<p>Breast cancer is one of the most commonly diagnosed cancers in women, with 1 in 8 women diagnosed during her lifetime. Distant metastatic breast cancer accounts for a majority of deaths in breast cancer patients. Changes in both the architecture and the biochemical composition of the extracellular matrix (ECM) occur during metastatic dissemination at both the primary tumor and the early metastatic niche. These changes play a significant role in the cell fate, and can alter proliferation, migration, and quiescence of cancer cells. This study utilizes tandem mass spectrometry to study ECM protein changes, specifically in the lungs, using an immune competent murine model of metastatic breast cancer. Liquid chromatography-tandem mass spectrometry was used to identify and quantify key ECM proteins in the primary tumors, lungs, and metastatic tumors during cancer progression. Fibronectin (FN) was upregulated in the primary tumor, suggestive of a more invasive mesenchymal-like cell. However, FN was decreased in abundance in metastatic tumors, which is favorable for a more epithelial phenotype, prompting tumor growth. The diseased lungs appear to have highly collagenous proteins, suggesting an increased stiffness in the matrix. This increase in stiffness would reduce physiologically induced strains, and potentially facilitate growth of metastatic lesions in the lungs. Characterization of the changes in the ECM during cancer progression will aid in development of future therapies as well as guide the design of relevant <i>in vitro</i> models, ultimately enhancing the knowledge of this phenomenon.</p>

The opioid epidemic has become a serious public health problem in the United States. The increasing abuse of synthetic opioids has raised concerns in the society. Fentanyl is a synthetic opioid analgesic which has resulted in an increasing number of drug overdoses since 2013. In addition, fentanyl analogs, originally manufactured for use as analgesics or animal tranquilizers, have emerged in the United States drug market. Fentanyl and its analogs, similar to other opioids, work as full µ-agonists, binding with µ-receptors in the brain. Fentanyl and its analogs elicit more potent effects compared to the traditional opioids being abused such as morphine or heroin. With the emergence of fentanyl analogs in the drug market, identifying and differentiating those analogs becomes a challenge due to their structural similarities to fentanyl. The purpose of this research was to develop a method of identifying and quantifying nine fentanyl analogs in urine and oral fluid using the QSight® Triple Quad LC-MS/MS, coupled with a Halo® C18, 2.7µm column. The method was validated based on AAFS Standards Board (ASB) Standard 036, Standard Practices for Method Validation in Forensic Toxicology. The analytes in this research included fentanyl, norfentanyl, acetyl fentanyl, carfentanil, cyclopropyl fentanyl, methoxyacetyl fentanyl, valeryl fentanyl, furanyl fentanyl and 4-anilino-N-phenethylpiperdine (4ANPP). All samples, calibrators, and quality controls (QC) were prepared by spiking certified reference standards into donated human urine or human oral fluid. Supported liquid extraction (SLE) was performed as the sample preparation method using ISOLUTE® SLE+ 1mL columns followed by evaporation. All samples were reconstituted with 200 µL methanol. The mobile phases used in this method were 5mM ammonium formate in Millipore water with 0.1% formic acid and methanol with 0.1% formic acid. A 10-minute LC method achieved complete resolution of the analytes, with specific retention times ranging from 3.5 to 5.7 minutes. For urine and oral fluid analysis, the calibration range for all analytes was established from 1 to 70 ng/mL. The resulting r2 values were greater than 0.988 for all analytes. Bias and precision were evaluated at 3, 25 and 60 ng/mL, and bias and percent coefficient of variation (%CV) for within and between run precision had acceptable values within ±20%. The limit of detection (LOD) was 0.1 ng/mL for most fentanyl analogs, with a LOD of 0.01 ng/mL for valeryl fentanyl and furanyl fentanyl. Carryover was not detected for any analytes in either matrix. Recovery of all compounds following SLE for both urine and oral fluid was above 50%. For urine, the ion enhancement and suppression of all analytes was within 25%. For oral fluid, the ion enhancement and suppression of most analytes was within 25% except valeryl fentanyl, which experienced suppression of 35%. The matrices analyzed had no interference effect on the detection or quantitation of analytes in this method. The interference effects of different commonly encountered drugs were studied and showed minimal impacts on the results generated from this method. All analytes were stable for up to 72 hours at room temperature, except cyclopropyl fentanyl. In conclusion, using the QSight® Triple Quad LC-MS/MS following SLE effectively identified and quantified fentanyl analogs present in both urine and oral fluid. This method has shown its potential to be applied to casework samples for fentanyl analogs detection.

Introduction. Popularity of electronic cigarettes (e-cigarettes) has increased dramatically in recent years, especially among adolescents. The most recent data from the National Institute on Drug Abuse (NIDA) cites that >16% of 12th graders have tried e-cigarettes, and >30% of those individuals will start smoking within 6 months1. E-cigarettes are available in a variety of ‘strengths’ indicating the labeled nicotine concentration in the product. In this project, we sought to investigate the accuracy of nicotine labeling found in some commercially available e-liquids. As such, we developed and validated a liquid chromatography-mass spectrometry (LC-MS) assay to determine the nicotine concentration in these products. Methods. A literature search revealed that the two most commonly used chromatographic approaches for nicotine are hydrophilic interaction liquid chromatography (HILIC) and reversed phase chromatography (RP). Both options were evaluated, and nicotine peak quality and reproducibility were assessed. Mass spectrometric conditions for positive electrospray (+ESI) ionization were optimized, including collision energy and ion accumulation time. The optimized method included a gradient separation using a UCT C18 column (2.1 x 100 mm; 1.8 micron) with acetonitrile as the organic phase and 0.1% formic acid in water as the aqueous phase. Nicotine stock solutions were prepared in 100% ethanol and diluted in acetonitrile to achieve calibration concentrations (5 – 75 micrograms/mL). Deuterium labeled nicotine (d4) was used as the internal standard at a concentration of 10 micrograms/mL. The method was evaluated for precision, reflected by percent relative standard deviation (%RSD) and accuracy, or percent error, at each concentration for three days. The method was applied to the assessment of nicotine concentration in samples of e-liquids labeled as 3 mg/mL nicotine. Results. Reversed phase chromatography outperformed HILIC separation for nicotine under the conditions tested. The final RP-LC-MS/MS method involves direct monitoring of m/z 163.1219 for quantification of nicotine (167.1219 for d4-nicotine). The method exhibits < 15% RSD and < 15% error for all concentrations in the calibration range (< 20% at the lower limit of quantification). The developed method allows for rapid throughput, with a run time of 5 minutes. The lower limit of detection was determined to be 1 microgram/mL. Of the e-liquids evaluated, variations of up to 37% from the labeled amount of 3 mg/mL were detected. Additionally, a product labeled ‘zero nicotine’ contained no detectable nicotine. Conclusions. A fast, accurate, and reproducible LC-MS/MS assay has been developed and validated for the determination of nicotine in e-cigarette liquids. This method was applied to the evaluation of e-liquids, which showed significant variability in nicotine content from the labeled amount. 1 https://www.drugabuse.gov/related-topics/trends-statistics/infographics/teens-e-cigarettes

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany and Ecology Candidate: Bc. Alžběta Vopelková Supervisor: doc. Ing. Lucie Cahlíková, Ph.D. Title of diploma thesis: Using of techniques of mass spectrometry in the analysis of natural compounds This diploma thesis deals with the topic " Using of techniques of mass spectrometry in the analysis of natural compounds ". The aim of the work was to create a research work and describe the profile of alkaloids in individual genera of the family Amaryllidaceae. The introduction to the theoretical part summarizes the analyzes gas chromatography (GC). Alkaloids have been classified according to their heterocyclic parts and the family Amaryllidaceae has been described. The work was based on professional texts by Czech and foreign authors. Used sources focused on the analysis of alkaloids in bulbs, or in the aboveground parts of plants in this family, using gas chromatography with mass detection (GC-MS). Key words: mass spectometry, analysis, natural compounds

Cannabidiol (CBD) is one of over 80 active cannabinoids found in Cannabis Sativa and is the second most abundant cannabinoid derived from the plant following d(9)-Tetrahydrocannabinol (THC). As opposed to THC, CBD does not appear to have any psychotropic effects, rather CBD is often utilized for its therapeutic properties, which include effects such as antinociception, anti-convulsion, and anti-inflammation. During the extraction of CBD from plant material, THC may be co-extracted. Therefore, screening and quantitating potential THC levels in individuals using CBD products is important in instances where the legality of use of THC does not match that of CBD. In recent years, oral fluid has gained recognition as a non-invasive and expedient matrix for both drug testing and forensic casework. Due to its relevance, oral fluid was selected for analysis. This project evaluated the detection and quantitation of CBD, THC, and two primary metabolites in oral fluid samples of a therapeutic-use cannabidiol donor using Biotage Supported Liquid Extraction (SLE) and subsequent testing by PerkinElmer QSight 220 CR LC-MS/MS in positive ionization mode All calibrators and quality controls were prepared by fortifying synthetic oral fluid with certified reference standards. Standards and samples were prepared in a 1:3 dilution with extraction buffer. Calibrators were prepared at 0.25, 0.5, 1, 5, 10, 50, 100, 200, 300, 400, and 500 ng/mL, with quality controls analyzed at 0.75, 70, and 425 ng/mL. Internal standard was added to the appropriate samples to account for any variation produced by sample preparation. SLE was performed using ISOLUTE SLE+ 1 mL columns with elution in hexane:ethyl acetate:methyl tert-butyl ether (80:10:10), followed by evaporation using an Organomation Multivap Nitrogen Evaporator (Berlin, Massachusetts). All samples were reconstituted in 100 uL of 0.1% formic acid in deionized water:0.1% formic acid in acetonitrile (70:30). Validation parameters were assessed using Academy Standards Board (ASB) Standard 036-Standard Practices for Method Validation in Forensic Toxicology, including linear dynamic range (LDR), limit of detection (LOD), limit of quantitation (LOQ), analyte recovery, ion suppression/enhancement, and carryover. Following reconstitution, samples were placed onto the autosampler for injection and subsequent chromatographic separation using a PerkinElmer Brownlee C18 2.1x50 millimeter (mm), 2.7 micrometer (um) column. Analysis of the samples by mass spectrometry was performed in positive mode with multiple reaction monitoring (MRM). Total run time including equilibration was 10.5 minutes. All compounds were quantified using linear calibration models with 1/X weighting (1/concentration) and measured values were normalized by their respective internal standards. The LDR was determined to be 0.25 to 500 ng/mL. For all analytes, LOD was assessed and determined to be 0.25 ng/mL with an LOQ of 1 ng/mL. Carryover was assessed by running a double blank following a sample spiked at 500 ng/mL with no analytes observed. The donor samples were collected at several timepoints around the oral administration of an 8 mg dose of CBD. These timepoints included: prior to administration, at the time of administration, 30 minutes post-administration, 45 minutes post-administration, 60 minutes post-administration, 90 minutes post-administration, and 120 minutes post-administration. CBD was quantified within the diluted oral fluid samples from below LOQ to 325.75 ng/mL. THC was detected above LOD but below LOQ, concentrations which lie below typical cut-offs used in both workplace drug testing and forensic casework. The two metabolites were not detected above the LOD. As such, the CBD product can be concluded to be of reasonable purity as it relates to legal implications. Overall, the use of laminar flow mass spectrometry was effective in detecting various cannabinoids in oral fluid samples following SLE sample extraction.

Image charge detector (ICD) technology has existed for decades. However, not until recently has an ICD been proposed for use in space exploration, specifically for studying the characteristics of the dust on Mars. Characterizing the dust on Mars is crucial for designing equipment to aid manned missions. It also improves our understanding of Mars' climate and weather systems. An ICD utilizing printed circuit board (PCB) electrodes, coupled with a custom differential amplifier, is best suited for this type of measurement because of its light weight, simplicity, and noise performance. The noise floor of our particular amplifier is measured to be 1030 e- and simulated to be as low as 140 e-. Both of these measurements are taken without averaging. To further verify and understand this device, I developed a novel simulation method using ANSYS Maxwell 3D to simulate the interaction between the charged particle and the electrodes of the ICD. The results from this simulation are then easily passed to Cadence where we can clearly see the response of the custom amplifier to the charged particle. This knowledge is used to study various types of electrode geometry for improved noise performance, as well as understand how particle trajectory affect the resulting signal. Once the validity of the Maxwell simulation is established, I use it, along with experimental data and a mathematical model based on conformal mapping, to optimize the ICD for noise performance. I find that the maximum noise performance does not lie in simply increasing the number of sensing stages, as was previously thought. The optimum number of stages is a function of the parasitic capacitance of the amplifier, with the greater parasitic capacitance leading to the greater number of stages for the optimum.

Analzying questioned documents in a nondestructive nature has been an issue for the forensic science community. Using nondestructive techniques such as video spectral comparator does not give reliable information due to the variations in gray or color levels that are distinguished differently by analysts. Destructive techniques such as chromatography give dependable, qualitative and quantitative, information but involves altering the evidentiary value of these questioned documents. The paradox of document examination becomes a problem when document evidence is involved, especially when trying to preserve its evidentiary value and critical data is needed. Thus, a nondestructive technique has been developed to solve the loopholes in document examinations. Direct analyte-probed nanoextraction (DAPNe) is a nanomanipulation technique that extracts ink directly off the document for further examination. A watermark is left, at most, post-extraction. DAPNe utilizes a tip emitter, pre-filled with a solvent, which is controlled in x-, y-, and z-coordinates via joystick controller and aspirates/extracts using a pressure injector. The versatility of this technique lies within the solvent chemistry and its capability to be coupled to various types of instrumentation. The extraction solvent can be altered to target specific components in the ink. For example, a chelator may be added to target metal ions found in ancient inks or methanol may be added to target certain organic resins and binding agents found in modern inks. In this study, DAPNe has been coupled to nanospray ionization mass spectrometry, fluorescence microscopy, Raman spectroscopy, matrix-assisted laser desorption ionization mass spectrometry, and laser ablation to solve questioned document concerns in the area of falsified or forged documents, redacted documents, and aging studies.

Membrane inlet mass spectrometry was used to observe nitric oxide in the well-studied reaction of nitrite with hemoglobin. The membrane inlet was submerged in the reaction solutions and measured NO in solution via its flux across a semipermeable membrane leading to the mass spectrometer detecting the mass-to-charge ratio m/z 30. This method measures NO directly in solution and is an alternate approach compared with methods that purge solutions to measure NO. Addition to deoxy-Hb(FeII) (near 38 μM heme concentration) of nitrite in a range of 80 μM to16 mM showed no accumulation of either NO or N2O3 on a physiologically relevant time scale with a sensitivity near 1 nM. The addition of nitrite to oxy-Hb(FeII) and met-Hb(FeIII) did not accumulate free NO to appreciable extents. These observations show that for several minutes after mixing nitrite with hemoglogin, free NO does not accumulate to levels exceeding the equilibrium level of NO. The presence of cyanide ions did not alter the appearance of the data; however, the presence of 2 mM mercuric ions at the beginning of the experiment with deoxy-Hb(FeII) shortened the initial phase of NO accumulation and increased the maximal level of free, unbound NO by about twofold. These experiments appear consistent with no role of met-Hb(FeIII) in the generation of NO and an increase in nitrite reductase activity caused by the presumed binding of mercuric to cysteine residues. These results raise questions about the ability of reduction of nitrite mediated by deoxy-Hb(FeII) to play a role in vasodilation.

Indiana University-Purdue University Indianapolis (IUPUI) / The nuclear protein SETMAR has been reported to be involved in many processes such as non-homologous end joining (NHEJ), di-methylation (arguably) of K36 of histone H3, restart of stalled replication forks, chromosome decatenation, enhancing of TOPII inhibitors which results in resistance to chemotherapeutics in cancer patients, etc. All these purported functions are impossible to execute without interaction with other proteins. It is established that SETMAR binds specifically to DNA at terminal inverted repeat sequences and can loop DNA. This DNA sequence specific pull-down exploits this attribute to identify possible protein interactors of SETMAR. As a result of this experiment several proteins have been identified for further research: BAG2, c12orf45, PPIA, XRCC5/6, and ZBTB43, all of which are found in higher statistical abundances in full length SETMAR samples.

Accelerator Mass Spectrometry (AMS) is a sensitive technique for the analysis of rare isotopes. Optimizing the output of the cesium-sputter ion source is a fundamental method for improving measurement precision, efficiency, and reliability. Several strategies for improving the ion source are discussed and lead to an understanding of the electrodynamics within the ion source to inform further improvement in design and operating parameters. At the Andr´e E. Lalonde Accelerator Mass Spectrometry Laboratory (Lalonde AMS), the High Voltage Engineering Europa (HVEE) SO-110C ion source was modelled using Integrated Engineering Software (IES)’s Lorentz-2E ion trajectory simulation software. Lorentz-2E incorporates the mutual space-charge interaction between the positively charged cesium ion beam and the sputtered negative ion beam. A critical component of this work was the development of the Rijke code. Rijke communicates with Lorentz-2E to initiate, generate, and run varied sequences of simulations, as well as analyze and record the input and output data in formats convenient for timely analysis. This software and its interconnection with Lorentz- 2E is described in extensive detail for a prospective user. Initial simulation work examined the effects of modifying various electrode geometries within the source such as the extraction cone, the target aperture, a simple cratered sample model as well as examining the effects of varying the cesium ion current. The self-repulsion of cesium was found to be important at currents of 250 µA and above. At high enough cesium currents, the expansion of the cesium beam is such that parts of it impinge outside the extents of the sample material. Through both simulation and experiment, it was demonstrated that this effect can be mitigated by either recessing (translating along the axis of symmetry away from the ionizer) the target holding the sample or by adjusting the potential difference between the target and ionizer. Experimentally, at routine settings (6 kV target to ionizer potential, 115 ◦C cesium oven temperature, and 35 keV output energy), a target recess of 1 mm gave the most stable and sustained output of 12C from graphite blanks. While the peak current was less than the unrecessed case, the total measured charge from the recessed target was higher. Cesium currents at these routine settings were found to be below the theoretical space-charge limited maximum. Using 10Be standards, a multi-dimensional experimental study examined the effects of increasing the cesium current, adjusting the target-ionizer potential from 4 to 11 kV, while also examining target recesses of 0 to 4 mm. Multiple combinations of these settings produced enhanced currents of 9Be2+, measured at the high-energy offset Faraday cup, as high as 13.5 µA. This was higher than previously observed, resulting in the most precise measurement of 10Be performed to date at Lalonde AMS. The electrodynamics within the ion source can be characterized as three competing processes: a) a strong locus of positive space charge located at the centre of the sample, depending primarily on the focusing of the cesium beam, which draws negative ions across the axis of symmetry; b) a bulk positive space charge external to the negative ion beam, depending primarily on the magnitude of the cesium current, draws the outer-most negative ions away from the axis; and c) the raw field from the electrode potentials and geometry which is mainly defocusing for negative ions. These effects are mitigated the most when the cesium beam is distributed across the entire sample surface with the additional critical benefit of maximizing the sample material accessed for sputtering. This thesis work has demonstrated that both the mutual and self space-charge interaction of the cesium and negative ion beams were critically important and that the use of the simulation software can inform both improved design and operation settings of the ion source.