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Metabolomics deals with analysis of highly complex biological samples resulting in insufficient separation of relevant metabolites from matrix substances with one-dimensional liquid chromatography. Comprehensive twodimensional liquid chromatography (2D-LC) is a promising technique for the analysis of complex samples because of its increased peak capacity. Comprehensive 2D-LC-MS/MS has been applied for a targeted metabolomics approach regarding metabolites from RNA catabolism. Combination of a Zorbax Eclipse Plus C18 column with a Zorbax Bonus-RP column resulted in increased peak capacity, minimisation of matrix effects and excellent reproducibility.