Date of Award

2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry

Department

Chemistry

First Advisor

Jimmie Oxley

Abstract

Compound identification, characterization and quantification has been the key elements behind development of analytical chemistry as a field. Mass spectrometry (MS) is one of the predominant techniques used for such purposes. To enhance its capabilities, it is often integrated with orthogonal methods such as gas and liquid chromatography, which offer analytes separation prior to MS detection. Another approach is to combine multiple mass spectrometers into tandem, e.g. triple quadrupole, or hybrid, e.g. linear ion trap-Orbitrap, designs, which provide another level of characterization, usually via compound fragmentation. All these designs exist because none, on their own, offer a perfect solution for analytes detection and characterization.

Energy resolved mass spectrometry (ERMS) adds a second energy dimension to mass spectral data analysis. Survival Yield (SY) was invented as a quick tool for analysis of such data. It fundamentally relies on the production of fragment ions for compound differentiation. Most explosive compounds do not produce detectable fragments (> m/z 50) within the linear ion trap-Orbitrap detection range; thus, SY method fails to differentiate these compounds. To overcome this issue, a new method, Fragmentation Resilience (FREMS) has been invented and presented in this thesis. Each precursor and its fragment ions are analyzed and quantified separately, using a four-parameter logistic function. In the process of method establishment, several remarkable discoveries were made. The normalization and overlay of fragmentation curves uncovered hidden properties of correlated ions, as they tend to overlap at the inflection points. This simple approach permitted the use of a non-statistical “cross-intersect” method, which then was used to improve so-called “modified-SY” method. FREMS also distinguishes unrelated fragments and contamination via simple visual inspection.

The FREMS methods were qualified using a full panel of testing on a Thermo LTQ-Orbitrap, using 4-chlorobenzylpyridinium ion. This revealed that breakdown energies depend only on three controllable parameters – number of ions inside the ion trap, Maximum Inject and Activation Times. A fairly linear relationship (R2 > 0.95) with proposed FR50, m-SY50 and C-I metrics provides reliable adjustment mechanisms for these variables via calibrations. Consequently, as long as ions are produced, any atmospheric pressure ionization processes are irrelevant for this application and can be treated as exclusively in vacuo experiments.

After a full investigation of input parameter effects, FREMS was successfully employed for structural elucidation, using multi-stage MSn experiments and fully characterizing [M+H]+ precursor ions of two model compounds – bupropion and glutathione. Isolation and fragmentation was performed in the linear ion trap-Orbitrap instrument, which allowed separation of the analysis into individual stages. The FREMS methodology produced fragments in sequential manner, with each MSn stage being its own experiment. Moreover, akin to cross-peaks in NMR spectroscopy, “cross-branching,” resulting from a merging of two distinct FREMS pathways, enhanced the analyst’s ability to define fragment structures based solely on MS data. Cross- branching anchored certain structural conformations in place, provided greater confidence in structural identification, and pointed to certain structural connectivity, making it a quasi-3D-MS technique.

Set of experiments using atmospheric pressure chemical ionization and electrospray ionization, in both positive and negative modes, were performed to establish the correlation between accurate, Orbitrap, and nominal mass, Faraday, detectors. For most cases, no statistical significance was found between FREMS energies of the two detectors (p-value > 0.05 at 95 % confidence level). This extended the use of FREMS to nominal mass measurements and enhanced the applications of compound differentiation and structural elucidation for these instruments.

FREMS was used to determine dissociation energies of ion-neutral complexes, adding an energy dimension to enhance their characterization. The experimental results were highly reproducible within ± 0.2 % NCE of a mean value for each ion-complex. All nitrate (or chloride) species of the same compound showed no statistical significance among different solvents or concentrations at similar signal intensity levels. At the same time, nitrate and chloride species of the same compound were easily differentiated. Theoretical predictions of nitrate esters ion complexes were conducted to explore the possibility of using computational approaches to forecast experimental results. Among multiple DFT theory models tested, ωB97X-D/6-31G*, provided the most correlated relationship (ρ = 0.94) with experimental FREMS values of up to 5-carbon chain nitrate esters adducts. This indicates the use of predictive analytics is possible to forecast dissociation energies for a small subset of closely related structures.

The potential of FREMS has only begun to be explored. Can additional information be obtained from the nominal mass detectors when more than one species are present and their energies are averaged? Can FREMS be used to identify energetic properties or differentiate between explosive and non-explosive compounds? Have shown that nominal and accurate mass detectors produce the same energies, can the FREMS approach be used on a spectrum of mass spectrometers and generate comparable output, thus providing a new way for compound libraries creation? One thing is clear, mass spectrometry continues to evolve and surprise scientists with new ways it can perform compound discovery and analysis.

Available for download on Friday, January 05, 2024

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