The main aim of this work was to prepare and characterize a new chiral stationary phase (CSP). The CSP was prepared by dynamic coating of sulfobutylether- β-cyclodextrin (SBE-β-CD) on a strong anion-exchange stationary phase (SP). The selectivity and stability of the newly prepared CSP were tested on the sets of chiral and achiral analytes. The next separation system used was composed of C18 SP with the addition of SBE-β-CD as a chiral selector into the mobile phase. The set of chiral analytes contained amino acids phenylalanin, tyrosine, tryptophan, their derivatives and dipeptides glycine-DL-tryptophan and glycine-DL- phenylalanin. Four groups of mixtures of blocked and unblocked dipeptide isomers were tested for achiral separations. Dipeptides used for the mixtures had the same molecular formula but the sequence of amino acids was reversed. Measurements were carried out in reversed phase separation mode and hydrophilic interaction liquid chromatography. Mobile phases composed of methanol as an organic modifier and four different aqueous parts: (i) deionized water, (ii) aqueous solution of formic acid (pH 2.10), (iii) 20mM ammonium acetate buffer (pH 4.70) and (iv) 10mM ammonium acetate buffer (pH 8.80) in various volume ratios. Newly prepared SBE-β-CD CSP was more suitable for separations of...

The aim of this diploma thesis was to prepare two new chiral stationary phases by dynamic coating of sulphobutylether-β-cyclodextrin (SBE-β-CD) with varying degrees of substitution onto strong anion-exchange stationary phases. The enantioselective potential and stability of the newly prepared chiral stationary phases were tested using a set of chiral analytes. The set contained structurally diverse analytes, i.e. benzodiazepines (oxazepam, lorazepam), phenothiazines (thioridazine, promethazine), β-blockers (labetalol, pindolol, propranolol, alprenolol), profens (carprofen, fenoprofen, flurbiprofen, indoprofen), flavanones (6-hydroxyflavanone, 7-hydroxyflavanone), DL-tryptophan and its derivatives (5-OH-DL-tryptophan, 5-F-DL-tryptophan, DL-tryptophan butylester and blocked aminoacid (t-Boc-DL-tryptophan)), dipeptides (glycyl-DL-phenylalanine, glycyl-DL-tryptophan) and Troger's base. Measurements were carried out in reversed-phase high-performance liquid chromatography. Mobile phases consisted of methanol/formic acid (pH 2.10) and methanol/10mmol l-1 ammonium acetate buffer (pH 4.00) in various volume ratios. The chiral stationary phase containing hexasubstituted SBE-β-CD was suitable for enantioseparation of eleven analytes. Four of them were baseline enantioresolved and seven partially. The chiral...

- 4 - Abstract in English This diploma thesis is focused on the evaluation and comparison of the enantioselective potential of two columns CHIRAL ART Amylose-SA and CDShell-RSP using a set of 29 chiral drugs in high performance liquid chromatography. The separations of enantiomers were performed in three modes: reversed-phase, normal-phase, and polar- organic mode. The CHIRAL ART Amylose-SA column was tested in normal-phase mode, the CDShell-RSP column in reversed-phase and polar-organic modes. The CHIRAL ART Amylose-SA column contains amylose tris(3,5- dimethylphenylcarbamate) immobilized on 3 µm porous silica gel particles. The CDShell-RSP column contains a chiral selector hydroxypropyl-β-cyclodextrin, which is covalently bonded on 2.7 µm superficially porous particles. In the normal-phase mode, mobile phases composed of hexane and propane-2-ol were used. Furthermore, the effect of various additives (triethylamine, diethylamine, trifluoroacetic acid, and the mixture of diethylamine and trifluoroacetic acid) in the mobile phase on the enantioseparation of chiral drugs was tested. The most universal additive was a mixture of diethylamine and trifluoroacetic acid. A total of 22 chiral drugs were enantioseparated on the CHIRAL ART Amylose-SA column, 10 of them were baseline separated. Mobile phases for...

OBJECTIVE: Methylphenidate is commonly used in the treatment of Attention Deficit Hyperactivity Disorder and narcolepsy. Methylphenidate is administered as a racemic mixture of the d- and l-threo enantiomers; however, the d-enantiomer is primarily responsible for the pharmacologic activity. Previous studies of the behavioral effects of methylphenidate have highlighted sex differences in the responsiveness to the drug, namely an increased sensitivity of females to its stimulatory effects. These differences may be due to differences in the uptake, distribution, and elimination of methylphenidate from male and female brains. Therefore, we compared the pharmacokinetics of d- and l-threo methylphenidate in the brains of male and female rats. MATERIALS AND METHODS: Adult male and female Sprague-Dawley rats were injected with 5 mg/kg d, l-threo methylphenidate, and whole brains were collected at various time points following injection. We measured methylphenidate concentrations utilizing chiral high pressure liquid chromatography followed by mass spectrometry. RESULTS: Females exhibited consistently higher brain concentrations of both d- and lmethylphenidate and a slower clearance of methylphenidate from brain as compared to males, particularly with the active d-enantiomer. CONCLUSIONS: The increased sensitivity of females to methylphenidate may be partially explained by an increase in total brain exposure to the drug

The primary focus of this research is to utilize cellular and polymeric membranes for biomedical applications: To date, several organic and inorganic materials have been used to synthesize nanoparticles (NPs). The question arises as to which criteria and design principles should be considered while selecting the best material. Based on the results of testing, three key roles of NPs have been identified. First, NPs need enough circulation time to reach their target. Then these NPs must be able to target diseased tissue while leaving healthy tissue unaffected. Finally, NPs must be biodegradable and easily eliminated from the body. Biomimetic nanoparticles based on cell membranes have been developed as an efficient way to fulfill the needs of drug delivery goals and achieve targeted delivery by actively interacting and communicating with the biological environment. In the first project, genome editing machinery was delivered to particular cells using biomimetic cancer cell coated zeolitic imidazolate frameworks. MCF-7 cells demonstrated the highest uptake of C3-ZIFMCF compared to HeLa, HDFn, and aTC cells. In terms of genome editing, MCF-7 cells transfected with C3-ZIFMCF showed 3-fold EGFP repression compared to C3-ZIFHELA cells transfected with 1-fold EGFP repression. In vivo tests demonstrated C3-ZIFMCF's affinity for MCF-7 tumor cells. This demonstrates the biomimetic approach's ability to target cells specifically, which is definitely the most essential step in future genome editing technology translation. In the second project, multimodal therapeutic nanowires (NWs D-ZIF) MCF-7 cancer cells were developed. D-ZIF coated NWs had higher cellular uptake and photothermal treatment efficiency than non-coated NWs. (NWs D-ZIF) MCF accumulates in MCF-7 tumor cells and enhances photothermal capability. On the other hand, chiral separation of enantiomers is becoming more important, particularly in pharmaceuticals. Because enzyme activities and other biological processes are stereoselective, chiral drugs' enantiomers often have different metabolic effects, pharmacological activity, metabolic rates, and toxicities. In an attempt to address this issue, we decided in the final project to study the capability of chiral polyamide membrane for efficient and energy-free chiral separation. In particular, to separate essential amino acid critical to all living organisms (DL-tryptophan).

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This dissertation describes the simulation and optimization of adsorptive and chromatographic separation processes. The first part focus on the simulation and comparison of operational modes in simulated moving bed (SMB) chromatography for separation and purification in bioprocesses. The second part includes the simulation of gas-phase adsorptive processes by pressure swing adsorption and temperature swing adsorption technologies. The applications of SMB chromatography are popular in separating and purifying enantiomers, petrochemicals, pharmaceuticals and biochemicals with higher yield and lower solvent consumption. We simulate and compare several operational modes of simulated moving bed (SMB) for a binary and a ternary bioprocess using Aspen Chromatography. These operational modes are able to improve the separation efficiency of the basic SMB process by our simulation and optimization. We compare their separation performances and identify heuristics that will guide the selection of operational modes across a variety of systems. Pressure swing adsorption (PSA) and temperature swing adsorption (TSA) are two of the main technologies for gas-phase adsorption separation processes. We simulate and demonstrate a PSA model for air separation system and a TSA model for CO2 capture system in Aspen Adsorption. We present their separation performance plots to provide the physical insights of these two systems. / Ph. D.

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.

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.

A duloxetina é um potente inibidor duplo da recaptação de serotonina e norepinefrina, disponível como enantiômero puro, sob a forma S-duloxetina e comercializado como pellets em cápsulas. O R enantiômero da duloxetina é também um inibidor da recaptação, no entanto, este é menos potente que seu isômero, sendo considerado como impureza enantiomérica. Este trabalho teve como objetivos o desenvolvimento e a validação de métodos analíticos para o controle de qualidade da duloxetina e de sua respectiva impureza enantiomérica por cromatografia líquida de alta eficiência (CLAE), e por eletroforese capilar (EC). A resolução dos enantiômeros da duloxetina foi realizada a partir da utilização de fase estacionária quiral, baseada celulose, por CLAE. A separação por EC foi desenvolvida a partir da utilização de hidroxipropil-β-ciclodextrina (HPβCD) como seletor quiral. A validação dos métodos foi efetuada de acordo com os guias de validação disponíveis na literatura e os métodos propostos foram considerados específicos, lineares, precisos, exatos e robustos. A análise comparativa entre os métodos desenvolvidos demonstrou não haver diferença estatisticamente significativa na quantificação do enantiômero S-duloxetina. / Duloxetine is a double potent inhibitor of serotonin and norepinephrine reuptake, available as a pure enantiomer, in the S-duloxetine form and marketed as pellets into capsules. The R enantiomer of duloxetine is also an inhibitor of reuptake, however, less potent and being considered enantiomeric impurity. This work aimed the development and validation of analytical methods for quality control of duloxetine and its respective enantiomeric impurity by high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). The resolution of the enantiomers of duloxetine was performed with the use of chiral stationary phase, based on cellulose, by HPLC. The separation by CE was developed with the use of hydroxypropyl-β-cyclodextrin (HPβCD) as chiral selector. The method validation was performed according to the guides available in the literature and the proposed methods were considered specific, linear, precise, accurate and robust. The comparative analysis between the methods developed showed no statistically significant difference in the quantification of S-duloxetine enantiomer.