Owing to subtle differences in the structures, separation of allthe compounds under isocratic conditions was not feasible. There-fore, various gradient elution programs were tried using ACN,MeOH and their mixtures as the organic phase. Because all theexamined compounds (apart from TAD) contain one or more basicnitrogen centers, the pH of the mobile phase had to be optimized.The effect of pH was studied in the range from 2.7 through 7.0using mixtures of ammonium acetate and acetic acid. Fig. 2 showschromatograms as a function of pH of mobile phase A. Regardlessof the organic phase used, increasing the pH of mobile phase Aresulted in an improvement of peak shape and resolution. For fur-ther method optimization pH 7.0 (200 mM ammonium acetate) wasapplied. Comparing the two systems at pH 7.0, neither ACN, norMeOH alone as mobile phase B were able to separate all the com-pounds (see Fig. 3). Using ACN as mobile phase B, ACE and NDV aswell as SIL and VAR co-eluted, while the use of MeOH resulted ina co-elution of VAR and HS. The elution order was also altered, themost significant being the case of ACE and TAD. The latter could beexplained by the different solubility of these substances in ACN andMeOH as compared to the other compounds. The poor separationwith both 100% ACN and 100% MeOH suggested the use of theirmixtures as mobile phase B. The most suitable system containeda 1:1 (v/v) mixture as mobile phase B, applying 200 mM ammo-nium acetate as mobile phase A using a gradient elution programas described in Section 2. It is noted that low salt concentrationin mobile phase A decreased the resolution between ACE and NDV.Elevated column temperatures showed a negative effect on the sep-aration of all compounds, therefore, no column heating was appliedand room temperature (25◦C) was chosen. 5 l injection volumeand 0.5 ml/min flow rate was found to be optimal. Fig. 4 shows arepresentative chromatogram obtained with the optimized system.