The development of a method to determine felinine in body fluids by capillary electrophoresis : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Chemistry at Massey University

Ion-exchange, paper-chromatography and high performance liquid chromatography were used in earlier studies for the determination of felinine in biological fluids. These methods were either inadequate and/or need laborious sample pre-treatments. A new method for the determination of felinine by capillary zone electrophoresis has been developed. Preliminary investigations were carried out to address the conditions required for the separation of felinine. The separation of felinine can be performed on a fused-silica capillary with a 20 mM phosphate buffer (pH 2.0) and detection wavelength 200 nm. The separation principle was based on the different migration times due to the different molecular weights, molecular sizes and charges under an applied potential field. The quantitative determination of felinine levels in cat urine has been achieved. The cat urine analysis was performed directly on the capillary electrophoresis without making any felinine derivative(s). The levels of felinine in different cat genders are reported. The results were compared with the results of an HPLC felinine derivatization method. Felinine levels in entire male cat urine were much higher than those in female and castrated male cat urine. A synthetic felinine was employed as standard felinine. Linear relationships between peak area and concentration of synthetic felinine calibrations are reported. Mean felinine recovery in cat urine was 95.9%. Taurine, urea, creatine and creatinine, which exist in large amounts in cat urine, showed no interference with the analysis of felinine by this method. The new capillary zone electrophoresis method was then applied to the study of felinine stability. Conditions reported to influence the stability of felinine were investigated. These conditions included oxidation, storage temperatures and times, heating, acidic and alkaline solutions. Both synthetic felinine and felinine in cat urine were investigated. Storage temperature (-20°C to 20°C) had no significant influence on the stability of felinine while higher temperatures increased the decomposition of felinine. Felinine degraded at strong acid and base conditions but was relatively stable under mild acid and base conditions. A similar stability of felinine in human urine is also reported. The capillary zone electrophoresis method was also employed to study felinine in plasma and serum. Plasma and serum as well as urine can be analysed directly on the capillary electrophoresis after sufficient dilution. Conditions (eg. protein clean up, changing of injection time, 37°C heating) that might influence of felinine behaviour in plasma and serum are discussed. This study indicated that no traces felinine be found in cat plasma, within the detection limits of this new capillary electrophoresis method

Capillary electrophoresis characterisation of a rapid prototyped PMMA chip for particle analysis

Màster en Nanociència i NanotecnologiaA rapid and cheap method has been developed for the fabrication of a capillary electrophoresis chip for the preliminary characterization of particles. The microfluidic chips were fabricated using polymethyl methacrylate (PMMA) with integrated platinum electrodes without the need of using high technology microfabrication techniques. The chips were characterized using electroosmotic flow (EOF) with different channel treatments. The electrodes were characterised with impedance and conductivity measurements using both static and electrophoretic flow, respectively. Nine micron diameter particles were detected and their electrophoretic mobility determined using capillary electrophoresis and conductivity detection

An investigation into the sample preparation procedure and analysis of cyanoacrylate adhesives using capillary electrophoresis

In this study, the trace acid profile of cyanoacrylate adhesives was studied using capillary electrophoresis. Liquid–liquid extraction was employed as the sample preparation step before separation by capillary electrophoresis. The solubility of the adhesives was investigated using various organic solvents, e.g. hexane and dichloromethane, and chloroform was determined to be the optimum solvent as it enabled the full dissolution of the adhesive. A comprehensive stability study was performed over a 3-year period and results indicate that the adhesives were stable for 2 years after which their stability and performance degraded

Use of capillary electrophoresis as a method development tool for classical gel electrophoresis

Capillary electrophoresis (CE) was used to optimize the buffer pH, ionic strength and sulfated cyclodextrin concentrations for enantiomeric separation of piperoxan. These enantioseparation conditions were then applied to a classical gel electrophoresis system. Binding constants of the sulfated beta-cyclodextrin–piperoxan couple were approximated using CE and the effects of organic solvents on the system were also investigated

Strongly nonlinear waves in capillary electrophoresis

In capillary electrophoresis, sample ions migrate along a micro-capillary filled with a background electrolyte under the influence of an applied electric field. If the sample concentration is sufficiently high, the electrical conductivity in the sample zone could differ significantly from the background.Under such conditions, the local migration velocity of sample ions becomes concentration dependent resulting in a nonlinear wave that exhibits shock like features. If the nonlinearity is weak, the sample concentration profile, under certain simplifying assumptions, can be shown to obey Burgers' equation (S. Ghosal and Z. Chen Bull. Math. Biol. 2010, 72(8), pg. 2047) which has an exact analytical solution for arbitrary initial condition.In this paper, we use a numerical method to study the problem in the more general case where the sample concentration is not small in comparison to the concentration of background ions. In the case of low concentrations, the numerical results agree with the weakly nonlinear theory presented earlier, but at high concentrations, the wave evolves in a way that is qualitatively different.Comment: 7 pages, 5 figures, 1 Appendix, 2 videos (supplementary material

A new multiplexed absorbance detection approach for high-throughput PCR analysis, enzyme assay, peptide mapping and combinatorial homogeneous catalysis screening using 96-capillary array electrophoresis

The purpose of this research is to develop a UV-vis absorbance detection approach for multiplexed capillary electrophoresis, and explore the use of multiplexed capillary electrophoresis with UV-vis absorbance detection as a high-throughput analysis technique for various types of applications;We first introduced a novel absorbance detection approach for 96-capillary array electrophoresis. The approach involved the use of a 1024-pixel linear photodiode array on which a 96-capillary array was imaged by a camera lens. The detection limit for Rhodamine 6G for each capillary in the multiplexed array was about 1.8 x 10--8 M (S/N = 2). The cross talk between adjacent capillaries is less than 0.2%. Uniform separation efficiency and signal to noise ratio were obtain across the capillary array. Simultaneous analysis of 96 samples using capillary zone electrophoresis (CZE) was demonstrated;We further successfully applied the 96-capillary array electrophoresis system with UV-vis absorbance detection for high-throughput HIV diagnosis, genetic typing, enzyme assay, protein analysis by peptide mapping, and combinatorial screening of homogeneous catalytic reactions

Gel versus capillary electrophoresis genotyping for categorizing treatment outcomes in two anti-malarial trials in Uganda

Abstract Background Molecular genotyping is performed in anti-malarial trials to determine whether recurrent parasitaemia after therapy represents a recrudescence (treatment failure) or new infection. The use of capillary instead of agarose gel electrophoresis for genotyping offers technical advantages, but it is unclear whether capillary electrophoresis will result in improved classification of anti-malarial treatment outcomes. Methods Samples were genotyped using both gel and capillary electrophoresis from randomized trials of artemether-lumefantrine (AL) vs. dihydroartemisinin-piperaquine (DP) performed in two areas of Uganda: Kanungu, where transmission is moderate, and Apac, where transmission is very high. Both gel and capillary methods evaluated polymorphic regions of the merozoite surface protein 1 and 2 and glutamine rich protein genes. Results Capillary electrophoresis detected more alleles and provided higher discriminatory power than agarose gel electrophoresis at both study sites. There was only moderate agreement between classification of outcomes with the two methods in Kanungu (kappa = 0.66) and poor agreement in Apac (kappa = 0.24). Overall efficacy results were similar when using gel vs. capillary methods in Kanungu (42-day risk of treatment failure for AL: 6.9% vs. 5.5%, p = 0.4; DP 2.4% vs. 2.9%, p = 0.5). However, the measured risk of recrudescence was significantly higher when using gel vs. capillary electrophoresis in Apac (risk of treatment failure for AL: 17.0% vs. 10.7%, p = 0.02; DP: 8.5% vs. 3.4%, p = 0.03). Risk differences between AL and DP were not significantly different whether gel or capillary methods were used. Conclusions Genotyping with gel electrophoresis overestimates the risk of recrudescence in anti-malarial trials performed in areas of high transmission intensity. Capillary electrophoresis provides more accurate outcomes for such trials and should be performed when possible. In areas of moderate transmission, gel electrophoresis appears adequate to estimate comparative risks of treatment failure

Kapilarna elektroforeza u farmaciji

Capillary electrophoresis is a new separation technique. It is an analytical technique that usually complements or replaces high performance liquid chromatography. Basic principle of the technique is the migration of charged species under applied electric field towards one of the electrodes through a narrow capillary filled with electrolyte solution. Main advantages over other chromatographic methods are short analysis time, high efficiency, small sample and solvent volumes, low costs, simplicity and ecological acceptability. Capillary electrophoresis has several modes of separation which allows analysis of different types of analytes. Capillary electrophoresis has found its application in the drug analysis of large molecules like proteins, peptides and nucleic acids, small organic molecules such as drugs, hormones, plant metabolites, food product ingredients, small inorganic ions etc. It is a method of choice when rapid results are needed. Capillary electrophoresis has become the predominant technique for the analysis of both basic and chiral pharmaceuticals, it is unavoidable in biotechnology and promises to be a valuable tool in troubleshooting in proteome-wide analysis, DNA sequencing and genotyping. Despites of all advantages and application possibilities, capillary electrophoresis is still not enough explored and used in routine analysis. A review of different capillary electrophoresis techniques principles and applications in pharmacy is presented in this article