The rise of nutraceutical health formulations has increased the need for more stringent analytical testing methods. Complex matrices present a new problem when determining concentration of compounds of interest. The presented method uses LC-MS analysis with a novel sample preparation method in the determination of Beta-hydroxybutyric acid in health formulations. The use of an aqueous analytical column allows for separations of polar compounds after non-polar compounds are removed through C18 packed column filtration. The samples were analyzed through time-of-flight mass spectrometry and results show that this is an effective method for the presented samples with a range of expected concentrations of total BHB was from 11.80% to 38.92%. It was seen that all samples exhibited a less than 10% percent deviation from the expected concentrations of the nutraceutical health samples with the highest being 9.74 % for sample 9 and the lowest being sample 3 with a deviation of 0.08 % from expected values.

Liquid chromatography-mass spectrometry (LC-MS) based proteomic methods have provided archaeologists with a powerful tool for the discovery and identification of proteins within artifacts. Traditionally, discovery-based methods have utilized a non-targeted full mass scan method in an attempt to identify all proteins present within a given sample. However, increased sensitivity is often needed to target specific proteins in order to test hypotheses. Proteins present within archaeological materials present a unique challenge, as they are often subjected to a variety of chemical transformations both before and after burial. Any preserved proteins will be present within a complex mixture of compounds, and full mass scans often fail to detect less abundant proteins of interest. Consistent and reliable targeted methods are needed to detect protein biomarkers. Taphonomic experimentation was employed as a means to identify the effect of particular processes and conditions on the preservation of mare's milk proteins. In addition, three LC-MS methods were evaluated for their efficiency in identifying mare's milk-specific peptide biomarkers from experimental pottery samples. The ability to reliably detect the presence of these species-specific peptides can help provide evidence about past cultural groups, including the origins of dairying and animal domestication.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease, with a five-year survival rate of only 13%. Patients are most commonly diagnosed in late stages, once the tumor has metastasized to distal organs, limiting treatment options. For PDAC patients, metastases most often go to the liver. However, there has been limited studies profiling matching human primary tumors and liver metastases because of the difficulty in procuring such samples. To understand the system in which primary tumors spread to the liver, an omics approach is most appropriate. Proteins are the final effectors of the cell, making mass spectrometry-based proteomics an extremely useful tool in understanding the metastatic landscape of PDAC. Here, we present a novel study that offers a comprehensive proteomic characterization of matching human primary PDAC tumors and liver metastases. Using mass spectrometry-based proteomics, we were able to elucidate immune signatures on both the global proteomic and phosphoproteomic level in PDAC, as well as. In addition, this work can serve as a useful resource to the community to help generate more hypotheses and follow-up experiments. Finally, we demonstrate the versatility and potential of mass spectrometry as a tool to probe various biological systems by offering a systematic comparison of the two main mass spectrometry acquisition types used in bottom-up proteomics. We found that data-dependent acquisition (DDA) and data-independent acquisition (DIA) can produce comparable results. However, DDA, in combination with isobaric labeling, disproportionally suffers from ratio compression, and DIA’s incompatibility with isobaric labeling for multiplexing can be a big drawback when working with limited sample amounts.

Prostate cancer (PCa) is the second most common cancer in men with more than 1 million new cases worldwide each year. While some of the genomic, genetic and molecular events characterizing PCa have been functionally associated with tumor onset, development and resistance to therapy, the meaning of many other molecular alterations remains poorly understood. Recent development of organoids technology and prostate organoid cultures has established an innovative and valuable model for the study of adult tissue homeostasis, physiology and disease. In this project we combined prostate organoids technology with genetic engineering and CLICK-chemistry coupled Mass Spectrometry approaches in order to better characterize molecular features of wild type and genetically engineered mouse prostate organoids modeling early steps of human prostate tumorigenesis. In details, by manipulating mPrOs to proxy ETS-related gene (ERG) precursor PIN/HGPIN lesions of human prostate, we identified possible novel pro-tumorigenic roles of ERG which unleashes cells proliferation from the tight control of growth stimuli, and, even more interesting, corrupts immune system components to escape immune surveillance. In conclusion, this project shows that coupling innovative biological systems and technological approaches can lead to significant improvements in the analysis and understanding of disease mechanisms.

Micellar electrokinetic chromatography (MEKC), one of the major capillary electrophoresis (CE) modes, has been interfaced to mass spectrometry (MS) to provide high sensitivity and selectivity for analysis of chiral compounds. The research in this dissertation presents the development of novel polymeric glucopyranoside based molecular micelles (MoMs) (aka. polymeric surfactants) and their application in chiral MEKC-MS. Chapter 1 is a review of chiral CE-MS - in the period 2010-2015. In this chapter, the fundamental of chiral CE and CE-MS is illustrated and the recent developments of chiral selectors and their applications in chiral EKC-MS, CEC-MS and MEKC-MS are discussed in details. Chapter 2 introduces the development of a novel polymeric α-D-glucopyranoside based surfactants, n-alkyl-α-D-glucopyranoside 4,6-hydrogen phosphate, sodium salt. In this chapter, polymeric α-D-glucopyranoside-based surfactants with different chain length and head groups have been successfully synthesized, characterized and applied as compatible chiral selector in MEKC-ESI-MS/MS. or the enantioseparation of ephedrines and β-blockers. Chapter 3 continues to describe the employment of polymeric glucopyranoside based surfactants as chiral selector in MEKC-MS/MS. The polymeric β-D-glucopyranoside based surfactants, containing charged head groups such as n-alkyl β-D-glucopyranoside 4,6-hydrogen phosphate, sodium salt and n-alkyl β-D-glucopyranoside 6-hydrogen sulfate, monosodium salt were able to enantioseparate 21 cationic drugs and 8 binaphthyl atropisomers (BAIs) in MEKC-MS/MS, which promises to open up the possibility of turning an analytical technique into high throughput screening of chiral compounds. Physicochemical properties and enantioseparation capability of polymeric β-D-glucopyranoside based surfactants with different head groups and chain lengths were compared. Moreover, the comparison of polymeric α- and β-D-glucopyranoside 4,6-hydrogen phosphate, sodium salt were further explored with regard to enantioseparations of ephedrine alkaloids and b-blockers. The concept of multiplex chiral MEKC-MS for high throughput quantitation is demonstrated for the first time in scientific literature.

Mass spectrometry is a versatile analytical tool for chemical and biomolecule identification, quantitation, and structural analysis. Tandem mass spectrometry further expands the applications of mass spectrometry, making it more than a mere detector. With tandem mass spectrometry, the mass spectrometer is capable of probing reaction mechanisms, monitoring reaction processes, and performing fast analysis on complex samples. In tandem mass spectrometry, after activation the precursor ions fragment into small fragment ions through one or more pathways, which are affected by the ion’s inherit property, the ion type, and the activation method. To obtain complementary information, one can alter the fragmentation pathway by changing the ion via ion charge manipulation and covalent modification to the ion. Gas-phase ion/ion reactions provide an easy approach to changing ion type and facile modification to the analyte ions. It has been extensively used for spectrum simplification and analyte structural studies. In this dissertation, ion/ion reaction facilitated mass spectrometry methods are studied, and explorations into the method development involving front-end mass spectrometer are discussed.<br>The first work demonstrates a special rearrangement reaction for gas-phase Schiff-base-modified peptides. Gas-phase Schiff-base modification of peptides has been applied to facilitate the primary structural characterization via tandem mass spectrometry. A major or minor fragment pathway related to the novel rearrangement reaction was observed upon in-trap collisional activation of the gas-phase Schiff-base-modified peptides. The rearrangement reaction involves the imine of the Schiff base and a nucleophile present in the polypeptide. The occurrence of the rearrangement reaction is affected by several factors, such as ion polarity, identity of the nucleophile in the peptide (e.g., side chains of lysine, histidine, and arginine), and the position of the nucleophile relative to the imine. The rearrangement reaction does not affect the amount of structural information that can be obtained by collisional activation of the Schiff-base-modified peptide, but when the rearrangement reaction is dominant, it can siphon away signal from the structurally diagnostic processes.<br>Efforts have also been put into the method development of peptide and protein aggregation detection via electrospray ionization mass spectrometry (ESI-MS). People have studied peptide and protein aggregation processes to understand the mechanism of amyloid-related diseases and to control the quality of the peptide and protein pharmaceuticals. ESI-MS is suitable for solution aggregation studies because of its compatibility with solution samples and the straightforward result of the analyte’s oligomeric state on the mass spectrum. However, peak overlap issue and nonspecific aggregation in the ESI process can obscure the result. Here, the application of proton transfer ion/ion reaction to the analyte has been found useful to reduce or eliminate the peak overlap issue. A statistical model based on Poisson statistics has been proposed to deal with the ESI-induced nonspecific aggregation in the droplet and to differentiate the solution-phase aggregation from the droplet-induced aggregation. Factors that affect the accuracy of the statistical model have been discussed with MATLAB simulations.<br>In the era of biological system studies, sample complexity is a challenge every analytical chemist has to face. The analysis of complex sample can be facilitated by the combination of separation techniques outside the mass spectrometer (such as differential mobility spectrometry (DMS)) and ion structure probing techniques inside the mass spectrometer (such as tandem mass spectrometry and gas-phase ion/ion reactions). Here the coupling method between DMS and ion/ion reaction is developed and tested with model peptide systems to demonstrate its possible application in complex sample characterization such as isomer identification.<br>

Lau, Wai Hung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 141-153). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Orientador: Wagner Vilegas / Resumo: Apesar dos diversos relatos do uso de Rhipsalis Gaërtn (Cactaceae) por populações tradicionais no tratamento de uma gama de enfermidades, há pouquíssimos estudos fitoquímicos com esse grupo de cactos e por vezes estes são superficiais, apenas indicando a possível presença de algumas classes de produtos naturais. Ademais, estudos avaliando a atividade biológica de Rhipsalis são escassos. Neste viés, este trabalho objetivou investigar a composição química do gênero Rhipsalis e avaliar possíveis atividades ligadas à sua composição, subsidiando desta forma seu uso popular. Esta dissertação foi dividida e organizada da seguinte forma: a primeira parte apresenta uma introdução geral sobre plantas medicinais e sua importância, assim como uma revisão sobre estudos fitoquímicos em Cactaceae. Em seguida, foram redigidas três seções: na primeira, investigamos a composição química de Rhipsalis teres (Vell.) Steud identificando 5 saponinas derivadas do ácido oleanólico, 2 flavonoides C-glicosilados derivados da apigenina e 2 ácidos fenólicos; na segunda, investigamos a composição química do gênero Rhipsalis identificando 28 saponinas cujo core é o ácido oleanólico e 8 flavonoides, também sugerimos fingerprints para cada espécie avaliada com base nas substâncias majoritárias, os resultados quantitativos obtidos por UPLC-MS foram eficientes na identificação das espécies com base na filogenia; na terceira, avaliamos as atividades antioxidante, antifúngica e anti-inflamatória de espécies de R... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Despite the several reports of the use of Rhipsalis Gaërtn (Cactaceae) by traditional populations in the treatment of a range of diseases, there are very few phytochemical studies with this group and sometimes these are superficial, only indicating the possible presence of some classes of natural products. In addition, there are very few studies evaluating the biological activity of Rhipsalis. Therefore, this work aimed to investigate the chemical composition of the genus Rhipsalis and to evaluate possible activities related to its composition, thus subsidizing its popular use. This dissertation was divided and organized as follows: the first part presents a general introduction on medicinal plants and their importance as well as a review on phytochemical studies in Cactaceae. Then, three sections were written: in the first one, we investigated the chemical composition of Rhipsalis teres, identifying 5 saponins derived from oleanolic acid, 2 C-glycosilated flavonoids derived from apigenin and 2 phenolic acids; in the second section, we investigate the chemical composition of the genus Rhipsalis identifying 28 saponins whose core is oleanolic acid and 8 flavonoids, we also suggested fingerprints for each species evaluated on the basis of the majority compounds, the quantitative results obtained by UPLC-MS were efficient in the identification of the species based on the phylogeny; in the third, we evaluated the antioxidant, antifungal and anti-inflammatory activities of Rhipsal... (Complete abstract click electronic access below) / Mestre

A cocaína (COC) é um alcaloide presente nas folhas de espécies do gênero Erytroxylum novagranatense e Erytroxylum coca. Possui grande efeito estimulante sobre o Sistema Nervoso Central (SNC) e por isso é utilizada como droga de abuso. Dentre os principais metabólitos estão a benzoilecgonina (BZE), éster metil anidroecgonina (EMA) e anidroegonina (AEC), sendo que os dois últimos são provenientes de pirólise (consumo do crack através do fumo ou inalação). Ainda, o cocaetileno (CE) é biotransformado após a ingestão conjunta de COC e etanol. O consumo de substâncias psicoativas (SPA), dentre elas a COC e seus derivados, traz diversos prejuízos à saúde dos usuários, principalmente quando se considera a interação causada pelos compostos adulterantes, adicionados à droga para aumentar lucros e mimetizar os efeitos desejados. Ainda, o uso associado ao trânsito, é uma preocupação crescente em toda a sociedade, pois eleva em números consideráveis a chance da ocorrência de acidentes de trânsito. A detecção de SPA pode ser feita em uma variedade de matrizes biológicas, sendo que cada uma possui suas particularidades, incluindo diferentes janelas de detecção. Análises realizadas em fluido oral (FO), urina e sangue são recomendadas por guias nacionais e internacionais para a detecção de SPA e se complementam pois identificam as drogas intactas e seus produtos de biotransformação em diferentes concentrações e períodos de tempo. A correlação de concentração entre essas matrizes não é bem estabelecida, sendo que diversos estudos reportados na literatura trazem resultados controversos. A validação de métodos analíticos ou bioanalíticos é etapa crucial para a realização de análises seguras e que não deixem margem de dúvida na interpretação dos resultados, caracterizando-se como de extrema importância em todas as áreas da toxicologia. Nesse sentido, foram desenvolvidos e validados métodos para a detecção simultânea de COC, BZE, CE, EMA e AEC em FO, urina e plasma, utilizando cromatografia líquida acoplada a detector de massas (CL-EM), além de três métodos para análise de COC e compostos adulterantes em amostras de apreensão, sendo dois deles qualitativos, utilizando cromatografia gasosa acoplada à detector de massas (CG-EM) e cromatografia gasosa portátil acoplada à detector de massas íon trap (CG-EM-IT) e um quantitativo utilizando cromatografia líquida acoplada à detector de massas sequencial (CL-EM/EM). Ainda, um estudo de correlação foi aplicado a fim de avaliar a concentração dos metabólitos entre as matrizes biológicas coletadas simultaneamente. As etapas de preparação das amostras nos métodos bioanalíticos compreenderam precipitação de proteínas com acetonitrila, seguida de filtração para urina e plasma e diluição em tampão seguida de filtração para FO. As curvas de calibração foram lineares entre 4,25 e 544,00 ng/mL para FO, e entre 5,00 e 320,00 ng/mL para urina e plasma. Os limites inferiores de quantificação foram iguais à menor concentração das curvas de calibração. Os limites de precisão e exatidão intra e inter-dias mantiveram-se dentro dos limites de ±20% para o limite de quantificação e ±15% para os demais controles preconizados pelas guias regulatórias. Os métodos desenvolvidos foram aplicados satisfatoriamente em 110, 116 e 113 amostras de FO, urina e plasma, respectivamente, coletadas de usuários de múltiplas drogas. Os resultados mostraram alta prevalência destes analitos nesta população, especialmente COC e BZE, presentes em 75,8 e 75,0% dos casos, respectivamente. Em relação ao método analítico quantitativo, este se mostrou preciso, exato e linear na faixa de 50 – 2000 ng/mL, todos os demais parâmetros se mantiveram dentro dos limites preconizados. Levamisol (LEV), fenacetina (FEN), cafeína (CAF), hidroxizina (HDZ) e benzocaína (BZC) foram os adulterantes mais encontrados dentre as 166 amostras de apreensão analisadas. O método qualitativo por CG-EM foi efetivo para ser utilizado como método de screening para todos os compostos, equanto que o método por CG-EM-IT apresentou restrições de aplicação para alguns compostos. Os capítulos apresentados nesse trabalho abrangem de uma maneira geral a análise de COC e seus derivados em amostras biológicas e material apreendido, assim como trazem uma abordagem em relação aos problemas relacionados ao uso e tráfico de drogas em diferentes cenários. Os métodos desenvolvidos neste trabalho podem ser aplicados, além da área de trânsito e controle de materias apreendidos, em diferentes áreas de conhecimento como laboratórios de toxicologia clínica, forense e no próprio meio científico. / Cocaine (COC) is an alkaloid which is found in the leaves of Erytroxylum novagranaense and Erytroxylum coca. COC has a strong stimulant effect on central nervous system (CNS) and this is the reason for its classification as drug of abuse. Benzoylecgonine (BZE), anhydroecgonine methyl ester (AEME) and anhydroecgonine (AEC) are among its main metabolites. AEME and AEC are pyrolysis products that forms as a result of crack-cocaine consumption through smoking or inhalation. Additionally, cocaethylene (CE) is biotransformed after the ingestion of cocaine and alcohol. The use of psychoactive substances (SP), including cocaine and its derivatives, can cause several issues to the health of its users specially when the interactions caused by cutting agents added to the drugs to increase the profits and mimic the effects are taken into consideration. Furthermore, the use of SP combined with driving is an emerging problem since it increases the chances of traffic accidents. The detection of SP can be done in a variety of biological matrices that each has their own particularities such as different windows of detection. The guidelines recommend analysis in oral fluid (OF), urine, and blood for the detection of SP. These three matrices complement each other because it is possible to identify drugs and the products of biotransformation in varying concentrations and periods of time. The coefficient of correlation between those three matrices is not very well established since many studies report conflicting results. The validation of analytical or bioanalytical methods is an important step to ensure accurate results and it is considered essential in every area of toxicology. Therefore, methods for the detection of COC, BZE, AEME, AEC and CE in OF, urine and plasma were developed and validated using liquid chromatography coupled to mass spectrometry (LC-MS). Likewise, three methods (two qualitative and one quantitative) for the detection of COC and known cutting agents in seized drugs were developed using gas chromatography coupled to mass spectrometry (GC-MS), portable gas chromatography toroidal ion trap mass spectrometry (GC-TMS), and liquid chromatography tandem mass spectrometry (LC-MS/MS). A correlation study was done to evaluate the drug concentrations in the three matrices collected simultaneously. OF was diluted in buffer while urine and plasma were precipitated using acetonitrile. Calibration curve ranges were prepared at 4.25 – 544 ng/mL for oral fluid, and 5 – 320 ng/mL for urine and plasma. The lowest concentration of the calibration curves were designated as the lower limit of quantification. The calculated precision and accuracy values were within the limits stipulated by the guidelines (±20% for the limit of quantification and ±15% for the rest of the quality controls). The methods were fully validated and proved to be suitable for analysis of 110, 116, and 113 samples of OF, urine, and plasma, respectively, that were collected from drug users. The results showed high prevalence of SP drugs in this population. Particularly of note, COC and BZE were found in 75.8 and 75.0% of the cases, respectively. The analytical quantitative method proved to be precise, accurate, and linear in the range of 50 – 2000 ng/mL; all the other parameters were within the limits stipulated. Levamisole (LEV), phenacetin (PHN), caffeine (CAF), hydroxyzine (HYDZ), and benzocaine (BZC) were the adulterants most prevalent in the 166 samples analyzed. The analytical qualitative method by GC-MS was shown to be effective as screening method for all the compounds, while the analytical method by GC-TMS was limited due to its incompatibility with certain target compounds. The chapters presented in this work comprise the analysis of cocaine and its derivatives in biological matrices and seized material, as well as an approach relating to the analysis of samples from impaired driving and similar scenarios. The methods developed in this work can be further applied to other areas of science and research including clinical toxicology, general laboratories and forensic laboratories.

<div>Amongst the various classes of mass analyzers, the quadrupole ion trap (QIT) is by far the most versatile. Although it can achieve only modest resolution (unit) and mass accuracy (101-102 ppm), it has high sensitivity and selectivity, can operate at pressures exceeding 10-3 torr, is tolerant to various electrode imperfections, and has single analyzer tandem mass spectrometry (MS/MS) capabilities in the form of product ion scans. These characteristics make the QIT ideal for mass spectrometer miniaturization, as most of the fundamental performance metrics of the QIT do not depend on device size. As such, the current drive in miniature systems is to adopt miniature ion traps in various forms – 3D, linear, toroidal, rectilinear, cylindrical, arrays, etc.</div><div><br></div><div>Despite being one of the two common mass analyzers with inherent MS/MS capabilities (the other being the Fourier transform ion cyclotron resonance mass spectrometer), it is commonly accepted that the QIT cannot perform one-dimensional precursor ion scans and neutral loss scans - the other two main MS/MS scan modes - or two-dimensional MS/MS scans. The former two are usually conducted in triple quadrupole instruments in which a first and third quadrupole are used to mass select precursor and product ions while fragmentation occurs in an intermediate collision cell. The third scan can be accomplished by acquiring a product ion scan of every precursor ion, thus revealing the entire 2D MS/MS data domain (precursor ion m/z vs. product ion m/z). This, however, is not one scan but a set of scans. Because the ion trap is a tandem-in-time instrument rather than a tandem-in-space analyzer, precursor ion scans, neutral loss scans, and 2D MS/MS are, at best, difficult.</div><div><br></div><div>Yet miniature mass spectrometers utilizing quadrupole ion traps for mass analysis would perhaps benefit the most from precursor scans, neutral loss scans, and 2D MS/MS because they generally have acquisition rates (# scans/s) an order of magnitude lower than their benchtop counterparts. This is because they usually use a discontinuous atmospheric pressure interface (DAPI) to reduce the gas load on the backing pumps, resulting in a ~1 scan/s acquisition rate and making the commonly-used data-dependent acquisition method (i.e. obtaining a product ion scan for every abundant precursor ion) inefficient in terms of sample consumption, time, and instrument power. Precursor and neutral loss scans targeting specific molecular functionality of interest - as well as 2D MS/MS – are more efficient ways of moving through the MS/MS data domain and thus pair quite readily with miniature ion traps.</div><div><br></div><div>Herein we demonstrate that precursor ion scans, neutral loss scans, and 2D MS/MS are all possible in a linear quadrupole ion trap operated in the orthogonal double resonance mode on both benchtop and portable mass spectrometers. Through application of multiple resonance frequencies matching the secular frequencies of precursor and/or product ions of interest, we show that precursor ions can be fragmented mass-selectively and product ions ejected simultaneously, preserving their relationship, precursor ion -> product ion + neutral, in the time domain and hence allowing the correlation between precursor and product ions without prior isolation. By fixing or scanning the resonance frequencies corresponding to the targeted precursor and product ions, a precursor ion scan or neutral loss scan can be conducted in a single mass analyzer. We further show that 2D MS/MS - acquisition of all precursor ion m/z values and a product ion mass spectrum for every precursor ion, all in a single scan - is possible using similar methodology. These scan modes are particularly valuable for origin-of-life and forensic applications for which the value of miniature mass spectrometers is readily evident.</div>