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Novel psychoactive substances (NPS) are synthetic drugs that pose serious public health and safety concerns as their ingestion by recreational drug users continues to cause adverse events and death. A multitude of NPS have been implicated in forensic investigations in the United States, but the identification of these emerging substances is challenging and complex, requiring advanced analytical capabilities and novel analysis workflows. The most common and effective manner for identifying NPS is by the use of mass spectrometry, while the true utility of this technology lies within non-targeted acquisition techniques. This research sought to utilize novel drug screening technologies and customized methodologies to characterize current NPS use in high risk populations through the analysis of biological sample extracts discarded from a partnering forensic toxicology reference laboratory. Specifically, NPS detection, identification, and characterization were the primary foci to produce increased awareness and education on a national level. To accomplish these goals, two novel workflows were developed: sample mining and data mining. A liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) assay was developed, validated, and implemented for forensic toxicology analytical testing. A SCIEX TripleTOF™ 5600+ QTOF-MS with SWATH® acquisition coupled to a Shimadzu Nexera XR UHPLC was used. Resulting data were compared against an extensive in-house library database containing more than 800 analytes. The LC-QTOF-MS assay was applied to the re-analysis of biological sample extracts to discover emergent NPS, their metabolites, and trends in use patterns. In total, 3,543 biological sample extracts were analyzed during this research and 21 emerging NPS were detected, some for the first time, through sample mining. Among these emerging substances were the NPS opioids: isopropyl-U-47700, 3,4-methylenedioxy-U-47700, and fluorofuranylfentanyl; the NPS opioid precursors: N-methyl norfentanyl and benzylfuranylfentanyl; the NPS hallucinogens: 2F-deschloroketamine, methoxy-PCP, and hydroxy-PCP; the NPS stimulants: 3,4-methylenedioxy-alpha-PHP, eutylone, and N-ethyl hexedrone; and the NPS benzodiazepine: flualprazolam. With respect to trends, NPS opioid positivity declined over time during this research; however, fentanyl positivity was persistent. Heroin and 3,4-methylenedioxymethamphetamine (MDMA) positivity appeared to decline slightly, but further temporal evaluation is necessary. NPS were less likely to be found in combination with other NPS; only one NPS substance was found in 82.5% of NPS-positive samples. Fentanyl poly-drug use was common, including concurrent or proximate use with traditional opioids (42.8%), NPS opioids (27.3%), cocaine (26.4%), methamphetamine (13.1%), NPS stimulants (4.2%), and other substances. The evaluation of in vitro metabolism for five emerging NPS detected for the first time during this research (3,4-methylenedioxy-U-47700, ortho-fluorofuranylfentanyl, 2F-deschloroketamine, eutylone, and N-ethyl hexedrone) resulted in the characterization of major metabolic pathways and the identification of metabolites presence in vivo by data mining of extract datafiles. These major metabolites provide utility for forensic laboratories to prolong detection windows for NPS. The primary metabolite identified for 3,4-methylenedioxy-U-47700 was N-demethyl-3,4-methylenedioxy-U-47700; the primary metabolite identified for ortho-fluorofuranylfentanyl was fluoro-4-ANPP; the primary metabolite identified for 2F-deschloroketamine was 2F-deschloro-norketamine; and the primary metabolites identified for eutylone and N-ethyl hexedrone were products of hydrogenation to the beta-ketone. As shown through this research, NPS continue to appear in forensic toxicology casework and novel assays for their detection and characterization are critical to remaining at the forefront of emerging drug trends and recreational drug use. LC-QTOF-MS was a vital piece of the analytical puzzle for discovering and characterizing emerging NPS and their metabolites. Analytical chemists must continue research involving NPS to broaden our understanding of synthetic drugs and their public health and safety impacts. / Chemistry

Successful methods for the separation of biomolecules like amino acids, proteins, peptides, and DNA have been developed previously using HPLC, GC, GC-MS, and CE. Recently CE has become a routine laboratory technique in the analysis of biological molecules. Even though high-resolution separations with small sample volumes is the main advantage, CE is limited by lower sensitivity detection of analytes when universal detectors like UV absorption or refractive index detectors are used. Therefore, sensitivity enhancement can be obtained by either using different detection schemes or electrophoretically based pre- or on-line concentration methods. These can be grouped into two categories. The first category includes IEF, CGF or TGF where sensitivity is achieved through equilibrium electrofocusing. In these methods, electrophoresis and bulk solution is combined in the capillary or separation column to form a null velocity point, a point at which the net velocity of the analyte is zero. Using these methods 10-10,000 fold sensitivity enhancement is achieved. The second category uses velocity gradients but not the nul velocity for the enrichment of samples. These methods include FASS, LVSS, NSM, etc., which are applied for the analysis of small molecules, and 10-10,000 fold sensitivity enhancement is reported by using these methods. In this work, first GEITP an on-line preconcentration technique is applied for the detection of amino acids (using Trp and Tyr as model analytes). This work also established the effects of different parameters on enrichment. The parameters studied include effect of current flow acceleration across capillary inner diameter, the effect of leading electrolyte (LE) concentration on current density, and the effect of applied electric fields on the current density. To explore the application of GEITP in biological fluids, optimized parameters were developed for the detection and separation of Trp and Tyr in artificial cerebrospinal fluid (aCSF). Next, GEITP was applied for enrichment and separation of physiologically relevant concentrations of chromophore-derivatized Asp and Glu in high conductivity samples like artificial cerebrospinal fluid (aCSF). It was concluded from this work that the major factors which influence the enrichment is the ratio of current density to sample conductivity. Finally, GEITP is applied as a prior step before CZE to increase the resolution between analytes without using ampholyte mixtures. In this method GEITP was combined to CZE to achieve resolution adjustment between amino acids mixture using low pressure hydrodynamic flow during CZE without changing the separation column, field strength, or electrolyte system. In this work, a rapid CE method for extraction and analysis of amino acids in planarians, labeled with 4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), was developed. This method was applied to detect the changes in the levels of amino acids when planarians were fed and starved. This method can be applied to study pharmacological effects in planaria, as it can monitor different amino acid levels with respect to feeding. Finally, ssDNA photoproducts of different lengths (11-mer and 63-mer) were separated using two different matrices, a traditional C18 and a PV/DBS (PLRP-S) matrix. A faster separation (within ~ 10 mins) was achieved for a 11-mer by the PLRP-S column. A separation was achieved in the PLRP-S column for the 63-mer while there was no separation in C18 column. Baseline resolution was not achieved. Therefore, C18 can best be used for small length DNA while PLRP-S can be applied for longer length DNA, as it is more hydrophobic than C18 column. Parameters can still be optimized for a baseline separation. / Chemistry

Molecular electronics based on bottom-up electronic circuit design is a potential solution to meet the continuous need to miniaturize electronic devices. The development of highly conductive molecular wires, especially for long distance charge transfer, is a major milestone in the molecular electronics roadmap. A challenge presented by single molecule conductance is to define the relative influence of the molecular "core" and the molecular "interconnects" on the observed currents. Much focus has been placed on designing conductive, conjugated molecules. However, the electrode-molecule contacts can dominate the responses of metal-molecule-metal devices. We have experimentally and theoretically probed charge transfer through single phenyleneethynylene molecules terminated with thiol and carbodithioate linkers, using STM break-junction and non-equilibrium Green's function methods. The STM break-junction method utilizes repeatedly formed circuits where one or a few molecules are trapped between two electrodes, at least one of which has nanoscale dimensions. The statistical analysis of thousands of measurements yields the conductance of single molecules. Experimental data demonstrate that the carbodithioate linker not only augments electronic coupling to the metal electrode relative to thiol, but reduces the barrier to charge injection into the phenyleneethynylene bridge. The theoretical analysis shows that sulfur hybridization provides the genesis for the order-of-magnitude increased conductance in carbodithioate-terminated systems relative to those that feature the thiol linker. Collectively, these data emphasize the promising role for carbodithioate-based connectivity in molecular electronics applications involving metallic and semi-conducting electrodes. One of the strategies for building molecular wires that can transfer charge over long distance is to incorporate metal ions into the conductive molecular core. Peptide nucleic acid (PNA) is a great candidate for this purpose. Studying the conductivity of PNA can not only contribute to a better understanding of charge transfer through biomolecules, but can also help develop better molecular wires and other building blocks of molecular electronics. We study the charge transfer of PNA molecules using the STM break-junction technique and compare with traditional macroscopic voltammetric measurements. By measuring the resistance of different PNA molecules, we hope to develop a deep understanding of how charge transport though PNA is affected by factors such as the number and type of natural and artificial bases, embedded metal ions, pH, etc. Self-assembled monolayers (SAMs) of porphyrins are of great interest due to their diverse applications, including molecular devices, nano-templates, electrocatalysis, solar cells, and photosynthesis. We combined a molecular level study of the redox reactions using electrochemical scanning tunneling microscopy (EC-STM) with a macroscopic electrochemical technique, cyclic voltammetry (CV), to study two redox active porphyrin molecules, TPyP (5,10,15,20-Tetra(4-Pyridyl)-21H,23H-Porphine) and 5, 10, 15, 20-tetrakis (4-carboxylphenyl)-21H, 23H-porphine (TCPP). We showed that the adsorbed oxidized TPyP molecules slowly change to brighter contrast, consistent with the appearance of the reduced form of TPyP, under reduction condition (0.0VSCE). The time scale of the slow reduction is in the order of tens of minutes at 0.0VSCE, but accelerates at more negative potentials. We propose that protonation and deprotonation processes play an important role in the surface redox reaction due to geometric restriction of the molecules adsorbed on the surface. EC-STM and CV experiments were performed at various pH values to investigate the mechanism of this anomalously slow redox reaction. Our results show that the increased concentration of H+ hinders the reduction of porphyrins, a feature that has not been reported preciously. This provides insight into the details of the surface redox reaction. / Chemistry