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1 Polish Journal of Chemistry

1 2009

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Direct Liquid Chromatography with Photodiode and Fluorescence Detection for Simultaneous Quantification of Gonadotropin-Releasing Hormone (GnRH), Its Complex with Cu2+, and Catabolites

Michaluk A., Gajewska A., Kulon K., Kozłowski H., Kochman K., Kowalczyk J., Czauderna M.

Polish Journal of Chemistry

2009

Vol. 83, nr 3

383-390

Gonadotropin-releasing hormone (GnRH) and its complex with Cu2+(Cu-GnRH) were separated on a Nova Pak C18 column (4 m, 150 3.9 mm I.D., Wa ters). Analyses of underivatized GnRH and Cu-GnRH were per formed by a gradient elution program (HPLC method I), UV detection at 280 nm and fluorescence detection (gammexcitation = 280 nm/gammaemis sion = 360 nm). The mobile phases used were: acetonitrile with 0.08% trichloroacetic acid (w/w) and water with 0.1% trichloroacetic acid (w/w). Elutions were carried out in a binary gradient mode with a flow-rate of 1 mL/min and column temperature of 28°C. The proposed gradient elution program with UV and fluorescence detection allowed satisfactory fraction ation of GnRH (at 31.5 plus minus 0.2 min) from Cu-GnRH (at 30.3 plus-minus 0.2 min) and endogenous species present in samples of cytosol and subcellular organelles from the hypothalamus. The proposed reversed-phase HPLC method I with fluorescence detection provides a more sensitive analytical tool for routine and simultaneous quantification of GnRH, Cu-GnRH and their enzymatic degradation products (catabolites) in all biological samples as compared with HPLC method I with UV detection. To avoid problems due to overlap ping peaks corresponding to GnRH, Cu-GnRH, and the respective enzymatic degradation products in samples of cytosol and subcellular organelles from the hypothalamus, we propose a very shallow binary gradient elution program (method I). The separation efficiency of GnRH and Cu-GnRH peaks in standards and biological samples was assessed based on purity analysis of UV spectra (250-300 nm) and on the values of ratios of the fluorescence response to UV response at 280 nm. Our second reversed-phase liquid chromatographic method (HPLC method II) with pre-column derivatization of aminoacids in catabolites of GnRH and Cu-GnRH enabled investigations of the degradation pattern as well as of the yield of enzymatic degradation of GnRH and its Complex with Cu2+ in pituitary cytosol and sub-cellular organelles.