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

• A HILIC method is presented for the quantification of ephedrines in doping control.
• We compare HILIC and high pH RPLC for accurate and robust quantification.
• Similar results for accuracy and precision were found between the two methods.
• Sample loading capacity, linearity and ESI sensitivity were increased with HILIC.
• Specific and sensitive analysis is achieved with QToF 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 < 7.5% error, HILIC < 7.6% error) and precision (RPLC < 7.0% RSD, HILIC < 10.3% RSD) for all analytes. Matrix effects were shown to have a negligible effect on ionisation variability in each mode, with inter-day retention times also being repeatable (<0.17% RSD). HILIC gave increased sensitivity with ESI-MS, giving a 6-fold increase in signal over the RPLC approach. In this application, we demonstrate the use of UHPLC technology coupled with a hybrid quadrupole time-of-flight (QToF) mass analyser. This approach provides fast scanning medium-resolution accurate mass detection for reliable identification and quantification purposes.