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

Abstract

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