The effect of eluent pH and compound acid-base character on the design of generic-gradient reversed-phase high-performance liquid chromatography (RP-HPLC) methods for use in drug discovery.

The design of generic reversed-phase high-performance liquid chromatography (RP-HPLC) gradient methods for the analysis of compound mixtures or 'cocktails' has been investigated with particular reference to the eluent pH and the type of compound (acid, base or neutral) analysed. The use of eluents with an acidic eluent pH, an approach which is widely employed, can lead to non-retention of polar bases resulting in 'failure' of the method. This problem is aggravated where the majority of compounds submitted for analysis are bases, which is typical of many drug discovery programs. The problem can be ameliorated through the use of eluents with near neutral pH. Although these neutral pH eluents can lead to co-elution when cocktails are analysed and possibly ion-suppression where mass spectrometry (MS) is the detection method, this can be avoided through optimisation of the gradient shape.