One way to identify peaks in a chromatogram is by their relative retention time (RRT). However, because of a lack of harmonization in the calculation of RRT, interpretation of results may be difficult. In both the European Pharmacopoeia (Ph. Eur.) and the United States Pharmacopeia, much confusion remains because no method for the determination of hold-up times is prescribed. Also, the influence of the stationary phase must be taken into account. In this study, five separations prescribed by the Ph. Eur. were performed on 59 different reversed-phase liquid chromatography C18 columns and the variability of RRTs between them was studied. It is shown that the RRT alone is not sufficient for correct peak identification.