title: High-performance liquid chromatographic determination of deoxycytidine monophosphate and methyldeoxycytidine monophosphate for DNA demethylation monitoring: experimental design and artificial neural networks optimisation.
abstract: The optimal conditions for the separation of 2'-deoxycytidine-5'-monophosphate and 5-methyl-2'-deoxycytidine-5'-moncphosphate in the matrix of other natural occurring nucleotides after digest of DNA were investigated. Using experimental design combined with artificial neural networks, efficient optimisation of the HPLC separation conditions was performed. The mobile phase composition was optimised on the basis of its three components (concentration of phosphate, content of methanol and pH). The best separation was obtained with a mobile phase containing 50 mM phosphate, pH 5.5 and 6% methanol. The final resolution achieved between 2'-deoxycytidine-5'-monophosphate and 5-methyl-2'-deoxycytidine-5'-monophosphate was equal to 2.78. Finally, the optimised system was successfully tested on the nucleotide mixture solution to determine the methylation state of 2'-deoxycytidine-5'-monophosphate in DNA in the search for FMR1 gene changes.
authors.affiliation: Department of Analytical Chemistry, Faculty of Science, Masaryk University, Brno, Czech Republic. jdqh@chemi.muni.cz
authors.forename: J
authors.initials: J
authors.lastname: Havlis
authors.forename: J E
authors.initials: JE
authors.lastname: Madden
authors.forename: A L
authors.initials: AL
authors.lastname: Revilla
authors.forename: J
authors.initials: J
authors.lastname: Havel
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019175:DNA Methylation; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000737:chemistry*; D003843:Deoxycytidine Monophosphate / Q000737:chemistry*; D016571:Neural Networks, Computer; D012107:Research Design
chemical_list: D003841:Deoxycytidine; D003843:Deoxycytidine Monophosphate; C016569:5-methyldeoxycytidine