Ambient ionization mass spectrometry, which has witnessed a flurry of recent developments, is a set of useful techniques for the analysis of samples under open-air conditions. It allows direct, rapid, real-time, high-throughput analysis with little or no sample pretreatment for the chemicals in solids or liquids. In this study, thermal desorption electrospray ionization mass spectrometry ( TD-ESI/MS ) involving direct insertion probe ( DIP ), thermal desorption ( TD ) and electrospray ionization ( ESI ) was used for the rapid screening of various types of samples. The source mainly consists of the sampling probe device, thermal desorption heating device, electrospray ionization device, ion source and temperature controller. A novel strategy involved in TD-ESI/MS processes where sampling, desorption, and ionization are separated as three independent events. The sampling probe is first used for the sampling of analytes and then inserted into a heat unit for thermal desorption. The desorbed analytes are finally carried into a reaction region with a stream of nitrogen gas, where charged methanol droplets were generated continuously by electrospray for post-ionization. Total analysis time is less than 10 seconds. Traditionally, three standard methods are used for the analysis for pesticide residues, biochemical, immunoassay and instrument. And, the instrument analysis is the most widely used because it provides lots of advantages in particularly accurate quantitative approach. However, its complicated steps take a long period of time for preparation. Here, we used TD-ESI/MS to rapidly screen the pesticide residues on the surface of fruits and vegetables. The MS/MS analysis was also performed to confirm those detected compounds. The experimental results of the standard deviation for reproducibility is 13.2% (n = 10), and the detection limit is approximately 10 ppb. Furthermore, several fruits and vegetables purchased from local market were used as test samples and pesticide residues on the surface of samples can be successfully detected via TD-ESI/MS. In addition, the TD-ESI/MS technique was also applied to the analysis of illegal additives or phthalates in food. In this study, the TD-ESI/MS technique emerges lots of advantages such as direct, rapid, real-time analysis of sample surface and sample pretreatment is not necessary, and shows highly potential for rapid screening of chemicals in food safety.

Desorption Electrospray Ionization Mass spectrometry Imaging (DESI-MSI) is an area of great interest and a promising tool in the field of chemical imaging. It is a powerful, label-free technique, which can determine, map and visualize different molecular compounds on a sample surface. The amount of information acquired in a single DESI-MSI experiment is enormous compared to other techniques, as it can simultaneously detect different compounds with their spatial distribution on the surface. The experiment can be used to produce two-dimensional and three-dimensional images. Chapter 2 focuses on the design and optimization of the setup for performing DESI-MS imaging on various substrates. The proposed setup was tested for its lateral spatial resolution. To provide proof-of-concept of the design, preliminary tests were performed to generate images from commercial thin layer chromatographic plates and photographic paper. Chapter 3 focuses on demonstrating the compatibility of novel microfabricated Thin Layer Chromatography plates (M-TLC plates) for detection with DESI-MSI.

<p>Ambient ionization mass spectrometry enables rapid <i>in-situ</i> analysis of a plethora of analytes that are relevant to the forensic and defense communities. As the arsenal of ambient ionization techniques, aimed at solving specific targeted problems, continues to expand, the adoption of these techniques into non-academic settings has been relatively slow. At times, although the technique can provide answers in a more rapid and cheaper manner, the technique does not pass all of the required legal rules for a particular analysis when dealing with forensic evidence. This can be demonstrated with the rapid detection of drugs by paper spray ionization mass spectrometry. Paper spray ionization mass spectrometry can have drugs deposited onto the paper substrate, the paper can wipe a surface for trace analytes, and there are commercial and automated ionization sources for this process. While analysis by paper spray is rapid, the Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG) states that a minimum of two instrumental techniques need to be utilized. Utilizing paper substrates that have nanoparticles embedded for surface enhanced Raman spectroscopy, that can also be utilized for paper spray ionization mass spectrometry, makes ambient ionization more appealing as it completes that first legal requirement. </p> <p>Other times, the slow adoption of these new ambient ionization techniques is due to specific communities not being aware of ambient ionization, and specific applications have not yet been demonstrated. Swab touch spray ionization mass spectrometry follows similar processes as paper spray ionization, as the swab acts both as the sampling substrate and the ionization source and can swab for analytes in a manner where the paper substrate may be damaged and unable to perform the ionization for analysis. This can be seen for the swabbing of organic gunshot residues and explosives, both of which current methods already use a swab for sampling but then need lengthy extraction techniques. The applicability of paper spray ionization and swab touch spray ionization for these forensic and defense analyses is only furthered by the fact that they both couple extremely well with portable mass spectrometers for analysis in the field.</p> <p>There are also many fields that ambient ionization is just starting to take its place in the analytical toolbox. Two such defense fields that are just beginning to expand into ambient ionization are the analysis of pyrotechnics and microelectronics. Pyrolysis gas-chromatography mass spectrometry methods have been developed and utilized for environmental tests for pyrotechnic formulation, but they are slow and there is an abundance of cleaning steps between analyses to prevent carry over and contamination. Using paper and swabs as the collection device and ionization source for environmental analysis of these pyrotechnics allow for them to be functioned at ambient conditions at the scale at which will be utilized in the field by the Warfighter. Similarly, authenticating microelectronics by desorption electrospray ionization mass spectrometry removes the subjectivity of the current methods, while rendering the integrated circuit intact enabling future use if deemed as a genuine part. By taking slower or more subjective tests, in a field that has not utilized ambient ionization heavily in the past and adding these new capabilities to their tool chest expands the acceptance and future applications of the technique.</p> <p>As acceptance and utilization of ambient ionization grows, the next generation of scientists need to have hands on training in these techniques. Through the development of new teaching laboratories that couple both the fundamentals of the technique at hand, while also examining an interesting application to better engage the students, a number of laboratory exercises have been developed. The creation of new laboratory exercise utilizing the next generation of instrumentation and analytical techniques is vital for the future and rapid application of these techniques. The work discussed herein chronicles the utilization and demonstration of ambient ionization mass spectrometry in monitoring clandestine activities, supporting the Warfighter, and redeveloping chemical laboratory education. </p>

<div> Advancements in cancer treatments have increased rapidly in recent years, but cures remain elusive. Surgical tumor resection is a central treatment for many solid malignancies. Residual tumor at surgical margins leads to tumor recurrence. Novel tools for assessing residual tumor at surgical margins could improve surgical outcomes by helping to maximize the extent of resection. Ambient ionization-mass spectrometry (MS) methods generate and analyze ions from minimally prepared samples in near-real-time (e.g. seconds to minutes). These methods leverage the high sensitivity and specificity of mass spectrometry for analyzing gas phase ions and generating those ions quickly and with minimal sample preparation. Recent work has shown that differential profiles of ions, corresponding to phospholipids and small metabolites, are detected from cancerous and their respective normal tissue with ambient ionization-MS methods. When properly implemented, ambient ionization-MS could be used to assess for tumor at surgical margins and provide a molecular diagnosis during surgery. </div><div><br></div><div>The research herein reports efforts in developing rapid intraoperative ambient ionization-MS methods for the molecular assessment of cancerous tissues. Touch spray (TS) ionization and desorption electrospray ionization (DESI) were utilized to analyze kidney cancer and brain cancer.</div><div><br></div><div> As a demonstration of the applicability of TS-MS to provide diagnostic information from fresh surgical tissues, TS-MS was used to rapidly analyze renal cell carcinoma and healthy renal tissue biopsies obtained from human subjects undergoing nephrectomy surgery. Differential phospholipid profiles were identified using principal component analysis (PCA), and the significant ions were characterized using multiple stages of mass spectrometry and high resolution/exact mass MS. The same TS-MS analyzed renal tissues were subsequently analyzed with DESI-MS imaging to corroborate the TS-MS results, and the significant DESI-MS ions were also characterized with MS.</div><div><br></div><div>Significant efforts were made in developing and evaluating a standalone intraoperative DESI-MS system for analyzing brain tissue biopsies during brain tumor surgery. The intraoperative DESI-MS system consists of a linear trap quadrupole mass spectrometer placed on a custom-machined cart that contains all hardware for operating the mass spectrometer. This instrument was operated in the neurosurgical suites at Indiana University School of Medicine to rapidly analyze brain tissue biopsies obtained from glioma resection surgeries. A DESI-MS library of normal brain tissue and glioma was used to statistically classify the brain tissue biopsies collected in the operating room. Multivariate statistical methodologies were employed to predict the disease state and tumor cell percentage of the samples. A DESI-MS assay for detecting 2-hydroxyglutarate (2HG), the oncometabolic product of the isocitrate dehydrogenase (IDH) mutation (a key glioma prognostic marker), was developed and applied to determine the IDH mutation status during the surgical resection. The strengths, weaknesses, and areas of future work in this field are discussed. </div><div><br></div>

<p>My research has focused on the development and translation of ambient ionization mass spectrometry (MS)-based platforms in clinical and surgical settings, specifically in the area of brain cancer diagnostics and surgical decision making. Ambient ionization MS methods, such as those described herein, generate and analyze gas phase ions with high sensitivity and specificity from minimally prepared samples in near-real-time, on the order of seconds to minutes, rendering them well suited to point-of-care applications. We used ambient ionization MS methods, specifically desorption electrospray ionization mass spectrometry (DESI-MS) and extraction nanoelectrospray ionization mass spectrometry (nESI-MS) to molecularly characterize brain cancer biopsies. The characterization was made using diagnostic compounds identified as markers of disease state, tissue composition, tumor type, and genotype in human brain tissue. Methods were developed and validated offline in the laboratory and translated to clinical and surgical settings, thereby generating chemical information on prognostic features intraoperatively and providing valuable information that would be otherwise unavailable. We believe that, with approval, the methodologies described can assist physicians and improve patient outcomes by providing analytical tools and molecular information that can inform surgical decision making and adjuvant treatment strategies, complementing and not interfering with standard of care protocols.</p> <p><br></p> <p>We have successfully demonstrated the use of desorption electrospray ionization mass spectrometry (DESI-MS) for the expedient molecular assessment of human glioma tissue biopsies based on lipid profiles and prognostic metabolites, both at the tumor core and near surgical margins, in two small-scale, clinical studies. Maximal surgical resection of gliomas that avoids non-infiltrated tissue is associated with survival benefit in patients with glioma. The infiltrative nature of gliomas, as well as their morphological and genetic diversity, renders treatment difficult and demands an integrated imaging and diagnostic approach during surgery to guide clinicians in achieving maximal tumor resection. Further, the estimation of tumor cell percentage (TCP), a measure of tumor infiltration at surgical margins, is not routinely assessed intraoperatively. </p> <p>We have previously shown that rapid, offline molecular assessment of tumor infiltration in tissue biopsies is possible and believe that the same assessment performed intraoperatively in biopsied tissue near surgical margins could improve resection and better inform patient management strategies, including postoperative radiotherapy. Using a DESI-MS spectral library of normal brain tissue and glioma biopsies to generate a statistical model to classify brain tissue biopsies intraoperatively, multivariate statistical approaches were used to predict the disease state and tumor cell percentage (TCP) of each biopsy, thereby providing an measure of tumor infiltration at surgical margins via molecular indicators. In addition to assessment of tumor infiltration, we have developed DESI-MS assays for detecting the oncometabolite 2-Hydroxyglutarate (2HG) to detect isocitrate dehydrogenase (IDH) mutations in gliomas intraoperatively. Knowledge of IDH genotypes at the time of surgical resection could improve patient outcomes, as more aggressive tumor resection of IDH-mutated gliomas is associated with increased survival. While assessments of IDH genotype are typically not available until days after surgery, we have demonstrated the ability to provide this information is less than five minutes. An intraoperative DESI-MS system has successfully been used in a proof-of-concept clinical study and intraoperative performance validation of this platform is ongoing. The findings of these two studies as well as strengths, weaknesses, and areas of improvement for upcoming future iterations of the research are discussed.</p> <p><br></p> <p>Point-of-care applications necessitate the adaptation of MS methodologies to smaller devices. Miniature mass spectrometers (Mini MS) boast small footprints, simple operation, and low power consumption, noise levels, and cost, making them attractive candidates for point-of-care use. In a small-scale clinical study, we demonstrated the first application of a Mini MS for determination of IDH mutation status in gliomas intraoperatively. This study paves a path forward for the application of Mini MS in the OR. With its small footprint and low power consumption and noise level, this application of miniature mass spectrometers represents a simple and cost-effective platform for an important intraoperative measurement. </p> <p><br></p> <p>While MS-based methods of tissue analysis can detect molecular features of interest and rapidly produce large quantities of data, their inherent speed is rarely utilized because they are traditionally coupled with time-consuming separation techniques (e.g., chromatography). Ambient ionization MS, specifically DESI-MS, is well suited for high-throughput applications due to its lack of sample preparation and purification techniques. In an attempt to rapidly characterize microarrays of tissue biopsies, we developed a high-throughput DESI-MS (HT-DESI-MS) method for the rapid characterization of disease state, human brain tumor type, glioma classification, and detection of IDH mutations in tissue microarrays (TMA) of banked and fresh human brain tissue biopsies. We anticipate that HT-DESI-MS analysis of TMAs could become a standard tool for the generation of spectral libraries for sample classification, the identification of biomarkers through large-scale studies, the correlation of molecular features with anatomical features when coupled to digital pathology, and the assessment of drug efficacy. </p>