Impurity profiling of drug candidates: Analytical strategies using reversed-phase and mixed-mode high-performance...

The development of new active pharmaceutical ingredients (API) requires accurate impurity profiling. Nowadays, reversed-phase HPLC (RPLC) on C18 stationary phase is the method of first choice for this task and usually employed in generic screening methods. However, this method sometimes fails, especially when the target analyte is not sufficiently retained, making impurity analysis difficult or even impossible. In such cases, a second method must be available. In the present paper, we compare the merits of RPLC on C18 phase to those of previously optimized alternative methods, based on the analysis of a large and diverse set of small-molecule drug candidates. Various strategies are considered: RPLC on C18 phase but with different mobile phase composition (acidic or basic), RPLC with a pentafluorophenyl stationary phase, or mixed-mode HPLC with both bimodal and trimodal stationary phases. First, method performances were compared in terms of response rate (proportion of compounds eluted) and peak shapes for a large set of synthetic drugs (140) with structural diversity and their orthogonality was evaluated. Then a subset of compounds (25) containing varied impurity profiles was used to compare the methods based on the capability to detect impurities and evaluate the relative purity of the API.