Approach
The incompatibility of generic reverse-phase chromatography necessitated the rapid development of an LC-MS/MS method using hydrophilic interaction chromatography (HILIC). The new method employed a conventional HILIC column and resulted in good retention and chromatographic peak shape for all analytes under investigation.
However, for some of the compounds, interference from co-eluting metabolites necessitated further method development, resulting in the use of a fused core pentafluoro- phenyl (PFP) column with an isocratic mobile phase of 95% acetonitrile/5% ammonium formate (200mM). In this way, excellent retention, separation and peak shape was achieved for all analytes and all samples were turned around within four working days of compound receipt.