Chemistry & Chemical Biology Department Theses and Dissertations

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https://hdl.handle.net/1805/2052

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Chemometric Comparison Of GC-MS And GC-VUV For The Trace Analysis Of Methamphetamine
(2024) Lyle, Grant; Goodpaster, John; Sardar, Rajesh; Manicke, Nicholas
Chemometrics, the application of mathematical or statistical algorithms to make inferences on the state of a chemical system from physical measurements of it, is a powerful tool that can be used to re-read what previously was observed as ‘noise’ in analytical measurements. Instruments such as spectrophotometers can take thousands of measurements over a predefined interval, but the spectra are only of great use when reference libraries exist, or if large trends occur that allow for visual matching, such as with a particular functional group. Application of statistical techniques to these data, such as principal component analysis (PCA) and linear discriminant analysis (LDA), can help to spot underlying variances, and differentiate between similar spectra by using linear combinations of these variables for classification. Methamphetamine (MA) is a member of the phenethylamines, a group of compounds that act as central nervous system stimulants, which are highly addictive and often the subject of law enforcement efforts at the local and federal level. Use of derivatization agents in analysis of seized narcotics is common practice, as it increases volatility/thermal stability of analytes, and improves peak shape for chromatographic resolution. In this analysis, we looked to investigate the difference in instrumental response for MA in its native form, as well as derivatized with two common agents, acetic anhydride and trifluoroacetic anhydride. These three forms were analyzed both on a gas chromatograph- mass spectrometer (GC-MS) and a gas chromatograph- vacuum ultraviolet spectrometer (GC-VUV). The raw GC-MS and GC-VUV data were separately normalized, and the dimensionality of the data was reduced through PCA, which uses orthogonal linear transformations of the data to capture most of the variance between datasets while simultaneously reducing the dimensionality for further analysis. Linear discriminant analysis was utilized to look at the principal components from PCA, and a classification model was built for use in discriminating between forms of methamphetamine from compressed datasets.
Development of Polymer Gel-Supported Lipid Bilayer Using Capillary-Assisted Assembly
(2024-12) Chuduang, Kridnut; Naumann, Christoph A.; Long, Eric C.; Sardar, Rajesh; Lin, Chien-Chi
The modern view of the plasma membrane is that of a complex, highly dynamic, compartmentalized system that critically impacts multiple important cellular functions. Supported model membranes of well-defined compositions have emerged as attractive experimental platforms to determine the underlying molecular processes that regulate membrane-associated cellular functions using advanced biophysical detection methods with up to single molecule resolution. However, membrane properties of previously employed supported membrane systems, such as solid-supported lipid bilayer (SLB) and polymer-supported lipid bilayer with a polymer layer thickness of several nm, were found to be perturbed by the nearby solid substrate. To overcome this limitation, the present work describes for the first time the capillary-assisted formation of a lipid bilayer (CA-PGB) on the surface of a fully hydrated, several micrometers thick polyacrylamide gel. CA-PGB formation can be accomplished by physisorption or specific chemical linkages (tethering) between polymer gel and bilayer. Not dissimilar to conditions found in plasma membranes, membrane properties of CA-PGB are found to be solely influenced by the attached polymer layer. The successful formation and lipid fluidity of CA-PGB is confirmed using confocal microscopy and fluorescence correlation spectroscopy (FCS). Lipid bilayer spreading on the hydrogel surface by capillary-assisted assembly is not altered when the polymer gel stiffness or bilayer bending stiffness are varied, illustrating the robustness and versatility of the assembly process. This work also shows that, unlike other supported membrane systems, the capillary-assisted assembly approach causes the formation of a lipid reservoir at the edge of the capillary. This lipid reservoir provides a lipid supply for the CA-PGB, enabling bilayer self-healing and superior bilayer stability relative to SLB. Experimental data are presented that support an assembly process, in which bilayer spreading on the gel surface inside the water capillary between two substrates is caused by monolayer collapse of suddenly accumulated lipids at the air-water interface of the capillary during sandwiching. A key aspect of the monolayer collapse-induced bilayer spreading is its rapid kinetics, which appears to be faster than the polymer gel swelling kinetics. The importance of the fast kinetics of bilayer spreading during capillary-assisted assembly is supported by the observation that attempts to build polymer gel-supported lipid bilayer using traditional lipid assembly methods [i. e., Langmuir-Blodgett (LB)/Langmuir-Schaefer (LS), LB- vesicle fusion, and spontaneous bilayer spreading from a hydrated lipid source], characterized by slower bilayer spreading kinetics, are unable to form a homogeneous fluid lipid bilayer on the polymer gel surface. The experimental results obtained in this work strongly suggest that the CA-PGB not only represents a powerful experimental model membrane platform for the analysis of membrane-associated processes relevant in cellular membranes, but also serves as promising cell surface mimetic to probe the cellular mechanosensitivity of adherend cells.
Expanding the Capabilities of Paper Spray Mass Spectrometry: High-Throughput Drug Screening and On-Paper Electrokinetic Stacking
(2024-12) Rydberg, Magnus Per; Manicke, Nicholas; Deiss, Frédérique T.; Web, Ian K.; Naumann, Christoph T.
Paper spray mass spectrometry (PS-MS) offers advantages in simplicity and rapid analysis but faces challenges that have limited its widespread adoption. These include insufficient sensitivity for certain analytes, susceptibility to matrix effects in complex samples, and inconsistent quantitative performance. Such limitations have restricted the application of PS-MS in fields requiring high sensitivity in analysis of complex biological matrices. This dissertation explores approaches to enhance the capabilities and applications of PS-MS, with a particular focus on overcoming sensitivity limitations. Chapter 2 demonstrates the potential of PS-MS in forensic toxicology through an automated high-throughput urine drug screening method. Chapter 3 investigates the negative impact of laser cutting paper spray substrates on sensitivity and provides practical remedies. The core of this work revolves around the development and application of electrokinetic stacking coupled to PS-MS. Chapter 4 describes the integration of field-amplified sample stacking and faradaic ion concentration polarization into paper-based MS ionization cartridges. Chapter 5 presents mathematical modeling to complement the experimental work, offering a theoretical framework for understanding system behavior. Chapter 6 demonstrates practical applications of the technique, utilizing stacking devices to detect low levels of per- and polyfluoroalkyl substances (PFAS) in tap water and drugs of abuse in urine. These applications demonstrate substantial enhancements in sensitivity over conventional PS-MS, while preserving the technique's advantage of minimal sample preparation. Moreover, this work also identifies a potential path towards incorporating electrokinetic separations in PS-MS, addressing a longstanding limitation of the technique and potentially broadening its applicability in complex sample analysis.
Photoinduced Pyridine N-Oxide Catalyzed Functionalization of Unactivated Olefins and Alkylboronic Acids
(2024-12) Ascenzi Pettenuzzo, Cristina; Deng, Yongming; Minto, Robert; Laulhé, Sébastien; Sardar, Rajesh
Primary alcohols are fundamental substrates in organic synthesis, and widely used in pharmaceutical, agrochemical, and bulk/fine chemical industries. Chapter one describes the current practices in industry and discoveries in academia for primary aliphatic alcohol synthesis. The methods discussed in chapter one include hydroboration-oxidation, the Ziegler process, hydroformylation/hydrogenation, transition metal-catalyzed hydrogenation of epoxides. Catalytic methods are described as well, both in transition metal catalysis and photoredox catalysis. Chapter two introduces a photoinduced pyridine N-oxide-catalyzed method for carbohydroxylation of unactivated olefins. The extensive reaction optimization process is shown, including the screening of different pyridine N-oxides, photocatalysts, and solvents. In this chapter, the applicability of the method is confirmed for a broad scope of unactivated olefins, both mono- and di-substituted. The regioselectivity of the transformation is confirmed by X-ray crystallography. Chapter three offers mechanistic insights regarding the carbohydroxylation of unactivated olefins. The proposed mechanism is corroborated through different experiments. Cyclic voltammetry and Stern-Volmer fluorescence quenching analysis, revealed that the photocatalyst directly oxidized pyridine N- oxide but does not oxidize the olefin substrate. The substitution step that ultimately affords the primary alcohol is confirmed with the use of different oxygen nucleophiles, that generate the corresponding carbo-oxygenated products. The 18O labeling experiments provide solid evidence that the oxygen source is not the pyridine N-oxide, rather it is the added nucleophile. Radical trapping experiments confirm the existence of the carbon radical generated, after pyridine N-oxy radical addition to the olefin. Chapter four delves in the development of a protocol for the generation of alkyl carbon radicals from alkylboronic acids, wherein photoexcited 4-nitropyridine N-oxide biradical features a catalyst to promote the nucleo-homolytic substitution of boronic acids. With a wide range of readily available aliphatic boronic acids, including methyl boronic acid, the developed catalytic system demonstrates broad applicability for alkylation, amination, and cyanation.
Development Of Paper Spray Mass Spectrometry Cartridges With Solid Phase Extraction For Drug Screening Applications
(2024-12) Jakstonyte Ren, Greta ; Manicke, Nicholas; Goodpaster, John; Naumann, Christoph; Deiss, Frédérique
The rise in overdoses, especially due to synthetic drugs like fentanyl has demonstrated a need for a rapid and simple drug detection method. This work describes the development and optimization of a paper spray mass spectrometry (PS-MS) with integrated solid-phase extraction (SPE) cartridges that can detect a wide range of drugs in plasma and whole blood The results can be obtained providing results in under five minutes with minimal sample preparation. The method is highly adaptable, allowing for rapid response to new emerging drugs. The initial focus on synthetic cannabinoids demonstrated sub-0.1 ng/mL detection limits for eight compounds in 100 µL of plasma. This work was expanded to screen thirty-five analytes from drug classes including fentanyl analogs, cathinones, benzodiazepines, and traditional illicit drugs. Validated according to SWGTOX guidelines, all drugs were detected in low ng/mL ranges. A streamlined data analysis method was also developed using a decision tree algorithm and an in-house library of nearly 200 compounds. This enabled retrospective analysis and detection of emerging drugs, such as 4F-MDMB-BINACA and brorphine, from previous samples. In a study of 400 authentic overdose plasma samples, 102 unique drugs were identified, mostly fentanyl-related. To further simplify the process, an "all-in-one" SPE cartridge was developed for whole blood, effectively pre-concentrating over 20 drugs of abuse. This device showed single and sub-ng/mL detection limits in 70 µL of blood. Samples were stable for 14 days, demonstrating the system’s potential for rapid, practical applications in forensic and clinical settings.
Mass Spectrometry Assay Development for Small Molecule Drug Therapies and Drugs of Abuse
(2024-12) Zimmerman-Federle, Hannah MacKenzie; Manicke, Nicholas; Goodpaster, John; Deiss, Frédérique; Deng, Yongming
Mass spectrometry (MS) is a comprehensive and adaptable technique that is useful for addressing a wide range of complex analytical challenges. In this work MS-based assays were developed to address issues relating to the synthetic drug epidemic and failures in point-of-care situations. Chapter one is an overview of the history of mass spectrometry, the fundamental operation of a mass spectrometer, and the advancements made in ionization methods. Chapter two is a review focusing on drug surveillance programs, or lack thereof, and their impact on combating the synthetic drug epidemic in the United States. The work in chapter three describes the development of a novel untargeted semiquantitative paper spray mass spectrometry assay for synthetic drug screening in human plasma. This work implemented a 3D printed solid phase extraction cartridge that allowed for sample preconcentration. This method was tested using remnant patient plasma samples collected from local downtown Indianapolis hospitals. Chapter four introduces a comparison of mass spectrometry acquisition methods to determine the most sensitive and specific techniques. The acquisitions that are compared are parallel reaction monitoring (PRM), sequential windowed acquisition of all theoretical mass (SWATH), and data dependent acquisition (DDA). A small subset of synthetic drugs with known properties were chosen for this comparison and a confirmation study was conducted with real patient samples. Chapter five describes the development of a paper spray mass spectrometry assay for the detection and semi quantitation of � -Lactam antibiotics to improve point-of-care therapeutic drug monitoring in critical care patients in the pediatric population. A liquid chromatography mass spectrometry method was also developed to compare limits of detection between these two methods. Single lot patient plasma samples were tested using both methods and the lower limits of detection and quantitation were compared. Chapter six details a combined approach for the determination of methylpyridine derivative isomers utilizing gas chromatography infrared spectroscopy (GC-IR), gas chromatography mass spectrometry (GC-MS), and density functional theory (DFT) calculations. The projects detailed herein show the extensive range with which mass spectrometry can be used for the detection of a wide range of drugs and synthetic compounds.
The Influence of Structure on the Donor-Acceptor Properties of Metallodithiolene Complexes
(2024-08) Colston, Kyle J.; Basu, Partha; Deng, Yongming; Pu, Jingzhi; Laulhé, Sébastien
The intrinsic charge transfer properties of a given system are dictated by their electronic structure. The movement of electrons from electron rich to electron deficient moieties of a system can spur useful photophysical properties that have been utilized in the development of materials science. Such systems take advantage of redox-active ligands, which can actively participate in electron transfer, and have the versatility to function as either electron donors or acceptors in charge transfer processes. One of the most widely used family of ligands in the development of such materials is dithiolene (Dt), which can exists in two redox extremes; reduced ene-1,2-dithiolate(2-) (Dt2-) and oxidized dithione (Dt0). Dt ligands draw inspiration from the molybdenum cofactor (Moco) found in molybdenum containing enzymes that are present in all phyla of life. The Dt2- and Dt0 ligands play contrasting roles in charge transfer, however, characterization of their electronic structure when both are incorporated into a coordination complex is underexplored. Detailed computational and experimental interrogation of such complexes are presented to highlight the importance of molecular and electronic structures on their charge transfer properties. Such complexes containing a Mo core are also relevant towards the comprehension of the electronic structure of Moco. This investigation focuses on the fundamental understanding of the charger transfer properties of metallodithiolene complexes containing both Dt2- and Dt0 ligands, and progress towards the synthesis of the closest Moco analogs.
Greener Photoredox-Catalyzed Phosphonations of Aryl Halides
(2024-05) Kelley, Alexandra S.; Laulhe, Sébastien; Minto, Robert; Deng, Yongming
Aromatic phosphonates and phosphine oxides are highly desirable synthetic targets used in pharmaceuticals, natural products, agrichemicals, catalysis, and materials science. While a variety of aromatic precursors have been used to access these motifs, aryl halides remain one of the most desirable coupling partners owing to their low cost, commercial availability, and regioselective reactivity. Traditional phosphonation often requires the use of harsh reductants in the presence of liquid ammonia, which are caustic and pose incredible environmental concerns. Milder, transition metal-catalyzed approaches have been developed, but can be limited by air sensitivity, cost, low reaction selectivity, and low functional group compatibility. Photoredox catalysis has been significantly advanced in the past decade in the pursuit of greener, more sustainable avenues to facilitate desirable reaction transformations under mild conditions. These methods most commonly use a dual catalytic strategy in which a metal is paired with an organocatalyst. While these approaches enable facile phosphonation of a variety of aromatic precursors, the metals and organocatalysts used are often expensive and toxic. Indeed, there remains unexplored chemical space for transition metal-free photoredox-catalyzed aryl C-P bond formations. Herein, we present a series of transition metal-free, photoredox-catalyzed approaches to the phosphonation of aryl halides. The approaches and mechanistic works will be discussed in the following order: First, the discovery that 10H-phenothiazine (PTZ) enables the transition metal-free phosphonation of aryl halides using trialkyl phosphites will be presented. PTZ serves as a photocatalyst capable of reducing the aryl halide to access aryl radicals, which readily couple with phosphite esters. This transformation exhibits broad functional group tolerance in good to excellent yields. Then, photoredox catalysis by PTZ enables the formation of unsymmetrical aromatic phosphine oxides using triphenylphosphine (PPh3) and aryl halides. This is the first work in which PPh3 has been used as the starting material, and the reaction proceeds via the alkaline hydrolysis of quaternary phosphonium salts. The final work exhibits novel photocatalytic activity of N-heterocyclic carbenes (NHC) to activate aryl halides, form aryl radicals, and enable phosphonation. This method displays broad functional group tolerance under mild conditions and highlights its untapped synthetic utility as a photocatalyst.
Investigating the Photophysical Properties of Potential Organic Lead Sensors
(2023) Quinones, Carlos; Basu, Partha; Deng, Yongming; Pu , Jingzhi
LeadGlow (LG) was reported in 2009 for its ability to both sensitively and selectively detect Pb2+ in aqueous solutions. Utilizing the synthetic approach of LG, it is possible to generate a class of novel fluorophores. A derivative of first-generation LG was synthesized and reported here for the first time, intuitively named LG2. Both compounds contain interesting photophysical properties that have not been extensively researched prior to this work. Because of this, photophysical properties of both LG and LG2 are unveiled here for the first time. These properties were investigated by determinations of quantum yield (QY), average fluorescence lifetime, and DFT calculations. LG was found to have a higher QY (0.057) than LG2 (0.011); however, LG2 displays an average fluorescence lifetime (3.186 ns) 5x greater than that of LG. Both LG and LG2 are synthesized via Hg2+-facilitated desulfurization of their respective thiocarbonyls, resulting in a turn-on fluorescence feature. The thiocarbonyl-containing fluorophores (SLG and SLG2) display quenched fluorescence compared to their oxo-derivatives (LG and LG2), this work attempts to investigate the mechanism(s) responsible. A whole class of LeadGlow compounds can be synthesized and could be potentially used as fluorescence-based sensors.
Development of Mass Spectrometry-Based Analytical Assays for Environmental and Chemical Defense Applications
(2023-12) Dowling, Sarah Naciye; Manicke, Nicholas; Goodpaster, John; Laulhé, Sébastien; Sardar, Rajesh
Mass spectrometry (MS) is a powerful and versatile technique that is useful for addressing a wide range of complex analytical challenges. In this work, mass spectrometry-based assays were developed to address issues relating to environmental contamination and for detecting analytes of interest to the defense industry. Chapter one is an overview of the history of mass spectrometry, the fundamental operation of a mass spectrometer, as well as, advancements in chromatographic separation and ionization methods. Chapter two focuses on the development of an assay that uses blow flies as environmental sensors of chemical weapon release. In that work, a liquid chromatography – tandem mass spectrometry (LC-MS/MS) method was developed to detect chemical warfare agent simulants and chemical warfare agent hydrolysis products in flies exposed to the chemicals in controlled feeding experiments. The work in chapter three describes the development of a surface enhanced Raman spectroscopy assay coupled to paper spray mass spectrometry for a more fieldable and environmentally friendly approach to detect organophosphorus compounds. Chapter four describes the development of a paper spray mass spectrometry assay for the detection and semi-quantitation of per- and polyfluoroalkyl substances in whole blood without sample cleanup or chromatographic separations. This method would be useful in detecting high levels of these carcinogenic compounds in individuals highly exposed via their occupations. The final chapter (chapter five) returns to using blow flies as environmental sensors, but this time to detect insensitive munitions in the environment. The work focuses on the development of two different liquid chromatography mass spectrometry methods for the detection of insensitive munitions, which are less shock sensitive explosives, and their transformation products in the environment. Controlled feeding experiments were also performed where flies were exposed to contaminated soil and water sources to show the feasibility of this method in a more realistic scenario. The projects detailed herein show the extensive range with which mass spectrometry can be used for the detection of harmful chemistries of environmental concern.

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