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- W930586490 abstract "Gonadotropin-releasing hormone (GnRH) and its complex with Cu 2+ (Cu-GnRH) were separated on a Nova Pak C 18 column (4 μm, 150x3.9 mm I.D., Waters). Analyses of underivatized GnRH and Cu-GnRH were performed by a gradient elution program (HPLC method I), UV detection at 280 nm and fluorescence detection (λ excitation = 280 nm/λ emission = 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 of28°C. The proposed gradient elution program with UV and fluorescence detection allowed satisfactory fractionation of GnRH (at 31.5±0.2 min) from Cu-GnRH (at 30.3±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 overlapping 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 amino acids 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 Cu 2+ in pituitary cytosol and sub-cellular organelles." @default.
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- W930586490 date "2009-01-01" @default.
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- W930586490 title "Direct Liquid Chromatography with Photodiode and Fluorescence Detection for Simultaneous Quantification of Gonadotropin-Releasing Hormone (GnRH), Its Complex with Cu2+, and Catabolites" @default.
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