Rapid separation of biologically important low molecular weight compounds by microchip electrophoresis and ultra-fast performance liquid chromatography

The research work in this thesis reports rapid separation of biologically important low molecular weight compounds by microchip electrophoresis and ultrahigh liquid chromatography. Chapter 1 introduces the theory and principles behind capillary electrophoresis separation. An overview of the history, different modes and detection techniques coupled to CE is provided. The advantages of microchip electrophoresis are highlighted. Some aspects of metal complex analysis by capillary electrophoresis are described. Finally, the theory and different modes of the liquid chromatography technology are presented. Chapter 2 outlines the development of a method for the capillary electrophoresis of (R, S) Naproxen. Variable parameters of the separation were optimized (i.e. buffer concentration and pH, concentration of chiral selector additives, applied voltage and injection condition).The method was validated in terms of linearity, precision, and LOD. The optimized method was then transferred to a microchip electrophoresis system. Two different types of injection i.e. gated and pinched, were investigated. This microchip method represents the fastest reported chiral separation of Naproxen to date. Chapter 3 reports ultra-fast separation of aromatic amino acid by capillary electrophoresis using the short-end technique. Variable parameters of the separation were optimized and validated. The optimized method was then transferred to a microchip electrophoresis system where the separation time was further reduced. Chapter 4 outlines the use of microchip electrophoresis as an efficient tool for analysis of aluminium complexes. A 2.5 cm channel with linear imaging UV detection was used to separate and detect aluminium-dopamine complex and free dopamine. For the first time, a baseline, separation of aluminium dopamine was achieved on a 15 seconds timescale. Chapter 5 investigates a rapid, ultra-sensitive and highly efficient method for quantification of histamine in human psoriatic plaques using microdialysis and ultrahigh performance liquid chromatography with fluorescence detection. The method utilized a sub-two-micron packed C18 stationary phase. A fluorescent reagent, 4-(1-pyrene) butyric acid N-hydroxysuccinimide ester was conjugated to the primary and secondary amino moieties of histamine. The dipyrene-labeled histamine in human urine was also investigated by ultrahigh pressure liquid chromatography using a C18 column with 1.8 μm particle diameter. These methods represent one of the fastest reported separations to date of histamine using fluorescence detection.

Effect of roasting on the antioxidant activity of coffee brews

Colombian Arabica coffee beans were roasted to give light, medium, and dark samples. Their aqueous extracts were analyzed by gel filtration chromatography, UV-visible spectrophotometry, capillary electrophoresis, and the ABTS(.+) assay. A progressive decrease in antioxidant activity (associated mainly with chlorogenic acids in the green beans) with degree of roasting was observed with the simultaneous generation of high (HMM) and low molecular mass (LMM) compounds possessing antioxidant activity. Maximum antioxidant activity was observed for the medium-roasted coffee; the dark coffee had a lower antioxidant activity despite the increase in color. Analysis of the gel filtration chromatography fractions showed that the LMM fraction made a greater contribution to total antioxidant activity than the HMM components.

Structuring reductive media containing protic ionic liquids and their application to the formation of metallic nanoparticles

Herein, we present a facile method for the formation of monodispersed metal nanoparticles (NPs) at room temperature from M^(III)Cl₃ (with M = Au, Ru, Mn, Fe or V) in different media based on N,N-dimethylformamide (DMF) or water solutions containing a protic ionic liquid (PIL), namely the octylammonium formate (denoted OAF) or the bis(2-ethyl-hexyl)ammonium formate (denoted BEHAF). These two PILs present different structures and redox-active structuring properties that influence their interactions with selected molecular compounds (DMF or water), as well as the shape and the size of formed metal NPs in these solutions. Herein, the physical properties, such as the thermal, transport and micellar properties, of investigated PIL solutions were firstly investigated in order to understand the relation between PILs structure and their properties in solutions with DMF or water. The formation of metal NPs in these solutions was then characterized by using UV–vis spectroscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and dynamic light scattering (DLS) measurements. From our investigations, it appears that the PILs structure and their aggregation pathways in selected solvents affect strongly the formation, growths, the shape and the size of metal NPs. In fact by using this approach, the shape-/size-controlled metal NPs can be generated under mild condition. This approach suggests also a wealth of potential for these designer nanomaterials within the biomedical, materials, and catalysis communities by using designer and safer media based on PILs.

Tunable gold nanoparticles shape and size in reductive and structuring media containing protic ionic liquids

Herein, a facile method was developed for preparing high concentration of monodispersed gold nanoparticles (NPs) at room temperature from gold(III) chloride by using different media based on N,N-dimethylformamide or water solutions containing a protic ionic liquid (PIL), namely, the octylammonium formate or the bis(2-ethyl-hexyl)ammonium formate, based on which both PILs were used as redox-active structuring media. The formation of gold NPs in these systems was then characterized using UV-visible spectroscopy, transmission electron microscopy, and dynamic light scattering. From these investigations, it appears that the structure and aggregation pathway of PILs in selected solvents affect strongly the formation, growth, the shape, and the size of gold NPs. In fact, by using this approach, the shape-/ size-controlled gold NPs (branched and spherical) can be generated under mild condition. This approach suggests also a wealth of potential for these designer nanomaterials within the biomedical, materials, and catalysis communities by using designer and safer media based on PILs.

L’étude des stratégies de séparations préparatrices de protéines par électrophorèse capillaire

La protéomique est un sujet d'intérêt puisque l'étude des fonctions et des structures de protéines est essentiel à la compréhension du fonctionnement d'un organisme donné. Ce projet se situe dans la catégorie des études structurales, ou plus précisément, la séquence primaire en acides aminés pour l’identification d’une protéine. La détermination des protéines commence par l'extraction d'un mélange protéique issu d'un tissu ou d'un fluide biologique pouvant contenir plus de 1000 protéines différentes. Ensuite, des techniques analytiques comme l’électrophorèse en gel polyacrylamide en deux dimensions (2D-SDS-PAGE), qui visent à séparer ce mélange en fonction du point isoélectrique et de la masse molaire des protéines, sont utilisées pour isoler les protéines et pour permettre leur identification par chromatographie liquide and spectrométrie de masse (MS), typiquement. Ce projet s'inspire de ce processus et propose que l'étape de fractionnement de l'extrait protéique avec la 2D-SDS-PAGE soit remplacé ou supporté par de multiples fractionnements en parallèle par électrophorèse capillaire (CE) quasi-multidimensionnelle. Les fractions obtenues, contenant une protéine seule ou un mélange de protéines moins complexe que l’extrait du départ, pourraient ensuite être soumises à des identifications de protéines par cartographie peptidique et cartographie protéique à l’aide des techniques de séparations analytiques et de la MS. Pour obtenir la carte peptidique d'un échantillon, il est nécessaire de procéder à la protéolyse enzymatique ou chimique des protéines purifiées et de séparer les fragments peptidiques issus de cette digestion. Les cartes peptidiques ainsi générées peuvent ensuite être comparées à des échantillons témoins ou les masses exactes des peptides enzymatiques sont soumises à des moteurs de recherche comme MASCOT™, ce qui permet l’identification des protéines en interrogeant les bases de données génomiques. Les avantages exploitables de la CE, par rapport à la 2D-SDS-PAGE, sont sa haute efficacité de séparation, sa rapidité d'analyse et sa facilité d'automatisation. L’un des défis à surmonter est la faible quantité de masse de protéines disponible après analyses en CE, due partiellement à l'adsorption des protéines sur la paroi du capillaire, mais due majoritairement au faible volume d'échantillon en CE. Pour augmenter ce volume, un capillaire de 75 µm était utilisé. Aussi, le volume de la fraction collectée était diminué de 1000 à 100 µL et les fractions étaient accumulées 10 fois; c’est-à-dire que 10 produits de séparations étaient contenu dans chaque fraction. D'un autre côté, l'adsorption de protéines se traduit par la variation de l'aire d'un pic et du temps de migration d'une protéine donnée ce qui influence la reproductibilité de la séparation, un aspect très important puisque 10 séparations cumulatives sont nécessaires pour la collecte de fractions. De nombreuses approches existent pour diminuer ce problème (e.g. les extrêmes de pH de l’électrolyte de fond, les revêtements dynamique ou permanent du capillaire, etc.), mais dans ce mémoire, les études de revêtement portaient sur le bromure de N,N-didodecyl-N,N-dimethylammonium (DDAB), un surfactant qui forme un revêtement semi-permanent sur la paroi du capillaire. La grande majorité du mémoire visait à obtenir une séparation reproductible d'un mélange protéique standard préparé en laboratoire (contenant l’albumine de sérum de bovin, l'anhydrase carbonique, l’α-lactalbumine et la β-lactoglobulin) par CE avec le revêtement DDAB. Les études portées sur le revêtement montraient qu'il était nécessaire de régénérer le revêtement entre chaque injection du mélange de protéines dans les conditions étudiées : la collecte de 5 fractions de 6 min chacune à travers une séparation de 30 min, suivant le processus de régénération du DDAB, et tout ça répété 10 fois. Cependant, l’analyse en CE-UV et en HPLC-MS des fractions collectées ne montraient pas les protéines attendues puisqu'elles semblaient être en-dessous de la limite de détection. De plus, une analyse en MS montrait que le DDAB s’accumule dans les fractions collectées dû à sa désorption de la paroi du capillaire. Pour confirmer que les efforts pour recueillir une quantité de masse de protéine étaient suffisants, la méthode de CE avec détection par fluorescence induite par laser (CE-LIF) était utilisée pour séparer et collecter la protéine, albumine marquée de fluorescéine isothiocyanate (FITC), sans l'utilisation du revêtement DDAB. Ces analyses montraient que l'albumine-FITC était, en fait, présente dans la fraction collecté. La cartographie peptidique a été ensuite réalisée avec succès en employant l’enzyme chymotrypsine pour la digestion et CE-LIF pour obtenir la carte peptidique.

Anionic metabolic profiling of urine from antibiotic-treated rats by capillary electrophoresis–mass spectrometry

A recently developed capillary electrophoresis (CE)-negative-ionisation mass spectrometry (MS) method was used to profile anionic metabolites in a microbial-host co-metabolism study. Urine samples from rats receiving antibiotics (penicillin G and streptomycin sulfate) for 0, 4, or 8 days were analysed. A quality control sample was measured repeatedly to monitor the performance of the applied CE-MS method. After peak alignment, relative standard deviations (RSDs) for migration time of five representative compounds were below 0.4 %, whereas RSDs for peak area were 7.9–13.5 %. Using univariate and principal component analysis of obtained urinary metabolic profiles, groups of rats receiving different antibiotic treatment could be distinguished based on 17 discriminatory compounds, of which 15 were downregulated and 2 were upregulated upon treatment. Eleven compounds remained down- or upregulated after discontinuation of the antibiotics administration, whereas a recovery effect was observed for others. Based on accurate mass, nine compounds were putatively identified; these included the microbial-mammalian co-metabolites hippuric acid and indoxyl sulfate. Some discriminatory compounds were also observed by other analytical techniques, but CE-MS uniquely revealed ten metabolites modulated by antibiotic exposure, including aconitic acid and an oxocholic acid. This clearly demonstrates the added value of CE-MS for nontargeted profiling of small anionic metabolites in biological samples.

Versatile microanalytical system with porous polypropylene capillary membrane for calibration gas generation and trace gaseous pollutants sampling applied to the analysis of formaldehyde, formic acid, acetic acid and ammonia in outdoor air

The analytical determination of atmospheric pollutants still presents challenges due to the low-level concentrations (frequently in the mu g m(-3) range) and their variations with sampling site and time In this work a capillary membrane diffusion scrubber (CMDS) was scaled down to match with capillary electrophoresis (CE) a quick separation technique that requires nothing more than some nanoliters of sample and when combined with capacitively coupled contactless conductometric detection (C(4)D) is particularly favorable for ionic species that do not absorb in the UV-vis region like the target analytes formaldehyde formic acid acetic acid and ammonium The CMDS was coaxially assembled inside a PTFE tube and fed with acceptor phase (deionized water for species with a high Henry s constant such as formaldehyde and carboxylic acids or acidic solution for ammonia sampling with equilibrium displacement to the non-volatile ammonium ion) at a low flow rate (8 3 nLs(-1)) while the sample was aspirated through the annular gap of the concentric tubes at 25 mLs(-1) A second unit in all similar to the CMDS was operated as a capillary membrane diffusion emitter (CMDE) generating a gas flow with know concentrations of ammonia for the evaluation of the CMDS The fluids of the system were driven with inexpensive aquarium air pumps and the collected samples were stored in vials cooled by a Peltier element Complete protocols were developed for the analysis in air of NH(3) CH(3)COOH HCOOH and with a derivatization setup CH(2)O by associating the CMDS collection with the determination by CE-C(4)D The ammonia concentrations obtained by electrophoresis were checked against the reference spectrophotometric method based on Berthelot s reaction Sensitivity enhancements of this reference method were achieved by using a modified Berthelot reaction solenoid micro-pumps for liquid propulsion and a long optical path cell based on a liquid core waveguide (LCW) All techniques and methods of this work are in line with the green analytical chemistry trends (C) 2010 Elsevier B V All rights reserved

Development and Validation of a Mice liar Electrokinetic Chromatography Method for Quantitative Determination of Butenolides in Piper malacophyllum (C. Presl) C. DC.

Introduction - A large number of natural and synthetic compounds having butenolides as a core unit have been described and many of them display a wide range of biological activities. Butenolides from P. malacophyllum have presented potential antifungal activities but no specific, fast, and precise method has been developed for their determination. Objective - To develop a methodology based on micellar electrokinetic chromatography to determine butenolides in Piper species. Methodology - The extracts were analysed in an uncoated fused-silica capillaries and for the micellar system 20 mmol/L SDS, 20% (v/v) acetonitrile (ACN) and 10 mmol/L STB aqueous buffer at pH 9.2 were used. The method was validated for precision, linearity, limit of detection (LOD) and limit of quantitation (LOQ) and the standard deviations were determined from the standard errors estimated by the regression line. Results - A micellar electrokinetic chromatography (MEKC) method for determination of butenolides in extracts gave full resolution for 1 and 2. The analytical curve in the range 10.0-50.0 mu g/mL (r(2) = 0.999) provided LOD and LOQ for 1 and 2 of 2.1/6.3 and 1.1/3.5 mu g/mL, respectively. The RSD for migration times were 0.12 and 1.0% for peak area ratios with 100.0 +/- 1.4% of recovery. Conclusions - A novel high-performance MEKC method developed for the analysis of butenolides 1 and 2 in leaf extracts of P. malacophyllum allowed their quantitative determined within an analysis time shorter than 5 min and the results indicated CE to be a feasible analytical technique for the quantitative determination of butenolides in Piper extracts. Copyright (C) 2010 John Wiley & Sons, Ltd.

Environmental formaldehyde analysis by active diffusive sampling with a bundle of polypropylene porous capillaries followed by capillary zone electrophoretic separation and contactless conductivity detection

Compared to other volatile carbonylic compounds present in outdoor air, formaldehyde (CH2O) is the most toxic, deserving more attention in terms of indoor and outdoor air quality legislation and control. The analytical determination of CH2O in air still presents challenges due to the low-level concentration (in the sub-ppb range) and its variation with sampling site and time. Of the many available analytical methods for carbonylic compounds, the most widespread one is the time consuming collection in cartridges impregnated with 2,4-dinitrophenylhydrazine followed by the analysis of the formed hydrazones by HPLC. The present work proposes the use of polypropylene hollow porous capillary fibers to achieve efficient CH2O collection. The Oxyphan (R) fiber (designed for blood oxygenation) was chosen for this purpose because it presents good mechanical resistance, high density of very fine pores and high ratio of collection area to volume of the acceptor fluid in the tube, all favorable for the development of air sampling apparatus. The collector device consists of a Teflon pipe inside of which a bundle of polypropylene microporous capillary membranes was introduced. While the acceptor passes at a low flow rate through the capillaries, the sampled air circulates around the fibers, impelled by a low flow membrane pump (of the type used for aquariums ventilation). The coupling of this sampling technique with the selective and quantitative determination of CH2O, in the form of hydroxymethanesulfonate (HMS) after derivatization with HSO3-, by capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-(CD)-D-4) enabled the development of a complete analytical protocol for the CH2O evaluation in air. (C) 2008 Published by Elsevier B.V.

Fabrication and integration of planar electrodes for contactless conductivity detection on polyester-toner electrophoresis microchips

In this report, we describe the microfabrication and integration of planar electrodes for contactless conductivity detection on polyester-toner (PT) electrophoresis microchips using toner masks. Planar electrodes were fabricated by three simple steps: (i) drawing and laser-printing the electrode geometry on polyester films, (ii) sputtering deposition onto substrates, and (iii) removal of toner layer by a lift-off process. The polyester film with anchored electrodes was integrated to PT electrophoresis microchannels by lamination at 120 degrees C in less than 1 min. The electrodes were designed in an antiparallel configuration with 750 mu m width and 750 gm gap between them. The best results were recorded with a frequency of 400 kHz and 10 V-PP using a sinusoidal wave. The analytical performance of the proposed microchip was evaluated by electrophoretic separation of potassium, sodium and lithium in 150 mu m wide x 6 mu m deep microchannels. Under an electric field of 250 V/cm the analytes were successfully separated in less than 90 s with efficiencies ranging from 7000 to 13 000 plates. The detection limits (S/N = 3) found for K+, Na+, and Li+ were 3.1, 4.3, and 7.2 mu mol/L, respectively. Besides the low-cost and instrumental simplicity, the integrated PT chip eliminates the problem of manual alignment and gluing of the electrodes, permitting more robustness and better reproducibility, therefore, more suitable for mass production of electrophoresis microchips.

A rapid and reliable bonding process for microchip electrophoresis fabricated in glass substrates

In this report, we describe a rapid and reliable process to bond channels fabricated in glass substrates. Glass channels were fabricated by photolithography and wet chemical etching. The resulting channels were bonded against another glass plate containing a 50-mu m thick PDMS layer. This same PDMS layer was also used to provide the electrical insulation of planar electrodes to carry out capacitively coupled contactless conductivity detection. The analytical performance of the proposed device was shown by using both LIF and capacitively coupled contactless conductivity detection systems. Efficiency around 47 000 plates/m was achieved with good chip-to-chip repeatability and satisfactory long-term stability of EOF. The RSD for the EOF measured in three different devices was ca. 7%. For a chip-to-chip comparison, the RSD values for migration time, electrophoretic current and peak area were below 10%. With the proposed approach, a single chip can be fabricated in less than 30 min including patterning, etching and sealing steps. This fabrication process is faster and easier than the thermal bonding process. Besides, the proposed method does not require high temperatures and provides excellent day-to-day and device-to-device repeatability.

Simultaneous voltammetric determination of paracetamol and caffeine in pharmaceutical formulations using a boron-doped diamond electrode

A simple and highly selective electrochemical method was developed for the single or simultaneous determination of paracetamol (N-acetyl-p-aminophenol, acetaminophen) and caffeine (3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione) in aqueous media (acetate buffer, pH 4.5) on a boron-doped diamond (BDD) electrode using square wave voltammetry (SWV) or differential Pulse voltammetry (DPV). Using DPV with the cathodically pre-treated BDD electrode, a separation of about 550 mV between the peak oxidation potentials Of paracetamol and caffeine present in binary mixtures was obtained. The calibration curves for the simultaneous determination of paracetamol and caffeine showed an excellent linear response, ranging from 5.0 x 10(-7) mol L(-1) to 8.3 x 10(-7) mol L(-1) for both compounds. The detection limits for the simultaneous determination of paracetamol and caffeine were 4.9 x 10(-7) mol L-1 and 3.5 x 10(-8) mol L(-1), respectively. The proposed method Was Successfully applied in the simultaneous determination of paracetamol and caffeine in several pharmaceutical formulations (tablets), with results similar to those obtained using a high-performance liquid chromatography method (at 95% confidence level). (C) 2008 Elsevier BY. All rights reserved.

Análise químico-farmacêutica de norflocacino comprimidos e estudos de complexação com β-Ciclodextrina e polimorfismo

Pós-graduação em Ciências Farmacêuticas - FCFAR

Análise químico-farmacêutica e estudo de estabilidade e de dissolução de cápsulas de flucloxacilina sódica

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Comparative study over methods developed for quantification of darunavir in tablets by environmental friendly infrared and capillary electrophoretic techniques

Darunavir, a protease inhibitor used in the treatment of HIV infection, presents few methods for its determination in pharmaceuticals. Infrared (IR) spectroscopy offers the possibility of obtaining spectra relatively quickly, providing interesting information, analytically, qualitatively or quantitatively. Capillary electrophoresis (CE) performs separations of high efficiency in shorter time with reagents and samples in small quantity. These two methods are cost-benefitted when we evaluate the green level and the cost of analysis. Faster and cheaper methods without generating organic waste by IR and CE for the quantification of darunavir were developed and validated, focusing socioeconomic impact of analytical decisions. If the cost of acquisition, maintenance, production, analysis and conditioning of drugs and pharmaceuticals is high, consequently the price of this product in the market will be higher and it cannot be accessible to the patient. Treatment failure not only affects the quality of life of patients, but also contributes significantly to the economic burden of the health system. In this context there is a tool called Analysis of the Life Cycle, which comes to make us think in a multidimensional way focusing the whole, the parts and especially the interaction among the parts of a system.