Comparison of reversed-phase and hydrophilic interaction liquid chromatography for the quantification of ephedrines using medium-resolution accurate mass spectrometry

The separation and quantification of hydrophilic basic compounds continues to challenge reversed-phase chromatography. Ephedrines are an example where the optimal separation of their isomers and related substances is complicated due to both their hydrophilicity and basic nature. Here we study two potential ultra-high pressure liquid chromatography (UHPLC) methods and present the merits and limitations of a high pH reversed-phase and a hydrophilic interaction liquid chromatography (HILIC) approach for the separation and quantification of ephedrines for doping control analysis. The study compares a hybrid silica material used for the HILIC separations with a C18 reversed-phase material produced from the same hybrid silica. While both analytical approaches provide good retention and resolution, HILIC offers benefits in terms of peak shape, sample loading capacity and enhanced sensitivity with electrospray ionisation-mass spectrometry (ESI-MS). HILIC permits favourable kinetic performance owing to the low viscosity mobile phase and hence better mass transfer characteristics. Common problems associated with HILIC including retention shifts and undesirable peak shapes are investigated and overcome using a suitable re-equilibration time and injection solvent. Validation data show both approaches provide good linearity (r>0.995), accuracy (RPLC