High-performance liquid chromatographic determination of deoxycytidine monophosphate and methyldeoxycytidine monophosphate for DNA demethylation monitoring: experimental design and artificial neural networks optimisation

The optimal conditions for the separation of 2'-deoxycytidine-5'-monophosph ate and 5-methyl-2'-deoxycytidine-5'-monophosphate in the matrix of other n atural occurring nucleotides after digest of DNA were investigated. Using e xperimental design combined with artificial neural networks, efficient opti misation of the HPLC separation conditions was performed. The mobile phase composition was optimised on the basis of its three components (concentrati on of phosphate, content of methanol and pH), The best separation was obtai ned with a mobile phase containing 50 mM phosphate, pH 5.5 and 6% methanol, The final resolution achieved between 2'-deoxycytidine-5'-monophosphate an d 5-methyl-2'-deoxy cytidine-5'-monophosphate was equal to 2.78. Finally, t he optimised system was successfully tested on the nucleotide mixture solut ion to determine the methylation state of 2'-deoxycytidine-5'-monophosphate in DNA in the search for FMR1 gene changes. (C) 2001 Elsevier Science B.V. All rights reserved.