Within the scope of research for target and non-target LC-MS/MS analysis of membrane degradation products of polymer electrolyte membrane fuel cells, a systematic method development for the separation of structurally similar compounds was performed by phase optimized liquid chromatography. Five different stationary phases with different selectivities were used. Isocratic separation for 4-hydroxybenzoic acid, isophthalic acid, terephthalic acid, 4-hydroxybenzaldehyde and 4-formylbenzoic acid was achieved on a C18 and a Phenyl phase. Using the PRISMA model the separation efficiency was optimized. This was achieved on a serially connected mixed stationary phase composed of 30 mm C18, 150 mm Phenyl and 60 mm C30. For the LC-MS screening of unknown degradation products from polymer electrolyte membranes in the product water of a fuel cell, a solvent gradient is mandatory for less polar or later eluting compounds. By means of 4-mercaptobenzoic acid it could be shown that a solvent gradient can be applied in order to elute later eluting compounds in a short time. The adaptability of this method for the qualitative analysis by target and non-target LC-MS/MS screening has been shown by means of 4-hydroxybenzoic acid. The combination of solvent gradient and isocratic conditions makes this approach attractive for the purpose of a screening method for known and unknown analytes in a water sample.