Capillary electrochromatography (CEC) is a major capillary electrophoresis (CE) mode that have been interfaced to mass spectrometry (MS) for sensitive and selective analysis of chiral compounds. This research expands CEC applications in cancer biomarker and chiral CE analysis. Chapter 1 is a review of liquid chromatography-mass spectrometry (LC/MS), gas chromatography-mass spectrometry (GC/MS), and capillary electrophoresis mass spectrometry (CE/MS) for analysis of metabolites in prostate cancer diagnostics and therapies. In this chapter, a literature survey was performed within the databases PubMed, 4 Caplus/Webline and Web of Sciences. A total 17 studies reporting on various analytical platforms for metabolite identification in prostate cancer research, which often include case-control comparison were identified and reviewed. Chapter 2 described the analysis of metabolite biomarkers in prostate cancer tissues by capillary electrochromatography mass spectrometry. In this chapter, a capillary CEC–MS/MS method was developed for the simultaneous determination and separation of eight proofs of concept (POC) metabolites (betaine, malate, proline, N-acetyl aspartate, N-acetylglucosamine, uracil, xanthine, and alanine) as potential prostate cancer diagnostic markers. A polymeric monolith column with a hydrophilic crosslinker and strong anion-exchange mixed-mode has been fabricated by an in situ copolymerization of vinyl benzyl trimethylammonium chloride, and bisphenol A glycerolate dimethacrylate (BisGMA) in the presence of methanol and dodecyl alcohol as porogens and AIBN as initiator. After CEC separation, samples were analyzed by a triple–quadrupole mass spectrometer operated in positive ion mode. After optimization, the data showed that the CEC-MS/MS method using monolithic column achieved a much better chromatographic selectivity compared to coated columns and increased sensitivity than bare fused silica column The effect of mobile phase pH, ACN percentage and additive were studies. Under the optimum mobile phase conditions, this method was carried out to separate and detect eight metabolites in the biopsy sample. The LOD for the metabolites is between 50nM-100nM. This method has successfully used to examine patients’ prostate cancer with an accuracy of 95%. Chapter 3 demonstrates Insights into Chiral Recognition Mechanisms in CEC using linear salvation energy relationship. By varying the linker (amide and carbamate), head group (alanine, leucine, and valine) and chain length (C8, C10 and C12) of the amino acid bound surfactants; monolithic column was made to ultimately understand the factors governing chiral stationary solid phase. Through the comparison of system parameters, we can see that surfactant head group, linker and chain length affect the separation of achiral and chiral compounds. Also, with the same type surfactant, data was presented to show how the trend of LSER parameters and how it affects separation between in CEC. This study showed the predictive capability of LSER to understand the aforementioned intermolecular processes controlling retention and by doing so, be able to quantitatively predict the experimental conditions to achieve an acceptable chiral separation.

Analysis of biological samples can be a difficult task. This thesis covers a broad aspect of the analytical areas of capillary electrophoresis (CE) and capillary electrochromatography (CEC) in combination with mass spectrometry (MS) that are of great importance for achieving fast, accurate and sensitive bioanalyses. A significantly time reduced and automated system for sample cleanup was developed to greatly simplify the pretreatment process of biological samples with a complex matrix. Desalting and preconcentration of species in urine was conducted and the limit of detection for the antidepressant escitalopram was lowered 10 times. This extraction devise was also successfully incorporated in a chip based platform for the possibility to be a part of multidimensional separation systems. The reduced risk of sample loss leads to improved detection limits, which are usually one the most challenging parts when working with bioanalyses. In the area of separation, a monomer surface with tailored hydrophobicity was developed to achieve rapid, high efficient separations of complex mixtures. Within five minutes a tryptic digest of a protein could be separated and then identified by a Mascot search. The applications addressed have been focused on medical conditions which are of highest interest for both physicians and patients. A high throughput analysis of the kynurenine metabolites with CE-MS offers a new method to rapidly examine samples from patients with neurological disorders. A screening study of possible biomarkers for the two different types of appendicitis, gangraenous and phlegmonous was conducted. Indicative patterns were found for both pre and post surgery of the two types of inflammation as well as between them. The divergences were traced back to the MS peaks obtained in the CE- and CEC-MS setups as possible biomarkers for the two forms of appendicitis. A preliminary study of polycystic ovary syndrome also offered some valuable results for future biomarker identification.

The research presented in this dissertation involves the development of chiral and achiral analysis using capillary electrochromatography (CEC) and CEC coupled to mass spectrometry (CEC-MS). Chapter 1 briefly reviews CEC fundamentals and latest development on chiral CEC and CEC-MS. The CEC-UV enantioseparations for several acidic compounds are described in Chapter 2. The optimum resolutions for these acidic enantiomers are achieved in ion-suppression mode, i.e. with an acidic mobile phase. One of major drawback in coupling CEC with MS is the bubble formation at the column outlet end, resulting in irreproducible retention time and erratic baseline, or even current breakdown. By introducing internal tapered columns, the aforementioned limitations of CEC-MS are successfully overcome in Chapter 3. The CEC-MS enantioseparation of warfarin and coumachlor is carefully investigated and applied to quantify R- and S-warfarin in human plasma. For individual enantiomers, a concentration of 25 ng/mL is detectable. To further improve the robustness of CEC-MS column, a new procedure of fabricating internal tapered columns is reported in Chapter 4. These internal tapered columns demonstrate excellent ruggedness, low background noise, and good compatibility in reversed-phase and polar organic modes of CEC-MS. In chapter 5, the feasibility of using internal tapered columns packed with vancomycin chiral stationary phase (CSP) is explored for simultaneous enantioseparation of eight â-blockers using CEC-MS. After a careful optimization of the mobile phase composition, sheath liquid and spray chamber parameter, 15 out of 16 enantiomers could be simultaneously resolved with excellent efficiency and detection sensitivity. The synthesis and characterization of sulfated and sulfonated cellulose phenylcarbamate CSPs is described in Chapter 6. The use of these CSPs, especially the sulfonated one, significantly enhances the EOF profile and sample throughput but maintain high enantiomeric resolving power under various modes of CEC and CEC-MS. By combining CEC and atmospheric pressure photo-ionization (APPI) MS, Chapter 7 demonstrates the separation and detection of mono-methylated benzo[a]pyrene (MBAP) isomers with ~100 times enhancement on detection sensitivity than CEC-UV. In Appedix 2, monolithic columns are synthesized through photopolymerized sol-gel approach and utilized for CEC and CEC-APPI-MS of polyaromatic hydrocarbons, and alkyl phenyl ketones.

Capillary electrophoresis (CE) is a modern analytical technique, which is electrokinetic separation generated by high voltage and taken place inside the small capillaries. In this dissertation, several advanced capillary electrophoresis methods are presented using different approaches of CE and UV and mass spectrometry are utilized as the detection methods. Capillary electrochromatography (CEC), as one of the CE modes, is a recent developed technique which is a hybrid of capillary electrophoresis and high performance liquid chromatography (HPLC). Capillary electrochromatography exhibits advantages of both techniques. In Chapter 2, monolithic capillary column are fabricated using in situ photoinitiation polymerization method. The column was then applied for the separation of six antidepressant compounds. Meanwhile, a simple chiral separation method is developed and presented in Chapter 3. Beta cycodextrin was utilized to achieve the goal of chiral separation. Not only twelve cathinone analytes were separated, but also isomers of several analytes were enantiomerically separated. To better understand the molecular information on the analytes, the TOF-MS system was coupled with the CE. A sheath liquid and a partial filling technique (PFT) were employed to reduce the contamination of MS ionization source. Accurate molecular information was obtained. It is necessary to propose, develop, and optimize new techniques that are suitable for trace-level analysis of samples in forensic, pharmaceutical, and environmental applications. Capillary electrophoresis (CE) was selected for this task, as it requires lower amounts of samples, it simplifies sample preparation, and it has the flexibility to perform separations of neutral and charged molecules as well as enantiomers. Overall, the study demonstrates the versatility of capillary electrophoresis methods in forensic, pharmaceutical, and environmental applications.