Separation of Cyclic GMP AND Cyclic AMP from Other Nucleotides by Reverse Phase Hplc

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Abstract
An accurate, specific and sensitive method to identify and quantify both cyclic CMP and cyclic AMP by HPLC is described and applied to standards mixtures and lichen extracts. Separation is achieved by reverse phase HPLC on a MCH-10 column by using methanol:water:acetic acid (80:19.5:0.5 v/v), isocratically, as mobile phase. Selectivity and resolution coefficients are always higher than 1.0. Cyclic nucleotides conveniently separate from CMP, AMP and adenosine. Recovery of cyclic CMP from biological samples has been estimated as about 93% after extraction procedure whereas that of cyclic AMP varies from 92.25% to 55.61 % depending on phosphodiesterase activity. By using this method, cyclic nucleotides have been identified from lichen species, Himantormia lugubris and Usnea aurantiaco-atra. Whereas cyclic AMP is the main cyclic nucleotide contained in the first species, cyclic CMP is the most abundant in the second one. In addition, apothecia of Usnea seem to contain an active phosphodiesterase which hydrolyzes mainly cyclic AMP.