Semester

Spring

Date of Graduation

2020

Document Type

Dissertation

Degree Type

PhD

College

Eberly College of Arts and Sciences

Department

Forensic and Investigative Science

Committee Chair

Glen Jackson

Committee Co-Chair

Luis Arroyo

Committee Member

Tatiana Trejos

Committee Member

Stephen Valentine

Abstract

The identification of well-characterized seized drugs is performed thousands of times a day in the United States; however, the expanding use of emerging synthetic drugs is creating a growing problem for both toxicological and seized drug analyses. Two of the most rapidly growing areas of emerging synthetic drugs are synthetic cathinones and fentanyl-related compounds (FRCs). In this work we demonstrate the combination of multi-stage mass spectrometry (MSn), accurate mass measurements with high-resolution mass spectrometry (HRMS), and isotopic labeling for the structural characterization of synthetic cathinones and fentanyl analogs. The deliverables of this research include the identification of conserved fragmentation pathways for synthetic cathinones and fentanyl analogs, proposed mechanisms for the formation of characteristic ions through both protonated tandem mass spectrometry (MS/MS) and electron ionization mass spectrometry (EI-MS), and a discussion about how to apply the broadened understanding of the fragmentation behavior to the identification of novel synthetic cathinones and fentanyl analogs.

The first major finding about the fragmentation behavior of synthetic cathinones is that the tropylium ion (m/z 91), or substituted derivative thereof, forms through different oxygen-containing intermediates that do not contain a formal C=O bond but instead contain a phthalane-like core structure. The phthalane-like intermediates were elucidated through gas-phase ion spectroscopy measurements and density functional theory (DFT) calculations. Likewise, the use of stable isotope labeling revealed the unprecedented finding that, during collision-induced dissociation (CID) of α-pyrrolidinophenone synthetic cathinones, the α-carbon is retained almost exclusively in the tropylium ion and the carbonyl carbon is not retained in the tropylium ion. Isotope labeling also identified competitive pathways for the loss of CO and ethylene (C2H4) from a primary intermediate ion, which provides support for the direct loss of CO from the alkyl side chain.

A second major finding was the identification of characteristic protonated MS/MS fragmentation pathways and proposed mechanistic origins for both protonated MS/MS and EI-MS fragmentation for α-pyrrolidinophenone and N-alkylated synthetic cathinones. For MS/MS spectra of protonated α-pyrrolidinophenone synthetic cathinones the dominant fragmentation pathways are through 4-center hydrogen rearrangements to produce pyrrolidine ring cleavage, characteristic iminium ions and diagnostic ions at m/z 91 and m/z 105. For EI mass spectra, radical-directed α-cleavages result in dominant iminium ions. In contrast to α-pyrrolidinophenone synthetic cathinones, MS/MS of protonated N-alkylated synthetic cathinones provided abundant radical losses from both the N-alkylated and aliphatic side chains, a dominant loss of H2O for 2° amines and the formation of abundant alkylphenones for 3° amines. These findings help advance our current understanding of the MS/MS analysis of synthetic cathinones, and they help analysts better understand and defend their observations and interpretations in existing and future casework.

For FRC’s, a combination of isotope labeling, HRMS and MSn experiments identified a novel isobaric product ion at m/z 188, which is elementally distinct from the two previous known isobars at m/z 188 and forms through an intermediate product ion at m/z 216 (for fentanyl). These studies also confirmed the pathways through which the three nominal isobars are formed and how substitutions to the aniline ring and amide moieties result in remarkably conserved fragmentation pathways. In contrast, substitutions to the piperidine ring, the N-alkyl chain, and the cyclic substituent of FRCs resulted in distinct differences in fragmentation pathways, the abundance of which is related to the identity of the specific substitution. For example, the presence of a hydroxyl group on the N-alkyl chain results in the MS/MS spectrum being dominated by the neutral loss of water, whereas the presence of a methyl group favors the formation of the tropylium ion. By understanding the fragmentation behavior of fentanyl and the impact of substitutions to the core fentanyl structure, toxicologists and seized drug analysts will be better prepared to identify emerging FRCs, which are increasingly common and deadly adulterants in the growing opioid epidemic.

The final major contribution from this work was the comparison between in-source CID and beam-type CID experiments of the same synthetic cathinones and FRCs on the same instrument. Whereas the relative abundance of certain fragments were often readily distinguishable between in-source CID and beam-type CID, the fragment m/z values and the overall pattern of fragmentation were sufficiently consistent that the spectra from the two different activation methods could serve as proxies for one another. However, because in-source CID involves fragmentation of all precursors from the source region of the mass spectrometer rather than through isolation and fragmentation in the collision cell, caution should be used when analyzing potential mixtures or complex biological samples where strict control of precursor ions present in the source region may not be possible.

Download

Share

COinS