as organic polyradicals.</div><div><br></div><div>The research described in this dissertation mainly focuses on the generation and gas-phase reactivity studies of different organic biradicals. Their reactions with various organic reagents are studied, and the reactivity-controlling factors are discussed. For example, the reactivity of several substituted pyridine-based biradical cations with 2,6-topology are discussed (all with singlet ground states), and their special reactivity from their excited triplet states are illustrated. Besides, several quinoline-based biradicals and cyano-substituted pyridine-based <i>para</i>-benzyne cations are also discussed. Some of the radicals (or ions) described in this dissertation are generated for the first time, i.e. the quinoline-based oxenium cations. Their structural characterization and gas-phase reactivity toward some organic molecules are discussed in the dissertation. Further, an electrophilic anion, <i>closo</i>-[B₁₂X₁₁]^- (X = Cl, Br) and its application in the activation of C-H and C-C bonds in hydrocarbon molecules are described in the dissertation.</div>

<div> <div> <p>Continuous flow chemistry for organic synthesis is an emerging technique in academia and industry because of its exceptional heat and mass transfer ability and, in turn, higher productivity in smaller reactor volumes. Preparative electrospray (ES) is a technique that exploits reactions in charged microdroplets that seeks to accelerate chemical synthesis. In Chapter 2, the flow synthesis of atropine, a drug which is included in the WHO list of essential of medicines and currently in shortage according to the U.S Food and Drug Administration (FDA)is reported.The two steps of atropine synthesis were initially optimized separately and then continuously synthesized using two microfluidic chips under individually optimized condition.The telescoped continuous-flow microfluidics experiment gave a 55% conversion with an average of 34% yield in 8 min residence time. In Chapter 3, a robotic HTE technique to execute reactions in 96-well arrays was coupled with fast MS analysis. Palladium-catalyzed Suzuki-Miyaura (S-M) cross-coupling reactions were screened in this system and a heat map was generated to identify the best reaction condition for downstream scale up in continuous flow. <br></p><p><br></p><p>In Chapter 4, an inexpensive and rapid synthesis of an old anticancer drug, lomustine,was synthesized. Using only four inexpensive commercially available starting materials and a total residence time of 9 min, lomustine was prepared via a linear sequence of two chemical reactions performed separately in two telescoped flow reactors. Sequential offline extraction and filtration resulted in 63% overall yield of pure lomustine at a production rate of 110 mg/h. The primary advantage of this approach lies in the rapid manufacture of lomustine with two telescoped steps to avoid isolation and purification of a labile intermediate, thereby decreasing the production cost significantly. A high throughput reaction screening approach based on desorption electrospray ionization mass spectrometry (DESI-MS) is described in Chapter 4 and 5 for finding the heat-map from a set of reaction conditions. DESI-MS is used to quickly explore a large number of reaction conditions and guide the efficient translation of optimized conditions to continuous flow synthesis that potentially accelerate the process of reaction optimization and discovery. Chapter 5 described HTE ofSNAr reactions using DESI-MS and bulk techniques with 1536 unique reaction conditions explored using both in DESI-MS and bulk reactors. The hotspots from the HTE screening effort were validated using a microfluidic system that confirmed the conditions as true positives or true.<br></p> </div> </div>

The function of many biological processes depends on the structure and composition of the biomolecules involved. Both spectroscopy and mass spectrometry provide complimentary information regarding the three-dimensional conformation and the composition, respectively, as well as many other things. Here, double resonance conformer specific spectroscopy coupled with the latest ab inito computational methods is used to make structural assignments at the atomic resolution as well obtain information regarding propensities of intramolecular interactions. Additionally, rapid cooling in conjunction with IR excitation to modulate and measure the relative populations of conformers present in the expansion. Two different designer peptide systems are studied, including an achiral acylated 𝛼-aminoisobutryic acid dipeptide (Ac-AIB2-R) with various C-terminal protecting groups (R=NHBn, NHBnF, 𝛼-methylbenzylamine) and an acylated 𝛾4-phenylalanine (Ac-𝛾4Phe-NHMe) with the a methyl amine C-terminal protecting group. Mass spectrometry is used to determine the kinetics of gas-phase covalent tagging reactions used to enhance the sequence coverage. The covalent modification reactions utilize click chemistry between NHS or HOBt substituted sulfobenzoic acid tags with nucleophiles present on the residues of the amino acids composing the backbone. Effective temperatures are approximated using the Tolmachev model, which relates the statistical average internal energy of the molecule to a temperature.