The pKa values of organic acids (HCOOH, CH3CH2COOH, HOCH2COOH, HSCH2COOH and FCH2COOH) in a liquid chromatography (LC) eluent mainly influenced the generation of adduct ions with corticosteroids. The relative abundance of [M + R] adduct ions increased as the pKa value of the organic acid decreased. An interaction between the functional groups of the organic acids in the LC eluent and those of the corticosteroid affected the generation of fragment ions. When HCOOH was present in the mobile phase, the sensitivity to endogenous hydrocortisone and cortisone in human urine was maximized.