Several reversed-phase columns were evaluated for HPLC. In view of the inherent matrix effects on the electrospray ionisation process of the analytes of interest, the Inertsil® column exhibited the best separation characteristic for analytes and the majority of the matrix components with the given mobile phase composition. Besides, the mixture of methanol and water (50:50, v/v) with 10 mM ammonium acetate which was chosen for the experiments gave the best compromise with regard to short analysis time and sufficient chromatographic separation for 2-NBA semicarbazone and 4-NBA semicarbazone which was used as internal standard (IS). Acetonitrile as organic modifier showed no advantages concerning sensitivity or stability of MS detection while the addition of ammonium acetate improved the intensity of the MS response significantly.